1
MECHANISM FOR DIFFERENTIAL DESICCATION TOLERANCE IN
PORPHYRA SPECIES
A Dissertation presented
by
Yen-Chun Liu
to
The Department of Biology
In partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the field of
Biology
Northeastern University
Boston, Massachusetts
November, 2009 2
MECHANISM FOR DIFFERENTIAL DESICCATION TOLERANCE IN PORPHYRA
SPECIES
A Dissertation presented
by
Yen-Chun Liu
ABSTRAC OF DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Biology
in the Graduate School of Arts and Sciences of
Northeastern University, November, 2009 3
Abstract
Drought has been the cause of most agricultural losses, and therefore it is not surprising that desiccation has been one of the most studied stresses on plants. As a result, the mechanisms that desiccation tolerant terrestrial plants use to survive drought conditions are well understood today. This is not the case for those macrophytic marine algae (or seaweeds) that grow in the intertidal zone on rocky shores, which experience far more rapid and severe losses of water content than terrestrial plants. The goal of this study was to determine the mechanism of desiccation tolerance in seaweeds using the Red algae
Porphyra umbilicalis and P. yezoensis as a model system. While these two algae have very different tolerances to desiccation, this study showed that both species lost about
95% of their water in the first two hours of dehydration and their final relative water content is virtually the same. Massive membrane leakage, reduced respiration and reduced oxygen evolution were observed in P. yezoensis after desiccation, but not in P. umbilicalis. TEM observation revealed extensive membrane disruption only in P. yezoensis after desiccation.
Reactive oxygen species (ROS) defense, repression of membrane phase transition and formation of cellular glass are the three major desiccation tolerance mechanisms reported in land plants. ROS defense is not the key to the difference between P. umbilicalis and P. yezoensis for several reasons. First, desiccation in the dark did not alleviate the desiccation damage in P. yezoensis, as light has been shown to stimulate
ROS damage. P. yezoensis also had higher superoxide dismutase activity. There was a small decrease in ascorbate content in P. umblicalis after desiccation, but such decrease 4 was not found in P. yezoensis. Furthermore, neither species showed an increase in membrane peroxidation after desiccation. Repression of membrane phase transition cannot explain the different response because the membranes of both species remained in liquid crystalline when desiccated. Our data suggest that the cytoplasm of P. umbilicalis forms a more stable glass when the organism is desiccated, and that the molecular mobility is lower in the drying P. umbilicalis. A dehydrin-like protein was detected in great abundance in P. umbilicalis and could play a key role in the better desiccation tolerance of this species. 5
Acknowledgements
I would like to thank Dr. Donald Cheney for giving me the opportunity to conduct this study in his laboratory, for his patience, tolerance, and guidance throughout my studies. Otherwise, I could not finish this study.
I would also like to thank my committee members: Dr. Cheney, Dr. Crowe, Dr.
Scheirer, Dr. Bracken, and Dr. Vollmer for their advice throughout this research.
I would like to thank the past members in the laboratory, including Andy, Angela,
Jon, Chris, and Tim. Without their company, I can't go this far and stay sane.
Most important of all, I would like to thank my parents, my sister, and my friends for their unconditional love and support throughout my life, especially in the past nine years. 6
Table of Contents
Abstract 3
Acknowledgements 5
Table of Contents 6
List of Figures 8
List of Tables 10
Chapter 1: Introduction 11
A. The problems with drought and damage caused by desiccation 11
B. Proposed desiccation tolerance mechanisms in land plants 13
C. Knowledge about desiccation tolerance in macroalgae 18
D. Why Porphyra is a good model system of study of desiccation tolerance 31
E. Objective 33
Chapter 2: The role of ROS defense in desiccation tolerance of P. umbilicalis 35
Abstract 36
Introduction 37
Materials and Methods 40
Results 45
Discussion 48
References 55
Chapter 3: How intertidal seaweeds dry but don’t die: the mechanism of desiccation 7
tolerance in Porphyra 69
Abstract 70
Introduction 71
Results and Discussion 74
Materials and Methods 82
References 87
Chapter 4: Determination of saturation irradiance level of laboratory Porphyra cultures
and characterizing field sample of P. umbilicalis and P. yezoensis 101
Objective 102
Materials and Methods 102
Results and Disccusion 105
Conclusion 108
Chapter 5: Discussion 123
References 130 8
List of Figures
Chapter 2
Figure 1: Relative water contents of Porphyra species during dehydration 58
Figure 2: Effect of desiccation on photosynthesis 59
Figure 3: Effect of desiccation on respiration 60
Figure 4: Effect of dehydration membrane leakage 61
Figure 5: Effect of light on membrane leakage 62
Figure 6: SOD activities of Porphyra species during dehydration 63
Figure 7: Effect of desiccation on ascorbate contents 64
Figure 8: Effect of desiccation on peroxide concentration 65
Chapter 3
Figure 1: Absolute water contents of Porphyra species during dehydration 92
Figure 2: Effect of desiccation on phycobiliprotein fluorescence 93
Figure 3: Relationship between water content and membrane leakage 94
Figure 4: Effect of desiccation on thylakoid membranes 95
Figure 5: FTIR spectra of Porphyra species 96
Figure 6: Shift of νOH band with increasing temperature 97
Figure 7: Molecular mobility in the drying Porphyra species 98
Figure 8: Western blot of dehydrin-like protein in Porphyra species 99
Chapter 4
Figure 1: Oxygen evolution and P-I curve for laboratory-grown Porphyra
yezoensis 110 9
Figure 2: Oxygen evolution and P-I curve for laboratory-grown Porphyra
umbilicalis 111
Figure 3: Relative water contents of field collected Porphyra species during
dehydration 112
Figure 4: Effect of dehydration membrane leakage of field collected Porphyra
species 113
Figure 5: Superoxide dismutaste activities of field collected Porphyra species
during dehydration 114
Figure 6: Western blot of dehydrin-like protein in laboratory-grown and field
collected Porphyra species 115 10
List of Tables
Chapter 1
Table 1: Review of studies on dehydration and desiccation of intertidal
macroalgae 26
Table 2: Examples of studies on mechanisms of desiccation tolerance in
macroalgae 30
Chapter 2
Table 1: ANOVA table for tests on relative water contents during desiccation,
effects of desiccation on photosynthesis, respiration, amino acid leakage,
ascorbate contents, and peroxide concentrations, and effects of light
on amino acid leakage 66
Chapter 3
Table 1: ANOVA table for tests on water contents during desiccation, the effects
of water contents on amino acid leakage, the effect of temperature on the
shift of νOH stretch band and the effect of water content on molecular
mobility 100
Chapter 4
Table 1: ANOVA table for tests on water contents during desiccation, the effects
of water contents on amino acid leakage, and the effect of water contents
on superoxide dismutase activity 116 11
Chapter 1
Introduction
A. The problems with drought and damage caused by desiccation
Water deficit and drought is the single most widespread factor that limits agriculture production (Chaves & Oliveira 2004). Boyer (1982) pointed out that drought affected more regions of the United States than any other environmental limitation. It also affects agriculture on every other continent (Kogan 1997). For example, in 2001 drought resulted in more than $5 billion dollars loss in agriculture in Canada (Phillips 2001), and it impacted 80% of the arable land of Henan, the most important province for winter wheat in China (R. Liu et al. 2006). Furthermore, drought is aggravated by human activities and global warming (Guobin & S. Chen 2006; Mittler 2006). Therefore, there is a great deal of interest in drought related issues in plant biology today (Chaerle et al.
2005; Parry et al. 2005; Flexas et al. 2006).
Desiccation has several adverse effects on the cell. For example, dehydration of plant cells leads to the decrease of the availability of CO2 for photosynthesis and causes unbalanced photosynthesis (Bota et al. 2004; Flexas et al. 2006). In excess light, the unbalanced photosynthesis results in a high concentration of oxygen, a high level of photorespiration and excited chlorophyll molecules, which all in turn favor the accumulation of reactive oxygen species (ROS) (Smirnoff 1993). The high concentration of oxygen from photosynthetic oxygen evolution and low availability of CO2 enhance 12 photorespiration. Hydrogen peroxide is generated when glycolate, a byproduct of photorespiration, is metabolized (Bob B. Buchanan et al. 2000). When CO2 fixation is limited at low CO2 concentration, the levels of reduced electron carriers in the photosynthetic electron transfer chain increase and oxygen molecules are reduced by these electron carriers more easily. For example, reduced ferredoxin in photosystem I converts oxygen to superoxide in such conditions and NAD(P)H stimulates superoxide formation in microsomal, peroxisomal and glyoxysomal membranes (Smirnoff 1993).
Also, singlet oxygen is generated when the excited chlorophyll molecule transfers its energy to the oxygen molecule and this ROS is believed to be more damaging to the membranes because it initiates lipid peroxidation (Smirnoff 1993).
ROS can cause oxidation of protein sulfhydryl groups, pigment loss, photosystem damage, lipid peroxidation and free fatty acid accumulation in membranes. Among the damage caused by ROS, lipid peroxidation has been discussed the most in the literature
(Black & Pritchard 2002). Polyunsaturated fatty acids (PUFAs) are susceptible to lipid peroxidation because it is easier to extract hydrogen atoms from such molecules. The resulting peroxyl radicals can combine with each other, attack membrane proteins, or extract hydrogen atoms from neighboring fatty acids and propagate the chain reaction of lipid peroxidation (Halliwell & Chirico 1993).
Desiccation also damages membranes directly. When water content becomes lower than
0.3 g H2O / g dry weight, the Van der Waal's interactions between adjacent membrane lipids increase, leading to the rigidification of the membrane and an increase in its 13 transition temperature (Tm) (Hoekstra et al. 1991). Membranes with higher Tm will attain gel state first and separate from membranes with lower Tm. This causes lateral phase separations of membranes and the aggregation of proteins when they are excluded from different domains (Hoekstra et al. 2001). Membrane phase transitions are responsible for imbibition damage in the dried cells upon rehydration (Crowe et al. 1989;
Golovina et al. 1998).
Another possible effect of desiccation on the cell is membrane fusion. Cellular volume reduction during dehydration causes a compaction of cytoplasmic components.
Membranes in close contact with other membranes are susceptible to fusion. Membrane fusion is also promoted by defects in the bilayer due to the vicinity of lipid phase transition, lateral phase separation, or high local membrane curvature. Phase separation is also more probable in the system where at least one of the membrane components is in the gel phase (Cevc & Richardsen 1999).
B. Proposed desiccation tolerance mechanisms in land plants
Although desiccation may cause severe damage to most plants, there are a few mosses, such as Tortula ruralis (Oliver et al. 2005) and about 330 vascular plants, such as
Craterostigma plantagineum and Myrothamnus fabellifolia, that can withstand desiccation (Scott 2000; Porembski & Barthlott 2000). It has been proposed that plant tissues must meet 3 criteria to be desiccation tolerant: 1) the plant must limit the damage to a repairable level, 2) the plant must maintain its physiological integrity in the dry state 14 and 3) The plant must mobilize repair mechanisms upon rehydration (Oliver & Bewley
1997). From past studies on these plants, three mechanisms for desiccation tolerance have been proposed: ROS defense, repression of membrane phase transition, and vitrification
(Crowe et al. 1992; Smirnoff 1993; Hoekstra et al. 2001).
ROS defense is an important desiccation tolerance mechanism in land plants.
There are two lines of ROS defense: ROS scavenging and ROS production reduction.
Superoxide dismustases (SOD) catalyzes dismutation of superoxide to hydrogen peroxide and oxygen. The hydrogen peroxide produced is then broken down to water by catalase in peroxysomes and glyoxysomes. In the chloroplast and cytoplasm, this function is carried out by ascorbate peroxidase. Ascorbate is the antioxidant used in this pathway and it is regenerated by NAD(P)H-dependent monodehydroascorbate reductase (MDHAR) and glutathione (GSH) in a reaction catalyzed by dehydroascorbate reductase (DHAR). The oxidized glutathione is reduced to GSH by NADP-dependent glutathione reductase. In the chloroplasts, hydrogen peroxide is reduced to water by this series of reactions using
NADPH in the Halliwell-Asada cycle (Smirnoff 1993). The carotenoids are effective scavengers of singlet oxygen. Carotenoids with 9 or more conjugated double bonds quench triplet chlorophyll or singlet oxygen. In both cases, triplet carotenoids decay harmlessly by heat emission. Protection of the thylakoid membrane from singlet oxygen is also provided by alpha-tocopherol. After scavenging lipid peroxyl or lipoxyl radicals, the alpha-tocopherol radical can be reduced back to its original form by ascorbate
(Smirnoff 1993). The importance of ROS scavengers can be clearly seen by the revival of the resurrection plant Myarothamnus flabellifolia after desiccation. After 4 months of 15 desiccation, M. flabellifolia still has substantial amounts of α-tocopherol and regenerates other antioxidants rapidly upon rehydration. After 8 months of desiccation, this plant depletes its ROS scavengers and abscises all its leaves before new leaves developed after rehydration (Kranner et al. 2002).
The other line of ROS defense in desiccation tolerant plants is to diminish the production of ROS during drying by down-regulation of metabolism. For example,
Leprince et al. (Leprince et al. 2000) showed that cucumber radicles that were treated to induce desiccation tolerance respire less before drying than untreated sensitive ones. The sensitive specimens also emitted ethanol and acetaldehyde, which enhances lipid peroxidation in membranes. Another example of controlled downregulation of metabolism is the reversible loss of chloroplasts and chlorophyll, known as poikilochlorophylly (Oliver et al. 2000; Proctor & Tuba 2002). Since photosynthesis is a major source of ROS, some resurrection plants, such as Xerophyta scabrida and X. humilis, dismantle their photosynthesis apparatus during drying and recover their photosynthetic ability upon rehydration (Tuba et al. 1993; Dace et al. 1998).
The second mechanism, repression of membrane phase transition, prevents phase separation and membrane leakage caused by desiccation (Crowe et al. 1987). One common strategy resurrection plant tissues employ to repress membrane phase transition is accumulation of a high concentration of sugars (Black & Pritchard 2002). Sugars, such as trehalose or sucrose, replace water molecules during dehydration by forming hydrogen bonding with phosphate groups of phospholipids. The interaction between sugars and phosphate groups prevents the packing of membrane lipids and membrane phase 16 transition at low water contents (Crowe et al. 1989; Hoekstra et al. 1991). Another group of compounds known to prevent membrane phase transition in resurrection plants during desiccation are amphiphiles. The most discussed amphiphile in the literature is arbutin, which accounts for 25% dry weight of Myrothamnus flabellifolia, a resurrection plant.
Arbutin has a phenol ring connecting to a glucose molecule (Bianchi et al. 1993; Oliver et al. 1998). Arbutin and other amphiples reside in the cytoplasm in the hydrated state but partition into the membrane during desiccation. The partitioning of amphiphiles impedes the alignment of acyl chains, increases membrane fluidity, and represses lipid phase transition (Golovina et al. 1998).
Desiccation tolerant plants also avoid damage through vitrification. Vitrification is a process in which cytoplasm enters a glassy state. Unlike in the crystalline state, molecules in a glassy state do not have an ordered orientation. A glass can be regarded as a liquid with extreme high viscosity. Vitrification occurs at a water content of about 0.1 g
H2O / g DW, at which viscosity of cytoplasm is very high. In this state, cytoplasm appears as a solid material but still keeps the disorder and physical properties of the liquid state
(Hoekstra et al. 2001). The glassy state prevents crystallization of cellular solutes, membrane fusion, and denaturation of proteins. In general, more viscous glasses better reduce deleterious reactions in the cell (Buitink et al. 2000). Glasses significantly reduce the rate of chemical reactions and therefore extend the life span of desiccated cells. The glassy state is characterized by the transition temperature (Tg) at which the cytoplasm goes from glass to liquid state. The glasses with higher Tg were shown to protect dry liposomes better (Sun et al. 1996). The viscosity of cytoplasmic glasses does not decrease 17 when the temperature goes above the Tg immediately. Instead, the collapse temperature,
Tc, of cytoplasmic glasses is usually 50 degrees higher than Tg (Hoekstra et al. 2001).
Oligosaccharides are found to be a better glass forming material than disaccharides because the Tg of oligosaccharide glasses is usually higher than that of monosaccharide and disaccharride glasses (Hincha et al. 2003). Wolkers et al. (Wolkers et al. 2004) studied the properties of glasses formed by different sugars (eg. glucose, sucrose, trehalose, raffinose and dextran) and suggested sugars with higher Tg pack more loosely in glasses and have weaker strength of hydrogen bonds. In desiccated cells, glasses are not composed by sugars only. Proteins, most likely late embryogenesis abundant (LEA) proteins, also play an important role in the formation of glasses. and even enhance the stability of the glasses (Sun & Leopold 1997; Wolkers et al. 1999). LEA proteins were initially discovered in developing seeds (Hoekstra et al. 2001), and the accumulation of LEA proteins in the developing seeds coincides with the acquisition of desiccation tolerance of seed embryos (Bochicchio et al. 1994). Among the LEA proteins, dehydrins, which belong to the LEA D11 family, are the most studied group (Close 1996;
Allagulova et al. 2003). Dehydrins are found in various photosynthetic organisms, including cyanobacteria, algae, bryophytes and terrestrial vascular plants and their expression is associated with water stresses (Campbell & Close 1997). These proteins are free of cysteine and tryptophan and low in hydrophobic amino acids but high in polar amino acids, e.g. glycine, alanine and proline. Dehydrins have conservative amino acid motifs such as K-, S- and Y- segments, and the molecular weights of dehydrins range from 9 to 200 kDa because of numerous combinations of these motifs (Close 1996). 18
Dehydrins are mostly unstructured when hydrated but adapt an α-helical conformation after desiccation (Close 1996; Wolkers et al. 2001). LEA proteins increase the stability of cytoplasmic glasses by forming tight hydrogen bonding networks with sugars during dehydration (Wolkers et al. 2001).
C. Knowledge about desiccation tolerance in macroalgae
It has been known for more than a century that different species of macroalgae tend to occupy different locations in the intertidal zone, and there have been several reviews focusing on this phenomenon (Zanevelt 1937; Southward 1958; Zaneveld 1969;
Davison & Peason 1996). Zaneveld (1937) studied the relationships between tidal height and emersion time, and he found that brown algae in the higher intertidal zones (e.g.,
Fucus spiralis and Ascophyllum nodosum) were exposed to air longer than the species in the lower intertidal zones (e.g., F. serratus). A latter review by Southward (1958) stated that the cause of zonation of intertidal organisms is the different tolerance to environmental stresses, and the differences in dehydration level is the most important determining factor. Probably the most thorough early reivew on desiccation of algae is that of Zaveneld (1969), who reviewed the studies on the physiological and morphological responses of intertidal macroalgae to the environmental stresses. In his review, Zaveneld (1969) pointed out that while the lower limit of intertidal algal distribution is controlled by light, competition with other algae, and grazing, the upper limit of intertidal algal distribution is mostly decided by the tolerance to abiotic stresses, such as extreme change in salinity, temperature, and desiccation. The most recent review, 19 by Davison and Pearson (1996), reviewed the general tolerance of intertidal macrogalgae to a wide range of stresses. They pointed out that that the ability to tolerate emersion is a key factor determining whether an alga can survive in the intertidal zone and that there is a positive correlation between the tolerances of intertidal macroalgae to different environment stresses they face during low tides. However, they also pointed out that the mechanisms for stress tolerances in macroalgae are not well understood (Davison &
Pearson 1996).
Among the resistances to abiotic stresses that determine the upper limit of algae distribution, Zaneveld (1969) reported that desiccation tolerance is the most important factor defining the upper limit at which an alga can grow in the intertidal zone. Indeed, even as long ago as early 1900's Baker (1909) in a comparison of six intertidal brown algae (Table 1), found that species that occupied the highest intertidal zones (Fucus ceranoides and Ascophyllum nodosum) showed the highest survival rates after 24 days of
1 hour in water and 11 hours in air cycles. In addition to the adult plants, Baker
(1910) also found that the embryos of the brown alga that occupied the highest intertidal zone (Fucus spiralis) had the highest germinating rate after four weeks of cycles consisted of 1 hour incubation in water and 11 hour exposure to air. In addition to reducing the survival rates of lower intertidal brown algae, drying also causes more severe membrane leakage in the lower intertidal brown algae. Moebus (1974) found that the lower intertidal Fucus vesiculosus leaked 50 times more organic carbon as the higher intertidal Ascophyllum nodosum did when they both lost 70% of their water. Studies on red algae also showed that desiccation limits the distribution of intertidal red algae. For 20 example, a study on Gastroclonium coulteri showed that the light intensities and temperatures that the plants encountered in the field didn't damage the photosynthesis of this plant, but losing 70% of its water content caused irreversible damage to photosynthesis (Hodgson 1981). Porphyra linearis in the uppermost intertidal zone had been shown to resume photosynthesis even after three weeks of desiccation (at 5% relative water content) (Lipkin et al. 1993). Such long term desiccation tolerance is essential for the survival of P. linearis in the field because the longest emersion this seaweed encounters in nature is two weeks, a period between two successive spring tides
(Lipkin et al. 1993).
High intertidal macroalgae have several physiological adaptations to their stressful habitats. One such adaptation is recovery of nutrient uptake after desiccaton. For example, the high intertidal brown alga Pelvetia limitata showed a two-fold increase in the ammonium and nitrate uptake after it lost 30% of its water, but the uptake of ammonium and nitrate of species from lower intertidal zones (Gigartina papillata and
Entromorpha intestinalis) did not increase after such water loss (Thomas et al. 1987).
Kim et al. (2008) showed that nitrate uptake of high intertidal Porphyra umbilicalis and
P. leucostica recovered to the level of control specimens within 3 hours after a 90% water loss, but that the nitrate uptake of the low intertidal P. yezoensis failed to recover after 90% of water loss.
The most frequently used method of evaluating adaptation of intertidal macroalgae to intertidal environment has been the recovery of photosynthesis after emersion. Dring and Brown (1982) compared 5 species of intertidal brown algae (Table 21
1) in Ireland, and found that the Fucus species showed similar repression in photosynthesis during desiccation, despite their different intertidal heights and that the photosynthesis apparatus of the high intertidal F. spiralis was not more resistant to desiccation than the lower intertidal F. serratus and F. vesiculosus. Further, the high intertidal species Pelvetia canaliculata and F. spiralis recovered from a 80 - 90% water loss while the subtidal species Laminaria digitata could not fully recover from a water loss beyond 60%. The extent of water loss that the mid to low intertidal species F. vesiculosus and F. serratus could recover from was between 60 - 70% (Dring & Brown
1982). Similarly, Brown (1987) tested the effect of desiccation on 6 macroalgae (Table 1) found in New Zealand. The results showed that only the seaweeds occupying the higher intertidal zones (Bostrychia arbuscula and Apophloea lyallii) showed nearly complete recovery after a 95% of water loss. Thus, Brown (1987) showed that the critical water content for complete photosynthesis recovery was higher for the species that occupied the lower intertidal zones. Schonbeck and Norton (1978) tested 4 intertidal brown algae from
Scotland (Table 1) and also observed the highest intertidal species, Pelvetia canaliculata had the best recovery in photosynthesis after drying. Abe et al. (2001) tested 18 intertidal seaweeds in Japan (Table 1) and found that Porphyra detata, the species collected from the uppermost intertidal zone, can fully recover photosynthetic activity after being desiccated at a water potential of -158 MPa, and that those collected from lower intertidal zones could only recover from less sever drying conditions.
In a review of stress tolerance of intertidal seaweeds, Davison and Pearson (1996) suggest that the tolerance to emersion in intertidal macroalgae depends upon a tolerance 22 to desiccation and not on the avoidance of water loss. Since seaweeds at higher intertidal zones showed better tolerance to emersion and desiccation, if the tolerance is related to the ability to keep water during emersion, seaweeds growing at higher intertidal zones should lose water more slowly during emersion. However, this hypothesis is not the case.
Dorgelo (1976) compared the rate of water loss in six intertidal brown algae (Table 1) and found that the most desiccation tolerant, the high intertidal Pelvetia canaliculata lost water faster than any other species investigated in the study, and that the subtidal species
Laminaria saccharina had the lowest rate of water loss. There was no clear relationship between the rates of water loss and location in tidal zones for the rest of intertidal brown macroalge in the Dorgelo (1976) study. Schonbeck and Norton (1979) did a similar study on intertidal brown algae (Table 1) and they also found that the highest intertidal species
Pelvetia canaliculata lost water faster than the lower intertidal species Fucus spiralis and
F. serratus. Finally, Dromgoole (1980) tested another 15 intertidal macroalgae in New
Zealand (Table 1), and also found no clear relationship between the rate of dehydration of these species and their intertidal positions.
Reports on the mechanisms of desiccation tolerance in intertidal seaweeds have mostly focused on the role of reactive oxygen species (ROS) defense. For example,
Sibbald and Vidaver (1987) showed that the desiccation tolerant species Porphyra sanjuanensis protected itself from photodamage by inactivating the photochemistry and electron transport system in high light. Harker (1999) showed that the high intertidal
Pelvetia canaliculata had higher levels of xanthophyll cycle carotenoids and higher de- epoxidation ratio than the subtidal Laminaria saccharina when they were dried to 40% of 23 their fresh weight. Protection of photosystems is important in terms of ROS defense because the damaged photosystems is one of the major sources of ROS (Smirnoff 1993).
In addition to protection of photosystems, several studies have shown that desiccation tolerant species have higher antioxidative activities and accumulate fewer ROS than less tolerant species. For example, Collén and Davison (1999a) found desiccation caused higher production of reactive oxygen in the less tolerant Fucus distichus than in the higher intertidal, more tolerant Fucus spiralis. The ROS production was detected by 2',7'- dichlorohydrofluorescein diacetate (DCFH-DA). DCFH-DA can diffuse into cells freely and be deacetylated by esterases, forming the polar 2',7'-dichlorohydrofluorescein
(DCFH) (Bass et al. 1983). DCFH is a polar molecule and is trapped in cell after its formation. Hydroxyl radical and hydrogen peroxide are thought to be the ROS that oxidizes DCFH and the product, 2',7'-dichlorofluorescein, is fluorescent (Zhu et al.
1994). The inverse correlation between ROS formation and degree of desiccation in
Fucus evanescens indicated that decreased metabolism caused by desiccation slows down
ROS production. Collén and Davison (1999b) also suggest there is a correlation between tolerance to high light and tolerance to desiccation. In a different report, Collén and
Davison (1999a), also showed that the least stress tolerant Fucus species, F. distichus, had the lowest activity of ROS scavenging enzymes expressed on the basis of chlorophyll contents. The most stress tolerant species, F. spiralis, had higher activity of superoxide dismutase and ascorbate peroxidase than lower intertidal F. evanescens. In addition, the level of ascorbate in F. spiralis was almost twice as high as in the F. evanescens on a chlorophyll basis (Collén & Davison 1999a). 24
In another paper, Collén and Davison (1999c) also reported that the high intertidal red alga Mastocarpus stellatus scavenges exogenous H2O2 faster and is more resistant to oxidative stress induced by H2O2 and Rose Bengal than another lower intertidal red alga
Chondrus crispus. The better antioxidative ability of M. stellatus corresponded to its higher levels of ascorbate and β-carotene and higher activities of glutathione reductase and catalase (Collén & Davison 1999c). Finally, Burritt et al. (2002) reported a difference in specimens of the same species but collected from different tidal heights. In the red alga
Stictosiphonia arbuscula, they found that specimens from a higher intertidal location showed less membrane leakage and produced less H2O2 and lipid hydroperoxides than specimens collected from lower intertidal zone.
One constraint with past studies on the algal desiccation tolerance is that the specimens were exposed to strong light at the same time they were desiccated to mimic the intertidal environment (e.g., Collén & Davison 1999a; Collén & Davison 1999b;
Collén & Davison 1999c; Burritt et al. 2002). Furthermore, the algae used in past studies were collected from the field and might have been exposed to other environmental stresses, including strong light. Since strong light itself induces ROS production (e.g.,
Collén & Pedersén 1996), the role of ROS defense in desiccation tolerance has to be further examined with laboratory-grown algae specimens in the conditions without strong lights.
In conclusion, although desiccation tolerance in macroalgae has received considerable attention for decades, scientists still don't understand the actual mechanism(s) behind this phenomenon (Davison & Pearson 1996). Most reports have 25 suggested a relationship between intertidal height and antioxidative activity, but this correlation should not be regarded as proof that ROS defense is the mechanism conferring desiccation tolerance in intertidal seaweeds. Furthermore, past studies usually measured antioxidant activity in the hydrated or partially dehydrated states and did not test specimens after rapid and extreme desiccation. ROS defense may not explain the survival of intertidal seaweeds under such extreme stress. Lastly, desiccation tolerance is a complex phenomenon and several protection mechanisms other than ROS defense have been proposed for land plants. However, these hypotheses have not be tested in intertidal seaweeds. Hence, this study will focus on elucidating how a desiccation-tolerant seaweed, P. umbilicalis survives rapid and extreme desiccation. 26
Table 1. Review of studies on dehydration and dessication of intertidal macroalgae
Species studied Key findings Location References Fucus ceranoides, High intertidal adult Bembridge, Isle Baker 1909 Ascophyllum nodosum, macroalgae showed higher of Wight, United F. vesiculosus, F. survival rates after long, Kindom serratus, Halidrys repetitive drying cycles siliquosa, Laminarias Fucus serratus, F. The embryos of high Bembridge, Isle Baker 1910 vesculosus, F. spiralis, intertidal F. spiralis had the of Wight, United Ascophyllum nodosum highest germinating rate after Kindom repetitive desiccation Fucus serratus, F. Algal species occupying Den Helder, Zaneveld vesculosus, F. spiralis, higher intertdal have longer Holland 1937 Ascophyllum nodosum emersion time Ascophyllum nodosum, A. nodosum (higher intertidal) Narragansett, Moebus 1974 Fucus vesiculosus leaked less organic carbon Rhode Island than lower intertidal F. vesiculosus after drying Ulva expansa, Prionitis Mild dehydration enhanced Mussel Point, Johnson et al. lanceolata, Iridaea photosynthesis of middle to Pacific Grove, 1974 flaccida, Porphyra high intertidal macroalgae California perforata, Fucus (Iridaea flaccida, Porphyra distichus, Endocladia perforata, Fucus distichus, muricata Endocladia muricata) Pelvetia canaliculata, No correlation between the Kattendijke, Dorgelo 1976 Fucus spiralis, ability to maintain water and Netherland Ascophyllum nodosum, the intertidal levels of the F. vesiculosus, F. species studied serratus, Laminaria saccharina Pelvetia canaliculata, High intertidal P. Port Loy, Isle of Schonbeck Fucus spiralis, canaliculata showed best Cumbrae in the and Norton Ascophyllum nodosum, recovery in photosynthesis Firth of Clyde, 1978 F. serratus after drying Scotland F. serratus transferred to higher intertidal zone grew poorly Pelvetia canaliculata, No correlation between the Great Cumbrae Schonbeck 27
Fucus spiralis, ability to maintain water and Island, Scoland and Norton Ascophyllum nodosum, the intertidal levels of the 1979 F. serratus species studied Hormosira banksii, No correlation between the Auckland, New Dromgoole Scytothamnus australis, ability to maintain water and Zealand 1980 Codium fragile, the intertidal levels of the Carpophyllum species studied maschalocarpum, Ecklonia radiata, Gastroclonium coulteri Desiccation stress determined Monterey Bay, Hodgson the upper limit of distribution California 1981 of Gastroclonium coulteri Pelvetia canaliculata, Photosynthetic activity of Belfast, Ireland Dring and Fucus spiralis, F. High intertidal Fucus spiralis Brown 1982 serratus,F. vesiculosus, recovered faster from Laminaria digitata desiccation Porphyra perforata, Limits of dehydration Pacific Grove, Smith and Rhodoglossum affine, tolerance correlated well with California Berry 1986 Gelidium coulteri, the distribution of species Smithora naiadum studied Porphyra perforata, P. Desiccation ruptured the Pacific Grove, Smith et al. nereocystis organelles of subtidal P. California 1986 nereocystis, but did not damage high intertidal P. perforata Bostrychia arbuscula, Photosynthesis of high Dunedin, South Brown 1987 Apophyloea lyallii intertidal B. arbuscula, A. Island, New Porphyra columbina, lyallii recovered completely Zealand Hormosira banksii, from desiccation but Xiphophora photosynthesis of the lower chondrophylla, intertidal species was Durvillaea willana impaired Gigartina papillata, High intertidal species Diana island, Thomas et al. Entormorpha (Fucus distichus, Pelvestia British 1987 intestinalis, Fucus limitata) showed increased Columbia, distichus, Pelvestia uptake of nitrate and Canada limitata ammonium after desiccation Porphyra linearis Long term desiccation Nathaniya, Lipkin et al. tolerance is essential for the Israeli 1993 survival of Porphyra linearis in high intertidal zone 28
Fucus evanescens Longer emersion resulted in Schoodic Point, Collén and more ROS production Maine Davison 1997 Pelvetia canaliculata, P. canaliculata (high Roscoff and Harker et al. Laminaria saccharina intertidal) showed better Wimereux, 1999 protection of photosystems France when dehydrated Fucus evanescens, F. Low intertidal F. distichus Schoodic Point, Collén and spiralis, F. distichus produced more ROS after Maine Davison desiccation than high 1999a intertidal F. spiralis and F. evanescens Pelvetia canaliculata Prolonged emersion caused Spiddal Pier, Pfetzing et al. irreversible damage to Ireland 2000 photosynthesis Porphyra dentata, Porphyra dentata,the species Nabeta Bay, Izu Abe et al. Monostroma nitidum, occurred at the highest Peninsula, Japan 2001 Gloiopeltis intertidal level, was the only complanata, species showing complete Entromorpha linza, recovery in photosynthesis Ulva pertusa, from desiccation at water Myelophycus simplex, potential at -158 MPa Gracilaria asiatica, Ishige okamurae, Gloiopeltis furcata, Chondrus verrucosus, Ishige sinicola, Petalonia fascia, Sargassum thunbergii, Hizikia fusiformis, Padina arborescens, Boodlea coata Ahnfeltiopsis flabelliformis, Gelidium elegans Stictosiphonia Specimens from lower Brighton Beach, Burritt 2002 arbuscula intertidal zone accumulated Otago, New more hydrogen peroxide and Zealand lipid peroxidation after desiccation Pelvetia canaliculata, Only high intertidal species East Haven, Skene 2004 Fucus sprialis, (Pelvetia canaliculata, Fucus Scotland Ascophyllum nodosum, sprialis, Ascophyllum 29
Porphyra umbilicalis, nodosum, Porphyra F. vesiculosus, umbilicalis,) showed fully Mastocapus stellatus, recover in photosynthesis Palmaria palmata, after 24 hr dehydration and Rhododymenia, 48 hr rehydration palmata, Ulva lactuta, F. serratus Ulva lactuca Subtidal individuals secreted Fort Pierce, Ross and Van more hydrogen peroxide after Florida Alstyne 2007 desiccation than the intertidal individuals Ulva lactuca Emersion under strong light Nanao Island, Zou et al. inhibited photosynthesis in Shantou, China 2007 Ulva lactuca Ascophyllum nodosum, Photosynthesis declined Pointe-Mitis, Lamote et al. Fucus vesiculosus, F. faster in the low intertidal F. Québec, Canada 2007 disticus, disticus than the other two higher intertidal species Ulva lactuca Desiccation reduced Nanao Island, Zou et al. respiration of Ulva lactuca, Shantou, China 2007 especially at a high temperature (33ºC) Porphyra umbilicalis, Nutrient uptake of low Rye New Kim et al. P. leucosticata, P. intertidal P. yezoensis was Hampshaire, 2008 yezoensis more susceptible to emersion Groton, than that of P. umbilicalis, Connecticut, and P. leucosticata Weekapaug, Rhode Island 30
Table 2. Examples of studies on mechanisms of desiccation tolerance in macroalgae.
Species studied Key findings Location References Porphyra sanjuanesis P. sanjuanesis inactivated the Dundarave Sibbald and photochemistry and electron beach, West Vidaver 1987 transport system in high Vancouver, lights to avoid photodamage Canada Fucus spiralis, F. The activities of superoxide Schoodic Collén and evanescens, F. distichus dismutase and ascorbate Point, Davison 1999a persoxidase in less Maine desiccation tolerant F. evanescens were lower than in more stress tolerant F. spiralis Mastocarpus stellatus, The higher intertidal, more Long Cove Collén and Chondrus, crispus stress tolerant Mastocarpus Point, Davison 1999c stellatus had higher levels of Chamberlai ascorbate and β-carotene and n Maine higher activities of catalase glutathion reductatse Pelvetia canaliculata, The high intertidal P. Roscoff and Harker et al. Laminaria saccharia canaliculata had higher Wimereux, 1999 photoprotection capabilities France than L. saccharia Stictosiphonia arbuscula The high intertidal specimens Brighton Burritt 2002 had higher regeneration of Beach, reduced ascorbate. Otago, New Zealand Ulva lactuca The intertidal specimens had Fort Pierce, Ross and Van higher activities of ROS Florida Alstyne 2007 scavenging enzymes. 31
D. Why Porphyra is a good model system of study of desiccation tolerance
The unique habitat, presence of congenerics at different tidal heights, simple construction, and culture ability of Porphyra make it an ideal model for desiccation studies. For example, it takes days for resurrection plants to develop desiccation tolerance, since natural drying occurs slowly on land (Black & Pritchard 2002). However, the intertidal algae Porphyra is emersed twice a day and experiences a drastic change of water content during emersion. This is very different from what happens in land plants and it seems likely that Porphyra may possess unique mechanisms in desiccation tolerance.
Because of the possibility of these unique mechanisms, it is informative to use a
“compare and contrast” approach between related organisms with different desiccation tolerances. Porphyra has high and low intertidal species (Tajiri & Aruga 1984; Kornmann
& Sahling 1991; Lipkin et al. 1993; Davison & Pearson 1996; Abe et al. 2001) and is thus a good candidate for such a strategy.
The simple construction of Porphyra is another advantage of this system. Its gametophytes have a sheet-like morphology that are either 1 or 2 cells thick. Such a simple structure prevents uneven water loss during dehydration, so the results are not confounded by structural protection. Its thin, planar morphology also allows for very rapid and complete desiccation, which is not commonly seen in resurrection plants.
Another advantage of Porphyra is that it can be easily cultivated in the laboratory.
Because one stress can enhance tolerance to other stresses (Davison & Pearson 1996), it can be helpful to culture specimens in controlled environments to minimize this effect. In 32 addition, because of its economic importance, Porphyra has been widely cultivated and studied biochemically and physiologically. Most importantly, there are reports describing responses of Porphyra to environmental stresses, such as high light (Bose et al. 1988), osmotic stress (Wiencke & Lõuchli 1980; Smith et al. 1986) and emersion (Tajiri &
Aruga 1984; Gao & Aruga 1987). In addition, much research has been published on establishing cultures of various Porphyra species, characterizing its development, and controlling its life history (Kapraun & Luster 1980; L. Chen 1989; Mitman & Meer 1994;
Kitade et al. 1998; Yamazaki et al. 1998). Since some Porphyra species are consumed as food, its chemical composition has been analyzed in considerable detail. For example, information on its carbohydrate and PUFA composition is well known (Su & Hassid
1962; Craigie et al. 1968; McLachlan et al. 1972; Kayama et al. 1985; Araki et al.
1986; Araki et al. 1987; Karsten et al. 1993; Graham 2003). Although no nuclear genome of any Porphyra species has been completely sequenced, the genomes of the chloroplast and mitochondrion of P. purpurea have been sequenced (Reith & Munholland 1993;
Burger et al. 1999) and some nuclear genes have been cloned and characterized (Stiller &
Hall 1998; Kitade et al. 2002; Fukuda et al. 2003). In addition, more than 10,000 EST sequences of P. yezoensis are available publicly (Nikaido et al. 2000; Asamizu et al.
2003). Several groups have also attempted to transform Porphyra species and observed transient expression of reporter genes (Kubler et al. 1994; Okauchi & Mizukami 1999;
Liu et al. 2003; Mizukami et al. 2004). 33
E. Objective
The overall goal of this study is to determine the mechanism(s) responsible for desiccation tolerance in Porphyra umbilicalis and to elucidate the protecting molecules by comparing P. umbilicalis, a desiccation resistant species, to P. yezoensis, a desiccation sensitive species. Preliminary data show that while both P. umbilicalis and P. yezoensis lose water at similar rates, only P. umbilicalis survives extreme desiccation.
Since there have not been many reports on extreme desiccation tolerance in seaweeds, the first part of this study will be the characterization of the responses of both species to desiccation. By doing so, the primary sites of cell damage in P. yezoensis can be identified. Next, this study will test the aforementioned tolerance mechanisms common in resurrection plants and seeds, namely ROS defense, repression of membrane phase transition and vitrification, to determine if these mechanisms are also present in macroalgae. To further validate the proposed mechanism, the last part of this study will be to identify the protecting molecules in the identified mechanism.
In summary, the specific goals of this study are as follows:
1. to characterize the effects caused by desiccation on photosynthesis, respiration,
and cellular structures in both species,
2. to test whether ROS defense, repression of membrane lipid phase transition and
vitrification is the tolerance mechanism in P. umbilicalis,
3. to determine the key molecules in the identified protection mechanism, such as
antioxidants and antioxidative enzymes for ROS defense, or carbohydrates for
lipid phase transition repression or vitrification. 34
The results of this study are presented in two chapters in a manuscript format.
Chapter Two, submitted to Journal of Phycology, describes the impact of desiccation on photosynthesis, respiration, and membrane integrity. Furthermore, the role ROS defense in the difference between the two species will be also investigated. Chapter Three, submitted to Proceedings of the National Academy of Sciences, describes the role of repression of membrane phase transition and vitrification, which have not been covered by other studies on algal desiccation tolerance. 35 Chapter 2
THE ROLE OF ROS DEFENSE IN DESICCATION TOERANCE OF
PORPHYRA UMBILICALIS
Yen-Chun Lin and Donald Cheney*
Department of Biology, Northeastern University, 360 Huntington Avenue,
Boston, Massachusetts, 02115
*: Corresponding author: Donald Cheney. Address: 134 Mugar Hall,
Department of Biology, Northeastern University, 360 Huntington Avenue,
Boston, Massachusetts, 02115. E-mail: [email protected]; telephone number: 617-373-2489.
Abbreviations: ROS: reactive oxygen species; SOD: superoxide dismutase;
DW: dry weight; FW: fresh weight. 36 Abstract
Zonation of seaweeds is a widely observed phenomenon on rocky coastlines, and seaweeds that occurring in the high intertidal zone have been shown to more stress tolerant. Reactive oxygen species (ROS) defense has been believed to be the reason for better stress tolerance in the intertidal seaweeds, however, little is known about the exact mechanisms. To investigate the role of ROS defense in desiccation tolerance of the seaweed Porphyra, we conducted a series of experiments with lab cultured high intertidal
P. umbilicalis and low intertidal P. yezoensis without strong light. The controlled experimental conditions and materials eliminated the influences of other environmental stresses. The results showed that both species experienced rapid dehydration in the experiments, but P. umbilicalis was much more desiccation tolerant than P. yezoensis.
ROS defense could not explain the difference in the tolerance between the two species as
P. yezoensis had higher ROS scavenging enzyme activity and antioxidant contents, and no sign of higher ROS damage to the membrane after desiccation. Our findings suggest that the key mechanism for extreme desiccation tolerance should be other than ROS defense ,and the role of the key mechanism, reduced molecular mobility, will be described elsewhere. 37 Introduction
The zonation of intertidal seaweeds on rocky coastlines has been noticed for over one hundred years. The differential tolerance to desiccation and other stresses of intertidal seaweeds has long been believed to be a cause of the distribution of intertidal seaweeds
(Baker 1909; Baker 1910; Zaneveld 1937; Zaneveld 1969). Because of the tidal movement, intertidal seaweeds experience repetitive emersion and submersion daily, and seaweeds in higher intertidal zones are exposed to air for longer than those in lower intertidal zones during each tidal cycle. When seaweeds are exposed to air, they face multiple environmental stresses such as desiccation, freezing, strong light, and nutrient limitation (Davison & Pearson 1996). Therefore, it is not surprising that seaweeds growing in the high intertidal zone have been found to recover better from drying than those growing at low intertidal zone (Dring & Brown 1982; Brown 1987; Abe et al.
2001).
Most studies on the tolerance of intertidal seaweed to environmental stresses have suggested that ROS defense is correlated with the better tolerance of higher intertidal species or populations. For example, high intertidal Fucus spiralis produced less ROS than low intertidal F. distichus when they were frozen or dried (Collén & Davison
1999a). Similarly, high intertidal populations of Stictosiphonic arbuscula had lower ROS accumulation and higher ROS scavenging enzyme activities (Burritt et al. 2002). The major source of ROS in plants is impaired photosynthesis under stress, and the first free radical produced in the sequential reduction of oxygen is superoxide (Smirnoff 1993). 38 Superoxide dismustases (SOD) catalyzes dismutation of superoxide to hydrogen peroxide and oxygen. Hydrogen peroxide can also be produced by reduction of superoxide. The hydrogen peroxide is then broken down to water by catalase in peroxysomes and glyoxysomes. In the chloroplast and cytoplasm, this function is carried out by ascorbate peroxidase. Ascorbate is the antioxidant used in this pathway and it is regenerated by
NAD(P)H-dependent monodehydroascorbate reductase, or in a reaction catalyzed by dehydroascorbate reductase using glutathione as electron donor. The oxidized glutathione is reduced to GSH by NADP-dependent glutathione reductase (Smirnoff 1993). If not removed, superoxide and hydrogen peroxide can form highly reactive hydroxyl radicals that initiate lipid peroxidation and hydroxylates nucleic acids (Kranner & Lutzoni 1999).
The aim of this study is to investigate the role of ROS defense specifically in desiccation tolerance in the intertidal red alga Porphyra. In the above studies regarding the relationship between ROS defense and algal desiccation tolerance, light of high intensity was applied to specimens during the experiments (Collén & Davison 1999a;
Burritt et al. 2002). The purpose of using bright light during drying was probably to mimic field conditions. However, the seaweeds in such experimental conditions were actually experiencing dual stresses because strong light alone is enough to induce ROS accumulation (Collén & M. Pedersén 1996; Choo et al. 2004). Furthermore, the tested seaweeds in these studies were collected from field and might have adapted to multiple stresses before collection (Davison & Pearson 1996). Acclimation to multiple stresses confounds elucidation of mechanisms specific to a single stress as it would be difficult to 39 decide the actual role of tolerance mechanism against a certain stress. In this study, we tested laboratory cultured P. umbilicalis and P. yezoensis to avoid the influence other environmental stresses prior to and during the experiments. Our findings demonstrate that the high intertidal P. umbilicalis is more tolerant to desiccation that the low intertidal P. yezoensis, and that ROS defense cannot explain the difference in tolerance between the two species. 40 Materials and Methods
Plant Material
Porphyra umbilicalis was originally collected from Cape Cod Canal,
Massachusetts (41°46'N, 70°29'W) in June 2004. Wild type P. yezoensis (strain U51) was collected from Hokkaido, Japan in 2000. Both species were grown, maintained and reproduced asexually via monospores at 15°C in filter-sterile seawater supplemented with
1% ESS (Kitade et al. 1996) in aerated flasks at a light intensity of 120 μmol photons · m-
2· s-1 on a 12:12 (L:D) cycle. Both species have been propagated for several generations continuously in seawater, and no prior desiccation was applied to the algae before experiments.
Desiccation and Measurement of Relative Water Content (RWC)
Water loss was measured by weighting algae before and after desiccation. Algae were desiccated in a Baker Edgegard EG 6320 hood at 23 to 25°C, 30 to 35% RH. The rate of air flow was 30.5 meters per minute. The light intensity was 30 µmol photons · m-2
· s-1. RWC was calculated as follows: RWC = 100 * (W(t) – DW) / (FW – DW), where
W(t) is the weight of specimen after t hour desiccation, FW is the fresh weight of the specimen and DW is the dry weight of specimens dried in oven for 72 h at 110°C.
Oxygen evolution measurement
Photosynthetic activity was estimated by the rate of oxygen evolution. Oxygen 41 released by algae was measured by a Clark type electrode at room temperature.
Experimental specimens were desiccated for specified time length and rehydrated in sea water for 10 minutes before the measurement. The assay was carried out in seawater at a light intensity of 500 μmol photons · m-2 · s-1.
Respiration activity assay
The respiration activity of algae was measured by 2,3,5-triphenyltetrazolium chloride (TTC), which was adapted from Nam et al. (Nam et al. 1998). TTC is a water- soluble compound that diffuses into cells freely, and it is reduced by succinate dehydrogenase to a red and insoluble triphenylformazan (Roberts 1951; Nachlas et al.
1960). Desiccated samples were rehydrated in sterile sea water for 10 minutes before the assay. One hundred milligrams of algae were incubated in 0.8% TTC in sea water at 15
°C for one hour in the dark. After incubation, each specimen was rinsed in sterile sea water three times. The specimen was then blotted dry and ground in liquid nitrogen. One hundred milligrams of powder was extracted in 1 ml 95% ethanol at 100°C for 15 minutes. After centrifuging at 13,000 g for 1 minute, the supernatant was diluted 10 times with ethanol before measuring absorbance at 545 nm. Relative viability was calculated as follows: (A545 of experimental sample – A545 of dead tissue) / ( A545 of control specimen -
A545 of dead tissue) * 100. Dead tissue was obtained by boiling the algae at 100°C for 5 minutes. 42 Amino Acid Leakage Assay
Plasma membrane damage was assayed by an amino acid leakage method described by Rosen (1957). Specimens with a fresh weight of 0.5g to 1g were desiccated and then rehydrated in 10 ml filtered seawater for 10 minutes. The concentration of released amino acids in the sea water was measured with ninhydrin stain using glutamate as standard.
Superoxide dismutase (SOD) activity assay
SOD activity of algal samples was measured by SOD Assay Kit from Sigma.
Algal samples were ground in liquid nitrogen and extracted in 100mM potassium phosphate buffer, pH 7.6. After centrifugation, the protein concentration was determined with Bio-Rad Protein Assay and was adjusted to 0.5 mg/ml before measuring SOD activity.
Ascorbate concentration assay
The assay of ascorbate was described by Gillespie and Ainsworth (Gillespie &
Ainsworth 2007). In brief, ascorbate reduced ferric ion to ferrous ion, which formed a red complex with 2, 2'-bipyridyl. Speciments were ground in liquid nitrogen, and approximately 250 mg powder was used in each reaction. Absorbance at 525nm was recorded and the concentration of ascorbate in the extract was calculated from a standard curve of the ascorbate standards. Desiccated samples were dried in a hood for 3 hours and 43 rehydrated in sterile sea water for 10 minutes before the assay.
Lipid peroxidation assay
The detection of lipid peroxidation was based on the oxidation of Fe2+ to Fe3+ by lipid peroxide, which then reacts with xylenol orange to form a measurable chromophore
(Jiang et al. 1991). Desiccated samples were dried in hood for 3 hours and rehydrated in sterile sea water for 10 minutes before the assay. Specimens were ground in liquid nitrogen, and 100 mg powder was extracted with 1 ml methanol with 4mM butylated hydroxytoluene (BHT) for 5 minutes at room temperature. The extract was diluted 10 fold, and 500 µl diluted extract was added to 500 µl reagent: 4mM BHT, 100 µM xylenol orange, 250 µM ammonium ferrous sulfate, 25 mM H2SO4 in methanol. Absorbance at
560nm was recorded after incubation at room temperature for 30 minutes, and the peroxide concentration was calculated from a standard curve of hydrogen peroxide standards. The concentration of lipid peroxide was expressed as µmol equivalent H2O2 per gram fresh weight.
Statistical analyses
Statistical analyses were performed using the SAS system for Windows v 9.2. The data were analyzed by repeated-measures ANOVAs with general linear models. The results of effect of light on membrane leakage (Fig. 5) and effect of desiccation on ascorbate contents (Fig. 7) were transformed by log (1+ value) before analyses to meet the 44 assumption of ANOVA. 45 Results
Effects of desiccation on Porphyra umbilicalis and Porphyra yezoensis
While P. umbilicalis (high intertidal) and P. yezoensis (low intertidal) grow at different tidal heights in the field, results showed they lost water at similarly rates (Table
1). The relative water content of both species decreased in a non-linear pattern: 80% of water was lost in the first 30 minutes of drying, and relative water content dropped to approximately 5% in the next 30 minutes. Both species did not lose any more weight after two more hours of drying and were considered to have reached equilibrium with the atmosphere at that point (Fig. 1).
Although P. umbilicalis and P. yezoensis lost water at similar rates, the two species showed very different photosynthetic and respiration responses to the water loss.
For example, photosynthetic activity of the rehydrated P. yezoensis decreased as desiccation stress increased and approached zero after three hours of drying (Fig. 2).
Although the oxygen evolution rate of P. umbilicalis was only about 50% of that of P. yezoensis prior to desiccation, it remained similar throughout the three hours of drying
(Fig. 2). The relative respiration rate during drying showed a similar difference between the species. The respiration rate of P. yezoensis increased after 30 minutes of drying but decreased sharply to zero after two hours of drying. On the other hand, the respiration rate of P. umbilicalis showed no significant difference in the three hours of drying. In addition to the difference in physiological responses, the better tolerance to desiccation in 46 P. umbilicalis was also observed in membrane integrity. The amino acid leakage in P. yezoensis increased rapidly after only 30 minutes of drying, and the leakage became more severe with increasing length of drying time. The membrane integrity of P. umbilicalis was not compromised by desiccation, as amino acid leakage remained insignificant even when the specimens reached the equilibrium with the atmosphere of 35% RH (Fig. 4).
Porphyra yezoensis had higher ROS defense than Porphyra umbilicalis
Desiccating P. yezoensis and P. umbilicalis in the dark did not alleviate or aggravate the damage to membrane integrity (Fig. 5, Table 1). The level of amino acid leakage of P. yezoensis and P. umbilicalis desiccated in the dark was (mean ± SE) 5.18 ±
0.19 and 0.16 ± 0.03 µmol / g FW, respectively, and the level of leakage of P. yezoensis and P. umbilicalis desiccated in light (approximate 30 µE/m2/s ) was (mean ± SE) 4.69 ±
1.74 and 0.17 ± 0.03 µmol / g FW, respectively. This observations indicates that the presence of light does not promote membrane damage. While the activity of superoxide dismutase of P. umbilicalis slightly decreased after one hour of drying, that of P. yezoensis was not affected by the increasing desiccation stress (Fig. 6). The data also showed that desiccation sensitive P. yezoensis had higher superoxide dismutase activity than desiccation tolerant P. umbilicalis prior to and throughout the three hours of drying
(Fig. 6). The ascorbate contents in these two species had a pattern similar to that observed for superoxide dismutase acitivity. P. yezoensis had a much higher ascorbate content than
P. umbilicalis, and the level of ascorbate in P. yezoensis did not decrease after 47 desiccation, while there was a small decrease in the ascorbate content in P. umbilicalis after desiccation (Fig. 7). The peroxide assay showed that lipid peroxide levels decreased in P. yezoensis after desiccation but were not significantly different in P. umbilicalis after three hours of drying (Fig. 8). In other words, lipid peroxidation did not happen when P. yezoensis was badly damaged by desiccation. 48 Discussion
Terrestrial vascular plants have evolved effective strategies to avoid water loss, such as a water-proof cuticle and the closing of stomata upon water stress. Plants grown in arid areas even have special morphologies to reduce water evaporation. For example,
Borya nitida, a resurrection plant growing in the Western Australia, has needle-shaped leaves to minimize the surface area, and its stems are covered by overlapping scales (Gaff
& Churchill 1976). Unlike terrestrial plants, P. umbilicalis and P. yezoensis do not have structural protection against water loss because they are sheets consisting of one cell layer. Our data on water loss clearly showed that both species cannot retain water when they are exposed to air, and P. umbilicalis and P. yezoensis experienced desiccation after merely three hours of air drying (Fig. 1, Table 1). In past work, it has been shown that the rate of water loss does not relate to the ability to tolerate desiccation or to the intertidal distribution of different algal species (Dorgelo 1976; Dromgoole 1980). This study reinforced this past observation by showing that P. umbilicalis and P. yezoensis had very distinct physiological responses to desiccation despite their similar rates of water loss.
The difference in desiccation tolerance between the two species was demonstrated by the assessment of photosynthetic and respiratory activity after 10 minutes of rehydration (Fig. 2, Fig. 3 and Table 1). The fastest decrease in photosynthetic and respiratory activities of P. yezoensis occurred in the first hour of drying, which corresponded to the fastest decrease in water content during the drying (Fig. 1). After one hour of drying, the decrease in oxygen evolution of P. yezoensis became lower as the rate 49 of water loss leveled off, and reached 6% of the initial rate of oxygen evolution after 3 hours of drying (Fig. 2). The change in respiratory activity of P. yezoensis was not detectable when the water activity in this species reached equilibrium with atmosphere.
The increase in respiratory activity after the first 30 minutes of drying might indicate the species managed to repair the damage, but further drying caused damage beyond repair.
In addition to the physiological damage, desiccation also compromised the membrane integrity of P. yezoensis. Similar to the damage to respiration and photosynthesis, the leakage of amino acids raised quickly in the first hour of drying (Fig. 4). These three assays clearly showed that P. yezoensis was sensitive to desiccation. On the other hand,
P.umbilicalis showed extraordinary desiccation tolerance. Its photosynthesis, respiration and membrane integrity were not affected by desiccation (Fig. 2, Fig. 3 and Fig. 4).
Although resurrection plants can also survive desiccation, they cannot survive desiccation that completes in 24 hours (Gaff & Churchill 1976; Schwab et al. 1989; Sherwin &
Farrant 1996). Lower plants like Tortula ruralis can be dried in a few hours, but such rapid desiccation caused more serious damage to them (Bewley et al. 1993; Melvin J
Oliver et al. 2000). In this study, P. umbilicalis was desiccated in 2 hours, which was faster than any resurrection plants can tolerate. Furthermore, its photosynthesis and respiration recovered within 10 minutes after rehydration, and the amino acid leakage was not significantly different from control samples. Since the P. umbilicalis used in this study was cultured in the lab and had not ever been exposed to desiccation stress before experiments, its tolerance to desiccation is constitutive. This constitutive and robust 50 tolerance to desiccation is crucial for the survival of P. umbilicalis in the high intertidal zone, because it loses water rapidly while exposed to air and it is only submerged for several hours before the arrival of the next low tide. The intertidal environment does not allow a long induction time for the establishment of desiccation tolerance or extensive repair after a previous drying event.
Desiccation tolerance in algae has long been associated with ROS defense. For example, higher intertidal Fucus spiralis produced less ROS and were more resistant to oxidative stress than the lower Fucus species when they are desiccated (Collén &
Davison 1999a). The higher intertidal algal species or population had been found to have higher antioxidant contents and higher ROS scavenging enzyme activities (Collén &
Davison 1999b; Burritt et al. 2002). However, the data in our study showed that ROS defense could not explain the difference in desiccation tolerance between P. umbilicalis and P. yezoensis.
Impaired photosynthesis during dehydration is a major source of ROS, especially when the plants are exposed to light (Smirnoff 1993). If the observed damage of P. yezoensis was caused by ROS, desiccation of this species in the dark should have alleviated the damage significantly. However, the data showed that the presence of light
(30 µmol photons · m-2 · s-1) did not affect the level of amino acid leakage after desiccation, and therefore, ROS, at least from photosynthesis, were not the primary reason for the damage in this study (Fig. 5). To further explore the role of ROS defense in the difference in desiccation tolerance between the two Porphyra species, we compared 51 the activity of SOD and ascorbate level of both species in the 3-hour desccation (Fig. 6 and Fig. 7). SOD was selected because its activity has been shown to be highly correlated with desiccation tolerance (Seel et al. 1992). To our surprise, P. umbilicalis did not have higher SOD activity, and the activity of this enzyme in P. yezoensis did not decrease with the increase of desiccation stress (Fig. 6). This observation indicated that the removal of superoxide, the first free radical of successive univalent reduction of oxygen (Smirnoff
1993), was not the key to the differential desiccation tolerance between the two Porphara species.
Hydrogen peroxide is produced by the reduction of superoxide, and the conversion of superoxide to hydrogen peroxide by SOD. Ascorbate peroxidase is the primary enzyme to remove hydrogen peroxide (Collén & Pedersén 1996), probably because catalase has a low affinity to hydrogen peroxide (Scandalios et al. 1972). The detoxification of hydrogen peroxide by ascorbate peroxidase requires ascorbate as the electron donor, and it has been shown that the level of ascorbate decreases rapidly after desiccation in red alga Stictosiphonia arbuscula grown at high intertidal zone and in the resurrection plant Myrothamnus flabellifolia after dehydration (Burritt et al. 2002;
Kranner et al. 2002). Our data showed that the ascorbate content in P. umbilicalis decreased slightly after desiccation, but no such decrease was observed in P. yezoensis after desiccation (Fig. 7). Therefore, although the desiccation treatment used in this study caused severe damage to P. yezoensis, it did not increase oxidative stress and the damage was unlikely to be the result of ROS accumulation. 52 This conclusion is further supported by the change of lipid peroxidation level after desiccation. The level of lipid peroxidation was measured because the amino acid leakage data (Fig. 4) and TEM observations (Liu et al. submitted) show that membranes in P. yezoensis were badly damaged, while those in P. umbilicalis was not damaged. One common cause of membrane damage due to ROS is lipid peroxidation. However, we did not find a significant increase of lipid hydroperoxide after desiccation in either species.
This result suggests that the membrane damage in P. yezoensis after desiccation is not caused by ROS.
It appeared counter-intuitive that the desiccation tolerant P. umbilicalis had lower ascorbate content and lower SOD activity than the desiccation sensitive P. yezoensis, but this observation can be explained by the low metabolic rate of this species. ROS scavenging systems are found in every aerobic organisms because ROS are constantly being generated in various metabolic pathways. It is also known that the desiccation tolerant plant species are generally less productive than the desiccation sensitive species
(Bates 1997; Alpert 2006). The lower metabolism in P. umbilicalis was demonstrated by its lower photosynthesis activity (Fig. 2). We think that high intertidal algal species showed higher antioxidant levels and ROS scavenging enzyme activities in the past studies because the specimens were collected from field before the experiments, and they might have been subject to other environmental stresses, such as high light intensities or extreme temperatures (Collén & Davison 1999b; Burritt et al. 2002). This could contribute to higher ROS defense because these stresses have also been shown to increase 53 the concentrations of ROS in algae (Collén & M. Pedersén 1996; Collén & Davison
1999a). The decreased lipid peroxidation level in P. yezoensis after can also be explained by the lower metabolism after desiccation. The electron transfer chains in photosynthesis and respiration are the important sources of ROS, but the activities of these two metabolic pathways in P. yezoensis were reduced to nil after desiccation (Fig. 2 and Fig.
3). Since the major sources of ROS were shut down after desiccation in P. yezoensis, the level of lipid peroxidation in this species immediately after desiccation would be expected to be lower.
We believe the reasons why ROS defense plays an insignificant role in this study are rapid dehydration and elimination of other environmental stresses. The generation of
ROS is promoted by an imbalanced metabolism at higher water contents (Alpert 2000), but not by a shutdown of metabolism, as Collén and Davison showed that Fucus at lower water contents produced less ROS (Collén & Davison 1999a). Both Porphyra species in this study lost 95% of their water in one hour of drying (Fig. 1), and therefore, the window for ROS production was very narrow. Furthermore, all the assays in this study were carried out in a short time after rehydration to minimize ROS propagation. The other factor promoting ROS production is high light intensity. High light intensities induced hydrogen peroxide production in a study of Ulva rigida (> 100 µmol/m2/s)
(Collén & M. Pedersén 1996), and the effect of desiccation on the production of ROS in
Fucus species was studied in the presence of strong light (1600 µmol/m2/s) (Collén &
Davison 1999a). The dehydration carried out in this study was at a much lower light 54 intensity (30 µmol/m2/s), which did not aggravate membrane damage (Fig. 5, Table 1).
ROS defense is definitely required for the survival of intertidal algae in the field as they face multiple stresses during low tide (Davison & Pearson 1996). However, the multiple stresses challenging the intertidal algae also confound the elucidation of mechanisms specifically for desiccation tolerance. Because the culturability and simple construction of Porphyra allow for the elimination of other stresses and rapid rates of desiccation / rehydration, we think Porphyra provides an ideal system to study desiccation tolerance of intertidal seaweed. With this system, we have been able to identify that reduced molecular mobility in the cytoplasm of P. umbilicalis is the key to superior desiccation tolerance of this species (Liu et al. submitted). 55 References
Abe, S. et al., 2001. The cellular ability of desiccation tolerance in Japanese intertidal seaweeds. Botanica Marina, 44(2), 125-131.
Alpert, P., 2006. Constraints of tolerance: why are desiccation-tolerant organisms so small or rare? J Exp Biol, 209(9), 1575-1584.
Alpert, P., 2000. The discovery, scope, and puzzle of desiccation tolerance in plants. Plant Ecology, 151(1), 5-17.
Baker, S.M., 1909. On the causes of the zoning of brown seaweeds on the seashore. New Phytologist, 8(5/6), 196-202.
Baker, S.M., 1910. On the Causes of the Zoning of Brown Seaweeds on the Seashore. II. The Effect of Periodic Exposure on the Expulsion of Gametes and on the Germination of the Oospore. New Phytologist, 9(1/2), 54-67.
Bates, J.W., 1997. Effects of Intermittent Desiccation on Nutrient Economy and Growth of Two Ecologically Contrasted Mosses. Ann Bot, 79(3), 299-309.
Bewley, J.D., Reynolds, T.L. & Oliver, M.J., 1993. Evolving strategies in the adaptation to desiccation. In Close, T. J. & Bray, E. A., eds. Plant responses to cellular dehydration during environmental stress; Current topics in plant physiology: American society of plant physiologists series. pp. 193–201.
Brown, M., 1987. Effects of desiccation on photosynthesis of intertidal algae from a southern New Zealand shore. Botanica Marina, 30, 121-127.
Burritt, D.J., Larkindale, J. & Hurd, C.L., 2002. Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation. Planta, 215(5), 829- 38.
Choo, K., Snoeijs, P. & Pedersén, M., 2004. Oxidative stress tolerance in the filamentous green algae Cladophora glomerata and Enteromorpha ahlneriana. Journal of Experimental Marine Biology and Ecology, 298(1), 111-123.
Collén, J. & Davison, I., 1999a. Reactive oxygen production and damage in intertidal Fucus spp. (Phaeophyceae). Journal of phycology, 35, 54-61.
Collén, J. & Davison, I., 1999b. Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus. Plant Cell 56 and Environment, 22(9), 1143-1151.
Collén, J. & Pedersén, M., 1996. Production, scavenging and toxicity of hydrogen peroxide in the green seaweed Ulva rigida. European Journal of Phycology, 31(3), 265-271.
Davison, I. & Pearson, G., 1996. Stress tolerance in intertidal seaweeds. Journal of Phycology, 32, 197-211.
Dorgelo, J., 1976. Intertidal fucoid zonation and desiccation. Aquatic Ecology, 10, 115- 122.
Dring, M. & Brown, F., 1982. Photosynthesis of Intertidal Brown Algae During and After Periods of Emersion: A Renewed Search for Physiological Causes of Zonation. Marine Ecology Progress Series, 8, 301-308.
Dromgoole, F.I., 1980. Desiccation resistance of intertidal and subtidal algae. Bot. Mar, 23, 149-159.
Gaff, D. & Churchill, D., 1976. Borya nitida Labill.—An Australian Species in the Liliaceae With Desiccation-tolerant Leaves . Aust. J. Bot., 24(2), 209-224.
Gillespie, K.M. & Ainsworth, E.A., 2007. Measurement of reduced, oxidized and total ascorbate content in plants. Nat. Protocols, 2(4), 871-874.
Jiang, Z., Woollard, A. & Wolff, S., 1991. Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric method. Lipids, 26(10), 853-6.
Kitade, Y., Yamazaki, S. & Saga, N., 1996. A Method for extraction of high molecular weight DNA from the macroalga Porphyra yezoensis (Rhodophyata). Journal of Phycology, 32(3), 496-498.
Kranner, I & Lutzoni, F, 1999. Evolutionary Consequences of Transition to a Lichen Symbiotic State and Physiological Adaptation to Oxidative Damage Associated with Poikilohydry. In H. R. Lerner, ed. Plant Responses to Environmental Stresses from Phytohormones to Genome Reorganization. Marcel Dekker, pp. 591-628.
Kranner, I. et al., 2002. Revival of a resurrection plant correlates with its antioxidant status. Plant Journal, 31(1), 13-24. 57 Nachlas, M.M., Margulies, S.I. & Seligman, A.M., 1960. Sites of Electron Transfer to Tetrazolium Salts in the Succinoxidase System. J. Biol. Chem., 235(9), 2739- 2743.
Nam, B. et al., 1998. Quantitative viability of seaweed tissues assessed with 2,3,5- triphenyltetrazolium chloride. Journal of applied phycology, 10, 31-36.
Oliver, M.J., Tuba, Z. & Mishler, B.D., 2000. The evolution of vegetative desiccation tolerance in land plants. Plant Ecology, 151(1), 85-100.
Roberts, L.W., 1951. Survey of Factors Responsible for Reduction of 2,3,5- Triphenyltetrazolium Chloride in Plant Meristems. Science, 113(2946), 692-693.
Rosen, H., 1957. A modified ninhydrin colorimetric analysis for amino acids. Archives of Biochemistry and Biophysics, 67, 10-15.
Scandalios, J.G., Liu, E.H. & Campeau, M.A., 1972. The effects of intragenic and intergenic complementation on catalase structure and function in maize: a molecular approach to heterosis. Archives of Biochemistry and Biophysics, 153(2), 695-705.
Schwab, K.B., Schreiber, U. & Heber, U., 1989. Response of photosynthesis and respiration of resurrection plants to desiccation and rehydration. Planta, 177(2), 217-227.
Seel, W.E., F. Hendry, G.A. & Lee, J.A., 1992. Effects of Desiccation on some Activated Oxygen Processing Enzymes and Anti-Oxidants in Mosses. J. Exp. Bot., 43(8), 1031-1037.
Sherwin, H.W. & Farrant, J.M., 1996. Differences in rehydration of three desiccation- tolerant angiosperm species. Ann Bot, 78(6), 703-710.
Smirnoff, N., 1993. Tansley Review No. 52: The role of active oxygen in the response of plants to water deficit and desiccation. New Phytologist, 125(1), 27-58.
Zaneveld, 1969. Factors controlling the delimitation of littoral benthic marine algal zonation. Amer. Zool., 9(2), 367-391.
Zaneveld, J.S., 1937. The Littoral Zonation of Some Fucaceae in Relation to Desiccation. Journal of Ecology, 25(2), 431-468. 58
Figure 1. Mean (±1 SE) relative water content of P. umbilicalis and P. yezoensis during a
3 hour desiccation. ANOVA: time * species effect, P > 0.05. H: high intertidal species; L: low intertidal species. 59
Figure 2. Mean (±1 SE) rate of oxygen evolution of P. umbilicalis and P. yezoensis during a 3 hour desiccation (ANOVA, time*species effect, P < 0.01). H: high intertidal species;
L: low intertidal species. 60
Figure 3. Mean (±1 SE) relative respiratory activity of P. umbilicalis and P. yezoensis during a 3 hour desiccation (ANOVA, time*species effect, P < 0.01). H: high intertidal species; L: low intertidal species. 61
Figure 4. Mean (±1 SE) amino acid leakage of P. umbilicalis and P. yezoensis during a 3 hour desiccation (ANOVA, time*species effect, P < 0.01). H: high intertidal species; L: low intertidal species. 62
Figure 5. Mean (±1 SE) amino acid leakage of P. yezoensis and P. umbilicalis after a 3 hour desiccation in light and dark (ANOVA, light*species effect, P > 0.05). H: high intertidal species; L: low intertidal species. 63
Figure 6. Mean (±1 SE) superoxide dismutase activity of P. umbilicalis and P. yezoensis during a 3 hour desiccation (ANOVA, time*species effect, P < 0.01). H: high intertidal species; L: low intertidal species. 64
Figure 7. Mean (±1 SE) ascorbate contents of P. umbilicalis and P. yezoensis after a 3 hour desiccation in light and dark (ANOVA, hydration*species effect, P < 0.01). 65
Figure 8. Mean (±1 SE) peroxide concentration of P. umbilicalis and P. yezoensis after a 3 hour desiccation (ANOVA, hydration*species effect, P < 0.01). 66 Table 1. ANOVA table for tests on relative water contents during desiccation, effects of desiccation on photosynthesis, effects of desiccation on respiration, effects of desiccation on amino acid leakage, effects of light on amino acid leakage, effects of desiccation on superoxide dismutase (SOD) activities, effects of desiccation on ascorbate contents and effects of desiccation on peroxide concentrations. 67 df Sum of squares Mean square F Relative water contents during desiccation speciesa 1 0.11 0.11 0.00 timeb 3 2737.21 912.40 26.43 ** time*species 3 69.82 23.28 0.67
Effect of desiccation on photosynthesis speciesa 1 0.01 0.01 0.26 timec 4 1.47 0.37 21.18 ** time*species 4 1.50 0.38 21.66 **
Effects of desiccation on respiration speciesa 1 2602.04 2602.04 7.33 timec 4 26411.08 6602.77 63.27 ** time*species 4 33720.28 8430.07 80.77 **
Effect of desiccation on amino acid leakage speciesa 1 48.27 48.27 16.23 * timec 4 25.70 6.43 13.80 ** time*species 4 21.62 5.40 11.61 **
Effect of light on amino acid leakage speciesa 1 1.47 1.47 331.16 ** lightd 1 0.0016 0.0016 0.37 light*species 1 0.0019 0.0019 0.44
Effect of desiccation on SOD activities speciesa 1 3579.58 3579.58 661.37 ** timec 4 367.44 91.86 44.70 ** time*species 4 124.58 31.15 15.15 **
Effect of desiccation on ascorbate contents speciesa 1 5.36 5.36 3077.45 ** hydratione 1 0.05 0.05 28.3 ** hydration*species 1 0.04 0.04 24.98 **
Effect of desiccation on peroxide concentrations speciesa 1 15552.00 15552.00 1.64 hydratione 1 115248.00 115248.00 12.15 ** hydration*species 1 241968.00 241968.00 25.51 ** 68 aPorphyra umbilicalis and P. yezoensis b0.5, 1, 2, and 3 hours c0, 0.5, 1, 2, and 3 hours d0 and 30 µmol photons · m-1 · s-1 econtrol and rehydrated
*: P < 0.05; **: P < 0.01 69 Chapter 3
How Intertidal Seaweeds Dry But Don’t Die: The Mechanism of
Desiccation Tolerance in Porphyra
Yen-Chun Liua, Jamie Lawtonb, David Budilb, Milos Miljkovicb, Jonathan Wonga, Andrew Carya, and Donald Cheneya* a: Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts, 02115. b: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts, 02115.
*: Corresponding author: Donald Cheney. Address: 134 Mugar Hall, Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts, 02115. E-mail: [email protected]; telephone number: 617-373-2489.
Abbreviations: DW: dry weight; EPR: electron paramagnetic resonance; FTIR: Fourier transform infrared spectroscopy; LEA: late embryogenesis abundant; ROS: reactive oxygen species; TEM: transmission electron microscopy. 70 Abstract
Intertidal macroalgae (seaweeds) experience daily far more rapid and severe desiccation and rehydration than terrestrial plants. For this reason, they provide an unique model system for the study of desiccation tolerance. Although high intertidal seaweeds have been known to exhibit greater desiccation tolerance than low intertidal seaweeds for nearly a century, the exact mechanism of their greater desiccation tolerance has never been explained. This study examined desiccation in two species of the simply-constructed red algae Porphyra umbilicalis from the high intertidal zone and P. yezoensis from the low intertidal zone. Although both species lose water at similar rates and have similar final relative water contents, they are very different in their desiccation tolerance. P. yezoensis exhibited extensive membrane damage and leakage following desiccation whereas P. umbilicalis showed no damage. Repression of membrane phase transition cannot explain the difference in desiccation tolerance between the two species because the membranes of both species remain in liquid crystalline when desiccated. Instead, our data suggest that the cytoplasm of P. umbilicalis forms a more stable glass when desiccated, and that lower molecular mobility in the drying P. umbilicalis is probably the key to its better desiccation tolerance. A dehydrin-like protein of 17 kDa was detected in great abundance in P. umbilicalis but not P. yezoensis, which appears to play a key role in the former species' better desiccation tolerance. 71 Introduction
All terrestrial plants and aquatic algae require water to survive. Terrestrial plants that regularly experience drought have a number of adaptations to reduce water loss, including the presence of a thick, water-proof cuticle and leaves with small surface areas and reduced numbers of stomata. However, only approximately 330 of the 265,000 vascular plants (less than 0.2%) are able to survive without water, or in a condition known at anhydrobiosis. Known as resurrection plants, such plants have the ability to survive with only a 10% relative water content for more than 6 months (1).
Because of the importance of desiccation and drought to agricultural production, the mechanisms behind the desiccation tolerance of resurrection plants as well as those of a few drought-resistant mosses and ferns have been intensively studied. Key to their ability to survive without water is their ability to protect their membranes and prevent them from fusing during desiccation (2). This is accomplished by two mechanisms, namely, repression of membrane phase transition and vitrification. Several types of molecules are believed to play an important role in the formation of such a state, including the sugars trehalos and sucrose, LEA proteins, and dehydrins. These molecules are believed to be involved in preventing membrane phase transition by interacting directly with membrane lipids and preventing membrane fusion by increasing the stability of cellular glass (2, 3).
Interestingly, there is a group of marine algae that are capable of tolerating even more 72 severe and more rapid desiccation than resurrection plants, but the mechanism behind their tolerance is not understood. These algae belong to the marine macrophytes (or seaweeds) that inhabit the intertidal zone of rocky shores. Resurrection plants typically take one to two days to reach an equilibrium with an atmosphere of 50% RH or below during droughts, with drying periods of less than 12 hours being generally lethal.
Similarly, rehydration in resurrection plants generally takes place from 5 hours to several days (4). In comparison, intertidal macroalgae are repeatedly exposed to rapid desiccation and then rehydration during the daily changes in tidal levels. Depending upon their tidal height, intertidal algae are typically desiccated for periods of 1 hr (for lower intertidal species) to 6 hrs (for upper intertidal species) every 12 hrs or twice daily. Those species that occupy the highest intertidal levels frequently spend as much time, if not more, out of water as in water.
For nearly a century, it has been recognized that the intertidal zone is composed of bands of different macroalgal species with different degrees of desiccation tolerance (5-7).
Macroalgae that occupy the high intertidal zone have been shown to have a greater ability to recover after desiccation than those growing in the lower intertidal zone (8, 9).
However, such differences in desiccation tolerance have been shown to be unrelated to differences in moisture content or the rate of water loss (6, 10). For example, in a study by Dorgelo (6) that compared the rates of water loss of several intertidal brown algae, the species that occurred the highest (Pelvetia canaliculata) lost water faster than any other 73 species tested, while, the species that grew at the subtidal fringe had the least tolerance to desiccation (Laminaria saccharina) and retained the highest water content. Thus, it appears that the tolerance to desiccation found in intertidal macroalgae is unrelated to the avoidance of water loss.
To date, the mechanism intertidal seaweeds use to survive repeated desiccation and rehydration has not been explained. Studies on desiccation tolerance of intertidal macroalgae have so far focused only on ROS defense and have not eliminated the possibility of high light effects from desiccation effects. (11-13). In this study, we employed a “compare and contrast” strategy to study two species of the red macroalga
Porphyra, P. umbilicalis and P. yezoensis, which grow in the high and low intertidal zones, respectively. Because of Porphyra’s extremely simple construction (i.e., one cell thick blades) and the existence of both desiccation tolerant and desiccation sensitive species, we think Porphyra provides an excellent model system for studying how intertidal macroalgae tolerate desiccation. In addition, since the plants used throughout this study came entirely from laboratory culture, there is no possibility of environmental adaptations influencing our results. In this paper, we use Fourier transform infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) to demonstrate the role of membrane phase transition repression and vitrification in P. umbilicalis’s greater desiccation tolerance. Furthermore, we have identified a dehydrin-like protein that appears to play a crucial role in the superior desiccation tolerance of P. umbilicalis. 74 Results and Discussion
Since the first experimental work on intertidal zone algal composition was done nearly
100 years ago (14), it has been recognized that macroalgal species growing at different heights in the intertidal zone have different levels of tolerance to desiccation, and that these differences can not be explained by their rates of water loss (8-10, 12). Our study of high and low intertidal species of Porphyra found similar results. Fully hydrated high intertidal P. umbilicalis and low intertidal P. yezoensis both had water contents of approximate 3.5 g H2O / g DW. After exposure to air, they both lost water at similar rates
(Fig. 1), and reached equilibrium with the atmosphere after two hours of desiccation with a final water content below 0.2 g H2O / g DW. Although P. umbilicalis and P. yezonesis shared similar rates and levels of water loss, the latter showed significant damages after just three hours of desiccation. This can be seen in a comparison of their phycoerythrin fluorescence (Fig. 2), which has been used as an indicator of cell damage in Red algae in the past (15). As shown in Fig. 2D, rehydrated P. yezoensis showed strong fluorescence under UV after desiccation, whereas rehydrated P. umbilicalis and control specimens of both species did not fluoresce under the same condition (Fig. 2 A, B and C).
The cause of P. yezoensis's poor tolerance to desiccation appeared related to its loss of membrane integrity. The leakage of electrolytes and amino acids from cells has been used as an indicator of plasma membrane integrity in the past (13, 16). We assayed the impact 75 of water loss on membrane integrity by rehydrating both Porphyra species after exposure to air for different amounts of time and recording the relationship between amino acid leakage and water content. We found the critical water content for P. yezonesis was 0.7 g
H2O / g DW and that amino acid leakage increased sharply when this species was dried below this level. However, amino acid leakage in P. umbilicalis remained low throughout the entire desiccation process. No clear relationship between water content and amino acid leakage was observed in P. umbilicalis (Fig. 3). This result suggests that the plasma membrane of P. yezoensis was damaged at a water content below 0.7 g H2O / g DW, while that of P. umbilicalis was insensitive to desiccation.
We assayed membrane integrity after desiccation further by examining the cellular membrane structure using TEM. Typical Porphyra cells contain one large chloroplast with many internal parallel thylakoid membranes (17). Consistent with this, we saw parallel thylakoid membranes in both Porphyra species in the fully hydrated condition
(Fig.4 A and C). However, after rehydration from a 97% water loss, the thylakoid membranes in P. yezoensis did not have constant spacing between them and appeared fused in places, whereas those of P. umbilicalis appeared parallel and normal (Fig. 4 B and D).
In order to determine how P. umbilicalis protects its membranes during desiccation, we examined if it uses similar methods that desiccation-tolerant terrestrial plants and seeds 76 use to protect their membranes, namely by repression of membrane phase transition and formation of cytoplamic glasses (2). The membrane fluidity was compared in the two
Porphyra species using FTIR. FTIR has been widely used to monitor the process in model membranes, seeds and plant vegetative tissues because of its ability to detect the vibrations of chemical bonds and functional groups of dry materials (18, 19). Past reports have shown that when membranes are in a liquid crystalline state, the CH2 groups produce a symmetrical stretching band of a wavenumber above 2853.5 cm-1. Whereas when membranes are in a gel state, the wavenumber of symmetrical CH2 stretching band was below 2852.0 cm-1 (18, 19). We examined desiccated plants of both Porphyra species with FTIR and found both species had a symmetrical CH2 stretching band close to 2854 cm-1 (Fig. 5). Thus, the majority of membranes in both specimens remained in liquid crystalline state when desiccated, and inhibition of membrane phase transition can not account for the differences in desiccation tolerance observed between the two species.
Trehalose and sucrose have been shown to interact directly with the headgroups of the membrane lipids and repress membrane phase transition when membranes are desiccated
(20, 21). A high degree of fatty acid unsaturation has also been shown to hinder the packing of acyl chains in the membrane (22). Although Porphyra does not contain trehalose or sucrose (23-25), it does have a high fluoridoside content (24, 26) and high polyunsaturated fatty acid content in its membranes (27, 28), which may be important for keeping the membranes from entering gel state when the cells are desiccated. 77 The last mechanism we tested for was the formation of cytoplasmic glasses, or vitrification. In this mechanism, membrane movement and fusion are prevented by the high viscosity of being in a glassy state (2, 29). It has been shown that the viscosity of cytoplasm increases dramatically when the water content of cells goes below 0.8 g H2O / g DW (30), and that cytoplasm enters a glassy state when the water content drops below
0.1 g H2O / g DW (29). We tested whether there is a difference in viscosity between dry
P. umbilicalis and P. yezoensis by assaying the strength of the hydrogen bonding of their glasses. This was done by monitoring the shift of the OH stretching band (vOH) present in FTIR spectra with increasing temperature, since this shift has been reported to correlate with the strength of hydrogen bonding (31). The shift of OH stretching band is expressed as the wavenumber-temperature coefficient (WTC), which is determined from the slope of the vOH-temperature plot. A high WTC indicates a weak hydrogen bonding networks and thus, a weaker glass (32). The WTC of the desiccated P. yezoensis extract was significantly higher than that of the P. umbilicalis extract (0.30 cm-1/ ºC vs 0.13 cm-1/
ºC) (Fig. 6). Higher WTC values have also been reported in desiccation sensitive
Arabidopsis thaliana mutant seeds and carrot somatic embryos compared to their desiccation tolerant counterparts (33, 34). The WTC differences between the Porphyra species suggest that the cytoplasmic glass of desiccation tolerant P. umbilicalis has a stronger hydrogen bonding network than that of desiccation sensitive P. yezoensis.
Additional evidence that vitrification plays a key role in the desiccation tolerance of P. 78 umbilicalis comes from direct determination of the molecular mobility of its glass. We measured molecular mobility using EPR, which has been used to monitor molecular mobility in seeds and pollen after desiccation (35, 36). EPR measures the rotational mobility of nitroxide spin-labeled probes (37) and signals from the cytoplasm can be specifically detected with the use of broadening agents such as K3Fe(CN)6 (38). The rotational correlation time of the probe is longer if the probe has lower molecular mobility (37). The cytoplasmic molecular mobility in intact specimens of both Porphyra species started to decrease rapidly when the water content went below 1.25 g H2O / g DW
(Fig 7). However, the molecular mobility in P. umbilicalis was lower than in P. yezoensis during dehydration, and the largest difference between the two species occurred around
0.7 g H2O / g DW (Fig. 7), which is the approximate water content where P. yezoensis showed severe membrane damage and leakage of amino acids (Fig. 3). This discovery strongly suggests that reduced molecular mobility is crucial for desiccation tolerance in
P. umbilicalis, which is similar to the findings of ESR studies conducted on desiccation tolerant seeds (3, 39).
Vitrification on its own cannot account for the greater desiccation tolerance of P. umbilicalis, compared to P. yezoensis. This is because a glassy state is not likely to exist at 0.7 g H2O / g DW (40), the water content level at which P. yezoensis' plasma membrane began to leak while that of P. umbilicalis was intact. A study conducted on the desiccation tolerant pollen of Typha latifolia and desiccation sensitive pollen of Zea mays 79 had similar findings (41). The difference in the loss of viability of the two pollens during dehydration was observed before vitrification occurred (41). Based on our data, the reduction in molecular mobility during desiccation seems to be the principal reason for the difference in desiccation tolerance between P. umbilicalis and P. yezoensis.
Vitrification perhaps is best viewed as a special case of reduced molecular mobility in cytoplasm at low water contents (< 0.1 g H2O / g DW) because it protects desiccated cells by preventing the approach of subcellular structures, including membranes (42).
Therefore, the existence of cellular glass itself should not be used as a requirement for desiccation tolerance. Rather, the reduction of molecular mobility during dehydration might be a better indicator of desiccation tolerance because the difference between being damaged and being protected appeared at this stage. We speculate that cytoplasm components have to form a hydrogen bonding framework that keeps the molecular mobility below a threshold that varies with water contents to prevent membranes from compressing and fusing during dehydration.
Since both P. umbilicalis and P. yezoensis have similar sugar compositions (23-26), we investigated whether dehydrin-like proteins might be playing a key molecular role in the greater desiccation tolerance of P. umbilicalis. Dehydrins belong to the D11 family of
LEA proteins and have been discovered in a wide range of organisms experiencing water stresses, including vascular plants, algae, cyanobacteria, and animals (43, 44). Dehydrins have several conserved domains, including Y, S and K segments (45). These proteins may 80 play an important role in desiccation tolerance because glasses found in desiccation tolerant seeds that are rich in LEA proteins have shown lower molecular mobility than pure sugar glasses (33, 46).
In this study, we preformed Western blots to test the existence of any dehydrin-like proteins in both Porphyra species with an antibody specific to the conserved K segment.
We detected a dehydrin-like protein in P. umbilicalis, but not in P. yezoensis (Fig. 8). The apparent molecular weight of this dehydrin-like protein is 17 kDa. Since the protein extracts were obtained from lab-grown P. umbilicalis thalli that had never experienced desiccation, the expression of this dehydrin-like protein is constitutive and does not even require desiccation. Given that wild P. umbilicalis encounters a large change in water content during each tidal cycle, a constant expression of protective proteins is a necessity.
Constitutive cellular protection is considered a primitive mechanism for desiccation tolerance and found only in lower plants (47). The only other report of dehydrin-like proteins in macroalagae is that of Li et al (48) who reported the constitutive expression of dehydrin-like proteins in several species of intertidal fucoid macroalgae. Although they used the same antibody from the same vendor as we did, the dehydrin-like proteins that
Li et al (48) found in fucoid algae were generally of much larger size in vegetative tissue
(e.g., 92 kDa). The dehydrin-like protein we found in P. umbilicalis was stable after a 15 minute heating at 100°C (Fig. 8, lane 3 and 4) and remained water soluble after ethanol precipitation (Fig. 8, lane 4). Such high heat stability and hydrophilicity are typical of 81 dehydrins (43). This protein was present in the extract of P. umbilicalis used for our FTIR study and could be responsible for the strong hydrogen bonding networks observed in the dry extract (Fig. 6), as LEA proteins have been shown to bind tightly to sugars (49) and to form intracellular filamentous networks during desiccation (50). Therefore, we believe the dehydrin-like protein found in P. umbilicalis may contribute to the faster decrease in molecular mobility during dehydration of P. umbilicalis, which in turn inhibits membrane fusion.
Desiccation tolerance is a complex phenomenon and multiple mechanisms have to work in synergy for organisms to survive desiccaiton (29, 51). This study confirms the importance of these mechanisms, and more importantly, highlights the pivotal role of low molecular mobility and dehydrins in this phenomenon. By using a “compare and contrast” strategy, we eliminated the influence of other stresses, and since only one dehydrin-like protein was found in the tolerant P. umbilicalis, this species is ideal for further studies on the role of dehydrins in lowering molecular mobility and vitrification in vivo. Dehydrin is also of interest because of the extremely ancient position of
Porphyra and its close relative Bangia, for which a fossil record dating back 1.2 bya exists (Butterfield, 2000). 82 Materials and Methods
Plant Material
Porphyra umbilicalis was collected from Cape Cod Canal, Massachusetts
(41°46'N, 70°29'W) in June 2004. P. yezoensis (strain U51) was collected from
Hokkaido, Japan. Both species were grown, maintained and reproduced asexually via monospores at 15°C in filter-sterile seawater supplemented with 1% ESS (52) in aerated flasks at a light intensity of 120 μmol m-2s-1 on a 12:12 (L:D) cycle. Both species have been propagated for many generations continuously in seawater, and no prior desiccation was applied to the algae before experiments.
Desiccation and Measurement of Water Content
Water loss was measured by weighting algae before and after desiccation. Algae were desiccated in a Baker Edgegard EG 6320 hood at 23 to 25°C, 30 to 35% RH. The rate of air flow was 30.5 meters per minute. The light intensity was 30 µmol photons · m-2
· s-1. Water content was calculated as follows: Water content = (W(t) – DW) / DW, where W(t) is the weight of specimen after t hour desiccation, and DW is the dry weight of specimens dried in oven for 72 h at 110°C.
Amino Acid Leakage Assay
Plasma membrane damage was assayed by an amino acid leakage method described by Rosen (53). Specimens with a fresh weight of 0.5g to 1g were desiccated 83 and then rehydrated in 10 ml filtered seawater for 10 minutes. The concentration of released amino acids in the sea water was measured with ninhydrin stain using glutamate as standard.
Transmission Electron Microscopy
The algal samples were first fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 at 4°C for 1 hour, washed in cacodylate buffer, and then fixed in 1% osmium tetraoxide in cacodylate buffer and dehydrated in a series of 30%, 50%,
70%, 85% and 95% ethanol for 15 minutes each, then in 100% ethanol for 1 hour with 2 changes. Samples were washed 3 times in n-butyl-glycidyl ether before being incubated in 1:1 Spurrs / Quetol resin to 100% ethanol for 2 hours. The samples were subsequently moved to 100% resin for 6 hours with 3 changes. Next, samples were embedded in fresh resin in BEEM capsules and placed in polymerization oven at 60°C for 24 hours.
Sectioning was performed on a Leica UltraCut E microtom, and sections were stained with 2% uranyl acetate and Reynolds' lead citrate with 10 second wash in DI water after each staining. Transmission electron microscopy was performed on a JEM-1010.
Fourier-Transform Infrared spectroscopy (FTIR)
FTIR measurements were carried out on Perkin-Elmer Spectrum Spotlight 400
FTIR Microscope System at 2 cm-1 resolution. For membrane fluidity measurements, the algae were rinsed in DI water for 2 seconds and dried on Low-e microscope slides 84 (Kevley Technologies). The measurements were done at room temperature. For each background and sample spectrum, 512 interferograms were averaged. The deconvolved spectra were calculated with interactive Perkin-Elmer deconvolution routine with γ = 2 and a length factor = 30. The wavenmuber of each peak was labeled by the software automatically and the state of the membranes was determined by the wavenumbers of symmetrical CH2 stretching peak.
To estimate the strength of hydrogen bonding in the dry cytoplasm, algal samples were ground in liquid nitrogen and extract with DI water at 5°C. Extracts of 200µl were added into 800µl 100% ethanol and centrifuged at 10,000 g for 10 minutes to remove polysaccharides. The supernatant was spotted on the Low-e microscope slides and dried before measurement. Thirty two interferograms were collected every 3 to 4 °C from 23°C to 120°C for each sample. The spectra were smoothed by a Perkin-Elmer smoothing routine with a block average algorithm (the number of points = 5). The rate of shift of
OH-stretching band with increase in temperature is a measure of strength of hydrogen bonding in the specimens (33).
Electron Paramagentic Resonance (EPR)
Molecular mobility in vivo was measured by EPR. The algae samples were incubated in seawater containing 1 mM 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPONE) spin probe and 120 mM of broadening agent potassium ferric cyanide for 2 85 hours before measurement. The treated algae were then spread on cover glasses and the spectra were obtained on an X band Bruker EMX at room temperature. Three scans of
2048 points were averaged for each spectrum. The magnetic field modulation was 0.2 G at 100 kHz, the time constant was 20.48 ms and a conversion time was 81.92 ms.
The fitting method utilized a MATLAB (MathWorks) based version of EPRLL, the slow-motional lineshape program of Freed and co-workers (54, 55), which can be used to fit multiple component spectra. The parameters that were varied during the fitting procedure included isotropic Gaussian inhomogeneous broadening, gib0, and Azz tensor components. Azz tensor components were then used to determine values for gxx and gyy, as described previously (56) from observations that the relation between the isotropic g- value, giso, and aN (or that between gxx and Azz) is approximately linear (57), and the
approximation that Δgx = (gx−gz) is linearly related to Δgy = (gy−gz) for which a linear fit gives Δgy = 0.20 Δgx (58). The remaining parameters were fixed at the values gzz =
2.0023, Axx/γe = 5.0 G, and Ayy/γe = 5.5 G. The isotropic rotational correlation time was
−1 calculated from the isotropic rotational diffusion constant R as τc = (6R) .
Western Blot
Crude protein extracts (24 µg protein per lane) and extracts prepared for FTIR experiments (6 µg protein per lane) were boiled in Laemmli buffer (59) for 15 minutes and separated on 4-20% SDS-PAGE (Bio-Rad) at 100V for one hour. Proteins were 86 transferred onto a PVDF membrane with pore size of 0.2 µm (Millipore, Cat.
ISEQ08100), at 350mA for one hour in Towbin buffer (25 mM Tris Base, 192 mM glycine and 10% methanol). The membrane was blocked in 5% skim milk in TBST buffer (10 mM Tris, 192 mM NaCl and 0.05% Tween 20, pH 7.5) for 3 hours. The membrane was then incubated in 1000-fold diluted anti-dehydrin antibody (StressGen,
Cat. PLA-100) in TBST buffer at 4°C overnight, washed in TBST buffer 3 times, and incubated in 10,000-fold diluted anti-rabbit IgG peroxidase conjugate (Sigma, Cat.
A4914) in TBST buffer for 1 hour. The dehydrin-like proteins were detected with
Immobilon Western HRP substrate (Millipore, Cat. WBKLS0100). The blot was then exposed to an X-ray film (Kodak, Cat. 864 6770) from 1 to 10 seconds and the film was scanned in a flat bed scanner.
Statistical analyses
Statistical analyses were performed using the SAS system for Windows v 9.2. The data were analyzed by ANOVAs and repeated-measures ANOVAs with general linear models.
The results of the effect of water contents on amino acid leakage (Fig. 3) were transformed by log (1+ value) before analyses to meet the assumption of ANOVA. 87 References
1. Gaff DF (1977) Desiccation tolerant vascular plants of southern Africa. Oecologia 31:95-109.
2. Oliver AE, Crowe LM, Crowe JH (1998) Methods for dehydration-tolerance: Depression of the phase transition temperature in dry membranes and carbohydrate vitrification. Seed Science Research 8:211-221.
3. Buitink J, Leprince O (2004) Glass formation in plant anhydrobiotes: survival in the dry state. Cryobiology 48:215-228.
4. Proctor MCF, Pence V (2002) in Desiccation and Survival of in Plants: Drying Without Dying, eds Black M, Pritchard HW (CABI Publishing), pp. 207-238.
5. Baker SM (1909) On the causes of the zoning of brown seaweeds on the seashore. New Phytologist 8:196-202.
6. Dorgelo J (1976) Intertidal fucoid zonation and desiccation. Aquatic Ecology 10:115- 122.
7. Chapman ARO (1986) Population and community ecology of seaweeds. Advances in marine biology 23:1-161.
8. Brown M (1987) Effects of desiccation on photosynthesis of intertidal algae from a southern New Zealand shore. Botanica Marina 30:121-127.
9. Abe S, Kurashima A, Yokohama Y, Tanaka J (2001) The cellular ability of desiccation tolerance in Japanese intertidal seaweeds. Botanica Marina 44:125-131.
10. Dromgoole FI (1980) Desiccation resistance of intertidal and subtidal algae. Bot. Mar 23:149-159.
11. Collén J, Davison I (1999) Reactive oxygen production and damage in intertidal Fucus spp. (Phaeophyceae). Journal of phycology 35:54-61.
12. Collén J, Davison I (1999) Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus. Plant Cell and Environment 22:1143-1151.
13. Burritt DJ, Larkindale J, Hurd CL (2002) Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation. Planta 215:829-38. 88 14. Baker SM (1910) On the Causes of the Zoning of Brown Seaweeds on the Seashore. II. The Effect of Periodic Exposure on the Expulsion of Gametes and on the Germination of the Oospore. New Phytologist 9:54-67.
15. Dudgeon SR, Davison IR, Vadas RL (1989) Effect of freezing on photosynthesis of intertidal macroalgae: relative tolerance of Chondrus crispus and Mastocarpus stellatus (Rhodophyta). Marine Biology 101:107-114.
16. Sherwin HW, Farrant JM (1996) Differences in rehydration of three desiccation- tolerant angiosperm species. Ann Bot 78:703-710.
17. Wiencke C, Lõuchli A (1980) Growth, cell volume, and fine structure of Porphyra umbilicalis in relation to osmotic tolerance. Planta 150:303-311.
18. Hincha D, Zuther E, Heyer A (2003) The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions. Biochim Biophys Acta. 10:172-177.
19. Oldenhof H et al. (2006) Freezing and desiccation tolerance in the moss Physcomitrella patens: An in situ Fourier transform infrared spectroscopic study. Biochimica et Biophysica Acta (BBA) - General Subjects 1760:1226-1234.
20. Crowe LM, Crowe JH, Chapman D (1985) Interaction of carbohydrates with dry dipalmitoylphosphatidylcholine. Arch Biochem Biophys 236:289-96.
21. Leslie S et al. (1995) Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl. Environ. Microbiol 61:3592-3597.
22. Cevc G (1991) How membrane chain-melting phase-transition temperature is affected by the lipid chain asymmetry and degree of unsaturation: an effective chain-length model. Biochemistry 30:7186-7193.
23. Majak W, Craigie JS, McLachlan J (1966) Photosynthesis in algae: I. Accumulation products in the rhodophyceae. Canadian Journal of Botany 44:541-549.
24. Holligan PM, Drew EA (1971) Routine analysis by gas-liquid chromatography of soluble carbohydrates in extracts of plant tissues. II. Quantitative analysis of standard carbohydrates, and the separation and estimation of soluble sugars and polyols from a variety of plant tissues. New Phytologist 70:271-297.
25. Kremer BP (1982) Biosynthesis of photosynthates and taxonomy of algae. Z Naturforsch 37c:761-771. 89 26. McLachlan J, Craigie J, LCM Chen L, Ogetze E (1972) Porphyra linearis Grev: An edible species of nori from Nova Scotia. Proc Int Seaweed Symp 7:473-476.
27. Araki S et al. (1986) Lipid and fatty acid composition in the red alga Porphyra yezoensis. Japanese Journal of Phycology 34:94-100.
28. Fleurence J, Gutbier G, Mabeau S, Leray C (1994) Fatty acids from 11 marine macroalgae of the French Brittany coast. Journal of Applied Phycology 6:527- 532.
29. Hoekstra F, Golovinab E, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends in Plant Science 6:431-438.
30. Leprince O, Hoekstra FA (1998) The responses of cytochrome redox state and energy metabolism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance. Plant Physiology (Rockville) 118:1253-1264.
31. Wolkers WF, Oliver AE, Tablin F, Crowe JH (2004) A fourier-transform infrared spectroscopy study of sugar glasses. Carbohydrate research 339:1077-1085.
32. Wolkers WF, Oldenhof H, Alberda M, Hoekstra FA (1998) A Fourier transform infrared microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells. Biochimica et Biophysica Acta (BBA) - General Subjects 1379:83-96.
33. Wolkers WF et al. (1998) Properties of proteins and the glassy matrix in maturation- defective mutant seeds ofArabidopsis thaliana. The Plant Journal 16:133-143.
34. Wolkers WF, Tetteroo FA, Alberda M, Hoekstra FA (1999) Changed properties of the cytoplasmic matrix associated with desiccation tolerance of dried carrot somatic embryos. An in situ fourier transform infrared spectroscopic study. Plant Physiol 120:153-164.
35. Buitink J, Hemminga MA, Hoekstra FA (1999) Characterization of molecular mobility in seed tissues: an electron paramagnetic resonance spin probe study. Biophys J 76:3315-22.
36. Buitink J, Dzuba SA, Hoekstra FA, Tsvetkov YD (2000) Pulsed EPR Spin-Probe Study of Intracellular Glasses in Seed and Pollen. Journal of Magnetic Resonance 142:364-368.
37. Hemminga M, Dries I (2002) in Biological Magnetic Resonance, Spin Labeling: The 90 Next Mellennium., ed Berliner, L. J. (New York: Plenum Publishing Corporation), pp. 339-366.
38. Golovina EA, Hoekstra FA, Van Aelst AC (2001) The competence to acquire cellular desiccation tolerance is independent of seed morphological development. J. Exp. Bot. 52:1015-1027.
39. Buitink J, Claessens MM, Hemminga MA, Hoekstra FA (1998) Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen. Plant Physiol. 118:531-541.
40. Williams RJ, Leopold AC (1989) The glassy state in corn embryos . Plant Physiol. 89:977-981.
41. Buitink J, Walters-Vertucci C, Hoekstra FA, Leprince O (1996) Calorimetric properties of dehydrating pollen: Analysis of a desiccation-tolerant and an intolerant species. Plant Physiology (Rockville) 111:235-242.
42. Sun WQ, Leopold AC (1997) Cytoplasmic vitrification and survival of anhydrobiotic organisms. Comparative Biochemistry & Physiology A-Comparative Physiology 117:327-333.
43. Campbell SA, Close TJ (1997) Dehydrins: Genes, proteins, and associations with phenotypic traits. New Phytologist 137:61-74.
44. Lapinski J, Tunnacliffe A (2003) Anhydrobiosis without trehalose in bdelloid rotifers. FEBS Letters 553:387-390.
45. Close TJ (1996) Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins. Physiologia Plantarum 97:795-803.
46. Buitink J et al. (2000) High critical temperature above Tg may contribute to the stability of biological systems. Biophys J 79:1119-28.
47. Oliver MJ, Tuba Z, Mishler BD (2000) The evolution of vegetative desiccation tolerance in land plants. Plant Ecology 151:85-100.
48. Li R, Brawley SH, Close TJ (1998) Proteins immunologically related to dehydrins in Fucoid algae. Journal of Phycology 34:642-650.
49. Walters C, Ried JL, Walker-Simmons MK (2008) Heat-soluble proteins extracted from wheat embryos have tightly bound sugars and unusual hydration properties. 91 Seed Science Research 7:125-134.
50. Goyal K et al. (2003) Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein. Journal of Biological Chemistry 278:12977-12984.
51. Smirnoff N (1993) Tansley Review No. 52: The role of active oxygen in the response of plants to water deficit and desiccation. New Phytologist 125:27-58.
52. Kitade Y, Yamazaki S, Saga N (1996) A Method for extraction of high molecular weight DNA from the macroalga Porphyra yezoensis (Rhodophyata). Journal of Phycology 32:496-498.
53. Rosen H (1957) A modified ninhydrin colorimetric analysis for amino acids. Archives of Biochemistry and Biophysics 67:10-15.
54. Schneider DJ, Freed JH (1989) Calculating slow motional magnetic resonance spectra: a user’s guide. Biological Magnetic Resonance 8:1–76.
55. Schneider DJ, Freed JH (1989) Spin relaxation and motional dynamics. Adv. Chem. Phys 73:119.
56. Lawton JS, Smotkin ES, Budil DE (2008) Electron spin resonance investigation of microscopic viscosity, ordering, and polarity in nafion membranes containing methanol- water mixtures. J. Phys. Chem. B 112:8549-8557.
57. Smirnova TI et al. (2007) Local polarity and hydrogen bonding inside the sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies. Biophys. J. 92:3686-3695.
58. Lebedev YS (1990) High-frequency continuous wave electron spin resonance, in “Modern Pulsed and Continuous-Wave Electron Spin Resonance”(L. Kevan and MK Bowman, Eds.) (Wiley, New York).
59. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. 92
Fig. 1. Rate of water loss in P. umbilicalis and P. yezoensis. Thalli of Porphyra were blotted dry and exposed to 30 to 35% RH air. The water contents were determined by the formula in Materials and Methods. Error bars are ± 1s.d. (n=3). ANOVA: time*species effect, P < 0.01. H: high intertidal species; L: low intertidal species. 93
Fig. 2. The influence of desiccation and rehydration on phycobiliprotein fluorescence in
Porphyra species was observed in UV using Fisher Scientific / Biotech TIV-88A UV transilluminator. The diameter of the petri dish is 9 cm. The UV intensity was 8000
µw/cm2. The desiccated thalli were rehydrated in sterile sea water for 10 minutes before fixation. A: P. umbilicalis before desiccation; B: P. umbilicalis after rehydration; C: P. yezoensis before desiccation; D: P. yezoensis after rehydration. 94
Fig. 3. Levels of amino acid leakage of P. umbilicalis and P. yezoensis at various water contents. Thalli of various water contents were rehydrated in 10 ml sterile sea water for
10 minutes and the concentration of amino acids in water was determined by ninhydrin assay. Error bars are ± 1s.d. (n=3). ANOVA: water content*species effect, P < 0.05 H: high intertidal species; L: low intertidal species. 95
Fig. 4. The influence of desiccation and rehydration on thylakoid membranes in
Porphyra species was observed by transmission electron microscopy. The desiccated thalli were rehydrated in sterile sea water for 10 minutes before fixation. A: P. umbilicalis before desiccation; B: P. umbilicalis after rehydration; C: P. yezoensis before desiccation;
D: P. yezoensis after rehydration. Note the parallel thylakoid membranes in A, B, and C and membrane fusion in D. 96
Fig. 5. Membrane states in desiccated Porphyra species. Deconvolved FTIR spectra of the 2800-3000 cm-1 region of desiccated P. umbilicalis and P. yezoensis showed that the
-1 wavenumber of the CH2 symmetrical band in both species was close to 2854 cm . 97
Fig. 6. Hydrogen bonding strength of dry cell extracts from P. umbilicalis and P. yezoensis was presented by plotting the shift of the OH-stretching vibration band as temperature increases. The WTC of dried P. umbilicalis and P. yezoensis extracts before melting was 0.13 and 0.30, respectively. ANOVA: temperature*species effect, P < 0.05
H: high intertidal species; L: low intertidal species. 98
Fig. 7. Molecular mobility in the drying P. umbilicalis and P. yezoensis thalli was monitored by EPR. Each data point is a sum of three scans. Longer rotational correlation time represents lower molecular mobility. ANOVA: water content*species effect, P <
0.01 H: high intertidal species; L: low intertidal species. 99
Fig. 8. The presence of a dehydrin-like protein in the cell extracts of P. umbilicalis and P. yezoensis was tested with Western blotting. Lane1: crude P. yezoensis extract; lane 2: P. yezoensis cell extract after ethanol precipitation; lane 3: crude P. umbilicalis extract; lane
4: P. umbilicalis cell extract after ethanol precipitation. 100 Table 1. ANOVA table for tests on water contents during desiccation, the effects of water contents on amino acid leakage, the effect of temperature on the shift of νOH stretch band and the effect of water content on molecular mobility.
df Sum of squares Mean square F Water contents during desiccation 1 0.49 0.49 17.58 * speciesa 4 51.58 12.89 1902.14 ** timeb 4 0.76 0.19 27.97 ** time*species
Effect of water contents on amino acid leakage speciesa 1 6.81 6.81 76.6 ** water content 1 1.13 1.13 12.74 ** water content*species 1 0.66 0.66 7.44 *
Effects of temperature on νOH shift (<80ºC) speciesa 1 59.54 59.54 2.53 temperature 1 343.53 343.53 14.58 ** temperature*species 1 110.94 110.94 4.71 *
Effect of water contents on molecular mobility speciesa 1 0.41 0.41 42.45 ** water content 1 7.16 7.16 744.31 ** water content*species 1 0.11 0.11 11.61 ** aPorphyra umbilialis and P. yezoensis b0, 0.5, 1, 2, and 3 hours
*: P < 0.05; **: P < 0.01 101 Chapter 4
Determination of Saturation Irradiance Level of Laboratory Porphyra
Cultures and Characterizing Field Samples of P. umbilicalis and P.
yezoensis 102 Objective
The goal of the experiments described in this chapter was to determine whether the observed differences in desiccation tolerance between the laboratory-grown Porphyra umbilicalis and P. yezoensis were due to the artificial growing conditions. To achieve this goal, I first measured the saturating irradiance level (Ik) of laboratory-grown Porphyra specimens. Second, I tested the dehydration rate, amino acid leakage, superoxide dismutase activities and the presence of dehydrin of P. umbilicalis and P. yezoensis collected from field.
Materials and Methods
Plant materials
The field sample of Porphyra umbilicalis was collected from the high intertidal zone at No Name Point, Nahant, Massachusetts, on November 20th, 2009. The field sample of P. yezoensis was collected from the very low intertidal zone at Dover Point,
New Hampshire on November 24th, 2009. Dover Point was selected for the collection of
P. yezoensis after the discussion with Dr. Chirs Neefus at University of New Hampshire, who recently published a report on introduced Asiatic Porphyra species along the
Northeastern Atlantic ocean (Neefus et al. 2008). The sources of laboratory-grown
Porphyra specimens were desicribed in the Materials and Methods in Chapter 2, page 37. 103 Desiccation and Measurement of Relative Water Content (RWC)
Water loss was measured by weighting algae before and after desiccation. Algae were desiccated in a Baker Edgegard EG 6320 hood at 23 to 25°C, 30 to 35% RH. The rate of air flow was 30.5 meters per minute. The light intensity was 30 µmol photons · m-2
· s-1. RWC was calculated as follows: RWC = 100 * (W(t) – DW) / (FW – DW), where
W(t) is the weight of specimen after t hour desiccation, FW is the fresh weight of the specimen and DW is the dry weight of specimens dried in oven for 72 h at 110°C.
Determination of saturating irradiance levels (Ik)
The procedure for measuring oxygen evolution rate of laboratory-grown
Porphyra umbilicalis and P. yezoensis was described in the Materials and Methods in
Chapter 2, page 37. To determine Ik, oxygen evolution rates were measured at various
-2 -1 light intensities (0 to 300 µmol photons ·m ·s ). Ik is defined as described in Lobban and
Harrison (1994)
Amino Acid Leakage Assay
Plasma membrane damage was assayed by the same amino acid leakage method described in Chapter 2 (Rosen 1957). Specimens with a fresh weight of 0.5g to 1g were desiccated and then rehydrated in 10 ml filtered seawater for 10 minutes. The concentration of released amino acids in the sea water was measured with ninhydrin stain using glutamate as standard. 104 Superoxide dismutase (SOD) activity assay
SOD activity of algal samples was measured with the same method described in
Chapter 2 using a SOD Assay Kit from Sigma. Algal samples were ground in liquid nitrogen and extracted in 100mM potassium phosphate buffer, pH 7.6. After centrifugation, the protein concentration was determined with Bio-Rad Protein Assay and was adjusted to 0.5 mg/ml before measuring SOD activity.
Western Blot
Crude protein extracts obtained with the same procedure as described in Chapter
3, and the extracts were purified with ethanol precipitation and boiled in Laemmli buffer
(59) for 15 minutes and separated on 4-20% SDS-PAGE (Bio-Rad) at 100V for one hour.
Proteins were transferred onto a PVDF membrane with pore size of 0.2 µm (Millipore,
Cat. ISEQ08100), at 350mA for one hour in Towbin buffer (25 mM Tris Base, 192 mM glycine and 10% methanol). The membrane was blocked in 5% skim milk in TBST buffer (10 mM Tris, 192 mM NaCl and 0.05% Tween 20, pH 7.5) for 3 hours. The membrane was then incubated in 1000-fold diluted anti-dehydrin antibody (StressGen,
Cat. PLA-100) in TBST buffer at 4°C overnight, washed in TBST buffer 3 times, and incubated in 10,000-fold diluted anti-rabbit IgG peroxidase conjugate (Sigma, Cat.
A4914) in TBST buffer for 1 hour. The dehydrin-like proteins were detected with
Immobilon Western HRP substrate (Millipore, Cat. WBKLS0100). The blot was then exposed to an X-ray film (Kodak, Cat. 864 6770) for 30 seconds to 1 minute. 105 Statistical analyses
Statistical analyses were performed using the SAS system for Windows v 9.2. The data were analyzed by ANOVAs and repeated-measures ANOVAs with general linear models.
The results of the effect of water contents on amino acid leakage (Fig. 4) were transformed by log (1+ value) before analyses to meet the assumption of ANOVA.
Results and Discussion
Photosynthesis properties of laboratory-grown P. umbilicalis and P. yezoensis
Because the light intensity in the incubators used in this study (120 µmol photons
·m-2·s-1) is lower than the light intensities at noon in the field, I wanted to determine if the lack of dehydrin-like protein in the laboratory-grown P. yezoensis could be due to light limitation. To test this hypothesis, I measured the saturating irradiance levels (Ik) of the laboratory-grown P. yezoensis and P. umbilicalis to see how close they were to the light level used to grow Porphyra in the laboratory. The results showed that the laboratory- grown P. yezoensis and P. umbilicalis have very similar values of Ik, which were both approximately 170 µmol photons ·m-2·s-1 (Fig. 1 and Fig. 2). The light intensity in the
-2 -1 incubators (120 µmol photons ·m ·s ) is about 70% of Ik of both laboratory-grown P. yezoensis and P. umbilicalis, and therefore, the laboratory Porphyra cultures were not light limited. In the literature, the Ik of P. yezoensis and P. umbilicalis collected from field was about 140 µmol photons ·m-2·s-1 (Gao & Aruga 1987) and 280 µmol photons ·m-2·s-1 106 (Johansson & Snoeijs 2002), respectively. Therefore, it is clear that both laboratory- grown Porphyra species have adapted to the irradiance level in the incubator after long term cultivation in laboratory. In addition, this finding also showed that the lack of dehydrin-like protein in the laboratory-grown P. yezoensis could not be due to the shortage of irradiance, because the laboratory-grown P. umbilicalis had similar Ik (Fig. 1;
Fig. 2), was cultivated in the same incubator, and yet it showed a strong dehydrin-like protein band in the Western blot (Fig. 8, Chapter 3; Fig. 6).
Comparison of field P. umbilicalis and P. yezoensis
The field collected P. umbilicalis and P. yezoensis plants showed similar rates of water loss (Fig. 3, Table 1). This is not surprising as the rate of water loss is determined by the ratio of surface area to the volume of the specimens (Schonbeck & Norton 1979;
Dromgoole 1980), and both P. umbilicalis and P. yezoensis are one-cell thick sheets. The rates of water loss of the field collected P. umbilicalis and P. yezoensis were also very close to those of the laboratory-grown P. umbilicalis and P. yezoensis (Fig.1, Chapter 2).
Field P. umbilicalis and P. yezoensis showed differences in terms of amino acid leakage during drying (Fig. 4, Table 1) and superoxide dismutase activities (Fig. 5, Table
1). When exposed to desiccation, the membrane integrity of field P. yezoensis was compromised as amino acid leakage became prominent after one hour of drying (Fig. 4).
This result showed that the field P. yezoensis is as susceptible to desiccation as the laboratory-grown P. yezoensis (Fig. 4, Chapter 2). Therefore, reactive oxygen species 107 (ROS) defense is probably not the cause for the difference in desiccation tolerance between the field P. umbilicalis and P. yezoensis, because the field P. yezoensis had a very high superoxide dismutase (SOD) activity, and yet was sensitive to desiccation.
Such differences in the amino acid leakage and SOD acitivity were also observed in the laboratory-grown P. umbilicalis and P. yezoensis (Fig. 4 and Fig. 6, Chapter 2). The field plants had higher SOD activities than the laboratory-grown plants (Fig. 6, Chapter 2;
Fig.5). This is probably because they must have higher ROS defense systems to adapt to the stronger light, which can stimulate ROS accumulation (Collén & Pedersén 1996;
Choo et al. 2004)
A difference was also found in the presence of a dehydrin-like protein between the field collected P. umbilicalis and P. yezoensis. Fig. 8 showed that both laboratory-grown and field P. umbilicalis had dehydrin-like protein (lane 3, 4, 5, and 6), but no such band was found for laboratory-grown and field P. yezoensis (lane 1, 2, 7, and 8). The reason for weaker signals of the dehydrin-like protein in the field P. umbilicalis compared to laboratory-grown P. umbilicalis is likely due to differences in the growth conditions between the field and the laboratory. First, when intertidal seaweeds are exposed to air during low tide, the uptake of nutrients, such as nitrate and phosphate, is limited (Davison
& Pearson 1996). On the other hand, the laboratory cultures are always submerged and the uptake of nutrients is not interrupted. Because P. umbilicalis is a high intertidal macroalgae, the difference between the two conditions is significant. Second, synthesizing and accumulating proteins requires input of nitrogen sources. The nitrate 108 concentration in the seawater at the time the plants were collected was probably less than
5 µM (Espinoza & Chapman 1983; Yap et al. 2005; Zhang & Fischer 2006). The laboratory-grown Porphyra was cultivated in filtered seawater supplemented with 1%
ESS (Kitade et al. 1996), which made the nitrate concentration greater than 700 µM in the culture medium. Such high nitrate concentration, combined with continuous immersion, allows laboratory grown P. umbilicalis to produce much higher levels of dehydrin-like protein. Since field P. yezoensis did not have the band of dehydrin-like protein and the laboratory grown P. yezoensis was also cultivated in the medium supplemented with 1% ESS, the lack of dehydrin-like protein in the extract of P. yezoensis is not due to the culture conditions or the shortage of nutrients.
Conclusion
Based on the data of Ik determination (Fig. 1 and Fig. 2) and Western blotting of laboratory-grown and field Porphyra umbilicalis and P. yezoensis (Fig. 6), the lack of dehydrin-like protein in P. yezoensis is unlikely the result of limited nutrients or culture conditions. The field Porphyra umbilicalis and P. yezoensis showed similar differences in the rates of water loss, amino acid leakage during dehydration, and superoxide dismutase activities as were observed between the laboratory-grown P. umbilicalis and P. yezoensis (Chapter 2 and Chapter 3). The experiments in this chapter show that the difference in desiccation tolerance observed in laboratory-grown P. umbilicalis and P. 109 yezoensis was also observed in P. umbilicalis and P. yezoensis collected from field, and that the difference in the presence of a dehydrin-like protein between P. umbilicalis and
P. yezoensis is intrinsic. 110
Fig. 1. Oxygen evolution (circles) and P-I curve for laboratory-grown Porphyra yezonesis. 111
Fig. 2. Oxygen evolution (circles) and P-I curve for laboratory-grown Porphyra umbilicalis. 112
Fig. 3. Mean (± 1 SE) relative water content of field samples of Porphyra umbilicalis and P. yezoensis during a 3 hour desiccation. ANOVA: time*species effect, P > 0.05 H: high intertidal species; L: low intertidal species. 113
Fig. 4. Mean (± 1 SE) amino acid leakage of field samples of Porphyra umbilicalis and
P. yezoensis during a 3 hour desiccation. ANOVA: time*species effect, P < 0.01 H: high intertdial species; L: low intertidal species. 114
Fig. 5. Mean (± 1 SE) superoxide dismutase activity of field samples of Porphyra umbilicalis and P. yezoensis during a 3 hour desiccation. ANOVA: time*species effect, P
< 0.01. H: high intertidal species; L: low intertidal species. 115
Fig. 6. The presence of dehydrin-like protein in the cell extracts of laboratory-grown
(lane 1 to lane 4) and field Porphyra specimens. All extracts were purified by ethanol precipitation as described in Chapter 3. Lane 1: laboratory-grown P. yezoensis extract, without desiccation; lane 2: laboratory-grown P. yezoensis extract, after desiccation; lane
3: laboratory-grown P. umbilicalis extract, without desiccation; lane 4: laboratory-grown
P. umbilicalis extract, after desiccation; lane 5: field P. umbilicalis extract, without desiccation; lane 6: field P. umbilicalis extract, after desiccation; lane 7: field P. yezoensis extract, without desiccation; lane 8: field P. yezoensis extract, after desiccation. Note the presence of dehydrin-like protein in the extracts of field P. umbilicalis (lane 5 and lane 6). 116 Table 1. ANOVA table for tests on water contents during desiccation, the effects of water contents on amino acid leakage, and the effect of water contents on superoxide dismutase activity.
df Sum of squares Mean square F Relative water contents during desiccation speciesa 1 27.19 27.19 2.24 timeb 3 1138.97 379.66 78.87 ** time*species 3 4.88 1.63 0.34
Effect of desiccation on amino acid leakage speciesa 1 5 5 203.42 ** timec 4 6.11 1.53 20.28 ** time*species 4 6.03 1.51 20.03 **
Effect of desiccation on SOD activities speciesa 1 9742.66 9742.66 6487.62 ** timec 4 361.77 90.44 108.98 ** time*species 4 513.06 138.26 154.56 ** aPorphyra umbilicalis and P. yezoensis collected from field b0.5, 1, 2, and 3 hours c0, 0.5, 1, 2, and 3 hours
*: P < 0.05; **: P < 0.01 117 Chapter 5
Discussion
It has been long appreciated that some intertidal seaweeds have greater desiccation tolerance than others, but the exact mechanisms have not been explained.
This was the first study to examine whether the mechanisms that function in desiccation tolerant terrestrial plants, such as ROS defense, repression of membrane phase transition and vitrification, are also at work in macrophytic marine algae. Perhaps the most important finding of this study was to show that reducing molecular mobility in the cytoplasm, in instead of widely believed ROS defense, was the key to the superior desiccation tolerance in P. umbilicalis.
This study has confirmed that high intertidal P. umbilicalis is more desiccation tolerant than the lower intertidal P. yezoensis, and this finding is very similar to what researchers have known for over a century (Baker 1909; Baker 1910; Brown 1987; Abe et al. 2001). As shown in previous studies, the rate of water loss was not related to desiccation tolerance of high intertidal seaweeds (Dorgelo 1976; M. W. Schonbeck & T.
A. Norton 1979; Dromgoole 1980). This study also found that rates of water loss of this two were similar (Fig. 1, Chapter 2; Fig. 1 Chapter 3). Our data revealed that membrane protection was the key difference between these two Porphyra species. The initial indication of membrane damage in rehydrated P. yezoensis was the strong fluorescence in
UV (Fig. 2D, Chapter 3). Red algae have an unique antenna complex called a 118 phycobilisome. Phycobilisomes are attached to chlorophyll a molecules and transfer the light energy they absorb to chlorophyll in the internal reactions of photosynthesis that takes place in Photosystem II. When phycobilisomes are detached from Photosystem II, it shows strong fluorescence in UV. Because of this property, the fluorescence of phycobilisomes has been used to indicate cell damages in red algae (Dudgeon et al.
1989). The fluorescence of rehydrated P. yezoensis indicated that chloroplasts of this species was disrupted after desiccation (Fig. 2D, Chapter 3). The damage to P. yezoensis' chloroplasts was observed by TEM. The control specimens of both species had parallel thylakoid membranes, which were also observed in the rehydrated P. umbilicalis.
However, in the rehydrated P. yezoensis the thylakoid membranes were obviously disrupted (Fig. 4D, Chapter 3). This observation explained the fluorescence of rehydrated
P. yezoensis in UV. The damage to membranes was not limited to thylakoid membranes, because the integrity of plasma membrane of P. yezoensis was also compromised by dehydration, especially when the water content dropped below 0.6 g water / g DW (Fig.
3, Chapter 3). The structural damages correlated well with the loss of function in P. yezoensis. Photosynthesis activity, represented by oxygen evolution, decreased in P. yezoensis as its water content decreased (Fig. 2, Chapter 2). P. yezoensis' photosynthesis activity was virtually non-exist after three hours of drying, when the fluorescence in UV and disruption of thylokoid membranes were observed (Fig. 2 and Fig. 3, Chapter 2).
Mitochondria also have elaborated membranes and it is not surprising that the respiration activity in P. yezoensis was also severely damaged by desiccation. In fact, the electron 119 transfer was not detectable when P. yezoensis was completely desiccated (Fig. 3, Chpater
1). These data showed that P. yezoensis suffered from extensive membrane damage after desiccation and that protection of membranes is the key factor differentiating the ability of these two species to survive desiccation.
ROS defense has been the focus of algal desiccation tolerance studies in the past
(Collén & Davison 1999a; Collén & Davison 1999c; Burritt et al. 2002). However, the present study did not indicate that ROS defense is the key to superior desiccation tolerance in P. umbilicalis. Since ROS accumulation was stimulated by strong light
(Collén & Pedersén 1996), desiccating P. yezoensis in the dark should alleviate membrane damage if ROS defense is the key to the difference between P. umbilicalis and
P. yezoensis. However, my results rejected this hypothesis. The amino acid leakage data showed that the absence of strong light (30 µmol photons · m-2 ·s-1) did not alter the result of desccaion (Fig. 5, Chapter 2). The contents of ROS scavengers also indicated that ROS defense was not the key factor in this study because P. yezoensis had higher contents of
ROS scavengers (Fig. 6 and Fig. 7, Chapter 2). The difference in the findings between this study and those in the past likely results from the source of specimens and how the experiments were performed. The specimens used in this study were cultured in the laboratory for several generations without being exposed to environmental stresses such as dehydration and high light intensities. On the other hand, past studies used specimens collected from the field, where high intertidal species experienced more severe 120 environmental stresses. Because high light intensities alone can cause accumulation of
ROS, the high intertdal species might be induced to accumulate higher ROS scavenger contents in response to their environment. In the past studies, the accumulation of ROS during desiccation was tested in the presence of high light levels, eg. 1600 µmol m-2 s-1
(Collén & Davison 1999b). Although desiccation sensitive species had more ROS at the end of experiments, the data did not support the hypothesis that ROS was the cause the desiccation damage. Disrupted electron transfer chains are the major source of ROS, and strong light just promotes the production of ROS. Therefore, ROS accumulation may simply be the result of desiccation damage instead of the cause of it. In our study, P. yezoensis was severely damaged without the depletion of ROS scavengers and the accumulation of lipid peroxide, a common ROS linked to membrane damage. The results of this study showed that ROS were not responsible for the membrane damage in the desiccated P. yezoensis and ROS defense cannot explain the better desiccation tolerance of P. umbilicalis.
The two most important mechanisms for protecting membranes in the dry state has been shown to be repression of membrane phase transition and vitrification (Oliver et al. 1998). FTIR results of this study showed that the majority of membranes in both desiccated species were still in liquid crystalline state (Fig. 5, Chapter 3). Sugars keep desiccated membranes in liquid crystalline state by direct interaction with phosphate groups of membrane lipids (Oliver et al. 1998) and high polyunsaturated fatty acid levels 121 in the membrane lipids also repress membrane phase transition by hindering the packing of phospholipids (Cevc 1991). Therefore, although P. umbilicalis and P. yezoensis don't have trehalose and sucrose, the high concentration of floridosides (Majak et al. 1966;
Holligan & Drew 1971; McLachlan et al. 1972; Kremer 1982), and high levels of polyunsaturated fatty acids in the membranes (Araki et al. 1986; Fleurence et al.
1994) may also keep the membranes in the fluid state when the seaweeds were dried.
Vitrification seemed a key to the difference between the two species because the shift of the OH stretching band with increasing temperature was mild in the dried P. umbilicalis cell extract (Fig. 6, Chapter 3). However, the critical water content for the membrane integrity of P. yezoensis was about 0.6 g H2O /g DW, which was too high for vitrification to occur (Williams & Leopold 1989). Because of this observation and the fact that vitrification is believed to protect desiccated cells by impeding the approach of subcellular structures, it was necessary to compare the molecular mobility in the cells of these two species during dehydration. The EPR data showed that molecular mobility in the cells of P. umbilicalis was higher than that of P. yezoensis during dehydration, and the biggest difference happened around 0.7 g H2O /g DW (Fig. 7, Chapter 3), which is very close to the critical water content for membrane integrity of P. yezoensis mentioned above. These two observations suggests that low molecular mobility is very important to protect membranes in the drying cells and that the difference occurred before vitrification happened. Cytoplasmic glass is a special case of high viscosity in cytoplasm because it is formed when the water content is lower than 0.1g H2O /g DW (Hoekstra et al. 2001). 122 Therefore, the existence of cytoplasmic glass itself should not be the predictor of desiccation tolerance. Instead, it is important to focus on the properties of glass related to high viscosity or the reduction of molecular mobility in the cytoplasm directly.
Sugars were probably not the key to the low molecular mobility in the drying P. umbilicalis cells because P. yezoensis actually had higher sugar contents than P. umbilicalis (Holligan & Drew 1971; McLachlan et al. 1972). However, dehydrin might be the key molecule. Wong (2009) found the existence of a dehydrin-like protein in P. umbilicalis but not in P. yezoensis. This protein was constitutively expressed, as the P. umbilicalis specimen was not exposed to desiccation before protein extraction. Dehydrins belong to the D11 family of LEA proteins and may play an important role in desiccation tolerance because glasses found in desiccation tolerant seeds that are rich in LEA proteins have lower molecular mobility than the pure sugar glasses (Wolkers et al. 1998; Buitink et al. 2000). Dehydrins can reduce molecular mobility and participate in the formation of strong glasses by binding tightly to sugars and forming intracellular filamentous networks during desiccation (Goyal et al. 2003; Walters et al. 2008).
In conclusion, this study showed that keeping molecular mobility below a threshold is the a factor differentiating the ability of P. umbilicalis and P. yezoensis to survive desiccation, but ROS defense and repression of membrane phase transition are also required for desiccation tolerance. In case one might question whether the results 123 obtained from laboratory-grown plants are not found in the field plants, field collected P. umbilicalis and P. yezoensis were also compared for rates of water loss, amino acid leakage, superoxide dismutase activities, and the presence of dehydrin-like proteins. The field collected P. umbilicalis and P. yezoensis showed the same differences as those observed in the laboratory-grown P. umbilicalis and P. yezoensis. Combined with the data of Ik determination, the data in Chapter 4 show that the differences observed in the laboratory-grown P. umbilicalis and P. yezoensis were not due to the culture conditions or nutrient limitation, and that the findings of this study can explain the differences in the desiccation tolerance of field collected P. umbilicalis and P. yezoensis. 124 References
Abe, S. et al., 2001. The cellular ability of desiccation tolerance in Japanese intertidal seaweeds. Botanica Marina, 44(2), 125-131.
Allagulova, C.R. et al., 2003. The Plant Dehydrins: Structure and Putative Functions. Biochemistry (Moscow), 68(9), 945-951.
Araki, S. et al., 1986. Lipid and fatty acid composition in the red alga Porphyra yezoensis. Japanese Journal of Phycology, 34, 94-100.
Araki, S. et al., 1987. Positional Distribution of Fatty Acids in Glycerolipids of the Marine Red Alga, Porphyra yezoensis. Plant Cell Physiol, 28(5), 761-766.
Asamizu, E. et al., 2003. COMPARISON OF RNA EXPRESSION PROFILES BETWEEN THE TWO GENERATIONS OF PORPHYRA YEZOENSIS (RHODOPHYTA), BASED ON EXPRESSED SEQUENCE TAG FREQUENCY ANALYSIS. Journal of Phycology, 39(5), 923-930.
Baker, S.M., 1909. On the causes of the zoning of brown seaweeds on the seashore. New Phytologist, 8(5/6), 196-202.
Baker, S.M., 1910. On the Causes of the Zoning of Brown Seaweeds on the Seashore. II. The Effect of Periodic Exposure on the Expulsion of Gametes and on the Germination of the Oospore. New Phytologist, 9(1/2), 54-67.
Bass, D. et al., 1983. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol, 130(4), 1910-1917.
Bianchi, G. et al., 1993. The unusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia. Physiologia Plantarum, 87, 223-226.
Black, M. & Pritchard, H., 2002. Desiccation and Survival in Plants : Drying Without Dying, CABI Publishing.
Bob B. Buchanan, Wilhelm Gruissem & Russell L. Jones, 2000. Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists.
Bochicchio, A. et al., 1994. Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: The questionable role of endogenous abscisic acid. Physiologia Plantarum, 91(4), 615-622. 125
Bose, S., Herbert, S.K. & Fork, D.C., 1988. Fluorescence Characteristics of Photoinhibition and Recovery in a Sun and a Shade Species of the Red Algal Genus Porphyra. Plant Physiol, 86(3), 946-950.
Bota, J., Medrano, H. & Flexas, J., 2004. Is photosynthesis limited by decreased Rubisco activity and RuBP content under progressive water stress? New Phytologist, 162(3), 671-681.
Boyer, J., 1982. Plant productivity and environment. Science, 218, 443-448.
Brown, M., 1987. Effects of desiccation on photosynthesis of intertidal algae from a southern New Zealand shore. Botanica Marina, 30, 121-127.
Buitink, J. et al., 2000. Molecular mobility in the cytoplasm: An approach to describe and predict lifespan of dry germplasm. PNAS, 97(5), 2385-2390.
Burger, G. et al., 1999. Complete Sequence of the Mitochondrial DNA of the Red Alga Porphyra purpurea: Cyanobacterial Introns and Shared Ancestry of Red and Green Algae. Plant Cell, 11(9), 1675-1694.
Burritt, D.J., Larkindale, J. & Hurd, C.L., 2002. Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation. Planta, 215(5), 829- 38.
Campbell, S.A. & Close, T.J., 1997. Dehydrins: Genes, proteins, and associations with phenotypic traits. New Phytologist, 137(1), 61-74.
Cevc, G. & Richardsen, H., 1999. Lipid vesicles and membrane fusion. Advanced Drug Delivery Reviews, 38, 207-232.
Cevc, G., 1991. How membrane chain-melting phase-transition temperature is affected by the lipid chain asymmetry and degree of unsaturation: an effective chain-length model. Biochemistry, 30(29), 7186-7193.
Chaerle, L., Saibob, N. & Van Der Straeten, D., 2005. Tuning the pores: towards engineering plants for improved water use efficiency. Trends in Biotechnology, 23, 308-315.
Chaves, M.M. & Oliveira, M.M., 2004. Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J. Exp. Bot, 55(407), 2365-2384. 126 Chen, L., 1989. Cell suspension culture from Porphyra linearis (Rhodophyta) a multicellular marine red alga. Journal of Applied Phycology, 1, 153-159.
Choo, K., Snoeijs, P. & Pedersén, M., 2004. Oxidative stress tolerance in the filamentous green algae Cladophora glomerata and Enteromorpha ahlneriana. Journal of Experimental Marine Biology and Ecology, 298(1), 111-123.
Close, T.J., 1996. Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins. Physiologia Plantarum, 97, 795-803.
Collén, J. & Davison, I., 1999a. Reactive oxygen metabolism in intertidal Fucus spp. (Phaeophyceae). Journal of Phycology, 35(1), 62-69.
Collén, J. & Davison, I., 1999b. Reactive oxygen production and damage in intertidal Fucus spp. (Phaeophyceae). Journal of phycology, 35, 54-61.
Collén, J. & Davison, I., 1999c. Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus. Plant Cell and Environment, 22(9), 1143-1151.
Collén, J. & Pedersén, M., 1996. Production, scavenging and toxicity of hydrogen peroxide in the green seaweed Ulva rigida. European Journal of Phycology, 31(3), 265-271.
Craigie, J., McLachlan, J. & Tocher, R., 1968. Some neutral constituents of the rhodophyceae with special reference to the occurence of the floridosides. Canadian Journal of Botany, 46, 605-611.
Crowe, J.H. et al., 1987. Stabilization of dry phospholipid bilayers and proteins by sugars. The Biochemical Journal, 242(1), 1-10.
Crowe, J.H., Hoekstra, F.A. & Crowe, L.M., 1992. Anhydrobiosis. Annual Review of Physiology, 54, 579-99.
Crowe, J.H., Hoekstra, F.A. & Crowe, L.M., 1989. Membrane Phase Transitions are Responsible for Imbibitional Damage in Dry Pollen. PNAS, 86(2), 520-523.
Dace, H. et al., 1998. Use of metabolic inhibitors to elucidate mechanisms of recovery from desiccation stress in the resurrection plant Xerophyta humilis. Plant Growth Regulation, 24(3), 171-177.
Davison, I. & Pearson, G., 1996. Stress tolerance in intertidal seaweeds. Journal of 127 Phycology, 32, 197-211.
Dorgelo, J., 1976. Intertidal fucoid zonation and desiccation. Aquatic Ecology, 10, 115- 122.
Dring, M. & Brown, F., 1982. Photosynthesis of Intertidal Brown Algae During and After Periods of Emersion: A Renewed Search for Physiological Causes of Zonation. Marine Ecology Progress Series, 8, 301-308.
Dromgoole, F.I., 1980. Desiccation resistance of intertidal and subtidal algae. Bot. Mar, 23, 149-159.
Dudgeon, S.R., Davison, I.R. & Vadas, R.L., 1989. Effect of freezing on photosynthesis of intertidal macroalgae: relative tolerance of Chondrus crispus and Mastocarpus stellatus (Rhodophyta). Marine Biology, 101(1), 107-114.
Espinoza, J. & Chapman, A.R.O., 1983. Ecotypic differentiation of Laminaria longicruris in relation to seawater nitrate concentration. Marine Biology, 74(2), 213-218.
Fleurence, J. et al., 1994. Fatty acids from 11 marine macroalgae of the French Brittany coast. Journal of Applied Phycology, 6(5), 527-532.
Flexas, J. et al., 2006. Keeping a positive carbon balance under adverse conditions: responses of photosynthesis and respiration to water stress. Physiologia Plantarum, 127(3), 343-352.
Fukuda, S. et al., 2003. Isolation and characterization of an elongation factor-1alpha gene in Porphyra yezoensis (Rhodophyta). Journal of Applied Phycology, 15(1), 81-86.
Gao, K. & Aruga, Y., 1987. Preliminary studies on the photosynthesis and respiration of Porphyra yezoensis under emersed conditions. Journal of the Tokyo University of Fisheries, 47, 51-65.
Golovina, E.A., Hoekstra, F.A. & Hemminga, M.A., 1998. Drying increases intracellular partitioning of amphiphilic substances into the lipid phase: Impact on membrane permeability and significance for desiccation tolerance. Plant Physiology (Rockville), 118(3), 975-986.
Goyal, K. et al., 2003. Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein. Journal of Biological Chemistry, 278(15), 12977-12984. 128 Graham, L., 2003. Polyunsaturated fatty acid production in the red alga Porphyra. Master's Thesis, Northeastern University.
Guobin, F. & Chen, S., 2006. Water Crisis in the Yellow River: Facts, Reasons, Impacts, and Countermeasures. Water Practice & Technology, 1, 1-8.
Halliwell, B. & Chirico, S., 1993. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr, 57(5), 715S-724.
Harker, M. et al., 1999. Effects of High Light and Desiccation on the Operation of the Xanthophyll Cycle in Two Marine Brown Algae. European Journal of Phycology, 34(01), 35-42.
Hincha, D., Zuther, E. & Heyer, A., 2003. The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions. Biochim Biophys Acta., 10, 172-177.
Hodgson, L.M., 1981. Photosynthesis of the red alga Gastroclonium coulteri (Rhodophyta) in response to changes in temperature, light intensity, and desiccation. Journal of Phycology, 17(1), 37-42.
Hoekstra, F., Golovinab, E. & Buitink, J., 2001. Mechanisms of plant desiccation tolerance. Trends in Plant Science, 6, 431-438.
Hoekstra, F.A., Crowe, J.H. & Crowe, L.M., 1991. Effect of sucrose on phase behavior of membranes in intact pollen of Typha latifolia L., as measured with fourier transform infrared spectroscopy. Plant Physiol, 97(3), 1073-1079.
Holligan, P.M. & Drew, E.A., 1971. Routine analysis by gas-liquid chromatography of soluble carbohydrates in extracts of plant tissues. II. Quantitative analysis of standard carbohydrates, and the separation and estimation of soluble sugars and polyols from a variety of plant tissues. New Phytologist, 70(2), 271-297.
Johansson, G. & Snoeijs, P., 2002. Macroalgal photosynthetic responses to light in relation to thallus morphology and depth zonation. Marine Ecology Progress Series, 244, 63–72.
Kapraun, D. & Luster, D., 1980. Field and culture studies of Porphyra rosengurtii Coll et Cox (Rhodophyta, Bangiales) from North Carolina. Botanica Marina, 23, 449- 457.
Karsten, U., Barrow, K.D. & King, R.J., 1993. Floridoside, L-Isofloridoside, and D- 129 Isofloridoside in the Red Alga Porphyra columbina (Seasonal and Osmotic Effects). Plant Physiol, 103(2), 485-491.
Kayama, M. et al., 1985. Effect of water temperature on the fatty acid composition of Porphyra. Bulletin of the Japanese Society of Scientific Fisheries, 51, 687.
Kim, J.K., Kraemer, G.P. & Yarish, C., 2008. Physiological activity of Porphyra in relation to eulittoral zonation. Journal of Experimental Marine Biology and Ecology, 365(2), 75-85.
Kitade, Y. et al., 2002. Isolation of a cDNA encoding a homologue of actin from Porphyra yezoensis (Rhodophyta). Journal of Applied Phycology, 14(2), 135-141.
Kitade, Y. et al., 1998. Porphyra monospore system (Bangiales, Rhodophyta): A model for the developmental biology of marine plants. Phycological Research, 46, 17- 20.
Kitade, Y., Yamazaki, S. & Saga, N., 1996. A Method for extraction of high molecular weight DNA from the macroalga Porphyra yezoensis (Rhodophyata). Journal of Phycology, 32(3), 496-498.
Kogan, F.N., 1997. Global Drought Watch from Space. Bulletin of the American Meteorological Society, 78, 621-636.
Kornmann, P. & Sahling, P., 1991. The Porphyra species of Helgoland (Bangiales, Rhodophyta). Helgolõnder Meeresuntersuchungen, 45, 1-38.
Kranner, I. et al., 2002. Revival of a resurrection plant correlates with its antioxidant status. Plant Journal, 31(1), 13-24.
Kremer, B.P., 1982. Biosynthesis of photosynthates and taxonomy of algae. Z Naturforsch, 37c, 761-771.
Kubler, J., Minocha, S. & Mathieson, A., 1994. Transient expression of the GUS reporter gene in protoplasts of Porphyra miniata (Rhodophyta). Journal of Marine Biotechnolgy, 1, 165-169.
Leprince, O. et al., 2000. Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles. Plant Physiology (Rockville), 122(2), 597-608.
Lipkin, Y., Beer, S. & Eshel, A., 1993. The ability of Porphyra linearis (Rhodophyta) to 130 tolerate prolonged periods of desiccation. Botanica Marina, 36, 517-523.
Liu, H. et al., 2003. Increasing the transient expression of GUS gene in Porphyra yezoensis by 18S rDNA targeted homologous recombination. Journal of Applied Phycology, 15(5), 371-377.
Liu, R. et al., 2006. Risk assessment model of drought-caused winter wheat yield loss and its applications in Henan Province. Proceedings of SPIE, 6031, 6310U1-6301U9.
Lobban, C. & Harrison, P., 1994. Seaweed ecology and physiology, Cambridge University Press.
Majak, W., Craigie, J.S. & McLachlan, J., 1966. Photosynthesis in algae: I. Accumulation products in the rhodophyceae. Canadian Journal of Botany, 44(5), 541-549.
McLachlan, J. et al., 1972. Porphyra linearis Grev: An edible species of nori from Nova Scotia. Proc Int Seaweed Symp, 7, 473-476.
Mitman, G. & Van der Meer, J.P., 1994. Meiosis, blade development, and sex determination in Porphyra purpurea (Rhodophyta). Journal of phycology, 30, 147- 159.
Mittler, R., 2006. Abiotic stress, the field environment and stress combination. Trends in Plant Science, 11(1), 15-19.
Mizukami, Y. et al., 2004. Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis. Journal of applied phycology, 16, 23-29.
Moebus, K., Johnson, K.M. & Sieburth, J.M., 1974. Rehydration of desiccated intertidal brown algae: Release of dissolved organic carbon and water uptake. Marine Biology, 26(2), 127-134.
Neefus, C.D. et al., 2008. The distribution, morphology, and ecology of three introduced Asiatic species of Porphyra (Bangiales, Rhodophyta) in the Northeastern Atlantic. Journal of Phycology, 44(6), 1399-1414.
Nikaido, I. et al., 2000. Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis. DNA Res, 7(3), 223-7.
Okauchi, M. & Mizukami, Y., 1999. Transient beta-glucuronidase (GUS) gene expression under control of CaMV 35S promoter in Porphyra tenera (Rhodophyta). Bulletin of National Research Institute of, Aquaculture(Suppl. 1), 13-18. 131
Oliver, A. et al., 1998. Interactions of arbutin with dry and hydrated bilayers. Biochim Biophys Acta., 1370, 87-97.
Oliver, A.E., Crowe, L.M. & Crowe, J.H., 1998. Methods for dehydration-tolerance: Depression of the phase transition temperature in dry membranes and carbohydrate vitrification. Seed Science Research, 8(02), 211-221.
Oliver, M.J., Tuba, Z. & Mishler, B.D., 2000. The evolution of vegetative desiccation tolerance in land plants. Plant Ecology, 151(1), 85-100.
Oliver, M.J., Velten, J. & Mishler, B.D., 2005. Desiccation Tolerance in Bryophytes: A Reflection of the Primitive Strategy for Plant Survival in Dehydrating Habitats? Integr. Comp. Biol., 45(5), 788-799.
Oliver, M.J. & Bewley, J.D., 1997. Desiccation-Tolerance of Plant Tissues: A Mechanistic Overview. HORTICULTURAL REVIEWS, 18, 171-214.
Parry, M., Flexas, J. & Medrano, .., 2005. Prospects for crop production under drought: research priorities and future directions. Annals of Applied Biology, 147(3), 211- 226.
Phillips, D., 2001. The top ten Canadian weather stories for 2001. CMOS Bull., 30, 19- 23.
Porembski, S. & Barthlott, W., 2000. Granitic and gneissic outcrops (inselbergs) as centers of diversity for desiccation-tolerant vascular plants. Plant Ecology, 151(1), 19-28.
Proctor, M.C.F. & Tuba, Z., 2002. Poikilohydry and homoihydry: Antithesis or spectrum of possibilities? New Phytologist, 156(3), 327-349.
Reith, M. & Munholland, J., 1993. A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea. Plant Cell, 5(4), 465-475.
Rosen, H., 1957. A modified ninhydrin colorimetric analysis for amino acids. Archives of Biochemistry and Biophysics, 67, 10-15.
Schonbeck, M. & Norton, T.A., 1978. Factors controlling the upper limits of fucoid algae on the shore. Journal of Experimental Marine Biology and Ecology, 31(3), 303- 313. 132 Schonbeck, M.W. & Norton, T.A., 1979. An investigation of drought avoidance in intertidal fucoid algae. Botanica marina, 22(3).
Scott, P., 2000. Resurrection Plants and the Secrets of Eternal Leaf. Ann Bot, 85(2), 159- 166.
Sibbald, P. & Vidaver, W., 1987. Photosystem I-mediated regulation of water splitting in the red alga, Porphyra sanjuanensis. Plant Physiology, 84, 1373-1377.
Smirnoff, N., 1993. Tansley Review No. 52: The role of active oxygen in the response of plants to water deficit and desiccation. New Phytologist, 125(1), 27-58.
Smith, C.M., Satoh, K. & Fork, D.C., 1986. The Effects of Osmotic Tissue Dehydration and Air Drying on Morphology and Energy Transfer in Two Species of Porphyra. Plant Physiol, 80(4), 843-847.
Southward, A., 1958. THE ZONATION OF PLANTS AND ANIMALS ON ROCKY SEA SHORES. Biological Reviews, 33(2), 137-177.
Stiller, J. & Hall, B., 1998. Sequences of the largest subunit of RNA polymerase II from two red algae and their implications for rhodophyte evolution. Journal of Phycology, 34(5), 857-864.
Su, J. & Hassid, W., 1962. Carbohydrates and Nucleotides in the Red Alga Porphyra perforata. I. Isolation and Identification of Carbohydrates. Biochemistry, 1, 468- 474.
Sun, W.Q. & Leopold, A.C., 1997. Cytoplasmic vitrification and survival of anhydrobiotic organisms. Comparative Biochemistry & Physiology A- Comparative Physiology, 117(3), 327-333.
Sun, W. et al., 1996. Stability of dry liposomes in sugar glasses. Biophys. J, 70(4), 1769- 1776.
Tajiri, S. & Aruga, Y., 1984. Effect of emersion on the growth and photosynthesis of the Porphyra yezoensis thallus. Japanese Journal of Phycology, 32, 134-146.
Thomas, T.E., Turpin, D.H. & Harrison, P.J., 1987. Desiccation enhanced nitrogen uptake rates in intertidal seaweeds. Marine Biology, 94(2), 293-298.
Tuba, Z. et al., 1993. Regreening of desiccated leaves of the poikilochlorophyllous Xerophyta scabrida upon rehydration. Journal of Plant Physiology, 142(1), 103- 133 108.
Walters, C., Ried, J.L. & Walker-Simmons, M.K., 2008. Heat-soluble proteins extracted from wheat embryos have tightly bound sugars and unusual hydration properties. Seed Science Research, 7(02), 125-134.
Wiencke, C. & Lõuchli, A., 1980. Growth, cell volume, and fine structure of Porphyra umbilicalis in relation to osmotic tolerance. Planta, 150, 303-311.
Williams, R.J. & Leopold, A.C., 1989. The glassy state in corn embryos . Plant Physiol., 89(3), 977-981.
Wolkers, W. et al., 2001. Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1544(1-2), 196-206.
Wolkers, W.F. et al., 1998. Properties of proteins and the glassy matrix in maturation- defective mutant seeds ofArabidopsis thaliana. The Plant Journal, 16(2), 133-143.
Wolkers, W.F. et al., 2004. A fourier-transform infrared spectroscopy study of sugar glasses. Carbohydrate research, 339(6), 1077-1085.
Wolkers, W.F. et al., 1999. Changed properties of the cytoplasmic matrix associated with desiccation tolerance of dried carrot somatic embryos. An in situ fourier transform infrared spectroscopic study. Plant Physiol, 120(1), 153-164.
Wong, J., 2009. Dehydrin-like proteins in desiccation tolerance in intertidal seaweeds. Master thesis. Northeastern University.
Yamazaki, A., Nakanishi, K. & Saga, N., 1998. Axenic tissue culture and morphogenesis in Porphyra yezoensis (Bangiales, Rhodophyta). Journal of Phycology, 34(6), 1082-1087.
Yap, C.K. et al., 2005. Nitrate Concentrations in the Surface Seawater of the Straits of Malacca. Asian Journal of Water, Environment and Pollution, 2(2), 45–49.
Zaneveld, J.S., 1937. The Littoral Zonation of Some Fucaceae in Relation to Desiccation. Journal of Ecology, 25(2), 431-468.
Zaneveld, J.S., 1969. Factors controlling the delimitation of littoral benthic marine algal zonation. Amer. Zool., 9(2), 367-391. 134 Zhang, J. & Fischer, C.J., 2006. A simplified resorcinol method for direct spectrophotometric determination of nitrate in seawater. Marine Chemistry, 99(1- 4), 220-226.
Zhu, H. et al., 1994. Oxidation pathways for the intracellular probe 2′,7′- dichlorofluorescin. Archives of Toxicology, 68(9), 582-587.