Photophysiological Adaptations and Acclimations of the Seagrass

Halophila stipulacea to Extreme Light Environments

Thesis submitted for the degree

"Doctor of Philosophy"

By Yoni Sharon

Submitted to the Senate of Tel Aviv University

This work was supervised by Prof. Sven Beer

In memory of my father [29.6.1936-4.11.2008]

Acknowledgements I would like to express my deep gratitude to my supervisor Prof. Sven Beer for his guidance, help and support in the last 4 years. Sven was an advisor and a friend who educated and supported me in both "life" and "science".

The Inter-University Institute (IUI) in Eilat hosted me for 3 experiencing years and gave me the wonderful opportunity to perform science and underwater exploration off-shore in the magical Red Sea. In the IUI many friends have been escorted me throughout my studies and I would like to thank personally Ada Alamaro, Inbal Ayalon, Oded Ben- Shaprut, Eran Brokovitz, Margarita Zurubin and Maya and Cam Veal for their friendship and help through the years.

I would like to thank the "Department of Plant Sciences" or the new "Department of Molecular Biology and Ecology of Plants" at Tel Aviv University for sponsoring me in the last 4 years of my studies. Among the departments members I would like to thank especially Prof. Amram Eshel and Prof. Danny Chamovitz for their constructive advises through the first years of my PhD work.

I would like to thank my collaborators: Dr. Ilana Berman-Frank, Orly Levitan and Dina Spungin, from Bar-Ilan University, for hosting me in their lab and assisting me during the last year. I also thank my collaborators Prof. Rui Santos and Dr. Joao Silva from the University d'Algarve in Faro, Portugal, for hosting me during two visits in the amazing south coast of Portugal.

Finally, I would thank my mother for supporting me and believing in me for the last 30 years, my deceased father who taught me all about "Windows and Doors", and to my beautiful loving wife whom I met during my studies on the magical IUI's Pier.

Summary

This PhD thesis started with field observations, leading my curiosity to investigate a marine angiosperm (or seagrass) that grows from the intertidal down to the great depths of >30 m. Investigating the photophysiology of this plant enabled me to learn the special characteristics in its acclimation and adaptation to extreme light environments. One can also use the scientific findings of this current work in order to investigate other marine plants and their eco-physiological responses to extreme (both high and low) irradiances.

In the marine environment, light quantity and quality change greatly along depth gradients. Photosynthetic organisms inhabiting those gradients thus need to acclimate or adapt in different ways in order to utilize low light (which is composed of mostly blue wavelengths) in deep waters and to avoid chronic damage in shallow waters where visible irradiances and UV radiation are high. Halophila stipulacea, a tropical and sub-tropical seagrass, is an important ecosystem-engineering macrophyte in the Gulf of Aqaba, northern Red Sea. Meadows of this plant serve as nurseries for many fish and invertebrates species, stabilize the sediment in their habitat and take a significant role in the oxygen, nitrogen and carbon cycles. This seagrass inhabits a wide depth gradient (0 - ~50 m), featuring more than an order of magnitude differences in irradiance, in the clear waters of the Gulf of Aqaba. In this work, its photosynthetic light utilization mechanisms were investigated using fluorometric, optic, microscopic, spectrophotometric and biochemical methods, in order to understand how this plant can grow under very dim light at depth and survive under high light and UVR stress in the intertidal. Thus, the main goal was to explain the photophysiological strategies that allow this plant to acclimate and adapt to conditions along a depth gradient where differences in irradiance are extreme.

Our first findings quantified and found a rationale for the observation of diurnal chloroplast movements in high-light growing Halophila stipulacea plants.

Along the morning hours till midday the chloroplasts clumped together and then dispersed evenly toward the late afternoon. These chloroplast clumping and dispersal were accompanied with changes in the absorption factor (AF) and, thus, affected the calculated electron transport rates (ETR) since the latter depends linearly on the amount of light absorbed by the photosynthetic tissues. This finding thus showed the importance of taking into account changing AF values along the day when calculating

ETRs of Halophila stipulacea, and perhaps other seagrasses too if featuring diurnally changing AFs under high-light conditions (Ch. 1).

In continuation with our first findings, we investigated the spectrum responsible for chloroplasts clumping in high-light growing Halophila stipulacea plants. Since potentially harmful UVR is present in the shallow waters of the Gulf of

Aqaba where part of its population thrives, we examined the effect of UVR on the clumping phenomenon. It was found that no chloroplast movements occurred when the plants were protected from 80% of the <400 nm wavelengths. In addition, UVR also affect the effective quantum and the ETRs. We conclude that the potentially harmful effects of UVR on the photosynthetic apparatus of Halophila stipulacea are mitigated by its activation of chloroplast clumping, which functions as a means of protecting most chloroplasts and perhaps the nucleus from high irradiances, including

UVR (Ch. 4).

Halophila stipulacea's successful growth under a broad irradiance gradient shows either a high plasticity or is caused by longer-term adaptations to the various depths, possibly resulting in the formation of ecotypes. In order to answer this question, we transplanted shoots of this seagrass between the extreme depths of its

3 distribution at the study time (8 and 33 m) in order to evaluate its acclimation potential to various irradiances. Our findings showed fast changes in photosynthetic responses and light absorption characteristics in response to changing light environments and suggested that Halophila stipulacea is a highly plastic seagrass with regard to irradiance, which may partly explain its abundance across a wide range of irradiances along the depth gradient that it occupies (Ch. 3). In addition, we used a novel PAM fluorometer to study the physiology of light-responses of native populations of Halophila stipulacea. We suggest that this seagrass acclimates to the widely varying irradiances across this depth gradient by regularly modulating down- regulation based non-photochemical quenching processes (Ch. 2).

Based on the extreme irradiance differences in Halophila's stipulacea light- environment along its depth gradient, we set out to estimate the potential contributions of PSII and PSI to light absorption, and photosynthetic ETRs, in plants growing at 1 and 48 m depth. Our results showed significant differences in the content of PSI in the two depth borders of Halophila stipulacea's meadow. The latter difference also affects the FII value (the PSII:(PSII+PSI) ratio) which effects linearly the photosynthetic

ETR calculations. We concluded that the ability of Halophila stipulacea to alter its amount of PSI relative to PSII according to the ambient irradiance and spectrum may be a basis for this organism's successful growth down to its exceptional depth limit in clear tropical waters (Ch. 5).

Index

Summary……………………………………………………………………………...1

1. General Introduction

1.1 Overview of relevant research fields

1.1.1 Seagrasses………………………………………………………..6

1.1.2 Light in the marine environment………………………………...9

1.1.3 Light absorption and utilization in marine plants………………10

1.1.4 Chloroplasts movement and clumping in plants……………….12

1.1.5 Light absorption under UVR and high light environments…….14

1.1.6 Acclimation processes of seagrasses………………………...…16

1.1.7 The photosystems' apparatus and stoichiometry………….……18

1.2 Research objectives………………………………………………………19

2. Research Chapters

2.1 Diurnal movements of chloroplasts in Halophila stipulacea and their

effect on PAM fluorometric measurements of photosynthetic rates…….21

2.2 Photosynthetic responses of Halophila stipulacea to a light gradient. I. In

situ partitioning of non-photochemical quenching…………………..…..25

2.3 Photosynthetic responses of Halophila stipulacea to a light gradient. II.

Acclimation following transplantations…………………………….....…35

2.4 Chloroplast clumping in the seagrass Halophila stipulacea is triggered by

UV radiation…………………………………………………………...…40

2.5 Photoacclimation of the seagrass Halophila stipulacea to the dim

irradiance at its 50 m depth limit………………………………………....61

2.6 Measuring seagrass photosynthesis: methods and applications………….67

3. Discussion……………………………………………………………………...82

4. Conclusions………………….……………………………………………...... 91

5. References……………………………………………………………………..92

1. General Introduction

1.1 Overview of relevant research fields

1.1.1 Seagrasses

Seagrasses (Angioperms: Monocotyledons) are a unique group of angiosperm that have adapted to the submerged marine environment. There, profoundly they influence the physical, chemical, and biological environments in coastal waters, performing as ecosystem engineers (Wright and Jones 2006) and providing several important ecological services to their environment (Costanza et al. 1997). Seagrasses modify water flow, nutrient cycling and food web structure (Hemminga and Duarte

2000). They are a vital food source for mega-herbivores such as green sea turtles, dugongs and manatees, and provide shelter and home for many animals, including commercially and recreationally important fishery species (Beck et al. 2001). In addition, seagrasses, through their root systems, stabilize sediments and generate large quantities of organic carbon through photosynthesis and carbon fixation

Seagrasses are distributed in marine environments across the globe, from arctic to tropical regions, but dissimilarly to other taxonomic groups with global distribution, they display a low taxonomic richness (approximately 60 species worldwide, compared with approximately 250,000 terrestrial angiosperms). The three seagrass families (Hydrocharitaceae, Cymodoceaceae and Zosteraceae) evolved from a single lineage of monocotyledonous sometimes between 70-100 million yeas ago

(Les et al. 1997). These plants are distinguished from other plant groups that have colonized the marine environment, such as salt marsh plants, mangroves, and marine algae, which are descended from multiple and diverse evolutionary lineages.

Regardless of their low species richness, seagrasses have successfully colonized all but the most polar seas. While seagrass meadows are widely distributed, other major

7 coastal marine habitats are geographically restricted to much smaller latitudinal ranges (mangroves and coral reefs in tropical regions, kelp beds and salt marshes in temperate regions).

Seagrasses have developed through their evolution unique ecological, physiological and morphological adaptations to live in the submerged marine environment. These adaptations include internal gas transport from the shoots to the roots in order to oxygenate the latter, epidermal chloroplasts so as to optimize light capture, submarine pollination and marine dispersal (den Hartog 1970, Les et al.

1997). To supply photosynthate to their roots and rhizomes, seagrasses need some of the highest light levels of any plant group worldwide (about 11% of the surface irradiance on average; Duarte 1991, but nearly 25% of incident radiation in some seagrass species, compared with 1% or less for other angiosperm species; Dennison et al. 1993). These extremely high light requirements mean that seagrasses at their deeper distribution level are intensely sensitive environmental changes such as water clarity.

Seagrass meadows have important ecological roles in marine coastal ecosystems and provide high-value ecosystem services compared with other marine and terrestrial habitats (Costanza et al. 1997). For example, primary production from seagrasses with their associated macro- and microepiphytes exceeds that of many cultivated terrestrial ecosystems (Duarte and Chiscano 1999). In their environment, seagrasses also provide a major source of carbon to the detrital pool, some of which is exported to the deep sea. There it provides an important supply of organic matter in an extremely food-limited environment (Suchanek et al. 1985). Much of the excess organic carbon produced is buried within seagrass sediments, which are hotspots for carbon sinks in the biosphere (Duarte et al. 2005). In addition, seagrass organs; leaves,

8 rhizomes and roots, modify the hydrodynamics around them, trapping and storing both sediments and nutrients, and effectively filter nutrient inputs back to the coastal marine environment (Hemminga and Duarte 2000).

Biodiversity and richness of associated fauna and flora in seagrass meadows is greater than in nearby non-vegetated areas, and faunal densities are orders of magnitude higher inside the meadows (Hemminga and Duarte 2000). Seagrasses also serve as a nursery land, often to juvenile stages of species of finfish and shellfish, although the species vary by region and climate (Beck et al. 2001, Heck et al. 2003).

The proximity of seagrass beds to other habitats, such as salt marshes (in temperate regions) or mangroves and coral reefs (in tropical regions), facilitates trophic transfers and exchange of habitat utilization by many species of fishes and invertebrates (Beck et al. 2001). It was suggested that this may be essential in maintaining the abundance of some coral reef fish species (Valentine and Heck 2005).

Seagrasses are often referred to being biological sentinels, or “coastal canaries". Changes in seagrass distribution and meadow density, such as an increase in water turbidity, change in the depth limit of the meadow (Abal and Dennison 1996) or widespread seagrass loss (Cambridge and McComb 1984), indicate important losses of ecosystem services that seagrasses provide. Seagrasses are sessile, and therefore are sensitive to water quality attributes such as chlorophyll-containing phytoplankton and turbidity, which affect the light reaching their leaves. In addition, seagrasses also live in shallow, protected coastal waters, directly in the path of watershed nutrient and sediment inputs. Thus they are highly susceptible to these inputs, unlike mangrove forests (which are largely unaffected by water quality) or coral reefs (which occur farther away from the inputs) (Orth et al. 2006). Another feature that makes seagrasses a precious biological indicator is that they integrate

9 environmental impacts over measurable and definable timescales (Longstaff and

Dennison 1999, Carruthers et al. 2002). For example, increased coastal development leading to nutrient inputs in Cockburn Sound, Australia, led to large-scale losses of seagrasses into the 1990s, and seagrasses remain at low levels in the area till this day

(Walker et al. 2006). The loss of seagrasses led to sediment re-suspension, which decreased fish populations.

1.1.2 Light in the marine environment

Light environments of the euphotic zone in coastal systems are highly extreme. In tropical mid-oceanic waters, irradiances can vary from 2300 µmol photons m-2 s-1 of photosynthetically active radiation (PAR) and high levels of UV radiation in surface waters to the 1% of surface irradiance necessary to maintain positive net photosynthesis of many algae at ca. 200 m depth (Lalli and Parsons

2002), while in coastal waters this 1% limit occurs at shallower depths. On a global average, the first 100 m are lighted with more than 1% of surface irradiance. This sunlit layer of the ocean, where 45% of the entire amount of photosynthesis in the world takes place (Falkowski and Raven 2007, ch.1), is called the euphotic zone.

A certain amount of incoming light is reflected away when it reaches the water surface, depending upon the state of the water itself. In calm, flat seas, less light will be reflected while in turbulent, wavy waters, more light will be reflected. Once the light penetrates the water, the irradiance decreases exponentially as light is absorbed and scattered along a depth gradient. The quality of light is thus shifted towards blue wavelengths at depth as photons with low energies (e.g. red) are absorbed by the water molecules in clear waters lacking particles. In turbid coastal waters, especially blue photons are scattered by particles (while the red photons are absorbed), such that

10 green light reaches deepest down. In Chapters 2.4 and 2.5, in situ measurements of coastal water spectrum will demonstrate these traits.

Light drives photosynthesis, leading to the production of oxygen and carbohydrates that are essential for plants to grow. For photosynthesis to occur in marine plants and in seagrass meadows specifically, the photons must penetrate the water column, enter the canopy of leaf blades, pass through a layer of possible epiphytes on the surface of the leaf and finally enter the leaf epidermis to reach the photosynthetic apparatus (Dalla Via et al., 1998). Light is attenuated at each of these steps. The level of light attenuation at the depth of the seagrass meadow is affected by external factors such as: (1) physical properties of the water (depth, dissolved organic matter content, sediment accumulation and re-suspension), (2) human activities

(increased sediment run-off, increased nutrient load leading to eutrophication and algal blooms, over-fishing, aquaculture and dredging) as well as (3) regional weather patterns (e.g. clouds, extreme storms and altered rainfall patterns) (Ralph et al. 2007).

1.1.3 Light absorption and utilization in marine plants

Leaf absorption is regulated by factors such as pigment content, morphology, chloroplast movements and dispersal and physical properties of the area (Ralph et al.

2007, Sharon and Beer 2008). Light reduction can cause an increase in chloroplast density, increase in chlorophyll content (Czerny and Dunton 1995, Longstaff et al.

1999, Sharon and Beer 2008, Sharon and beer 2009), decrease in the chlorophyll a to b ratio and a decrease in UVR blocking pigments (Abal et al., 1994). Yet, interestingly, large variations in chlorophyll content of seagrass leaves result in relatively small variations in leaf absorbance (Enríquez et al. 1992, 1994, Cummings and Zimmerman 2003, Enríquez 2005, Sharon and Beer 2008). This phenomenon has

11 been ascribed to the package effect, whereby the relationship between light harvesting efficiency and chlorophyll content is non-linear due to pigment self-shading among thylakoid membranes, chloroplasts and cell layers (Kirk, 1994).

The strong package effect observed in seagrasses is because chloroplasts are restricted to the leaf epidermis in these plants. Seagrass leaves do not have specialized tubular palisade cells that facilitate penetration of light in the leaf (Vogelmann and

Martin 1993), nor do they have a spongy mesophyll that increases the optical path length due to scattering at the interfaces between the air spaces and cells such as in terrestrial leaves (Terashima and Saeki 1983). In the face of these constraints, in most cases, seagrass leaves exhibit efficient and relatively uniform light harvesting capabilities across varying depths and water qualities (Cummings and Zimmerman

2003, Olesen et al. 2002) and significant differences in some others (Olesen et al.

2002, Enríquez 2005, Runcie et al. 2009). Meadow (i.e. height, complexity and density; Enríquez and Reyes 2005, Ralph et al. 2007) and leaf morphology (i.e. leaf thickness and leaf surface) may also effect the regulation of light absorption efficiency through changing the optical light path (Enríquez 2005).

Light absorption can be measured with an integrating sphere (Cummings and

Zimmerman 2003, Drake et al. 2003, Runcie and Durako 2004, Thorhaug et al. 2006,

Zimmerman 2006), using Shibata's (1959) spectroscopic determination of leaf absorbance (Enríquez et al. 1992, 1994, Enríquez and Sand-Jensen 2003, Enríquez

2005) or using the simplified method according to Beer (2001). Correct measurements and determinations of leaf absorbance should account for light that is transmitted, reflected and scattered as well as for light absorption by non-photosynthetic tissues.

As for the photosynthetic tissue, although pigments have a crucial effect on the plant photosynthesis processes, it seems that they don't affect the optical absorption of the

12 leaf significantly. Enríquez et al. (1994) measured light absorption properties of 13 seagrasses and reported that although the species examined ranged widely in chlorophyll a density and light absorption properties, they absorbed, on average, 59% of the incident PAR. These findings are comparable with an overall mean of 57±6%

PAR absorption reported for 8 seagrasses from the east and west coasts of Australia

(Durako 2007). Thus, despite structural restrictions and a strong package effect, seagrass leaves are relatively uniform and efficient light-capturing organs.

One special study case is the light absorption by the species Halophila stipulacea. This tropical seagrass features diurnal chloroplast movements which affect significantly its optical properties (Drew 1979, Sharon and Beer 2008) and its photosynthetic rates. These chloroplast movements will be discussed in the coming section.

1.1.4 Chloroplasts movement and clumping in plants

Chloroplasts contain the primary photosynthetic apparatus of plants, and their intracellular distribution depends on environmental factors, especially the availability and quality of light. Chloroplast photo-relocation movements can be classified and divided into two categories; 1) avoidance responses, where chloroplast move away from light, and 2) accumulation responses, where chloroplast move toward light

(Wada et al. 2003). Chloroplast accumulation responses are thought to maximize photosynthesis, whereas avoidance responses minimize photodamage to chloroplasts

(Kasahara et al. 2002).

For seagrasses, in addition to the general trend in changing pigment contents according to the incident irradiance, at least one species features the ability to clump its chloroplasts and, thus, reduce the light absorption of the leaves under high

13 irradiances (observed in the seagrass Halophila stipulacea, Drew 1979). Movements of chloroplasts within plant cells have been described for a few algae and angiosperms

(Marchant 1976, Furukawa et al. 1998, Kasahara et al. 2002, Kondo et al. 2004), including, as mentioned above, the seagrass Halophila stipulacea (Drew 1979). In the latter work, the clumping of chloroplasts was illustrated by drawings for intertidal plants under high-irradiance conditions, and this was suggested to be a mechanism for protecting the photosynthetic apparatus from excess light. In addition to protecting some of the chloroplasts, it is possible that the clumping phenomenon has a role in protecting also the cell nucleus from excess light, as it was found that the chloroplasts tend, in some cases, to clump around it (Kondo et al. 2004). Similar photoprotective movements of chloroplast were shown to be mediated by phototropin in mosses and algae (Suetsugu and Wada 2007) and by abscisic acid in a succulents plant (Kondo et al. 2004). It should be pointed out that most of the literature published so far is dealing mainly with molecular aspects of the signaling pathway that leads to chloroplast movements (Uenaka and Kadota 2007, Suestsugu and Wada 2007, Cho et al. 2007, Suestsugu et al. 2006, Luesse et al. 2006) or its biochemical-cellular mechanism (Shiihira-Ishikawa et al. 2007, Krzeszowiec et al. 2007, Paves and Truve

2007, Gabrys 2004), and not the eco-physiological role of this phenomenon.

Therefore, there is a need to explore the ecological role of the phenomenon.

Since Drew's initial observation, movements of chloroplasts in seagrasses have not been studied further. The clumping of chloroplasts has an apparent effect on leaf transparency as reflected in the visually much paler leaves of Halophila stipulacea plants growing in shallow waters (<1 m) during daytime characterized by high irradiances (Yoni Sharon and Sven Beer, personal observations). The possible effect

14 of such chloroplast movements on light absorption by the leaves was only indicated once (Schwarz and Hellblom 2002), but was never measured.

1.1.5 Light absorption under UVR and high-light environments

In addition to high photosynthetically active radiation (PAR, 400-700 nm), coastal waters, especially clear tropical waters, also feature high levels of UV radiation (UVR, 280-400 nm) in their upper layers (Dunne and Brown 1996; Agusti and Liabres 2007; this study). In general, adaptations to UVR and high light assume the existence of mechanisms that protect the organism or reduce their harmful effects.

Four basic mechanisms allow an organism to cope with stressful UVR and excess high light exposure; 1) avoidance, 2) reducing the stress by a physiological behavioral mechanism, e.g. through the synthesis of UV absorbing compounds or other protecting pigments, 3) repairing the damage produced and 4) acclimating to the stress

(Helbling and Zagarese 2003). More specifically, under extreme light conditions plants evolve mechanisms enabling them to reduce the light absorption and, thus, high-light damage in their photosynthetic tissues. These adaptive processes include reduce leaf area and increase leaf thickness (Dawson and Dennison 1996), the production of mycosporine-like amino acids (MAA; Whitehead and Vernet 2000; Gao et al. 2007) and other UV-absorbing compounds (flavones and flavone-glycosides in seagrasses; Durako et al. 2003, Kunzelman et al. 2005, Meng et al. 2008). The accumulation of these compounds occurs mainly in the epidermis, creating a UVR screen for the underlying tissues. Epidermis tissue enriched with UV-absorbing compound may reduce up to 99% of the UV radiation (Robberecht and Caldwell

1978). Flavonoids and related pigments are considered to account for a large percentage of this attenuation (Dawson and Dennison 1996).

Also changing morphologies (Monro and Poore 2004), changing pigment contents (Dawson and Dennison 1996; Gao et al. 2007), down-regulating photosystem

II (PSII; Figueroa et al. 2002) or shading by carbonate layers (Beach et al. 2006) can reduce the UVR levels and damage in the leave. The latter may change also the light absorption characteristics of the photosynthetic tissue. Generally, reflectance of light by CaCO3 has been largely ignored, or has been assumed to be minimal (Luning and

Dring 1985, Henely 1993, Markager 1993). However, a few studies have shown that

CaCO3 can increase the reflection and, thus, reduce light absorption by photosynthetic tissues (Littler and Littler 1980, Beach et al. 2006).

Under anthropogenic stress, leading to increased nitrogen levels in the water, epiphytes that thrive under such conditions may decrease the UVR (as well as PAR) levels reaching the leaves (Trocine et al. 1981; Brandt and Koch 2003). It was reported that periphyton (defined as the complex matrix of living and dead organisms as well as mucus and sediment particles) developing on seagrasses can reduce the light levels and screen off UVB radiation, thus protecting their leaves from this radiation in shallow waters (Brandt and Koch 2003).

Despite the fact that seagrasses are referred to be, in general, shade-adapted plants, e.g. Halophila stipulacea is growing under high levels of PAR and UVR in the intertidal of the Red Sea. While it was shown that this high level of PAR and UVR inhibits seed germination in shallow waters (Malm 2006), and different morphology of the leaves was found in the deeper waters (Schwarz and Hellblom 2002), there is not much more information available about the photophysiological adaptation of this plant neither in shallow nor in deeper waters. While some seagrasses are known to be sensitive to high levels of PAR while others are sensitive to high UVR levels

(Dawson and Dennison 1996), the cause which triggers adaptive mechanisms for high light and/or high UVR levels in Halophila stipulacea is still unknown.

Since many seagrasses thrive in tropical shallow waters, it may be that the midday chloroplast clumping found in at least one of them (Halophila stipulacea) has a protective role against UVR (in addition to high PAR). In light of this, the possible role of UVR, in addition to PAR, in mediating chloroplast movements in this seagrass, and the possible effects of UVR on some of its photosynthetic parameters will be discussed further in this work.

1.1.6 Acclimation processes of seagrasses (from the community to the individual)

While certain photosynthetic organisms have adapted to specific light regimes

(e.g. low irradiances at depth or very high irradiances in the tropical intertidal), others show a high plasticity as evidenced in their ability to inhabit areas with either low or high irradiances. The ability of such organisms to develop mechanisms to utilize and/or cope with different irradiances may be crucial for their functioning at spatially different and temporally dynamic surroundings (Sultan 2000, Stewart and Carpenter

2003).

Terrestrial plants use various strategies in their adaptation to extreme light environments. Diurnal morphological changes allow flowers or leafs to face the sun or to avoid it. Trees of an entire forest can be flat-topped in response to light and moisture requirements (Raven et al. 1992). Plants growing under extremely high light carry sometimes salt-secreting hairs and bladders on their leaves in order to reflect some of the light, thus reducing the photon flux reaching the leaves (Raven et al.

1992). Shade-growing plants use physiological mechanisms to enhance pigment

(chlorophylls) content and, thus, photosynthetic efficiency at low irradiances (Johnson

17 et al. 2000, Ralph et al. 2007). In contrast, high-light plants which are exposed also to high UV radiation can respond in a plastic way by producing UVB absorbing pigments, the so-called mycosporine-like amino acids (MAA) and flavonoids in greater amounts than low-light plants (Rozema et al. 2002). In addition, a higher stimulation of DNA repair genes is present in those high-light plants (Ries et al.

2000).

In the marine environment, many subtidal macroalgae have the capacity to detect variations in the amount of available light and its spectral proportions, and to respond to this variation in a plastic way. This is done both through changing morphologies (e.g. thallus shape and thickness) and physiology (e.g. pigment content, including those of the photoprotective xanthophyll cycle) in response to limiting or excess irradiances (Ralph et al. 2002, Monro and Poore 2004, Ralph et al. 2007).

Also seagrasses, are well-known for their plastic responses to different environments (Reusch 2001, French and Moore 2003, Peralta et al. 2005, Ralph et al.

2007, Sharon et al. 2009). They are distributed around the globe, from the arctic to tropical regions, where they inhabit areas with very differing irradiance regimes. In order to maximize light utilization in very dim environments, seagrasses use several mechanisms. On the community level, epiphytes may shade and protect seagrass leaves from over-exposure to damaging sun light (Trocine et al. 1981; Brandt and

Koch 2003). On the population level, one mechanism includes a canopy architecture that reduces self shading where light is absent. On the organism level, chloroplast and thylakoid membrane dispersal (Dawson and Dennison 1996, White and Critchley

1999) and a change in pigment content may enhance or decrease light absorption according to the plants' demand in its light environment. One relevant example of the adaptations to irradiance is the longer and wider leaves of Halophila stipulacea

18 growing in deeper waters (Lipkin 1979, Schwarz and Hellblom 2002). In this present work the plasticity in the acclimation to light in Halophila stipulacea is discussed (see

Chapter 2). Yet, though light-stress avoidance adaptations are known for seagrasses, the physiological adaptations/responses of deep-growing populations that photosynthesis under extreme low light conditions are still not clear. Here, I questioned the role of photosystem composition in the adaptation to low light at depth

(see Chapter 5).

1.1.7 The photosystems' apparatus and its stoichiometry

In photosynthesis, the energy of absorbed photons is used to modify the electronic structure of pigment molecules to the extent that, in the reaction centers, an electron can be physically transferred from a donor to an acceptor. Thus, the light reactions in photosynthesis are photochemically catalyzed oxidation-reduction reactions. The main "players" of the light absorption in the photochemical electron transport are photosystem I (PSI with chlorophyll P700 at its reaction center) and photosystem II (PSII with chlorophyll P680 at its reaction center). The efficiency of photosynthetic electron transport depends on the coordinated interactions of photosystem II (PSII) and photosystem I (PSI) in the electron transport chain. Each photosystem contains different pigment-protein complexes that harvest light from slightly different regions of the visible spectrum. The light energy is utilized to excite an electron which leads to an electron-transport sequence in each photosystem. Some evidence has shown a large variability in the PSII/PSI stoichiometry from its generally assumed ratio of 1:1 in plants grown under different environmental irradiance conditions in terrestrial habitats (reviewed in Fujita 1997).

Relatively to terrestrial plants, little is known on macro-marine plants' photosystem composition or stoichiometry. Among marine macrophytes, spectral changes were shown to influence the functionality of the two PSs as reported for the green macroalgae Ulva pertusa and Bryopsis maxima (Yamazaki et al. 2005).

Regarding seagrasses, only two studies have reported on changes in the Photosystems composition of shallow-growing plants during various seasons (Major and Dunton

2000) and along a depth gradient down to 1.6 m (Major and Dunton 2002). For seagrasses, it is still not known what are the stoichiometry and functionality of deep growing plants photosystems. In the current work (see chapter 5), protein analysis and

77K measurements are presented in order to investigate the mechanism of depth acclimation in the tropical seagrass, Halophila stipulacea. To our knowledge there are no studies on PS relations in seagrasses growing at such great depths where both the irradiance and light spectrum change drastically.

1.2 Research Objectives

Halophila stipulacea grows from the intertidal down to >50 m depth along a large light gradient. However, the mechanisms and characteristics of this plant to adapt and acclimate to this light gradient are not known. Accordingly, the specific aims of this work were to:

1. Quantify chloroplast movements and elucidate there importance on

photosynthetic rates.

2. Investigate the acclimation of Halophila stipulacea to different light

environments.

3. Investigate Halophila stipulacea's excitation energy allocation to

photochemistry along its light gradient.

4. Elucidate the role of UVR in chloroplast movements and photosynthetic rates

in Halophila stipulacea.

5. Characterize the photosystems' stoichiometry and functioning of shallow and

deep growing Halophila stipulacea plants.

Available online at www.sciencedirect.com

Aquatic Botany 88 (2008) 273–276 www.elsevier.com/locate/aquabot

Diurnal movements of chloroplasts in Halophila stipulacea and their effect on PAM fluorometric measurements of photosynthetic rates

Yoni Sharon a,b, Sven Beer a,*

a Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel b The Inter-University Institute in Eilat, P.O. Box 469, Eilat 88103, Israel Received i2 August 2007; received in revised form i9 November 2007; accepted 27 November 2007 Available online 4 December 2007

Abstract

Since diurnal chloroplast movements in Halophila stipulacea were described by Drew in i979, this phenomenon has not been studied further for seagrasses. In addition to an apparent photoprotective role, such movements may affect the measurements of photosynthetic rates based on pulse amplitude modulated (PAM) fluorometry. This is because calculations of electron transport rates (ETR) are directly affected by the light absorption of the leaves (or the so-called absorption factor, AF), the latter of which changes with the movements of the chloroplasts. In this work, we therefore determined chloroplast clumping and dispersal, and measured AFs, chlorophyll contents and PAM fluorescence diurnally for H. stipulacea grown under two irradiance regimes. Diurnal chloroplast clumping occurred in high-light grown (HL) plants ( 450 mmol photons m—2 s—i during midday), which was accompanied by a decrease in AF values (from 0.56 in the early morning to 0.34 at midday) but not in the chlorophyll content. Also, non-photochemical quenching (measured as NPQ) increased during the day in these plants. No such chloroplast movements and, thus, no diurnal changes in AF values (0.60 ± 0.04 throughout the day), and no changes in NPQ, were found in low-light grown (LL) plants ( i50 mmol photons m—2 s—i during midday). As a consequence of the chloroplast clumping in HL plants, and its effect on AF values, maximal ETRs did not differ significantly between HL and LL plants. This finding thus shows the importance of taking into account changing AF values along the day when calculating ETRs of H. stipulacea, and other seagrasses potentially featuring diurnally changing AFs, under high-irradiance conditions. # 2007 Elsevier B.V. All rights reserved.

Keywords: Chloroplast movement; Chloroplast clumping; Halophila stipulacea; PAM fluorometry; Photosynthesis

1. Introduction acterised by high irradiances (Yoni Sharon and Sven Beer, personal observations). The possible effect of such chloroplast Movements of chloroplasts within plant cells have been movements on light absorption by the leaves was only indicated described for a few algae and angiosperms (Marchant, i976; once (Schwarz and Hellblom, 2002), but was never measured. Furukawa et al., i998; Kasahara et al., 2002; Kondo et al., Pulse amplitude modulated (PAM) fluorometry has been 2004), including the seagrass Halophila stipulacea (Drew, used increasingly during the past decade for photosynthetic i979). In the latter work, the clumping of chloroplasts was measurements in seagrasses (cf. Beer et al., 200i). In order to illustrated by drawings for intertidal plants under high- measure photosynthetic electron transport rates (ETR) by this irradiance conditions, and this was suggested to be a method quantitatively, it is necessary to take into account the mechanism for protecting the photosynthetic apparatus from absorption of light by the photosynthetic pigments, or at least excess light. Since this initial observation, movements of the so-called absorption factor (AF), of the leaves (Beer et al., chloroplasts in seagrasses have not been studied further. The i998). If indeed chloroplast movement affects the leaf clumping of chloroplasts has an apparent effect on leaf transparency (i.e. AF value), and if this occurs on a diurnal transparency as reflected in the visually much paler leaves of H. basis, it follows that such continuously changing AFs need to be stipulacea plants growing in shallow waters (

* Corresponding author. Tel.: +972 3 6409i25; fax: +972 3 6409380. Given the potential importance of chloroplast movement on E-mail address: [email protected] (S. Beer). AF values and, thus, photosynthetic ETRs, we here set out to (a)

274 Y. Sharon, S. Beer / Aquatic Botany 88 (2008) 273–276

confirm microscopically chloroplast clumping and dispersal taken to the laboratory (within 10 min), placed on microscope throughout the day in low- and high-light grown H. stipulacea glass slides, examined and photographed. leaves, (b) measure AFs diurnally and (c) evaluate the diurnal Chlorophyll contents of leaves were determined after photosynthetic performance of H. stipulacea taking into extraction in dimethyl formamide (DMF) for 24 h in darkness. account the potential fluctuations in the AF values. In doing Absorbance was measured spectrophotometrically according to so, we confirm the importance of using diurnally changing, Moran (1982), and chlorophyll a and b contents were calculated chloroplast movement induced, AF values for assessing ETRs on a leaf area basis. correctly. Chlorophyll fluorescence was measured diurnally for individual leaves throughout cloudless days using the Diving 2. Materials and methods PAM (Walz, Germany). An intact leaf growing on the water table was fixed at a distance of 10 mm from the instrument’s Whole plants, including roots and rhizomes, of H. optical fibre using a so-called leaf distance clip (Walz, stipulacea from a monospecific bed were collected together Germany) such that it received the sunlight remaining under with their sediment at 30 m depth 200 m south of the Inter- the shading nets. The PAM fluorometer was programmed to University Institute (IUI) in Eilat, the Gulf of Aqaba, take a yield (Y) measurement, together with irradiance, every Northern Red Sea (29830F .01N, 34854F .57E). The seagrasses 30 min using the ‘‘rep clock’’ mode. ETR was calculated as of this bed grow between 7 and >40 m depth. The midday Y x PAR x 0.5 x AF. Non-photochemical quenching (NPQ) irradiance at 30 m was .60 mmol photons m—2 s—1 and the was calculated individually for each leaf as (Fm — FF m)/FF m; temperature was 21 8C during the time of this study Fm was measured early in the morning before sunrise. (February–March, 2007). The plants were transported to In order to determine AF diurnally, 10 leaves were removed an outdoor water table (a table with a 20 cm margin, forming at 5 different times during a cloudless day and absorption was a shallow container) located at the IUI, where they were measured under a lamp using the Diving PAM’s light sensor acclimated for 30 days in running seawater under shading calibrated against a LiCor LI-189 (USA) quantum sensor. nets providing either .450 (‘‘high-light’’ grown, HL) or These AF measurements were done in an aquarium such that —2 —1 .150 (‘‘low-light’’ grown, LL) mmol photons m s the reflection of the leaf was minimised in the water medium during midday, respectively. After this acclimation, the (cf. Beer and Bjo¨ rk, 2000). It is important to note that the vigour of the plants was verified by night-time Fv/Fm absorption of the thin leaves is mainly due to the photosynthetic measurements. pigments. This was ascertained by measuring AF values of Chloroplast clumping was investigated using both light- and leaves from which all pigments had been extracted several confocal microscopy (Nikon Eclipse TE2000-E, Japan). At times by DMF until they were close to transparent; these leaves each of the observation times along the day, leaves were quickly showed AF values <0.04.

Fig. 1. Micrographs of Halophila stipulacea epidermal leaf cells photographed at various times during the day. The plants were grown on a sunlit water table at .150 (panel a) and .450 (panel b) mmol photons m—2 s—1 during midday. Black bars represent 10 mm. 23

Y. Sharon, S. Beer / Aquatic Botany 88 (2008) 273–276

3. Results throughout the day (Fig. 2b). While the trend showed decreased values during midday, the diurnal variation was significant (one Night-time Fv/Fm values of the experimental plants were way ANOVA, p < 0.01) for the HL plants only. In the lack of found to be 0.73 ± 0.02 (n = 8) and 0.71 ± 0.03 (n = 8) for LL significant daily variations in chlorophyll content, we suggest and HL plants, respectively. In comparison, in situ measured that the daily oscillation in AF was due to the clumping and night-time Fv/Fm values of plants growing at 30 m were subsequent dispersal of chloroplasts. 0.79 ± 0.02 (n = 8). These results were taken to confirm that ETRs of LL plants, calculated using corrected AF values the plants were not suffering photosynthetically from the 30 for the various times of the day, followed the irradiance days of growth on the water table. throughout the day (Fig. 3a). The highest rate was The epidermal cells of LL H. stipulacea leaves did not show 29.2 mmol electrons m—2 s—1 at noon, which coincided with a any clumping of chloroplasts during the day (Fig. 1a). In peak in the irradiance reaching the water table. During this time, contrast, HL plant leaf cells did feature diurnal clumping and non-photochemical quenching values were below 0.3. This, and subsequent dispersal of chloroplasts as illustrated in Fig. 1b. the fact that photosynthesis paralleled irradiance, confirms a These results were confirmed using both light- and confocal virtual absence of photoinhibition. Under HL conditions, ETRs microscopy, but only light-microscopic pictures are presented followed the irradiance only during the morning hours (Fig. 3b). here. At 06:00, before sunrise, the chloroplasts were rather After 9:30, at irradiances above .350 mmol photons m—2 s—1, evenly dispersed in the cytoplasm of the cells. Migration started ETRs decreased while the irradiance remained stable (until with sunrise, and the beginning of clumping can be seen at 12:30). Thus, ETR and irradiance did not parallel one another 08:00. Full clumping was observed at midday, and continued from 9:30 until 15:30. NPQ increased during the day, reaching throughout the afternoon. In the late afternoon the chloroplasts maximum values of around 3 at 12:00–14:30 (400– dispersed throughout the cytoplasm, and full dispersal was 200 mmol photons m—2 s—1). NPQ values were >2 during the observed a few hours after sunset (not shown). time that the ETRs did not parallel irradiances, indicating Chlorophyll a + b contents showed no significant variations photoinhibition by heat dissipation, e.g. via the xanthophyll throughout the day (Fig. 2a). This was true for both LL and HL cycle. After 15:30, ETR decreased in parallel with irradiance plants. However, the average chlorophyll a + b contents pooled (below .75 mmol photons m—2 s—1). The form of photoinhibi- for plants from the two irradiances showed a significantly higher tion between 9:30 and 15:30 is dynamic as evidenced by a full (one way ANOVA, p < 0.00001) chlorophyll content in LL recovery of the effective quantum yield in the late afternoon to (14.86 ± 3.51 mg cm—2) than in HL (8.32 ± 3.37 mg cm—2) the same values as during the morning hours at similar plants. The absorption factor, on the other hand, did vary irradiances.

Fig. 2. Daily changes in chlorophyll content (panel a, n = 5) and absorption factor (AF, panel b, n = 10) of Halophila stipulacea grown on a sunlit water table under shading nets at .150 (open circles) and .450 (closed Fig. 3. Daily changes in irradiance (open circles), electron transport rate (ETR, circles) mmol photons m—2 s—1 during midday. Significant differences at p closed circles) and the non-photochemical quenching parameter NPQ (closed < 0.0001 (6:00 vs. 13:20, 13:20 vs. 21:00), p < 0.01 (6:00 vs. 10:00, 10:00 squares) of Halophila stipulacea grown on a sunlit water table under shading vs. 13:20, nets at .150 (panel a, n = 3) and .450 (panel b, n = 3) mmol photons m—2 s—1 13:20 vs. 17:00) (one way ANOVA) along the day are indicated with different during midday. Note that the NPQ scales show 10 x NPQ. letters. 24

276 Y. Sharon, S. Beer / Aquatic Botany 88 (2008) 273–276

One striking consequence of taking the diurnally fluctuating diurnal development of NPQ in high-light grown plants. In AF values of HL plants into consideration when calculating contrast, no such NPQ was observed for low-light grown plants. ETRs can be exemplified in determining daily maximal ETRs. This, together with the absence of chloroplast clumping in these If early morning AF values of LL and HL plants are used, then plants, indicated that there is no need for photoprotection in significantly different (one way ANOVA, p < 0.001) maximal deeper growing plants. ETRs are derived for the two plant types (34.7 ± 2.43 and The diurnal changes in AF caused by chloroplast movements 47.0 ± 1.08 mmol electrons m—2 s—1 for LL and HL plants, in HL leaves of H. stipulacea have obvious consequences when respectively). If, however, the AF values at the time of determining photosynthetic rates by PAM fluorometry. For the measurements are used, then no significant (one way ANOVA, latter, it is important to use correct AF values since ETRs p > 0.05) differences in maximal daily ETRs between plants strongly depend on them in a linear fashion (e.g. Beer et al., frown under the two irradiances are apparent (30.8 ± 3.01 and 1998). Thus, when leaves change their absorption through the 35.6 ± 1.93 mmol electrons m—2 s—1 for LL and HL plants, day, it is important to take such changes in AF into account respectively). when calculating photosynthetic ETRs. This corrective practice can even change conclusions regarding maximum photosyn- 4. Discussion thetic rates in low- and high-light plants: In this study, using, e.g. early morning AF values for the HL plants renders them One of H. stipulacea’s outstanding features is its wide depth significantly different maximal ETRs from LL plants. This distribution. In the Gulf of Aqaba, Northern Red Sea, it grows difference is, however, not apparent when using the corrected from the intertidal down to depths of 70 m (Lipkin, 1979). At AF values measured at the time of day when maximal ETRs depth, the thin leaves of this plant may be a characteristic of low- were recorded. light adaptations. In addition, the leaves at depth are larger than in shallow waters (Lipkin, 1979; Schwarz and Hellblom, 2002) and the above- to below-ground biomass ratio is higher (Sharon, References unpublished data). In shallow waters, the ability to withstand high irradiances may be facilitated by the ability of chloroplasts Beer, S., Bjo¨ rk, M., 2000. Measuring rates of photosynthesis of two tropical seagrasses by pulse amplitude modulated (PAM) fluorometry. Aquat. Bot. to move into clumps during the day and the apparently efficient 66, 69–76. mechanism for non-photochemical quenching. Beer, S., Vilenkin, B., Weil, A., Veste, M., Susel, L., Eshel, A., 1998. Measuring While chloroplast movement was previously described for photosynthetic rates in seagrasses by pulse amplitude modulated H. stipulacea in drawings (Drew, 1979), the present work shows (PAM) fluorometry. Mar. Ecol. Prog. Ser. 174, 293–300. for the first time photographs of this diurnally oscillating Beer, S., Bjo¨ rk, M., Gademann, R., Ralph, P., 2001. Measurements of photosynthetic rates in seagrasses. In: Short, F.T., Coles, R. (Eds.), Global phenomenon in high-light grown plants. In some algae (e.g. the Seagrass Research Methods. Elsevier, Amsterdam. diatom Pleurosira, Furukawa et al., 1998) as well as both Drew, E.A., 1979. Physiological aspects of primary production in seagrasses. submerged (Vallisneria, Sakurai et al., 2005) and terrestrial Aquat. Bot. 7, 139–150. (Arabidopsis, Kasahara et al., 2002) plants, similar movements Furukawa, T., Watanabe, M., Shihira-Ishikawa, I., 1998. Green- and blue-light- of chloroplasts were induced by relatively low levels of blue mediated chloroplast migration in the centric diatom Pleurosira laevis. Protoplasma 203, 214–220. light, and in the succulent CAM plant Zygocactus this Kasahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao, M., Wada, M., movement was mediated by abscisic acid (Kondo et al., 2002. Chloroplast avoidance movement reduces photodamage in 2004). Similar mechanisms may be triggers for chloroplast plants. Nature 420, 829–832. movements in H. stipulacea, but we observed that clumping of Kondo, A., Kaikawa, J., Funaguma, T., Ueno, O., 2004. Clumping and dispersal chloroplasts occurred only in plants growing at midday of chloroplasts in succulent plants. Planta 219, 500–506. —2 —1 Lipkin, Y., 1979. Quantitative aspects of seagrass communities, particularly of irradiances above .200 mmol photons m s . Therefore, it those dominated by Halophila stipulacea, in Sinai (Northern Red- seems that high irradiances are a prerequisite for chloroplast Sea). Aquat. Bot. 7, 119–128. clumping irrespective of the mechanism involved. Marchant, H.J., 1976. Mechanism of chloroplasts movement in green In the Red Sea area, which may experience the highest freshwater algae Coleochaete and Mougeotia. J. Cell Biol. 70, A56. irradiances in the world (e.g. Winters et al., 2003), there may be Moran, R., 1982. Formulas for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiol. 69, 1376–1381. a clear need for photoprotection of marine organisms growing Sakurai, N., Domoto, K., Takagi, S., 2005. Blue-light-induced reorganization of in shallow waters. H. stipulacea, like the genus Halophila in the actin cytoskeleton and the avoidance response of chloroplasts in general, is characterised by very thin leaves of a few cell layers epidermal cells of Vallisneria gigantea. Planta 221, 66–74. only. It is thus likely that the chloroplast clumping ability, Schwarz, A.M., Hellblom, F., 2002. The photosynthetic light response causing self shading of the chloroplasts, is an important of Halophila stipulacea growing along a depth gradient in the Gulf of Aqaba, the Red Sea. Aquat. Bot. 74, 263–272. photoprotective feature in this plant. In addition to chloroplast Winters, G., Loya, Y., Rottegers, R., Beer, S., 2003. Photoinhibition in shallow- clumping, an efficient non-photochemical quenching mechan- water colonies of the coral Stylophora pistillata as measured in situ. Limnol. ism, probably via the xanthophyll cycle, is evidenced by the Oceanogr. 48, 1388–1393. 25

Vol. 7: 143 –152, 2009 AQUATIC Published online October 22, 2009 doi: 10.3354/ab00164 BIOLOGY Aquat Biol

Contribution to the Theme Section ‘Primary production in seagrasses and macroalgae’ OPEN ACCESS

Photosynthetic responses of Halophila stipulacea to a light gradient. I. In situ energy partitioning of non-photochemical quenching

1, 2 2 3, 4 4 2 John W. Runcie *, Diogo Paulo , Rui Santos , Yoni Sharon , Sven Beer , João Silva

1 School of Biological Sciences, University of Sydney, New South Wales 2006, Australia 2ALGAE-Marine Plant Ecology Research Group, Center of Marine Sciences (CCMAR), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal 3 The Interuniversity Institute for Marine Sciences, POB 469, Eilat 88103, Israel 4Department of Plant Sciences, Tel Aviv University, Tel Aviv 60078, Israel

ABSTRACT: The quantum yield of photosystem II (φII, also termed ΔF /Fm’ or Fv /Fm in light- or dark- acclimated plants, respectively) of the tropical seagrass Halophila stipulacea was measured in situ using modulated fluorescence techniques over diel periods at a range of depths. Photosynthetic electron transport rates (ETRs), as derived from φII values at specific ambient photosynthetically available radiation (PAR) irradiances, increased in direct proportion to increasing irradiance in the morning and, at shallow sites (7 to 10 m), reached saturating rates and then declined in the afternoon with lower PAR-specific ETRs. On the other hand, plants at 32 to 33 m showed no saturation even at midday, and the percentage reduction in PAR-specific afternoon ETRs was less than that of the shallower plants. The use of an automated shutter in the measuring device enabled non-photochemical quenching due to down-regulation and basal intrinsic non-radiative decay to be distinguished. While midday values of down-regulation were lower in deeper water, basal intrinsic non-radioactive decay remained fairly constant at 30 to 40 % at all depths, with more variation in shallow waters. The maximal φII (i.e. Fv /Fm) reached similar values at midnight regardless of depth. H. stipulacea acclimates to the widely varying irradiances across this depth gradient by regularly modulating down-regulation- based non-photochemical quenching processes, while dissipating a large proportion of light energy through intrinsic decay regardless of depth.

KEY WORDS: Depth gradient · Halophila stipulacea · Irradiance · Nonphotochemical quenching · PE curves · Photosynthesis · Pigments

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INTRODUCTION that the net carbon balance is positive is vital for the survival and resilience of a meadow. Thus consider- Seagrass meadows dominate many shallow estuaries able research effort over the last half-century has and marine embayments, yet at a global scale sea- maintained a focus on seagrass growth and photosyn- grasses are in decline (Orth et al. 2006). While this thesis (e.g. Larkum 1976, Lee et al. 2007). decline has been largely attributed to human The seagrass Halophila stipulacea is an integral activity through increased light attenuation caused component of the the Gulf of Aqaba coral reef ecosys- largely by higher sediment loads and algal densities, tem in the Red Sea (Edwards & Head 1987), where it other factors such as eutrophication, heavy metals and can be found in extensive monospecific beds. H. stipu- petrochemi- cal pollution have a significant impact on lacea has a generally wide ecological range, growing seagrass survival (Orth et al. 2006, Ralph et al. 2007). from the intertidal to depths > 50 to 70 m (Hulings The capacity for a seagrass species to conduct 1979, Lipkin 1979, Beer & Waisel 1982). This is in con- photosynthesis such

© Inter-Research 2009 · www.int-res.com *Email: [email protected] 26

144 Aquat Biol 7: 143 –152, 2009

trast with the seagrass Halodule uninervis that coexists directed to non-photochemical processes when ab- in shallow waters in the northern Red Sea, Gulf of sorbed by non-stressed phototrophs; and (4) induced Aqaba, but does not extend as deeply (Beer & Waisel thermal dissipation is associated with PSII photo- 1982). Mechanisms that enable the survival of H. stip- inactivation and is sometimes termed photoinhibition. ulacea in shallow and deep waters may include an Kramer et al. (2004) pooled the 2 thermal dissipation insensitivity to pressure due to its lack of substantial components together and termed them basal intrinsic gas lacunae (Beer & Waisel 1982) and its capacity non-radiative decay. For simplicity we adopt this latter for chloroplast clumping (Schwarz & Hellblom 2002, term to describe thermal dissipation. Sharon & Beer 2008). As part of the recent Group for Aquatic Primary Since the advent of submersible modulated fluoro- Productivity (GAP) workshop in Eilat (April 2008, see meters in the 1990s, chlorophyll fluorescence techniques www.gap-aquatic.org/), we studied various photo- have been widely used in the examination of in situ physiological aspects of the seagrass Halophila stipu- seagrass photophysiology (e.g. Beer et al. 1998, 2000), lacea, in situ. In the present study, we determined how and the effects of varying irradiance with increasing irradiance energy absorbed by PSII is allocated to turbidity or depth have been examined in some detail photochemistry versus non-photochemical processes in (Lee et al. 2007). However, most in situ fluorescence plants exposed to very different irradiances along a studies have been constrained by the necessity for wide depth gradient. By determining diel changes in divers to make the measurements, limiting studies to a electron transport rates at various depths (8 to 33 m), combination of maximum depth, frequency and dura- we aimed to distinguish differential responses in terms tion of measurements at these depths. Fewer studies of photochemical and non-photochemical use of the have taken advantage of logging functions available in contrasting ambient photosynthetically available radi- commercially available instruments (e.g. Winters et ation (PAR) irradiances at each depth. The specific al. 2003, Schwarz et al. 2003); other studies have devel- objectives of the present study were to determine how oped custom devices for continuous measurements excitation energy is allocated within H. stipulacea to over time of multiple replicate samples (Runcie & photochemistry, down-regulation and basal intrinsic Riddle 2004, 2006, Runcie & Durako 2004). non-radiative decay processes when exposed to differ- The quantum yield (φ) of a light-dependent process ent irradiances (at various depths), and how the alloca- is defined as the ratio of the rate of that process to the tion to these processes varies during the onset of irra- rate of photon absorption. In unstressed green plants diance stress and recovery. A parallel study was also and algae, the light-dependent process of photo- conducted to examine acclimatory responses of H. stip- chemical energy conversion uses approximately 83 % ulacea transplanted across the 3 depths (Sharon et al. of light absorbed, thus the quantum yield of photosyn- 2009, this Theme Section). thesis is 0.83. Modulated fluorescence techniques are particularly useful for measuring the quantum yield of photochemical energy conversion in photosystem II MATERIALS AND METHODS (PSII), φII, and by difference, the quantum yield of non- photochemical energy conversion in PSII, φNPQ. Study sites and seagrass species. Halophila stipu-

Diel changes in φII describe how a lacea (Forsskål) Ascherson was examined in situ at 8, phototroph 18 and 33 m depth just south of the Inter-University responds to irradiance and can be used with other Institute (IUI) in Eilat, the Gulf of Aqaba, northern Red measurements to estimate net carbon gain (Beer et al. Sea (29° 30’ N, 34° 55’ E) during April 2008. Automated 2000, Longstaff et al. 2002). However, irradiance chlorophyll fluorescence measurements were set up at energy absorbed by PSII that cannot be directed to each depth using SCUBA. Individual leaves separated photochemistry is (by definition) directed to several from each other by ≥10 cm were examined. Selected classes of non-photochemical quenching (NPQ) pro- samples were taken to the laboratory for leaf ab- cesses. Of these, the generation of singlet oxygen can sorbance determinations. cause intensive damage to internal structures if the In situ irradiance and Kd measurements. Irradiance absorbed energy is not effectively dissipated through was measured in situ during fluorometer deployments other NPQ processes (Müller et al. 2001). using cosine-corrected irradiance sensors incorporated Kornyeyev & Holaday (2008) separated NPQ into 4 in the custom fluorometers, and is termed incident irra- components: (1) fluorescence is generally a very small diance or Ii. Data was compared with measurements in proportion of NPQ (a few percent at most) and is W m–2 obtained at the nearby research station and con- generally disregarded; (2) regulated, dark-reversible verted to µmol photons m–2 s–1 by multiplying by 4.6 dissipation is generally activated in response to irra- (McCree 1981). Occasional in-air measurements and diance stress; (3) constitutive thermal dissipation can depth profiles using a LiCor 190SA quantum sensor be regarded as the (assumed) invariant ~27 % of light 27 Runcie et al.: Depth-dependent seagrass photosynthesis 145

were also taken from a boat in the vicinity of the study site; those in-air irradiance values were similar to values measured at the research station. All light sensors were calibrated against the LiCor 190SA quantum sensor. The light attenuation coefficient Kd was calculated from depth profile data. Absorbance measurements. The absorption factor (AF) of 12 different leaves was assessed and averaged at each depth (8, 18 and 33 m) throughout the day (06:00, 10:00, 12:30, 16:00 and 18:00 h) by determining the reduction of ambient irradiance (or artificial irradiance at dawn and dusk) caused by a single leaf placed over the light sensor of a Diving-PAM fluorometer (Walz, Effeltrich). This reduction in irradiance was converted to a proportion. For example, a 50 % reduction in irradiance caused by placing a leaf over the light sensor Fig. 1. Custom fluorometer holding a Halophila stipulacea corresponded to an AF value for that leaf of 0.5 (Sharon leaf with automated shutter (left) and battery data/acquisition & Beer 2008). It was assumed that absorbance by photo- unit (right) synthetic pigments contributed 96 % of the overall absorbance in Halophila stipulacea (as found earlier, sequent values obtained from the seagrass samples Sharon & Beer 2008); the absorbance of other com- when the sensor head was in a closed position. The pounds was therefore ignored. The measured AF val- other fluorometer design simply positioned the sensing ues were regressed against time separately for morning part of the fluorometer close to the sample which was and afternoon measurements and thus 2 linear relation- held in a clear acrylic sample holder; samples were ships were determined for each depth. The trajectory of exposed to ambient irradiance throughout the course these regression lines indicated the direction and rate of measurements. Each fluorometer was connected by of change in absorbance throughout the day (a proxy cable to an independent submersible data-logger and for chloroplast clumping, Sharon & Beer 2008), and power supply; previous experiments (authors’ unpubl. enabled the estimation of AF by interpolation at each data) demonstrated that the system can operate for at depth for each fluorescence measurement. least 2 d on a single charge. Several fluorometers of Twelve absorbance values were obtained at each both designs were deployed at each site and a single depth at (approximately) each time; differences be- seagrass blade was held in the sample holder of the tween mean AF values at each depth at dawn and fluorometer with an upwards orientation; the irradi- dusk were compared using ANOVA after testing for ance sensor incorporated into each fluorometer like- homogeneity of variances. wise faced upwards. Care was taken during the In situ modulated fluorescence measurements. Two deployments to minimise any disturbance of the sea- designs of custom submersible fluorometer (Aquation, grasses. The fluorometers were left in position for at Australia) were used that excite chlorophyll a fluo- least 24 h. While the second design of fluorometer was rescence with a modulated blue light (470 nm light- programmed simply to measure ΔF/Fm’ every 15 min, emitting diode, LED) before and during a 0.8 s blue- the dark-acclimating shutter fluorometers were addi- enriched white saturating pulse provided by a 10 W tionally programmed to remain closed for a 15 min LED (Luxeon, Lumileds Lighting). A far-red LED interval once every 2 h, thereby also allowing 12 mea- (735 nm) incorporated into the fluorometers was acti- surements of Fv /Fm during a 24 h interval. Examination vated for 5 s prior to saturating pulse measurements to of the φII data directly after the Fv /Fm measurements facilitate the opening of PSII centres and hence deter- suggested no apparent effect of the 15 min dark treat- mine Fo' (i.e. minimal fluorescence intensity with all PS ment on subsequent φII measurements, which would II reaction centers open in a light-acclimated state). have manifested as an increase in φII values The sensor head of one fluorometer design closed over directly after each Fv /Fm measurement. the sample when taking a measurement, thus placing Diel photosynthesis – irradiance curves. ETRs were the sample in darkness (Fig. 1). The sensor head could calculated according to Genty et al. (1989) as the prod- then be left in place for a predetermined interval, uct of ambient irradiance, φII, AF at the time of mea- thereby dark-acclimating the sample. Fluorescence surement and 0.5 to account for electron sharing measurements made immediately prior to closing of between photosystem I (PSI) and PSII. Diel photosyn- the automated shutter provided background fluores- thesis – irradiance (P-I) curves (Longstaff et al. 2002), cence values which could then be subtracted from sub- (where ‘I ’ represents non-wavelength-specific irradi- 28

146 Aquat Biol 7: 143 –152, 2009

ance) were plotted using absorbed irradiance (Ia, Saroussi & Beer 2007) for the abscissa; I was calcu- a 0.7 lated as the product of incident irradiance (Ii), AF and 0.5, which describes the proportion of exciton energy 0.6 shared between PSI and PSII. P-I curves were gener- 0.5 ated using data acquired over 24 h intervals. The expo- nential model of Webb et al. (1974) was used to derive 0.4 values for ETRmax and the photon to electron conver- 0.3 sion efficiency (α) using non-linear least squares min- Absorbance 8 m imisation techniques and Sigmaplot software (Jandel 0.2 18 m Scientific). The saturation irradiance, I , was calcu- k 33 m lated from ETR and α by extrapolating a linear P-I 0.1 max curve with slope α to ETRmax, and errors associated 0.0 with Ik were determined by propagation from ETRmax 06:00 09:00 12:00 15:00 18:00 and α. Non-saturating curves generated from the deeper Time (h) plants were analysed using the Webb model, with ini- Fig. 2. Halophila stipulacea. Relative absorbance of leaves at tial parameter seed values obtained from the saturat- 8, 18 and 33 m depth measured from dawn to dusk. ing curves of shallow plants. Thus values of ETRmax Values are mean ± SD of 12 replicate measurements were extrapolated from the non-saturating data obtained in the field. As the above curves were calculated using data RESULTS pooled from both the morning and afternoon, estimates of ETRmax necessarily could not reflect the ob- Absorbance served maximum ETR at noon. Therefore, observed

ETRmax and Ik calculated using observed ETRmax were AF values of Halophila stipulacea at 8 m declined also presented. Values of α were assumed to be similar significantly from ~0.60 to a midday minimum of 0.34, for both calculated and observed curves. rising again in the afternoon to values similar to, Fluorescence parameters and quenching analysis. but slightly lower than, predawn values (Fig. 2). We Fluorescence parameters follow the notation of Van ascribe this difference in AF to the chloroplast clump- Kooten & Snel (1990). F or Ft denote steady state fluo- ing phenomenon as described by Sharon & Beer rescence under ambient irradiance. Fm’ and Fm are (2008). In contrast, AF values of the deeper growing maximal fluorescence obtained during a 0.8 s period of plants only declined slightly during the day, with mar- saturating light for samples exposed to ambient light or ginally greater predawn and dusk values compared to darkness, respectively. Fo and Fo’ represent minimal the 8 m plants. fluorescence of (respectively) dark-acclimated samples, or light-acclimated samples measured after a brief 0.04 dark exposure after the shutter had closed. Kramer et

) al. (2004) described the analytical techniques that –1 Morning enabled the examination of the allocation of excitation Afternoon 0.03 energy to photochemistry (φII) and 2 competing non- (A photochemical pathways (downregulation, φ ) and NPQ ty other energy losses (basal intrinsic nonradiative decay loci in the present study, φNO). 0.02

NPQ = (Fm – Fm’)/Fm’ ce qP = (Fm’ – Ft)/(Fm’ – Fo’) ban r0.01 eh F φNO = 1/{NPQ + 1 + qP[(Fm/Fo) – 1]} so 1 = φNO + φNPQ + φII

Ab qcu = (Fm’ – Ft)/[(Ft – Fo’) + Fm’ – Fo’] 0.00 where qP represents photochemical quenching, and 8 18 33 qcu represents the proportion of open reaction centres Depth (m) (Kramer et al. 2004), assuming a value of 0.5 as the Fig. 3. Halophila stipulacea. Rate of change of absorption fac- exciton transfer probability parameter, and assuming tor (AF) at 8, 18 and 33 m before and after midday. Values an intermediate model for reaction centre connectivity presented are absolute; morning values are negative and (Lavergne & Trissl 1995). afternoon values are positive 29 Runcie et al.: Depth-dependent seagrass photosynthesis 147

At each depth, the absolute value of the rate of decline reflecting the decline in midday irradiance with depth in AF during the morning was similar to that during the (Figs. 4 – 6). After sunset, φII of plants at all afternoon (Fig. 3). The magnitude was greatest for shal- depths increased rapidly to values around 0.6, low seagrasses and least for the deepest seagrasses, in- followed by a slow gradual increase throughout the dicative of a more marked response in shallow water. night. Fv/Fm, as measured after in situ dark- Data describing AF acquired at dawn satisfied the as- acclimation for 15 min, declined during the day with sumptions of homogeneity, and AF at 8 m (0.558 ± 0.03; declining φII and recov- ered rapidly at dusk all reported values are mean ± SD) was significantly less assuming the same values as φII during the night. than values at the deeper sites which did not differ sig- Fv/Fm continued to rise slightly after dawn while φII nificantly (33 m: 0.604 ± 0.05; 18 m: 0.632 ± 0.03; F = 14.2, commenced its decline p > 0.001). AF measured at dusk was significantly differ- ETRs tended to increase rapidly during the morning ent at the 3 sites (8 m: 0.509 ± 0.03; 18 m: 0.642 ± 0.04; and decline more slowly in the afternoon. This can be 33 m: 0.598 ± 0.06; F = 26.6, p > 0.000). seen more clearly in Fig. 7 where morning values of ETRs for shallow plants are far greater than afternoon values for the same absorbed irradiance. The reduced differences between morning and afternoon ETRs of Morning and afternoon fluctuation in φ II, Fv /Fm and ETR deeper plants is largely a consequence of the reduced irradiance, as can be seen when comparing ETRs where I is ~20 to 160 µmol photons m–2 s–1 for ex- Minimum midday values of φII at 8, 18 and 33 a m dropped to less than 0.1, 0.2 and 0.3, periments conducted at the 3 depths (Fig. 7). Clearly, respectively, ETRmax calculated from pooled morning and afternoon

8 m depth 18 m depth 20 20

) ) –1 s –1 15 s

15 –2 –2

m m – 10 – 10 e

mol 5 (µmol e (µmol µ ( 5

ETR 0 ETR 0

0.6 0.6

m 0.5 F / F

v v F

F 0.4 0.4

and and

and and 0.3 II II

II II Φ Φ 0.2 0.2 ΦII ΦII Fv /Fm

0.1 ) F /F

) v m –1 –1 s

400 –2 150 –2 m

m 300 100 200 50 100 0 0

06:00 09:00 12:00 15:00 18:00 21:00 00:00 03:00 06:00 09:00 12:00 15:00 18:00 21:00 00:00 03:00 06:00 09:00 12:00 21:00 12:00 15:00 12:00 15:00 18:00 00:00 03:00 06:00 09:00 18:00 21:00 (µmol quanta quanta (µmol (µmol quanta i i

I Time (h)

Fig. 4. Halophila stipulacea. Diel variation in electron trans- Fig. 5. Halophila stipulacea. Diel variation in electron trans-

port rate (ETR), quantum yield of PSII (φII) and incident irra- port rate (ETR), quantum yield of PSII (φII) and incident irradiance (Ii) of a single representative leaf at 8 m depth diance (Ii) of a single representative leaf at 18 m depth 30

148 Aquat Biol 7: 143 –152, 2009

) 12

–1 33 m depth

s 10 8 m

–2 10

m – 5 8

(µmol e 0 4

ETR 0.6 2

m 0.5 0 F /

v F 0.4 0 20 40 60 80 100 120 140 160 180 12 and and 0.3

II II ΦII 18 m

Φ 10 F /F )

0.2 v m –1

s 8 0.1 –2 m

)

– 6

–1 e s

–2 20 4

m 15 10 5 2 0 ETR (µmol

12:00 15:00 09:00 18:00 06:00 09:00 12:00 15:00 18:00 21:00 00:00 03:00 06:00

0 20 40 60 80 100 120 140 160 180

(µmol quanta quanta (µmol

i Time (h) I 12 Fig. 6. Halophila stipulacea. Diel variation in electron trans- 33 m port rate (ETR), quantum yield of PSII (φII) and incident irra- 10 diance (Ii) of a single representative leaf at 33 m depth 8

Table 1. Halophila stipulacea. Observed maximum electron 2 –2 –1 transport rate (ETRmax, µmol electrons m s ) and calculated photosynthetic parameters ETRmax, initial slope of the P-I 0 –1 curve (α; electrons photon ), saturation irradiance Ik (derived from calculated and observed ETRmax values) and –2 –1 0 20 40 60 80 100 120 140 160 180 incident irradiance Ii (µmol photons m s ) measured in situ –2 –1 at 8, 18 and 33 m. ETRs were derived from multiple Absorbed irradiance, Ia (µmol quanta m s ) modulated chlorophyll fluorescence measurements and Fig. 7. Halophila stipulacea. Representative photosynthesis – estimates of absorbed irradiance (I ) obtained over a irradiance curves measured over ≥ 24 h at 8, 18 and 33 m intervals ≥ 24 h. Each value represents parameter depth (see also Table 1). Absorbed irradiance (I × AF × 0.5) estimates (± SD) derived from a single experiment i was used to calculate electron transport rate and was used for conducted over a 24 h interval on an individual leaf. calc.: the abscissa. Closed and open circles represent morning and calculated; obs.: observed afternoon measurements, respectively Depth ETRmax α Ik Ii (m) (calc.) (obs.) (calc.) (obs.) (max)

8 7.04 ± 0.4 18.67 0.80 ± 0.4 8.75 ± 4.1 23.3 419 data underestimated observed ETRmax when noonday 7.44 ± 0.6 21.24 0.77 ± 0.4 9.65 ± 5.4 27.6 419 irradiance was high (Table 1, Fig. 7), while calculated 5.28 ± 0.2 8.60 0.70 ± 0.2 7.56 ± 1.9 12.3 419 values for the deeper plants more closely approxi- 18 17.05 ± 2.0 18.35 0.55 ± 0.1 30.84 ± 4.7 33.4 167 4.90 ± 0.4 8.84 0.41 ± 0.1 12.02 ± 2.3 21.6 167 mated observed values. Observed values of ETRmax –2 –1 33 9.87 ± 0.6 7.24 0.57 ± 0.0 17.25 ± 1.3 12.7 75 ranged from 7 to 21 µmol electrons m s , with lower 20.69 ± 2.8 9.22 0.58 ± 0.0 35.87 ± 4.9 15.9 75 values observed at all depths and higher values only observed at shallow and intermediate depths (Table 1). experiment ment experi- 8 m 18 tochemical quenching quenching tochemical cal units, (atomic 9. Fig. tochemical quenching cal 8. Fig. 8 m (atomic units, (atomic –2 –1 –2 –1) Quantum yield (a.u.) I (µmol quanta m s ) Quantum yield (a.u.)

I (µmol quanta m s quenching quenching by quenching depth. Incident depth. Incident irradiance 100 200 300 400 100 150 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 50 0 0 Halophila stipulacea Halophila Halophila stipulacea

depth. Incident depth. Incident irradiance 06:00 12:00 is

09:00 is a.u.) of a.u.) a.u.) of PSII of a.u.) by 15:00 presented presented 12:00 presented

down-regulation down-regulation down-regulation down-regulation 15:00 18:00

18:00 PSII ( ( φ φ 21:00 NO 21:00 NO in µmol in µmol in

o a of ) fluorescence fluorescence fluorescence fluorescence o a of ) . 00:00 . µmol

Diel Diel 00:00 18 m Diel Diel Time m Time

03:00 at the

single single depth

variation in 03:00 quanta quanta

06:00 variation in depth ( ( Runcie (h) φ φ (h) 09:00 NPQ depth NPQ

at the the at 06:00

representative representative ( representative representative ( 12:00 ) ) and ) φ φ

m m II II and other non-pho- ), ), 15:00 09:00 –2 –2 et al.: et al.: of non-photochemi- non-photochemi- non-photochemi-

s quantum yields quantum 18:00 quantum yields yields quantum –1 –1 the other

depth 12:00

21:00 Φ

Depth-dependent seagrass experiment experiment

00:00 NPQ

15:00 Φ non-pho- non-pho- Φ Φ

NPQ

leaf leaf 03:00 NO Φ leaf II of Φ

NO the

18:00

06:00 II

at 09:00 at

21:00 12:00 31 decline was also was decline 12 derived plants a onset, night. steady timated at < 0. At 33 m mal ably generally around φ around ous shallow ranged declined the (Fig. 8 0.3 at irradiance ( irradiance Fig. 10.Fig. cal photochemical quenching quenching photochemical (atomic units, (atomic NPQ NPQ leaf at 33 m 33 at leaf The daily daily The t m 8 At –2 –1

strong midday decline midday strong I (µmol quanta m s ) Quantum yield (a.u.) and

quenching 0.0 0.2 0.4 0.6 0.8 1.0 shallow differences between depths and and depths between differences value 20 40 60 80 the the er 0 similarly ). A

midday,

and then and

at 33 m At 8 m 8 At roneous) Halophila stipulacea 33 µmol 33 µmol at 0.6 to from from from 0.41 to 0.80, to 0.41 from experiment 09:00

Non-photochemical Non-photochemical site. site. with photosynthesis of 1at depth, about similar

I plants a.u.) of a.u.) k

by 12:00 mean initial slope observed ETR observed near ) depth. depth depth While r also were higher were dropped to dropped

educed depth, photons photons anomaly where anomaly where 0.4 in 0.4 midday midday φ

0.4 down-regulation down-regulation

pattern could could pattern incr 15:00 apparent, apparent,

NPQ NPQ to zero at zero is PSII dropping dropping

Incident irradiance irradiance Incident pr (Fig. 10). at eased calculated values values calculated about 18:00

esented depth,

declined declined

fluorescence fluorescence this midday irradiance irradiance plants m 33 m ( . Fig. 9 (Fig. φ night Diel Diel –2 NO Time near 21:00

was

max with ) of of ) value s 10 % for the for 10 % for but far but at to zero to zero at –1

φ depth quantum in variation in The at all at

NPQ NPQ , . ) be values showed showed values

00:00

(h) ( φ zer from from µmol except except

midday midday Fig. and deeper plants, deeper plants, values evident a NPQ NPQ of the of gr (

seen from from ( declined declined

range only rose rose only o single representative NPQ φ eater 03:00 at II

φ 8 depths quanta ), reached reached ranged midday ) ) less ). NO at the the at

non-photochemi- non-photochemi- night. yields 18m At for an at 18 m and about about

night,

quantum yields quantum 06:00 P of was to

of (Fig. values deep plants. - marked, marked, I

m φ Φ other

with during to about cur saturation NO depth

–2 between 09:00 φ NO values under (presum- (presum- its Φ

20 % for for 20 % NO

no obvi- no obvi- but was was but Φ s about

9 wher

NPQ ves, –1 values values depth, II ). maxi-

t the at

was 12:00 The light light non- non-

149 of the es- es-

0.5 of of for for e

15:00 32

150 Aquat Biol 7: 143 –152, 2009

DISCUSSION Axelsson 2004) as more electrons are diverted from photochemistry. Thus, Halophila stipulacea at 8 m has The rate of decline in absorbance of Halophila stipu- more than enough light to conduct photosynthesis dur- lacea was greatest in shallow waters, supporting previ- ing the day, and accordingly diverts the excess energy ous observations that high light exposure causes a absorbed into NPQ (Fig. 8). Also, the fact that ETRs physiological response in this species (Sharon & Beer were lower in the afternoon than before noon at com- 2008). This response was less marked for the deeper parable irradiances points towards a dynamic photo- plants, with progressively decreasing rates of change inhibition taking place after several hours of high over the day with increasing depth. These data sug- irradiance. A similar phenomenon of hysteretic diurnal gest that, with increasing depth, there is a diminishing ETRs was also described for shallow-growing corals in requirement for chloroplast translocation throughout the Gulf of Aqaba (Winters et al. 2003) and the sea- the day. Consequently, resources used for chloroplast grass Thalassia testudium (Belshe et al. 2007, 2008). At movement in shallow plants can be reallocated to other the 18 and 33 m sites, φII does not exceed this threshold processes. This capacity to acclimate during the day to value (Figs. 5 & 6) and accordingly diverts less excess elevated irradiance appears to be inducible, as shown energy into NPQ. Even at 33 m there is substantial by Sharon et al. (2009), where translocated seagrasses NPQ, with 30 % of exciton energy diverted to this sink assumed similar responses to those resident at each at noon (Fig. 10). However, this may not be surprising depth. as H. stipulacea has been observed at depths far P-I curves are commonly used in aquatic photosyn- in excess of 33 m (Lipkin 1979). thesis research to characterise the response of meta- The automated dark-acclimation function enables bolic rates to changing irradiance, and ETRs are often the measurement of dark-acclimated fluorescence in presented because chlorophyll fluorescence (from the field without user intervention, as well as the appli- which ETR is derived) can be easily measured. How- cation of a far-red light. Until now this not been possi- ever, recent research has demonstrated the impor- ble and, as pointed out by Maxwell & Johnson (2000) tance of diel changes in parameters often assumed to and others, this enables the significant limitations of be invariant (Belshe et al. 2007). For example, diel dark-acclimating samples in the field to be overcome. changes in absorbance of Halophila stipulacea due to Using this technique, our field measurements enable chloroplast clumping clearly influenced calculated us to determine the extent that long-term NPQ (qI, or ETRs (Sharon & Beer 2008), and Saroussi & Beer (2007) photoinhibitory quenching; Müller et al. 2001) con- argued that Ia rather than Ii should be used in tributes to the decline in photon conversion efficiency P-I curves when calculating α and Ik. Both studies relative to the other more rapidly relaxing NPQ com- demon- strated that apparent differences in ETR due ponents qE (energy-dependent quenching) and qT to seasonal or depth effects may be non-significant (state-transition quenching). As expected, all 3 forms when the P-I curve is constructed using Ia rather of NPQ are active during the day, hence the difference than Ii. The results of the present study show wide between effective and maximal quantum yield values. variation in maximum ETRs of P-I curves measured However, at night, short-term relaxation of qE and qT across the depth range (Table 1). The differences in has occurred and the remaining NPQ (qI) is evident for values of ETRmax and α derived from curves using both effective and maximal quantum yields. Here, qI pooled data versus observed values of ETRmax can be seen as the difference between the predawn demonstrate the additional complexity of analysing maximum quantum yield and nighttime quantum yield. diel P-I curves when representative parameter In addition to this ability to quantify these 3 different values are desired, and the use of observed data may phases of NPQ according to relaxation kinetics, in situ be generally advised if representative parameters are dark-acclimation also enables a detailed deconvolu- required. tion of in situ-derived NPQ components over a diel The wide variation in parameter estimates across the cycle and enables the direct estimation of NPQ- depth gradient in the present study may in part be parameters described in the literature (Kramer et al. due to variability in leaf condition and orientation, as 2004, Kornyeyev & Holaday 2008). Here, the relatively leaves were fixed in a horizontal position to enable straightforward deconvolution described by Kramer et measurement. As the propagated errors associated al. (2004) is employed, where the term for basal intrin- with Ik values were considerably greater than those sic non-radiative decay, φNO, includes both constitutive associated with ETRmax and α, Ik should be treated with thermal dissipation and thermal dissipation associated caution when used to compare treatments. with PSII photo-inactivation (Kornyeyev & Holaday

At 8 m depth, φII declined below 0.1 (Fig. 4). When 2008).

φII reaches this critical value (due to increasing φNO remained fairly constant at between 30 and 40 % irradiance), electron transport estimates become at all 3 depths, with slightly more variation in shallow less suitable as descriptors of photosynthetic rates (Beer & 33

Runcie et al.: Depth-dependent seagrass photosynthesis 151

waters and slightly lower values in deep waters. Thus, Genty B, Briantias JM, Baker NR (1989) The relationship Halophila stipulacea dissipates a large proportion of between the quantum yield of photosynthetic electron light energy through intrinsic decay apparently re- transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87– 92 gardless of depth within the depth range examined. Hulings NC (1979) The ecology, biometry and biomass of the The water clarity in the Gulf of Aqaba is such that pho- seagrass Halophila stipulacea along the Jordanian coast of tosynthetic organisms at 33 m are unlikely to be light the Gulf of Aqaba. Bot Mar 22:425 – 430 limited (Winters et al. 2003). As φ describes the dis- Kornyeyev D, Holaday AS (2008) Corrections to current NO approaches used to calculate energy partitioning in photo- sipation of excess energy and is relatively constant system 2. Photosynthetica 46:170 –178 throughout the diel period in the deepwater plants, it Kramer DM, Johnson G, Kiirats O, Edwards GE (2004) New partly represents a constitutive property of H. stipu- fluorescence parameters for the determination of QA redox lacea rather than a high-light-induced acclimatory state and excitation energy fluxes. Photosynth Res 79: 209 –218 mechanism. The reduced capacity for photosynthetic Larkum AWD (1976) Ecology of Botany Bay. I. Growth of Posi- photon conversion during the day in deep water leaves donia australis (Brown) Hook. f. in Botany Bay and other occurs at a depth where one might consider irradiance bays of the Sydney basin. Aust J Mar Freshw Res 27: to be limiting; clearly it is not and the daytime decline 117–127 Lavergne J, Trissl HW (1995) Theory of fluorescence induc- in φII of deepwater plants is apparently an acceptable tion in photosystem II: derivation of analytical expressions trade-off for the capacity to rapidly divert excess light in a model including exciton-radical-pair equilibrium and energy. If one were to extrapolate the daily maximum restricted energy transfer between photosynthetic units. Biophys J 68:2474 – 2492 φNO of H. stipulacea measured over a diel period to H. Lee KS, Park SR, Kim YK (2007) Effects of irradiance, temper- stipulacea at deeper depths, the less-constitutive por- ature, and nutrients on growth dynamics of seagrasses: a tion may become negligible at the depth limit of this review. J Exp Mar Biol Ecol 350:144 – 175 species; such an approach might be used to suggest Lipkin Y (1979) Quantitative aspects of seagrass communities a species’ depth limit. The present study invites the particularly those dominated by Halophila stipulacea, in Sinai (northern Red Sea). Aquat Bot 7:119 –128 question of the cost of maintaining a constitutive Longstaff BJ, Kildea T, Runcie JW, Cheshire A and others capacity to dissipate excitons versus the cost of main- (2002) An in situ study of photosynthetic oxygen exchange taining a down-regulatory capacity for dissipation. and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta). Photosynth Res 74:281–293 Maxwell K, Johnson GN (2000) Chlorophyll fluorescence — Acknowledgements. This work was conducted in conjunction a practical guide. J Exp Bot 51:659 – 668 with the Bat Sheva de Rothchild seminar on Gross and Net McCree KJ (1981) Photosynthetically active radiation. In: Primary Productivity within the framework of the 8th Interna- Lang OL, Novel P, Osmond B, Ziegler H (eds) Physiologi- tional workshop of Group for Aquatic Primary Productivity cal plant ecology. Encyclopedia of Plant Physiology, Vol (GAP). The authors thank the Inter-University Institute for 12A. Springer-Verlag, Berlin Marine Sciences of Eilat for logistic support, in particular Müller P, Li XP, Niyogi KK (2001) Non-photochemical quench- I. Berman-Frank, and O. Ben Shaprut and D. Tobias for ing: a response to excess light energy. Plant Physiol 125: diving support. 1558 –1566 Orth RJ, Carruthers TJB, Dennison WC, Duarte CM and others (2006) A global crisis for seagrass ecosystems. Bio- LITERATURE CITED science 56:987– 996 Ralph PJ, Durako MJ, Enríquez S, Collier CJ, Doblin MA Beer S, Axelsson L (2004) Limitations in the use of PAM fluo- (2007) Impact of light limitation on seagrasses. J Exp Mar rometry for measuring photosynthetic rates of macroalgae Biol Ecol 350:176 – 193 at high irradiances. Eur J Phycol 39:1– 7 Runcie JW, Durako MJ (2004) Among-shoot variability and Beer S, Waisel Y (1982) Effects of light and pressure on photo- leaf-specific absorptance characteristics affect diel esti- synthesis in two seagrasses. Aquat Bot 13:331– 337 mates of in situ electron transport of Posidonia australis. Beer S, Vilenkin B, Weil A, Veste M, Susel L, Eshel A (1998) Aquat Bot 80:209 –220 Measuring photosynthetic rates in seagrasses by pulse Runcie JW, Riddle MJ (2004) Measuring variability in chlo- amplitude modulated (PAM) fluorometry. Mar Ecol Prog rophyll-fluorescence-derived photosynthetic parameters Ser 174:293 – 300 in situ with a programmable multi-channel fluorometer. Beer S, Larsson C, Poryan O, Axelsson L (2000) Funct Plant Biol 31:559 – 562 Photosyn- thetic rates of Ulva (Chlorophyta) measured Runcie JW, Riddle MJ (2006) Photosynthesis of marine by pulse amplitude modulated (PAM) fluorometry. Eur J macroalgae in ice-covered and ice-free environments in Phycol 35: East Antarctica. Eur J Phycol 41:223 –233 69 – 74 Saroussi S, Beer S (2007) Alpha and quantum yield of aquatic Belshe EF, Durako MJ, Blum JE (2007) Photosynthetic rapid plants derived from PAM fluorometry: uses and misuses. light curves (RLC) of Thalassia testudinum exhibit diurnal Aquat Bot 86:89 – 92 variation. J Exp Mar Biol Ecol 342:253 – 268 Schwarz AM, Hellblom F (2002) The photosynthetic light Belshe EF, Durako MJ, Blum JE (2008) Diurnal light response of Halophila stipulacea growing along a depth curves and landscape-scale variation in photosynthetic gradient in the Gulf of Aqaba, the Red Sea. Aquat Bot 74: characteristics of Thalassia testudinum in Florida Bay. 263 –272 Aquat Bot Schwarz AM, Hawes I, Andrew N, Norkko A, Cummings V, 89:16 –22 Thrush S (2003) Macroalgal photosynthesis near the Edwards AJ, Head SM (eds) (1987) Key environments: Red Sea. Pergamon Press, Oxford 34

152 Aquat Biol 7: 143 –152, 2009

southern global limit for growth; Cape Evans, Ross Sea, Van Kooten O, Snel JFH (1990) The use of chlorophyll fluores- Antarctica. Polar Biol 26:789 – 799 cence nomenclature in plant stress physiology. Photosynth Sharon Y, Beer S (2008) Diurnal movements of chloroplasts in Res 27:121–133 Halophila stipulacea and their effect on PAM fluorometric Webb WL, Newton M, Starr D (1974) Carbon dioxide ex- measurements of photosynthetic rates. Aquat Bot 88: change of Alnus rubra: a mathematical model. Oecologia 273 –276 17:281–291 Sharon Y, Silva J, Santos R, Runcie J (2009) Photosynthetic re- Winters G, Loya Y, Rottgers R, Beer S (2003) Photoinhibition sponses of Halophila stipulacea to a light gradient. II. Accli- in shallow-water colonies of the coral Stylophora pistillata mations following transplantations. Aquat Biol 7:153 – 157 as measured in situ. Limnol Oceanogr 48:1388 – 1393

Submitted: November 28, 2008; Accepted: April 24, 2009 Proofs received from author(s): June 9, 2009 35

Vol. 7: 153 –157, 2009 AQUATIC Published online October 22, 2009 doi: 10.3354/ab00148 BIOLOGY Aquat Biol

Contribution to the Theme Section ‘Primary production in seagrasses and macroalgae’ OPEN ACCESS

Photosynthetic responses of Halophila stipulacea to a light gradient. II. Acclimations following transplantation

Yoni Sharon1, 2,*, João Silva3, Rui Santos3, John W. Runcie4, Mark Chernihovsky2, 1 Sven Beer

1 Department of Plant Sciences, Tel Aviv University, Tel Aviv 60078, Israel 2 The Interuniversity Institute for Marine Sciences, POB 469, Eilat 88103, Israel 3ALGAE - Marine Plant Ecology Research Group, Centre of Marine Sciences (CCMAR), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal 4 School of Biological Sciences, University of Sydney, New South Wales 2006, Australia

ABSTRACT: Halophila stipulacea is the dominant seagrass in the Gulf of Aqaba (northern Red Sea), where it grows from the intertidal to depths exceeding 50 m. Its successful growth under such a broad irradiance gradient shows either a high plasticity or is caused by longer-term adaptations to the various depths, possibly resulting in the formation of ecotypes. In April 2008 we transplanted shoots of this seagrass between the extreme depths of its distribution at the study site (8 and 33 m) in order to evaluate its acclimation potential to various irradiances. We compared photosynthetic parameters derived from light response curves generated by PAM fluorometry (so-called rapid light curves, RLC) and measured chlorophyll a and b concentrations. RLCs from the shallow (~400 µmol photons m–2 s–1 at midday) and deep (~35 µmol photons m–2 s–1 at midday) sites were characteristic for high- and low- light growing plants, respectively, and the transplanted seagrasses acclimated to their new environments within 6 d, at which time their RLCs resembled those of the original plants growing at the depths to which they had been transplanted. Concentrations of both chlorophyll a and b decreased or increased when the plants were transferred to high- vs. low-light environments, respectively, but the chlorophyll a:b ratios remained constant. These fast changes in photosynthetic responses and light absorption characteristics in response to changing light environments points to Halophila stipulacea as being a highly plastic seagrass with regard to irradiance, which may partly explain its abundance across a wide range of irradiances along the depth gradient that it occupies.

KEY WORDS: Acclimation · Depth gradient · Halophila stipulacea · Irradiance · Photosynthesis · Pigments · Seagrass

Resale or republication not permitted without written consent of the publisher

INTRODUCTION deed been shown to possess the capacity to cope with variation in irradiance as well as spectral composition In addition to diurnal changes in insolation, the marine in a plastic way (e.g. Monro & Poore 2005, Mata et environment is characterised by strongly attenuated al. 2006). Regarding submerged marine angiosperms irradiances along a relatively short depth gradient. (seagrasses), much information has been acquired The ability of marine macrophytes to develop mecha- regarding their photophysiological responses to irradi- nisms to utilise and/or cope with different irradiances ance (Olesen et al. 2002, Durako et al. 2003, Silva & may therefore be crucial for their functioning at tempo- Santos 2003, recently reviewed by Ralph et al. 2007), rally dynamic as well as spatially different surround- but whether these responses are the result of adapta- ings (Sultan 2000). Some marine macroalgae have in- tions (long-term selection processes resulting in eco-

*Email: [email protected] © Inter-Research 2009 · www.int-res.com 36

154 Aquat Biol 7: 153 –157, 2009

types) or acclimation processes (short-term plastic re- (possibly belonging to the same clone) and roots, from sponses) are largely unknown. the 2 depths were removed with their sediments (ca. Halophila stipulacea is the dominant seagrass in the 3 l) into mesh bags, and 3 such bags were placed into clear waters (light attenuation coefficient Kd [400 to dug-out depressions at each of the sites (both vertically 700 nm] = 0.1) of the Gulf of Aqaba (northern Red Sea). and horizontally for same-depth controls). Photosyn- There it grows abundantly in fine sediments from the thesis and pigment measurements were performed intertidal down to depths > 50 m (Lipkin 1979, Y. just prior to transplantation and following 6 and 14 d of Sharon pers. obs.) where, unlike shallow-growing spe- exposure to the various sites. cies, it is not affected by hydrostatic pressure (Beer & Chlorophyll fluorescence was measured on cloudless Waisel 1982). Its photosynthetic responses to irradi- days at 09:30 to 10:30 h at the 2 sampling depths using ance at various depths are characterised by decreasing an underwater fluorometer (Diving-PAM). Individual maximal photosynthetic rates (measured as relative leaves (from different shoots, n = 5) were placed into a electron transport rates, rETR) and decreasing onsets leaf-clip and rapid light curves (RLCs) were initiated of saturating irradiances (I k) at increasing depths from immediately. Each leaf was irradiated with increasing 7 to 30 m (Schwarz & Hellblom 2002). For plants grown pre-set photosynthetically active radiation (PAR) steps at higher irradiances, midday chloroplast clumping (0, 37, 56, 104, 160, 227, 339, 512 and 818 µmol photons was quantified by Sharon & Beer (2008), and it was m–2 s–1) for 5 s, and the effective quantum yield of suggested that this is an important photoprotective Photosystem II (PSII) was recorded after each step. It mechanism for shallow-growing plants. Thus, while ac- was found that 5, 10 and 30 s of exposure to each level climation to daily temporal changes in irradiance was of irradiance yielded the same results, so 5 s was shown for high-light plants of Halophila stipulacea, it chosen in order to minimise the diving time. Electron is not known whether the lower rETRs of this seagrass transport rates (ETR) were calculated as ETR = Y × I × at low irradiances such as demonstrated by Schwarz & AF × 0.5 where Y is the effective quantum yield of PSII, Hellblom (2002) are also due to acclimation processes I is the incident irradiance, AF is the absorption factor or can only be explained by longer-term adaptations. and 0.5 is the assumed proportion of light absorbed be- As part of the recent Group for Aquatic Primary Pro- tween PSII and PSI. AFs were estimated in situ by mea- ductivity (GAP, see www.gap-aquatic.org/) workshop suring the light absorbed by the leaves as described in Eilat (April 2008), we studied in situ various photo- elsewhere (Beer & Björk 2000, except that ambient physiological aspects of the seagrass Halophila stipu- light was used instead of a lamp), and assuming that all lacea. In the present study, we tested the photosyn- light was absorbed by the photosynthetic pigments of thetic plasticity of this plant as a response to in situ the leaves (Sharon & Beer 2008). ETR vs. PAR curves irradiance. This was done by comparing pigment con- were generated after correcting incident PAR with AF tents and photosynthetic parameters derived from so as to depict absorbed PAR on the x-axes (according pulse-amplitude modulated (PAM) fluorometry follow- to Saroussi & Beer 2007; this is logically called for since ing reciprocal transplantations from the 2 edges of an the ETRs of the y-axis also include AF values). The H. stipulacea meadow. maximal photosynthetic rate (Pmax) and onset of light saturation (I k) were calculated after fitting the ETR data to equations by Platt et al. (1980). The maximal MATERIALS AND METHODS effective quantum yield (Y0) was taken as the first yield measurement of the RLC (according to Saroussi & The Halophila stipulacea seagrass bed studied was Beer 2007; this value is equal to α as commonly calcu- located ca. 200 m south of the InterUniversity Institute lated from the initial slope of the RLC). Initially, in (IUI) in Eilat, the Gulf of Aqaba, northern Red Sea order to find out if the plants from the deep and (29° 30’ N, 34° 54’ E). The vertical extension of the bed shallow sites had the same potential photosynthetic varies between seasons, and was between 8 and 33 m at capacity, PSII maximum quantum yield of photo- the time of the present study (April 2008). Midday irradi- chemistry in PSII (Fv /Fm) of dark-adapted plants were ances at the 2 experimental depths at this time were recorded for those plants at dawn (06:00 h). ~400 and ~35 µmol photons m–2 s–1 at 8 and 33 m, respec- Chlorophylls a and b (chl a + b) were extracted in tively; most days were cloudless during the 2 wk exper- N ’N-dimethylformamide from cut-out leaf sections imental period. The water temperature was 23°C. All within 20 min of collection (n = 5). The absorbance plants were accessed by SCUBA diving using NITROX. was measured in a spectrophotometer at 664, 647 and In order to measure the plasticity of the photosyn- 625 nm and concentrations were calculated according thetic responses of Halophila stipulacea to irradiance, to Moran (1982). we transplanted plants reciprocally from one site to Statistical analyses were performed using Sigma Stat another. Some 30 to 50 shoots, including rhizomes 3.00. Differences were considered significant at p < 0.05. 37

Sharon et al.: Photosynthetic acclimation after transplantation of a seagrass 155

RESULTS 50

In situ Fv /Fm values (mean ± SD) of native ) 40 –1 s

plants were found to be 0.76 ± 0.02 (n = 10) and 0.76 ± 0.01 (n = 10) for shallow (8 m) and deep (33 m) –2 m plants, respectively. Thus, no significant difference was 30

found in the maximum photosynthetic efficiency of PSII in Halophila stipulacea at the different 20 bathymetric sites during the time of these measurements. 10 The average RLCs (n = 5) of Halophila stipulacea (µmol electrons from the 2 different sites are depicted in Fig. 1. The

max

Pmax, I k and Y0 (equalling α) values derived from P 0 –2 –1 0 3 6 9 12 15 these curves were 39.7 ± 3.8 µmol electrons m s , Time (d) 65.0 ± Fig. 2. Halophila stipulacea. Maximal photosynthetic rates –2 –1 7.1 µmol photons m s and 0.61 ± 0.03 mol electrons (Pmax) at 0, 6 and 14 d after transplantation from 8 to 33 mol photons–1, respectively, for the shallow plants and m depth (s, —–), from 33 to 8 m (d, —–) and the 20.6 ± 1.4 µmol electrons m–2 s–1, 41.1 ± 4.4 µmol pho- corresponding controls (– – – ). Data points are means ± SD –2 –1 of 4 replicates tons m s and 0.50 ± 0.04 mol electrons mol photons–1, respectively, for the deep plants. All parameters were significantly different between the shallow and deep plants (Mann-Whitney U-test, p < 0.001). (Fig. 3). Also, Ik of the transplanted

Pmax of the shallow plants decreased significantly seagrasses became quantitatively similar to that of (Mann-Whitney U-test, p < 0.001) 6 d after transplanta- the plants originally growing at the 2 depths. While tion to the deep site, and kept decreasing slightly after the Y0 values of the transplanted plants also 14 d of exposure to the dimmer environment (Fig. 2). showed a pattern of change with time such that they

Conversely, deep plants increased their Pmax came to resemble those of the plants originally significantly (p < 0.001) 6 d after transplantation, and growing at the 2 depths (Fig. 4), this change was

did not change over the remaining 8 d. Thus, Pmax slower than for the other parameters. values of shallow plants became quantitatively The chl a + b content of shallow plants increased similar to those of deep plants after 6 d and vice between 6 and 14 d following transplantation to the versa. The control plants, which were dug up and deeper site (Fig. 5). Conversely, the deep plants then replanted at the same depths in order to showed a decreased chl a + b content after 6 d follow- appraise whether mechanical uprooting had affected ing transplantation to the shallow site. There was no

them, showed much less of a change in Pmax over significant difference in chl a:b ratios following the time. transplantations (Fig. 6).

Ik changed similarly to Pmax at the different envi-

) ronments to which the plants were transferred

–1 s

–2

m 35 80

30 )

–1 s

25 –2

m 20 40 15 ETR (µmol ETR electrons

10 20 (µmol photons

k 5 l

0 0 0 100 200 300 400 500 0 3 6 9 12 15 –2 –1 Ia (µmol photons m s ) Time (d)

Fig. 1. Halophila stipulacea. Electron transport rates (ETR) as Fig. 3. Halophila stipulacea. Onset of light saturation (Ik) at 0, a function of absorbed irradiances (Ia) derived from rapid light 6 and 14 d after transplantation from 8 to 33 m depth (s, —–), curves of plants growing at 8 (s) and 33 m (d) depth. Data from 33 to 8 m (d, —–) and the corresponding controls (– – – ). points are means ± SD of 5 replicates Data points are means ± SD of 4 replicates 38

156 Aquat Biol 7: 153 –157, 2009

0.7

DISCUSSION ) –1 Our results indicate that Halophila stipulacea is a 0.6 highly plastic seagrass that can acclimate to various

photons light environments quickly. This was shown before for plants grown under high-light conditions, where mol 0.5 chloroplast movements were suggested as a means to

both capture photons under diurnal low-light periods (when chloroplasts are dispersed) and apparently pro- 0.4 tect the leaves from photodamage during midday (by

clumping the chloroplasts, Sharon & Beer 2008). In the (mol electrons 0 present study, we showed that the photosynthetic Y 0.3 0 3 6 9 12 15 apparatus also features plasticity and it can acclimate Time (d) to various irradiances along a depth gradient accord- Fig. 4. Halophila stipulacea. Maximal effective quantum yield ing to classical adaptation strategies of high- and low- (Y0) at 0, 6 and 14 d after transplantation from 8 to 33 m depth light plants. Further, this plasticity is in line with the (s, —–), from 33 to 8 m (d, —–) and the corresponding controls thought that specimens of H. stipulacea growing at (– – – ). Data points are means ± SD of 4 replicates various depths in a meadow are not ecotypes, although the maximal irradiance during midday spans at least 7 1 order of magnitude. ) 6 The apparent lack of ecotype formation at various –2 depths within a Halophila stipulacea meadow may stem 5 from its largely vegetative growth pattern (through rhi-

(mg cm zome elongation) resulting in large areas belonging to

b 4 the same clone. Further, we have observed that the ver- + a 3 tical extension of the meadow differs between seasons. During the summer, the studied meadow extends from phy

o2 ll 5 m to a depth of 45 m (Y. Sharon pers. obs.), while during the winter the meadow’s lower limit only extends to 1 Chlor ~30 m. This may be due to irradiance and/or temperature; the lowest irradiance in the Gulf of Aqaba is in Jan- 0 0 3 6 9 12 15 uary and the lowest temperature in March (Winters et al. Time (d) 2006). The present study suggests that the plasticity in Fig. 5. Halophila stipulacea. Chlorophyll a + b concentrations the response to seasonal irradiance levels would allow at 0, 6 and 14 d after transplantation from 8 to 33 m for the successful acclimation to the different depths as depth (s, —–), from 33 to 8 m (d, —–) and the corresponding controls (– – – ). Data points are means ± SD of 4 replicates observed throughout the year. This would then explain the dynamic seasonal meadow extensions as based on 3 clonal growth, although H. stipulacea meadows were

found to be highly polymorphic between depths in the Mediterranean (Procaccini et al. 1999). On the cellular level, the fast (within 6 d) adjustment of Pmax to irradiance points towards rapid changes in the ratio b carbon fixation reactions during the acclimation to high : a or low midday irradiances. These changes could involve 2 key enzymes of the Calvin cycle, e.g. Rubisco, or other reactions following those of the primary photochemistry. The fact that P values in the transplanted seagrasses max Chlorophyll Chlorophyll became similar to those of the plants growing originally at the transplant depths shows that these acclimations are not only qualitative but also quantitative. The finding 1 that I values varied plastically in concord with P is 0 3 6 9 12 15 k max Time (d) also in agreement with acclimation to high and low irradiances. While chl a + b content also showed plasticity, Fig. 6. Halophila stipulacea. Chlorophyll a:b ratio at 0, 6 and 14 d after transplantation from 8 to 33 m depth (s, —–), from the response in the plants transplanted from the high- to 33 to 8 m (d, —–). Data points are means ± SD of 4 replicates the low-light environment was slower than in the low- to 39

Sharon et al.: Photosynthetic acclimation after transplantation of a seagrass 157

high-light transplants, indicating a longer time is re- Beer S, Waisel Y (1982) Effects of light and pressure on photo- quired for the production of new chlorophyll than its loss. synthesis in two seagrasses. Aquat Bot 13:331– 337 The lack of changes in chl a:b ratios in all treatments Durako MJ, Kunzelman JI, Kenworthy WJ, Hammerstrom WJ (2003) Depth-related variability in the photobiology of two suggests that this parameter has no role in in situ accli- populations of Halophila johnsonii and Halophila decipi- mation processes, such as reported by Durako et al. ens. Mar Biol 142:1219 – 1228 (2003) for 2 other Halophila species. While terrestrial Lipkin Y (1979) Quantitative aspects of seagrass communities, shade-adapted plants often increase chl b contents rela- particularly of those dominated by Halophila stipulacea, in Sinai (Northern Red Sea). Aquat Bot 7:119 –128 tive to chl a, it may be that the spectral changes with Mata L, Silva J, Schuenhoff A, Santos R (2006) The depth prevent such a change in H. stipulacea. effects of light and temperature on the photosynthesis In addition to the differences in photosynthetic of the Asparagopsis armata tetrasporophyte responses at different irradiances, Halophila stipu- (Falkenbergia rufo- lanosa), cultivated in tanks. lacea has been reported to feature various leaf mor- Aquaculture 252:12 –19 Monro K, Poore AGB (2005) Light quantity and quality induce phologies with depth (e.g. longer and wider leaves in shade-avoiding plasticity in a marine macroalga. J Evol deeper waters, Lipkin 1979). Also, changes in Pmax and Biol 18:426 – 435

I k along a depth gradient have been reported (Schwarz Moran R (1982) Formulas for determination of chlorophyllous & Hellblom 2002). However, in neither case was it ver- pigments extracted with N,N-dimethylformamide. Plant Physiol 69:1376 – 1381 ified whether those changes could be due to acclima- Olesen B, Enriquez S, Duarte CN, Sand-Jensen K (2002) tion processes or were the outcome of longer-term Depth-acclimation of photosynthesis, morphology and adaptations possibly resulting in ecotype formations. demography of Posidonia oceanica and Cymodocea nodosa Olesen et al. (2002) have suggested that some sea- in the Spanish Mediterranean Sea. Mar Ecol Prog Ser 236: 89–97 grasses (i.e. Cymodocea nodosa) can acclimate and Platt T, Gallegos CL, Harrison WG (1980) Photoinhibition of respond differently to changing light conditions than photosynthesis in natural assemblages of marine phyto- others (i.e. Posidonia oceanica). While C. nodosa dis- plankton. J Mar Res 38:687– 701 played reduced light requirements with depth, both Procaccini G, Acunto S, Fama P, Maltagliati F (1999) Struc- species acclimated mainly at the population structure tural, morphological and genetic variability in Halophila stipulacea (Hydrocharitaceae) populations in the western level by reducing self-shading in deeper waters. Mediterranean. Mar Biol 135:181–189 This is the first work reporting that Halophila stipu- Ralph PJ, Durako MJ, Enriquez S, Collier CJ, Doblin MA lacea’s wide depth distribution across a broad range of (2007) Impact of light limitation on seagrasses. J Exp Mar irradiances can be explained by its plastic photosyn- Biol Ecol 350:176 – 193 Saroussi S, Beer S (2007) Alpha and quantum yield of aquatic thetic abilities allowing for successful acclimation both plants derived from PAM fluorometry: uses and misuses. spatially and seasonally. While most of the differences Aquat Bot 86:89 – 92 between shallow (i.e. high-light) and deeper (low- Schwarz AM, Hellblom F (2002) The photosynthetic light light) growing plants measured in the present study response of Halophila stipulacea growing along a depth follow classical sun- vs. shade-acclimation patterns, gradient in the Gulf of Aqaba, the Red Sea. Aquat Bot others do not. For the latter, the higher Y0 (i.e. α) 74:263 –272 in shallow-growing plants and the stable chl a:b Sharon Y, Beer S (2008) Diurnal movements of chloroplasts ratios may be a result of spectral changes along the in Halophila stipulacea and their effect on PAM fluoro- depth gradient in the water column, which still need metric measurements of photosynthetic rates. Aquat Bot to be elucidated. 88:273 –276

Silva J, Santos R (2003) Daily variation patterns in LITERATURE CITED seagrass photosynthesis along a vertical gradient. Mar Ecol Prog Ser 257:37– 44 Beer S, Björk M (2000) Measuring rates of photosynthesis of Sultan SE (2000) Phenotypic plasticity for plant develop- two tropical seagrasses by pulse amplitude modulated ment, function and life history. Trends Plant Sci 5: (PAM) fluorometry. Aquat Bot 66:69 – 76 537– 542 Winters G, Loya Y, Beer S (2006) In situ measured

seasonal variations in Fv /Fm of two common Red Sea corals. Coral Reefs 25:593 – 598

Submitted: November 28, 2008; Accepted: March 1, 2009 Proofs received from author(s): March 25, 2009 40

Chloroplast clumping in the seagrass Halophila stipulacea is triggered by UV radiation

Yoni Sharon1,2, Gal Dishon2,3 and Sven Beer1

1Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel

2The Inter-University Institute of Marine Sciences, POB 469, Eilat 88103, Israel

3Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel

Corresponding author: Yoni Sharon

E-mail: [email protected]

Tel: +972-3-6409125

Running title: UVR triggers chloroplast clumping in Halophila stipulacea

ABSTRACT

It was shown previously that high irradiances caused chloroplasts to clump in the seagrass

Halophila stipulacea during midday (Sharon and Beer 2008). Since potentially harmful UVR is present in the shallow waters of the Gulf of Aqaba where part of its population thrives, we examined the effect of UVR on the clumping phenomenon. This was done by acclimating plants to high midday irradiances under filters that did or did not transmit UVR. It was found that no chloroplast movements occurred when the plants were protected from 80% of the

<400 nm wavelengths. Accordingly, UVR caused the clumping, which resulted in a 44% reduction in the absorption cross section of the leaves and, therefore, a concomitant lowering of midday electron transport rates. In addition, the effective quantum yield decreased by 26% during midday and the pre-dawn maximal quantum yield decreased by 10%. On the other hand, UVR had no effect on chlorophylls contents or on the characteristic absorption spectra of flavones and flavone-glycosides. We conclude that the potentially harmful effects of UVR on the photosynthetic apparatus of Halophila stipulacea are mitigated by its activation of chloroplast clumping, which functions as a means of protecting most chloroplasts and perhaps the nucleus from high irradiances, including UVR.

Key words: Chloroplast clumping; Halophila stipulacea; Photosynthesis; UV radiation;

Seagrasses

INTRODUCTION

While the mechanisms underlying chloroplast movements in plants have been studied on the intracellular and molecular levels (Gabrys 2004; Sakuri et al. 2005; Luesse et al. 2006;

Krzeszowiec et al. 2007; Suetsugu and Wada 2007; Yamashita et al. 2007), their ecophysiological role(s) is less understood. These movements are in many plants induced by blue light (Gabrys 2004; DeBlasio et al. 2005; Sakuri et al. 2005; Luesse et al. 2006;

Krzeszowiec et al. 2007; Shiihira-Ishikawa et al. 2007). In a few studies, it was suggested that also UV, or "near to UV", wavelengths could trigger chloroplast movements (Yatsuhashi

1996; Rodrigues et al. 2007). Interestingly, Kondo et al. (2004) reported that in many cases the chloroplast clumps are formed near or around the nuclei.

The only seagrass so far for which chloroplast movements has been described is Halophila stipulacea (Drew 1979; Sharon and Beer 2008). In the latter work, it was found that chloroplast clumping occurred during midday only in plants growing under high irradiances

(450 µmol photons m-2 s-1), while lower-irradiance grown plants maintained their chloroplasts evenly dispersed during the day. It was then suggested that the chloroplast clumping during midday may have a protective role against high irradiances.

In addition to high photosynthetically active radiation (PAR, 400-700 nm), coastal waters, especially clear tropical waters, also feature high levels of UV radiation (UVR, 280-400 nm) in their upper layers (Dunne and Brown 1996; Agusti and Liabres 2007; this study). Various marine plants respond to such high UVR levels by producing mycosporine-like amino acids

(MAA; Whitehead and Vernet 2000; Gao et al. 2007) and other UV absorbing compounds

(flavones and flavone-glycosides in seagrasses; Durako et al. 2003, Kunzelman et al. 2005,

Meng et al. 2008), changing morphologies (Monro and Poore 2004), changing pigment contents (Dawson and Dennison 1996; Gao et al. 2007), down-regulating photosystem II

(PSII; Figueroa et al. 2002) or shading by carbonate layers (Beach et al. 2006). Also epiphytes

43 may decrease the UV (as well as PAR) levels reaching the leaves (Trocine et al. 1981; Brandt and Koch 2003). Since many seagrasses thrive in tropical shallow waters, it may be that the midday chloroplast clumping found in at least one of them (Halophila stipulacea) has a protective role against UVR (in addition to high PAR as suggested by Sharon and Beer 2008).

In light of this, we investigated the possible role of UVR in mediating chloroplast movements in this seagrass, and the possible effects of UVR on some of its photosynthetic parameters.

MATERIALS AND METHODS

Whole plants (including roots and rhizomes) of Halophila stipulacea, together with the surrounding sediments, were collected at 20 m depth from the densest part of a monospecific meadow (extending from 7 to 33 m at the time of this study; the tidal range is <40 cm) located ca. 200 m south of the Inter-University Institute (IUI) in Eilat, Gulf of Aqaba, northern Red

Sea (29o30'N, 34o54'E) during March and April, 2007. These plants were grown in cups containing natural fine-sandy sediment on an outdoor water table (a table with a 20 cm margin, forming a shallow container) through which natural seawater at 21°C was flowing.

Thus, the leaves were submerged 5-10 cm under the water surface. The water table was shaded by a neutral-density net allowing 50% of the natural sunlight to reach the plants.

Typical midday irradiances under the net were ~850 µmol photons m-2 s-1 during midday. The light under the shading net was further screened through UV-filters (supplied by the GKSS

Forschungszentrum, Germany) placed just above the water surface over some of the plants, while control-filters that transmitted UVR (from the same supplier) was used for the other plants. Both filters reduced PAR by ~20%, while the UV-filter reduced wavelengths <400 nm by >80% (see Table 1 and Fig. 2). The wavelength-dependent transmittance properties of the filters were verified using a miniature-fibre optical spectrometer (USB 2000, Ocean Optics,

USA). All plant measurements were done after 14 days of acclimation to the water-table

44 conditions (except measurements of Fv/Fm, which were performed more often). This time was found to be enough for the plants to feature diurnal chloroplast clumping under the chosen conditions, and was later also found to be sufficient for the acclimation of several photosynthetic parameters to changing light regimes (Sharon et al. 2009).

The solar irradiance (PAR and UVR) reaching the water surface was measured by a

CM11B (Kipp & Zonen, The Netherlands) sensor at the IUI pier (displayed also at www.meteo-tech.co.il/eilat-yam/eilat_daily.asp). Spectral radiation measurements were done using a profile reflectance radiometer (PRR-800, BioSpherical Instruments Inc., USA) equipped with both spectral channel (300-875nm, µW cm-2 nm-1) and PAR (400-700 nm,

µmol photons m-2 s-1) sensors. Underwater spectral measurements of downwelling UVR, PAR and infrared radiation were also done using the PRR-800, which was lowered from a boat offshore the IUI using a free-fall system (cf. Kirk 1994) in order to avoid boat shading and to keep the instrument in a vertical posture.

Chloroplast clumping was verified through microscopic observations on live leaves within

2 min of collection (using a Nikon YS100, Japan). The absorption cross sections (or absorption factor, AF) of the leaves (i.e. the fraction of incident irradiance absorbed by the photosynthetic pigments of the leaves) were estimated along the day by measuring the PAR above and below a submerged leaf with the Diving-PAM’s quantum sensor (while subtracting the absorption of non-chlorophyllous pigments, see Sharon and Beer 2008). Chlorophyll contents of the leaves were determined after extraction in dimethyl formamide for 24 h in darkness. Absorbance was then measured spectrophotometrically according to Moran (1982), and chlorophyll a and b contents were calculated on a leaf area basis. Total carotenoids were measured by their absorption maxima at 480 and 510 nm according to Gradinaru et al. (2000).

Absorption spectra (300-400 nm) with special attention to absorption at 340 and 350 nm of the UV absorbing pigments (flavones and flavone-glycosides; Meng et al. 2008) were

45 measured after extraction in methanol using an Ultraspec 2100 Pro (Amersham Biosciences,

UK) spectrophotometer.

Chlorophyll fluorescence was measured by pulse-amplitude-modulated (PAM) fluorometry using a Diving-PAM (Walz, Germany). Effective quantum yield (ΔF/Fm’) measurements were carried out throughout the day using a leaf-distance clip (Walz, Germany) and photosynthetic electron transport rates (ETR) were calculated as ΔF/Fm’ x PAR x AF x

0.5; PAR was measured with the Diving-PAM's quantum sensor calibrated against a quantum- sensor-equipped Li-250A light meter (LiCor, USA). Maximal quantum yields (Fv/Fm) were measured in darkness at pre-dawn hours every morning for the first week of acclimation and after 14 days.

RESULTS

The midday underwater spectra (300-800 nm) from two depths and the integrated total

PAR (400-700 nm) and UVR (300-400 nm) at those depths, as well as on the water-table under the control- or UV-filters, are shown in Fig. 1 and Table 1, respectively. The spectra

(Fig. 1) reveal high UVR values at 6 m depth that could hardly be detected at 30 m. Similarly, there were measurable amounts of red light (650-750 nm) available at 6 m but not at 30 m. In all, approximately 45% and 5% of surface PAR was present at 6 and 30 m depths, respectively, while the corresponding percentages for UVR were 50% and 3% (Table 1). The

PAR measured under the filters on the water-table was 45% of that of full sunlight, while

UVR was 41% and 7.4% under the control- and UV-filters, respectively (see Fig. 2 for the spectral transmission of these filters). Unlike for UVR, there was no reduction in red wavelengths by the UV-filter (Fig. 2).

The epidermal leaf cells of plants grown on the water-table under control-filtered conditions featured midday clumping of chloroplasts, followed by their dispersal during the

46 evening, while no such chloroplast movements could be detected in the UV-filtered plants

(Fig. 3). While peaking at midday, chloroplast aggregation and dispersal was observed between 09:00 and 15:00 and was paralleled by decreasing and increasing AF values, respectively. Because the only difference between the treatments was the presence and virtual absence of UVR, it is concluded that the UVR dose present for the control-filtered plants caused the chloroplast movements that resulted in their midday clumping. More precisely, wavelengths between 280 and 380 nm caused the clumping (since these wavelength were the absolute cut-of points for the control- and UV-filters, respectively). In comparison, also leaves of plants growing at 6 m depth clumped their chloroplasts during midday while those at 30 m did not (Runcie et al. 2009 and personal observations). These chloroplast movements resulted in a significant (44%) change in the AF of the control-filter-grown plant leaves from early morning to midday while the UV-filtered plants did not show any significant differences in

AF during the day (Fig. 4); the AF values of the plants growing under the control-filters were significantly lower than those of the UV-filtered plants between 10:00-14:00.

Chlorophyll a+b contents showed no significant differences between the UV- and control- filtered water-table plants after 14 days: Chlorophyll a contents were 2.3 ± 0.17 and 2.6 ± 0.27 mg cm-2 for the control- and UV-filtered plants, respectively, while chlorophyll b contents were 0.3 ± 0.1 mg cm-2 in both control- and UV-screened plants. Also carotenoid absorption spectra peaks (at 480 and 510 nm) showed no significant difference between the two types of water-table grown plants after 14 days of acclimation. Similarly, the absorption spectra in the region where flavones and flavone-glycosides absorb (340–350 nm), nor the peak maxima of anthocyanin (290 nm), showed significant differences between treatments with or without

UVR.

Photosynthetic ETRs of the plants grown on the water-table with or without UV screening are presented in Fig. 5. Since these ETRs were calculated according to their respective AF

47 values during the various times of the day (see Fig. 4, cf. Sharon and Beer 2008), it follows that the differences in ETRs between control- and UV-filtered plants are partly due to the differences in AFs (here termed an "indirect" UVR-effect). The maximum ETR reached at around midday by the UVR-protected plants coincided with the highest PAR reaching the water-table while the ETRs for the control-plants levelled off after ca. 09:00 in the morning.

This, and the fact that the midday decrease in ETR in the control-filtered plants was more than the 44% that could be accounted for by the decrease in AF, indicates an excessive decrease of effective photosynthetic quantum yields by UVR during the high-irradiance hours of the day.

Indeed, ΔF/Fm' values were significantly lower in the control-filtered than the UV-filtered plants from around 11:30 and throughout the afternoon (Fig. 6), confirming a higher down- regulation of PSII in the former as caused by UVR (here termed a "direct" UVR-effect).

Fv/Fm values of plants acclimated to control-filter conditions decreased slightly but significantly from 0.79 to 0.71 (± 0.02) after 14 days of acclimation (and a significant decrease was observed already after 3 days) while there was no significant change in the

Fv/Fm values of the plants growing under the UV-filters (Fig. 7).

DISCUSSION

In a previous study, we suggested that the midday chloroplast clumping observed in

Halophila stipulacea had a photoprotective role against high PAR during midday (Sharon and

Beer 2008). Here, we found that it is UVR that triggers the diurnal chloroplast movements that cause their midday clumping under high-PAR conditions. This was realised at midday

UVR values of ca. 2000 µW m-2 on the water table (while UV-screening under those same high PAR values prevented clumping). In nature, at depth where clumping does not occur, reduced UVR is usually accompanied with low PAR, but the UVR/PAR ratio may also change (here it was reduced by 46% from 6 to 30 m). Thus, it is possible that also the

UVR/PAR ratio has an effect on the clumping phenomenon in shallow waters, but this was not quantified here. While the surface irradiance characteristics of the region where this study was conducted (northern Red Sea) are among the highest in the world (Winters et al. 2003), we were surprised to find high UVR doses similar to those on the shaded water table also in situ at 6 m depth. Indeed, midday chloroplast clumping was observed at that depth (Runcie et al. 2009). Thus, apparently, the successful adaptation of this plant to shallow waters (~6 m at the site studied) is partly based on the ability of its chloroplasts to shade one another in order to protect them from high irradiances, including UVR. In addition, the surrounding chloroplasts may, besides shading one another, protect the nucleus and its genetic material from excess UV to prevent UV damage (such as suggested by Kondo et al. 2004).

Another distinction from our earlier work is that while PAR correlated negatively with chlorophyll concentrations (Sharon and Beer 2008), manipulating UVR under constant PAR did not cause any changes in chlorophyll nor carotenoid contents in Halophila stipulacea.

Supportively, also another Halophila species (Halophila johnsonii) did not show differences in absorption peak maxima for any photosynthetic and UV-protecting pigments when acclimated to various PAR and UV treatments (Kunzelman et al. 2005). Surprisingly, UVR apparently did not induce the formation of flavones and flavone-glycosides as indicated by similar UV absorption values in methanol extracts of UVR-exposed and UVR-protected plants. This is unlike other tropical shallow-growing seagrasses (Dawson and Dennison 1996;

Meng et al. 2008), corals (Dunlap and Shick 1998), microalgae (Geo et al. 2007) and macroalgae (Karsten et al. 1998)), many of which do protect their tissues from UVR by the formation of UV-absorbing pigments. Regarding seagrasses, one other Halophila species

(Halophila ovalis) did, however, produce less UV-blocking pigments than other genera

(Dawson and Dennison 1996).

While the reduction in midday ETRs was largely an indirect effect of UVR via the decreased AF values caused by the clumping of chloroplasts, UVR also affected photosynthesis directly by reducing quantum yields. The daily reduction in effective quantum yield (ΔF/Fm’) can be seen as a dynamic form of photoinhibition (or down-regulation of PSII) since values in the evening returned to morning values. One mechanism that can explain this midday reduction in effective quantum yields is the operation of the xanthophyll cycle, in which excess light energy is dissipated as heat (Demmig-Adams and Adams 2006). In addition, but to a lesser degree than the effective quantum yield, the maximal quantum yield

(Fv/Fm) was also reduced by UVR. Since Fv/Fm is measured in pre-dawn darkness, its lower values reflect an incomplete recovery from high irradiances that might indicate a photoacclimatory response to UV stress as resulting of a regulating reduction in the PSII repair mechanism. Thus, as part of the more general negative effects of UVR on phytoplankton

(Agusti and Liabres 2007) and other marine photosynthetic organisms (Sinha et al. 1998), the chloroplast-based photosynthetic apparatus of some seagrasses is also influenced by UVR.

Perhaps logically, this radiation also activates mechanisms such as chloroplast clumping and a dynamic down-regulation of photosystem II that protect at least Halophila stipulacea against it.

REFERENCES

Agusti, S. and M.L. Liabres. 2007. Solar radiation-induced mortality of marine pico-plankton

in the oligotrophic ocean. Photochem Photobiol 83: 793-801.

Beach, K.S., H.B. Borgeas and C.M. Smith. 2006. Ecophysiological implications of the

measurement of transmittance and reflectance of tropical macroalgae. Phycologia 45:

450-457.

Brandt, L.A. and E.W. Koch .2003. Periphyton as a UV-B filter on seagrass leaves: A result

of different transmittance in the UV-B and PAR ranges. Aquat Bot 76: 317-327.

Dawson, S.P. and W.C. Dennison. 1996. Effects of ultraviolet and photosynthetically active

radiation of five seagrass species. Mar Biol 125: 629-638.

DeBlasio, S.L., J.L. Mullen, D.R. Luesse and R.P. Hangarter. 2005. Phytochrome modulation

of blue light-induced chloroplasts movements in Arabidopsis. Plant Physiol 133: 1471-

1479.

Demmig-Adams, B. and W.W. Adams .2006. Photoprotection in an ecological context: The

remarkable complexity of thermal energy dissipation. New Phytol 172: 11-21.

Drew, E.A. 1979. Physiological aspects of primary production in seagrasses. Aquat Bot 7:

139-150.

Dunlap, W.C. and J.M. Shick. 1998. Ultraviolet radiation absorbing mycosporine like amino

acids in coral reef organisms: a biochemical and environmental respective. J Phycol 34:

418-430.

Dunne, R.P. and B.E. Brown. 1996. Penetration of solar UVB radiation in shallow tropical

waters and its potential biological effects on coral reefs; Results from the central Indian

ocean and Andaman sea. Mar Ecol Prog Ser 144: 109-118.

Figueroa, F.L., C. Jimenez, B. Vinegla, E. Perez-Rodriguez, J. Aguilera, A. Flores-Moya, M.

Altamirano, M. Lebert and D.P. Hader. 2002. Effects of solar UV radiation on

photosynthesis of the marine angiosperm Posidonia oceanica from southern Spain. Mar

Ecol Prog Ser 230: 59-70.

Gabrys, H. 2004. Blue light-induced orientation movements of chloroplasts in higher plants:

Recent progress in the study of their mechanisms. Acta Physiol Plant 26: 473-478.

Gao, K., Y. Wu, G. Li, H. Wu, V.E. Villafane and E.W. Helbing. 2007. Solar UV radiation

drives CO2 fixation in marine phytoplankton; a double-edged sword. Plant Physiol 144:

54-59.

Gradinaru, C.C., I.H.M. van Stokkum, A.A. Pascal, R. van Grondelle and H. van Amerongen.

2000. Identifying the pathways of energy transfer between carotenoids and chlorophylls

in LHCII and CP29. A multicolor, femtosecond pump−probe study. J Phys Chem 104:

9330-9342.

Karsten, U., T. Sawall, D. Hanelt, K. Bischof, F.L. Figueroa, A. Flores-Moya and

C. Wiencke. 1998. An inventory of UV-absorbing mycosporine-like amino acids in

macroalgae from polar to warm-temperate regions. Bot Mar 41: 443-453.

Kirk, J.T.O. 1994. Light and photosynthesis in aquatic systems. Cambridge University Press,

Cambridge.

Kunzelman, J.I., M.J. Durako, W.J. Kenworthy, A. Stapleton and J.L.C. Wright. 2005.

Irradiance-induced changes in the photobiology of Halophila johnsoni. Mar Biol 148:

241-250.

Krzeszowiec, W., B. Rajwa, J. Dobrucki and H. Gabrys. 2007. Actin cycoskeleton in

Arabidopsis thaliana under blue and red light. Biol Cell 99: 251-260.

Luesse, D.R., S.L. DelBlasio and R.P. Hangdarter. 2006. Plastid movement impaired 2: a new

gene involved in normal blue light-induced chloroplast movements in Arabidopsis. Plant

Physiol 141: 1328-1337.

Meng, Y., A.J. Krzysiak, M.J. Durako, J.I. Kunzelman and J.L.C. Wright. 2008. Flavones and

flavone glycosides from Halophila johnsonii. Phytochem 69: 2603-3608.

Monro, K. and A.G.B. Poore. 2004. Light quantity and quality induce shade-avoiding

plasticity in a marine macroalga. J Evol Biol 18: 426-435.

Moran, R. 1982. Formulae for determination of chlorophyllous pigments extracted with N,N-

dimethylformamide. Plant Physiol 69: 1376–1381.

Rodrigues, V., R. Bhandari, J.P. Kburana and P.K. Sharma. 2007. Movement of chloroplasts

in mesophyll cells of Garcinia indica in response to UV-B radiation. Curr Sci 92: 1610-

1613.

Runcie, J.W., D. Paulo, R. Santos, Y. Sharon. S. Beer and J. Silva. 2009. Photosynthetic

Responses of Halophila stipulacea to a Light Gradient: I – In situ Energy Partitioning of

Non-photochemical Quenching. Aquat Biol 7: 142-152.

Sakuri, N., K. Domoto and S. Takagi. 2005. Blue light-induced reorganization of the actin

filaments and the avoidence response of chloroplasts in epidermal cells of Vallisneria

gigatea. Planta 221: 66-74.

Sharon, Y. and S. Beer. 2008. Diurnal movements of chloroplasts in Halophila stipulacea and

their effect on PAM fluorometric measurements of photosynthetic rates. Aquat Biol 88:

272-276.

Sharon, Y., J. Silva, R. Santos, J. Runcie, M. Chernihovsky and S. Beer. 2009. Photosynthetic

Responses of Halophila stipulacea to a Light Gradient: II – Plastic Acclimations

Following Transplantation. Aquat Biol 7: 153-157.

Shiihira-Ishikawa, I., T. Nakamura, S.I. Higashi and M. Watanabe. 2007. Distinct responses

of chloroplasts to blue and green laser microbeam irradiations in the centric diatom

Pleurosira laevis. Photochem Photobiol 83: 1101-1109.

Sinha, R.P., M. Klisch, A. Groniger and D.P. Hader. 1998. Ultraviolet-absorbing/screening

substances in cyanobacteria, phytoplankton and macroalgae. J Photochem Photobiol B:

Biol 47: 83-94.

Suetsugu, N., M. Wada. 2007. Chloroplast photorelocation movement mediated by

phototropin family proteins is green plants. Biol Chem 388: 927-935.

Trocine, R.P., J.D. Riceand G.N. Wells. 1981. Inhibition of seagrass photosynthesis by

Ultraviolet-B radiation. Plant Physiol 68: 74-81.

Winters, G., Y. Loya, R. Rottgers and S. Beer. 2003. Photoinhibition in shallow water

colonies of the coral Stylophora pistillata as measured in situ. Limnol Oceanogr 48:

1388-1393.

Whitehead, K. and M. Vernet. 2000. Influence of mycrosporine-like amino acids (MAAs) on

UV absorption by particulate and dissolved organic matter. Limnol Oceanogr 45: 1788-

1796.

Yamashita, W., T. Kanegae and A. Kadota. 2007. Analyses on the actin dynamics during

chloroplasts photorelocation movement in the moss Physcomitrella patens by use of

tdTomato-talin. Plant Cell Physiol 48: 55.

Yatsuhashi, H. 1996. Photoregulation systems for light-oriented chloroplast movement. J

Plant Res 109: 139-146.

Table 1: Midday values of PAR and UVR and percentage transmittances (%) of the in situ downwelling (at 6 and 30 m) and 50%-shaded water-table (under the control- and UV-filters)

PAR (400-700 nm) and UVR (300-400 nm). The water-table values were measured on April

10, 2007, and the in situ values were measured on April 1, 2008 offshore the IUI, with the

PRR-800 profiling radiometer.

PAR UVR

(µmol photons m-2 s-1) (%) (µW m-2) (%)

In situ, 6 m 750 45 2116 50

In situ, 30 m 90 5 134 3.0

Water-table + control-filter 860 45 2132 41

Water-table + UV-filter 860 45 192 7.4

FIGURE LEGENDS

Fig. 1: In situ transmitted downwelling irradiance (Ed) at 6m (open circles) and 30m (closed circles) measured with the PRR-800 (BioSpherical Instruments) on April 1, 2008, offshore the

IUI.

Fig 2: Transmission spectra of control- and UV-filter (lines marked in graph) as measured using the USB-2000 (Ocean Optics).

Fig. 3: Light-microscopic photographs of epidermal cells of Halophila stipulacea during midday (at 850 µmol photons m-2 s- 1) grown for 14 days under a control-filter (a) and under a

UV-filter (b). Black bars represent 10 µm.

Fig. 4: Daily changes in the absorption cross section (or absorption factor, AF) of plants grown on the water-table for 14 days under control-filters (open circles) or UV-filters (closed circles), and PAR (400-700nm) measured above the plants on the water-table (full line) and

UVR (300-400nm) under the control-filter (dotted line; there was no measurable UVR under the UV-filter). Data points of AF are means of 10 replicates ± SE.

Fig 5: Daily changes in electron transport rates (ETR) of plants grown on the water-table for

14 days under control-filters (open circles) or UV-filters (closed circles), and PAR (400-

700nm) measured above the plants on the water-table (full line) and UVR (300-400nm) under the control-filter (dotted line; there was no measurable UVR under the UV-filter). Data points are means of 8 replicates ± SD.

Fig. 6: Daily changes in effective quantum yield (ΔF/Fm') of plants grown on the water-table for 14 days under control-filters (open circles) or UV-filters (closed circles) and PAR (400-

Fig. 7: Maximal quantum yields (Fv/Fm) as a function of time for acclimated to water-table conditions (~850 µmol photons m-2 s-1 PAR during midday) under control-filters (open circles) and UV-filters (closed circles). Data points are means of 8 replicates ± SD.

60 ) -1 nm -2 40 W cm μ Ed (

0 300 400 500 600 700 800 Wavelength (nm)

Fig. 1: In situ transmitted downwelling irradiance (Ed)

100 UV filter Control filter 80 Control filter

40 Transmittance (%)

20 UV filter

0 300 400 500 600 700 800 Wavelength (nm)

Fig. 2: Transmission spectra of control- and UV-filter

a b

Fig. 3: Light-microscopic photographs of epidermal cells of Halophila stipulacea

0.70 3000

0.65 2500

0.60 ) -1 s

2000 -2

0.55 ) -2

0.50 1500 W cm μ mol photons m μ 0.45 UVR ( 1000

0.40 PAR ( Absorption cross section (AF) 500 0.35

0.30 0 04:00:00 08:00:00 12:00:00 16:00:00 Time of day

Fig. 4: Daily changes in the absorption cross section

180 3000

160 2500 ) 140 ) -1 -1 s s -2 -2

120 2000 ) -2

100 W cm 1500 μ

80 mol photons m UVR ( UVR (μ mol electrons m electrons mol

μ 60 1000 PAR PAR

ETR ( 40 500 20

0 0 05:00:00 09:00:00 13:00:00 17:00:00 Time of day

Fig. 5: Daily changes in electron transport rates

1.0 3000

2500 0.8 ) -1 s

2000 -2 ) -2 F/Fm')

Δ 0.6 W cm

1500 μ mol photons m mol photons

0.4 μ UVR ( 1000 PAR ( Quantum yield ( yield Quantum 0.2 500

0.0 0 05:00:00 09:00:00 13:00:00 17:00:00 Time of day

Fig. 6: Daily changes in effective quantum yield

0.84

0.82

0.80

0.78

0.76 Fv/Fm 0.74

0.72

0.70

0.68 0 2 4 6 8 10 12 14 16 Time (days)

Fig. 7: Maximal quantum yields

Photoacclimation of the seagrass Halophila stipulacea to the dim irradiance at its 48-meter depth limit

Yoni Sharon,a,b,* Orly Levitan,c Dina Spungin,c Ilana Berman-Frank,c and Sven Beera

a Department of Plant Sciences, Tel Aviv University, Tel Aviv, Israel b The Inter-University Institute, Eilat, Israel c The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel

Abstract The seagrass Halophila stipulacea grows in the northern Red Sea from the intertidal to depths of ,50 m. Along that gradient, there is a ~ 1 order of magnitude difference in irradiance and the spectrum narrows from that of full sunlight to dim blue-green light. Based on these differences, we set out to estimate the molar ratios and potential contributions of photosystem II (PSII) and photosystem I (PSI) to light absorption, and photosynthetic electron transport rates (ETR), in plants growing at 1-m and 48-m depths. The amount of PsaC (a proxy for PSI) was three times higher in the deep-growing plants. On the other hand, the amount of PsbA (a proxy for PSII) did not differ significantly between the two depths. Thus, the PSII : (PSII + PSI) ratio (FII) was 0.62 in the shallow- and 0.41 in the deep-growing plants. Similar results were obtained by 77K emission fluorescence. Because ETR is linearly dependent on FII, it follows that the ETR vs. irradiance curves differed significantly if calculated based on the commonly used FII value of 0.5 or the FII values we found. As a result, the photosynthetic parameters ETRmax and a also differed when using the different FII values. Correct(ed) FII values should, therefore, be used in the calculation of photosynthetic ETRs. The ability of H. stipulacea to alter its amount of PSI relative to PSII according to the ambient irradiance and spectrum may be one reason why this organism can grow down to its exceptional depth limit in clear tropical waters.

In aquatic systems, irradiance is attenuated with depth, environment changes from high levels of photosynthetically and the light spectrum in clear waters shifts from that of active radiation (PAR) and ultraviolet radiation (UVR) to full sunlight in the intertidal to a narrower band of bluish a narrow blue-green spectrum containing virtually no red light at depth. Accordingly, plants growing along a depth light nor UVR. In the summer, the midday PAR decreases

gradient have to acclimate to these differences in the optical from , 2100 mmol photons m-2 s-1 at the water surface to -2 -1 properties of the water that surrounds them. In addition to , 100 mmol photons m s at 50 m, which is the depth changes in plant morphology (Schwarz and Hellblom 2002) limit for this plant. In previous studies we investigated how and meadow architecture (Dalla Via et al. 1998; Peralta et H. stipulacea copes with high PAR and high UVR. It was found that midday PAR irradiances of . 400 mmol al. 2002), marine angiosperms, or seagrasses, may also photons m-2 s-1, corresponding to depth of , 10 m, enhance chlorophyll (Chl) concentrations and photosyn- caused chloroplasts to clump together as an apparent thetic efficiencies at low irradiances (reviewed in Ralph et mechanism of photo-protection (Sharon and Beer 2008). al. 2007). Also spectral changes can affect light absorption This clumping also lowered the amount of incident PAR by altering the composition and ratio of the two absorbed by the leaves, thus altering their calculated

photosystems (PSs) and, thus, photosynthetic rates and photosynthetic electron transport rates (ETR). In a plant growth (Fujita 1997; Falkowski and Raven 2007). subsequent study, we found that UVR was responsible Among marine macrophytes, such spectral changes were for chloroplast movements in this organism (Y. Sharon and shown to influence the functionality of the two PSs as S. Beer unpubl.). found for the green macroalgae Ulva pertusa and Bryopsis Although we have frequently observed the presence of maxima (Yamazaki et al. 2005). Regarding seagrasses, only H. stipulacea at depths of . 30 m, diving limitations in two studies have reported on changes in the PS composi- bottom time at such depths prevented in situ photosyn- tion: one in shallow-growing plants during various seasons thetic measurements there. However, recent improvements (Major and Dunton 2000) and another along a narrow in diving techniques have now enabled the application of in depth gradient down to 1.6 m (Major and Dunton 2002). situ pulse amplitude modulated (PAM)–fluorometric mea- To our knowledge there are no studies on photosystems’ surements in order to quantify photosynthetic rates down acclimatory mechanism in seagrasses growing at greater to 50 m. In order to understand Halophila stipulacea’s depths where both the irradiance and light spectrum change photo-acclimation strategy to the low irradiances and the drastically. blue-green–shifted spectrum at its depth limit, several In the Gulf of Aqaba (northern Red Sea), the seagrass aspects of the photosynthetic units were studied: in situ Halophila stipulacea grows from the intertidal to 30–50 m PAM-fluorometric measurements were, thus, combined depending on season. Along such depth gradients, the light with determinations of Chl a and b concentrations, measurements of the abundance of core subunits of PSI (PsaC), PSII (PsbA, D1 protein), and the large subunit * Corresponding author: [email protected]

357 62

358 Sharon et al.

(RbcL) of ribulose-1,5-bisphosphate carboxylase and oxy- measured spectrophotometrically (using an Ultraspec 2100 genase (Rubisco), as well as energy transfer by the two PSs. Pro; Amersham Biosciences), and Chl a and b concentrations were calculated on a leaf-area basis according to Methods Moran (1982). Middle parts of leaves with a known surface area were Study site—The Halophila stipulacea meadows studied suspended in 250 mL cold 13 denaturing extraction buffer (during Jul and Aug 2009) are located , 200 m south and containing 140 mmol L21 tris(hydroxymethyl)aminomethane 300 m north of the Inter-University Institute (IUI) in Eilat, (Tris) base, 105 m mol L21 Tris–HCl, 0.5 m mol L21 the Gulf of Aqaba, northern Red Sea (29u309N, 34u559E). These meadows extend down to , 50 m during the Ethylenediaminetetraacetic acid, 2% lithium dodecyl sulfate, summer. 10% glycerol, and 0.1 mg : mL PefaBloc SC 4-(2-Aminoethyl) benzenesulfony fluoride hydrochloride protease inhibitor Spectral and irradiance measurements—PAR irradiance (Roche). These samples were then sonicated using a Fisher Scientific Model 100 Sonic dismembrator with a microtip and spectral composition at various depths were measured attachment at a setting of 30%. To avoid overheating and for seven cloudless middays during the study period with a improve extraction efficiency, the samples were then refrozen profile reflectance radiometer (PRR-800; BioSpherical immediately in liquid N . Two such cycles of freezing followed Instruments) equipped with both spectral (300–875 nm, 2 mW cm22 nm21) and PAR (400–700 nm, mmol photons by thawing were performed in order to maximize protein m22 s21) sensors. This instrument was lowered down from extraction with minimal degradation of representative mem- a boat through the water column above the seagrass beds branes and soluble proteins (Brown et al. 2008; Levitan et al. using a free-fall technique (Kirk 1994). 2010a). Following the disruption of the leaves, the samples were centrifuged for 3 min at 10,000 3 g to remove insoluble Chlorophyll fluorescence measurements—Chl fluores- material and unbroken cells. The total protein concentra- cence was measured in situ on cloudless days at 1-m and tion was measured with a modified Lowry assay (Bio-Rad DC) using bovine gamma globulin as a comparative protein 48-m depth by PAM fluorometry using a Diving-PAM standard. (Walz). This instrument was programmed to perform so- called rapid light curves (RLC) by irradiating a leaf with eight increasing irradiances (up to 1450 mmol photons Target protein quantification—Key photosynthetic pro- m22 s21 and 1827 mmol photons m22 s21 for the deep- and teins subunits of PSI (PsaC), PSII (PsbA), and Rubisco shallow-growing plants, respectively) for 10 s each, where (RbcL) were quantified using specific standards (AgriSera, in the end of each irradiation the effective quantum yield Sweden) following the procedure described in Brown et al. (DF : Fm9) was measured. Each sequence of measurements (2008) and Levitan et al. (2010a). Primary antibodies was preceded by minimal exposure to darkness (, 1 s) just (AgriSera, Sweden) were used at a dilution of 1 : 40,000 in after the opened dark-leaf clip (Walz) attached to the leaf 2% enzymatic chemiluminescence (ECL) advanced block- was connected to the instrument’s optical fiber, following ing reagent in Tris buffered saline (TBS-T) for PsbA, PsaC, incubated for 1 h with the protocol of Beer et al. (2001). The photosynthetic ETR and RbcL. The blots were at each given irradiance was calculated as DF : Fm9 3 PAR horseradish-peroxidase–conjugated rabbit anti-chicken sec- 3 absorption factor (AF) 3 FII, where PAR is the incident ondary antibody (Abcam) for RbcL primary antibodies irradiance given by the Diving-PAM’s actinic light source and with horseradish-peroxidase–conjugated chicken anti- (as premeasured with the Diving-PAM’s quantum sensor, rabbit secondary antibody (Abcam) for the PsbA and PsaC calibrated against a quantum-sensor–equipped Li-250A primary antibodies (diluted to 1 : 40,000 in 2% ECL). The light meter, LiCor), AF is the fraction of light absorbed by blots were developed with ECL Advance detection reagent the leaf as determined under water (Sharon and Beer 2008), (Amersham Biosciences, GE Healthcare) using a charged and FII (or the FII value) is the assumed fraction of coupled devise imager (DNR; M-ChemiBIS). Protein levels photons absorbed by PSII (normalized to (PSI + PSII)). FII of the immunoblots were quantified using the Quantity One is often assumed to be 0.5, but varied here with depth (see software (Bio-Rad) as calculated from standard curves for Results). The ETR values of the RLCs were depicted each blot (Brown et al. 2008). Assuming that PsaC and PsbA (pmol mg protein21) are against absorbed PAR (PARa; i.e., PAR 3 AF) as recommended by Saroussi and Beer (2007) and as proxies for PSI and PSII, respectively, the FII factor was performed previously for this plant (Sharon and Beer calculated as the amount of PSII relative to (PSI + PSII) 2008). according to PsbA : (PsaC + PsbA).

Pigment quantification and protein extraction—Plants Energy transfer by photosystems—The distribution of were collected for Chl and protein analysis. After the leaves excitation energy between PSI and PSII can be estimated by were separated from the shoots, some were used for Chl measuring the Chl fluorescence emission spectra at 77K (Butler 1978). The functionality of the two photosystems extraction while others were frozen in liquid N2 for further protein analyses. was estimated according to Schubert et al. (1995). A frozen Chl contents of the leaves were determined after glass cuvette containing a single leaf was scanned in the extraction in N,N-dimethyl formamide for 24 h in low-temperature holder of a spectrofluorometer (Cary darkness. Absorbance at 663, 647, and 625 was then Eclipse Fluorometer; Varian) with the excitation wavelength set to 435 nm. Emission of the leaf was measured at 63

Halophila dim irradiance acclimation 359

hardly detected at 48 m, where . 90% of the surface irradiance narrowed to blue-green wavelengths. Within this depth gradient, the PAR decreased . 1 order of magnitude, from , 1500 mmol photons m22 s21 at 1 m to , 100 mmol photons m22 s21 at 48 m, during midday (Table 1). The contents of Chl a and b per leaf area were significantly different between plants growing at 1 m and 48 m (Fig. 2). The deep-growing plants contained almost three times more of both Chl a and b than the shallow- growing ones. Chl a : b ratio did not differ between the shallow- and deep-growing plants because both Chls increased similarly with depth. Although there was no significant difference in the amount of PsbA between the shallow- and deep-growing plants, the PsaC concentration was three times higher in the deep-growing ones (Fig. 3). Thus, the PsaC : PsbA ratio increased , three-fold in the deep-growing plants. Assuming that the amount of PsaC and PsbA subunits are quantitative proxies for the presence of PSI and PSII, respectively (Brown et al. 2008), the PSI : PSII ratio (as reflected by the PsaC : PsbA ratio) increased similarly in deep-growing plants. In the latter, the quantity of the large subunit of the RbcL was reduced to 60% compared with that contained in the shallow-growing

plants. The RbcL : PsbA ratio was twice as high in the Fig. 1. In situ transmitted midday down-welling irradiance shallow-growing plants as in the deep-growing ones (2.23 at the surface (closed circles) and at 1-m (open circles), and 48-m vs. 1.12). (closed triangles) depths offshore the IUI, Eilat, Israel. Data The 77K Chl fluorescence emission spectra are depicted points are means 6 SD of 20 measurements during seven different in Fig. 4. The spectrum of the 48-m plants showed no days in July and August 2009. differences between peak heights at 686 nm (for PSII) and 717 nm (for PSI). On the other hand, the fluorescence 1-nm intervals from 650 nm to 750 nm. Ratios of PSI and emission spectrum of the shallow-growing plants’ Chl PSII emission were determined from the peak heights at revealed a significant difference between those peak 717 nm and 686 nm, respectively. Assuming that these peak heights. heights are proxies for the excitation energy of PSI and The FII factor (i.e., the probability of photons to be PSII, respectively, the FII factor was here calculated as the absorbed by PSII), was found to be 0.62 and 0.41 for the emission value of PSII relative to (PSI + PSII) according to 1-m and 48-m growing plants, respectively, according to the l686 : (l717 + l686). protein data. This parameter influences the calculated ETR linearly (see Methods). Thus, when using these FII factors Statistical analyses—For the protein data (n 5 4) we instead of the commonly used factor of 0.5, the difference used a t-test for analyzing equality of means between between the ETR vs. irradiance curve of plants from the groups (p , 0.05). For the 77K fluorescence data, Chl two depths is enhanced (Fig. 5). Accordingly, the photo- content, and RLC analysis (n 5 7–8), t-tests were used to synthetic parameters maximal electron transport rate assess their significance (p , 0.05). (ETR ), the initial slope of the ETR vs. irradiance curve max (a), and the onset of light-saturation point (Ik) based on the Results curves differ too. Extrapolating the values of those parameters by using the curve-fitting equation of Platt et The midday spectra (300–800 nm) from the surface and al. (1980) reveals that: (1) The ETR value of deep- 1-m and 48-m depths are presented in Fig. 1. These spectra max reveal high UVR (, 400 nm) and red (650–700 nm) growing plants is 60% vs. 30% that of the shallow-growing wavelengths values at the surface and at 1 m, which were plants when using 0.5 or the correct(ed) FII values (0.62

Table 1. Midday irradiance, photosynthesis maximum (Pmax), the initial slope (a), and onset of light-saturation point (Ik) value derived from RLC performed in situ on 1-m– and 48-m–depth growing leaves of Halophila stipulacea when using either 0.5 or the correctly acquired PSII : (PSII + PSI) ratio (FII) values at each depth. Data are means 6 SD of eight replicates. ns 5 not significant.

Midday p Ik p a p Pmax irradiance FII Depth (m) ,0.001 189.52641.12 ,0.0001 0.3660.03 ,0.05 68.66610.91 1498653 0.5 1 ,0.001 63.1767.12 ,0.0001 0.6160.016 ,0.05 38.7964.34 10768 0.5 48 ,0.001 189641.1 ns 0.4560.039 ,0.0001 103.68613.52 1498653 0.62 1 ,0.001 6567.3 ns 0.5060.018 ,0.0001 32.7463.49 10768 0.41 48 64

360 Sharon et al.

Fig. 2. Chlorophyll a, b, and a : b ratio concentrations of 1- m- (open bars) and 48-m-deep– (gray bars) growing Halophila Fig. 4. The 77K fluorescence emission spectra of 1-m- (open stipulacea plants. Data points are means 6 SD of eight replicates. circles) and 48-m-deep– (closed circles) growing Halophila Different letters represent statistically significant differences stipulacea plants. Data points are means 6 SD of eight replicates. between each pair in a group. Different letters represent statistically significant differences in peak heights between plants from the two depths. and 0.41), respectively; (2) a differs significantly between plants from the two depths when using 0.5 as the FII value Discussion but no such difference is apparent when using the correct(ed) FII values for each depth; and (3) the difference In this study, we investigated deep-growing plants of the in Ik is similar when using either 0.5 or the correct FII seagrass H. stipulacea, which, at their depth limit values (Table 1). Similar differences in these photosynthetic in the summer (, 50 m), received a down-welling PAR of parameters were found when using the 77K fluorescence , 5% of that at the surface. This low light requirement in spectra peak heights to calculate the FII values. comparison with the average one for seagrasses (e.g., 11% as reported by Duarte [1991]) could not be explained by a high photosynthetic : nonphotosynthetic biomass ratio because this plant has the same above- (leaves and stems) to below-ground (roots and rhizomes) biomass ratio (, 1 on a dry-weight basis, data not shown) as do many other seagrasses (Duarte and Chiscano 1999). Thus, the ability to modify its photosynthetic system(s; e.g., photosystem ratio and Chl content), may contribute to its abundant growth under very dim irradiances. Changes in Chl concentration are a known response of plants in their acclimation to variations in the light environment. We found that in deep-growing H. stipulacea leaves both Chl a and b were increased, as was also previously reported for this species (Sharon and Beer 2008; Sharon et al. 2009), as well as for other seagrasses (Wiginton and McMillan 1979; Dennison 1990; Abal et al. 1994). In terrestrial plants that grow within the shade of a leaf canopy, Chl b concentrations increase more than

those of Chl a because Chl b utilizes a slightly narrower Fig. 3. Amounts of the photosynthesis-related proteins wavelength span in accordance with the spectrum found PsbA, PsaC, and RbcL, and PsaC : PsbA ratios of 1-m- (open there (Martin and Churchill 1982; Marschall and Proctor bars) and 48-m-deep– (gray bars) growing Halophila stipulacea 2004). In clear marine waters there is not only a narrowing plants. Data points are means 6 SD of four replicates. Different of the spectrum with depth, but also a shift toward bluish letters represent statistically significant differences between each pair in a group. light because of the selective absorption of water. 65

Halophila dim irradiance acclimation 361

The almost three times higher PSI : PSII ratio in the deep- growing H. stipulacea plants was a result of changes mainly in the PSI content, similar to what was found for a cyanobacterium (Levitan et al. 2010b), some terrestrial plants and freshwater algae (Fujita 1997) and the macroalgae Ulva pertusa and Bryopsis maxima (Yamazaki et al. 2005). The latter authors suggested that the higher ratios of PSI : PSII may be derived from the need for a higher adenosine triphosphate and nicotinamide adenine dinucleotide phosphate (ATP : NADPH) ratio, which can be obtained by cyclic electron flow around PSI. In addition, those authors also found a higher level of excitation energy from light to PSI than to PSII as measured by 77K fluorescence. The high PSI : PSII ratio found here for H. stipulacea growing at its depth limit, together with the higher functionality of PSI as indicated by the 77K fluorescence emission, thus suggest a similar advantage to this plant under the extremely limiting light conditions found at 48-m depth. It is known that low- light grown chlorophytes usually change the number of the photosynthetic units (i.e., photosystems and Chl) rather than their size as a light-shade adaptation strategy (Falkowski and Owens 1980). Yet, we still do not know the reason why in this plant ATP vs. NADPH production at depth should or would be favored. The increase in Rubisco, as reflected by its large subunit protein pool, correlates with the higher photosynthetic ETR under high-light conditions (in shallow water). The RbcL : PsbA ratio can, thus, represent Ek, or light saturation index (Brown et al. 2008). At light saturation, the electron transport rate from water to CO2 is limited by the concentration of Rubisco per electron transport chain (Sukenik et al. 1987), here measured as RbcL : PsbA. For H. stipulacea, the RbcL : PsbA ratio was twice as high in the shallow-growing plants, probably reflecting higher carboxylation rates at the higher irradiance. This is similar to the findings of Schofield et al. (2003) for an algal symbiont during a season featuring high irradiances. Another aspect of our study is to emphasize the need of using correct FII values when calculating ETRs, especially for plants featuring a wide depth-distribution pattern such as exemplified for H. stipulacea. When using FII values based on our protein quantification, the ETRmax values were , 2.5 times higher in the shallow-growing plants as compared to only , 1.5 times higher when using the commonly accepted value of 0.5. Interestingly, the initial slope of the RLC (a) did not change between shallow- and deep-growing plants when using correct(ed) FII values, suggesting the same photosynthetic efficiency at low Fig. 5. Electron transport rates (ETR) derived from PAM irradiances for both plant types. We conclude that one fluorometry of 1-m- (open circles) and 48-m-deep– (closed circles) should be cautious when using 0.5 as the FII value based on growing Halophila stipulacea plants as calculated (A) with an FII our present findings that the amount, functionality, and of 0.5, and (B) with the correct(ed) FII values as derived from the protein analysis. light absorption probability for each photosystem is clearly not equal in plants growing at extreme irradiances.

Therefore, it may be that H. stipulacea does not feature the Acknowledgments typical terrestrial-plant response to shading. Also the This work is in partial fulfillment of the requirements for virtual absence of red light at the depth limit of this plant the Ph.D. thesis of Y. Sharon at Tel Aviv University. We would not provide Chl b with any advantage in that thank Cameron Veal and Oded Ben-Shaprut for assistance spectral region, while the blue light reaching that depth with the technical underwater work, Ada Alamaro for her would sustain the absorption of both Chl a and b in the help in the figures assembly, Danni Tchernov for the use of his lab blue-light region rather equally. 66

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fluorometers, Gal Dishon for the use of the underwater spectral pigments extracted with N,N-dimethylformamide. Plant radiometer, and two anonymous reviewers. Physiol. 69: 1376–1381, doi:10.1104/pp.69.6.1376 PERALTA, G., J. L. PEREZ-LLORENS, I. HERNANDEZ, AND J. J. References VERGARA. 2002. Effects of light availability on growth, architecture and nutrient content of the seagrass Zostera noltii ABAL, E. G., N. R. LONERAGAN, P. BOWEN, C. J. PERRY, J. W. Hornem. J. Exp. Mar. Biol. Ecol. 269: 9–26, doi:10.1016/ UDY, AND W. C. DENNISON. 1994. Physiological and morphological S0022-0981(01)00393-8 responses of the seagrass Zostera capricorni Aschers PLATT, T., C. L. GALLEGOS, AND W. G. HARRISON. 1980. to light intensity. J. Exp. Mar. Biol. Ecol. 178: 113–129, Photoinhibition doi:10.1016/0022-0981(94)90228-3 of photosynthesis in natural assemblages of marine BEER, S., M. BJO¨ RK, R. GADEMANN, AND P. RALPH. 2001. phytoplankton. J. Mar. Res. 38: 687–701. Measurements of photosynthetic rates in seagrasses, p. 138– RALPH, P. J., M. J. DURAKO, S. ENRIQUEZ, C. J. COLLIER, AND 198. In F. T. Short and R. G. Coles [eds.], Global seagrass M. A. DOBLIN. 2007. Impact of light limitation on seagrasses. research methods. Elsevier. J. Exp. Mar. Biol. Ecol. 350: 176–193, doi:10.1016/j.jembe. BROWN, C. M., J. D. MACKINNON, A. M. COCKSHUTT, T. A. 2007.06.017 VILLAREAL, AND D. A. CAMPBELL. 2008. Flux capacities and SAROUSSI, S., AND S. BEER. 2007. Alpha and quantum yield of acclimation costs in Trichodesmium from the Gulf of Mexico. aquatic plants derived from PAM fluorometry: Uses and Mar. Biol. 154: 413–422, doi:10.1007/s00227-008-0933-z misuses. Aquat. Bot. 86: 89–92, doi:10.1016/j.aquabot.2006. BUTLER, W. L. 1978. Energy distribution in the photochemical 09.003 apparatus of photosynthesis. Annu. Rev. Plant Physiol. 29: SCHOFIELD, S. C., D. A. CAMPBELL, C. FUNK, AND T. D. B. 345–378, doi:10.1146/annurev.pp.29.060178.002021 MACKENZIE. 2003. Changes in macromolecular allocation in DALLA VIA, J., C. STURMBAUER, G. SCHONWEGER, E. SOTZ, nondividing algal symbionts allow for photosynthetic acclimation S. MATHEKOWITSCH, M. STIFTER, AND R. RIEGER. 1998. in the lichen Lobaria pulmonaria. New Phytol. 159: Light gradients and meadow structure in Posidonia oceanica: 709–718, doi:10.1046/j.1469-8137.2003.00857.x Ecomorphological and functional correlates. Mar. Ecol. Prog. SCHUBERT, H., R. M. FORSTER, AND S. SAGERT. 1995. In situ Ser. 163: 267–278, doi:10.3354/meps163267 measurement of state transition in cyanobacterial blooms: DENNISON, W. C. 1990. Chlorophyll content, p. 83–85. In R. C. Kinetics and extent of the state change in relation to Phillips and C. P. McRoy [eds.], Seagrass research methods. underwater light and vertical mixing. Mar. Ecol. Prog. Ser. UNESCO. 128: 99–108, doi:10.3354/meps128099 DUARTE, C. M. 1991. Seagrass depth limits. Aquat. Bot. 40: SCHWARZ, A. M., AND F. HELLBLOM. 2002. The photosynthetic 363–377, doi:10.1016/0304-3770(91)90081-F light response of Halophila stipulacea growing along a depth ———, AND C. L. CHISCANO. 1999. Seagrass biomass and gradient in the Gulf of Aqaba, the Red Sea. Aquat. Bot. 74: production: A reassessment. Aquat. Bot. 65: 159–174, 263–272, doi:10.1016/S0304-3770(02)00080-3 doi:10.1016/S0304-3770(99)00038-8 SHARON, Y., AND S. BEER. 2008. Diurnal movements of FALKOWSKI, P. G., AND T. G.OWENS. 1980. Light–shade chloroplasts in Halophila stipulacea and their effect on PAM adaptation. fluorometric measurements of photosynthetic rates. Aquat. Plant Physiol. 66: 592–595, doi:10.1104/pp.66.4.592 Bot. 88: 273–276, doi:10.1016/j.aquabot.2007.11.006 ———, AND J. A. RAVEN. 2007. Aquatic photosynthesis, 2nd ed. ———, J. SILVA, R. SANTOS, J. W. RUNCIE, M. Princeton Univ. Press. CHERNIHOVSKY, FUJITA, Y. 1997. A study on the dynamic features of photosystem AND S. BEER. 2009. Photosynthetic responses of Halophila stoichiometry:Accomplishments and problems for future studies. stipulacea to a light gradient. II. Acclimations following Photosynth. Res. 53: 83–93, doi:10.1023/A:1005870301868 transplantation. Aquat. Biol. 7: 153–157, doi:10.3354/ab00148 KIRK, J. T. O. 1994. Light and photosynthesis in aquatic systems. SUKENIK, A., J. BENNETT, AND P. FALKOWSKI. 1987. Cambridge Univ. Press. Lightsaturated LEVITAN, O., C. BROWN, S. SUDHAUS, D. CAMPBELL, J. photosynthesis—limitation by electron transport LAROCHE, AND I. BERMAN-FRANK. 2010a. Regulation of or carbon fixation? Biochem. Biophys. Act. 891: 205–215, nitrogen metabolism in the marine diazotroph Trichodesmium doi:10.1016/0005-2728(87)90216-7 IMS101 under varying temperatures and atmospheric CO2 WIGINTON, J. R., AND C. MCMILLAN. 1979. Chlorophyll concentrations. Environ. Microbiol. 12: 1899–1912, doi:10. composition 1111/j.1462-2920.2010.02195.x under controlled light conditions as related to the ———, S. A. KRANZ, D. SPUNGIN, O. PRASIL, B. ROST, AND I. distribution of seagrass in Texas and US Virgin Islands. BERMAN-FRANK . 2010b. The combined effects of pCO2 and Aquat. Bot. 6: 171–184, doi:10.1016/0304-3770(79)90060-3 light on the N2 fixing cyanobacterium Trichodesmium YAMAZAKI, J. Y., T. SUZUKI, E.MARUTA, AND Y. KAMIMURA. IMS101: A mechanistic view. Plant Physiol. 154: 346–356, 2005. The stoichiometry and antenna size of the two photosystems doi:10.1104/pp.110.159145 in marine green algae, Bryopsis maxima and Ulva pertusa, in MAJOR, K. M., AND K. H. DUNTON. 2000. Photosynthetic relation to the light environment of their natural habitat. J. performance in Syringodium filiforme: Seasonal variation in Exp. Bot. 56: 1517–1523, doi:10.1093/jxb/eri147 light-harvesting characteristics. Aquat. Bot. 68: 249–264, doi:10.1016/S0304-3770(00)00115-7 Associate editor: John Albert Raven ———, AND ———. 2002. Variations in light-harvesting characteristics Received: 05 August 2010 of the seagrass, Thalassia testudinum: Evidence for Accepted: 19 October 2010 photoacclimation. J. Exp. Mar. Biol. Ecol. 275: 173–189, Amended: 02 November 2010 doi:10.1016/S0022-0981(02)00212-5 MARSCHALL, M., AND M. C. PROCTOR. 2004. Are bryophytes shade plants? Photosynthetic light responses and proportions of chlorophyll a, chlorophyll b and total carotenoids. Ann. Bot. 94: 593–603, doi:10.1093/aob/mch178 MARTIN, C. E., AND S. P. CHURCHILL. 1982. Chlorophyll concentrations and a/b ratios in mosses collected from exposed and shaded habitats in Kansas. J. Bryol. 12: 297–304 MORAN, R. 1982. Formulas for determination of chlorophyllous 69

Vol. 7: 127–141, 2009 AQUATIC Published online October 22, 2009 doi: 10.3354/ab00173 BIOLOGY Aquat Biol

Contribution to the Theme Section ‘Primary production in seagrasses and microalgae’ OPEN ACCESS

Measuring seagrass photosynthesis: methods and applications

1, 2, 3 1 3 João Silva *, Yoni Sharon , Rui Santos , Sven Beer

1 Marine Plant Ecology Research Group, Center of Marine Sciences (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal 2 The Interuniversity Institute for Marine Sciences, POB 469, Eilat 88103, Israel 3 Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel

ABSTRACT: This review originates from a keynote lecture given at the recent 8th Group for Aquatic Productivity (GAP) workshop held in Eilat, Israel. Here we examine the most important methodologies for photosynthetic measurements in seagrasses and evaluate their applications, advantages and disadvantages, and also point out the most relevant results. The most commonly used methodologies are based on oxygen (O2) evolution and chlorophyll fluorescence measurements. O2-based methodologies allowed for the first approaches to evaluate seagrass productivity, whereas chlorophyll a fluorescence has more recently become the choice method for in situ experiments, particularly in evaluating photosynthetic responses to light and assessing stress responses. New methodologies have also emerged, such as O2 optodes, underwater CO2 flux measurements, geo-acoustic inversion and the eddy correlation technique. However, these new methods still need calibration and validation. Our analysis of the literature also reveals several significant gaps in relevant topics concerning seagrass photosynthesis, namely the complete absence of studies on deep-growing populations that photosynthesise under extreme low light conditions and the uncertainties about the true degree of seagrass carbon limitation, which limits our ability to predict responses to global changes.

KEY WORDS: Seagrass photosynthesis · O2 evolution · Chlorophyll a fluorescence · Carbon uptake · CO2 flux

Resale or republication not permitted without written consent of the publisher

INTRODUCTION Seagrass photosynthesis was, until recently, most commonly measured in laboratory experiments, usu- Early studies on seagrass photosynthesis (e.g. Drew ally by incubating leaf segments in closed chambers 1978) usually began by explaining that seagrasses are and determining initial and end O2 values, or by fol- relatively primitive monocotyledons, closely related to lowing continuous O2 evolution in the chambers with freshwater plants but able to live in the marine envi- Clark-type electrodes. These methods have provided ronment. Presently, many introductions start by high- most of the fundamental information on seagrass lighting the importance of seagrasses in overall responses to factors such as light, temperature and marine productivity and their subsequent economical nutrients (comprehensively reviewed by Lee et al. importance (e.g. Duarte & Cebrián 1996, Costanza et 2007) and on the mechanisms of carbon uptake and al. 1997, Duarte & Chiscano 1999). This shift of em- fixation by these plants (Beer et al. 2002). However, phasis indicates the evolution of seagrass perception they are extremely intrusive, as they involve plant in global biological science and reflects the contribu- removal from the natural environment and a high tion of the increasing number of physiological and degree of manipulation (Beer et al. 2001). ecophysiological studies dealing with this plant In situ measurements of photosynthetic activity in group. seagrasses were made possible after the development

*Email: [email protected] © Inter-Research 2009 · www.int-res.com 68

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of a submersible pulse-amplitude modulated (PAM) & Robertson 1974, Grasshoff et al. 1983) usually involves fluorometer (applied initially by e.g. Beer et al. 1998, enclosing the plants in sealed containers (or ‘bottles’) and Ralph et al. 1998, Björk et al. 1999, Beer & Björk 2000). leaving them to incubate for a period of time. The in-

Recently, an infrared gas analysis (IRGA) technique crease or decrease of the O2 concentration during the in- has been adapted for in situ continuous dissolved CO2 cubation period provides a measure of either net photo- flux measurements using incubation chambers con- synthesis (in the illuminated transparent bottles) or nected to the analyser at the surface (Silva et al. 2008). respiration (in darkened bottles), respectively. Gross While PAM fluorometry determines photosynthetic photosynthesis can be calculated by correcting the net traits of individual plants, the IRGA method measures photosynthetic rates obtained with the rate of dark (mi- the rates of gas exchange at the community level. A tochondrial) respiration. The Winkler method remains combination of both methods may give real-time in- one of the most accurate ways of measuring dissolved O2 formation on both the photosynthetic characteristics of concentrations, arguably better than most commercial seagrass plants and the community CO2 exchange field-going O2 probes. Nevertheless, there are a number of undisturbed seagrass meadows. of downsides to this methodology, namely: (1) the need In situ experiments are less intrusive and deal with to detach and cut down plant samples (since whole the plants in their natural habitat, hence the photo- plants usually do not fit into the bottles), compromising synthetic measures incorporate the influences of all integrity and inducing stress (except when this method is ambient parameters. However, the control of experi- used to measure O2 before and after whole-community mental conditions is limited, and insights on specific incubations); (2) the difficulty in maintaining adequate processes are difficult to obtain. On the other hand, homogenization of the medium during incubations, lead- laboratory experiments allow for the control and ing to increased boundary layers and resulting in under- manipulation of most external parameters and are estimations of photosynthetic rates (Koch 1994); (3) the most suited to obtain very specific information, e.g. likelihood of O2 saturation and/or inorganic carbon de- data on biochemical processes related to photosynthe- pletion during the incubation period, both resulting in sis. The general downside of laboratory experiments is photosynthetic inhibition due to, for example, photo- the difficulty in extrapolating results to the natural respiration (Beer 1989) and carbon limitation, respec- environment, e.g. time-related patterns of photo- tively (neither verifiable in real-time as only initial and synthetic activity. final O2 concentrations are determined); and (4) the dif- We review the fundamental aspects of the most im- ficulty in ensuring a proper homogeneous or in situ-like portant methodologies used for photosynthetic mea- illumination of the plant sample. This method thus surements in seagrasses, detailing their field of appli- allowed for the very first approaches to in situ and labo- cation and highlighting the most relevant results ratory experiments of seagrass productivity: Drew achieved. Some new and promising techniques are (1978, 1979) investigated the seasonal variation in the presented and a critical analysis of the major advan- photosynthetic activity of Cymodocea nodosa and Posi- tages and disadvantages of each method is given. Sig- donia oceanica, generated photosynthesis-irradiance nificant gaps in this area of research are discussed and (P-E) curves and measured dark respiration in C. nodo- presented as potential pathways for future work. sa, Halophila stipulacea, Phyllospadix torreyi, Posidonia oceanica, Zostera angustifolia and Z. marina, determining the effects of temperature on photosynthesis and cal- MEASURING SEAGRASS PHOTOSYNTHESIS culating compensation and saturation irradiances for those species. Over the years, the effects of light, temper-

O2 measurements ature, salinity and pressure on seagrass photosynthesis were further investigated using this technique in Halo-

Measuring the O2 evolved during photosynthesis is dule uninervis, Halophila stipulacea and Halophila one of the oldest and simplest ways of quantifying the ovalis (Beer & Waisel 1982, Wahbeh 1983), Z. muelleri photosynthetic activity of plants. In the aquatic envi- (Kerr & Strother 1985), C. nodosa (Pérez & Romero 1992, ronment, O2 concentration can be determined: Zavodnik et al. 1998, Olesen et al. 2002) and Posidonia (1) chemically by Winkler titration, (2) oceanica (Olesen et al. 2002). Recently, a few studies polarographically using O2 electrodes, or (3) optically focusing on whole-community metabolism also used using O2 optodes. Winkler titrations to determine O2 concentrations before and after incubations in benthic chambers (Barrón et al. 2004, Gazeau et al. 2005a,b). Nevertheless, the long in- The Winkler method cubation times used in these studies raise the concern that photosynthesis may be underestimated due to the When applied to seagrasses, the Winkler method (see decreased carboxylase activity of Rubisco in response to Strickland & Parsons 1972, adapted to field use by Drew 69

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the increase in O2 and decrease in CO2 concentrations synthetic rates of Zostera marina and Ruppia maritima within the incubation chambers. leaf tips (from Chesapeake Bay) while Terrados & Ros (1995) investigated the seasonal variation of temperature effects on the photosynthesis and respiration of O2 electrodes shallow Mediterranean Cymodocea nodosa. Koch & Dawes (1991) searched for differences in P-E curves Electrochemical sensors for O2 measurements are between 2 ecotypes of R. maritima from Florida and commonly known as Clark-type O2 electrodes (after North Carolina and Enríquez et al. (1995) determined a their inventor, Leyland C. Clark). These electrodes are series of P-E relationships in plant sections in an composed of a platinum cathode and a silver anode attempt to identify a relationship between the photo- separated by a salt bridge. An electron flow is set in synthetic activity of several Mediterranean macro- motion when a control unit applies a small electric phytes and their morphological characteristics. Invers potential to the system. The current flow is propor- et al. (1997) investigated the effects of pH on photo- tional to the O2 consumed at the cathode and can be synthetic rates of Zostera noltii, C. nodosa and Posido- either analogically plotted or converted into a digital nia oceanica; Ramírez-García et al. (1998) incubated signal to be recorded by a control unit (Walker 1987). pre-desiccated leaves of Phyllospadix scouleri and Clark-type electrodes are available in a number of Phyllospadix torreyi in glass bottles to assess the options such as handheld units, bench oxymeters or effects of different previous air-exposure periods on incorporated in complete commercially available the photosynthetic activity; and Ruiz & Romero (2001) setups. Whereas handheld units, with low accuracy, compared light response curves among Posidonia are mainly useful for indicative measurements of dis- oceanica samples harvested from an in situ multilevel solved O2 (DO) concentrations, bench-top shading experiment. Finally, Alcoverro et al. (1998, oxymeters, with better performance, have been used 2001) examined the seasonal and age-dependence of in conjunction with customized incubation Posidonia oceanica photosynthetic parameters and its chambers, allowing for versatile and tailor-made annual carbon balance; Invers et al. (1999, 2001) eval- apparatuses (Smith et al. uated the role of carbonic anhydrase in the inorganic 1984, Fourqurean & Zieman 1991, Koch 1994, Masini carbon supply to Posidonia oceanica and C. nodosa; et al. 1995, Masini & Manning 1997). Complete sys- and Bintz & Nixon (2001) measured respiration and tems comprised of Clark-type electrodes attached to photosynthetic rates of whole-plant Z. marina seed- water-jacketed incubation chambers are also available lings at 3 different light levels. Hence this type of setup on the market, namely those by Hansatech Instru- has been used to determine photosynthetic light ments and Rank Brothers. These laboratory systems response curves and to evaluate the effects of season- provide the highest available resolution and accuracy ality, morphological characteristics, leaf age depen- and have become standard in photosynthetic research. dence, shading, temperature, desiccation and pH on O2 electrodes such as these are probably among the seagrass photosynthesis. best tools to investigate photosynthetic mechanisms Systems based on continuous O2 measurement have and determine the photosynthetic capacities of sea- been used both in laboratory and field experiments. grasses. Photosynthetic capacity refers strictly to the Most laboratory work has been done using the com- photosynthetic rate obtained under ideal conditions, mercially available integrated systems. Dennison & i.e. with non-limiting dissolved inorganic carbon (DIC) Alberte (1982) investigated the effects of light avail- supplies, nutrients and light and at optimum tempera- ability on Zostera marina photosynthesis and growth; ture (Jones 1994). Such demanding conditions are rel- seasonal variations in photosynthetic light responses atively easy to reproduce and maintain in a small, were evaluated in Z. noltii from the Netherlands (Ver- highly controlled environment such as an O2 elec- maat & Verhagen 1996), in Z. marina and Phyllospadix trode-coupled reaction vessel, but almost impossible to scouleri from California (Cabello-Pasini & Alberte achieve in any other environment. 1997) and in 2 Texan Thalassia testudinum populations O2 electrodes are mostly used in 2 general types of (Herzka & Dunton 1997); Kaldy & Dunton (1999) deter- experimental setups: (1) those in which whole plants or mined P-E relationships in T. testudinum laboratory- plant sections are incubated for a selected period of grown seedlings; Cabello-Pasini et al. (2002) compared time in a sealed container and O2 concentrations are the maximum photosynthetic rates of an open-ocean determined at the beginning and at the end of the Z. marina population with that from a coastal lagoon; incubations (endpoint incubations), and (2) those in Invers et al. (2001) measured continuous O2 evolution which an O2 electrode is coupled to the incubation in Z. marina and P. torreyi from the eastern Pacific; chamber and O2 evolution is continuously monitored. Peralta et al. (2000, 2005) compared photosynthetic Using endpoint incubations, Evans et al. (1986) eval- light response curves between 2 Z. noltii morphotypes; uated temperature acclimation effects on the photo- 70

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Silva & Santos (2004) correlated photosynthetic O2 (1984) used a custom-made 2-chambered apparatus release with electron transport rates in the same spe- connected to a Clark-type O2 electrode to demonstrate cies; and Cayabyab & Enríquez (2007) determined the and quantify the transport of O2 from the shoots to the photosynthetic light responses of T. testudinum plants root-rhizome system of Zostera marina. In a very ele- submitted to 3 distinct light treatments. These inte- gant experimental design, the authors incubated intact grated O2 measuring systems have the advantage shoots, separating the leaves from the root-rhizome of measuring net gas exchange, which may be system in 2 adjacent compartments with separate correlative to growth, but their often small size and media and illumination. By quantifying the O2 taken the fact that the plants (or usually only leaves) must up or released by the root-rhizome system in the be enclosed, limits the reliability of the results for absence or presence of shoot illumination, it was possi- extrapolation to true, unobstructed, in situ conditions. ble to elucidate the role of the light environment in the The above-mentioned systems have also been maintenance of root aerobiosis. This line of research widely used in experiments addressing the mecha- was further pursued by Greve et al. (2003) and Binzer nisms of carbon uptake, particularly those concerning et al. (2005), using microelectrodes inserted directly

– the use of HCO by seagrasses. Using Clark-type O into seagrass rhizomes. These studies significantly 3 2 – electrode-based experimental setups, the use of HCO3 advanced our understanding of metabolic integration as a DIC source was demonstrated for Zostera muelleri in seagrasses. They provided the first in situ measure- from Australia (Millhouse & Strother 1986), Thalassia ments of the fraction of photosynthetically evolved O2 testudinum from Florida (Durako 1993), Posidonia aus- that is channelled down internally to below-ground tis- tralis from Australia (James & Larkum 1996) and Z. sues and eventually leaked to ensure aerobiosis in the marina (Beer & Rehnberg 1997). Beer & Koch (1996) rhizosphere. Fourqurean & Zieman (1991) used a cus- used this kind of system to simulate primitive inorganic tom-made transparent acrylic chamber with a coupled carbon conditions in the Cretaceous and compare the Clark-type polarographic O2 probe to determine the photosynthetic performance of Z. marina and T. testu- light-response curves of whole Thalassia testudinum dinum under those conditions with that under present leaves from south Florida. Koch (1994) examined the day ocean conditions. Zimmerman et al. (1997) evalu- effects of hydrodynamics and boundary layer thick- ated the effects of CO2 enrichment on Z. marina pro- ness on the photosynthetic rates of T. testudinum and ductivity and light requirements with a similar ap- Cymodocea nodosa. In that study, plant samples were paratus. Björk et al. (1997) investigated inorganic incubated in a temperature-controlled microcosm with carbon use in 8 seagrass species from the Eastern an externally recirculated incubation medium. O2 evo- African coast, by measuring the response of photosyn- lution was continuously monitored by a Clark-type O2 thesis to increased inorganic carbon levels. In the same electrode installed at an intermediate point of the sys- study, Halophila ovalis, Cymodocea rotundata and tem. Masini et al. (1995) and Masini & Manning (1997) Syringodium isoetifolium also revealed their ability to determined photosynthetic light responses in Posido-

– use HCO3 as an inorganic carbon source. Hellblom et nia sinuosa, P. australis, Amphibolis griffithii and A. al. (2001) assessed the effects of commonly used antarctica in a custom-built tubular incubation cham- buffers on the photosynthetic rates of Z. marina and ber with an external water recirculation circuit. Water

– were able to identify 2 mechanisms of HCO3 utiliza- flowing in the circuit was passed through a high- tion, one of them firstly reported for seagrasses based resolution O sensor, recording O values every 15 s. 2 2 – on extruded protons-mediation of HCO3 uptake. Some researchers brought high precision O2 elec- Mercado et al. (2003) investigated the affinity for DIC trodes to the field, connecting them to benthic incuba- – and the presence of different mechanisms of HCO3 tion chambers to monitor the whole-community O2 use in 2 morphotypes of intertidal Z. noltii from southern release and/or consumption. Dunton & Tomasko (1994) Portugal. They found that this species had a low affin- and Major & Dunton (2000) incubated, respectively, – ity for HCO 3 , particularly the lower intertidal morpho- whole Halodule wrightii and Syringodium filiforme type. Further, photosynthetic pathways, mechanisms plants from south Texas, in situ, using transparent of inorganic carbon acquisition, effects of carbon acrylic chambers placed on the seabed. Continuous O2 enrichment and buffer additions and the molar rela- evolution within the chambers was followed using a tionships between O2 release and electron transport high-resolution dissolved O2 probe installed on a boat have been elucidated using such O2 electrode systems. at the surface. In both studies, the authors also con- On the other hand, extrapolation of the results ob- ducted laboratory measurements of P-E curves in leaf tained in these small-volume, laboratory-bound sys- segments using water-jacketed incubation chambers tems to in situ conditions may be rather incorrect. with coupled O2 electrodes. Field and laboratory mea- A few authors have built custom chambers with cou- surements were compared, and in both studies the pled electrodes for specific experiments. Smith et al. photosynthetic efficiency (α), expressed as the initial 71

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slope of P-E curves, was shown to be highly overesti- Both planar and tapered O2 optodes appear mated in laboratory measurements. Other significant as promising techniques for use in seagrass differences, observed in the compensation intensity (Ic) photosynthetic research. Their full potential is yet and in respiration, highlighted the advantages of in to be explored, and further studies are necessary, situ measurements. Benthic chambers with continuous particularly to compare and validate O2 optodes O2 monitoring were also used by Plus et al. (2001) to against other established methods. determine primary production and respiration of Zostera noltii beds in the Thau lagoon in South France. Chlorophyll fluorescence

O2 optodes When photons within the photosynthetically active radiation (PAR) region strike the photosynthetic pig- Optodes are sensors for optical detection of chemical ment molecules, these become excited. Their excitation species. The common measuring principle is the use of energy is, during the following de-excitation, trans- an indicator dye (immobilized on the tip of an optic ferred towards the reaction centre’s special chlorophyll a fibre or on a planar surface), the colour of which molecules, whose electrons also become excited and changes as a function of variations in the concentration are channelled to an electron acceptor molecule of the analyte. Optodes can be used to quantify a num- (Quinone A, QA). However, a significant portion of the ber of analytes, e.g. O2 and CO2. Several indicator dyes energy released by de-excitation (or decay) back to can be used to build O2 optodes, providing that ground-level does not enter the photochemical process their luminescence intensity and lifetime are affected and is, instead, released (or dissipated) as heat or fluo- by O2 concentration. A measurement is obtained rescence, i.e. the emission of photons of longer wave- when an excitation light is supplied via the fibre and length than the ones absorbed (Schreiber et al. 1998). It the luminescence quenching of the dye is guided in is the nature of an inverse relationship between chloro- the oppo- site direction and digitally imaged (Glud phyll a fluorescence and photochemistry that enables 2008). The quenching magnitude is then directly the use of the former to be used in the investigation of proportional to the O2 concentration (Kühl & many aspects of the photosynthetic process. Polerecky 2008). Op- todes, mostly in their planar Conventional fluorometers emit identical actinic (or configuration, were developed as a tool for 2- photosynthesis-causing) and excitation lights. The dimensional measurements of O2 profiles in benthic high-wavelength fluorescent light is separated from communities. The 2-dimensional O2 imaging allows a the actinic light by filters before reaching the instru- higher degree of spatial integration than single-point ment’s detector. This allows for a strong fluorescence microsensor measurements, and thus planar O2 optodes signal, but makes quenching analysis and quantum are seen as a powerful tool to describe spatial and yield measurements difficult. In modulated fluorome- temporal benthic O2 distribution patterns and their ters, light is emitted in a succession of alternate dynamics (Glud et al. 2001, Glud 2008). In recent light – dark periods, which enables a more clear sepa- studies (Jensen et al. 2005, Frederiksen & Glud 2006), ration of the fluorescence signal from ambient light. planar optodes were used for the first time to PAM fluorometers, the more recent type, emit continu- investigate O2 leakage in the rhizosphere of the sea- ous short measuring-light pulses of red or blue light. grass Zostera marina. In both cases, O2 As the fluorescence signal caused by this measuring imaging revealed high spatial and temporal dynamics light is captured during the very short pulse periods, of the O2 distribution in the rhizosphere and identified external disturbances, background signals and tran- root tips as the zone where the oxic areas were more sient artefacts are eliminated and do not mask the flu- signifi- cant, and thus where most leakage orescence signal (Schreiber et al. 1998). occurred. One apparent drawback of planar optodes In PAM fluorometers, the short pulses of measuring is that the measurements are conducted along an light induce the emission of a fluorescence signal artificial wall, which represents a significant termed Fo or Fs (depending on whether the plant was alteration of the natural sediment structure, with dark-adapted or not, respectively). When a saturating possible effects on O2 spatial dynamics. Tapered O2 light pulse of some 0.8 s duration is applied to the plant micro-optodes provide 1- dimensional measurements sample, all reaction centres become reduced (or and are seen as a possible alternative to micro- ‘closed’) and the fluorescence emission is maximal (Fm electrodes, with the advantage of not consuming O . 2 or Fm’, again dependent on the previous dark-adapta- Miller & Dunton (2007) essayed the use of micro- tion or not, respectively; complete notation of fluores- optodes to measure photosynthetic rates in incubated cence terms can be found in van Kooten & Snel 1990). Laminaria hyperboria discs, and their results agreed In the case of dark-adapted plants, the maximum pho- with published ones obtained by established methods. 72

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tochemical efficiency of PSII is then given by ΦPSII meters, mainly those produced by Walz. This instru-

= Fm – Fo /Fm or simply Fv /Fm. This parameter, ment allows measurements to be conducted in full sun- besides expressing the potential for PSII light, thanks to a special emitter-detector unit that sep- photochemistry, is commonly used to investigate the arates the fluorescence signal from ambient light onset and recovery of stress situations due to its (Schreiber et al. 1988). Most PAM fluorometers are sensitivity to plant stress (Beer et al. 2001). In portable and one model, the Diving-PAM, is adapted illuminated samples, the same measurement provides for underwater operation. The coupling of these char- the effective quantum yield (Y) which is given by Y = acteristics opened the way for autonomous in situ

Fm’ – Fs /Fm’ = ΔF/Fm’(Genty et al. measurements of effective quantum yields on plants 1989). When Y is measured at steady-state at a known exposed to natural conditions. Novel, automated, multi- irradiance (I), the rate at which electrons are carried channel chlorophyll fluorometers, first described by through the transport chain past Photosystem II (PSII) Runcie & Riddle (2004) and able to withstand prolonged (electron transport rate, ETR) can be estimated, ETR = deployments, were used to investigate light acclimation Y × I × AF × 0.5, where AF is the absorption factor (Beer processes of Halophila stipulacea (Runcie et al. 2009, et al. 2001) and 0.5 expresses an equal distribution of this Theme Section) during the recent 8th Group for the absorbed photons between PSII and PSI (Schreiber Aquatic Productivity (GAP) workshop. Those instru- et al. 1995, see however Grzymski et al. 1997 for alter- ments allow autonomous measurements to be con- native distribution values in some forms of macro- ducted for longer periods (> 24 h), opening new and ex- algae). Depending on the research-specific goals, or if citing possibilities in seagrass photosynthetic research. AF cannot be obtained, it may not always be necessary Whether in the field or in laboratory experiments, PAM or relevant to use absolute values of ETR. In such fluorescence has been used in the evaluation of time- or cases, relative ETRs (rETR), expressed as the product space-related variations in photosynthesis, with in- of the effective quantum yield by irradiance, can also sights on the dynamic behaviour of the photosynthetic be used (for a full review of the optical properties and apparatus of several seagrass species (Silva & Santos absorptances of seagrass leaves see Durako 2007). 2003). P-E curves can be obtained by PAM fluorescence Absolute ETRs are mandatory, for instance, when com- when Y is determined along an irradiance gradient. If parisons of ETR and gross photosynthetic O2 evolution this gradient is imposed by the fluorometer’s own ac- are made (Beer et al. 2001). Unlike terrestrial plants, tinic light source, usually only short (up to minutes) where an AF value of 0.84 is often accepted as a valid light steps are allowed, due to instrument limitations. value, seagrass leaves present high variability in this The resulting curves are named rapid light curves parameter (Durako 2007). (RLCs) due to this feature, and must be interpreted dif- Chlorophyll fluorescence measurements have been ferently from conventional light response curves, al- applied to seagrass research from the mid-1990s. Two though they are graphically similar. The critical differ- main types of fluorometers have been used to investi- ence between RLCs and conventional P-E curves is that gate a wide array of questions. A non-modulated or the ETRs used to draw the curves are not measured at continuous excitation fluorometer (PEA, Hansatech steady-state conditions, given the short exposure at Instruments) was used in laboratory experiments by each light step (Ralph & Gademann 2005 and refer- Dawson & Dennison (1996) to assess the effects of ences therein). Still, RLCs provide important ecophysi- increased ultraviolet radiation and elevated PAR on ological information, namely through the interpretation various morphological and physiological parameters of of curve defining parameters, similar to those of con- 5 Australian seagrass species. The same type of instru- ventional light response curves (Beer et al. 1998, Ralph ment was also used by Enríquez et al. (2002) to exam- & Gademann 2005, Saroussi & Beer 2007). ine variations in photosynthetic activity along the Conventional light response curves can be deter- leaves of Thalassia testudinum in the Caribbean Sea. mined in nature using chlorophyll fluorescence, as Continuous excitation fluorometers allow for high tem- long as an external light source supplies more poral resolution measurements, an essential condition to extended periods of illumination at each light step. investigate the kinetics of the Kautsky induction This type of curve has been obtained mainly with the curve (Buschmann & Lichtenthaler 1988, Enríquez et goal of comparing ETRs with gross O2 production in al. 2002). Given that this kind of fluorometry does not the search for an expedient and field-going methodol- allow the separation of fluorescence emission from ogy to estimate photosynthetic rates. In these experi- ambient light, measurements can only be performed in ments, O2 production and ETRs are simultaneously dark-adapted samples, which restricts their usefulness measured at several irradiances, always at steady to high quality Fv/Fm determinations and the above- state. Considering that 4 mol of electrons are carried mentioned induction kinetics analysis. through the transport chain for each mol of O2 pro- Most research involving fluorescence determination duced, there is a theoretical molar ratio of 0.25 of seagrasses has been conducted using PAM fluoro- 73

Silva et al.: Measuring seagrass photosynthesis 133

between O2 production and ETR (Walker 1987). In dressing similar questions have been conducted in situ, order for ETR measurements to be used as proxies for either in the intertidal area or underwater, using estimations of actual photosynthetic production, 2 SCUBA. Daily variation in seagrass photosynthesis, as basic conditions must be met: both a linear relationship influenced by ambient irradiance, has been investi- between O2 production and ETR, and a 0.25 O2 gated in situ for Zostera noltii and Cymodocea nodosa /ETR molar ratio must be observed, preferably along a in southern Europe (Silva & Santos 2003), Posidonia comprehensive range of light levels (Silva & Santos australis in Australia (Runcie & Durako 2004), Halo- 2004). Beer et al. (1998) published the first comparison phila stipulacea in the Red Sea (Sharon & Beer 2008) between photosynthetic O2 evolution and ETRs in and Thalassia testudinum in Florida (Belshe et al. 2008). seagrasses. The authors examined the O2/ETR Spatial variation in photosynthetic activity has also relationship in Cymodocea nodosa, Halophila been investigated, at scales ranging from within- to stipulacea and Zostera marina and found molar values among-shoot variability up to landscape-level patterns. of 0.3, 0.12 and Ralph & Gademann (1999) measured Fv /Fm ratios along 0.5, respectively, although only C. nodosa presented a the leaves of Posidonia australis while assessing the ef- clearly linear relationship between the evolved O2 and fect of epiphytes on the plants’ photosynthesis. Durako the transported electrons. Beer & Björk (2000) ob- & Kunzelman (2002) evaluated the variability of photo- served a linear relationship and a ratio of 0.28 in Halo- synthesis within the shoots of T. testudinum in Florida, phila ovalis, and a contrasting curvilinear relationship and went further, examining differences between and a ratio of 0.57 in Halodule wrightii. Silva & Santos shoots from healthy and die-off patches and looking at (2004) obtained a linear relationship and a ratio of 0.15 temporal variation among ca. 300 sampling stations dis- in Zostera noltii. Enríquez & Rodríguez-Román (2006) tributed among several basins. Turner & Schwarz verified a linear relationship and a ratio of 0.25 for Tha- (2006) conducted a similar study on Z. capricorni from lassia testudinum, but only for irradiances <170 µmol New Zealand. Cayabyab & Enríquez (2007) evaluated m–2s–1. Common to all these studies are: (1) the obser- the photoacclimatory responses of T. testudinum plants vation of quite disparate molar ratios, many of them far to 3 distinct light treatments, comparing the daily varia- from the theoretical stoichiometry and ranging from tion in photochemical efficiency between basal and 0.12 to 0.57, (2) the recording of different initial slopes apical leaf sections. of ETR and O2 response curves, and (3) the fact Light availability is often regarded as the most impor- that ETR tends to saturate at higher irradiances tant single parameter controlling seagrass distribution. than O2 evolution. Putative explanations for problems Responses to light have been widely explored by many concerning ETR calculations are electron-cycling authors and from a number of different perspectives. A around PSI and non-photochemical quenching in few studies have particularly addressed the relation- PSII reaction centres at saturating light levels ships between photosynthesis and both depth- and (Franklin & Badger turbidity-related light attenuation. Using the Diving- 2001). Uncertainties in O2 evolution may be related to PAM, Ralph et al. (1998) measured the in situ photo- photorespiration, which can be an important O2 sink synthetic responses of Posidonia australis, P. sinuosa, (e.g. Flexas & Medrano 2002 and references therein), Amphibolis antarctica, A. griffithii and Halophila ovalis or the Mehler reaction, in which O2 is photoreduced at from shallow Australian waters (0 to 6 m), comparing PSI with the production of superoxide radicals (Asada them with laboratory measurements of the deeper 1999). The Mehler reaction alone can represent up to growing P. angustifolia (27 m) and Thalassodendron 10 % of the ETR (Makino et al. 2002), corresponding to pachyrhizu (46 m). Despite the use of 2 contrasting ex- a similar deviation in the theoretical stoichiometry of perimental approaches, the authors found significantly the photosynthetic process. The conversion of net to different patterns of photosynthetic behavior between gross photosynthesis is also likely to introduce addi- shallow and deeper growing seagrasses. Schwarz & tional errors (discussed in Silva & Santos 2004), de- Hellblom (2002) measured the photosynthetic light re- pending on how dark respiration is estimated. sponses of Halophila stipulacea growing at different One of the most common applications of PAM fluo- depths in the Red Sea. They found patterns of acclima- rescence in seagrass research has been the study of tion to distinct light environments, even though they temporal and spatial variation in photosynthesis pat- only sampled a small depth range (7 to 30 m from a terns. In a set of laboratory experiments, Ralph (1996) known 0 to 70 m total range). The same approach was examined the effects of daily irradiance patterns in the used by Durako et al. (2003) to compare the photobiol- photosynthetic activity of Halophila ovalis, comparing ogy of Halophila johnsonii and H. decipiens along a laboratory-grown plants with wild ones, and observed depth gradient in Florida, with the goal of explaining very different patterns among the 2 groups in response their relative depth distributions. Additionally, Collier to different light regimes. Since these experiments, and et al. (2008) used Diving-PAM measurements to com- taking advantage of the portability and water resis- tance of the new Diving-PAM, most of the studies ad- 74

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plement their physiological characterization of Aus- tidal pools, given that the presence of the other 2 plants tralian Posidonia sinuosa along a very small vertical raises the pH above its compensation point. gradient down to 9 m depth. Comparing a broader The combined effects of desiccation and high tem- spectrum of irradiance controlling factors, Campbell et peratures were compared in Australian Posidonia aus- al. (2003, 2007) used PAM fluorescence to evaluate the tralis and Amphibolis antarctica by Seddon & Cheshire combination of light with other environmental factors (2001). Thalassia hemprichii and Halodule uninervis on the distribution of several tropical seagrass species, from Taiwan were also compared by Lan et al. (2005) comparing 4 major Australian habitats (estuarine, regarding their relative capacity to deal with both high coastal, reef and deepwater). irradiances and air exposure. The stress responses to Chlorophyll fluorescence has provided detailed de- high irradiance regimes have been mostly investigated scriptions of variation in seagrass photosynthetic per- in laboratory experiments, under well-controlled con- formance at a number of spatial and temporal scales, ditions, namely for Halophila ovalis (Ralph & Burchett essentially through the analysis of the quantum effi- 1995, Ralph 1999a) and Zostera marina (Ralph et al. ciency fluctuations. Photosynthetic responses to light 2002). Figueroa et al. (2002) and Kunzelman et al. have also been elucidated across a wide range of nat- (2005) approached UV radiation stress by evaluating ural conditions of light quantity and quality. the responses of Posidonia oceanica and Halophila PAM fluorescence has also been used to investigate johnsonii to combinations of PAR with UV-A and/or inorganic carbon limitations to seagrass photosyn- UV-B, respectively. Other types of stress, whose effects thesis. Schwarz et al. (2000) used a Diving-PAM con- have been evaluated through the PAM fluorescence nected to a small chamber to evaluate in situ the in- technique, include thermal stress (Halophila ovalis, organic carbon limitation to Halophila ovalis and Ralph 1998; H. ovalis, Zostera capricorni, Syringodium Cymodocea serrulata photosynthesis. The underwater isoetifolium, Campbell et al. 2006), osmotic stress (H. operating chamber was used to hold and isolate still- ovalis, Ralph 1998), herbicide toxicity (H. ovalis, Z. attached leaves, while permitting the addition of inor- capricorni, Cymodocea serrulata, Haynes et al. 2000; ganic carbon, buffers or inhibitors to the leaf-adjacent H. ovalis, Ralph 2000), ammonium toxicity (Brun et al. medium. PAM fluorescence was also used by Enríquez 2008) or even a combination of factors such as temper- & Rodríguez-Román (2006) to analyze the photo- ature, light and salinity (Ralph 1999b). synthetic response of Thalassia testudinum to carbon Overall, PAM fluorometry has the advantage of limitation, as influenced by water flow, in a set of labo- being quick and non-intrusive, and since no enclo- ratory experiments. sures are needed, it is particularly suited for in situ Chlorophyll fluorescence has proven to be a very measurements. On the other hand, since respiration is useful tool in assessing the onset and recovery of ignored in the measurements (as only photosynthesis numerous types of stress on seagrasses. As discussed per se is measured), it is often impossible, or at least above, the potential quantum yield of PSII, Fv/Fm, is a difficult, to compare such measurements with growth very useful indicator of stress conditions, and therefore rates. Indeed, the most suitable questions to ask using is widely used in studies addressing a wide variety of PAM fluorometry are those concerning the photo- stress situations. Desiccation is a major stressor in synthetic light responses to the whole range of ambient intertidal seagrasses. Hanelt et al. (1994) investigated parameters. low-tide stress in Thalassia hemprichii in South China and observed a midday depression in Fv/Fm, particularly when low tide occurred around solar noon, when Dissolved inorganic carbon uptake and CO2 fluxes desiccation effects were enhanced by high irradiances. The desiccation tolerance of several tropical intertidal Gas molecules, with the exception of those com- seagrasses was evaluated by Björk et al. (1999) in posed by 2 identical atoms, absorb radiation at ex- Zanzibar. Interestingly, in that study, shallow intertidal tremely narrow bandwidths within the infrared region species were overall more resistant to desiccation than of the spectrum. CO2 has its main absorption peak at deeper growing ones, leading the authors to conclude λ = 4.25 µm, with 3 secondary peaks at 2.66, 2.77 and that desiccation tolerance is not the key factor in deter- 14.99 µm (Long & Hallgren 1985). Infrared gas analysis mining the vertical positioning of seagrass species in (IRGA) has therefore long been used to measure, with the intertidal zone, but rather their ability to withstand high accuracy, the evolution of CO2 exchanged in high irradiances. In a subsequent investigation in either the photosynthetic or respiratory process in ter- Zanzibar, Beer et al. (2006) compared the photo- restrial plants. A number of laboratory and portable synthetic responses of H. ovalis, Cymodocea rotundata gas analyzers are available, with varying configura- and Thalassia hemprichii in low-tide conditions, to find tions, from closed to semi-closed or even open systems, that the first species can only survive in monospecific depending on the type of enclosure and air pathway 75 Silva et al.: Measuring seagrass photosynthesis 135

(Long & Hallgren 1985). Compared to the previously (4) Air – sea CO2 fluxes have also been used to esti- discussed methodologies, direct assessments of DIC mate the net community production of seagrass- uptake or CO2 flux measurements have rarely been dominated coastal systems (Gazeau et al. 2005b). The used to estimate seagrass productivity; 4 distinct types air – sea CO2 flux is computed from the air – sea gradi- of approaches have been described. ent of pCO2, the gas transfer velocity and the solubility

(1) Direct measurement of CO2 uptake by individual coefficient of CO2. These fluxes can be determined leaves, which are enclosed in a mini-cuvette with tem- either by following pCO2 evolution inside a perature and humidity control coupled to a portable floating bell system (Frankignoulle & Distèche 1984) infrared gas analyzer. Given the considerable exten- or by simultaneous measurements of both air and sions of intertidal seagrass meadows worldwide, and water pCO2 (Gazeau et al. 2005a, 2005b). the array of information likely to be obtained in situ by these gas-exchange instruments (to which PAM fluorometry can also be coupled), it is surprising that only Other methods a few studies have used this approach (Leuschner & 14 14 – Rees 1993, Leuschner et al. 1998, both with Zostera Radioactively labelled carbon ( CO2 or H CO3 marina and Z. noltii). These highly controlled and ) was one of the first (Beer et al. 2001) methods to 14 accurate systems are, however, limited to intertidal determine CO2 uptake. The C technique has habitats, as no adaptations are yet made to operate mostly been used to investigate the mechanisms of them underwater. carbon uptake in seagrasses (Beer & Waisel 1979, Abel (2) DIC uptake at the community level has been 1984) and its productivity (Williams & McRoy 1982), mostly investigated using benthic incubations in but nowadays it is only rarely used. closed chambers, where water is retained for a few New methodological possibilities are emerging, par- hours. Initial and final DIC values are derived from ticularly those addressing large-scale evaluations of variations in pH and alkalinity. As long incubation seagrass communitiy production. Hermand et al. times are required for pH and alkalinity variations to (2001) and Hermand (2006) summarized the applica- occur, photosynthesis tends to saturate as Rubisco car- bility of geo-acoustic inversion techniques to monitor boxylase activity decreases in response to the increas- photosynthetic O2 release in Posidonia oceanica mead- ing O2 and decreasing CO2 concentrations within the ows. The working principle, verified in this technique, chambers. No assessment has yet been made of how was that the presence of non-dissolved gases in the underestimated the carbon uptake measurements may leaves’ aerenchyma and the production of O2 micro- be when using this technique. Using both transparent bubbles sticking to the blade surface interfere with the and dark chambers, the net community production and impulse response of broad-band acoustic transmis- dark respiration are measured, providing an overall sions and can be correlated with whole-meadow gross community production value. Studies based on photosynthetic activity. This method may provide con- this approach have been conducted on Zostera marina tinuous measurements of oxygen produced by sea- (Ibarra-Obando et al. 2004, Martin et al. 2005) and grasses, and may be useful in monitoring seagrass pro- Posidonia oceanica communities (Barrón et al. 2006), ductivity in large coastal areas. However, more complemented with O2 measurements. developments of this technique, including calibration (3) Silva et al. (2005, 2008) proposed a variation to with more established methods and extrapolation to the benthic incubation method by introducing water different seagrass species, are necessary. recirculation in the chambers and reducing the length The eddy correlation technique, used in terrestrial of the incubations to minutes instead of hours. In this ecosystems, was adapted to aquatic environments by system, water from the chambers is recirculated by a Berg et al. (2003). This technique determines the sedi- peristaltic pump at the surface and flows through an ment – water fluxes of dissolved O2. The general oper- equilibrator, which allows the partial pressure of CO2 ating principle lies in the simultaneous measurement

(pCO2) to be continuously monitored in the gas phase of vertical water column velocity and O2 concen- by an infrared gas analyzer. It is important that pH and tration at a point a given distance from the sediment alkalinity are measured before and after the incuba- surface (Berg & Huettel 2008). This is a completely tion period, so as to provide an estimate of the total DIC non-intrusive approach, capable of integrating con- flux. This method may also underestimate seagrass siderable sediment areas (Berg et al. 2007). Although community production as CO2 uptake is being regen- the technique’s full potential is yet to be explored, it erated through carbonate equilibrium dynamics. Fur- appears very promising, particularly in heterogeneous ther discussion on the technical and analytical aspects environments where its integrating capacity is an of this method can be found in Abril (2009) and Silva & advantage. If used above seagrass meadows, this Santos (2009). method may provide whole-ecosystem metabolism 76

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information. The first attempt to determine oxygen rhizome respiration is rather difficult to measure in fluxes and ecosystem metabolism in seagrasses was situ, and highly unrealistic if measured in laboratory made by Hume et al. (unpubl. data) in a Zostera conditions. On the other hand, a significant portion of marina community. the photosynthetically evolved O2 is conveyed down from the leaves to the below-ground tissues in order to support respiration and also to maintain some CONCLUSIONS degree of aerobiosis in the rhizosphere, which is often sur- rounded by anoxic sediment (Larkum et Table 1 summarizes the applications, advantages al. 1989). In this context, despite several laboratory and disadvantages of the methods presented above to studies (Smith et al. 1984, Connell et al. 1999), it measure seagrass photosynthesis and production. In was Greve et al. (2003) and Binzer et al. (2005) roughly 3 decades of research on seagrass photo- who firstly provided accurate in situ measurements of synthesis, much information has been gained at differ- O2 leakage by seagrass roots and rhizomes, using ent levels through the use of the various methodologies O2 micro-electrodes. O2 optodes (Glud 2008) added a described here. O2 measurements, either by second dimension to these measurements. Whereas Winkler titration or by electrodes, provide accurate respiration measurements at the plant level are fairly rates of net photosynthetic gas exchange in the light simple in the laboratory, their extension to the and respiration in darkness. However, limitations community level has low value. On the other hand, include the need to use enclosures which, invariably, community measurements still present a great number alter natural irradiances and water flow conditions. of uncertainty factors (out- lined in Middelburg et al. O2 electrodes are also usually employed in the 2005). One of the paths to explore further in seagrass laboratory, with obvious limitations related to the photosynthetic production research is the assessment removal of plants from their natural environments. of the metabolic contribution of other autotrophic

Measurements of CO2 employ- ing IRGA are much and heterotrophic components of seagrass more sensitive than O2 measurements and thus communities. Process discrimination and separate require lower incubation times. While the IRGA metabolic evaluations are essential, in particular technique is probably the best for measurements of sediment respiration, to avoid considering the intertidal seagrasses exposed to the air, its recent sediment as just a black box within the community. This adaptation for measuring dissolved CO2 fluxes of will help obtain accurate metabolic budgets for underwater communities may underestimate them. seagrass communities, thus closing the gap between Currently, the most employed method for in situ photo- gross and net primary production. synthetic measurements is PAM fluorometry. It is fast (a This review of the published literature on seagrass measure of ETR can be obtained within 1 s), and no photosynthesis, although oriented to methodological enclosures are necessary. One major restriction is that aspects, also provided an assessment of the status of respiration rates cannot be obtained by PAM fluorom- seagrass photosynthesis research. We agree with a etry, and photosynthetic rates thus cannot be readily recent review of the impact of light limitation on sea- converted to production rates. However, this method is grasses (Ralph et al. 2007): although a great deal is superb for investigating the stresses that impede pho- known on seagrass ecophysiology, much information is tosynthesis (by measuring Fv/Fm) as well as for estimat- still missing, including many fundamental photo- ing photosynthetic responses to irradiance (including biological data. Our analysis of the literature revealed, the use of RLCs). In the future, it is desirable that for example, that even though seagrasses have a wide autonomous instruments that measure both chloro- depth distribution gradient, down to 50 – 70 m (den phyll fluorescence and CO2 gas exchange Hartog 1970), hardly any work has been done on deep become available, so that many of the relevant seagrass populations. This is a major gap in knowledge, espe- photo- synthetic parameters can be measured cially given the importance of understanding the biol- simultaneously over extended time periods. ogy of plants living in extreme environments and tak- Being rooted plants, seagrasses present the most ing into account that seagrass declines worldwide are complex physical structure among marine autotrophs, attributed largely to reductions in light availability as plants simultaneously occupy 2 distinct environ- (Ralph et al. 2007 and references therein). The avail- ments, the water column and the sediment. The inte- ability of underwater instruments such as the Diving- grated physiology of above- and below-ground tissues PAM, and the fact that such deep populations are adds a considerable degree of complexity to metabolic within the depth range for technical diving, provides studies, namely in the accurate determination of CO2 an opportunity for narrowing these gaps in knowl- fixation and O2 production rates. In fact, edge. although it may represent more than half of the With the development of the new large-scale assess- whole-plant O2 consumption (Fourqurean & Zieman ment techniques described above, research questions 1991), root- 77

Silva et al.: Measuring seagrass photosynthesis 137

Table 1. Applications, advantages and disadvantages of the most common methods used in seagrass photosynthesis and community metabolism studies

Method Applications Advantages Disadvantages

O2 titration (Winkler) • Photosynthesis and dark • High accuracy • Intrusive (if plants are respiration of whole plants • Low price incubated in bottles) or leaf cuts incubated in • Problems related to bottles (laboratory or in containment in closed

situ) chambers • O analysis of water • Initial and final O 2 2 samples from benthic concentrations only chambers (field) • Cumbersome

O2 electrodes • Photosynthesis and dark • High resolution • Intrusive coupled to small respiration of leaf cuts • High accuracy • Highly artificial

reaction chambers (laboratory) • Continuous O2 measure- • Spectral quality of arti- ments ficial light sources • Highly controlled conditions • Possibility to manipulate the incubation medium

O2 microelectrodes • O2 consumption by below- • Not very intrusive (small • Small spatial resolution ground tissues (in situ) diameter electrodes) • Fragile in field condi- • O2 leakage into the • Fast response time tions rhizosphere (in situ) • Positioning possibilities • O production or con- 2 sumption in custom-made chambers

• Used in the eddy correlation technique (in situ)

O2 optodes • O2 mapping of • Not very intrusive • Slower response than seagrass rhizosphere • 2-dimensional microelectrodes (in situ) measurements • Technique still under

• Very sensitive at low O2 development concentrations

• Does not consume O2 • Long-term stability

PAM fluorescence • Non-intrusive • Measures light reactions • In situ and laboratory • Portability only measurements of photo- • Autonomous • Does not allow respira- synthetic efficiency at the underwater equipment tion measurements or plant level • Possibility of continuous thus production esti- measurements mates

CO2 evolution • Non-intrusive • Possibility of underesti-

• In situ measurements of • Integration of whole- mating CO2 uptake in community uptake and community metabolism underwater conditions

release of CO2, in incu- • Highly reliable in air- • Problems related to bation chambers exposed conditions containment in closed chambers

Geo-acoustics • Large-scale • Underdeveloped • In situ large-scale estima- application, suitable for technique

tion of community O2 prod- ecosystem level studies uction • Continuous measurements

Eddy correlation • Non-intrusive • In situ sediment – water • Autonomous • Underdeveloped

fluxes of dissolved O2 underwater equipment technique • Community level O2 • Good surface integrating fluxes metabolic studies capacity • Continuous measurements 78

138 Aquat Biol 7: 127–141, 2009

pertaining to seagrass meadows as global CO2 sinks Mar Ecol Prog Ser 141:199 – 204 may be answered in the coming years. The effects of Beer S, Rehnberg J (1997) The acquisition of inorganic carbon by the seagrass Zostera marina. Aquat Bot 56:277–283 global warming on seagrass metabolism also require Beer S, Waisel Y (1979) Some photosynthetic carbon fixation further research, particularly concerning the effects of properties of seagrasses. Aquat Bot 7:129 –138 respiration. Additionally, ocean acidification with the Beer S, Waisel Y (1982) Effects of light and pressure on photosynthesis in two seagrasses. Aquat Bot 13:331– 337 concomitant CO2 increase is expected to positively Beer S, Vilenkin B, Weil A, Veste M, Susel L, Eschel A (1998) affect seagrass photosynthetic rates, but experimental Measuring photosynthetic rates in seagrass by pulse studies are scarce (Palacios & Zimmermann 2007). amplitude modulated (PAM) fluorometry. Mar Ecol Prog Another interesting aspect to explore related to ocean Ser 174:293 – 300 acidification is the interaction between seagrass photo- Beer S, Björk M, Gademann R, Ralph P (2001) Measurements synthesis and the calcification rates of other organisms of photosynthetic rates in seagrasses. In: Short FT, Coles RG (eds) Global seagrass research methods. Elsevier, within the community (e.g. Semesi et al. 2009). We Amsterdam, p 183 –198 thus expect future research to bridge the gap between Beer S, Bjork M, Hellblom F, Axelsson L (2002) Inorganic car- the photosynthetic behaviour of individual plants and bon utilization in marine angiosperms (seagrasses). Funct the various aspects of community-level metabolism. Plant Biol 29:349 – 354 Beer S, Mtolera M, Lyimo T, Bjork M (2006) The photosynthetic performance of the tropical seagrass Halophila Acknowledgements. This review was presented as a keynote ovalis in the upper intertidal. Aquat Bot 84:367– 371 lecture at the 8th International Workshop of the Group for Belshe EF, Durako MJ, Blum JE (2008) Diurnal light Aquatic Primary Productivity (GAP) and Batsheva de curves and landscape-scale variation in photosynthetic Roth- schild Seminar on Gross and Net Primary Productivity, characteristics of Thalassia testudinum in Florida Bay. held at the Interuniversity Institute for Marine Sciences, Eilat, Aquat Bot Israel, in April 2008. We thank the Batsheva de Rothschild 89:16 –22 Founda- tion, Bar Ilan University, the Moshe Shilo Center for Berg P, Huettel M (2008) Monitoring the seafloor using the Marine Biogeochemistry and the staff of the Interuniversity noninvasive eddy correlation technique: integrated ben- Institute for funding and logistic support. J.S. thanks the thic exchange dynamics. Oceanography 21:164 – 167 organizers of the workshop, in particular I. Berman-Frank, for Berg P, Røy H, Janssen F, Meyer V, Jørgensen BB, Huettel M, the opportunity to give this talk and for the wonderful De Beer D (2003) O2 uptake by aquatic sediments welcome in Eilat. measured with a novel non-invasive eddy correlation technique. Mar Ecol Prog Ser 261:75 – 83 Berg P, Røy H, Wiberg PL (2007) Eddy correlation flux mea- LITERATURE CITED surements: the sediment surface area that contributes to the flux. Limnol Oceanogr 52:1672 – 1684 Abel KM (1984) Inorganic carbon source for photosynthesis in Bintz JC, Nixon SW (2001) Responses of eelgrass the seagrass Thalassia hemprichii (Ehrenb) Aschers. Plant Zostera marina seedlings to reduced light. Mar Ecol Prog Physiol 76:776 – 781 Ser 223: Abril G (2009) Comments on: ‘Underwater measurements of 133 –141 carbon dioxide evolution in marine plant communities: a Binzer T, Borum J, Pedersen O (2005) Flow velocity new method’ by J. Silva and R. Santos [Estuarine, Coastal affects internal oxygen conditions in the seagrass and Shelf Science 78(2008) 827– 830]. Estuar Coast Shelf Cymodocea nodosa. Aquat Bot 83:239 –247 Sci 82:357– 360 Björk M, Weil A, Semesi S, Beer S (1997) Photosynthetic utili- Alcoverro T, Manzanera M, Romero J (1998) Seasonal and sation of inorganic carbon by seagrasses from Zanzibar, age-dependent variability of Posidonia oceanica (L.) Delile East Africa. Mar Biol 129:363 – 366 photosynthetic parameters. J Exp Mar Biol Ecol 230:1–13 Björk M, Uku J, Weil A, Beer S (1999) Photosynthetic Alcoverro T, Manzanera M, Romero J (2001) Annual toler- ances to desiccation of tropical intertidal seagrasses. metabolic carbon balance of the seagrass Posidonia Mar Ecol Prog Ser 191:121–126 oceanica: the importance of carbohydrate reserves. Mar Brun FG, Olivé I, Malta EJ, Vergara JJ, Hernandez I, Pérez- Ecol Prog Ser 211:105 – 116 Lloréns JL (2008) Increased vulnerability of Zostera noltii to Asada K (1999) The water-water cycle in chloroplasts: scav- stress caused by low light and elevated ammonium levels enging of active oxygens and dissipation of excess under phosphate deficiency. Mar Ecol Prog Ser 365:67– 75 photons. Annu Rev Plant Physiol Plant Mol Biol 50:601– Buschmann C, Lichtenthaler HK (1988) Complete fluores- 639 cence emission spectra determined during the induction Barrón C, Marbá N, Terrados J, Kennedy H, Duarte CM kinetic using a diode-array detector. In: Lichtenthaler HK (2004) Community metabolism and carbon budget along a (ed) Applications of chlorophyll fluorescence. Kluwer Aca- gradient of seagrass (Cymodocea nodosa) colonization. demic Press, Dordrecht, p 77– 84 Limnol Oceanogr 49:1642 – 1651 Cabello-Pasini A, Alberte RS (1997) Seasonal patterns of Barrón C, Duarte CM, Frankignoulle M, Borges AV (2006) photosynthesis and light-independent carbon fixation in Organic carbon metabolism and carbonate dynamics in a marine macrophytes. J Phycol 33:321– 329 Mediterranean seagrass (Posidonia oceanica) meadow. Cabello-Pasini A, Lara-Turrent C, Zimmerman RC (2002) Estuar Coast 29:417– 426 Effect of storms on photosynthesis, carbohydrate content Beer S (1989) Photosynthesis and photorespiration in marine and survival of eelgrass populations from a coastal lagoon angiosperms. Aquat Bot 34:153 –166 and the adjacent open ocean. Aquat Bot 74:149 –164 Beer S, Björk M (2000) Measuring rates of photosynthesis of Campbell S, Miller C, Steven A, Stephens A (2003) two tropical seagrasses by pulse amplitude modulated Photo- synthetic responses of two temperate seagrasses (PAM) fluorometry. Aquat Bot 66:69 – 76 across a water quality gradient using chlorophyll Beer S, Koch E (1996) Photosynthesis of marine fluorescence. J Exp Mar Biol Ecol 291:57– 78

macroalgae and seagrasses in globally changing CO2 Campbell SJ, McKenzie LJ, Kerville SP (2006) Photosynthetic environments. 79 Silva et al.: Measuring seagrass photosynthesis 139

responses of seven tropical seagrasses to elevated sea- Evans A, Webb K, Penhale P (1986) Photosynthetic tempera- water temperature. J Exp Mar Biol Ecol 330:455 – 468 ture acclimation in two coexisting seagrasses, Zostera Campbell SJ, McKenzie LJ, Kerville SP, Bite JS (2007) Patterns marina L. and Ruppia maritima L. Aquat Bot 24:185 – 197 in tropical seagrass photosynthesis in relation to light, Figueroa FL, Jiménez C, Viñegla B, Pérez-Rodriguez E and depth and habitat. Estuar Coast Shelf Sci 73:551– 562 others (2002) Effects of solar UV radiation on photosynthe- Cayabyab NM, Enríquez S (2007) Leaf photoacclimatory sis of the marine angiosperm Posidonia oceanica from responses of the tropical seagrass Thalassia testudinum southern Spain. Mar Ecol Prog Ser 230:59 – 70

under mesocosm conditions: a mechanistic scaling-up Flexas J, Medrano H (2002) Energy dissipation in C3 study. New Phytol 176:108 – 123 plants under drought. Funct Plant Biol 29:1209 – 1215 Collier CJ, Lavery PS, Ralph PJ, Masini RJ (2008) Physiologi- Fourqurean JW, Zieman JC (1991) Photosynthesis, respiration cal characteristics of the seagrass Posidonia sinuosa along and whole plant carbon budget of the seagrass Thalassia a depth-related gradient of light availability. Mar Ecol testudinum. Mar Ecol Prog Ser 69:161–170

Prog Ser 353:65 – 79 Frankignoulle M, Distèche A (1984) CO2 chemistry in the Connell EL, Colmer TD, Walker DI (1999) Radial oxygen loss water columm above a Posidonia seagrass bed and related from intact roots of Halophila ovalis as a function of dis- air – sea exchanges. Oceanol Acta 7:209 –219 tance behind the root tip and shoot illumination. Aquat Bot Franklin LA, Badger MR (2001) A comparison of photo- 63:219 –228 synthetic electron transport rates in macroalgae measured Costanza R, d’Arge R, de Groot R, Farber S and others (1997) by pulse amplitude modulated chlorophyll fluorometry The value of the world’s ecosystems services and natural and mass spectrometry. J Phycol 37:756 – 767 capital. Nature 387:253 – 260 Frederiksen MS, Glud RN (2006) Oxygen dynamics in the Dawson SP, Dennison WC (1996) Effects of ultraviolet and rhizosphere of Zostera marina: a 2-dimensional planar photosynthetically active radiation on five seagrass spe- optode study. Limnol Oceanogr 51:1072 – 1083 cies. Mar Biol 125:629 – 639 Gazeau F, Borges A, Barrón C, Duarte CM and others (2005a) den Hartog C (1970) Seagrasses of the world. North-Holland Net ecosystem metabolism in a micro-tidal estuary (Ran- Publishing, Amsterdam ders Fjord, Denmark): evaluation of methods. Mar Ecol Dennison WC, Alberte RS (1982) Photosynthetic responses of Prog Ser 301:23 – 41 Zostera marina L (eelgrass) to in situ manipulations of Gazeau F, Duarte CM, Gattuso JP, Barrón C and others

light intensity. Oecologia 55:137–144 (2005b) Whole-system metabolism and CO2 fluxes in Drew EA (1978) Factors affecting photosynthesis and its sea- a Mediterranean Bay dominated by seagrass beds sonal variation in the seagrasses Cymodocea nodosa (Palma Bay, NW Mediterranean). Biogeosciences 2:43 – 60 (Ucria) Aschers, and Posidonia oceanica (L.) Delile in the Genty B, Briantais J, Baker N (1989) The relationship Mediterranean. J Exp Mar Biol Ecol 31:173 – 194 between the quantum yield of photosynthetic electron Drew EA (1979) Physiological aspects of primary production transport and quenching of chlorophyll fluorescence. in seagrasses. Aquat Bot 7:139 –150 Biochim Biophys Acta 990:87– 92 Drew EA, Robertson WAA (1974) A simple field version of the Glud RN (2008) Oxygen dynamics of marine sediments. Mar Winkler determination of dissolved oxygen. New Phytol Biol Res 4:243 –289 73:793 – 796 Glud RN, Tengberg A, Kuhl M, Hall POJ, Klimant I, Host G

Duarte CM, Cebrián J (1996) The fate of marine autotrophic (2001) An in situ instrument for planar O2 optode measure- production. Limnol Oceanogr 41:1758 – 1766 ments at benthic interfaces. Limnol Oceanogr 46: Duarte CM, Chiscano CL (1999) Seagrass biomass and pro- 2073 –2080 duction: a reassessment. Aquat Bot 65:159 –174 Grasshoff K, Ehrhardt M, Kremling K (1983) Methods of sea- Dunton KH, Tomasko DA (1994) In situ photosynthesis in the water analysis. Verlag Chemie, Weinheim seagrass Halodule wrightii in a hypersaline Greve TM, Borum J, Pedersen O (2003) Meristematic oxygen subtropical lagoon. Mar Ecol Prog Ser 107:281–293 variability in eelgrass (Zostera marina). Limnol Oceanogr

Durako MJ (1993) Photosynthetic utilization of CO2 48:210 –216 and – HCO3 in Thalassia testudinum (Hydrocharitaceae). Mar Grzymski J, Johnsen G, Sakshaug E (1997) The significance Biol 115:373 – 380 of intracellular self-shading on the bio-optical properties Durako MJ (2007) Leaf optical properties and photosynthetic of brown, red and green macroalgae. J Phycol 33:408 – 414 leaf absorptances in several Australian seagrasses. Aquat Hanelt D, Li J, Nultsch W (1994) Tidal dependence of photo- Bot 87:83 – 89 inhibition of photosynthesis in marine macrophytes of the Durako MJ, Kunzelman JI (2002) Photosynthetic characteris- South China Sea. Bot Acta 107:66 – 72 tics of Thalassia testudinum measured in situ by pulse- Haynes D, Ralph P, Pranges J, Dennison B (2000) The impact amplitude modulated (PAM) fluorometry: methodological of the herbicide diuron on photosynthesis in 3 species of and scale-based considerations. Aquat Bot 73:173 –185 tropical seagrass. Mar Pollut Bull 41:288 –293 Durako MJ, Kunzelman JI, Kenworthy WJ, Hammerstrom KK Hellblom F, Beer S, Bjork M, Axelsson L (2001) A buffer sen- (2003) Depth-related variability in the photobiology of two sitive inorganic carbon utilisation system in Zostera populations of Halophila johnsonii and Halophila decipi- marina. Aquat Bot 69:55 – 62 ens. Mar Biol 142:1219 – 1228 Hermand JP (2006) Continuous acoustic monitoring of physi- Enríquez S, Rodríguez-Román A (2006) Effect of water flow ological and environmental processes in seagrass prairies on the photosynthesis of three marine macrophytes from a with focus on photosynthesis. In: Caiti A, Chapman NR, fringing-reef lagoon. Mar Ecol Prog Ser 323:119 – 132 Hermand JP, Jesus S (eds) Acoustic sensing techniques for Enríquez S, Duarte CM, Sand-Jensen K (1995) Patterns in the the shallow water environment: inversion methods and photosynthetic metabolism of Mediterranean macro- experiments. Springer, Dordrecht, p 183 –196 phytes. Mar Ecol Prog Ser 119:243 – 252 Hermand JP, Nascetti P, Cinelli F (2001) Inverse acoustical Enríquez S, Merino M, Iglesias-Prieto R (2002) Variations in determination of photosynthetic oxygen productivity of the photosynthetic performance along the leaves of the Posidonia seagrass. In: Caiti A, Hermand JP, Jesus S, tropical seagrass Thalassia testudinum. Mar Biol Porter MB (eds) Proc Workshop Exp Acoustic Inversion 140: Meth Explor Shallow Water Environ. Kluwer Academic 891– 900 80

140 Aquat Biol 7: 127–141, 2009

Press, Dordrecht, p 125 –144 productivity and photosynthesis. Pergamon Press, Oxford, Herzka SZ, Dunton KH (1997) Seasonal photosynthetic pat- p 62 – 94 terns of the seagrass Thalassia testidinum in the western Major KM, Dunton KH (2000) Photosynthetic performance Gulf of Mexico. Mar Ecol Prog Ser 152:103 – 117 in Syringodium filiforme: seasonal variation in light- Ibarra-Obando SE, Smith SV, Poumian-Tapia M, Camacho- harvesting characteristics. Aquat Bot 68:249 –264 Ibar V, Carriquiry JD, Montes-Hugo M (2004) Benthic Makino A, Miyake C, Yokota A (2002) Physiological functions metabolism in San Quintin Bay, Baja California, Mexico. of the water – water cycle (Mehler reaction) and the cyclic Mar Ecol Prog Ser 283:99 –112 electron flow around PSI in rice leaves. Plant Cell Physiol Invers O, Romero J, Perez M (1997) Effects of pH on seagrass 43:1017–1026 photosynthesis: a laboratory and field assessment. Aquat Martin S, Clavier J, Guarini JM, Chauvaud L and others Bot 59:185 –194 (2005) Comparison of Zostera marina and maerl commu- Invers O, Perez M, Romero J (1999) Bicarbonate utilization in nity metabolism. Aquat Bot 83:161–174 seagrass photosynthesis: role of carbonic anhydrase in Masini RJ, Manning CR (1997) The photosynthetic responses Posidonia oceanica (L.) Delile and Cymodocea nodosa to irradiance and temperature of four meadow-forming (Ucria) Ascherson. J Exp Mar Biol Ecol 235:125 – 133 seagrasses. Aquat Bot 58:21– 36 Invers O, Zimmerman RC, Alberte RS, Pérez M, Romero J Masini RJ, Cary JL, Simpson CJ, McComb AJ (1995) Effects (2001) Inorganic carbon sources for seagrass photosynthe- of light and temperature on the photosynthesis of temper- sis: an experimental evaluation of bicarbonate use in spe- ate meadow-forming seagrasses in Western Australia. cies inhabiting temperate waters. J Exp Mar Biol Ecol 265: Aquat Bot 49:239 –254 203 –217 Mercado JM, Niell FX, Silva J, Santos R (2003) Use of James PL, Larkum AWD (1996) Photosynthetic inorganic car- light and inorganic carbon acquisition by two bon acquisition of Posidonia australis. Aquat Bot 55: morphotypes of Zostera noltii Hornem. J Exp Mar Biol 149 –157 Ecol 297:71– 84 Jensen SI, Kühl M, Glud RN, Jørgensen LB, Priemé A (2005) Middelburg JJ, Duarte CM, Gattuso JP (2005) Respiration in Oxic microzones and radial oxygen loss from roots of coastal benthic communities. In: Williams PJB, del Giorgio Zostera marina. Mar Ecol Prog Ser 293:49 – 58 PA (eds) Respiration in aquatic ecosystems. Oxford Uni- Jones HG (1994) Plants and microclimate. Cambridge Univer- versity Press, New York, p 206 –224 13 sity Press, New York Miller HL III, Dunton KH (2007) Stable isotope ( C) and O2 Kaldy JE, Dunton KH (1999) Ontogenetic photosynthetic micro-optode alternatives for measuring photosynthesis in changes, dispersal and survival of Thalassia testudinum seaweeds. Mar Ecol Prog Ser 329:85 – 97 (turtle grass) seedlings in a sub-tropical lagoon. J Exp Mar Millhouse J, Strother S (1986) The effect of pH on the inorganic Biol Ecol 240:193 – 212 carbon source for photosynthesis in the seagrass Zostera Kerr EA, Strother S (1985) Effects of irradiance, temperature muelleri Irmisch ex Aschers. Aquat Bot 24:199 –209 and salinity on photosynthesis of Zostera muelleri. Aquat Olesen B, Enriquez S, Duarte CM, Sand-Jensen K (2002) Bot 23:177–183 Depth-acclimation of photosynthesis, morphology and Koch EW (1994) Hydrodynamics, diffusion-boundary layers demography of Posidonia oceanica and Cymodocea and photosynthesis of the seagrasses Thalassia testudi- nodosa in the Spanish Mediterranean Sea. Mar Ecol Prog num and Cymodocea nodosa. Mar Biol 118:767– 776 Ser 236:89 – 97 Koch EW, Dawes CJ (1991) Ecotypic differentiation in popula- Palacios SL, Zimmermann RC (2007) Response of eelgrass

tions of Ruppia maritima L. germinated from seeds and Zostera marina to CO2 enrichment: possible impacts cultured under algae-free laboratory conditions. J Exp of climate change and potential for remediation of Mar Biol Ecol 152:145 – 159 coastal habitats. Mar Ecol Prog Ser 344:1–13 Kühl M, Polerecky L (2008) Functional and structural imaging Peralta G, Pérez-Lloréns JL, Hernández I, Brun F and others of phototrophic microbial communities and symbioses. (2000) Morphological and physiological differences be- Aquat Microb Ecol 53:99 –118 tween two morphotypes of Zostera noltii Hornem. from Kunzelman JI, Durako MJ, Kenworthy WJ, Stapleton A, the south-western Iberian Peninsula. Helgol Mar Res 54: Wright JLC (2005) Irradiance-induced changes in the pho- 80–86 tobiology of Halophila johnsonii. Mar Biol 148:241–250 Peralta G, Brun FG, Hernandez I, Vergara JJ, Perez-Llorens Lan CY, Kao WY, Lin HJ, Shao KT (2005) Measurement of JL (2005) Morphometric variations as acclimation mecha- chlorophyll fluorescence reveals mechanisms for habitat nisms in Zostera noltii beds. Estuar Coast Shelf Sci 64: niche separation of the intertidal seagrasses Thalassia 347– 356 hemprichii and Halodule uninervis. Mar Biol 148:25 – 34 Pérez M, Romero J (1992) Photosynthetic response to light Larkum AWD, Roberts G, Kuo J, Strother S (1989) Gaseous and temperature of the seagrass Cymodocea nodosa and movement in seagrasses. In: Larkum AWD, McComb AJ, the prediction of its seasonality. Aquat Bot 43:51– 62 Shepherd SA (eds) Biology of seagrasses. Elsevier, Ams- Plus M, Deslous-Paoli JM, Auby I, Dagault F (2001) terdam, p 686 – 722 Factors influencing primary production of seagrass beds Lee KS, Park SR, Kim YK (2007) Effects of irradiance, temper- (Zostera noltii Hornem.) in the Thau Lagoon (French ature, and nutrients on growth dynamics of seagrasses: a Mediter- ranean coast). J Exp Mar Biol Ecol 259:63 – 84 review. J Exp Mar Biol Ecol 350:144 – 175 Ralph PJ (1996) Diurnal photosynthetic patterns of Halophila

Leuschner C, Rees U (1993) CO2 gas exchange of two ovalis (R. Br.) Hook. f. In: Kuo J, Philips RC, Walker DI, Kirk- intertidal seagrass species, Zostera marina L. and man H (eds) Seagrass biology: Proc Int Workshop, Rottnest Zostera noltii Hornem., during emersion. Aquat Bot 45:53 – Island, Western Australia, 25 –29 Jan 1996, p 197–202 62 Ralph PJ (1998) Photosynthetic responses of Halophila ovalis Leuschner C, Landwehr S, Mehlig U (1998) Limitation of car- (R. Br.) Hook. f. to osmotic stress. J Exp Mar Biol Ecol 227: bon assimilation of intertidal Zostera noltii and Z. marina 203 –220 by desiccation at low tide. Aquat Bot 62:171–176 Ralph PJ (1999a) Light-induced photoinhibitory stress re- Long SP, Hallgren JE (1985) Measurement of CO2 assimila- sponses of laboratory-cultured Halophila ovalis. Bot Mar tion by plants in the field and the laboratory. In: Coombs J, 42:11–22 Hall DO, Long SP, Scurlock JMO (eds) Techniques in bio- Ralph PJ (1999b) Photosynthetic response of Halophila ovalis 81 Silva et al.: Measuring seagrass photosynthesis 141

(R. Br.) Hook. f. to combined environmental stress. Aquat gradient in the Gulf of Aqaba, the Red Sea. Aquat Bot 74: Bot 65:83 – 96 263 – 272 Ralph PJ (2000) Herbicide toxicity of Halophila ovalis assessed Schwarz AM, Björk M, Buluda T, Mtolera M, Beer S (2000) by chlorophyll fluorescence. Aquat Bot 66:141–152 Photosynthetic utilisation of carbon and light by two tropical Ralph PJ, Burchett MD (1995) Photosynthetic response of the seagrass species as measured in situ. Mar Biol 137:755 – 761 seagrass Halophila ovalis (R. Br.) Hook. f. to high Seddon S, Cheshire AC (2001) Photosynthetic response of irradiance stress, using chlorophyll a fluorescence. Amphibolis antarctica and Posidonia australis to tempera- Aquat Bot ture and desiccation using chlorophyll fluorescence. Mar 51:55 – 66 Ecol Prog Ser 220:119 – 130 Ralph PJ, Gademann R (1999) Photosynthesis of the seagrass Semesi IS, Beer S, Björk M (2009) Seagrass photosynthesis Posidonia australis Hook. f. and associated epiphytes, controls rates of calcification and photosynthesis of cal- measured by in situ fluorescence analysis. In: Walker DI, careous macroalgae in a tropical seagrass meadow. Mar Wells FE (eds) The seagrass flora and fauna of Ecol Prog Ser 382:41– 47 Rottnest Island, Western Australia. Western Australian Sharon Y, Beer S (2008) Diurnal movements of chloroplasts in Museum, Perth, p 63 – 71 Halophila stipulacea and their effect on PAM fluorometric Ralph PJ, Gademann R (2005) Rapid light curves: a powerful measurements of photosynthetic rates. Aquat Bot 88: tool to assess photosynthetic activity. Aquat Bot 82: 273 – 276 222 –237 Silva J, Santos R (2003) Daily variation patterns in Ralph PJ, Gademann R, Dennison WC (1998) In situ seagrass seagrass photosynthesis along a vertical gradient. Mar photosynthesis measured using a submersible, pulse- Ecol Prog Ser 257:37– 44 amplitude modulated fluorometer. Mar Biol 132:367– 373 Silva J, Santos R (2004) Can chlorophyll fluorescence be used Ralph PJ, Polk SM, Moore KA, Orth RJ, Smith WO (2002) to estimate photosynthetic production in the seagrass Operation of the xanthophyll cycle in the seagrass Zostera Zostera noltii? J Exp Mar Biol Ecol 307:207–216 marina in response to variable irradiance. J Exp Mar Biol Silva J, Santos R (2009) Reply to comments of G. Abril on Ecol 271:189 – 207 ‘Underwater measurements of carbon dioxide evolution in Ralph PJ, Durako MJ, Enriquez S, Collier CJ, Doblin MA marine plant communities: a new method’ by J. Silva and (2007) Impact of light limitation on seagrasses. J Exp Mar R. Santos [Estuarine, Coastal and Shelf Science Biol Ecol 350:176 – 193 78(2008) Ramírez-García P, Lot A, Duarte CM, Terrados J, Agawin 827– 830]. Estuar Coast Shelf Sci 82:361– 362 NSR (1998) Bathymetric distribution, biomass and growth Silva J, Santos R, Calleja ML, Duarte CM (2005) Submerged dynamics of intertidal Phyllospadix scouleri and Phyllo- versus air-exposed intertidal macrophyte productivity: spadix torreyi in Baja California (Mexico). Mar Ecol Prog from physiological to community-level assessments. J Exp Ser 173:13 –23 Mar Biol Ecol 317:87– 95 Ruiz JM, Romero J (2001) Effects of in situ experimental shad- Silva J, Feijóo P, Santos R (2008) Underwater measurements of ing on the Mediterranean seagrass Posidonia oceanica. carbon dioxide evolution in marine plant communities: a Mar Ecol Prog Ser 215:107–120 new method. Estuar Coast Shelf Sci 78:827– 830 Runcie JW, Durako MJ (2004) Among-shoot variability and Smith RD, Dennison WC, Alberte RS (1984) Role of seagrass leaf-specific absorptance characteristics affect diel esti- photosynthesis in root aerobic processes. Plant Physiol 74: mates of in situ electron transport of Posidonia australis. 1055 – 1058 Aquat Bot 80:209 –220 Strickland JDH, Parsons TR (1972) A practical handbook Runcie JW, Riddle MJ (2004) Measuring variability in chloro- of seawater analysis. Fish Res Board Canada 167, Ottawa phyll-fluorescence derived photosynthetic parameters in Terrados J, Ros JD (1995) Temperature effects on photosynthesis situ with a programmable multi-channel fluorometer. and depth distribution of the seagrass Cymodocea nodosa Funct Plant Biol 31:559 – 562 (Ucria) Ascherson in a mediterranean coastal lagoon: the Runcie JW, Paulo D, Santos R, Sharon Y, Beer S, Silva J (2009) Mar Menor (SE Spain). PSZNI Mar Ecol 16:133 –144 Photosynthetic responses of Halophila stipulacea to a light Turner SJ, Schwarz AM (2006) Biomass development and gradient. I. In situ energy partitioning of non-photochemical photosynthetic potential of intertidal Zostera capricorni in quenching. Aquat Biol 7:143 –152 New Zealand estuaries. Aquat Bot 85:53 – 64 Saroussi S, Beer S (2007) Alpha and quantum yield of aquatic van Kooten O, Snel JFH (1990) The use of chlorophyll fluores- plants derived from PAM fluorometry: uses and misuses. cence nomenclature in plant stress physiology. Photosynth Aquat Bot 86:89 – 92 Res 25:147–150 Schreiber U, Bilger W, Klughammer C, Neubauer C (1988) Vermaat JE, Verhagen FCA (1996) Seasonal variation in the Application of the PAM fluorometer in stress detection. In: intertidal seagrass Zostera noltii Hornem.: coupling demo- Lichtenthaler HK (ed) Applications of chlorophyll fluores- graphic and physiological patterns. Aquat Bot 52:259 –281 cence in photosynthesis research, stress physiology, hydro- Wahbeh MI (1983) Productivity and respiration of three sea- biology and remote sensing. Kluwer Academic Press, grass species from the Gulf of Aqaba (Jordan) and some Dordrecht, p 151–155 related factors. Aquat Bot 15:367– 374 Schreiber U, Bilger W, Neubauer C (1995) Chlorophyll fluo- Walker D (1987) The use of the oxygen electrode and fluores- rescence as a nonintrusive indicator for rapid assessment cence probes in simple measurements of photosynthesis. of in vivo photosynthesis. In: Schulze ED, Caldwell MM Oxygraphics, Sheffield (eds) Ecophysiology of photosynthesis. Springer-Verlag, Williams SL, McRoy CP (1982) Seagrass productivity: the Berlin, p 49 – 70 effect of light on carbon uptake. Aquat Bot 12:321– 344 Schreiber U, Bilger W, Hormann H, Neubauer C (1998) Zavodnik N, Travizi A, De Rosa S (1998) Seasonal variations Chlorophyll fluorescence as a dioganostic tool: basics and in the rate of photosynthetic activity and chemical compo- some aspects of practical relevance. In: Raghavendra AS sition of the seagrass Cymodocea nodosa (Ucr.) Asch. Sci (ed) Photosynthesis: a comprehensive treatise. Cambridge Mar 62:301– 309 University Press, Cambridge, p 320 – 336 Zimmerman RC, Kohrs DG, Steller DL, Alberte RS (1997)

Schwarz AM, Hellblom F (2002) The photosynthetic light Impacts of CO2 enrichment on productivity and response of Halophila stipulacea growing along a depth light requirements of eelgrass. Plant Physiol 115:599 – 607

Submitted: December 29, 2008; Accepted: June 19, 2009 Proofs received from author(s): September 15, 2009 82

Discussion

One of Halophila stipulacea’s outstanding features is its wide depth distribution. In the summer time, its plant meadows can extend from the intertidal down to a depth of ~50 m.

Along this gradient, the irradiance changes from high levels of PAR and UV radiation in shallow water to very dim and spectrally blue-shifted light at depth. In order to grow under such differences in irradiance, we hypothesised that Halophila stipulacea must use mechanisms in its light utilization and, accordingly, in its photosynthetic apparatus that are adapted to the specific conditions at which it grows.

In shallow waters, the ability to withstand high irradiances may be facilitated by the ability of the plant's chloroplasts to move into clumps during the day and the apparently efficient mechanism for non-photochemical quenching (Chapter 1). While chloroplast movement was previously described for Halophila stipulacea in drawings (Drew, 1979), the present PhD work (Chapter 1) shows for the first time this phenomenon in photographs, as well as its diurnal oscillations in high-light grown plants. We observed that clumping of chloroplasts occurred only in plants growing in shallow water, and the importance of such self-shading of the chloroplasts should be seen especially against the background that the Gulf of Aqaba-area of the Red Sea may experience one of the highest irradiances on earth on a yearly basis (Winters et al. 2003).

In addition to the obvious photoprotective role of chloroplasts movements, the latter was also shown to have an effect on photosynthetic parameters. Midday chloroplast clumping decreased the light absorbed by the plant leafs and, thus, decrease the co-called absorption factor (AF) and, accordingly, influenced the calculation of electron transport rates (ETR).

When using PAM fluorometry and using ETR as an estimator for photosynthetic rates we therefore suggested the use of a corrected AF, as a result of changes in leaf absorption: The latter changes diurnally in shallow-growing plants and affects the calculation of 83 photosynthetic rates differently along the day depending on the degree of chloroplast clumping at a certain time of the day, and we device a practical way of taking this into account when calculating such rates (Chapter 1).

In addition to high photosynthetically active radiation (PAR, 400-700 nm), coastal waters, especially clear tropical waters such as in the Red Sea, also feature high levels of UV radiation (UVR, 280-400 nm) in their upper layers (Chapter 4). It was found that chloroplast movements that cause their midday clumping under high-PAR conditions was present only in plants acclimated on a water-table to control-filters that allowed UV to pass through them:

The clumping phenomenon was absent in plants that were protected from UV by UV-filters but exposed to high PAR levels. This strongly indicated that UVR (rather than PAR), or UVR in the presence of PAR, was the trigger of chloroplast movements in Halophila stipulacea. As mentioned previously, the surface irradiance characteristics of the studied region (northern

Red Sea) are among the highest in the world (Winters et al. 2003). We found high UVR doses similar to those on the shaded water table also in situ at 6 m depth (where >50% of surface-

UV doses remained). Indeed, 6-m growing plants featured midday chloroplast clumping as verified by us (Chapters 2 and 4). Thus, apparently, the successful adaptation of this plant to shallow waters (<6 m at the sites studied) may be partly based on the ability of its chloroplasts to shade one another (and perhaps the nucleus too) in order to protect them from high irradiances and/or UVR.

While the reduction in midday ETRs were largely an indirect effect of UVR via the decrease leaf absorption caused by the clumping of chloroplasts, thus causing a lower AF,

UVR also affected photosynthesis directly by reducing quantum yields. The daily reduction in effective quantum yield (ΔF/Fm’) can be seen as a dynamic form of photoinhibition (or down- regulation of PSII) since values in the evening returned to morning values. One mechanism that can explain this midday reduction in effective quantum yields is the operation of the 84 xanthophyll cycle, in which excess light energy is dissipated as heat (Demmig-Adams and

Adams 2006), as indeed shown in Chapters 1 and 2. Thus, as part of the more general negative effects of UVR on phytoplankton (Agusti and Liabres 2007) and other marine photosynthetic organisms (Sinha et al. 1998), including seagrasses (Meng et al. 2008), the photosynthetic apparatus of some seagrasses is also influenced by UVR. Logically, this radiation activates mechanisms such as chloroplast clumping and a dynamic down-regulation of photosystem (PS) II that protect at least Halophila stipulacea against it.

PAM fluorometry has been suggested as a method to measure photochemical and non- photochemical rates back in 1986 (Schreiber et al. 1986). Since then, an in situ approach (via the DivingPAM, Walz, Germany) to study marine photosynthesis in macrophytes was established and tested also for seagrass photosynthesis (Beer et al. 1998, Ralph et al. 1998).

Nowadays, in situ PAM fluorometry is widely use as a robust method to study seagrass photosynthesis and metabolism (reviewed by Silva et al. 2009). While quantifying the quantum yield using PAM fluorometry is under broad agreement among researchers, the calculation of true ETR values is controversial (Evans 2009, Dan Tchernov, personal communications). Indeed there are several challenges and potential errors when calculating

ETRs, especially when failing to measure accurate AF and FII values. The current work suggested a few methods and applications to overcome these errors (Sharon and Beer 2008,

Sharon et al. 2011; Chapters 1 and 5). (It should be mentioned that another possible inaccuracy when using fluorescence to study photosynthesis is the possibility of electron radiation from PSII [while Walz have developed the PAM 2000 which should overcome this problem, there still isn't a submergible device available on the market]).

In addition to the need of using correct parameters to calculate ETRs, it is recommended, in some cases, to test whether the studied organism (Halophila stipulacea in our study) is showing reasonable correlation between calculated ETRs and oxygen evolution 85

(Beer et al. 1998, Dan Tchernov, personal communications). This is a challenging fundamental question since oxygen evolution tests net photosynthesis while measuring quantum yields and calculating ETR examines gross photosynthesis (this could be amended partly by calculating and subtracting dark-respiration). Nevertheless, this correlation was examined for several seagrass species and PAM fluorometry was suggested as a robust method to assess photochemical study in Halophila stipulacea after gaining a strong correlation coefficient when using a third degree polynomial function (r2=0.95, Beer et al.

1998) when comparing oxygen evolution to ETR calculations. Initially, it was suggested that photorespiration was the cause of the oxygen evolution deviation from linearity for Halophila stipulacea at high irradiances. In addition, the authors explain the correlation deviation by 1) an experimental flaw causing different PPF (photosynthetic photon flux) reaching the plants leafs when using both methods and 2) differences in the calculation of AF's.

In all, the molar ration of O2/ETR in Halophila stipulacea is close to the theoretical

0.25, and it was concluded that the Diving-PAM is suitable for measurements of photosynthetic ETRs in seagrasses (Beer et al. 1998). Nevertheless, there is a need to expand our knowledge in gaining a more exact O2/ETR values in future research. Evans (2009) questions the use of fluorescence for ETR determinations since several fluorescence signals are emitted from chlorophyll in the multilayer leave which might cause false measurements.

We believe that future fluorescence studies can contribute and improve our understating while still, for in-situ seagrass photosynthesis research, PAM fluorometry is a favourable method.

Deep-growing plants of the seagrass Halophila stipulacea received at their depth limit in the summer ( ~ 5 0 m ) a down-welling PAR of ~5% of that at the surface, UVR was virtually absent and the spectrum narrowed to blue-green light. At this depth chloroplast clumping is not induced and the chloroplasts are evenly dispersed in the cytoplasm volume to maximize light absorption along the day. In addition to wider and longer leafs at depth 86

(Schwartz and Hellblom 2002), the current PhD work found some biochemical and physiological adaptations that may explain the successful growth of Halophila stipulacea under dim irradiance at its lower growth limit as follows:

Changes in Chl concentration are a known response of photosynthetic organisms, including seagrasses, in their acclimation to variations in the light environment. Here

(Chapter 5), it was found that in deep-growing Halophila stipulacea leaves both Chl a and b were increased, as was also previously reported in our studies for this species (Chapter 1,

3 and 4), as well as for other seagrasses (Wiginton and McMillan 1979, Dennison 1990,

Abal et al. 1994). However, unlike in most other studies, the ratio of chlorophyll a:b did not change between the different depths (1 and 48 m), and therefore cannot explain the adaptive mechanism that enables Halophila stipulacea to grow under very dim irradiances.

Unlike for the chlorophyll a:b ratios, there was a marked change of almost three times in PSI : PSII ratio in the deep- growing Halophila stipulacea plants. This change was a result of changes in the PSI content (Chapter 5), similar to previous studies in various photosynthetic organisms (Fujita 1997, Yamazaki et al. 2005, Levitan et al. 2011). Yamazaki et al. (2005) suggested that the higher ratios of PSI:PSII may be derived from the need for a higher ATP:NADPH ratio, which can be obtained by cyclic electron flow around PSI, increasing ADP phophorylation around the cytochrome b6/f complex. In addition we found, similarly to those authors, a higher level of fluorescence emission in PSI than is PSII after excitation as measured by 77K fluorescence. The high PSI:PSII ratio found here for

Halophila stipulacea growing at its depth limit, together with the higher functionality of

PSI as indicated by the 77K fluorescence emission, thus suggest an advantage to this plant under the extremely limiting light conditions found at 48-m depth. It is known that low-light grown chlorophytes usually change the number of the photosynthetic units (i.e. photosystems and Chl) rather than their size as a light-shade adaptation strategy (Falkowski 87 and Owens 1980). Yet, we still do not know how the favour of a higher ATP:NADPH production provides and adaptive advantage for extremely deep-growing Halophila stipulacea plants.

The increase in Rubisco in shallow-growing Halophila stipulacea plants correlates with the higher photosynthetic ETR under high-light conditions. The RbcL:PsbA ratio was twice as high in the shallow-growing plants, probably reflecting higher carboxylation rates at the higher irradiance. This is similar to the findings of Schofield et al. (2003) for an algal symbiont during a season featuring high irradiances.

Another aspect of our study on extremely deep-growing plants (Chapter 5) is to emphasize the need of using correct FII (the probability of a photon to be absorbed by PSII) values when calculating ETRs, especially for plants featuring a wide depth-distribution pattern such as exemplified for Halophila stipulacea. When using FII values based on our protein analysis (both protein concentration and functionality), the maximum photosynthetic rate (Pmax) is higher in the shallow-growing plants as compared when calculated using the commonly accepted value of 0.5. However, the initial slope (α) of the rapid light-curve (RLC) did not change between shallow- and deep-growing plants when using correct(ed) FII values, suggesting the same photosynthetic efficiency at low irradiances for both plant types. Based on our finding from Chapter 5, we conclude that one should be cautious when using 0.5 as the FII value as based on our present findings that the amount, functionality, and light absorption probability for each photosystem is clearly not equal in plants growing at extreme irradiances.

Once we found and explained different physiological mechanisms enabling Halophila stipulacea to grow under high light and UV radiation in shallow waters and under dim environments at depth, we were curious to question whether these traits are plastic or evolve from two ecotypes at the two depths. Our results indicate that Halophila stipulacea is a highly 88 plastic seagrass that can acclimate to various light environments quickly (Chapter 3). This was shown before for plants grown under high-light conditions, where chloroplast movements were suggested as a means to both capture photons under diurnal low-light periods (when chloroplasts are dispersed) and apparently protect the leaves from photodamage during midday (by clumping the chloroplasts, Chapter 1). Later we showed that the photosynthetic apparatus also features plasticity and can acclimate to various irradiances along a depth gradient according to classical adaptation strategies of high- and low-light plants (e.g. both chlorophyll contents and Pmax changed according to dogma within a week). Further, this plasticity is in line with the thought that specimens of Halophila stipulacea growing at various depths in a meadow are not ecotypes, although the maximal irradiance during midday spans at least one order of magnitude.

The apparent lack of ecotype formation at various depths within a Halophila stipulacea meadow may stem from its largely vegetative growth pattern (through rhizome elongation) resulting in large areas belonging to the same clone. Further, we have observed that the vertical extension of the meadow differs between seasons. During the summer, the studied meadow extends from 5 m to a depth of 50 m, while during the winter the meadow’s lower limit only extends to ~30 m. This may be due to irradiance and/or temperature; the lowest irradiance in the Gulf of Aqaba is in January and the lowest temperature in March

(Winters et al. 2006).

Chapter 3 suggests that the plasticity in the response to irradiance levels would allow for the successful acclimation to the different depths as observed throughout the year. This would then explain the dynamic seasonal meadow extensions as based on clonal growth, although Halophila stipulacea meadows were found to be highly polymorphic between depths in the Mediterranean (Procaccini et al. 1999). On the cellular level, the fast (within 6 d) adjustment of Pmax to irradiance points towards rapid changes in the carbon fixation 89 reactions during the acclimation to high or low midday irradiances. These changes could involve key enzymes of the Calvin cycle, e.g. Rubisco, or other reactions following those of the primary photochemistry. The fact that Pmax values in the transplanted seagrasses became similar to those of the plants growing originally at the transplant depths shows that these acclimations are not only qualitative but also quantitative. In summary, it is proposed that the plant’s adaptive abilities to change its photosystem compositions, light utilisation, pigment accumulation and carboxylation rates may be involve also in its acclimation process to changed light environments as shown in Chapter 3. While chl a + b content also showed plasticity, the response in the plants transplanted from the high- to the low-light environment was slower than in the low- to high-light transplants, indicating that a longer time is required for the production of new chlorophyll than its loss. In addition, it is possible that maintaining a lower concentration of light-absorbing pigments in shallow water may decrease the potential for photodamage or/and the damage from reactive oxygen species.

The lack of changes in chlorophyll a:b ratios in all treatments of the transplantation experiment, together with the finding from Chapter 5 (for both shallow- and deep-growing plants), suggests that this parameter has no role in in situ acclimation processes, in contrast to what was reported by Durako et al. (2003) for 2 other Halophila species. While terrestrial shade-adapted plants often increase chlorophyll b contents relative to chlorophyll a, it may be that the spectral changes with depth prevent such a change in Halophila stipulacea.

For Halophila stipulacea, some previous studies have reported changes in light utilisation, through pigments concentration and leafs size, yet, in neither case was it verified whether those changes could be due to acclimation processes or were the outcome of longer- term adaptations possibly resulting in ecotype formations. Olesen et al. (2002) have suggested that some seagrasses (i.e. Cymodocea nodosa) can acclimate and respond differently to changing light conditions than others (i.e. Posidonia oceanica). While Cymodocea nodosa 90 displayed reduced light requirements with depth, both species acclimated mainly at the population structure level by reducing self-shading in deeper waters.

This is the first work reporting that Halophila stipulacea’s wide depth distribution across a broad range of irradiances (from ~2000 to <100 μmol photons m-2 s-1) can be explained by its plastic photosynthetic abilities allowing for successful acclimation both spatially and seasonally. These plastic abilities include chloroplasts movements, changes in light absorbing pigments, photosystems composition and functionality and Rubisco concentration. Still, there remain some unanswered issues, like the energetic advantage of favouring ATP vs. NADPH production and other accessories pigments’ involvements in the plant’s light utilisation under extreme light environments.

4. Conclusions

The present research provided several novel findings regarding Halophila

stipulacea's adaptations and accordingly acclimations to the wide light gradient in

which it grows:

(i) We found a rationale for the observation of diurnal chloroplast movements in

high-light growing Halophila stipulacea plants;

(ii) It was shown that the potentially harmful effects of UVR on the

photosynthetic apparatus of Halophila stipulacea are mitigated by its activation of

chloroplast clumping, which functions as a means of protecting most chloroplasts

from high irradiances, including UVR;

(iii) The fast changes in photosynthetic responses and light absorption

characteristics in response to changing light environments suggest that Halophila

stipulacea is a highly plastic seagrass with regard to irradiance, which may partly

explain its abundance across a wide range of irradiances along the depth gradient that

it occupies;

(iv) Novel dive techniques allowed us to successfully use underwater PAM

fluorometry in order to study the physiology of light-responses of native populations

of Halophila stipulacea, including those at depths that have never been investigated

before. It is suggested that this seagrass acclimates to the widely varying irradiances

across its depth gradient by regularly modulating a down-regulation type of non- photochemical quenching processes;

(v) It is concluded that the ability of Halophila stipulacea to alter its amount of

PSI relative to PSII according to the ambient irradiance and spectrum may be a basis for this organism's successful growth down to its exceptional depth limit in clear tropical waters.

5. References

Abal EG, Dennison WC (1996) Seagrass depth range and water quality in Southern Moreton Bay, Queensland, Australia. Marine and Freshwater Research 47: 763–771.

Abal EG, Loneragan NR, Bowen P, Perry CJ, Udy JW, Dennison WC (1994) Physiological and morphological responses of the seagrass Zostera capricorni (Aschers) to light intensity. Journal of Experimental Marine Biology and Ecology 178: 113–129.

Agusti S, Liabres ML (2007) Solar radiation-induced mortality of marine pico- plankton in the oligotrophic ocean. Photochemistry and Photobiology 83: 793- 801.

Beer S, Bjork M, Gademann R, Ralph P (2001) Measurements of photosynthetic rates in seagrasses. Global seagrass Research Methods. Elsevier Science B.V.

Beer S, Vilenkin B, Weil A, Veste M, Susel L, Eshel A (1998) measuring photosynthetic rates in seagrasses by pulse amplitude modulated (PAM) fluorometry. Marine Ecology Progress Series 174: 293-300

Beach KS, Borgeas HB, Smith CM (2006) Ecophysiological implications of the measurement of transmittance and reflectance of tropical macroalgae. Phycologia 45: 450-457.

Beck MW, Heck KL, Able KW, Childers DL, Eggleston DB, Gilllanders BM, Halpern B, Hays CG, Hoshino K, Minello TJ, Orth RJ, Sheridn PF, Weinstein MP (2001) The identification, conservation, and management of estuarine and marine nurseries for fish and invertebrates. BioScience 51: 633–641.

Brandt LA, Koch EW (2003) Periphyton as a UV-B filter on seagrass leaves: A result of different transmittance in the UV-B and PAR ranges. Aquatic Botany 76: 317-327.

Cambridge ML, McComb AJ (1984) The loss of seagrasses in Cockburn Sound,Western Australia, 1: The time course and magnitude of seagrass decline in relation to industrial development. Aquatic Botany 20: 229–243.

Carruthers TJB, Dennison WC, Longstaff BJ, Waycott M, Abal EG, McKenzie LJ, Long WJL (2002) Seagrass habitats of Northeast Australia: Models of key processes and controls. Bulletin of Marine Science 71: 1153–1169.

Cho HY, Tseng TS, Kaiserli E, Sullivan S, Christie JM, Briggs WR (2007) Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis. Plant Physiology 143: 517-529.

Costanza R, d'Arge R, de Groot R, Farber S, Grasso M, Hannon B, Limburg K, Naeem S, O'Neill RV, Paruelo J, Raskin RG, Sutton P, van den Belt M (1997)

The value of the world’s ecosystem services and natural capital. Nature 387: 253–260.

Cummings ME, Zimmerman RC (2003) Light harvesting and the package effect in the seagrasses Thalassia testudinum Banks ex König and Zostera marina L.: optical constraints on photoacclimation. Aquatic Botany 75: 261–274.

Czerny AB, Dunton KH (1995) The effects of in situ light reduction on the growth of two subtropical seagrasses, Thalassia testudinum and Halodule wrightii. Estuaries 18: 418–427.

Dalla Via J, Sturmbauer C, Schonweger G, Sotz E, Mathekowitsch S, Stifter M, Rieger R (1998) Light gradients and meadow structure in Posidonia oceanica: ecomorphological and functional correlates. Marine Ecology Progress Series 163: 267-278.

Dawson SP, Dennison WC (1996) Effects of ultraviolet and photosynthetically active radiation on five seagrass species. Marine Biology 125: 629-638. den Hartog C (1970) The Seagrasses of the World. Amsterdam: North-Holland.

Dennison WC, Orth RJ, Moore KA, Stevenson JC, Carter V, Kollar S, Bergstrom PW, Batiuk RA (1993) Assessing water quality with submersed aquatic vegetation. BioScience 43: 86–94.

Drake LA, Dobbs FC, Zimmerman RC (2003) Effects of epiphyte load on optical properties and photosynthetic potential of the seagrasses Thalassia testudinum Bank ex König and Zostera marina L. Limnology and Oceanography 48: 456– 463.

Drew EA (1979) Physiological-aspects of primary production in seagrasses. Aquatic Botany 7: 139-150.

Duarte CM, Middelburg J, Caraco N (2005) Major role of marine vegetation on the oceanic carbon cycle. Biogeosciences 2: 1–8.

Duarte CM, Chiscano CL (1999) Seagrass biomass and production: a reassessment. Aquatic Botany 65: 159-174.

Duarte CM (1991) Seagrass depth limits. Aquatic Botany 40: 363-377.

Dunne RP, Brown BE (1996) Penetration of solar UVB radiation in shallow tropical waters and its potential biological effects on coral reefs; Results from the central Indian ocean and Andaman sea. Marine Ecology Progress Series 144: 109-118.

Durako MJ (2007) Leaf optical properties and photosynthetic leaf absorptances in several Australian seagrasses. Aquatic Botany 87: 83-89.

Durako MJ, Kunzelman JI, Kenworthy WJ, Hammerstrom KK (2003) Depth-related variability in the photobiology of Halophila johnsonii and Halophila decipiens. Marine Biology 142: 1219–1228.

Enríquez S (2005) Light absorption efficiency and the package effect in the leaves of the seagrass Thalassia testudinum. Marine Ecology Progress Series 289: 141– 150.

Enríquez S, Pantoja-Reyes NI (2005) Form-function analysis of the effect of canopy morphology on leaf self-shading in the seagrass Thalassia testudinum. Oecologia 145: 235–243.

Enríquez S, Sand-Jensen K (2003) Variation in light absorption properties of Mentha aquatica L. as a function of leaf form: implications for plant growth. International Journal of Plant Sciences 164: 125–136.

Enríquez S, Agustí S, Duarte CM (1994) Light absorption by marine macrophytes. Oecologia 98: 121–129.

Enríquez S, Agustí S, Duarte CM (1992) Light absorption by seagrass Posidonia oceanica leaves. Marine Ecology Progress Series 86: 201–204.

Evans JR (2009) Potential errors in electron transport rates calculated from chlorophyll fluorescence as revealed by a multilayer leaf model. Plant and Cell Physiology 50: 698-706.

Falkowski PG, Raven JA (2007) Aquatic Photosynthesis, second edition. Princeton University Press, Princeton and Oxford.

Figueroa FL, Jimenez C, Vinegla B, Perez-Rodriguez E, Aguilera J, Flores-Moya A, Altamirano M, Lebert M, Hader DP (2002) Effects of solar UV radiation on photosynthesis of the marine angiosperm Posidonia oceanica from southern Spain. Marine Ecology Progress Series 230: 59-70.

French GT, KA Moore (2003) Interactive effects of light and salinity stress on the growth, reproduction, and photosynthetic capabilities of Vallisneria americana (wild celery). Estuaries 26: 1255-1268.

Fujita Y (1997) A study on the dynamic features of photosystem stoichiometry: Accomplishments and problems for future studies. Photosynthesis Research 53:83-93.

Furukawa T, Watanabe M, Shihira-Ishikawa I (1998) Green- and blue-light-mediated chloroplast migration in the centric diatom Pleurosira laevis. Protoplasma 203: 214-220.

Gao K, Wu Y, Li G, Wu H, Villafane VE, Helbing EW (2007). Solar UV radiation drives CO2 fixation in marine phytoplankton; a double-edged sword. Plant Physiology 144: 54-59.

Heck KL, Hays C, Orth RJ (2003) A critical evaluation of the nursery role hypothesis for seagrass meadows. Marine Ecology Progress Series 253: 123–136.

Helbling and Zagarese 2003 (2003) UV effects in Aquatic Organisms and Ecosystems. In: Hader DP, Jori G (Eds.) Comprehensive Series in Photochemical and Photobiological Sciences, Royal Society of Chemistry.

Hemminga M, Duarte CM (2000) Seagrass Ecology. Cambridge (United Kingdom): Cambridge University Press.

Henley WJ (1993) Measurement and interpretation of photosynthetic light-response curves in algae in the context of photoinhibition and diel changes. Journal of Phycology 29: 729-739.

Johnson GN, Rumsey FJ, Headley AD, Sheffield E (2000) Adaptations to extreme low light in the fern Trichomanes speciosum. New Phytologist 148: 423-431.

Kasahara M, Kagawa T, Oikawa K, Suetsugu N, Miyao M, Wada M (2002) Chloroplast avoidance movement reduces photodamage in plants. Nature 420: 829-832.

Kirk JTO (1994) Characteristics of the light-field in highly turbid waters - a Monte- Carlo study. Limnology and Oceanography 39: 702-706.

Kondo A, Kaikawa J, Funaguma T, Ueno O (2004) Clumping and dispersal of chloroplasts in succulent plants. Planta 219: 500-506.

Krzeszowiec W, Rajwa B, Dobrucki J, Gabrys H (2007) Actin cytoskeleton in Arabidopsis thaliana under blue and red light. Biology of the Cell. 99: 251- 260.

Kunzelman JI, Durako MJ, Kenworthy WJ, Stapleton A, Wright JLC (2005) Irradiance-induced changes in the photobiology of Halophila johnsoni. Marine Biology 148: 241-250.

Lalli CM, Pearsons TR (2002) Biological oceanography - an introduction, Second edition. Elsevier Science.

Les DH, Cleland MA, Waycott M (1997) Phylogenetic studies in the Alismatidae, II: Evolution of the marine angiosperms (seagrasses) and hydrophily. Systematic Botany 22: 443–463.

Lipkin Y (1979) Quantitative aspects o seagrass communities, particularly of those dominated by Halophila-Stipulacea, in Sinai (northern Red-Sea). Aquatic Botany 7: 119-128.

Littler MM, Littler DS (1980) The evolution of thallus form and survival strategies in benthic marine macroalgae - field and laboratory tests of a functional form Model. American Naturalist 116: 25-44.

Longstaff BJ, Dennison WC (1999) Seagrass survival during pulsed turbidity events: The effects of light deprivation on the seagrasses Halodule pinifolia and Halophila ovalis. Aquatic Botany 65: 105–121.

Longstaff BJ, Loneragan NR, O'Donohue MJ, Dennison WC (1999) Effects of light deprivation on the survival and recovery of the seagrass Halophila ovalis. Journal of Experimental Marine Biology and Ecology 234: 1–27.

Luesse DR, DeBlasio SL, Hangarter RP (2006) Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis. Plant Physiology 141: 1328-1337.

Luning K, Dring MJ (1985) Action spectra and spectral quantum yield of photosynthesis in marine macroalgae with thin and thick thalli. Marine Biology 87: 119-129.

Malm T (2006) Reproduction and recruitment of the seagrass Halophila stipulacea. Aquatic Botany 85: 347-351.

Major KM, Dunton KH (2000) Photosynthetic performance in Syringodium filiforme: seasonal variation in light-harvesting characteristics. Aquatic Botany 68: 249- 264.

Major KM, Dunton KH (2002) Variations in light-harvesting characteristics of the seagrass, Thalassia testudinum: evidence for photoacclimation. Journal of Experimental Marine Biology and Ecology 275: 173-189.

Marchant HJ (1976) Mechanism of chloroplasts movement in green freshwater algae Coleochaete and Mougeotia. The Journal of Cell Biology 70: A56.

Markager S (1993) Light-absorption and quantum yield for growth in 5 species of marine macroalgae. Journal of Phycology 29: 54-63.

Meng Y, Krzysiak AJ, Durako MJ, Kunzelman JI, Wright JLC (2008) Flavones and flavone glycosides from Halophila johnsonii. Phytochemistry 69: 2603-3608.

Monro K, Poore AGB (2005) Light quantity and quality induce shade-avoiding plasticity in a marine macroalga. Journal of Evolutionary Biology 18: 426- 435.

Olesen B, Enriquez S, Duarte CM, Sand-Jensen K (2002). Depth-acclimation of photosynthesis, morphology and demography of Posidonia oceanica and Cymodocea nodosa in the Spanish Mediterranean Sea. Marine Ecology Progress Series 236: 89-97.

Orth RG, Carruthers TJB, Dennison WC, Duarte CM, Fourqurean JW, Heck KL, Hughes AR, Kendrick GA, Kenworthy WJ, Olyarnik S, Short FT, Waycott M, Williams SL (2006) A Global Crisis for Seagrass Ecosystems. Biosciences 56: 987-996.

Paves H, Truve E (2007) Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells. Protoplasma 230: 165-169.

Peralta G, Brun FG, Hernandez I, Vergara JJ, Perez-Ilorens JL (2005) Morphometric variations as acclimation mechanisms in Zostera noltii beds. Estuarine Coastal and Shelf Science 64: 347-356.

Ralph PJ, Durako MJ, Enriquez S, Collier CJ, Doblin MA (2007) Impact of light limitation on seagrasses - A Review. Journal of Experimental Marine Biology and Ecology 350: 176-193.

Ralph PJ, Polk SM, Moore KA, Orth RJ, Smith WO (2002) Operation of the xanthophyll cycle in the seagrass Zostera marina in response to variable irradiance. Journal of Experimental Marine Biology and Ecology 271: 189- 207.

Ralph PJ, Gademann R, Dennison C (1998) In situ seagrass photosynthesis measured using a submersible, pulse amplitude modulated fluorometer. Marine Biology 132: 367-373.

Raven PH, Ray FE, Eichhorn SE (1992) Biology of plants. Worth Publishers.

Ries G, Heller W, Puchta H, Sandermann H, Seidlitz HK, Hohn B (2000) Elevated UV-B radiation reduces genome stability in plants. Nature 406: 98-101.

Reusch TBH (2001) New markers - old questions: population genetics of seagrasses. Marine Ecology-Progress Series 211: 261-274.

Robbereeht R, Caldwell MM (1978) Leaf epidermal transmittance of ultraviolet radiation and its implications for plant sensitivity to ultraviolet-radiation induced injury. Oecologia 32: 277-287.

Rozema J, Bjorn LO, Bornman JF, Gaberscik A, Hader DP, Trost T, Germ M, Klisch M, Groniger A, Sinha RP, Lebert M, He YY, Buffoni-Hall R, de Bakker NVJ, van de Staaij J, Meijkamp BB (2002) The role of UV-B radiation in aquatic and terrestrial ecosystems - an experimental and functional analysis of the evolution of UV-absorbing compounds. Journal of Photochemistry and Photobiology B-Biology 66: 2-12.

Runcie JW, Paulo D, Santos R, Sharon Y. Beer S, Silva J (2009) Photosynthetic Responses of Halophila stipulacea to a Light Gradient: I – In situ Energy Partitioning of Non-photochemical Quenching. Aquatic Biology 7: 142-152.

Runcie JW, Durako MJ (2004) Among-shoot variability and leaf specific absorptance characteristics affect diel estimates of in situ electron transport of Posidonia australis. Aquatic Botany 80: 209–220.

Schreiber U, Schliwa U, Bilger W (1986) Continuous recording of photochemical and non photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Research 10: 51-62.

Schwarz AM, Hellblom F (2002) The photosynthetic light response of Halophila stipulacea growing along a depth gradient in the Gulf of Aqaba, the Red Sea. Aquatic Botany 74: 263-272.

Sharon Y, Levitan O, Spungin D, Berman-Frank I, Beer S (2011) Photoacclimation of the seagrass Halophila stipulacea to the dim irradiance at its 48-meter depth limit. Limnology and Oceanography 56: 357-362.

Sharon Y, Silva J, Santos R, Runcie JW, Chernihovsky M, Beer S (2009) Photosynthetic responses of Halophila stipulacea to a light gradient. II. Acclimations following transplantation. Aquatic Biology 7: 153-157.

Sharon Y, Beer S (2008) Diurnal movements of chloroplasts in Halophila stipulacea and their effect on PAM fluorometric measurements of photosynthetic rates. Aquatic Botany 88: 273-276.

Shibata K (1959) Spectrophotometry of translucence biological materials: opal glass transmission method. Methods of Biochemical Analysis 7: 77–109.

Shiihira-Ishikawa I, Nakamura T, Higashi SI, Watanabe M (2007) Distinct responses of chloroplasts to blue and green laser microbeam irradiations in the centric diatom Pleurosira laevis. Photochemistry and Photobiology 83: 1101-1109.

Stewart HL, Carpenter RC (2003) The effects of morphology and water flow on photosynthesis of marine macroalgae. Ecology 84: 2999-3.

Suchanek TH, Williams SW, Ogden JC, Hubbard DK, Gill IP (1985) Utilization of shallow-water seagrass detritus by Caribbean deep-sea macrofauna: δ 13C evidence. Deep Sea Research 32: 2201–2214.

Suetsugu N, Wada M (2007) Chloroplast photorelocation movement mediated by phototropin family proteins in green plants. Biological Chemistry 388: 927- 935.

Sultan SE (2000) Phenotypic plasticity for plant development, function and life history. Trends in Plant Science 5: 537-542.

Terashima I, Saeki T (1983) Light environment within a leaf. I. Optical properties of paradermal sections of Camellia leaves with special reference to differences in the optical properties of palisade and spongy tissues. Plant and Cell Physiology. 24: 1493–1501.

Thorhaug A, Richardson AD, Berlyn GP (2006) Spectral reflectance of Thalassia testudinum (Hydrocharitaceae) seagrass: low salinity effects. American Journal of Botany 93: 110–117.

Trocine RP, Riceand JD, Wells GN (1981) Inhibition of seagrass photosynthesis by Ultraviolet-B radiation. Plant Physiology 68: 74-81.

Uenaka H, Kadota A (2007) Functional analyses of the Physcomitrella patens phytochromes in regulating chloroplast avoidance movement. The Plant Journal 51: 1050-1061.

Valentine JF, Heck KL (2005) Perspective review of the impacts of overfishing on coral reef food web linkages. Coral Reefs 24: 209–213.

Vogelmann TC, Martin G (1993) The functional significance of palisade tissue: penetration of directional vs. diffuse light. Plant Cell and Environment. 16: 65–72.

Wada M, Kagawa T, Sato Y (2003) Chloroplast movements. Annual Review of Plant Biology 54: 455-468.

Walker DI, Kendrick GA, McComb AJ (2006) Decline and recovery of seagrass ecosystems-the dynamics of change. Larkum AWD, Orth RJ, Duarte CM, eds. Seagrasses: Biology, Ecology and Conservation. Dordrecht (The Netherlands): Springer.

White AJ, Critchley C (1999) Rapid light curves: A new fluorescence method to assess the state of the photosynthetic apparatus. Photosynthesis Research 59: 63-72.

Whitehead K, Vernet M (2000) Influence of mycrosporine-like amino acids (MAAs) on UV absorption by particulate and dissolved organic matter. Limnology and Oceanography 45: 1788-1796.

Winters G, Loya Y, Rottgers R, Beers S (2003) Photoinhibition in shallow-water colonies of the coral Stylophora pistillata as measured in situ. Limnology and Oceanography 48: 1388-1393.

Wright JP, Jones CG (2006) The concept of organisms as ecosystem engineers ten years on: Progress, limitations, and challenges. BioScience 56: 203–209.

Yamazaki JY, Suzuki T, Maruta E, Kamimura Y (2005) The stoichiometry and antenna size of the two photosystems in marine green algae, Bryopsis maxima and Ulva pertusa, in relation to the light environment of their natural habitat. Journal of Experimental Botany 56: 1517-1523.

Zimmerman RC (2006). Light and photosynthesis in seagrass meadows. In: Larkum, A.W.D., Orth, R.J., Durate, C.M. (Eds.), Seagrasses: Biology, Ecology and Conservation. Springer.

תקציר

עבודת הדוקטוראט הנוכחית החלה בתצפיות שדה שעוררו את סקרנותי לחקור צמח בעל פרחים ימי (או (

עשב ים) הגדל מאזור הגאות ושפל ועד לעומק הגדול מ -30 מ .' חקר הפוטו -פיזיולוגיה של אותו צמח אפשרה לי

ללמוד מאפיינים מיוחדים בהתאמה ובאקלום לסביבות אור קיצוניות. ניתן להשתמש בממצאים המדעיים של עבודה

זו לצורך מחקר צמחים ימיים אחרים ואת תגובותיהם האקו -פיזיולוגיות לעוצמות תאורה (גבוהות ונמוכות)

קיצוניות.

בסביבה הימית איכות ועוצמת האור משתנה לאורך מדרון העומק. יצורים פוטוסינתתים המאכלסים מדרונות אלו

צריכים, לכן, להתאים עצמם בדרכים שונות על מנת לנצל עוצמות אור נמוכות (במורכב בעיקר מאורכי גל כחולים)

במים עמוקים ולהימנע מנזק כרוני במים רדודים בהם עוצמת האור הנראה וקרינת UV גבוהות.

Halophila stipulacea, עשב ים טרופי וסאב-טרופי, הוא מאקרופיט מהנדס -סביבה חשוב במפרץ אילת , צפון ,

הים האדום. אוכלוסיות עשב הים הזה משמשות כבית גידול לדגים וחסרי חוליות רבים, מספקות הגנה לשלבים

הצעירים של יצורים אלה, מייצבות את הסדימנט החולי ולוקחות תפקיד משמעותי במחזורי החמצן , חנקן והפחמן ,

בבית הגידול. עשב ים זה מאכלס מדרון עומק גדול (0-50 מ )' , המאופיין בהבדל של יותר מקנה מידה אחד בעוצמת

הקרינה. בעבודה זו, מנגנוני ניצול האור לצורך פוטוסינתזה נחקרו תוך שימוש בשיטות פלורוסנטיות, אופטיות,

מיקרוסקופיות, ספקטרא-סקופיות וביוכימיות, על מנת להבין כיצד צמח זה גדל תחת עוצמות אור מאוד נמוכות

בעומק ושורד תחת עקת אור ו-UV באזור הגאות והשפל . על כן, מטרת העבודה הראשית הנה להסביר את

האסטרטגיות הפוטו -פיזיולוגיות המאפשרות לצמח זה להתאים עצמו לתנאים השוררים לאורך מדרון עומק עם

הבדלי תאורה קיצוניים.

הממצא הראשון שלנו כימת ונתן הסבר לתצפית תנועת הכלורופלסטיים היומית בצמחי Halophila stipulacea הגדלים תחת אור גבוה. לאורך שעות הבוקר עד לאמצע היום הכלורופלסטיים התאגדו בצברים

והתפזרו בצורה שווב לקראת אחר הצהריים המאוחרים. תופעת צברי הכלורופלסטיים ופיזורם לוותה בשינויים

במקדם הבליעה וכך, השפיעו על חישוב קצב העברת האלקטרונים מאחר והאחרון תלוי בצורה ישרה בכמות

האור הנבלעת על ידי הרקמה הפוטוסינתתית. ממצא זה הדגיש את חשיבות השינויים היומיים במקדם הבליעה כאשר מחשבים קצבי העברת אלקטרונים ב -Halophila stipulacea ואולי בעשבי ים נוספים המראים שינויים

יומיים במקדם הבליעה תחת אור גבוה (פרק )1 .

בהמשך לממצאינו הראשוניים, חקרנו את הספקטרום האחראי לתנועת הכלורופלסטים בצמחי

Halophila stipulacea הגדלים באור גבוה . מאחר וקרינת UV (המועדת לגרום נזק) מצויה בשפע מים הרדודים

במפרץ אילת, בו חלק מאוכלוסיות הצמח משגשגות, בחנו את השפעת קרינה זו על תופעת יצירת צברי

הכלורופלסטיים. נמצא כי תנועת כלורופלסטים אינה מתרחשת כאשר הצמחים הוצלו ב -80% מאורכי גל נמוכים

מ -400nm . בנוסף , קרינת UV השפיעה על היעילות הקוונטית היחסית וקצבי העברת האלקטרונים . הסקנו כי .

ההשפעות המזיקות של קרינת UV על המערך הפוטוסינתתי ממותנות על ידי ההפעלה של התקבצות

הכלורופלסטים, המשמשת כאמצעי להגנה על רוב הכלורופלסטים ואולי הגרעין מעוצמת קרינה גבוה , כולל קרינת ,

UV (פרק 4).

הגדילה המשגשגת של Halophila stipulacea תחת מדרון רחב של תאורה מצביעה על יכולת פלסטית (גמישות)

גבוהה או שהיא נגרמת כתוצאה מהתאמות ארוכות טווח לעומקים שונים, שיכולות להוביל ליצירת אקוטיפים

שונים. על מנת לענות על שאלה זו, שתלנו נצרים של עשב הים הזה בין גבולות העומקים בהם הוא גדל בתקופת

הניסוי 8( ו -33 מ ') על מנת להעריך את פוטנציאל האקלום לעוצמות אור שונות. ממצאינו הראו שינויים מהירים

בתגובות הפוטוסינתתיות ובמאפייני בליעת האור כתגובה לשינויי סביבת האור והציעו כי Halophila stipulacea

הוא עשב ים מאוד פלסטי בתגובותיו לאור, ממצא היכול להסביר את תפוצתו במדרון האור הרחב לאורך מדרון

העומק הרחב אותו הוא מאכלס (פרק 3). בנוסף, השתמשו בפלורומטר (מסוג PAM) חדיש שאפשר לנו ללמוד את

התגובות הפיזיולוגיות לאור באוכלוסיות טבעיות של Halophila stipulacea. הצענו כי עשב הים מתאקלם

לסביבות אור שונות לאורך מדרון העומק בו הוא מאוכלס על ידי מערכת בקרה המבוססת על תהליכי ניתוב אנרגיה

שאינם פוטו -כימיים (פרק ).2

בהתבסס על ההבדלים הקיצוניים בסביבות האור המאכלסות את Halophila stipulacea לאורך מדרון העומק בו

הוא משגשג, ניסינו להעריך את תרומתם האפשרית של PSII ו-PSI בבליעת אור ובקצב מעבר אלקטרונים

פוטוסינתתי בצמחים שגדלו במטר וב -48 מ '. תוצאותינו הראו הבדלים משמעותיים בכמות PSI בשני עומקי הגבול

לתפוצת Halophila stipulacea. הנ"ל השפיע גם על ערך ה-FII (יחס (PSII(PSII+PSI) המשפיע ישירות על

חישוב קצבי מעבר אלקטרוניים פוטוסינתתים. סיכמנו כי היכולת של Halophila stipulacea לשנות את הכמות היחסית של PSI ל-PSII בהתאם לתאורת הסביבה והספקטרום יכולה להוות את הבסיס לגידול המצליח של יצור

זה בעומק מחייתו היוצא דופן מים טרופיים צלולים (פרק 5 ). ). 5 לזכרו של אבי [29.6.1936-4.11.2008]

התאמות פוטו-פזיולוגיות של עשב הים

Halophila stipulacea לסביבות אור קיצוניות

חיבור לשם קבלת התואר "דוקטור לפילוסופיה"

מאת:

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ספטמבר 2010

עבודה זו נעשתה בהדרכת פרופסור סוון בר התאמות פוטו-פזיולוגיות של עשב הים

Halophila stipulacea לסביבות אור קיצוניות

חיבור לשם קבלת התואר "דוקטור לפילוסופיה"

מאת:

יהונתן שרון

הוגש לסנאט אוניברסיטת תל -אביב

ספטמבר 2010