42 Plant Stress Physiology: Physiological and

Stress Physiology: 42 Plant Physiological and Biochemical Strategies Allowing Plants/Crops to Thrive under Ionic Stress Hans-Werner Koyro, N. Geissler, R. Seenivasan, and Bernhard Huchzermeyer CONTENTS 42.1 Introduction ........................................................................................................................ 1052 42.2 Physiological Aspects ......................................................................................................... 1053 42.2.1 Halophytes: Plants Able to Thrive on Saline Substrates ........................................ 1053 42.2.2 Experimental Proof for the Suitability of Plants .................................................... 1054 42.2.3 Threshold of Salinity Tolerance ............................................................................. 1055 42.2.4 Major Constraints of Plant Growth on Saline Substrates ....................................... 1056 42.2.5 Regulation of the Water Relations and Optimization of the Gas Exchange........... 1057 42.2.6 Maintenance of Ion Homeostasis: Avoidance of Ion Excess and Ion Imbalance ... 1058 42.2.6.1 Protection of the Cytoplasm .................................................................... 1059 42.2.6.2 Selective Ion Accumulation ..................................................................... 1060 42.2.7 Morphological Adaptation ...................................................................................... 1061 42.2.8 Sustainable Use of Halophytes ............................................................................... 1063 42.3 Biochemical Aspects .......................................................................................................... 1063 42.3.1 Inhibition of Primary Reactions of Photosynthesis ................................................ 1063 42.3.1.1 Photosynthetic Conversion of Energy ...................................................... 1064 42.3.1.2 Salt Effects on Photosynthetic Energy Conversion ................................. 1065 42.3.1.3 Salt Effects on Chloroplast Structure and Metabolite Transfer ............... 1066 42.3.2 Salt-Induced Production of Potential Toxic Intermediates of Photosynthesis........ 1067 42.3.2.1 ROS .......................................................................................................... 1067 42.3.2.2 Photorespiration ....................................................................................... 1070 42.3.2.3 Non-Photochemical Quenching of Energy .............................................. 1072 42.3.3 Protecting from Direct Salt Effects on Protein and Membrane Structure and Function ........................................................................................................... 1073 42.3.3.1 Control of Mechanisms Improving Stress Tolerance: Compatible Solutes ................................................................................................. 1073 42.3.3.2 Polyols and Sugars ................................................................................... 1075 42.3.3.3 Nitrogen-Containing Compatible Solutes ................................................ 1077 42.3.3.4 Aspect of Energy Consumption by N- and S-Pathways .......................... 1078

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Handbook of Plant and Crop Stress

42.3.4 Salt Effects on Metabolite Transport and Bioenergetics of Cells ........................... 1079 42.3.4.1 Sugar Phosphate Export Out of the Chloroplasts .................................... 1079 42.4 Summary ............................................................................................................................ 1081 42.5 Future Perspective .............................................................................................................. 1081 References .................................................................................................................................... 1082

42.1

INTRODUCTION

Abiotic stresses, such as drought and salinity, are serious threats to agriculture and the natural status of the environment. They are recurring features of nearly all the world’s climatic regions since various critical environmental threats with global implications have linkages to water crises (Gleick, 1994, 1998, 2000). These threats are collaterally catalyzed by global warming and population growth. The latest scientific data confirm that the earth’s climate is rapidly changing. Global temperatures have increased by about 1°C over the course of the last century, and will likely rise even more rapidly in the coming decades (Intergovernmental Panel on Climate Change, 2007). Scientists predict that temperatures could rise by another 3°C–9°C by the end of the century with far-reaching effects. Increased drought and salinization of arable land are expected to have devastating global effects (Wang et al., 2003a). The current amount of annual loss of arable area could double by the end of the century because of global warming (Evans, 2005). Simultaneously, rapid population growth increasingly generates pressure on existing cultivated land and other resources (Ericson et al., 1999). Population migration to those arid and semiarid areas increase the problems of water shortage and worsens the situation of land degradation in the destination, and in turn causes severe problems of poverty, social instability, and population health threats (Figure 42.1, Moench, 2002). Water scarcity and desertification could critically undermine efforts for sustainable development, introducing new threats to human health, ecosystems, and national economies of several countries. Therefore, solutions to these problems are desperately needed, such as the improvement of salinity tolerance of crops. Two different experimental approaches to increase crop salt tolerance are in use: (1) growing plants in saline conditions and comparing their performance to a control group grown under optimal conditions and (2) applying salt stress after plants have been grown under optimal conditions for a while. These approaches investigate how an individual plant species adapts to a saline environment and what its stress response is like, respectively. This second approach is widely used, but its Global hunger on the rise The number of undernourished people in the world surpassed 1 billion, according to a U.N. report released Wednesday. Undernourished people 1.05 billion

1.02 billion

1.00 0.95

Regional breakdown In millions Sub-Saharan Africa: 265 Asia/ Pacific: 642 Latin America/ Caribbean: 53

0.90 0.85 0.80 1995– 1997

FIGURE 42.1

2000– 2004– 2008 2009 2002 2006

Developed countries: 15

Mideast/ North Africa: 42

Global hunger on the rise. (From Food and Agriculture Organization of the United Nations.)

Plant Stress Physiology: Physiological and Biochemical Strategies

Salt water (sea water or saline ground water)

+

Wasteland, dry or saline habitat

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Salt tolerant plants (halophytes)

+

Food Chemicals including pharmaceutics Biomass Aster tripolium

Salicornia fruticosa Forage crops Wood and fibers

Greenification and CO2-sequestriation

Mangal Atriplex nummularia

FIGURE 42.2

Avicennia marina

Sesuvium portulacastrum

Sustainable use of cash-crop halophytes.

meaning has been questioned a lot as well: As a rule, salt stress does not occur suddenly in the field. Moreover, the combination of this approach with current micro-array technique provided too many hits to be handled in subsequent breeding approaches. Apparently most of the genes would respond to any type of stress. They are activated in response to disturbed homeostasis rather than specific response to salt stress (Kawasaki et al., 2001). Some eco-physiologists have analyzed performance of plants differing in their salt tolerance and have introduced the term “halophytes” for plants showing high salt tolerance. Some of these plants can tolerate seawater irrigation (Lieth and Menzel, 1999); most of these plants are obligate halophytes, i.e., they show optimal growth only in the presence of some salt (100–200 mM, these are concentrations already toxic for many other plants). It is a matter of ongoing research to breed halophytes for cropping purpose (Figure 42.2, sustainable use of so-called cash-crop halophytes). Another approach under discussion is, whether genes from halophytes can be transferred to regular crops to make them more salt tolerant (Flowers et al., 1997; Glenn et al., 1998). A more recent idea is to take advantage of the genetic potential crops still have (Glenn et al., 1998). This idea requires to first identify accessions of a crop species showing enhanced salt tolerance. In a second step, genes (enzymes or metabolic pathways) have to be identified that result in stress tolerance. Finally, early indicators of salt tolerance have to be identified allowing successful breeding (Ashraf, 1999; Koyro et al., 2009). Moreover, understanding the physiological and biochemical basis of stress tolerance can help to identify proper culture conditions. This may allow extending the cultivation area of an individual species to regions having suboptimal environmental conditions.

42.2 42.2.1

PHYSIOLOGICAL ASPECTS HALOPHYTES: PLANTS ABLE TO THRIVE ON SALINE SUBSTRATES

Despite the importance of salinity in shaping the composition of coastal plant communities, our understanding of how different species respond physiologically to variable salinities is limited (Touchette et al., 2009). Halophytes are plants that are able to complete their life cycle in a substrate rich in NaCl (Schimper, 1891). Approximately 2600 halophytes are known worldwide, and they constitute 1% of the world’s flora (Lieth and Menzel, 1999; Flowers and Comer, 2008).

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FIGURE 42.3

Salt excretion of the mangrove Avicennia marina L.

In contrast to most glycophytic crops, they still have not lost resistance mechanisms to salt stress conditions. These may take the form of salt avoidance or tolerance (Yeo, 1983, 1998; Touchette et al., 2009). They are classified as euhalophytes (true halophytes), pseudohalophytes (salt avoiders), or crinohalophytes (salt excreters), according to their apparent adaptation to environmental salinity (Figure 42.3, Ungar, 1991). Some plants (the Mojave Desert Star, for instance) avoid the effects of high salt by fancy tricks such as by completing the reproductive life cycle during rainy seasons (facultative halophytes). Nevertheless, the bandwidth of resistance mechanisms is larger in obligatory halophytes or xerohalophytes (drought-tolerant halophytes). These are plants tolerating salinities higher than 0.5% NaCl (Koyro and Lieth, 1998). For obligate halophytes, saline substrates even offer advantages for the competition with glycophytes. Although the terms glycophytes and halophytes inseminate the impression that there are general qualitative differences in adaptation, in reality things are more complex. There is a fluent passage between these two groups or may be a just stereotype thinking owed to the human wish of differentiation. Additionally, almost all of the salt adaptive mechanisms underlie the physiological and ecological complexity as well as structural changes. Because of that, information about the salinity tolerance of glycophytes and halophytes needs partially careful checking. Furthermore, a prerequisite for the sustainable utilization of plants (such as suitable halophytes) on saline sites is the precise knowledge about the various mechanisms enabling a plant to grow at (their natural) saline habitats (Marcum, 1999; Warne et al. 1999; Weber and D’Antonio, 1999; Winter et al., 1999). This chapter reviews the eco-physiological mechanisms.

42.2.2

EXPERIMENTAL PROOF FOR THE SUITABILITY OF PLANTS

As freshwater resources will become limited in near future (Lieth, 1999), it is necessary to develop sustainable biological production systems, which can tolerate higher water salinity. A precondition is the identification and/or development of salinity-tolerant crops. First of all, halophytes have to be studied in their natural habitat and a determination of all environmental demands has to be completed. On the basis of this information, the selection of potentially useful plants should begin (Lieth, 1999). The first step of this identification list contains the characterization and classification of the soil and climate, under which potentially useful halophytes grow. Only artificial conditions in sea water irrigation systems in a growth cabinet under photoperiodic conditions offer the possibility to study potentially useful halophytes under reproducible experimental growth and substrate conditions. The supply of different degrees of sea water salinity


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FIGURE 42.4 Quick check system of halophytes. Gravel/hydroponic quick check system (QCS) with automatic drip irrigation under photoperiodic conditions in a growth cabinet (plant species: Beta vulgaris ssp. maritima). Controls are visible in the foreground, the sea water salinization treatment in the background.

[0%, 25%, 50%, 75%, 100% (and if necessary higher) sea water salinity] to the roots in separate systems under otherwise identical or/and close to natural conditions gives the necessary preconditions for a comparative study in a quick check system (QCS) for potential cash crop halophytes (Figure 42.4; Koyro and Huchzermeyer, 1999a). It is well known that salinity tolerance depends on the stage of development and period of time over which the plants have grown in saline conditions (Munns, 2002). Plants were exposed to salinity in the juvenile state of development and were studied until achieving the steady state of adult plants. Variable applicable QCS seems to be valuable for the selection of useful plants and it suggests itself as a first step for the controlled establishment of “cash crop halophytes” because it provides detailed information about three major goals as there are the threshold of salinity tolerance at idealized growth conditions, how to uncover the individual mechanisms for salt tolerance and about the potential of utilization for the preselected halophytic species.

42.2.3

THRESHOLD OF SALINITY TOLERANCE

In correspondence with the definition for the threshold of salinity tolerance according to Kinzel (1982), the growth reaction and the gas exchange are used during the screening of halophytes as objective parameters for the description of the actual condition of a plant (Ashraf and O’Leary, 1996). Reliable information is now available about studies with several halophytic species from different families such as Aster tripolium, Inula crithmoides, Plantago cf. coronopus, Laguncularia racemosa, Limoniastrum articulatum, Beta vulgaris ssp. maritima, Atriplex nummularia, Atriplex leucoclada, Atriplex halimus, Chenopodium quinoa, Batis maritima, Puccinellia maritima, Spartina townsendii, and Sesuvium portulacastrum (Pasternak, 1990; Koyro and Huchzermeyer, 1997, 1999a; Koyro et al., 1999; Lieth and Menzel, 1999; Koyro, 2000; Koyro and Huchzermeyer, 2004a; Geissler et al., 2009a). The substrate concentration leading to a growth depression of 50% (refer to freshweight, in comparison to plants without salinity) is easy to calculate with the QCS (by extrapolation of the data) and it leads to a precise specification of a comparative value for the threshold of salinity tolerance. Dramatic differences are found between halophytic plant species. The threshold of salinity tolerance amounts to 300 mol m−3 NaCl in Aster tripolium, 375 mol m−3 in Beta vulgaris ssp. maritima, 500 mol m−3 in Spartina townsendii, and 750 mol m−3 in Sesuvium portulacastrum (Figure 42.5). These results prove that it is essential to quantify differences in salinity tolerance between halophytic species as one basis for assessment of their potential of utilization.

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100

% Growth rate

80

60

40

Aster tripolium Beta vulgaris ssp. maritima Spartina townsendii Sesuvium portulacastrum

20

0 0

20

40 60 % Sea water salinity

80

100

FIGURE 42.5 Threshold of salinity tolerance. Dramatic differences are found between halophytic plant species. The threshold of salinity tolerance amounts to 300 mol m−3 NaCl in Aster tripolium, 375 mol m−3 in Beta vulgaris ssp. maritima, 500 mol m−3 in Spartina townsendii, and 750 mol m−3 in Sesuvium portulacastrum.

42.2.4

MAJOR CONSTRAINTS OF PLANT GROWTH ON SALINE SUBSTRATES

The salinity tolerance of halophytic plants is in most cases multigenic; it comprises a wide range of morphological, physiological, and biochemical mechanisms on whole plant, tissue, and cellular/ molecular levels (Wang et al., 2003a,b; Ashraf and Harris, 2004). Only rarely a single parameter is of major importance for the ability to survive at high NaCl salinity. A comprehensive study with the analysis of at least a combination of several parameters is a necessity to get a survey about the mechanisms which in the end leads to the salinity tolerance of individual species. These mechanisms are connected to the four major constraints of plant growth on saline substrates: (1) water deficit, (2) restriction of CO2 uptake, (3) ion toxicity, and (4) nutrient imbalance. Plants growing in saline habitats face the problem of a low water potential in the soil solution and high concentrations of potentially toxic ions such as chloride and sodium. Salt tolerance involves physiological and biochemical adaptations for maintaining protoplasmic viability while cells compartmentalize electrolytes. Salt avoidance involves structural and physiological adaptations to minimize salt concentrations of the cells or physiological exclusion by root membranes. In principle, salt tolerance can be achieved by salt exclusion or salt inclusion. Salt exclusion minimizes ion toxicity but accelerates water deficit and indirectly diminishes CO2 uptake. Salt absorption facilitates osmotic adjustment but can lead to toxicity and nutritional imbalance. The following physiological mechanisms to avoid salt injury (and to protect the symplast) are known as major plant responses to high NaCl salinity (Marschner, 1995; Mengel and Kirkby, 2001; Munns, 2002; Koyro and Huchzermeyer, 2004a): 1. Regulation of the water potential, decrease of the osmotic and matrix potential, enhanced synthesis of organic solutes. 2. Optimization of the gas exchange (H2O and CO2), high water use efficiency (of photosynthesis (H2O loss per net CO2 uptake), ion radical scavenging, or/and switch to CAM-type of photosynthesis.


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3. Maintenance of ion homeostasis especially in the cytoplasm of vital organs a. Selective uptake or exclusion (e.g., salt glands). b. Selective ion transport in the shoot, in storage organs, to the growing parts and to the flowering parts of the plants, re-translocation in the phloem. c. Compartmentation of Na and Cl in the vacuole or cell wall. 4. High storage capacity for NaCl in the entirety of all vacuoles of a plant organ, generally in old and drying parts (e.g., in leaves supposed to be dropped later) or in special structures such as hairs. The dilution of a high NaCl content can be reached in parallel by an increase in tissue water content (and a decrease of the surface area, succulence). 5. Tolerance of high NaCl concentrations in the symplast. 6. Conversion of whole plant metabolism to high NaCl concentrations (synthesis of NaCltolerant enzymes, osmolyte biosynthesis, ion-protecting agents such as proline and glycinebetaine). 7. Restricted diffusion of NaCl in the (root-) apoplast. 8. Anatomical modification. These mechanisms will be reviewed in more detail in the following sections.

42.2.5

REGULATION OF THE WATER RELATIONS AND OPTIMIZATION OF THE GAS EXCHANGE

According to Munns, plants show a two-phase growth response to salinity (Munns, 1993, 2002; Munns et al., 2002). The first phase of growth reduction is essentially a water stress or osmotic phase and presumably regulated by hormonal signals coming from the roots. Terrestrial plants in saline habitats are often surrounded by low water potentials in the soil solution and atmosphere. For water to flow through the soil–plant–atmosphere continuum, a gradient of decreasing water potentials (Ψ) must be established. The Ψ of pure water is defined as 0 MPa; increasing salinity or concentrations of other solutes will decrease Ψ. Thus, any sharp rise in salinity could effectively hold water osmotically away from plants that lack any physiological or morphological modifications (Larcher, 2003). To limit restrictions on water uptake, plants must generate increasingly lower Ψ to allow continued water flux into belowground structures (Touchette et al., 2009). It is also important to prevent water loss by transpiration from being higher than the influx rate. This is only possible if the water potential remains lower in the plant than in the soil. However, data demonstrated clearly that leaf water potential of halophytes does not correlate alone as a single factor with salinity tolerance. Plant species with different levels of salt tolerance such as Aster tripolium, Beta vulgaris ssp. maritima, Spartina townsendii, and Sesuvium portulacastrum, have a sufficient adjustment mechanism even at high salinity treatment. Clearly, tolerance in the form of osmotic adjustment plays an important role in halophytes residing in saline environments (Flowers and Colmer, 2008). However, in addition were the osmotic potentials of all four halophytes (and many others) up to sea water salinity level sufficiently low to explain the full turgescence of the leaves (results not shown). Except for differences in concentration and type of osmotica used in plant tissues (ions are more prevalent in halophytes), physiological responses in plants to salt stress were remarkably similar to those employed during drought (Touchette et al., 2009). For plants with limited water availability, physiological adjustments often involve avoidance and tolerance, with most plants using some combination of the two (Yue et al., 2006; Romanello et al., 2008). Assuming there is no interruption of the water supply, water can flow passively from the root to the shoot and there seems to be no reason for growth reduction by water deficit for any of the studied species. However, by regulating the extent of apoplastic barriers and their chemical composition (long-distance response coordination), plants can effectively regulate the uptake or loss of water and solutes (by structures such as barriers in the hypo- or exodermis). This appears to be an additional or compensatory strategy of plants to acquire water and solutes (Hose et al., 2001) and at the extremes of growth under conditions of drought and high salinity make the exodermis an

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absolute barrier for water and ions in the strict sense (Azaizeh and Steudle, 1991; North and Nobel, 1991; Nublat et al., 2001). Thus, the rate of supply of water to the shoot can be restricted due to the coupling between the flows of water and solutes (Na and Cl) even if the leaf water potential is low. Therefore, the balance between water flow (sum of water accumulation and transpiration) and the decrease in the amounts of nutrients or unfavorable nutrient ratios (e.g., Na+/K+) are important factors for impaired leaf elongation (Lynch et al., 1988; Munns et al., 1989; Neves-Piestun and Bernstein, 2001; Cramer, 2003) and plant growth. In any case, plant water loss has to be minimized at low soil water potentials, since biomass production depends mainly on the ability to keep a high net photosynthesis by low water loss rates. In this field of tension, biomass production of a plant has always to be seen in connection with the CO2/H2O-gas exchange, which can be estimated by the water use efficiency (WUE) of photosynthesis. A critical point for the plant is reached if the CO2 fixation (apparent photosynthesis) falls below the CO2 production (compensation point). Therefore, one crucial aspect of the screening procedure is the study of growth reduction, water consumption, and net photosynthesis especially at the threshold of salinity tolerance (Geissler et al., 2009a). Several halophytic plants such as Aster tripolium, Beta vulgaris ssp. maritima, Chenopodium quinoa, or Spartina townsendii reveal a combination of low (but positive) net photosynthesis, minimum transpiration, high stomatal resistance, and minimum internal CO2 concentration at their threshold salinity tolerance (Koyro, 2000; Koyro and Huchzermeyer, 2004a). However, there is a big bandwidth among halophytes, especially for succulent halophytes such as Sesuvium portulacastrum or Avicennia marina, which have alternatives if the water balance is still positive (water uptake minus water loss) and not the limiting factor for photosynthesis. In case of S. portulacastrum, net photosynthesis and WUE increase but stomatal resistance decreases. These results show that it is quite important to describe the regulation of gas exchange at high salinity in strong reliance with other parameters (such as water relations). Water deficit is one major constraint at high salinity and can lead to a restriction of CO2 uptake and to the development of radical oxygen species. The balance between water loss and CO2 uptake helps to find weak spot in the mechanism of adjustment (of photosynthesis) to high salinity (Badawi et al., 2004).

42.2.6

MAINTENANCE OF ION HOMEOSTASIS: AVOIDANCE OF ION EXCESS AND ION IMBALANCE

There is a second phase of growth response to salinity which takes time to develop, and results from internal injury (Munns, 1993, 2002; Mengel and Kirkby, 2001; Boström et al., 2003). It is due to ion-specific effects, i.e., salts accumulating in transpiring leaves to excessive levels. Ion toxicity and nutrient imbalance are two major constraints of growth at saline habitats and therefore of special importance for the salt tolerance of halophytes. For some ions (such as Na+ and K+), either their excess or deficiency has been found to be toxic to freshwater and marine organisms. Adverse effects can occur in plants on saline substrates when common ions exceed a certain concentration, when the normal composition (ratio) of ions is not correct, or in some cases, when ion concentrations are too low. Data of additional scientific studies have shown that halophytes exhibit very different ways of adjustment to high NaCl salinity. There are several known examples, where salt-tolerant plants differ from salt-sensitive relatives in having a lower rate of Na+ and Cl− transport to leaves (Munns, 2002). However, some halophytes even need an excess of salts for maximum growth and for attaining low solute potentials (Flowers et al., 1977; Greenway and Munns, 1980). This is much less energy consuming than synthesizing organic substances (Yeo, 1983; Chaves et al., 2009). Nevertheless, high substrate salinities can lead to toxic effects of salt even inside these includers (Munns, 2005). The cause of injury is probably the salt load exceeding the ability of cells to compartmentalize salts in the vacuole. Salts would then build up rapidly in the cytoplasm and inhibit enzyme activity. Alternatively, they might build up in the cell walls and dehydrate the cell.


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High Na+ and Cl− concentrations can be avoided by filtering out most of the salt. Plants with this ability are often called excluders in literature (in contrast to includers). The use of these terms is highly debated because they are mainly used to provide a clearer understanding although they are again just stereotypes with a limited validity. However, ion-excluding halophytes have to synthesize osmotically active solutes within the plant to meet turgor pressure demands (Mengel and Kirkby, 2001). This adaptive feature can be of importance even in species that have salt glands or bladders. For the sake of completeness, it has to be said that it is quite important to distinguish between both ions to uncover the individual mechanisms for salt tolerance. The mechanisms of salt exclusion are discussed in literature mainly as if a common reaction of both ions (Na+ and Cl−) is leading to salt injury. This is not always the case. Some halophytes such as Laguncularia racemosa (with salt glands) is a typical Na excluder but with high Cl− accumulation in the leaves (Koyro et al., 1997), and Beta vulgaris ssp. maritima is a typical Cl− excluder with high Na+ accumulation in the leaves (Kinzel, 1982; Koyro and Huchzermeyer, 1999a). Halophytes are able to distinguish precisely between the metabolic effects of both ions Cl− and Na+: Some halophytes such as Scirpus americanus, Avicennia marina (salt excreter with salt glands), or Rhizophora mangle are able to exclude Na+ and Cl− (see literature in Kinzel, 1982) from the leaves, Laguncularia racemosa (salt excreter with salt glands) diminish the Na+ but not the Cl− concentration in the leaves (Koyro et al., 1997), Beta vulgaris ssp. maritima, Suaeda brevifolia, Suaeda vera, Limoneastrum monopetalum, Allenrolfea occidentalis, or Spartina townsendii accumulate much higher Na+ than Cl− in the leaves (see literature in Kinzel, 1982; Koyro and Huchzermeyer, 1999a) and Salicornia rubra, Salicornia utahensis, Suaeda occidentalis, Atriplex vesicaria, Atriplex nummularia, Atriplex papula, Atriplex rosea, or Inula crithmoides accumulate Na+ and Cl− in the leaves in a range above the saline environment (salt includers). Typical halophytic adaptation includes in this case leaf succulence in order to dilute toxic ion concentrations (Kinzel, 1982; Mengel and Kirkby, 2001) or they perform salt excretion (e.g., with bladder hairs such as all listed Atriplex species). If plants accumulate Na+ and Cl− in concentrations not sufficient to balance the external waterpotential, a lack of solutes may result in adverse effects on water balance, so that water deficiency rather than salt toxicity may be the growth-limiting factor (Greenway and Munns, 1980; Mengel and Kirkby, 2001). To achieve a low water potential and/or a charge balance, the solute potential in these species is decreased by the synthesis of organic solutes such as sugar alcohol (e.g., mannitol in leaves of Laguncularia racemosa), soluble carbohydrates (e.g., sucrose in taproots of Beta maritima ssp. maritima), organic acids (incl. amino acids), or by reducing the matrical potential (e.g., with soluble proteins in leaves of Beta vulgaris ssp. maritima). However, the synthesis of organic solutes is energy demanding and the formation of these solutes decreases the energy status of the plant (Yeo, 1983; Chaves et al., 2009). Thus, for plant survival, growth depression is a necessary compromise in Na+ and/or Cl− excluding species and not a sign of toxicity or nutrient imbalance. 42.2.6.1 Protection of the Cytoplasm It can be distinguished between two salt-specific effects. One is leading to a reduction of the entry of salt into the plant and the other one regulates a low concentration of salt in the cytoplasm. Both effects contribute to root and leaf cytosolic Na+ and Cl− concentrations in the order of 10–30 mM (Wyn Jones and Gorham, 2002; Tester and Davenport, 2003; Koyro and Huchzermeyer, 2004b). The disturbance of metabolism by Na+ or Cl− has to be avoided if plants have to grow on saline habitats. Therefore, the protection of the responsible enzymes by maintaining low cytosolic sodium concentrations is of major importance (Borsani et al., 2003). Indeed, leaves being fed by the transpiration stream receive large quantities of sodium, which must be regulated. Plant cells respond to salt stress by increasing sodium efflux at the plasma membrane and sodium accumulation in the vacuole. For such a reason, the proteins, and ultimately genes, involved in these processes can be considered as salt-tolerance determinants. The cloning experiments of Na+/H+ antiporter have demonstrated the role of intracellular sodium (Ohta, 2002) compartmentalization in plant salt

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resistance. Such compartmentation of sodium and chloride in leaf vacuoles can only be attained with an active transport into the vacuole and low permeability of the tonoplast to these ions. The transport of ions across the plasma membrane and tonoplast requires energy, which is provided by plasma membrane and vacuolar ATPase, respectively (Koyro and Huchzermeyer, 1997; Leigh, 1997). Na+/H+ antiporters, for instance, take advantage of a proton gradient formed by these pumps. Salt stress was shown to increase Na+/H+ activity in glycophytes and halophytes (Apse and Blumwald, 2002). The activation of such antiporters is likely to be operating to reduce sodium toxicity in salt-tolerant plants under saline conditions. 42.2.6.2 Selective Ion Accumulation High salt concentrations can also cause intracellular ionic imbalances (such as of K+, Ca2+, Mg2+, NO3−, PO43−, and SO42−) (Mengel and Kirkby, 2001). The capacity of plants to maintain K+ homeostasis and low Na+ concentrations in the cytoplasm appears to be one important determinant of plant salt tolerance (Yeo, 1998; Läuchli, 1999). A possibility to find such limiting factors is the study of the relations inside single cells such as the compartmentation between cytoplasm and vacuole, the distribution of elements in different cell types or along a diffusion zone in a root apoplast and ultrastructural changes. Beta vulgaris ssp. maritima and Spartina townsendii (salt excreter) both have high Na+ accumulation in the leaves. Both species seem to react similar to salinity with changes in leaf water potential, gas exchange, and nutrients. However, this number of congruence does not allow to conclude analogical intracellular relations. The comparison of their intracellular ionic balance will be used to demonstrate the necessity of special physiological investigations. In contrast to water stress effects that occur in the meristematic region of younger leaves, the effects of ion toxicity predominantly arise in mature leaves (Mengel and Kirkby, 2001). This is because Na+ and Cl− are stored mainly in the shoot of halophytes such as Beta vulgaris ssp maritima and Spartina townsendii leading to a growth reduction of the aboveground parts much higher than of the root (Koyro, 2000; Koyro and Huchzermeyer, 2004a). These changes can be interpreted as signs of a critical load. Therefore, to distinguish between the individual mechanisms of salinity tolerance, further investigations of the intracellular ionic balance were performed first of all at epidermal leaf cells (the end of the transpiration stream) of both these species. However, the intracellular composition of the leaf epidermal cytoplasm and vacuoles of controls of Beta vulgaris ssp. maritima and Spartina townsendii show some more congruities of both species. The epidermal vacuoles of controls of both species contain most of the elements (with the exception of PO43−) in higher concentrations as the cytoplasm indicating the overall picture of a vacuolar buffer. The leaf vacuoles in its entirety can be described as a voluminous potassium pool with high storage capacity for sodium and chloride. This pool is needed in case of high NaCl salinity for the maintenance of the K-homeostasis in the cytoplasm. The dominant elements in the cytoplasm were PO43− and K+. The K+-concentrations were in the epidermal cytoplasm of control plants in an ideal range for enzymatic reactions (Wyn Jones et al., 1979; Wyn Jones and Pollard, 1983; Koyro and Stelzer, 1988). It is obvious that seawater salinity leads to a decrease of PO43−, SO42−, Mg2+, and K+ in the epidermal vacuoles of both species. The remaining K+, SO42−, and Mg2+ concentrations were only in Spartina two digit and especially for K+ much higher as in Beta. The vacuolar buffer of the latter one seems to be exhausted. NaCl salinity led to a significant decrease of the K+ and PO43− concentrations especially in the cytoplasm of Beta and to a breakdown of the homeostasis. This result points at a deficiency for both elements in the cytoplasm. Additionally, the concentrations of sodium and chlorine were at high NaCl salinity below 5 mol m−3 in the cytoplasm of the epidermal cytoplasm and the gradients between cytoplasm and vacuole were higher in comparison with the results of Spartina. In summary, these results support the hypothesis that the sea beet does not sustain ion toxicity but ion deficiency! It is hypothesized that such low K+ levels in the cytoplasm can lead to a reduction of protein


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synthesis, which is of utmost importance in the process of leaf expansion (Mengel and Kirkby, 2001). One possible consequence is the supply of sufficient fertilizers (especially K+ and PO43−) at high NaCl salinity to reduce the symptoms of K+ and PO43−-deficiency in Beta. The salt-induced reductions of the cytoplasmic K+ and PO43− concentrations were much less pronounced in Spartina as in Beta. The results of Spartina point at a working system to keep ionic homeostasis. However, there was one important exception: The sodium concentration increased significantly in the epidermal cytoplasm. Sodium could (to some extent) substitute potassium in its cytoplasmic functions or it could be the first sign of an intoxication. The results and interpretations are in agreement with the hypothesis that plant growth is affected by ion imbalance and toxicity and probably leads to the long-term growth differences between the salt-tolerant and salt-sensitive species. However, Beta and Spartina are also two excellent examples of how important it can be to validate intracellular ionic imbalances (K+, Ca2+, and Mg2+) at high salt concentrations to uncover the individual mechanisms for salt tolerance and to understand the threshold levels of individual species.

42.2.7

MORPHOLOGICAL ADAPTATION

In many cases, various mechanisms and special morphological structures are advantageous for halophytes since they help regulate tissue salt concentrations or minimize water loss and oxidative stress (Figure 42.6, Marschner, 1995; Breckle, 2002; Koyro, 2002; Geissler et al., 2009b). • Succulence and/or a high LMA (leaf mass-to-area ratio) is demonstrated in many genera of plants that inhabit saline environments, such as Suaeda maritima, Sesuvium portulacastrum, or even Aster tripolium. By depositing ions of salts in large vacuoles, the toxicity is partitioned from the cytoplasm and organelles of the cells. • A laterally extensive, shallow root system enables the plant to optimize water and nutrient uptake. • Many halophytes exhibit a rather rapid turnover of their leaves, e.g., in rosette species such as Aster tripolium salts are stored in older leaves and removed from the plant when these leaves are shed. • Excretive halophytes have glandular cells capable of excreting excess salts from plant organs (salt excreter). A simple system with two-celled trichomes that have evolved as collecting chambers for salts, e.g., in Spartina townsendii, Glaux maritima, Triglochin maritimum, and a complex type of salt glands is known e.g., in several mangroves such as Avicennia marina or Limonium vulgare. • Not only glands but also bladder hairs can remove salts from salt-sensitive metabolic sites. Some halophytes like Atriplex or Chenopodium have vesiculated trichomes on the leaf surface which release the salt back into the environment when they are ruptured. • Halophytes often exhibit reflecting surfaces (by wax such as Spartina ssp. or trichomes such as Avicennia marina) preventing ultraviolet light from reaching the leaf tissues and therefore minimizing the development of reactive molecules (reactive oxygen species, ROS, as well as nitrogen radicals). • Curled leaves, fine hairs, waxy cuticle, high stomate density, a small stomate size, sunken stomata, elevations on the surface such as bulliform cells support the buildup of an unstirred layer at the leaf surface and reduce transpiration and thus import of salt. For example, in Aster tripolium salinity leads to a significant increase in cuticle and cell wall thickness of the epidermal leaf cells. • A decrease in intercellular spaces is often observed with increasing salinity in order to reduce transpiration, such as in Aster tripolium or Beta vulgaris ssp. maritima. • An increased number of vesicles under saline conditions and transfer cells in the vascular bundles facilitate selective ion transport processes, such as in Aster tripolium.

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0.4 ± 0.013 µm*

Cut 0.3 ± 0.002 µm

Cut cw

cw 1.7 ± 0.26 µm

2.3 ± 0.44 µm

1 µm

1 µm (a)

(b) 100 μm

100 µm

ad

ad pp pp

vb sp sp

ab

ab (c)

(d)

tc

s d

d tc d 5 μm

200 nm

5 μm

(e)

300 nm

(f )

FIGURE 42.6 Structural features of Aster tripolium. (a) Cell wall and cuticle of the upper leaf epidermis, control; (b) cell wall and cuticle of the upper leaf epidermis, 375 NaCl; (c) cross section of leaf, control; (d) cross section of leaf, 375 NaCl; (e) transfer cell in leaf vascular bundle (phloem); (f) dilations of thylakoid membranes, 375 NaCl.


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Although halophytes have developed morphological structures (and physiological mechanisms) that help regulating tissue salt concentrations or to minimize water loss and oxidative stress, they may show structural symptoms of disorders under high salinity levels (Yamada et al. 2009). Dilations of cell membranes such as the thylakoid membranes in the chloroplasts are often found not only in salt-stressed glycophytes, but also in halophytes (Fidalgo et al., 2004; Paramanova et al., 2004; Geissler et al., 2009b). Such kind of damage has been discussed by many authors as a consequence of oxidative stress (Mitsuya et al., 2003; Oksanen et al., 2005), but it may also be the result of ion toxicity or imbalance (Keiper et al., 1998; Yamane et al., 2003; Geissler et al., 2009a,b).

42.2.8

SUSTAINABLE USE OF HALOPHYTES

Physiological studies using the sea water irrigation system have the potential to provide highly valuable means of detecting individual mechanisms of species against NaCl stress, and may also provide opportunities for the comparison and screening of different varieties for their adaptation to salinity (QCS for cash crop halophytes). After the selection of halophytic species suited for a particular climate and for a particular utilization, green house experiments at the local substrates (and climatic conditions) to select and propagate promising sites (Isla et al., 1997) have to be started. This must be followed by studies with lysimeters on field site to study the water consumption and ion movements. Last but not least, a design for a sustainable production system in plantations at coastal areas or at inland sites (for example, for economical use) has to be developed.

42.3 42.3.1

BIOCHEMICAL ASPECTS INHIBITION OF PRIMARY REACTIONS OF PHOTOSYNTHESIS

Limitation of plant growth by environmental factors is a matter of general concern especially with respect to crop production and food and feed supply. Photosynthesis is dominating plant growth and production of biomass. Therefore, the sequence of reactions leading to the phenomenon named photosynthesis is in the focus of interest when breeding for high crop yield. In order to allow detailed analysis of salt effects, several individual steps have to be distinguished (Figure 42.7). At first, there are primary reactions of photosynthesis, namely, absorption of light energy and (1) its conversion to redox energy, conserved in the coenzyme NADPH, and (2) energy of chemical Reaction site

Reaction

Energy conversion

Thylakoid membrane

Light absorption

Light energy

Electron transport ATP NADPH Ferredoxinred

Chloroplast stroma

Cytosol

Calvin cycle Nitrate reduction

Sugars

Amino acids

Redox energy Membrane potential

Energy of coenzymes

Energy of metabolites

Transport tissues Meristems and storage organs

Plant growth/biomass

Energy in biomass

FIGURE 42.7 Photosynthetic energy flow. Reaction sequence of photosynthesis can be described in terms of reaction sites (left column), sequence of reactions (center), conversion of energy forms (right column).

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bounds, conserved in the coenzyme ATP. On the second level, we find reactions of the Calvin cycle, nitrate and sulfate reduction as well as sugar, lipid, and amino acid metabolism. Typical reactions we have to discuss on the third level are trans membrane and inter tissues transport of metabolites. The fourth level of photosynthesis relates to physiological aspects of gas exchange, turgor homeostasis, etc.; these aspects already have been discussed above. In the literature, we find several publications on salt stress effects on photosynthesis (see, for example, Chaudhuri and Choudhuri, 1997; Rajesh et al., 1998; Soussi et al., 1998; AliDinar et al., 1999; Kurban et al., 1999; Kao et al., 2001; Romeroaranda et al., 2001). Mostly, it is stated that salt stress response of plants is a multifactorial trait. This statement is correct, for sure. But it does not provide sufficient help for breeders and farmers requesting advice what traits to look at and how to treat crops in the field properly. In many papers analyzing salt stress effects on photosynthesis, more precise answers could have been achieved, if authors would have described more precisely what they really have been measuring. The general impression is that most papers deal on salt effects on physiological aspects of biomass production rather than partial reactions of photosynthesis as defined above. As there are cross reactions as well as cell signaling involved in regulation of photosynthetic metabolic pathways, there are no strict correlations between biomass production and individual gene activities. As a consequence, predicted correlations, though they had been observed in laboratory experiments, could not be shown in subsequent field experiments. Here, we try a more differentiated approach and review analysis of salt stress effects at different levels of photosynthesis. It will become very obvious that no individual laboratory is able on its own to approach the problem of salt effects on plant growth in an appropriate way. Working on plant salt stress physiology requires cooperation among teams of different expertise. 42.3.1.1 Photosynthetic Conversion of Energy In plants active in photosynthesis, energy of light quanta is absorbed by chlorophyll. Just like all other pigments, activated chlorophyll can return from its activated state to the stable, nonactivated state by emitting heat. Other than most pigments, chlorophyll after absorption energy of a red light quantum is sufficiently stable in its activated state to allow transfer of energy to acceptor molecules of biological relevance (Strasser et al., 2004; Figure 42.8). In principle, there are the two options of biological relevance in addition to a third one we can use for monitoring of plant performance: (1) resonance energy transfer to activate other pigments and (2) transfer of an electron to an acceptor. The electron acceptor can be a component of the photosynthetic electron transport chain or any

Activated state Pigments (NPQ) Photosynthesis Light

O2

Heat

ROS Fluorescence Ground state

Chlorophyll

FIGURE 42.8 Competition for absorbed energy of light quanta. Photosynthetic electron transport is competing with “futile reactions” for energy of light-activated chlorophyll.


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other molecule having a less negative potential as compared to activated chlorophyll. This second option requires a “refill” of electrons, and it is well documented that in chloroplasts the water splitting system is functioning that way in order to have chlorophyll recycled to its ground state again (Strasser et al., 2004). (3) Another option, competing for energy with the already mentioned ones, is the emission of fluorescence energy (Schreiber, 1997; Schreiber et al., 2002; Strasser et al., 2004). As will be discussed later, any inhibition of an individual pathway will enhance the possibility of the other pathways to occur. In thylakoid membranes, photosystems I and II can be distinguished from other chlorophyllcontaining protein complexes, the light-harvesting complexes. Only the special pairs of chlorophylla located in the active centers of the two photosystems are involved in electron transport. Energy transfer among other pigments occurs via resonance energy transfer. In this section, we focus on electron transfer reactions. As known from the literature, half lifetime of activated state of chlorophyll is very short and depends on the environment of the pigment (Rees et al., 1990; Laible et al., 1994; Ma et al., 2009). It is obvious that efficiency of photosynthesis depends on the probability that activated chlorophyll will transfer electrons to an acceptor of the photosynthetic electron transport chain rather than “wasting” energy by using one of the other pathways mentioned above (Ruban et al., 1993; Horton et al., 1996; Horton, 2000; Allen and Forsberg, 2001). In order to meet this requirement, reaction partners are arranged in ideal neighborhood within protein complexes located in the thylakoid membranes. Moreover, it has been demonstrated that this structure undergoes permanent adjustment to match the requirement of chloroplast metabolism and to adapt to changes in the environment, i.e., changes of light quality and intensity, for instance (Allen and Bennett, 1981; Allen, 1992; Allen and Forsberg, 2001). By means of photosynthetic electron transport, energy of absorbed light quanta is converted to redox energy stored in the coenzyme NADPH and proton motive force (Mitchell, 1967) stored in a proton gradient across the thylakoid membranes. This proton gradient is the driving force for ATP synthesis catalyzed by the chloroplast F-type ATPase, called the CFOCF1-complex or the chloroplast coupling factor (Strotmann et al., 1976; Huchzermeyer and Strotmann, 1977; Boyer, 2000). The number of coenzyme molecules (NADP +/NADPH and ADP/ATP) is limited, and there is no exchange of coenzymes among cell compartments. Therefore, it is required for efficiency of this machinery that acceptor forms of coenzymes, NADP + and ADP, respectively, permanently are recycled by subsequent metabolic pathways. Otherwise, turnover of energy by the electron transport would be inhibited, and this inhibition finally would lead to an inhibition of electron release from activated chlorophyll, enhancing the probability of alternative routes mentioned above (Figure 42.8). Photosynthetic CO2 assimilation is the major consumer recycling both coenzymes in the reaction sequence of the Calvin cycle. Under physiological conditions, as a rule of thumb, in chloroplasts of nonwoody plants two-thirds of the electrons from the noncyclic electron transport pathway finally will be consumed by CO2 fixation while one-third will be used for nitrate reduction (see top part of Figure 42.10) (Schmidt and Jäger, 1992). But it has to be mentioned here that a significant portion of the absorbed light energy will be “wasted” in futile reaction sequences rather than be used for biomass synthesis. 42.3.1.2 Salt Effects on Photosynthetic Energy Conversion In order to understand individual reactions of photosynthesis, and finally get an overview of the principles how they interact, thylakoid membranes and protein complexes have been isolated and analyzed with respect to their structure and function. During preparation and subsequent tests of enzyme activities high salt concentrations (50 mM NaCl as a rule) have been applied without any inhibitory effect on individual enzyme activities (Strotmann et al., 1976). Such high salt concentrations are not found inside chloroplasts, neither under physiological conditions nor under salt stress. It therefore can be concluded that primary reactions of photosynthesis are not directly inhibited under salt stress (Richter et al., 2000; Huchzermeyer et al., 2004; Huchzermeyer and

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Koyro, 2005; Koyro and Huchzermeyer, 2005). But this conclusion disagrees with apparent inhibition of photosynthesis observed in whole plant experiments (Lawlor and Fock, 1978; Lawlor, 2002). Therefore, it has to be analyzed in more detail, at what extent the observed inhibition may be attributed to salt-dependent changes in thylakoid structure (Hesse et al., 1976) or other effects described in the physiology and transport section of this chapter. One first approach to answer this question could be a detailed analysis of the kinetics of fluorescence light emission subsequent to chlorophyll activation by light pulses. Application of the pulsed amplitude modulation technique (PAM) allowed a detailed analysis of the network of primary reactions in photosynthesis (Strasser et al., 2004). It became quite obvious that salt stress does not directly inhibit primary reactions of photosynthesis but inhibits product export and fine-tuning of primary reactions by interfering with optimal arrangement of proteins and membranes (Koyro and Huchzermeyer, 1999b; Huchzermeyer and Heins, 2000; Huchzermeyer, 2000). Accordingly, it was observed that maximal photochemical efficiency, indicated by high Fv/Fm values of chlorophyll fluorescence, remain high under tolerable salt stress, while growth rate of turf grass, for instance, was reduced under the same salinity level (Lee et al., 2004). It will be discussed in subsequent paragraphs how salt can inhibit export of products of photosynthesis and why proper function of the phosphate translocator, located in the inner envelop membrane, is essential for photophosphorylation (see Section 42.3.4). At this stage, we can state that inhibition of product export feeds back to primary reactions and finally will inhibit photosynthetic electron transport. One option to release energy from its activated state is blocked and chlorophyll will increase activity of fluorescence light emission, heat production, energy transfer to other pigments, and ROS production. This latter option will be discussed in the following paragraph (see Section 42.3.2). Though leguminosae like peas and beans are of high importance, we will not comment on special aspects of photosynthesis linked to symbiosis. From current literature it becomes obvious that in these crops nitrogen fixation in root nodules is more sensitive to salt stress as compared to CO2 fixation. In chick pea, for instance, it was found that this apparent high sensitivity is due to interference of incoming salt with malate transport to the bacteroides (Soussi et al., 1998). Discrimination of primary and secondary salt stress effects in general is complicated, and primary targets within the metabolic network of hosts and symbionts are not easy to identify. 42.3.1.3 Salt Effects on Chloroplast Structure and Metabolite Transfer Chloroplasts easily can be sedimented by centrifugation because of their very high protein content. Süss and coworkers argued that inside the chloroplast stroma, proteins tend to interact and form aggregates. Indeed, they were able to isolate such “super complexes” and found out that they contain enzymes belonging to individual metabolic pathways (Süss et al., 1993). Such an arrangement helps to increase substrate turnover, because diffusion distances among enzymes of a pathway are close to zero. For the same reason, formation of such aggregates prevents occurrence of side reactions, because intermediates are not available for other enzymes. As will be discussed in Section 42.3.3.1, salt on one hand can inhibit turnover of substrates by destroying enzyme aggregates. On the other hand, intermediates of sugar metabolism, for instance, can become available for other enzymes and alternative products (compatible solutes, for instance) can be formed. Though salt effects on metabolite patterns have been analyzed in several papers, no data linking these findings to the occurrence of protein aggregates are available to date. The occurrence of thylakoid grana stacks has attracted a lot of interest for years (Huchzermeyer et al., 1986; Lam and Malkin, 1989; Malkin and Braun, 1993; Romanowska and Albertson, 1994). It was calculated that about 70% of all PSII complexes are located within stacked regions of the thylakoid membranes, while most of the PSI complexes are found in un-stacked part of the thylakoid membranes (Schmidt and Malkin, 1993; Romanowska and Albertsson, 1994). From this, it was concluded that 70% of the PSII complexes must be in an inactive state, because the distance between PSII and PSI would be to far to allow sufficient turnover rates of photosynthetic electron transport


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(Malkin and Braun, 1993). Moreover, it was found that grana are unstable, permanently folding and unfolding structures. Apparently CFOCF1-complexes are initiating formation of membrane loops (Boekema et al., 1988) and grana are formed by interaction of membrane proteins (Staehelin, 1975). As active PSII has an extremely short half lifetime in the range of 20–30 min (Kuhn and Böger, 1990; Trebst and Soll-Bracht, 1996; Keren et al., 1997; Jansen et al., 2001), the function of this permanent modification of membrane structure may be, to initiate interaction among membrane proteins and to support building of aggregates of protein complexes interacting in photosynthetic electron transport chain. Such preferred neighborhood of membrane proteins has been found, indeed (Laszlo et al., 1984; Huchzermeyer and Willms, 1985). There are several publications indicating that such neighborhoods of protein complexes are controlling efficiency of energy conversion in primary reactions of photosynthesis (see papers on localized protons, for instance, Laszlo et al., 1984; Löhr and Huchzermeyer, 1985; Löhr et al., 1985; Allnutt et al., 1989). Moreover, they might be controlling photo-inhibition as well (Lu and Zhang, 1999; Lu et al., 2002). From the above description, it is obvious that formation of grana stacks is essential for optimal functioning and permanent repair of photosynthetic electron transport rate. It has been found that grana become destabilized if the ratio of monovalent and divalent cations is impaired (Hesse et al., 1976). Thus, it may be argued that salt stress does not directly inhibit individual primary reactions of photosynthesis but will impair adjustment to environmental conditions and destabilize structures allowing optimal energy use. Until now salt-tolerant and salt-sensitive plant species have not been analyzed for differences in this respect. Analysis of the situation becomes more complicated by recent findings: Apparently repair of light and ROS stressed photosystem II can be impaired on translation level of the D1 protein via interference with photorespiration (Takahashi et al., 2007). This way, inhibition of metabolite transfer between chloroplasts, peroxisomes, and mitochondria would feed back on photoinhibition. Data on salt stress effects on photosystem I of higher plants currently are not available. But investigation of primary events of photosynthesis in green algae indicate that assembly of PSI subunit structure is impaired by incoming salt (Allakhverdiev et al., 2000) (more recent publication in preparation).

42.3.2

SALT-INDUCED PRODUCTION OF POTENTIAL TOXIC INTERMEDIATES OF PHOTOSYNTHESIS

42.3.2.1 ROS 42.3.2.1.1 The Physiological Situation As described above, any reduction of electron transport rate, especially under high light conditions, will increase the risk of ROS production (see Figure 42.8). Concentration of these reactive compounds will build up in the light and eventually will reach concentrations toxic for cells active in photosynthesis. The redox potential of activated chlorophyll is more negative than the one of oxygen. Therefore electron transfer from activated chlorophyll can occur unless energy is abstracted from activated chlorophyll faster than electron transfer to oxygen can occur. There are further options of ROS production as several intermediates of photosynthetic electron transport are radicals. On the other hand, ROS can spontaneously convert or can be turned over under the control of enzymes (Figure 42.9). Accordingly, in the literature the occurrence of various forms of ROS is described (Halliwell and Gutteridge, 1986; Elstner, 1987). Cytotoxicity may be attributed to oxidative damage of membrane lipids (Fridovich, 1986; Wise and Naylor, 1987) as well as oxidation of proteins and nucleic acids (Fridovich, 1986; Imlay and Linn, 1988). In the field, salt stress results in severe damage especially in situations when its inhibitory effects occur in presence of high light intensity. Then PSII activity will result in high oxygen concentrations, especially if stomata are closed under stress. Concomitantly, chlorophyll will remain in its active state for a prolonged period of time, as electron transport rate is reduced by inhibited off flow of products. Thus, probability for a transfer of electrons from activated chlorophyll to molecular

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Handbook of Plant and Crop Stress Light 2O2 H2O 1 2

Activated chlorophyll 2H+ 2O2

O2

O2

Catalase

–

or H2O2

SOD

Toxic effects

2H2O

APX

2OH 2O2H Ascorbate

NADP

+

2GSH Glutathione reductase

NADPH

Monodehydroascorbate radical

Dehydroascorbate reductase

Monodehydroascorbate reductase

GSSG NADP

+

NADPH

Dehydroascorbate

FIGURE 42.9 ROS production and detoxication. Detoxication of ROS by sequential ascorbate and glutathione cycles will consume NADPH and, thus, result in a relief of NADP + shortage in high light. A prerequisite is that (1) enzymes involved are available at ample concentrations and (2) are positioned in ideal neighborhood to allow high turnover rates.

oxygen to form O2•− will increase. O2•− will rapidly dismutate to yield O2 and the less reactive ROS H2O2. But in the presence of some cations, like Cu and Fe, for instance, highly reactive OH•− may be formed (Imlay and Linn, 1988; Figure 42.9). H2O2 is one of the most important secondary messengers in plant tissues, modulating effects of hormones and involved in developmental control of cells and tissues (van Breusegem and Dat, 2006; van Breusegem et al., 2008). It has been shown that mitogen-activated protein kinases (MAP kinases) are involved in transduction of H2O2 signaling on cellular level (Pitschke and Hirt, 2009). Apparently, MAPK3 and MAPK6 are integrating stress signals that regulate stomatal development (Wang et al., 2008a,b). Moreover, in an earlier paper, Verslues et al. (2007) have shown that nucleoside diphosphate kinase 2 (NDPK2) can interact with salt stress signaling salt overlay sensitive 2 kinase as well as with catalase. Genes under the control of these pathways have to be identified. A mode of cell signal tuning may occur via regulation of transcript stability. In agreement with this assumption, it has already been found that stability of the SOS1 mRNA, induced under salt stress, is enhanced with increasing ROS concentration (Chung et al., 2008). Ascorbic acid is a major antioxidant in plants. It detoxifies reactive oxygen species and maintains photosynthetic function (Figure 42.9). Through its ascorbate recycling function, dehydroascorbate reductase affects the level of foliar reactive oxygen species and photosynthetic activity during leaf development. As a consequence, this enzyme influences the rate of plant growth and leaf aging (Chen and Gallie, 2006). ABA-induced closure of stomata and ABA-mediated inhibition of stomata opening are two ABA effects based on different reaction sequences (Mishra et al., 2006). Nevertheless, both processes


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are fine-tuned by ROS signaling, and it appears to be clear that ROS signals are transduced via a cascade of MAP kinase reactions (Gudesblat et al., 2007). 42.3.2.1.2 Detoxification of ROS As stated above, ROS production occurs permanently at a certain probability. It is well documented that ROS as well as nitrogen radicals are involved in cell signaling (Wilken and Huchzermeyer, 1999) in plant cells like in cells of most other organisms. But, as high ROS concentrations are toxic, plants are equipped at varying degrees with several systems to detoxify ROS species. With this respect, molecules having antioxidative potential can be discriminated from enzyme-catalyzed reaction sequences. Alpha-tocopherol is synthesized in the chloroplast (Schultz et al., 1976) and can be found in high concentrations in thylakoid membranes. Alpha-tocopherol disrupts lipid peroxidation cascades, reacts with O2•−, and is capable of scavenging hydroxyl-, peroxyl-, and alkoxyl-radicals (Halliwell, 1987). Oxidation of alpha-tocopherol leads to formation of an alpha-chromoxyl radical, which can be reduced by ascorbic acid. Ascorbic acid and several redox-active tri-peptides, called glutathiones, can be found in chloroplasts in millimolar concentrations (Halliwell, 1982). Several different types are known from plants (Schmidt and Jäger, 1992). This finding suggests that they are involved in different pathways controlled by specific enzymes. But there is only little information available to date. In chloroplasts, H2O2 can be detoxified by an ascorbate-specific peroxidase (Chen and Asada, 1989) involved in the ascorbate–glutathione cycle (Halliwell and Guteridge, 1986) (Figure 42.9) while in the cytosol H2O2 detoxification is catalyzed in a catalase-dependent reaction. Other enzymes involved in detoxification of ROS are superoxide dismutase, which converts O2•− to H2O2, and several peroxidases (Chang et al., 1984). Antioxidants as well as enzymes capable of detoxifying ROS are present in all plants and plant tissues. But their concentrations and catalytic activities, respectively, as well as their patterns differ a lot (Streenivasulu et al., 2000). Therefore plants differ in their capacities (1) to immediately detoxify ROS upon their occurrence and (2) to build up a detoxification potential under stress. If the balance between production of ROS and quenching capacity of the respective tissues is upset, oxidative damage will be produced (Harper and Harvey, 1978; Dhindsa and Matowe, 1981; Wise and Naylor, 1987; Spychalla and Desborough, 1990). In experimental approaches, it was demonstrated that (1) enzyme activities of antioxidative pathways increase as a salt stress response (Verma and Mishra, 2005) and that (2) the maximal level of salt tolerance correlated with maximal respective enzyme activities (Gossett et al., 1994; Hernandez et al., 1995, 2000; Sehmer et al., 1995; Kennedy and De Fillippis, 1999; Benavides et al., 2000; Lee et al., 2001; Mittova et al., 2002, 2003). Their importance in developing salt stress tolerance of individual antioxidants along with enzymes stimulating their turnover has been documented in several approaches using molecular genetic techniques. The Arabidopsis soz1 mutant contains only one-third of the ascorbate concentration of the wild type. As expected, the mutant is less tolerant toward ROS stress as compared to the wild type (Conklin et al., 1996). The observed positive correlation between a plant’s antioxidative capacity and salt stress tolerance was supported further by analysis of salt stress–induced changes of mRNA patterns. In citrus, for instance, not only enzyme activities of Cu/Zn-SOD, glutathione peroxidase, and cytosolic APX increase, but also the respective mRNAs were found at higher abundance (Holland et al., 1993; Gueta-Dahan et al., 1997). This result suggested that plants are able to adapt their antioxidative capacity to physiological needs. Moreover, de novo synthesis of enzymes rather than regulation of those already present appears to be essential. This idea was supported by Wilkens et al., who used antisense mutations to produce catalase-deficient tobacco plants. They found that catalase deficiency results in ROS sensitivity when salt stress is applied (Wilkens et al., 1997). In addition to the above-described negative controls, improving stress tolerance by overexpression or de novo implementation of genes has been tested as well. Successful approaches of this kind

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leading to improved stress tolerance increased the concentrations of (1) mitochondrial Mn-SOD, Fe-SOD, chloroplastic Cu/Zn-SOD, and glutathione-S-transferase/glutathione peroxidase (Bowler et al., 1991; Gupta et al., 1993a,b; Van Camp et al., 1996; Shikanai et al., 1998; Roxas et al., 2000) or (2) of Bet-A product leading to enhanced concentration of glycine betaine (Bhattacharya et al., 2004). In summary, these results indicate that salt stress tolerance may be improved by overexpression of genes involved in the antioxidant system of plants. The advantage of this approach is that mostly only one or a very limited number of genes have to be overexpressed to improve scavenging of ROS. A strategy to improve stress tolerance of plants might be to treat cultures with compounds stimulating the production of antioxidants or enzymes involved in detoxification pathways. Apparently, putrescine is such a compound. It was found that putrescine treatment of Brassica resulted in increased contents of glutathiones and carotenoids, and that this increased antioxidant content of leaves was paralleled by stimulated growth rates of mustard seedlings under stress (Verma and Mishra, 2005). 42.3.2.2 Photorespiration Closure of stomata thus inhibiting gas exchange is a secondary effect of salt stress, mostly brought about by ABA released from the plant roots. In the presence of light, O2/CO2 ratio will increase inside leaves and impair CO2 fixation especially in C3 plants. This happens as the enzyme Rubisco can bind O2 instead of CO2 to its reaction center, thus catalyzing synthesis of a C3 plus a C2 compound instead of two C3 compounds in the primary reaction of the Calvin cycle (see Figure 42.10). The C2 compound, 2-phosphoglycolate, will be converted to glycolate, a molecule that cannot be metabolized by chloroplasts. Concentration of this molecule eventually would become toxic. Nitrate Chloroplast Nitrate

Calvin cycle

NADPH NADP Ribulose 1,5-bisphosphate CO2 O2

NH3

ATP

6 Ferredoxinred Glutamate

Glutamine NADPH

NADP 3-Phosphoglycerate

Glycerate

Glycolate O2

NH3

2-Oxoglutarate

NAD

H2O2

NADH

Glyoxylate

Peroxisome

Glutamate

NAD NADH Glycine

NH3

Serine Mitochondrium

Aspartate 2-Oxaloacetate

CO2

Glycine

NH3 Glutamine

2-Oxaloacetate

2-Oxoglutarate

Glutamate

Hydroxypyruvate

Glucose

Glutamate

2-Phosphoglycolate

ATP

2 Glutamate

Cytoplasm

FIGURE 42.10 Linking metabolic pathways of cell compartments. Biochemical pathways of chloroplasts, peroxisoms, and mitochondria are linked via shuttle systems. With respect to energy flow, photorespiration and nitrate reduction are of focal interest. Like the Calvin cycle, nitrate reduction is consuming electrons released from photosystem I. Thus, nitrate reduction is recycling the cofactors ferredoxin and NADP +. Photorespiration, on the other hand, is transferring redox power to the mitochondria and is involved in shuttling of ammonia.


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Detoxification and recycling of the C3 compound 3-PGA are understood to be the major function of a pathway called photorespiration. Photorespiration takes place by metabolite transfer between three compartments: chloroplasts, peroxisomes, and mitochondria. The photorespiratory reaction cycle we know from our textbooks is a simplification allowing general estimates. We know that amino acids (glycine, serine, glutamic acid, and glutamine) may be subtracted from or fed into the cycle in vivo. Such reactions modify nitrogen flow among cell compartments. But, with respect to equilibrium of carbon flow, another aspect may be more important: It has been shown by Niessen et al. that mitochondrial glycolate oxidation contributes to photorespiration in higher plants as well (Niessen et al., 2007). This finding not only complicates photorespiratory reaction mechanism, it also provides further options of fine-tuning of the system: (1) the mitochondrial glycolate dehydrogenase reaction contributes to energy balance of oxidative phosphorylation (Paul and Volcani, 1976); (2) the turnover capacity is lower as compared to the peroxisomal pathway. Thus the total energy balance of photorespiration will vary with Rubisco oxygenase activity. Several efforts have been made to improve plant productivity. These include attempts to improve the efficiency of Rubisco by engineering or by prospecting known organisms for more efficient variants (Andrews and Whitney, 2003). The most ambitious approach would be to engineer C4 photosynthesis into a C3 cereal, because it requires targeting photosynthesis pathways in two different cell types. Moreover, it would include structural modification of leaf tissues. Therefore, it is not a surprise that no such experiments have been reported to be successful so far. But an impressive result has been presented by Kebeish et al. They managed to express a photorespiratory bypass inside chloroplasts (Kebeish et al., 2007). They took advantage of the glycolate catabolism from E. coli that is using NAD+ as an electron acceptor and does not produce ROS. Moreover, the bypass is releasing CO2 inside chloroplasts rather than inside mitochondria. This way refixation of CO2 is favored. Nevertheless, the success is stunning, because chloroplasts are not known to be capable of accumulating CO2. Therefore, the advantage probably is due to improved turnover rates rather than enhanced substrate (CO2) concentrations. Such an interpretation would be in line with our arguments. On this basis, it may be concluded that salt stress can interfere with protective effects of photorespiration by inhibition of its turnover capacity; by intermediate shuttling among compartments, for instance. It has to be kept in mind that photorespiration is a major source of H2O2 in illuminated C3 leaves. On the other hand, H2O2 production and interaction with pyridine nucleotide coenzymes make photorespiration an important player in cellular redox homeostasis. Furthermore, H2O2 is an important second messenger controlling cell development and tuning effects of hormones like ABA, for instance. Any interference with this reaction will have effects not only on immediate energy status and phosphorylation efficiency, but also on plant hormonal responsiveness, thus on development and plant life cycle (Foyer et al., 2009). In most textbooks, it is stated that photorespiration is a major problem of C3 plants rather than C4 and CAM plants. The latter ones can overcome the problem on the expense of extra energy consumption by using the C4 pathway, i.e., binding CO2 to PEP to form a C4 compound (that can be reduced to malate, for instance). In the final carbon fixation step, CO2 will be released from the C4 compound, and assimilation will be catalyzed by Rubisco in an environment characterized by low oxygen partial pressure. Based on these findings, it is argued that formation of significant concentrations of glycolate is not a problem of C4- and CAM plants. If salt stress would interfere with aggregate formation of chloroplasts, mitochondria, and peroxisomes, thus inhibiting by glycolate toxicity proper performance of C3 plants; such effects should not be observed in C4- and CAM plants. Nevertheless, salt stress might interfere with intermediate transfer among cells and cell compartments in C4- and CAM plants. This would inhibit photosynthetic activity but would not result in “classical” glycolate toxicity. But, again the situation apparently is not as simple as suggested in textbooks. Several studies have pointed out that a low rate of photorespiration takes place in C4 plants as well. In maize leaves

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grown at normal CO2 concentration, photorespiration may reach 5% of the rate found in tobacco grown under identical conditions (Zelitch, 1973). Though such a rate may appear to be low, glycolate oxidase activity obviously is essential for C4 plants. Maize plants showing less than 10% of glycolate oxidase activity of the wild type could grow at high CO2 concentrations, they became necrotic in normal air, and died within 2 weeks (Zelitch et al., 2009). There is evidence that glycolate can inhibit QA/QB electron transfer in PSII (Petrouleas et al., 1994). As stated above, this would lead to stimulated ROS production. This interpretation is in line with the observed bleaching of maize in presence of glycolate (s.a.). In several publications, it is suggested that the function of photorespiration is to serve as a sink to dissipate excess redox energy (Kozaki and Takebe, 1996; Wingler et al., 2000). In terms of our arguments, this would mean photorespiration is recycling coenzymes functioning as acceptors of photosynthetic electron transport chain. This would help preventing ROS production as well. But, to our understanding, this interpretation does not sufficiently take into account the compartmentation of coenzymes. 42.3.2.3 Non-Photochemical Quenching of Energy Under conditions that limit assimilation of CO2 the potential rate of NADPH production exceeds the actual rate of consumption of reductive power. In order to be able to grow under stressful conditions, plants have to be equipped with mechanisms preventing excess reducing power. But, these futile mechanisms compete with photochemistry for absorbed energy. They lead to a decrease in quantum yield of photosystem II (Genty et al., 1989). The photosystem II antenna is highly flexible in tuning delivery of excitation energy to the photosystem II reaction center (Horton et al., 1996). The principal adaptation mechanism in photosynthesis is the control of thermal dissipation of excess energy within the photosystem II antenna, thus matching physiological needs (Johnson et al., 2009). In C3 plants, losses by this mechanism, named non-photochemical energy quenching, may exceed the ones caused by photorespiration. Despite extensive investigations, the reaction mechanism of photochemical quenching is not completely understood by now, because (1) turnover of intermediates is fast and (2) reaction depends on intact structures of protein complexes and their in vivo arrangement inside the thylakoid membranes; i.e., reaction partners may not be extracted and individually analyzed. But some insight was achieved by a combination of molecular biological and biophysical techniques (Johnson et al., 2009). For experimental approaches investigating salt stress effects, it is important to know that non-photochemical quenching of excitation energy is comprised of a fast and a slow component, qE and qI, respectively. Both reactions are reversible. The trigger of qE is the ΔpH across the thylakoid membrane sensed by the PsbS subunit of the light-harvesting complex (Li et al., 2000, 2004). Full expression of qE is associated with the enzymatic de-epoxidation of violaxanthin to zeaxanthin. This reaction is part of the xanthophylls cycle (Havaux et al., 2007). Enzymes involved are pH controlled and function on the expense of NADPH (Demmig-Adams and Adams, 1996). This makes the cycle a futile reversible reaction sequence on its own. The majority of photoactive xanthophylls is bound to the light-harvesting complex. In addition to the ones involved in the xanthophyll cycle, lutein and lutein epoxide are bound there as well and can be turned over in a cycle on their own (Matsubara et al., 2001). Depending on distances among pigments, these two cycles can interact with soluble xanthophylls and control non-photochemical energy quenching in a synergistic, but not well-understood way (Johnson et al., 2009). Sensitivity of non-photochemical quenching to any experimental approach interfering with membrane structure and protein fine structure indicates that this reaction sequence of outstanding physiological importance will be highly sensitive to any reaction causing imbalance of ionic homeostasis. The threshold ion concentration resulting in significant inhibition of non-photochemical quenching will depend on availability of compatible solutes, for instance. Based on current understanding, it can be expected that such adverse effects can be monitored (1) by measuring pigment shifts due to altered ratios of xanthophylls and (2) by high-resolution chlorophyll fluorescence measurement.


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Thus, it should be possible to measure salt effects on non-photochemical quenching even in the field using noninvasive techniques. Especially, near the end of life cycle of leaves, masking of chlorophyll by anthocyanins becomes important. It prevents photooxidative damage and allows an efficient nutrient retrieval from leaves to storage organs (Field et al., 2001). Some plants use such mechanisms regularly when under salt stress. They can be identified, because leaf color will vary depending on their growth conditions like it can be observed with Salicornia and Sempervivum, for instance.

42.3.3 PROTECTING FROM DIRECT SALT EFFECTS ON PROTEIN AND MEMBRANE STRUCTURE AND FUNCTION 42.3.3.1 Control of Mechanisms Improving Stress Tolerance: Compatible Solutes As mentioned in the physiology section, sequestration of ions to the vacuole is a strategy leading to enhanced salt stress tolerance. This strategy requires compensation for osmotic and ionic potential of vacuolar ions. Part of the ionic component is delivered by surface charges of cytosolic proteins. As a rule, the Donan potential of proteins is beyond experimental access, and no reliable data are available to date (Gerendás and Sattelmacher, 2002). Some low molecular weight, nontoxic compounds have been identified to significantly contribute to ionic and osmotic balance inside cells. They are called “compatible solutes” (Yancey et al., 1982; Ford, 1984; Ashihara et al., 1997; Hasegawa et al., 2000; Zhifang and Loescher, 2003). Chemically they can be described to be poly-amines or poly-hydroxyls. In addition to their function in ionic and osmotic homeostasis, their important function is that they can replace water in its function to stabilize aggregates of soluble proteins and membrane–protein interactions, respectively (Yancey et al., 1982; Crowe et al., 1992). Depending on enzyme patterns and metabolic pathways preferred in individual plants, different compatible solutes have been found in plants (Figure 42.11). Among the compounds described in the literature are proline (Khatkar and Kuhad, 2000; Singh et al., 2000), glycine betaine (Rhodes and Hanson, 1993; Khan et al., 1998; Wang and Nil, 2000), sugars (Pilon-Smith et al., 1995; Bohnert and Jensen, 1996; Kerpesi and Galiba, 2000), di-, tri-saccharides and other sugar derived compounds (Hagemann and Murata, 2003), and polyols (Ford, 1984; Popp et al., 1985; Orthen et al., 1994; Bohnert et al., 1995). Overexpression of genes of metabolic pathways leading to production Compatible solutes Sorbitol

NADH Glucose NADH

Mannitol

3-P-glycerate 2-Oxoglutarate

Mannose ATP + NADH

Proline

Glycine betaine

Glutamate

Ethanolamine (from membrane lipids)

Serine (photorespiration)

FIGURE 42.11 Overview on metabolism of compatible solutes. Glucose can function as a substrate for synthesis of compatible solutes. As a rule, cytosolic synthesis occurs at the expense of NADH. Thus, synthesis of compatible solutes is a relief under conditions, when production of redox power and glucose are exceeding consumption of electrons and export of sugar. Cytosolic pattern of compatible solutes varies among plant species depending on respective enzyme activities. In addition to regulation of respective gene expression, synthesis rates are controlled by availability of precursors.

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of compatible solutes has been shown to improve salt tolerance. A prerequisite is that (1) substrates are available in ample amounts, and (2) overproduction of these compounds does not interfere with plant growth and development (Parida et al., 2002; Parida and Das, 2005). Apparently, there are three ways to explain the mechanism how compatible solutes become overproduced under stress. Under optimal growth conditions, these compounds are produced at low rates; therefore, their concentration is found to be low. Therefore, the question is, whether overproduction under stress is due to activation of already existing enzymes or de novo synthesis of such enzymes. It was observed that many stress conditions like drought and salt stress, for instance, initially inhibit export of products of photosynthesis from their source tissues. This will result in enhanced concentrations of primary products of the Calvin cycle in leaf cells. As shown by Koyro and Huchzermeyer (2005) the most compatible solutes derive from primary products of sugar metabolism. Therefore, increased concentrations of metabolites of photosynthesis will stimulate synthesis rate of compatible solutes. Such an increase will become significant, if metabolite concentrations under optimal growth conditions are below the Km values of enzymes catalyzing the initial reactions of the pathways leading to compatible solutes. This interpretation agrees with the findings of Soussi et al. (1998), who attributed enhanced concentrations of proline and carbohydrates under salt stress in chick pea to damage of metabolic pathways rather than to protective mechanisms. A second working hypothesis is based on findings of Süss et al. (1993). They found that enzymes of individual pathways in chloroplasts tend to form clusters. Such conditions would contradict free mobility of intermediates. Thus, substrate concentrations localized to catalytic centers of enzymes may significantly differ from respective bulk phase concentrations. Calvin cycle enzymes, for instance, tend to bind to one another and thus form aggregates of proteins. This allows substrates as well as products formed to be shuttled from one enzyme to the next one of the respective pathway. Süss postulated that modulation of pathways in this model is brought about by modulation of enzyme neighborhood. From analysis of internal signaling within cells, it is well documented that such modulations can be brought about by protein phosphorylation and de-phosphorylation, for instance. Indeed, such phosphorylation-dependent variations in protein–protein interactions have been observed by Allen and Horton, when analyzing protein localization in thylakoid membranes (Horton and Foyer, 1983; Pursiheimo et al., 2001; Allen, 2002). Moreover, effects of protein– protein interactions inside thylakoid membranes on the efficiency of primary reactions of photosynthesis have been analyzed in detail by the teams of Dilley and Huchzermeyer (Huchzermeyer and Löhr, 1984; Laszlo et al., 1984; Huchzermeyer and Willms, 1985; Löhr et al., 1985; Löhr and Huchzermeyer, 1985; Huchzermeyer et al., 1986; Allnutt et al., 1989). A third model refers to the observation that salt stress response of plants has been found to be under the control of hormones (ABA, for instance) and second messengers (sugar signaling, for instance). This implies that modification of enzyme patterns will result in modified metabolic activity of cells and tissues under stress. Such argumentation would explain the above observations on the level of gene activities. But we have to take into account that it has been observed that binding sites of motor proteins of the cell skeleton are under hormonal control as well. Thus, hormone action can influence neighborhoods of proteins. This interpretation will link the ideas of model 2 and 3. A similar interpretation holds true for sugar signaling that is known to include regulation of protein phosphorylation. It is well known that protein phosphorylation status controls formation of protein aggregates. These latter arguments may suggest not to discuss three different models of regulation of metabolic pathways leading to the synthesis of compatible solutes. It rather appears to us that hormone- and secondary messenger actions explain how formation of protein aggregates may be controlled in plants. Biochemical reaction sequences leading to improved salt tolerance are likely to act synergistically (Iyengar and Reddy, 1996). The reactions include (1) synthesis of compatible solutes, (2) stimulation of antioxidative enzyme activities, and (3) modifications of the photosynthetic pathway.


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42.3.3.2 Polyols and Sugars A considerable percentage of assimilated CO2 is found in polyols. As there is an equilibrium between polyol and sugar metabolism, a possible role of polyols in carbon storage under stress has been discussed (Vernon et al., 1993; Klages et al., 1999; Sun et al., 1999). Among their functions in cell physiology and biochemistry are compatible solutes, low molecular weight chaperones, scavengers of reactive oxygen species (Smirnoff and Cumbes, 1989; Bohnert et al., 1995), and compounds contributing to osmotic adjustment in the cytosol (Yancey et al., 1982; Ford, 1984; Ashihara et al., 1997; Hasegawa et al., 2000; Zhifang and Loescher, 2003). In celery, mannitol is synthesized by the action of the enzyme mannose-6-phosphate reductase (M6PR). Enhanced concentrations of this compatible solute is found under salt stress (Zhifang and Loescher, 2003). It was shown that M6PR from celery under the control of the CaMV35S promotor introduce into Arabidopsis, a plant not producing significant amounts of mannitol, significantly improves salt tolerance of mutants as compared to the wild type (Zhifang and Loesher, 2003). The mutants were able to grow, flower, and produce seeds in soil culture irrigated with up to 300 mM NaCl. Pinitol is synthesized by the sequential action of inositol-o-methyltransferase and ononitol epimerase when significant concentrations of myo-inositol have built up (Bohnert and Jensen, 1996). Sugars are synthesized in primary metabolism of plants. Therefore, bottlenecks in metabolic pathways and source-to-sink transport, respectively, translate to significant changes in sugar and starch concentrations in source and sink tissues. Sugar and starch concentrations may be used as indictors to localize cells, tissues, and plant organs most sensitive to salt stress. The problem in analysis of data from the literature is that in some papers data on sugar concentrations are not clearly aligned to specific cells or tissues but to “plant material,” roots or shoots. Moreover, not individual sugars are identified but “total sugar,” “soluble sugars,” “reducing sugars,” etc. Such information is of limited value, when it comes to analysis of the mechanism of salt stress effects on plant metabolism. However, relevant papers may help identify a promising experimental plant for analysis of a special salt stress effect on regulation of sugar metabolism. Obviously, there can be a broad spectrum in sensitivity of enzymes and transporters of sugar metabolism within accessions of a species. It was found, for instance, that under salt stress sugar concentration in some genotypes of rice increase, while it decreases in other ones (Alamgir and Ali, 1999). Moreover, salt stress obviously is targeting several enzymes and transporters. Though concentrations of sugars vary in the shoot, starch concentration was observed to be constant in the shoot, while it was reduced with increasing salt concentration in rice roots. There are several options to explain stress responsiveness of root starch concentration. But a final conclusion would require data on enzyme activities and their metabolite concentrations available. As rice is one of the most important crops, parallel analysis of respective data will be done soon. Then, promising targets for future breeding for stress tolerance can be identified. Such an analysis has been performed by Dubey and Singh. They found that under salt stress the sugar content and the activity of sucrosephosphate synthase increased, whereas the activity of starch phosphorylase decreased (Dubey and Singh, 1999). Similar investigations on other crops are reported in the literature. Like in rice, sugar concentrations have been found to increase in tomato leaves. Starch content of leaves was not affected by salt stress (Khavarinejad and Mostofi, 1998). Under similar experimental conditions, Gao et al. observed that the activity of sucrosephosphate synthase in leaves is increased while acid invertase is decreased under salt stress (Gao et al., 1998). As can be seen from Figures 42.11 and 42.12, such experimental approaches allow some more detailed analysis, but for identification of target enzymes (target genes for breeding) the complex regulation of sugar metabolism requires to measure more data in parallel. Another problem becomes obvious when analyzing anatomy and physiological differentiation of cells from the base to the tip of monocot leaves. Especially from field-grown cereals, it is well known that leaves located near

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Fructose 2, 6bisphosphate

Glucose 1-phosphate

Glucose 6-phoshate Pi

ATP

Fructose 1, 6bisphosphate

P Pi Pi

Pi Triose phosphate

Glucose 6-phosphate

Inhibiting

Triose phosphate + NADP NADPH

Glucose 1-phosphate

ADP Sucrose 6-phosphate Pi

UDP-glucose

ATP 3-Phosphoglycerate

ADP-gluc pyrophosphorylase

Fructose 6-phosphate

Stimulating ADP

Sucrose Photosynthesis

Starch

Export Cytosol

Chloroplast matrix

FIGURE 42.12 Regulation of sugar status inside the chloroplasts and in the cytosol. Levels of sugar metabolites are permanently monitored inside chloroplasts as well as in the cytosol of leaf cells. Phosphate is one of the most important ligands controlling starch and sucrose synthesis in chloroplast and the cytosol, respectively. Inside the chloroplasts, free phosphate is inhibiting the ADP-glucose pyrophosphorylase. In the cytosol, phosphate is stimulating fructose-6-phosphate 2-kinase, while it is inhibiting both fructose-2,6 bisphosphatase and sucrose-phosphate synthase. Thus, phosphate limitation (caused by competition for uptake with chloride, for instance) will result in an inhibition of sucrose synthesis in leaf cells. As a physiological response of chloride stress, an inhibition of sugar export via the phloem will be observed.

the top of the shoot contribute most of photosynthetic activity of a plant. Old lower leaves become senescent or function as storage organs (to sequester salt and wastes) rather than being active in photosynthesis. Like in dicots, young but full expanded photosynthetically active leaves contain young cells with young plastids as well as old cells with mature chloroplasts (Heintze et al., 1990). These cells differ in compartmentation of metabolic pathways as well as in stress sensitivity. Other than in dicots, where young and mature cells form patches all over the leaves, young cells are found preferentially at the leaf base while mature cells are found at the leaf tip in monocots (Heintze et al., 1990). This makes our cereals ideal experimental plants for the investigation of stress effects at different developmental states of leaf cells. Methods for analysis of primary and secondary metabolite synthesis are described in the literature (Heintze et al., 1990). Moreover, compartmentation of amino acids and the activity of amino acid transport into the vacuoles has been analyzed (Homeyer et al., 1989). The methods in use allow to analyze activities of amino acid transporters as well as ATPase and PPase activities (Homeyer et al., 1989). In summary, it can be stated that experimental results currently available clearly prove salt stress effects on metabolism and compartmentation of sugars. As a rule, increased sugar concentrations can be found in leaves as a salt stress response. But published data do not allow to identify the individual mechanisms improving salt tolerance of some accessions of plants and the bottle necks of less tolerant plants of the same species, respectively. The network of metabolic pathways and the various regulations of enzyme and transporter activities are too complex and the data sets too limited to immediately allow a direct approach. Moreover, there are three other aspects impairing


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direct comparison of experimental data from different publications (such an approach would help building a broader database): 1. Experimental material is not sufficiently described in many papers. Especially the developmental stage of plant material would need more precise description in many papers: It has been shown in several papers that permanent salt stress results in retarded development (late flowering, for instance) of crops (Bourque et al., 1975; Fahnenstich et al., 2008). This observation requires, especially in long-term experiments, to compare plants of identical developmental stage and not plants of the same age. For definition of developmental stages, activity patterns of marker genes may be used, for instance. 2. Salt stress not simply inhibits enzyme activities. It has been described that an increase of leaf sugar concentration is an immediate stress response. But, if plants can tolerate the applied stress, sugar concentrations will level off again and eventually will reach values of control plants (Fahnenstich et al., 2008). The period during which plants show elevated leaf sugar concentrations depends on the level of stress applied and the degree of salt tolerance of the plant. Therefore, it is essential for the comparison of experimental data to exactly know the timing of the experimental approaches. 3. It is well known that stress tolerance as well as responsiveness of plants varies with the developmental stage of plants. Plants showing high salt tolerance can have only limited tolerance in seedling stage (Mangroves, for instance). Like stated under (1), it will be essential to establish molecular markers or protein (membrane receptor) patterns for definition of developmental stages of experimental plants. 42.3.3.3 Nitrogen-Containing Compatible Solutes Like stated for polyols and sugars, the pattern of specific nitrogen-containing compatible solutes that accumulate under salt stress varies with plants species. In addition to the functions mentioned for other compatible solutes, nitrogen-containing ones are of special importance for maintenance of cytosolic pH. Due to its effects on ATPase and PPase activities, ionic stress impairs pH homeostasis. Therefore, it appears to be logical that the capacity to accumulate high cytosolic concentrations of nitrogen-containing compatible osmolytes is quite common among salt-tolerant plants (Mansour, 2000). Moreover, as nitrate and chloride compete for uptake at root level, storage of surplus nitrogen in organic molecules in periods of low stress might be a strategy to better prepare for subsequent harsh conditions. Stored “organic nitrogen” may lead to a delayed stress response of highly salttolerant plants. Especially, glycine betaine has been found to be the dominant compatible solute in highly salt-tolerant plants, named halophytes (Khan et al., 1998, 1999, 2000; Saneoka et al., 1999; Muthukumarasamy et al., 2000; Wang and Nil, 2000). Glycine betaine is synthesized from choline in a two-step reaction sequence catalyzed subsequently by choline monooxygenase and betainealdehyde dehydrogenase (Rhodes and Hanson, 1993). It may be postulated that salt tolerance of plants can be increased by a molecular genetic approach, if they have a sufficient potential to provide choline as a substrate. Indeed, it was reported by Sulpice and coworkers that transformation of plants with the codA gene, coding for choline oxidase, resulted in improved salt tolerance of the mutant (Sulpice et al., 2003). As expected, it was observed that the mutants accumulated enhanced cytosolic concentrations of glycine betaine. A similar positive result has been reported by Bhattacharya et al., who transferred the bacterial betA gene for the synthesis of glycine betaine to cabbage. The transformed plants showed higher tolerance to salt stress as compared to the wild type (Bhattacharya et al., 2004). Several amino acids have been found to increase in concentration under salt stress. The most prominent one is proline (Parida et al., 2002), but also valine, isoleucine, and aspartic acid have been observed to increase in concentration in leaf parenchyma cells of cereals (Mattioni et al., 1997; Elshintinawy and Elshourbagy, 2001). On the other hand, cysteine, methionine, and arginine are

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reported to reduce in concentration upon salt stress. This observation indicates that there have to be more aspects to be mentioned but increased buffer capacity and contribution to osmotic homeostasis in order to sufficiently explain the phenomenon of improve salt tolerance of plants. It is common to most of the dominant nitrogen-containing compatible osmolytes that they are zwitter ions having no net electrical charge at physiological pH. Therefore, they do not impair with surface charge patterns of proteins. On the other hand, they will bind to incoming salt ions and will form several layers of zwitter ionic shells around the ions. This way, the ion radius will significantly increase resulting in a significant decrease of charge density of each of the ion complexes. As one major aspect of ion toxicity is believed to be due to competition of ions with enzymes for their hydrate shell, reduction of charge density (reduced capacity to tightly bind water molecules) will reduce the apparent toxic effect of incoming salt ions. When analyzing salt-induced increase of cytosolic proline concentrations in more detail, it became obvious that the effect cannot sufficiently be explained by an increase of precursor concentration or allosteric effects but de novo synthesis of enzyme protein is involved. This makes proline effects contributing to salt tolerance an ideal target for a molecular genetic approach. It was found that in wheat activity of the enzyme delta-1-pyrroline-5-carboxylate reductase, involved in proline synthesis, is enhanced under drought stress as well as ionic stress, whereas activity of the enzyme proline dehydrogenase, involved in proline degradation, is inhibited exclusively under salt stress (Mattioni et al., 1997). It may be concluded that de novo synthesis of proline is the dominant regulatory mechanism of cytosolic proline concentration in wheat leaf cells. 42.3.3.4 Aspect of Energy Consumption by N- and S-Pathways As a rule, in nonwoody plants nitrate is reduced in chloroplasts, while it is reduced in root cell plastids of trees. As mentioned above in the physiology chapter already, nitrate competes with chloride (due to similar radii of the hydrated ions) for uptake via specific translocators. These translocators are regulated via the plant’s sugar pool and their activity thus matches the photosynthetic activity of source leaves. In this chapter, we focus on coupling of nitrate reduction to photosynthesis in chloroplasts of weeds. About one-third of the electrons released by PSII finally will be used for nitrate reduction. Therefore, nitrate reduction is a major sink for electrons and sufficient N fertilization contributes to prevention of primary reactions of photosynthesis from over-reduction in high light. Nitrate reduction and incorporation of reduced nitrogen into glutamate is shown in Figure 42.10. It is obvious that the first intermediates of nitrate reduction are toxic. Like in primary reactions of photosynthesis, building up of concentrations and occurrence of side reactions that eventually might be toxic for the cell are prohibited by fast turnover of products. Direct neighborhood of enzymes involved, i.e., prevention of prolonged diffusion or release of intermediates to the bulk phase, is an essential prerequisite for this reaction sequence. Again, we have to refer to the ideas of Süss et al. (1993). It has been shown in the literature that affinity for nitrate uptake from the soil can be improved by means of a molecular biological approach (Matt et al., 2001; Wang et al., 2003b; Lillo, 2004). Also, nitrate fertilization helps to reduce salt stress effects (Syvertsen et al., 1989; Murillo-Amador et al., 2006). In principle, the same arguments hold true that have been discussed at the end of the above paragraph. It is obvious that salt stress can have direct as well as indirect effects on nitrate reduction and amino acid biosynthesis. Direct effects mostly are due to competition among nitrate and chloride ions. Indirect effects are based on dependence of nitrate reduction on (1) electrons released by photosynthetic electron transport, (2) intact structure of enzymes and enzyme aggregates, and (3) dependence of amino acid biosynthesis on substrates that derive from sugar metabolism (i.e., sugar supply from photosynthesis). We have to keep in mind that sugars, sugar phosphates, and ROS are functioning as secondary messengers regulating expression of enzymes. Therefore, there will be another level of regulatory effects. Though a lot information on sugar signaling has been analyzed


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and a list of participants has been outlined in pretty much detail, the complex pathway structure of second messenger signaling is not well characterized to date (Sheen et al., 1999; Gibson, 2000; Sheen, 2002; Baena-González and Sheen, 2008). As nitrate reduction and subsequent amino acid biosynthesis depend on photosynthetic activity, there has to be some buffer capacity for intermediates to allow continuous metabolic activity of plants. In principle, there are two storage compartments in leaf cells: plastids and the vacuole. In storage organs, plastids can differentiate to protein storage compartments. In chloroplasts, only low amounts of storage proteins typical for storage plastids are found. But in some papers it is discussed whether the huge amounts of Rubisco accumulating in chloroplasts may function as storage proteins as well. In vacuoles, free amino acids and other low molecular weight molecules are stored rather than macro molecules like proteins. It has been shown that vacuoles from tissues active in photosynthesis can actively import amino acids at final concentrations several fold exceeding the ones in the cytosol (Homeyer et al., 1989). Amino acid import occurs at the expense of a transmembrane pH gradient. Both, v-Type ATPase and PPase of the tonoplast build this gradient at the expense of ATP and pyrophosphate, respectively (Homeyer et al., 1989). It is well known that activity of these two enzymes is regulated (Taiz, 1992; Niu et al., 1995; Davies, 1997; Zhang and Liu, 2002; Han et al., 2005; Park et al., 2005), and that amino acid content is affected by salt stress (Pahlich et al., 1983). But in-depth understanding of regulatory pathways and regulation of transport activities under salt stress is still missing. Regulation of gene expression under stress has been observed some 20 years ago already, but research in this field is continuing in order to elucidate the regulatory network (Narasimhan et al., 1991; Park et al., 2005). In contrast to nitrogen that is found exclusively in the reduced form in organic molecules, sulfur occurs in metabolites of biological relevance in several redox states. Only a minor portion of the photosynthetic electrons are used for the sulfate reduction pathway. As outlined by Schmidt and Jaeger (Schmidt and Jaeger, 1992, and citations therein), sulfate reduction pathway in chloroplasts differs from the one in bacteria that had been identified earlier. Inhibition of sulfur metabolism has major secondary effects, because SH bounds are essential for functioning of catalytic centers of many enzymes and reduced sulfur is found in coenzymes like CoA and liponic acid, for instance. Moreover, a pool of various glutathiones forms the dominant redox buffer of plant cells, and glutathiones are involved in detoxification of heavy metals and ROS. Therefore, enhanced salt stress tolerance of sulfur metabolism always goes along with improved metabolic activity and apparent tolerance of other essential functions as well.

42.3.4

SALT EFFECTS ON METABOLITE TRANSPORT AND BIOENERGETICS OF CELLS

Products of photosynthesis, sugars, amino acids, and lipids, have to be exported out of the chloroplasts into the cytosol of host cells and further on to sink organs. Compartmentation of metabolic pathways has been investigated in detail during the last 40 years (Lunn, 2007). Nevertheless, most information is available on sugar transport. 42.3.4.1 Sugar Phosphate Export Out of the Chloroplasts In the presence of light, sugars are exported as triose phosphates (DHAP and GAP) in exchange of free phosphate via the phosphate translocator (Riesmeier et al., 1993)]. Vmax of sugar phosphate export is too low to keep up with sugar phosphate synthesis under average light conditions. This would cause shortage of phosphate inside the chloroplast stroma, thus inhibiting primary reactions of photosynthesis and increasing the risk of ROS production. Starch production inside the chloroplast is a relief, because phosphate will be released and becomes available for the chloroplast coupling factor to produce ATP. Vmax of starch production has not been measured yet as physiological sugar concentrations are too low. The capacity to produce starch obviously is high enough to turn over any sugar concentration that might become available under physiological conditions. There is an equilibrium between starch synthesis and hydrolysis, respectively. Therefore, starch will be

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degraded at night and allow a permanent supplementation of the cytosol of the host cell. This system allows plant cells to grow permanently on a continuous flow of incoming sugar phosphates. In the cytosol of green leaf parenchyma cells, sugar phosphates are used to fuel cell metabolism. In a competing metabolic pathway sucrose is formed from glucose and fructose and exported into the phloem to feed sink organs. As shown in Figure 42.12, there is a complex regulation of sugar metabolism inside and outside the chloroplasts, respectively. Moreover, with respect to the ideas of Süss et al., there will be a second level of regulation by aggregation of enzymes, not sufficiently analyzed to date (Adler et al., 1993; Süss et al., 1993). More reliable data are available concerning the function of hexose phosphates as second messengers tuning gene expression to the physiological and biochemical needs of an individual cell (Sheen, 2002 and citations therein). From the above description it becomes clear that there are several alternatives, how incoming salt may affect functioning of cellular sugar metabolism. In principle, the situations in source and sink tissues are comparable. But, under moderate salt stress, salt concentrations have been found to differ a lot between root, leaf, and fruit tissues (see Sections 42.2.6.2 and 42.2.7). Therefore, the observed effect of salt stress also differs among tissues of plant organs. Currently, salt stress effects on aggregation of enzymes to restructure neighborhoods and affect equilibria among biochemical pathways are poorly investigated. But it has been documented that product patterns of secondary metabolism varies a lot under stress (Zuther et al., 2007). As mentioned above, compatible solutes are stabilizing enzyme aggregates thus compensating salt effects at least to some extent. This matter will be analyzed in more detail as soon as reliable methods will be available for screening for such effects. Salt effects on sugar metabolism and sugar export from the chloroplasts into the cytosol can be explained on the basis of a competition between Cl− and H2PO4− for binding sites on enzymes and receptors, respectively. This competition is due to similar diameters of these two ions when hydrated. From data currently available, it appears to us that the phosphate translocator is more chloride sensitive as compared to the other phosphate binding sites shown in Figure 42.13. This would mean that under salt stress the chloroplast stroma is at risk to run out of free phosphate. As mentioned above, this would inhibit ATP synthesis at the chloroplast coupling factor, which has an K of about 5 mM for phosphate (Groth et al., 2000). The observed effect would be quite Location of the phosphate translocator

Cytosol

Plastid

C3 chloroplast

Pi

C4 chloroplast

Pi

Leucoplast, amyloplast

Seed storage amyloplast developing fruit, non-phosphorylating leaf

Triose phosphates Phosphoenolpyruvate malate Glucose 1-phosphate Glucose 6-phosphate Pi Glucoe 6-phosphate

Triose phosphates (3-PGA) Triose phosphates (2-PGA, 3-PGA) phosphoenolpyruvate

Pi

Glucose 6-phosphate Pi

Cl–

FIGURE 42.13 Specificity of phospate translocators. Phosphate translocators of plastidal inner envelop membranes differ in their specificity toward sugars transported in exchange of phosphate. It may be expected that Cl− competes with phosphate at any of these translocators. This interpretation explains the observation that inhibition of sugar export from plastids is an early response under salt stress.


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similar to the one observed after energy transfer inhibitors like Nitrofen (Huchzermeyer and Löhr, 1990). Nitrofen is a herbicide used in rice cultures and it is known to stimulate light-dependent ROS production in plants. By the way, this observation again is a proof for the tight coupling between primary reactions of photosynthesis. With respect to reducing stress effects at a given salt concentration, thus improving crop yield, phosphate fertilization should help. On the other hand, stress tolerance of plant species could be improved if phosphate uptake at higher rate or higher specificity could be engineered by molecular biological methods. Any method preventing chloride import into photosynthetic active tissues would have a similar effect, of course. This consideration provides a good example demonstrating that it is not easy to decide how salt stress and stress relief, respectively, are acting on molecular level.

42.4

SUMMARY

The need to feed a fast-growing world population calls for ever-increasing food production. But increase of agricultural productivity is leveling off and per capita cereal production, therefore, is reducing year by year since the early 1990s. An alternative would be to tap areas currently not used for agriculture, salt contaminated soils, for instance. But such an approach calls for information on plant performance under salt stress in order to be in the position to design appropriate cropping and breeding strategies. In the first part of this review, we have summarized information on plant performance under salt stress. Halophytes, plants of the coastal ecosystems, can teach us strategies how to survive and finish life cycle under salt stress. As a rule, enzymes and cell organelles isolated from halophyte tissues are salt sensitive, similar to samples isolated from our regular salt-sensitive crops. Halophytes have developed strategies to avoid uptake of excess salt or to sequester salt from the cytosol. Moreover, halophytes are able to keep ion homeostasis and to regulate nutrient and water relations. In the second part of this presentation, we focus on biochemical aspects, i.e., the basis of all physiological observations. As photosynthesis is a prerequisite for biomass production, we concentrate on information related to this essential sequence of reactions. After some 40 years of research, it is not possible to present information on all aspects treated in the literature. Another reason to concentrate on salt stress effects on photosynthesis relates to applied aspects: Salt stress–induced reduction of photosynthetic efficiency directly relates to several biochemical “signals” that easily can be monitored like chlorophyll fluorescence, for instance. A better understanding of plant metabolism under salt stress will allow to design monitoring systems to predict crop yield or to identify promising crop accessions at an early stage of their development. Last, but not least, we should mention that identification of enzymes that are under stress the bottlenecks of metabolic pathways, means to identify targets for salt-resistance breeding.

42.5

FUTURE PERSPECTIVE

Several papers recently have shown that accessions of plants species selected from environments differing in environmental stress differ in their tolerance. Marker-assisted selection of promising accessions, therefore, can be a promising approach to identify stress-tolerance strategies of individual plant species (Tuberosa et al., 2002). Such an approach will lead to a better understanding of physiological and biochemical needs thus allowing to design strategies to engineer salt stress tolerance of individual plant species. Induced resistance of plants (IR) has been first described in the context of pathogen resistance. Subsequent to an infection, plants can develop enhanced resistance to a broad spectrum of pathogens. It was found that this type of resistance is under the control of the hormone salicylic acid (Ryals et al., 1996; Durrant and Dong, 2004). This type of resistance also can be induced by some natural or synthetic compounds. These compounds apparently induce a general stress resistance in

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plants (Janda et al., 1999; Senaratna et al., 2000; Kohler et al., 2002). In general, it can be found that plant cells and tissues respond faster and develop a broad spectrum of stress response reactions. The basis of this latent capacity to react in Arabidopsis has been identified by Beckers et al. They found that occurrence of chemically induced resistance correlates with enhanced cellular concentrations of inactive proteins of MPKs, especially of MPK3 and MPK6 as well as the respective mRNAs (Beckers et al., 2009). It has been found in experiments with grape (Vitis vinifera) as well as Arabidopsis thaliana that ROS release is among the first detectable signals subsequent to application of abiotic stress. It may be assumed that like in animal tissues H2O2 is functioning as a signaling molecule leading to downstream responses (Mittler et al., 2004). Currently, it is an unanswered question, whether NO signaling in plants is capable of neutralizing H2O2 signals like it has been observed in animal neurons (see for reference: Wilken and Huchzermeyer, 1999). The activation of two mitogen-activated protein kinases, MPK3 and MPK6, has been found to be essential for signal transduction in response to H2O2 in Arabidopsis (Kovtun et al., 2000). Moreover, Rentel and Knight could show that OXI1 kinase acts as a H2O2 sensor and activates MPK3 and MPK6 (Rentel and Knight, 2004). Skopelitis et al. have drawn our attention to another aspect (Skopelitis et al., 2006) Under continuous salt stress, increased proteolytic activity is found especially in mature tissues. Degradation of proteins will produce enhanced cytosolic ammonia concentrations that may reach toxic levels if not efficiently removed (Lutts et al., 1999). In plant cells, the mayor pathway for ammonium detoxification is the GS/GOGAT system leading to the production of glutamine and glutamate (Lea and Miflin, 1974). In addition, glutamate dehydrogenase, an enzyme abundant in plant tissues, can show aminating activity and catalyze reductive amination of 2-oxoglutarate. It has been shown that abiotic stress induced enhanced cytosolic ammonium concentration results in enhanced GDH activity (Lutts et al., 1999; Hoai et al., 2003). In salt-tolerant rice cultivars, GDH activity was found to increase subsequent to stress, and decreased in salt-sensitive rice (Kumar et al., 2000). These results indicate that GDH is a salt (abiotic) stress responsive protein involved in ammonia detoxification under stress. Indeed, it could be shown in experiments with vine and tobacco that GDH gene expression can be induced by ammonium ions as well as ROS that were generated as a salt stress response (Hassan and Fridovich, 1979). In more detailed analysis of salt-stress response of Nicotiana tabacum, it was found that in addition to GDH genes, isocitrate dehydrogenase genes were up-regulated as well (Skopelitis et al., 2006). This means that 2-oxoglutarate production will be enhanced under salt stress as well. In in vitro experiments with cell and callus cultures, it could be shown that addition of ascorbate interferes with salt-induced gene regulation. (Skopelitis et al., 2006). This may indicate that ROS are functioning as signaling molecules.

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