Ecophysiological Needs of the Potential Biomass Crop Spartina Townsendii Grov

KOYRO & HUCHZERMEYER 123

Tropical Ecology 45(1): 123-139, 2004 ISSN 0564-3295 © International Society for Tropical Ecology

Ecophysiological needs of the potential biomass crop Spartina townsendii Grov.

HANS-WERNER KOYRO1 & BERNHARD HUCHZERMEYER2

1Institute for Plant Ecology, Justus Liebig University Gießen, Heinrich–Buff–Ring 26-32, D-35392, Giessen; 2Botany Institute, Hannover University, Herrenhäuser Str. 2, D-30 419 Hannover

Abstract: The aim of this study was to determine the level of salinity tolerance of Spartina townsendii and to study the mechanisms by which this species survives salinity and the specific conditions at the intertidal zone. Therefore, Spartina was irrigated with five different saline levels ranging from nutrient solution (control) to 100% seawater (480 mM NaCl) in a “quick check” system (QCS). The salinity tolerance was measured on the basis of the two parameters maximum yield, and gas exchange. a) The maximum yield of Spartina was reduced by 50% (C50-value) at 480 mM NaCl salinity. b) Gas exchange parameters such as net photosynthesis (µmol * m-2 * s-1), stomatal conductance (mol * m-2 * s-1), transpiration (mol * m-2 * s-1), and water use efficiency of the photosynthesis (µmol CO2 * mmol-1 H2O) were lowest at 500 mM NaCl-salinity. The strategies employed by Townsend´s cordgrass for avoiding salt injury depend on adaptation to low water potential and high Na and Cl concentrations. Spartina has sufficient adjustment mechanisms even at high NaCl salinity suggesting that there was no reason for growth reduction by water deficit. The low external water potential was balanced e.g. by a decrease of the osmotic potential. Especially Na and Cl were accumulated in high concentrations in the root and shoot. The main defence of Spartina townsendii to elevated salinity regimes is the activation of salt glands. These salt glands are working highly selective and eliminate relatively large quantities of salt by secretion to the leaf surface. However, the salt glands were neither able to balance the high burden especially of Na in the leaf tissues at the high salinity treatment, nor useful in maintaining a constant supply of essential elements such as K, Mg and Ca. The gap between a sufficient nutrient supply and NaCl accumulation grows with increasing salinity and seems to limit the salinity tolerance of Spartina by nutrient imbalance and/or ion toxicity. The high salinity tolerance and the ecological engineering of Spartina townsendii can produce various ecological (e.g. highly effective nutrient cycling of N, Fe and S) and economical (e.g. biomass crop) benefits. The QCS offers a reliable basis to define this species as a potentially useful cash crop halophyte.

Resumen: El propósito de este estudio fue determinar el nivel de tolerancia a la salinidad de Spartina townsendii y estudiar los mecanismos que permiten a esta especie sobrevivir a la salinidad y a las condiciones específicas de la zona intermareal. Para ello, Spartina fue regada con cinco diferentes niveles salinos, variando desde solución de nutrientes (control) hasta agua de mar al 100% (480 mM NaCl) en un sistema de verificación rápida (QCS por sus siglas en in- glés). Las estrategias utilizadas por esta planta (conocida como ‘pasto cuerda de Townsend’) para evitar el daño por la sal dependen de la adaptación a un bajo potencial hídrico y a altas concentraciones de Na y Cl. Spartina tiene suficientes mecanismos de ajuste, incluso con una alta salinidad de NaCl, lo que sugiere que no existe ninguna razón para una reducción del creci- miento debida a un déficit hídrico El bajo potencial hídrico externo estuvo balanceado, p.ej. por un decremento del. potencial osmótico. En especial, el Na y el Cl se acumularon en grandes concentraciones tanto en la raíz como en la parte aérea. La principal defensa de Spartina townsendii contra regímenes de alta salinidad es la activación de glándulas de sal que eliminan can-

124 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

tidades relativamente grandes de sal por secreción hacia la superficie foliar. La brecha entre una suficiente disponibilidad de nutrientes y la acumulación de NaCl crece conforme aumenta la salinidad y parece limitar la tolerancia a la salinidad de Spartina por un desequilibrio nutri- cional y/o por toxicidad de iones. La alta tolerancia a la salinidad y la ingeniería ecológica de Spartina townsendii pueden producir varios beneficios ecológicos (p.ej. un reciclaje muy eficien- te de N, Fe y S) y económicos (p.ej. cosecha de biomasa). El QCS ofrece una base confiable para definir a esta especie como una halófita potencialmente útil como cultivo comercial.

Resumo: O objectivo deste estudo foi o de determinar o nível de tolerância da Spartina townsendii ao sal e estudar os mecanismos pelos quais esta espécie sobrevive à salinidade e as condições específicas na zona entre marés. Nesse sentido a Spartina foi irrigada com cinco níveis diferentes de solução salina num intervalo que variou entre uma solução nutriente (con- trolo) a 100% de água do mar (480 mM NaCl) utilizando um sistema de “avaliação rápida” (QCS). As estratégias empregues por “Townsend’s cordgrass” para evitar os danos do sal de- pende na adaptação ao baixo potencial hídrico e elevada concentração em Na e Cl. A Spartina tem mecanismos de adaptação suficientes, mesmo para valores elevados de NaCl, sugerindo que não há razão para a redução do crescimento pelo deficit hídrico. O baixo potencial hídrico externo foi balanceado, p.e. por um decréscimo do potencial osmótico. O Na e o Cl foram espe- cialmente acumulados em elevadas concentrações nas raízes e nos lançamentos. A defesa principal da Spartina townsendii para regiões salinas elevadas é a activação das glândulas de sal as quais eliminam quantidades relativamente elevadas de sal por secreção da superfície das folhas. A diferença entre uma oferta nutritiva suficiente e a acumulação de NaCl cresce com o acréscimo de salinidade e parece limitar a tolerância à salinidade da Spartina por um dese- quilíbrio nos nutrientes e/ou toxicidade iónica. A elevada tolerância ao sal e o “engineering” ecológico da Spartina townsendii pode produzir vários benefícios ecológicos (p.e. reciclagem al- tamente efectiva de nutrientes quanto ao N, Fe e S) e económicos (p.e. produção de biomassa). O QCS oferece uma base fiável para definir esta espécie como uma cultura halófita potencialmente útil.

Key words: Spartina townsendii, cash crop halophytes, gas exchange, NaCl salinity, nutrients, “quick check” system, salinity tolerance, water relations, water potential.

Introduction increasing shortage of living space and availability of freshwater. It is, therefore, necessary to develop Background information sustainable systems in presently waste or deserted areas. Besides the naturally occurring salt-affected The economic basis of the existence of more soils, the extent of man-made salinized soils as a than 1 billion of people in 100 countries is threat- consequence of improper irrigation management is ened by the expansion of the deserts. Every year 6 significant (Choukr-Allah 1996). It should be noted million ha arable land are lost for agricultural use that salinity problems are liable to spread as irriga- in developing countries. Reasons for the desertifica- tion is intensified and irrigated areas are extended tion are climatic changes, intensive pasture, defor- (Choukr-Allah & Harrouni 1996). Therefore, a new estation and unsuited irrigation practices. The ex- concept of saline irrigation had to be developed and tension of irrigated agriculture and the intensive the research into management practices with salt use of water resources combined with high rates of resistant species became increasingly essential. The evaporation in arid and semiarid regions, have in- technology is available now to use unconventional evitably given rise to the problems of salinity in the water resources or habitats for salinity-tolerant soil and in underground water. Among the most plant production systems for many purposes. pressing problems for the growing mankind are the

KOYRO & HUCHZERMEYER 125

Introduction of halophytes component of new coastal management practices and useful in developing strategies for the stabili- The standard approach to this problem would zation of deteriorating marshes (in marsh restora- be to increase salinity salt tolerance of conven- tion projects; Lieth 1999; Miller et al. 2001; tional crop plants, but the gained yield is generally Pezeshki & DeLaune 1997; Simas et al. 2001), low (Koyro & Huchzermeyer 1999b). The alterna- - can tolerate oil spills (its growth is even tive approach is to make use of the plants that al- stimulated by crude oils) and are hosts of microbial ready have the required level of salt tolerance, and degraders promoting oil spill cleanup in coastal are still productive at high external salinity levels: wetlands (Lin et al. 1999; Lindau et al. 1999; Ny- the halophytes. Halophytes are plants, able to man 1999; Pezeshki et al. 2001; Smith & Proffitt complete their life cycle in a substrate rich in NaCl 1999), (Lieth 1999; Schimper 1891). This substrate offers - support biodiversity and the production of advantages for obligate halophytes in the competi- marsh fauna (e.g. fishes, benthic invertebrates; tion with salt sensitive-plants (glycophytes). Angradi et al. 2001; Connolly 1999; Riera et al. Some of 2600 species of halophytes occur in sa- 1999; San Leon et al. 1999; Waide et al. 1999; line coastal environment and inland deserts. In- Weinstein et al. 2000), creasing attention has been paid to research and - support bioremediation of recalcitrant com- development of halophytes and several authors plex carbohydrate biopolymers by marine bacteria proposed utilising undiluted seawater on a large (Ensor et al. 1999). scale for irrigation (Koyro & Huchzermeyer 1999a; Spartina itself Lieth & Al Masoom 1993). The sustainable use of - is a potential biomass crop (e.g. grown for halophytic plants is a promising approach to valor- fodder; Beale et al. 1999; Lieth 1999) in poor soil ize strongly salinised zones unsuitable for conven- conditions, tional agriculture and mediocre waters (Boer & - is highly effective in nutrient cycling (e.g. N- Gliddon 1998; Choukr-Allah 1996; Lieth et al. fixation, Fe-reduction, sulfate-reduction, sulfide- 1999; Pasternak 1990). There are already many oxidation, Se-biotransformation to DMSeP, Si- halophytic species used for economic interests reservoir; Ansede et al. 2001; de Bakker et al. (human food, fodder) or ecological reasons (soil de- 1999; Hines et al. 1999; Lee 1999; Norris & Hack- salinisation, dune fixation, CO2-sequestration). ney 1999), One plant species with a high potential to be- - reduces toxic metal bioavailability (e.g. Cd, come a cash crop halophyte is Spartina townsendii. Pb, Cu, Cr, Hg and Zn), by sequestering a larger Townsend´s Cordgrass was first found at the end proportion of its metal burden in its belowground of the 19th century on intertidal mud and sand tissues which are likely to be permanently buried flats at the coasts of the English Channel. It was (Burke et al. 2000; Heller & Weber 1998; Klap et bigger than the native Spartina alternifolia and al. 1999; Patra & Sharma 2000; Windham et al. colonised these sites immediately. Halophytes such 2001), as Spartina alterniflora (Cordgrass) and Spartina - is a biomonitor for environmental toxicants townsendii (Townsend´s Cordgrass) introduce the from municipal and industrial wastes, agricultural intertidal zone of temperate estuaries and lagoons runoff, recreational boating, shipping and coastal as a sustainable system for cash crop halophytes development (Lewis et al. 2001; Lytle & Lytle (Beale et al. 1999). Salt marshes dominated by 2001; Padinha et al. 2000), Spartina species are among the most productive - is used as an indicator for estuarine sediment ecosystems known, despite nitrogen limitation quality (Lewis et al. 2001), (Bagwell & Lovell 2000). Spartina ecological engi- Biomineral liquids extracted from Spartina neering can produce various ecological and eco- culms have a number of health functions (e.g. car- nomic benefits (Qin et al. 1998): diotonic, enhance of life span), the total flavonoids Salt marshes dominated by Spartina species: of Spartina can be separated and used to resist - help to reduce atmospheric CO2 enrichment blood coagulation and encephalon thrombus (Qin (Matamala & Drake 1998), et al. 1998). - have a low vulnerability against sea level The C-4 perennial grass Spartina townsendii change and protect the estuaries against the ef- and other species of this genus are potential bio- fects of global changes, are ‘therefore’ an important

126 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII mass crops. However, it is necessary to produce the necessary preconditions for a reliable quick utilisable biomass with unconventional irrigation check system (QCS). (up to seawater salinity) in a way that is ecologi- cally sustainable and economically feasible. The threshold of salinity tolerance Spartina spec. is also a perennial grass and Generally, classification of the salinity toler- dreaded for its impact on estuarine flora and ance (or sensitivity) of crop species is based on the fauna. Spartina can rapidly colonize the intertidal threshold EC (electrical conductivity) and the per- zone of temperate estuaries and lagoons. Conse- centage of yield decrease beyond threshold quently beside all economic interests there is con- (Greenway & Munns 1980; Marschner 1995). The siderable concern about its impact on estuarine substrate concentration leading to a growth de- flora and fauna especially in regions where its not pression of 50% (reference to fresh weight, in com- an indigenous species (Hedge & Kriwoken 2000). parison to plants without salinity) is widely used Therefore, detailed information are essential for by ecophysiologists as a definition for the thresh- the controlled establishment especially in regions old of salinity tolerance (Kinzel 1982), because it is where its not a native species. Current theories as difficult to fix the upper limit of salinity toler- differ in their prediction concerning the effects of ance. The agreement to the above-mentioned interspecific interaction on species growth and dis- growth depression is comparatively arbitrary, but tribution along environmental gradients (La Peyre it leads to a precise specification of a comparative et al. 2001). The goal of this research is to provide value for halophytic species and is especially ex- a reliable, ecophysiological basis as the first step pressive for applied aspects such as economic po- for the development of a ecological sustainable and tentials of suitable halophytes. economically feasible cash crop Spartina town- The threshold of salinity tolerance according to sendii. Kinzel (1982) is used in the quick check system (QCS) for halophytes as an objective parameter for Reproducable experimental growth and the description of the actual condition of a plant substrate conditions (Ashraf & O´Leary 1996; Koyro 2000). There are There are major problems to be overcome in now reliable informations available about studies determining and selecting the best species and with several species such as Plantago cf. ecotypes to be used in the vast areas of degraded coronopus, Beta vulgaris ssp. maritima, Laguncu- salt-affected soil or other saline habitats around laria racemosa and Batis maritima (Koyro & the world. A precondition for a sustainable utilisa- Huchzermeyer 1997, 1999a; Koyro et al. 1999; tion of suitable halophytes is the precise knowl- Koyro 2000). The substrate concentration leading edge about their salinity tolerance and the various to a growth depression of 50% (refer to fresh- mechanisms enabling a plant to grow at (their weight) is easy to calculate with the QCS (by ex- natural) saline habitats (Koyro & Huchzermeyer trapolation of the data) and it leads to a precise 1997; Marcum 1999; Warne et al. 1999; Weber & specification of a comparative value for halophytic D´Antonio 1999; Winter et al. 1999). species. It will be used for the reasons listed above The study in the natural habitat represents a to compare the threshold of salinty tolerance of the mean behaviour but the major constraints can experimental plants. vary this much that a precise definition of the salinity tolerance of a species (and a selection of use- Ecophysiological parameters of the QCS ful plants) is not possible. Only artificial conditions The threshold of salinity tolerance describes in sea water irrigation systems in a growth cabinet the limitations of productivity, but not the physio- under photoperiodic conditions offers the possibil- logical mechanisms of salinity-induced growth re- ity to study potentially useful halophytes under duction. Therefore, a selection of parameters with reproducible experimental growth and substrate a close connection to the four major constraints of conditions (Koyro & Huchzermeyer 1999a). The plant growth on saline substrates is necessary: a) supply of different degrees of sea water salinity water deficit, b) restriction of CO2-uptake, c) ion [0%, 25%, 50%, 75%, 100% (and if necessary toxicity d) nutrient imbalance. Plants growing in higher) sea water salinity] to the roots in separate saline habitats face the problem of encountering a systems under otherwise identical conditions gives

KOYRO & HUCHZERMEYER 127 low water potential in the soil solution and high sity was in the range of 1500 µmol * m-2 * s-1 at concentrations of potentially toxic ions such as plant level. chloride and sodium (Marschner 1995). Salt exclu- The stepwise addition of NaCl to the basic nu- sion minimizes ion toxicity but accelerates water trient solution began after a period of 6 months by deficit and diminishes indirectly the CO2 uptake. raising salinity of the solution in steps of 40 mM Salt absorption (includer) facilitates osmotic ad- NaCl each day. There were altogether six treat- justment but can lead to toxicity and nutritional ments: Control, 1, 40, 240, 360 and 480 mM NaCl imbalance. The QCS of potential crop halophytes (equivalent to 0, 2, 8, 50, 75 and 100 % NaCl). The comprises general scientific ecophysiological data highest salinity treatment was reached after 12 and some special physiological examinations days. The “quick check” system was programmed (Koyro 2000). The collected data comprise informa- by a timer to water the plants every 4 hours for 30 tion about morphology, photosynthesis (incl. gas min starting at midnight, 4 a.m., 8 a.m., 12 noon, exchange), water relations (leaf water potential, 16 p.m. and 20 p.m. daily and allow the saline so- osmotic potential), mineral content, content of os- lutions to drain freely from the pots. Solutions motically active organic substances (such as car- were recycled and changed every 2 weeks to avoid bohydrates and amino acids) and growth. nutrient depletion. The experiment was performed for a total period of twelve months. Three weeks Aim of the study before the harvest, the leaves were washed prop- erly with distilled water and not touched anymore The aim of this study is to provide reliable, until the end of the experiment. ecophysiological parameters of Spartina townsendii to get a survey of the mechanisms leading Growth parameters and quantitative e.g. to the salinity tolerance of this species. It will be shown that the salinity-induced multiplicity chemical analysis (network) of structural and functional changes The number, the LMA (leaf mass per area ra- constitutes a group of indicators for the salinity tio defined as weight per surface area) of the tolerance and growth potential of this species and leaves and the weight of plants, leaves and roots gives an impression of its potential as a cash crop (main and adventitious roots) were noted. A piece halophyte. of 4 ± 1 mm (width) and 80 mm (length) was cut out of the middle region of an adult leaf. The ad- Material and methods axial and abaxial leaf surface was rinsed with 10 ml distilled water and stored in a refrigerator until Plant material and culture conditions the quantitative chemical analysis (see below). The plant was devided into two parts (under Spartina townsendii is an intertidal, estuarine and above ground). Subsamples were taken for (a) saltmarsh grass which can grow up to 130 cm in the determination of the dry weight and water height. Its leaves are narrow, usually 45 cm long content and for (b) the quantitative chemical and 1.5 cm wide. It is a perennial grass spreading analysis (QCA). by underground stems. Cuttings of Spartina town- To (a) The samples were weighed and dried for sendii (Townsends Cordgrass, original habitat We- 48 h at 90 °C in an oven. ser marshes in Germany) were transplanted into a To (b) The samples for the QCA were washed soilless (gravel/hydroponics) culture quick check separately for 1 minute in ice-cooled 0.2 mol . m-3 system (Fig. 1, Koyro & Huchzermeyer 1999a). CaSO4 solution, 1 min in distilled water and blot- The free surface of the substrate was covered with ted carefully with tissue paper. Representative a black foil to hinder the spattering of the plants parts of the leaves and roots were weighed and with the nutrient solutions. The plants were irri- extracted with 0.5% HNO3 in a water bath (80°C) gated with a basic nutrient solution as modified by for 12 h. Na, K, Mg and Ca were determined di- Epstein (1972) under photoperiodic conditions (16 rectly from the extract with an atomic absorption h light/ 8 h dark) in an environment controlled spectrophotometer (Perkin Elmer PE3300) and Cl- greenhouse. Temperatures were 25 ± 2 °C during by electrochemical titration (AMINCO COTLOVE the day and 15 ± 2 °C during the night. Relative chloride titration). humidity ranged from 45 % to 70 %. Light inten-

128 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

Fig. 1. (a) Hydroponic quick check system (QCS) of Spartina townsendii under photoperiodic conditions in a growth cabinet; (b) Leaves of the control plants (1 mM NaCl); (c) Salt crystals on the leaves of the high salinity treatment (480 mM NaCl).

Chlorophyll, total carbohydrate spectrophotometrically after Lichtenthaler & and total protein Wellburn (1983). Freeze dried sample (100 mg) was homoge- Leaf discs (0.78 cm2) of fresh material were ex- nized, extracted in 6 ml phosphate buffer (pH 7.4), tracted in 80% ethanol at 80 °C (carbohydrates) or and centrifuged at 4°C for 30 min at 30 000 x g. in 80% acetone (for the determination of chloro- The soluble proteins in the supernatant were pre- phyll a and b). Carbohydrates were measured cipitated with 400 µl TCA [10% (w/v), for 1 h on spectrophotometrically with the Molisch reaction ice]. The sediment was resolved with 100 µl 1 N (Wild 1999), chlorophyll a and b were determined NaOH (for 24 h). The soluble protein was meas-

KOYRO & HUCHZERMEYER 129 ured photometrically after Bradford (1976) against cal fluid) after the critical point method and a thin a standard with bovine serum albumin. gold layer was sputtered on the surface to enhance the electrical conductivity (Robinson et al. 1985). CO2-gas exchange Statistical treatment The closed photosynthesis measurement system LI-COR 6200 (LI-COR, Lincoln, NE, USA) Data (n > 5) were subjected to one-way was used to determine the response of analysis of variance using pc-stat computer photosynthesis to substrate salinity (Von Willert et software. An analysis of variance was conducted, al. 1995). An initial concentration of 400 + 14 ppm and least significant differences (LSD) were was adjusted using a gas-tight Hamilton syringe determined with F test (p=0.05). filled with pure CO2. Photosynthesis reduced the concentration in the closed loop until the Results compensation point was reached. Calculation of the gas exchange parameters was corrected for Morphological adaptation leaks as described by LI-COR Inc. Net Spartina townsendii reduces the salt photosynthesis (µmol * m-2 * s-1) stomatal conductance (mol * m-2 * s-1), transpiration (mol * concentrations of the active photosynthetic tissue m-2 * s-1) and water-use efficiency (µmol * mmol-1) by various mechanisms such as the secretion by were measured on youngest, fully emerged leaf salt glands (Figs. 1 & 2). The leaf anatomy of blades. All measurements were taken at light Spartina shows some additional adjustment to its saturation (PPFD = 1500 µmol * m-2 * s-1) and 24- natural habitat - the changing tide. The adaxial 26 °C. leaf surface has longitudinal incisions giving evidence for a structural adaptation to drought Osmotic potential (typical for gramineae such as Bouteloua curtipendula, Oryzopsis canadiensis and Stipa Leaf samples were frozen with liquid nitrogen tenacissima, Eschrich 1995). The stomates are and homogenized in a mortar. After thawing the located near the bottom of these incisions samples were centrifuged (at 4 °C, for 5 min at (Sutherland & Eastwood 1916) and can be 3000 x g). The osmotic potential was determined in protected against water loss by rolling up of the the supernatant (leaf sap) with the freeze-point leaf longitudinally. The salt glands are inserted depression method by an cryo-osmometer into the leaf just beyond the laminar edges, and (GONOTEC). NaCl salinity leads to an expansion of the lateral papillas enabling the plant to use it as a zip and to Light (LM), transmission- (TEM) and control influx and efflux of substances between the scanning electron microscopy (SEM) longitudinal incisions and the atmosphere (or seawater). This seems to be extremely important Leaf sections were fixed for 3 h with 4% glu- when the tide gets higher than the plant and the taraldehyde in 50 mM Pipes-buffer (pH 7). After water attempts to flood the longitudinal incisions washing the leaf sections were rinsed three times of the leaf. The laminar edges can be used to in buffer and subsequently postfixed for 4 hours control the stream of water into this region and to with a 2% OsO4 solution in buffer. After a few prevent a flooding of the stomatas. This has two washings in distilled water, the leaf sections were advantages: a) Transpiration can be diminished dehydrated in acetone, imbedded in Durcupan and photosynthesis can continue without ACM (Fluka) or ERL—4206 (Spurr) and polymer- significant water loss. b) The salt excreted from ized at 70 °C for 8 h. The embedded material was the salt glands can still be washed off the surface. sectioned for the light microscopy with a glass knife on a LKB ultramicrotome. The sections were stained with methylene blue (in 2% ethanol) on a Growth heating plate. For a closer examination of the leaf In contrast to glycophytes, Spartina town- surface by scanning electron microscopy (Philips sendii was able to complete its life cycle at high SEM XL20), dehydrated leaf sections (see above) salt concentrations (Fig. 3). Spartina showed the were dried with CO2 at high pressure (supercriti- overall growth response (transient increase) to ele-

130 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

Fig. 2. Adaxial leaf surface of Spartina townsendii (Gramineae). (a), (b) and (c) SEM micrograph of the adaxial surfaces of a leaf of plants grown at 0, 40 and 240 mM NaCl salinity. Elevated salinity led to an expansion of the lateral papilles (please compare a, b and c); (d) SEM micrograph of the salt glands (Sg) and stomates (St) on the adaxial surface of a control leaf (see arrows); (e) Cross sections of a leaf of Spartina townsendii (240 mM NaCl). Saltglands are inserted into the leaf beyond the laminar edges but above the stomates; (f) SEM micrograph of a trichome on the abaxial surface of a leaf. La = laminar edge, Sg = saltgland, St = stomate vated salinity typical for halophytes or halophytic Leaf water potential, osmotic potential and crops (Greenway & Munns 1980). However, the mineral content halophyte Spartina townsendii has a physiological The leaf water potentials ( ) of Spartina requirement for salt in a range of 1 mM NaCl (see Ψ townsendii plants were generally lower than the squares in Fig. 3). The plants showed negative water potentials in the accessory five seawater di- growth without NaCl addition after 6 months of lutions (Koyro 2002). The reduction of the leaf wa- culture. The salinity threshold (initial significant ter potential was mainly reached by an increase of reduction in the expected maximum yield, Shan- the concentrations of osmotically active solutes non & Grieve 1999) of Spartina townsendii was (Fig. 4). The osmolality in adult leaves was gener- reached at 50% seawater salinity, the growth was ally higher than in juvenile leaves. However, there reduced by NaCl treatment to 50% at 480 mM was only a minor increase of the osmolality in seawater salinity.

KOYRO & HUCHZERMEYER 131

60 cumstances were much higher than in the nutrient solution. Elevated salinity led to a transient in- 50 crease of the potassium and to an increase of the magnesium, calcium, sodium and chloride concen- 40 trations. The sodium and the chloride concentrations in the root tissues were in the same range 30 and always above the salinity level in the nutrient solutions. Chloride, potassium, sodium, but also Freshweight in [g] 20 magnesium and calcium, were important osmotically active substances in the shoots of control 10 plants (1 mM NaCl, Fig. 5b). However, especially the potassium and calcium concentrations in the 0 0 100 200 300 400 500 shoot were significantly higher than in the root NaCl-concentration in the nutrient solution tissues. With the increase of the NaCl salinity the potassium, calcium and magnesium concentrations Fig. 3. Development of the plant fresh weight [whole declined and especially the sodium, but also the plant () and (z); shoot () and (z); root ( ) and ({)] chloride concentrations reached levels far above at treatments with different percentages of sea water those present in the nutrient solutions or in the salinity. The squared symbols document the fresh root tissues. However, sodium was accumulated to weight of plants grown without additional NaCl supply. a much higher level in the leaf tissues than chlo- The crossover of the dotted and the black line reflects ride. The difference between sodium and chloride the NaCl salinity where the growth depression falls concentration in leaves reached a value of nearly down to 50% of the control plant. 40 % at 480 mM NaCl salinity. It corresponds to

the osmolalities in adult and juvenile leaves that adult leaves between 50 and 100 % seawater salin- these changes were much more pronounced in the ity (240 and 480 mM NaCl). latter ones (results not shown). The difference be- Potassium and magnesium were the major tween chloride and sodium accumulation in the cations in the root tissues of plants at 0.2% sea- leaves of the high salinity treatment cannot be ex- water salinity (Control, Fig. 5b). However, it was plained by differences in the excretion. There was much more conspicuous that even the Na and Cl only a minor increase of the sodium and chloride concentrations in the root tissue under these cir-

2500 Juvenile leaf Adult leaf

2000

1500

1000 Osmotic value [mOsmol]

500

0 tip base tip base

Fig. 4. Osmotic values (mOsmol) in the tip or basis of juvenile and adult leaves of Spartina townsendii.

132 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

800 indications of ion imbalance: Elevated salinity root

a 700 Root t

t promotes the excretion of essential elements such h

h 600

g g i

i as K, Ca and Mg and these values are still re-

e e

500

w w

h markable at the high salinity treatment. s

s 400

e e

r r

f f

300

1 1

- -

g g

200 Dry matter, protein and carbohydrate content

* *

l l o

o 10 0 m

m The reduction in fresh weight (Fig. 3) at µ µ 0 [Cl] [K] [Na] [Ca] [Mg] elevated salinity was partially compensated in root element and shoot tissues by an increase of the dry matter 1200 b shoot (in % fresh weight, Fig. 6a; ratio Control/100%

1000 Shoot seawater salinity in the root 1.5 and in the shoot

t t

h h

g g

i i

i i 1.3) and of the protein content of the shoot (in % e

e 800

e e

w w

w dry matter, Fig. 6b; ratio Control/100% seawater

h h h h

s s s s

e e e e 600

freshweight

r r r r

-1 salinity 1.2). Spartina townsendii showed an

f f f f

1 1 1 1

- - - -

g g g

g 400

obvious shift from storage of carbohydrates (Fig.

* * * *

µmol * g * µmol

l l l l

o o o o 6c) to the synthesis of proteins.

200

m m m m

µ µ µ µ

0 [Cl] [K] [Na] [Ca] [Mg] Photosynthesis and chlorophyll content element NaCl salinity affected [beside changes of the c metabolism (such as increases of dry matter and Salt crystals protein content)] also the chlorophyll content. The

enhancement of the NaCl salinity led in Spartina

t t

h h

g g i

i to an increase of the chlorophyll a/b ratio in the

e e

w w

juvenile and adult leaves (Fig. 6d) but also to a

h h

s s

e e r

r decrease of the chlorophyll a and even more to a

f f

1 1

- -

g g

decrease of the chlorophyll b content. The CO2-net

* *

l l o

o assimilation rate showed a similar curve as the

m m µ µ carbohydrate content (Table 1 and Fig. 6c). An (insignificant) increase at low salinity was followed by a steep decrease at higher salinities. Additionally, a steep decrease between 240 and 480 mM NaCl was a common reaction of growth, Fig. 5. Chloride-, potassium- , sodium- , calcium and carbohydrate content in the leaves CO2-net magnesium concentrations in mM in root (a), shoot assimilation rate, stomatal conductance and tissues (b) and excreted salt crystals (c) of Spartina transpiration. The water-use efficiency of the townsendii treated with 1 ( ), 40 ()),, 240 () or 480 () mM NaCl-salinity. photosynthesis decreased with elevated salinity from 5.24 (control) to 3.68 (100 % seawater salinity) -1 excretion between 50 % and 100 % seawater salin- µmol CO2 * m mol H2O. ity (Fig. 5c). The chloride accumulation seems to be bal- Discussion anced even at high salinity level with the existing mechanisms (a) selective uptake in the root, (b) Photosynthesis selective translocation from the root to the shoot, In the present study, there was evidence for a and (c) via excretion through the salt gland. This is correlation between salinity tolerance and the pho- not the case for sodium. The sodium concentration tosynthesis and growth responses of Spartina in the shoot in the high salinity treatment reaches townsendii. Similar results were found also for a level twice as high as in the nutrient solution. Spartina patens, S. maritima and S. densiflora This overflooding with sodium cannot be balanced populations collected in Lousiana Gulf Coast by the excretion through the salt glands. Beside marshes or on the coasts of SW Europe (Nieva et evidence for sodium toxicity there are also some al. 1999; Pezeshki & DeLaune 1997). The sensitiv-

KOYRO & HUCHZERMEYER 133

)

t ductance in this and in many other species (Pearcy

h 35

a i & Ustin 1984; Rozema & van Diggelen 1991).

e 30 w In agreement with the results presented for

s 25

r Spartina townsendii in this paper, other species of

f 20 % the genus Spartina, such as S. foliosa, S. maritima

(

r 15

t and S. densiflora, also present high water use

a 10

m efficiency (WUE) at low salinity, with a decline at

y r 5 higher salinity (Mahall & Park 1976; Nieva et al.

D 0 1999). However, the WUE of all these C4 plants 1 40 240 480 mentioned above at elevated salinity were still

) 6

e high in comparison with other halophytic C3

t b a 5 species (Nieva et al. 1999).

r 4 The WUE of photosynthesis decreased in

( Spartina townsendii leaves with elevated salinity

t 3

e and in response to low salinity levels mainly

o 2 because of increasing stomatal conductance and

n waterloss. It is not clear whether the water-use i 1

o efficiency of photosynthesis would be similar for

r P 0 Spartina townsendii in its natural habitat (the 1 40 240 480 intertidal zone under various flooding-salinity 20

) combinations). The gas exchange of Spartina r 18

e c t t 16 maritima was independent of elevation of salinity

m 14 as were its chlorophyll fluorescence parameters

r 12 d (Castillo et al. 2000). In contrast, in S. densiflora

% 10

(

t 8 the rate of CO2 uptake declined, and stress to

e t 6 photosystem II increased at lower salinities. It is

o 4

not known whether transpiration is diminished in h 2 C S. townsendii during flooding enabling the T 0 photosynthesis to operate without significant 1 40 240 480 water loss. Further investigations are necessary to 3,5 examine the differences in physiological and

d o 3

a growth response to distinct flooding-salinity

2,5

/ combinations. The influence of flooding on gas

l 2 l exchange could be also an explanation for

h 1,5 p considerable variation in the performance among

l 1 certain populations of Spartina in response to

C 0,5 salinity regimes (Pezeshki et al. 1993). In Spartina townsendii the lower net photo- 0 synthesis rate under salinity is not solely due to a 1240480 decrease in the contribution of CO2 by reduction in mM NaCl in the nutrient solution the stomatal conductance (see Table 1). Non- stomatal limitations of CO2-assimilation capacity Fig. 6. Dry matter ( : root, : shoot) protein content were shown also for Spartina maritima and S. (shoot), total leaf carbohydrate content (TCh, : densiflora (Nieva et al. 1999). All three Spartina juvenile, : adult) and chlorophyll a/b ratio in leaves ( : species maintained higher intercellular CO2 con- juvenile, : adult) of Spartina townsendii. centrations under saline irrigation (results not shown for Spartina townsendii). The variation in ity in the response of the net photosynthetic rate is the net photosynthesis rate could be related with an ideal indicator of salt tolerance for Spartina biochemical changes due to ion toxicity that affect townsendii and seawater salinity irrigation is as- carboxylase activity of the ribulose-1,5- sociated with a decrease in the net photosynthesis bisphosphate carboxylase/oxygenase and the size rate, the transpiration rate and the stomatal con-

134 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

Table 1. Transpiration (mmol * m-2 * s-1), stomatal conductance (mmol * m-2 * s-1), CO2 net-assimilation rate (µmol * m-2 * s-1) and water use efficiency of the photosynthesis, (µmol CO2 * mmol-1 H2O) of leaves of Spartina townsendii. 0,2% sea water salinity (sws) = 1NaCl, 8% sws = 40NaCl, 50% sws = 240NaCl and 100% sws = 480NaCl

Nutrient solution Transpiration Stomatal Conductance CO2 Net assimilation Water use efficiency of the [mM NaCl] [mmol * cm-2 *s-1] [mmol * m-2 *s-1] rate [µmol * cm-2 *s-1] photosynthesis [µmol CO2 * mmol-1 H2O] 1 2.632 ± 0.782 58.100 ± 5.110 13.78 ± 1.27 5.24 ± 0.28 40 3.006 ± 0.383 70.077 ± 4.071 14.88 ± 1.64 4.95 ± 0.02 240 3.388 ± 0.384 75.453 ± 7.015 12.95 ± 1.91 3.82 ± 0.02 480 2.499 ± 0.307 56.671 ± 5.169 9.20 ± 1.69 3.68 ± 0.03 of the phosphate triose pool (Wyn Jones & Pollard townsendii to elevated salinity regimes is the acti- 1983; Ziska et al. 1990; Antolin & Sánchez-Díaz vation of salt glands. It was shown in the previous 1993; Nieva et al. 1999). study that these salt glands are working highly NaCl salinity had an significant effect on the selective and eliminate relatively large quantities chlorophyll a and b content and the chlorophyll b/a of salt by secretion to the leaf surface, where it can ratio of Spartina townsendii and on chlorophyll be washed off by seawater, rain or dew (Marcum et fluorescence in S. densiflora (Castillo et al. 2000; al. 1998). However, the salt glands were neither Nieva et al. 1999) with significantly higher values able to balance the high burden especially of Na in in freshwater irrigation, probably indicating a the leaf tissues at the high salinity treatment, nor greater size of the PSII electron acceptor pool, useful in maintaining a constant supply of essen- which would lead to a decrease in non- tial elements such as K, Mg and Ca. The gap be- photochemical quenching. tween a sufficient nutrient supply and NaCl accumulation grows with increasing salinity and seems Leaf water potential to limit the salinity tolerance of Spartina town- sendi by nutrient imbalance and/or ion toxicity. Data of the leaf water potentials demonstrated This hypothesis was also confirmed and clearly that Spartina townsendii, S. matritima and specified by studies of the element compositions S. densiflora (Koyro 2002; Nieva et al. 1999) have (with EDXA analysis of bulk frozen samples in a sufficient adjustment mechanisms even at high scanning electron microscope) in the cytoplasm of salinity, suggesting that there was no reason for single epidermal leaf cells of Spartina townsendii growth reduction by water deficit. Thus, if the (Koyro 2002). NaCl at 100 % seawater salinity led rate of supply of water to the shoot is not only to a minor decrease of the major elements K restricted, the depression of the shoot growth and P (in comparison with the control) but to a shown in Fig. 3 is likely to depend mainly on significant increase of the Na and Cl mineral nutrition. It seems to be only a matter of concentrations in this compartment. The author controversy as to whether a decrease in the discussed these findings as a beginning Na and Cl amounts of nutrients or unfavourable nutrient toxicity in the cytoplasm supporting the hypothesis ratios (e.g. Na+/K+) are important factors for that a major reason for the limitation of salinity impaired leaf elongation (Lynch et al. 1988; Munns tolerance was ion toxicity. It was recommended to et al. 1989). reduce the ion toxicity by the supply of sufficient fertilizers at high NaCl salinities (Koyro 2002). Nutrients The osmotic adjustment in the root and shoot Nitrogen metabolism tissues in our experiment mainly based on the ac- The rhizosphere sediments of cordgrasses are cumulation of Na and Cl (Figs. 5a & b). It is, there- generally a site of intense nitrogen fixation activity fore, not astonishing that there was also a correla- and a major source of biologically available nitro- tion between salinity tolerance and ion composi- gen providing a significant source of nitrogen (e.g. tion in the shoot. The main defence of Spartina

KOYRO & HUCHZERMEYER 135

91 % for Spartina maritima) for the growth of the tentially useful cash crop halophytes (Koyro 2000; plants (Nielsen et al. 2001; Welsh 2000). Nitrogen Isla et al. 1997). fixation in the rhizosphere is regulated by photo- The results of this study confirm again that synthetically driven release of oxygen and fixed the final selection of halophytic species suited for a carbon by the plant roots and rhizomes. In addi- particular climate and for a particular utilisation, tion, plant-associated nitrogen fixation could sup- a sustainable production system has to be ply more than 37% of the nitrogen needed by the designed in plantations at coastal areas or at sulfate-reducing community. Sucrose stimulated inland sites. A cultivation of Spartina townsendii nitrogen fixation and sulfate reduction signifi- in foreign salt marshes needs to be accompanied cantly in root and rhizome of Spartina maritima by studies about the interaction between plant and (Nielsen et al. 2001). Elevated salinity led to an habitat, especially the physiological range, increased demand for proteins (nitrogen see Fig. 6) ecological optimum and yield characteristics. in Spartina townsendii, and in other species of this Selecting the best adapted species or populations genus also for glycine betaine (nitrogen), proline for wetland creation and restoration involves e.g. (nitrogen) and dimethyl-sulphoniopropionate the matching of the planting stock with the site [(DMSP) sulfur], an osmoprotectant accumulated conditions (Igartua 1995; Rozema 1996). Among by the cordgrass at elevated salinity (Kocsis & the other characters that determines survival and Hanson 2000; Mulholland & Otte 2001). The in- growth are phenology (early or late emergence of creased demand for S and N can be one explana- sprouts), responses to diseases and herbivory, tion for the reduction of the total carbohydrate responses to other chemical and physical factors pool in the leaves (Fig. 6). A higher demand for the such as fluctuation of soil salinity, waterlogging, proteins can lead also to a switch in the metabo- nutrient enrichment, periods of drought and lism from carbohydrate storage to the synthesis of periods of frost (Rozema 1996). The importance of proteins. The increased uptake of S and N into the these investigations should not be underestimated. plant at elevated salinity points to a higher meta- It is well known that e.g. salinity and waterlogging bolic activity in the rhizosphere of Townsend´s conditions are the major factors underlying salt Cordgrass. Spartina is a potential biomass crop marsh vegetation zonation and succession and that (e.g. grown for fodder; Beale et al. 1999; Lieth lower marsh species such as Salicornia and 1999). This effective activation of N and S com- Spartina are well adapted to seawater salinity and pounds in flooded soils by the mechanisms de- waterlogged conditions. scribed above can be a major reason for the inva- Finally, it is important to balance the potential sive growth of Spartina townsendii in the poor nu- benefits and risks of nonindigenous species. A trient conditions (especially for N and S) of the salt precondition for an introduction of nonindigenous marsh ecosystem. species should be the sustainability as described by Ewel et al. (1999) with the eight key areas of Development of cash crop halophytes consensus. The results presented in this paper inform about the eco-physiological needs of the Spartina References townsendii at high salinity. However, this study is Angradi, T.R., S.M. Hagan & K.W. Able. 2001. only the first step for the development of cash Vegetation type and the intertidal crops or other usable plants from existing halo- macroinvertebrate fauna of a brackish marsh: phytes. It is of major importance for all further Phragmites vs. Spartina. Wetlands 21: 75-92. investigations that the potential benefits and risks Ansede, J.H., R. Friedman & D.C. Yoch. 2001. of nonindigenous species are balanced carefully. Phylogenetic analysis of culturable dimethyl sulfide- This OCS offers a reliable base, but not more than producing bacteria from a Spartina-dominated salt the first step, for the selection of economically im- marsh and estuarine water. Applied and portant cash crop halophytes or other usable Environmental Microbiology 67: 1210-1217. plants from existing halophytes. It is necessary to Antolin, M. C. & M. Sánchez-Díaz. 1993. Effects of stepwise approach the propagation at promising temporary drought on photosynthesis of alfalfa sites under controlled conditions to establish po- plants. Journal of Experimental Botany 44: 1341- 1349.

136 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

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