THE UNIVERSITY OF WESTERN AUSTRALIA

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2 THE UNIVERSITY OF WESTERN AUSTRALIA STATEMENT OF ORIGINAL CONTRIBUTION The research presented in this thesis is an original contribution to the field of plant physiology. The thesis has been completed during the course of enrolment in a PhD degree at the University of Western Australia, and has not been used previously for a degree or diploma at any other institution. This thesis contains work accepted to be published and/or work prepared for publication, all of which has been co-authored. I contributed all of hands-on work for all papers. Professor Zed Rengel and Dr Olga Babourina provided overall supervision and advice with experimental designs and critical comments and corrections on the manuscripts. Kunmei Guo Signature: Candidate Professor Zed Rengel Signature: Coordinating Supervisor Jan i

3 PUBLICATIONS ARISING FROM THIS THESIS 1. Guo KM, Babourina O, Christopher DA, Borsics T, Rengel Z The Cyclic Nucleotide-gated Channel, AtCNGC10, Influences Salt Tolerance in Arabidopsis Physiologia Plantarum 134, (Chapter 4) 2. Guo KM, Babourina O, Christopher DA, Borsics T, Rengel Z The Cyclic Nucleotide-gated Channel, AtCNGC10, is Involved in Transporting Ca 2+ and Mg 2+ : Fluorescence Lifetime Imaging and Ion Fluxes Analysis. (Submitted to Functional Plant Biology) (Chapter 5) 3. Guo KM, Babourina O, Rengel Z Na + /H + Antiporter Activity of SOS1 Gene: Lifetime Imaging Analysis and Electrophysiological Studies on Arabidopsis Seedlings. (Submitted to Physiologia Plantarum) (Chapter 6) ii

4 ABSTRACT Control of intracellular ion homeostasis is pivotal to plant salt tolerance. Plants have developed a number of mechanisms to keep ions at appropriate concentrations. Both transporters and channels on the plasma membrane play important roles in this function. Plant cyclic nucleotide-gated channels (CNGCs) in the plasma membrane are non-selective monovalent and divalent cation channels. So far, most studies on plant CNGCs have been conducted on heterologous systems. In planta, reverse genetic studies revealed the role of different CNGCs in cation uptake, transport and homeostasis. However, there is little information available about the functional characteristics of plant CNGCs. Among the 20 members of this protein family in Arabidopsis, only AtCNGC2 has been functionally identified as an ion channel; therefore, more functional characterization needs to be done on other members of this protein family. Several CNGCs were suggested to be involved in K +, Ca 2+ and Na + uptake and transport, but available information is scarce. This study investigated the relationship between CNGC10 and ion transport in Arabidopsis, with a particular emphasis on the involvement of CNGC10 in salt tolerance. Arabidopsis thaliana wild type (WT) and two AtCNGC10 antisense lines (A2 and A3) were used to characterise the impact of different level of salt stress on (i) root growth, ion concentration in tissues, ion fluxes across the root surface and intracellular ion concentration and ph at the seedling stage, and (ii) photosynthesis and ion concentration in tissues at the flowering stage. Plants of both antisense lines had higher K + and lower Ca 2+ and Mg 2+ concentrations in shoots than WT plants when grown in non-salt control 1/4 Hoagland solution. Altered K +, Ca 2+ and Mg 2+ internal concentrations in AtCNGC10 antisense lines compared with WT plants under non-salt conditions indicated disturbed long distance ion transport, especially xylem loading/retrieval and/or phloem loading. The results of ion fluxes across the root surface also suggested that AtCNGC10 might be involved in transport of K +, Ca 2+ and Mg 2+ in tissue. Under sudden salt exposure, higher Na + efflux and smaller K + efflux in both antisense lines suggested that AtCNGC10 channels are involved in Na + and K + transport. The shoots of AtCNGC10 antisense lines A2 and A3 contained higher Na + concentrations and significantly higher Na + /K + ratios compared to WT, resulting in impaired iii

5 photosynthesis and increased salt sensitivity in A2 and A3 than in WT plants. In contrast, seedlings of both antisense lines exposed to salt stress had lower shoot Na + /K + ratios and longer roots than WT seedlings, indicating that A2 and A3 were more salt-tolerant than WT in the seedling stage, likely because growth is less dependent on photosynthesis in the seedling than in the flowering stage. These results suggested CNGC gene might play a different role during different developmental stages and in various plant organs. The salt overly sensitive gene (SOS1) codes for the Na + /H + antiporter in the root-cell plasma membrane; previous studies revealed that (i) overexpression of SOS1 improved salt tolerance in Arabidopsis and (ii) the activity of the SOS1 antiporter can be modified by an SOS3-SOS2 protein kinase complex. In this study, the functional characterization of Arabidopsis salt-overly-sensitive mutant (sos1) was investigated, including the work on sos2 and sos3 mutants for comparison. The sos1 mutants demonstrated H + efflux and intracellular alkalinisation of the meristem zone after the salt stress application; such results were in contrast to H + influx and slight acidification of intracellular ph in the meristem zone of sos2 and sos3 mutants and WT after salt stress application. Amiloride, inhibitor of Na + /H + antiporter activity, was applied either prior to (pre-treatment) or together with (treatment) NaCl stress to WT and sos1 mutant plants. The 1-h pre-treatment with 1 mm amiloride caused significant acidification of intracellular ph in roots of both WT and sos1 mutants exposed to salt stress, but no significant changes were found in Ca 2+ fluxes across the plasma membrane and intracellular Ca 2+ activity ([Ca 2+ ] cyt ) in roots. WT plants pretreated with amiloride did not demonstrate H + influx and acidification of intracellular ph in roots upon exposure to salt stress; in contrast, intracellular ph shifted to higher values after NaCl application in both WT and sos1 mutants pre-treated with amiloride; however, there was a significantly lower H + fluxes in sos1 pre-treated plants 10 minutes after salt stress application in comparison with WT pre-treated plants, which suggests that amiloride does not affect the SOS1 transporter (Na + /H + antiporter), but other transporters located in the plasma membrane of the meristem zone of plant roots. Future experiments should characterise the role of AtCNGC10 in xylem loading and retrieval and/or phloem loading in Arabidopsis plants as well as the tissue-specific activity iv

6 of AtCNGC10. Further functional characterization of other members of the Arabidopsis CNGC family, especially functional studies of their roles in plant nutrient acquisition and resistance to environmental stresses, is eagerly awaited. A clear relationship between salt tolerance and regulation of ion transport in roots under salinity at the molecular level would be useful for plant breeders selecting for new salt-tolerant genotypes. Other transporters in the plasma membrane that regulate intracellular ph in sos1 mutants need to be investigated in the future. v

7 ACKNOWLEDGEMENT The work presented in this thesis was completed between August 2004 and July 2008 at the School of Earth and Environment, Faculty of Natural and Agricultural Sciences, The University of Western Australia. I gratefully acknowledge the financial support of International Postgraduate Research Scholarship and University Postgraduate Award (International Student) from the University of Western Australia. I would like to take this opportunity to thank the people who have helped me over the past four years. First, I am very grateful to my supervisors, Professor Zed Rengel and Dr Olga Babourina, for their experienced supervision, and constructive criticism, and helpful suggestions throughout the course of my study. To Professor Zed Rengel, thank you very much for the opportunity you gave me to expand my professional and personal development at UWA. Thanks for spending many hours reviewing draft manuscripts, and providing much needed advice on writing manuscripts to a standard suitable for thesis and publication. To Dr Olga Babourina, your patient guidance in everything, especially for hands-on technique with confocal microscope and MIFE systems, was unforgettable; your guidance was always available when I sought it. I have been and will be very grateful forever for that, thanks for spending so much time on my thesis writing and publication preparation, thank you very much! To Dr Emmanuel Mapfumo, your useful suggestions and your help with soil sampling (although I changed research direction afterwards, it does not affect my gratefulness to you) were in time; I am very grateful for that. I would also like to take this opportunity to thank the Australian Research Council for the financial support for my experiments, and to The Centre for Microscopy, Characterisation and Analysis, The University of Western Australia for their facilities and technical support. I extend thanks and appreciation to the staff of School of Earth and Environment-Soil Science and Plant Nutrition Group. I gratefully acknowledge Michael Smirk and Paul Damon for analytical and technical assistance. Thanks also goes to IT support office vi

8 members for their timely and patient help with computer technique and problems, and to office members for their routine help with my study. I extend my appreciation to those friends who helped me with my study and life here in Australia. I am deeply indebted to my loving family: to my daughter, when I came to study in Australia four years ago, she was just four months old, I owed her so much; and to my husband, Dr Heping Han, thank you very much for you constant support, inspiration and encouragement during my study, I am also very grateful to my parents-in-law for their support and looking after my daughter while I was studying in Australia, and to my parents, my sisters and brothers for their spiritual support and encouragement throughout my long education. vii

9 TABLE OF CONTENTS DECLARATION. i PUBLICATIONS ARISING FROM THIS THESIS....ii ABSTRACT.....iii ACKNOWLEDGEMENTS...vi TABLE OF CONTENTS...viii CHAPER 1 GENERAL INTRODUCTION..1 General introduction CHAPTER 2 LITERATURE REVIEW Salt stress occurrence and effects on plants Disturbance of ion homeostasis in plants by salinity Transporters and ion channels involved in plant response to salinity Transporters in plasma membrane and tonoplast that are affected by salt stress Ion channels involved in plant salt tolerance Summary Photosynthesis under salt stress: chlorophyll fluorescence and chlorophyll content Summary Outline of the study General Aims Structure of the thesis..25 CHAPTER 3 GENERAL MATERIALS AND METHODS General Material and methods..27 viii

10 CHAPTER 4 THE CYCLIC NUCLEOTIDE-GATED CHANNEL, AtCNGC10, INFLUENCES SALT TOLERANCE IN ARABIDOPSIS Introduction Materials and methods Results Discussion...52 CHAPTER 5 THE CYCLIC NUCLEOTIDE-GATED CHANNEL AtCNGC10 TRANSPORTS Ca 2+ AND Mg 2+ IN ARABIDOPSIS Introduction Materials and methods Results Discussion.. 72 CHAPTER 6 Na + /H + ANTIPORTER ACTIVITY OF sos1 GENE: LIFETIME IMAGING ANALYSIS AND ELECTROPHYSIOLOGICAL STUDIES ON ARABIDOPSIS SEEDLINGS Introduction Materials and methods Results Discussion..85 CHAPTER 7 GENERAL DISSCUSION AND CONCLUSIONS General discussion AtCNGC10 antisense lines differ in ion composition AtCNGC10 is involved in plant salt tolerance Na + /H + antiporter SOS1 demonstrated H + transport Na + /H + antiporter SOS1may be involved in the transport of other ions Conclusions REFERENCES.. 99 ix

11 CHAPTER 1 GENERAL INTRODUCTION 1.1 General introduction Salinity is a main constraint of crop productivity because high salt concentration in the substrate results in large decreases in growth and yield of a wide variety of crops all over the world (Tester and Davenport, 2003). Approximately 1 billion ha of land is affected by soil salinity (Szabolcs, 1994), which is 7% of the total land area. About 5% of cultivated land (77 million ha) is affected by salt (Munns et al., 1999). However, due to bad agricultural practices, this problem is becoming increasingly severe in many areas. For example, one third of irrigated land is being significantly affected by salinity (Munns, 2002). Although the area of this kind of salinisation is relatively small, one third of world s food is produced from irrigated land, making the problem particularly critical. In Australia, irrigation-related salinisation affects about 4 million hectares (Niknam and McComb, 2000). Moreover, dryland salinity resulting from disturbances in the hydrological cycles and re-distribution of salt in the landscape is also an increasingly important problem in some areas of the world, such as in southern Australia, where an estimated 17 million ha of Australia s agricultural land will be significantly affected by salinity by 2050, accounting for 25% of Australia s wheat belt (see The problem of salinity tolerance can be tackled by either improving farming practices to prevent soil salinizaion, or by implementing schemes to ameliorate salinized soils (Tester and Davenport, 2003). The alternative option is growing salinity-tolerant crops. Breeding crops for higher salinity tolerance (Grattan and Oster, 2003) should combine genetic, physiological and molecular approaches; functional genomics may serve an important role in that respect (Jain et al., 2003). Screening genotypes in breeding for increased adaptation to saline environments (Munns and James, 2003) should be based on the sound understanding of the biochemical and physiological basis of tolerance to salinity stress

12 Plant tolerance mechanisms consist of restricted uptake and extrusion of harmful ions, or storage in particular tissues, particularly compartmentation into vacuoles (Apse and Blumwald, 2007). Plants may resist salinity by osmotic regulation, ion homeostasis and antioxidant protection (Apse and Blumwald, 2007). Recently, increasing attention is being paid to how ions are taken up and transported to different plant parts as a contribution to understanding nutrient toxicity problems at the whole plant and cellular levels. Mechanisms of ion acquisition by plants, transport and distribution within, and plant tolerance to abiotic stress inevitably depend on the membrane transport proteins that are responsible for the transmembrane movement of ions and partitioning within plant. Therefore, the study of such membrane transport proteins is important for understanding plant tolerance to abiotic stress (Maathuis, 2007). Plant membrane transport proteins include transporters, channels and pumps. Transporters and ion channels differ in structure and function. Transporters transport ions across a membrane against the electrochemical gradient, whereas channels are pores that allow specific ions to pass through them, and thereby cross the membrane, down the electrochemical gradient. Pumps operates as an antiporter, actively pump ions out of cell against its electrochemical gradient. The relative contribution of the three transport pathways varies with plant species and growth conditions (Zhu, 2002; Tester and Davenport, 2003; Ryan and Vandenberg, 2005; Mahajan et al., 2006). To establish which particular membrane transport protein takes part in a certain transport process is quite a difficult task. However, the previous and ongoing functional characterisation of specific transport proteins, combined with a large amount of data from genetic studies, provides the opportunity to carry out more detailed dissection of the role of these transport proteins and related gene families in plant nutrition (Maathuis, 2007), e.g. the contribution of specific protein families and gene isoforms to important physiological processes such as the uptake and long distance transport of nutrients, compartmentation of excessive ions and extrusion of harmful ions. A recently identified protein family of cyclic nucleotide-gated channels (CNGC) was suggested to act as cation pathway and thus play an important role in plants (Kaplan et al., 2007). Some studies have demonstrated that membrane transport proteins such as - 2 -

13 CNGC and the Arabidopsis Na + /H + antiporter SOS1 (salt overly sensitive) play a crucial role in plant tolerance to salt stress (Fox and Guerinot, 1998; Tester and Davenport, 2003; Brett et al., 2005; Ryan and Vandenberg, 2005), including restricting Na + entry into plants, increasing extrusion of Na + from cytoplasm into apoplast or the vacuole, improving K + nutrition under salinity to keep an optimal K + /Na + ratio, etc. The detrimental effects of salinity stress on plants have been characterized both at the whole plant and the cellular level (Maathuis, 2006). Osmotic stress resulting from excess salinity leads to water deficit with several consequences at the level of nutrition, e.g., reduced transpiration limits the long-distance transport of low-mobility ions, thus resulting in ion deficiency. Secondly, increases in salt concentrations in the environment can lead to toxic accumulation of Cl - and particularly Na + in the cytosol, hence exacerbating the negative effects on the acquisition and homeostasis of essential nutrients such as K + and Ca 2+. Thirdly, salt stress can cause oxidative stress damage and reactive oxygen species (ROS) generation (Smirnoff, 1993). A fourth mechanisms affected by high levels of salinity is ion transport itself. Some specific transport systems (Cerezo et al., 1997; Santa-Maria et al., 1997) are inhibited by the existence of significant amounts of Cl - and Na + (Maathuis, 2006). Thus, functionally identifying the role of membrane transport proteins in plant salinity tolerance can make a contribution to nutrient cycling in plants and, combined with genetic studies, may result in identification of candidate genes. New insights into physiological processes influencing salt toxicity and tolerance in plants can be expected, which may help plant breeders to further improve salt-tolerant genotypes. A series of experiments was undertaken to test the hypothesis that AtCNGC10 (Arabidopsis thaliana cyclic nucleotide-gated channel) gene is associated with salt tolerance in plants by examining the effects of different salt treatments on a variety of parameters in the AtCNGC10 antisense lines, with emphasis on ion content in tissue, ion fluxes and changes in cytosolic ion concentrations under salt stress. The activity of SOS1 gene coding for a Na + /H + antiporter was also tested directly on plants through Fluorescence Lifetime Imaging Measurements (FLIM) and electrophysiological measurements

14 CHAPTER 2 LITERATURE REVIEW 2.1 Salt stress occurrence and effects on plants Salinity is one of the most critical abiotic stresses affecting crop production in arid and semiarid regions. It occurs where soil salt content is naturally high and atmospheric precipitation can be insufficient for leaching; salinity can also be caused by bad agricultural practices, especially irrigation (Tester and Davenport, 2003). Soil salinity affects many morphological, physiological, and biochemical processes, including seed germination, plant growth, water and nutrient uptake and photosynthesis of plants (Willenborg et al., 2004; Hu and Schmidhalter, 2005). Salinity represents a major threat to the maintenance of crop yields due to the sensitivity of most crop species to Na + accumulation in shoot and leaf tissues, which leads to high Na + /K + ratio (Tester and Davenport, 2003). For many plants, Na + is the main cause of ion-specific damage of salinity. However, the capacity of plants to resist salt stress strongly depends on the status of their K + nutrition (Maathuis and Amtmann, 1999); thus, the maintenance of suitable Na + /K + ratio in cytosol is crucial for many plant species to survive salt stress. Na + -specific damage is linked with the build-up of Na + in leaf tissues and results in necrosis of older leaves, starting at the tips and margins and progressing back through the leaf. Growth and yield reductions are results of the shortening of the lifetime of individual leaves (Munns, 1993, 2002). The timescale of Na + -specific damage is associated with the rate of Na + accumulation in leaves, and with the effectiveness of Na + compartmentalization inside leaf tissues and cells. These Na + -specific effects are aggravated by the osmotic effects of NaCl, and Na + -specific effects demonstrate greater variation among species than osmotic effects (Munns, 2002). Osmotic damage is the result of the accumulation of high concentrations of Na + in the leaf apoplast due to entry of Na + into the xylem stream and water evaporation (Oertli, 1968; Flowers et al., 1991). Salinity not only causes Na + accumulation in plants, but also influences the uptake of essential nutrients such as K + and Ca 2+ through affecting ion selectivity (Grattan and - 4 -

15 Grieve, 1992). Thus, salinity can cause ion toxicity and ion imbalance in plants (Greenway and Munns, 1980). In this literature review, an emphasis will be placed on ion imbalance caused by salinity (particularly salt effects on transporters and channels), and on salt-related disturbance of photosynthesis, an important process which is often used as a tool to detect early environmental stresses. For other aspects of salt stress effects on mineral nutrition of plants, a reader is referred to (Hu and Schmidhalter, 2005), for Na + transport and Na + tolerance in higher plants, please see (Tester and Davenport, 2003). 2.2 Disturbance of ion homeostasis in plants by salinity Mineral nutrition is important to plant growth and development, and thus to agriculture and human health. Ion imbalance under salinity may result from the effect of salt on nutrient availability, competitive uptake, transport or partitioning inside plant (Grattan et al., 1994). High concentration of Na + and Cl - may limit nutrient-ion activities and result in extreme ratios of Na + /K +, Na + /Ca 2+, and Cl - /NO - 3. As a result, the plants become sensitive to osmotic and specific-ion injury as well as to nutritional disorders that may result in yield loss or quality reduction (Grattan and Grieve, 1999). Effects of salinity on K + uptake K + is the most prominent inorganic solute in plants; the maintenance of adequate content of K + is essential for plants to survive in saline conditions. Na + disturbs K + homeostasis in plants by altering K + uptake and transport. Reduction in K + uptake by Na + is the result of the following factors: First, competition for binding sites on transport systems that regulate K + uptake (Epstein, 1961; Rains and Epstein, 1965; Niu et al., 1995; Hasegawa et al., 2000). Secondly, high concentrations of Na + reduce the activity K +, making it less available for plants. Moreover, a significant membrane depolarization was generated when positively charged Na + crosses the plasma membrane (Shabala et al., 2003; Shabala et al., 2005a), making passive K + uptake through inward-rectifying K + channels thermodynamically impossible and, meanwhile, significantly increases K + leak through depolarization-activated outward-rectifying K + channels (KOR). Finally, the available ATP pool is severely reduced by increased de - 5 -

16 novo synthesis of various compatible solutes used for osmoprotection under saline conditions, making high-affinity K + uptake even more difficult (Shabala and Cuin, 2008). A low Na + /K + ratio in cytosol is more important for many plant species than simply maintaining a low concentration of Na + (Dubcovsky et al., 1996; Amtmann and Sanders, 1999; Cuin et al., 2003; Hu and Schmidhalter, 2005) under salt stress. Potassium has essential functions in plant metabolism (e.g. charge balance, osmotic adjustment, plant turgor, cellular homeostasis, protein synthesis and enzyme catalysis) and in growth and development (Maathuis and Sanders, 1996; Rigas et al., 2001; Elumalai et al., 2002). Sodium-induced K + deficiency has been demonstrated in growth and yield reductions of various crops, including tomato (Lopez and Satti, 1996; Song and Fujiyama, 1996), spinach (Chow et al., 1990), maize (Botella et al., 1997), eggplant (Savvas and Lenz, 2000), soybean (Essa, 2002) and plantain (Koyro, 2006). Although increasing leaf Na + concentrations may be beneficial for maintaining plant turgor, Na + cannot completely substitute for K + which is absolutely essential for protein synthesis and enzyme activation (Marschner, 1995). High K + concentrations in the chloroplast stroma are required to maintain optimum photosynthetic capacity under stress conditions; the K + demand in spinach leaves grown in solution cultures containing 250 mm NaCl was twice as high as in non-saline substrates (Chow et al., 1990). Therefore, K + is a particularly important nutrient element when plants are exposed to salinity. The presence of adequate Ca 2+ in the substrate affects the Na + /K + selectivity, leading to a shift in the uptake ratio in favour of K + at the expense of Na +, which effectively reduces Na + uptake and increases NaCl tolerance (Epstein, 1961; Cramer et al., 1987a; Rengel, 1992; Demidchik et al., 2002; Zhu, 2003; Shabala et al., 2006). The improvement in Ca 2+ -mediated membrane integrity results in reduction of K + leakage from root cells and a more favourable root-k + status (Alberico and Cramer, 1993; Cachorro et al., 1994; Shabala et al., 2003; Shabala et al., 2005b; Shabala et al., 2006). Hence, the beneficial effects of supplemental Ca 2+ on the K + status of salt-stressed plants are suggested to be more evident in root tissue than in the shoots, e.g. tomato (Lopez and Satti, 1996). These results suggests that Ca 2+ is important for controlling low Na + /K + ratio in plants; therefore, supplemental Ca 2+ can be one option to improve K + accumulation in some salt-stressed plants

17 Effects of salinity on calcium homeostasis With an increase in salt concentration in the root zone, plant requirement for Ca 2+ also increases (Bernstein, 1975). Meanwhile, the uptake of Ca 2+ from the substrate may be depressed due to ion interactions, precipitation and increased ionic strength, which decrease the activity of Ca 2+ in solution and therefore decrease availability of Ca 2+ to the plants (Cramer et al., 1986; Suarez and Grieve, 1988). Severity of the calcium disorder depends on (i) the ions that contribute to salinity and (ii) environmental conditions. Na + -related effects on the cell Ca 2+ homeostasis can be mediated through (i) changes in the plasma membrane composition and permeability (Kaya and Higgs, 2002; Kaya et al., 2003a; Kaya et al., 2003b; Shabala et al., 2003), (ii) altered activity of Ca 2+ channels and Ca 2+ -ATPase pumps embedded in the plasma membrane, or (iii) direct interaction of Na + and Ca 2+ at intracellular sites due to fast uptake of Na + across the plasma membrane (Lynch and Lauchli, 1988; Halperin and Lynch, 2003). Sodium ions reduced adsorption of Ca 2+ on isolated barley root cell walls through direct competition for the same binding sites (Stassart et al., 1981). Such displacement of Ca 2+ from critical sites in the apoplasm could be, at least partly, responsible for the observed salinity stress symptoms (Cramer et al., 1985; Yermiyahu et al., 1997). The Ca 2+ -displacement hypothesis has also been used to explain toxicities of H + (Yan et al., 1992; Kinraide, 1998) and Al (Rengel and Zhang, 2003). In Arabidopsis thaliana, mild salt stress (60 mm NaCl) inhibited Ca 2+ translocation from the relatively mature root parts (where transport is mostly symplastic), but not from young root parts (where Ca 2+ transport is mainly apoplastic). It appears that it is Ca 2+ translocation rather than Ca 2+ uptake that is deleteriously influenced by salinity (Halperin et al., 1997); only in the meristematic cells at the root tip Ca 2+ uptake may be inhibited by salinity. In barley cultivars, Na + also interfered with translocation rather than uptake of Ca 2+ because salinity stress decreased Ca concentration in leaves, but not in roots (Martinez and Lauchli, 1993). Because symplastic transport of Ca 2+ is inhibited by salinity, it is worth manipulating Ca 2+ uptake into root endodermal cells as a key to enhancing Ca 2+ translocation in salinity-affected plants

18 Symptoms of Ca deficiency can be seen in shoots due to severely deleterious effects of salinity on Ca 2+ uptake and translocation (Cramer et al., 1989; Francois et al., 1991), especially in salt-sensitive genotypes (Lynch and Lauchli, 1985; Grieve and Maas, 1988; Huang and Redmann, 1995). Salinity stress decreased Ca accumulation in leaves and shoots of a range of plant species tested so far, eg. legumes (Esechie and Rodriguez, 1999), cereals (Hawkins and Lewis, 1993; Fortmeier and Schubert, 1995; Huang and Redmann, 1995; Sultana et al., 2001), potato (Ghosh et al., 2001), cucumber (Kaya and Higgs, 2002), strawberry cultivars (Kaya et al., 2003a; Kaya et al., 2003b), okra (Ashraf et al., 2003) as well as in root tissues of sorghum (Koyro, 1997), cotton (Cramer et al., 1987b) and barley (Lynch and Lauchli, 1985). Therefore, the capacity to maintain high Ca 2+ uptake under salinity stress is as important for salinity tolerance as Na + exclusion. A cytosolic calcium ([Ca 2+ ] cyt ) increase occurs as a result of environmental stimuli due to the activation of the plasma membrane and/or endomembrane Ca 2+ channels (Rengel and Zhang, 2003; White and Broadley, 2003; Shabala et al., 2006). The increased [Ca 2+ ] cyt acts as a signal to elicit changes in a series of biochemical and physiological processes in the cell (Bush, 1995; Webb et al., 1996; Pandey et al., 2000; White and Broadley, 2003). Direct involvement of [Ca 2+ ] cyt in transduction of abiotic signals to changes in cellular metabolism has been demonstrated for salinity (Xiong et al., 2002; Zhu, 2003; Shabala et al., 2006) In contrast to all the published papers on the involvement of Ca 2+ in the signalling in salinity stress, no change in [Ca 2+ ] cyt was found in Arabidopsis thaliana root hair tips after 20-min exposure to salinity stress (Halperin et al., 2003). The tip-focussed [Ca 2+ ] cyt gradients decreased only after prolonged salinity stress (2 or 6 days). Hence, short-term signalling events in response of Arabidopsis thaliana root hairs to NaCl may operate by a means other than altering Ca 2+ dynamics at the root hair tips (Halperin et al., 2003). Ca 2+ plays an essential role in processes that preserve the structural and functional integrity of plant membranes, stabilise cell wall structures, regulate ion transport and selectivity, and control ion-exchange behaviour as well as cell wall enzyme activities (Rengel, 1992; Marschner, 1995). Because Ca 2+ plays a key role in cross-linking the pectic materials in the cell wall (Carpita and Gibeaut, 1993), the displacement of - 8 -

19 pectin-bound Ca 2+ would inevitably alter physical properties of cell wall, including extensibility, rigidity and permeability, which would be detrimental to cell extension as well as division. However, cell wall extensibility was not altered by salinity in a range of grass species (Cramer, 2003). Further work needs to be done to clarify these discrepancies. Effects of salinity on Mg 2+ Some studies showed that salinity decreased leaf Mg 2+ concentrations in sesame (Nassery et al., 1979), citrus (Ruiz et al., 1997), eggplant (Savvas and Lenz, 2000) and soybean (Essa, 2002). However, increases in salinity are not always linked with decreases in leaf Mg 2+ (Ruiz et al., 1997). Bernstein et al. (1974) found that increases in salinity only decreased leaf Mg 2+ concentration in beet and had little or no effect in leaves of five other vegetable crops tested. Salinity increased Mg 2+ content in sheath but had no effect on Mg 2+ in blade of wheat seedlings (Hu et al., 2006). Hence, effects of salinity on Mg 2+ are complex, and may be influenced by plant species, type of tissue, and various environmental factors. Further work on these aspects is warranted to clarify the effects of salinity on Mg 2+. Effect of salinity on micronutrient uptake by plants The availability of most micronutrients depends on ph of the root medium as well as the nature of binding sites on organic and inorganic matter. In saline conditions, the solubility of micronutrients (e.g. Cu, Fe, Mn, Mo and Zn) is relatively low, thus, plants often experience deficiencies in these elements (Page et al., 1990). However, differences in plant type, plant tissue, salinity level and composition, micronutrient concentration, growing conditions and the duration of study can cause different results. Therefore, the relationship between salinity and micronutrients is complex; salinity might increase, decrease, or have no effect on the micronutrient concentration in plant shoots (Grattan and Grieve, 1999). Salinity decreased Mn concentration in the shoots of bean (Doering et al., 1984) and maize (Izzo et al., 1991; Rahman et al., 1993); however, some studies with tomato indicated that salinity either had no effect (Al-Harbi, 1995) or increased (Mass et al., - 9 -

20 1972) Mn in leaf or shoot tissue. In addition, salinity was found to increase Mn concentration in sugar beet shoots (Khattak and Jarrell, 1989). Zinc concentration in shoot tissues in bean (Doering et al., 1984), citrus (Ruiz et al., 1997), maize (Rahman et al., 1993) and tomato (Knight et al., 1992) increased under salinity. However, in other studies, Zn concentration in tissues was not affected (Izzo et al., 1991) or actually decreased in cucumber leaves (Al-Harbi, 1995). Reports on the influence of salinity on Fe concentration in plants are similar to those on Zn and Mn concentration (Mass et al., 1972; Dahiya and Singh, 1976). Little attention has been paid to salinity effect on Cu and Mo uptake and accumulation in plants. Despite a large number of studies on the effect of salinity on mineral nutrient uptake and accumulation or nutrient partitioning within plant, the interaction between salinity and mineral nutrients still attracts attention of researchers due to the importance of mineral nutrients to yield, quality, and defense against pathogens and environmental stresses, especially when new plant material appears, such as nutritional mutants. Such mutants with changes in specific nutrient accumulation in plant tissues could provide valuable information on the potential mechanisms of salt toxicity as well as tolerance. 2.3 Transporters and ion channels involved in plant response to salinity Plants have developed a number of mechanisms to maintain appropriate intracellular levels of various cations with the main role attributed to transporters located within the plasma membrane and tonoplast, which mediate cation transport in and out of the cytoplasm and organelles (Fox and Guerinot, 1998). Many genes encoding cation transporters and channels in the plasma membrane or tonoplast that are affected by salt stress have been recently isolated from plants (Brett et al., 2005). Mostly, these transport systems are channels/transporters mediating Na + entry into and removal from the cytoplasm, including ion channels that are affected by changes in the plasma membrane potential, and ion channels that are voltage independent, but can be regulated ligand-gated (calmodulin, cyclic nucleotides, etc.)

21 Initial Na + influx is transported actively by differences in both concentration and voltage at the border of the cytoplasm/apoplast and the cytoplasm/vacuole and is mediated by channels. The Na + efflux from the cytosol to the apoplast under salt stress is mediated by an active transporter against Na + electrochemical gradient across a membrane. Under continuous salt stress, Na + concentration becomes high in the vacuole, and Na + uptake into the vacuole requires active transport as well. Therefore, there are two major classes of Na + transporters located in the plasma membrane and tonoplast: active transporters and channels; Na + net influx or efflux at the border of plant tissue depends on the relative contribution of all transporters (Tester and Davenport, 2003; Ryan and Vandenberg, 2005) Transporters in plasma membrane and at tonoplast that are affected by salt stress Plant cells use different transporters to decrease Na + concentration in the cytosol to avoid Na + toxicity. Na + /H + -antiporters are membrane proteins playing an important role in the cellular Na + homeostasis. They mediate the transport of Na + across the plasma membrane into apoplast (Tester and Davenport, 2003), or into vacuoles through a tonoplast-associated Na + /H + -antiporter (Apse et al. 1999). These antiporters are suggested to be the main determinants of salt tolerance in plants, and, therefore, studying the mechanisms of their regulation is very important in agriculture and biotechnology (Moffat, 2002; Shi et al., 2003). Plasma membrane transporters The physiological activity of Na + /H + -antiporters located in the plasma membrane has been reported for barley (Ratner and Jacoby, 1976), tomato (Mennen et al., 1990), and wheat (Allen and Sanders, 1995), Recently, genes encoding a Na + /H + -antiporter located in the plasma membrane have been identified and studied in the model systems Arabidopsis: salt overly sensitive gene (SOS1) (isolated by positional cloning) (Shi et al., 2000) Overexpression of SOS1 improved salt tolerance in Arabidopsis (Wu et al., 1996; Shi et al., 2003). In addition to the regulation of Na + concentration in the cytoplasm at the cytoplasm/apoplast border, Shi et al. (2002) demonstrated that SOS1 is essential for controlling long-distance Na + transport. Furthermore, the activity of the

22 SOS1 antiporter can be modified by an SOS3-SOS2 protein kinase complex (Zhu, 2002). SOS3 is a myristoylated calcium-binding protein that is considered to respond to salt-induced cytosolic Ca 2+ increases (Knight et al., 1997) by sensing the Ca 2+ signal due to salt stress and translating it to the downstream responses (Liu and Zhu, 1998; Ishitani et al., 2000). In particular, SOS3 activates SOS2, a serine/threonine protein kinase (Halfter et al., 2000; Liu et al., 2000). One of the targets of this signaling pathway is SOS1. This progress in understanding the signal transduction pathway for the ion homeostasis and salt tolerance of higher plants will help to develop salt-resistant crop plants in the future. The CAX (calcium/proton exchanger) family is also considered important in ion regulation of the plant cells during salt stress. Arabidopsis CAX1 and 2 are the first two CAX genes isolated (Hirschi et al., 1996); they could suppress Ca 2+ -sensitive defect, increase Ca 2+ accumulation in the cell and sensitivity to Ca 2+ -related stress such as chilling sensitivity when expressed in tobacoo(hirschi, 1999). A recent study demonstrated that a putative plasma membrane gene GmCAX1 from soybean conferred salt tolerance in Arabidopsis (Luo et al., 2005). Transgenic Arabidopsis plants overexpressing GmCAX1 accumulated less Na +, K + and Li +, and were more tolerant to elevated Li + and Na + levels during germination when compared with the controls. These results suggest that GmCAX1 may also function as an antiporter for Na +, K + and Li +. Therefore, the regulation of this antiporter could be helpful for the modulation of ion homeostasis and plant salt tolerance. The high-affinity K + transporter (HKT) has been identified in many plant species by its functioning as a Na + /K + symporter and Na + -selective transporter of both high and low affinity and Na + /Na + symporter (Rubio et al., 1995; Garciadeblas et al., 2003; Rus et al., 2004; Haro et al., 2005). The wheat TaHKT2;1 gene expressed in Xenopus oocytes functioned as a Na + /K + symporter (Rubio et al., 1995), and down-regulation of expression in planta decreased Na + accumulation in roots and improved growth in saline conditions (Laurie et al., 2002). Reduced expression of TaHKT1 decreased Na + uptake into wheat roots and increased plant tolerance to salt (Laurie et al., 2002), despite the results from electrophysiological measurements based on expression in heterologous systems that implicated a Na + and K + co-transport function for TaHKT1 (Rubio et al., 1995). Physiological characterization of hkt1 genotypes has identified that

23 Arabidopsis HKT1 (AtHKT1) limits plant root and shoot Na + content and prevents salt stress by decreasing Na + accumulation in leaves (Maser et al., 2002), suggesting AtHKT1 regulates Na + homeostasis in planta and through this function modulates K + nutrients status (Davenport et al., 2007). Another plasma membrane located transporter that could be affected by salt stress is the low-affinity cation transporter (LCT). LCT1 was cloned from a wheat cdna library by functional complementation of a yeast strain lacking high-affinity K + uptake (Anderson et al., 1992; Schachtman et al., 1997). Compared with HKT1, LCT1 increased K + uptake capacity only when K + concentration was 1 mm and above, indicating relatively low affinity for K +. The suggestion that LCT1 plays a role in salt sensitivity of wheat resulted from the observation that LCT1 mediates Na + influx (Schachtman et al., 1997), Suggesting that LCT1 is a potentially important pathway for Na + in wheat and hence a potential target for molecular engineering of salt tolerance in wheat. However its physiological role remains unclear in planta (Amtmann et al., 2001). Additionally, LCT1 likely plays a role in Ca 2+ uptake, and thus, if target is to produce salt-tolerant wheat plants by modifying this gene, the role of LCT1 in Ca 2+ uptake has to be taken into account to avoid a potentially damaging loss of Ca 2+ uptake capacity, therefore, modification of LCT1 has to be very careful.(amtmann et al., 2001) Detailed structure-function analysis of LCT1 will be necessary in future study. Transporters located at the tonoplast In plants, vacuolar Na + /H + -antiporters can pump Na + into the vacuole through the tonoplast to reduce Na + concentration and maintain a high K + /Na + ratio in the cytosol. They also keep osmotic balance by sequestering Na + into vacuole from the cytoplasm (Glenn et al., 1999). Overexpression of tonoplast-associated Na + /H + -antiporters conferred salt tolerance in transgenic plants of sugar beet (Blumwald and Poole, 1985), barley (Garbarino and DuPont, 1988), sunflower (Ballesteros et al., 1997), rice (Fukuda et al., 1998; Fukuda et al., 1999), Arabidopsis (Apse et al., 1999), tomato (Zhang and Blumwald, 2001), maize (Zorb et al., 2005), wheat (Xue et al., 2004), and halophytes Suaeda salsa (Ma et al., 2004; Li et al., 2007) and gouan (Zhang et al., 2007). These results implicate the importance of Na + sequestration in vacuoles for the plant salt tolerance

24 Importance of the regulation of the tonoplast Na + /H + antiporter activity for plant salt stress can be demonstrated in two ways. Firstly, overexpression of NAX1 (Na + /H + exchanger) in tomato (Zhang and Blumwald, 2001) and Arabidopsis (Apse et al., 1999) led to increased plant salt tolerance. Secondly, the activity of vacuolar Na + /H + -antiporters in rice was reported by Fukuda et al. (1998) to be lower than that in salt-tolerant crop barley. The study also suggested that the amount of the Na + /H + -antiporter was one of the most important factors affecting Na + removal from the cytoplasm and determining salt tolerance. In addition, the endogenous level of vacuolar Na + /H + -antiporters were lower in salt-sensitive wheat genotypes as compared to salt-tolerant ones when exposed to salt stress (Saqib et al., 2005). Some vacuolar Na + /H + -antiporters (NHX) such as Arabidopsis Na + /H + -antiporter (AtNHX1) and rice Na + /H + -antiporter (OsNHX1) were found regulating the transport of K + as well as Na + (Venema et al., 2002; Fukuda et al., 2004). Further studies revealed that in cellular physiology, the role of vacuolar Na + /H + -antiporters was not limited to ion homeostasis. The microarray expression profiles of a T-DNA insertion knockout Arabidopsis mutant of AtNHX1 and a rescued line demonstrated that, apart from responses to salt stress, AtNHX1 is also important to the key cellular processes such as calcium signalling, cell structure and cell growth as well as protein processing (Apse et al., 2003; Sottosanto et al., 2004; Sottosanto et al., 2007). All these results suggested that the Na + /H + -antiporters play an important role not only in ion homeostasis and salt tolerance, but also in plant development. However, the function of these Na + /H + antiporters in plants when they are not under salt stress conditions is not clear Ion channels involved in plant salt tolerance Channels permeable to Na + The most likely pathway for Na + influx was suggested to be mediated via non-selective cation channels (NSCCs) (Amtmann and Sanders, 1999; White, 1999; Davenport and Tester, 2000; Demidchik et al., 2002). However, there are many possible genes that might encode these NSCCs, and their precise molecular identification remains obscure (Demidchik and Tester, 2002). Two main candidates for NSCCs are the cyclic nucleotide-gated channels (CNGCs) encoded by cngc genes and the putative

25 glutamate-activated channels (the glutamate receptors, GLRs) encoded by glr genes. The channels encoded by these genes showed the properties of NSCCs (Cheffings, 2001; Lacombe et al., 2001; Leng et al., 2002), but the function in planta of specific gene products remains unclear before their targeted membranes and the cell types expressing these gene products are identified, and the effects on ion fluxes in plants expressing these genes are measured (Tester and Davenport, 2003). Plant glutamate receptors (GLRs) Genes encoding putative glutamate receptors in Arabidopsis thaliana were reported by Lam et al. (1998). Twenty glutamate receptor-like genes (AtGLRs) were found and divided into three subgroups according to their sequence similarity (Lacombe et al., 2001). However, the functional analysis is lacking so far. Addition of glutamate to intact Arabidopsis seedlings resulted in increases in the activity of cytosolic Ca 2+ (Dennison and Spalding, 2001). This observation was consistent with Ca 2+ entering through the plasma membrane, and the kinetics of the Ca 2+ increases were in line with a direct glutamate activation of cation channels. Similar results were obtained by patch-clamp measurements: protoplasts from Arabidopsis roots showed glutamate activation of cation currents (Demidchik and Tester, 2002). The possibility and magnitude of the observed glutamate-activated currents increased with increasing glutamate concentration. Moreover, this possibility and magnitude were positively proportional to the magnitude of the current occurring before glutamate was added, suggesting that glutamate-activated currents, to some extent, can also be responsible for the background current occurring before addition of glutamate. Activated by low millimolar concentrations of Na-glutamate, these voltage-independent channels displayed nonselective permeability for monovalent cations such as K +, Na +, and Cs + as well as large permeability to Ca 2+. Channel activity was inhibited by quinine and lanthanides (Demidchik and Tester, 2002). Loss of function mutations in rice GLR3.1 (Li et al., 2006) and AtGLR3.2 (Kim et al., 2001; Turano et al., 2002) demonstrated their involvement in Ca 2+ accumulation and Ca 2+ -regulated reactions such as stress responses, cell division and programmed cell death. Qi et al. (2006) found the strong evidence for GLR3.3 forming Ca 2+ -permeable channels. Overexpression of a radish GLR (Kang et al., 2006) in Arabidopsis increased glutamate-activated transient

26 [Ca 2+ ] cyt elevation and changed Ca 2+ -regulated mechanisms such as growth and development. In addition, it increased resistance to a fungal pathogen. Several other studies (Kang and Turano, 2003; Kang et al., 2004) found AtGLR1.1 expression was associated with seed germination and ABA-mediated processes. Glutamate also activated unidirectional influx of Na + into intact Arabidopsis roots (Demidchik et al., 2004). All these results suggest that GLRs play a role in plants that goes from salt stress to signaling and development. Plant cyclic nucleotide-gated channels (CNGCs) Plant CNGCs are a recently identified family of plant ion channels. They are structurally similar to Shaker-type K channels in structure with six membrane-spanning domains (S1-6) and a pore region situated between S5 and S6 (Varnum and Zagotta, 1997; Schuurink et al., 1998; Maser et al., 2001; Talke et al., 2003). A domain in the 4 th transmembrane span shows similarity to the shaker-type voltage sensor, and a C-terminal cyclic nucleotide-binding domain binds both cyclic nucleotides (CN) and calmodulin (CaM) (Arazi et al., 1999; Kohler and Neuhaus, 2000; Kaplan et al., 2007). CNGCs have been identified in many species (Talke et al., 2003); however, until recently, their functions in plants have not been clearly understood. Despite the first cloning and characterization of a CaM-bing putative CNGC from barley a decade ago (Schuurink et al., 1998), an elaborate functional characterization of plant CNGC properties is still not available (Ali et al., 2006). Studies suggested that plant CNGCs function as cation pathway, and play a role in selectivity for different cations, defenses against abiotic and biotic threats, and plant development (Arazi et al., 1999; Clough et al., 2000; Chan et al., 2003; Borsics et al., 2007). These results strongly suggest that plant CNGCs are involved in different physiological roles throughout plant development. In Arabidopsis, 20 channels from the CNGC family were found (Maser et al., 2001). Electrophysiological, molecular and genetic complementation studies have revealed that six of these 20 members are generally non-selective monovalent and divalent cation channels (Kohler et al., 1999; Li et al., 2005; Ali et al., 2006; Gobert et al., 2006;

27 Kaplan et al., 2007), although their selectivity profiles may differ (Leng et al., 1999; Leng et al., 2002; Balague et al., 2003; Hua et al., 2003a; Li et al., 2005). Among 20 CNGCs members, several of them have been suggested to be involved in K +, Na + and Ca 2+ uptake and transport, therefore contributing to plant salt tolerance (Clough et al., 2000; Chan et al., 2003; Gobert et al., 2006; Ma et al., 2006; Borsics et al., 2007). Regarding heterologous expression for functional characterization of plant CNGC, tolerance and sensitivity to various cations, and involvement in plant defense against pathogens and plant development, please see latest review by Kaplan et al. (2007). Recent studies suggested that AtCNGC1 gene was mainly expressed in roots of Arabidopsis seedlings (Ma et al., 2006), involved in Ca 2+ uptake into plants, and affected the growth in the primary root of Arabidopsis seedlings. Seedlings lacking this protein have slightly lower Ca 2+ content in shoots than WT plants. Similarly, knockout AtCNGC2 plants (both seedlings and mature plants) were hypersensitive to Ca 2+, but not to Na + and K +, compared with WT controls. However, this hypersensitivity is not related to Ca 2+ accumulation in these mutants, so Ca 2+ hypersensitivity might be caused by damaged signaling pathway at high Ca 2+ concentration (Chan et al., 2003). AtCNGC2 mutants suggested a role of AtCNGC2 in disease-resistance, programmed cell death, and plant development mediated by Ca 2+ (Clough et al., 2000). Gobert et al. (2006) demonstrated that AtCNGC3 plays an apparent role in ion homeostasis, and is linked with the non-selective uptake of Na + and K + and perhaps some other monovalent cations. Compared to WT, AtCNGC3 mutants are more sensitive to Na + toxicity during germination, having lower germination rates when subjected to mm NaCl. However, mutant seedlings were not different in Na + content, but 50 % lower in K + than WT, which suggested that CNGC3 played a role in salinity adaptation at germination stage. Combined with K + and Na + uptake experiments, it showed AtCNGC3 may participate in distribution or translocation of ions from the xylem in mature plants, and when the gene is knocked out, these ions accumulated excessively, which caused damage to plant development by altering water potential. In seedlings, AtCNGC3 might take part in the Na + uptake, and if the gene is knocked out, fewer ions accumulated, suggesting that seedlings have less sensitivity to high concentrations of Na + (Gobert et al., 2006)

28 AtCNGC10 was studied in the Arabidopsis akt-1 mutant (Borsics et al., 2007). The gene could partially rescue akt-1 mutant for K + uptake if transformed with a 35s::AtCNGC10 construct. However, if WT plants were transformed with an AtCNGC10 antisense construct, a 40% lower K + content was obtained as compared to non-transformed WT plants, suggesting that AtCNGC10 may be involved in plant K + uptake. So far, most studies on plant CNGCs have been conducted on heterologous systems. In planta, reverse genetic studies revealed the role of different CNGCs in cation uptake, transport, and homeostasis (Arazi et al., 1999; Clough et al., 2000; Sunkar et al., 2000; Chan et al., 2003; Ma et al., 2006; Borsics et al., 2007). There is paucity of information about the specific roles CNGCs play in plant response to external signals, selectivity of cations, growth, development and pathogen defense. Similarly, there is little information available about the functional properties of plant CNGCs. Among the 20 individual members of this protein family in Arabidopsis, only AtCNGC2 has been functionally identified as an ion channel (Leng et al., 1999); therefore, more functional characterization needs to be done on other members of this protein family. Voltage-regulated K + channels involved in plant salt tolerance Potassium (K + ) transport systems play crucial roles in maintenance of a high K + /Na + ratio, which is important in salt adaptation of plant cells (Maathuis and Amtmann, 1999; Kasukabe et al., 2006). Plant K + channels also play a variety of other important physiological roles in the plasma membrane of various tissues and organs (Schroeder et al., 1994; Maathuis et al., 1997; Fox and Guerinot, 1998). Given that salt stress can cause severe impacts on K + homeostasis in all plant organs, K + transport to cell compartments and tissues might be subject to different regulation under salinity (Maathuis, 2006). Based on their voltage-dependence, plant K channels can be classified into three functionally distinct types: i) hyperpolarization-activated inward-rectifying K + channels mediating K + uptake, ii) depolarization-activated outward-rectifying channels that regulate K + release, and iii) weakly-rectifying K + channels mediating both K uptake and release (Gambale and Uozumi, 2006)

29 Some studies demonstrated that Arabidopsis weakly voltage-dependent inward-rectifying K + channel AKT2 and its truncated version AKT3 are up-regulated during salinity. AKT2,3 proteins were identified in Arabidopsis phloem and suggested to function as a shunt conductance in phloem cells (Cao et al., 1995); hence, they may be associated with the recirculation of K + through phloem. As the essential part of K + homeostasis in plant, the recirculation of K + through phloem is possible to be influenced during salinity, particularly in relation to root/shoot partitioning of Na + and K +. Similarly, the gene encoding SKOR, the outward- rectifying K + channel that is located in the plasma membrane of stelar root tissue in Arabidopsis (Gaymard et al., 1998) and mediates K + release into the xylem, is also significantly up-regulated when the plant is exposed to salt stress. Thus, the rates of K + circulation through vascular tissues would be increased by the substantial up-regulation of both SKOR in roots and AKT2,3 in shoots under salt stress, leading to changes in a long-distance redistribution of K + between the roots and shoot (Maathuis, 2006). Previous studies (Murata et al., 1994a; Murata et al., 1994b) showed that the permeability of the plasma membrane outward-rectifying K + channels (TORK1) from tobacco was reduced to minimize K + loss from and Na + entry into the cytoplasm, leading to the adaptation of tobacco cells to salt stress. Further studies indicated that the outward K + currents decreased with increasing extracellular Ca 2+ (Murata et al., 1998; Kasukabe et al., 2006). Similar results were obtained in Arabidopsis root and leaf cells (Shabala et al., 2006), suggesting an additional mechanism of Ca 2+ in the amelioration of K + loss from the cell and therefore increased plant salt tolerance. Kasukabe et al. (2006) reported that the expression of outward-rectifying K + channel of tobacco (TORK1) was lower in NaCl-adapted than unadapted cells, suggesting that the reduction in the number of outward-rectifying K + channels might lead to tolerance to salinity stress through the decrease of outward K + currents. Both rice K + uptake channel OsAKT1 mrna and inward K + currents decreased during salt stress in the root of rice seedlings (Fuchs et al., 2005); however, it was not reported whether the activities of outward-rectifying K + channel were down-regulated under salt stress Summary So far, considerable progress has been made in understanding plant salt tolerance at a cellular level through electrophysiological and molecular studies (Rentsch et al., 1996;

30 Liu and Zhu, 1998; Nuccio et al., 1998; Amtmann and Sanders, 1999; Apse et al., 1999; Tyerman and Skerrett, 1999; Halfter et al., 2000; Liu et al., 2000; Shi et al., 2000; Zhu, 2002; Shi et al., 2003; Saqib et al., 2005; Zhang et al., 2007). Many genes encoding transporters and/or channels involved in plant salt tolerance have been identified and localized in the cell, with the complete genome sequences being available for some of them. The functional analysis for most of these genes was conducted after heterologous expression; hence, their functional characterization directly in plants as well as their precise localization at the organ and cell level is eagerly awaited. In addition, a clear relationship between salt tolerance and regulation of ion transport under salinity needs to be established. 2.4 Photosynthesis under salt stress: chlorophyll fluorescence, chlorophyll content and stomatal limitation Photosynthesis is an important process to be monitored in plant responses to abiotic stress. In recent years, the technique of chlorophyll fluorescence has become widely used in plant ecophysiological studies. Many plant breeders are using chlorophyll fluorescence as a convenient tool to distinguish between genotypes that are different in their sensitivity to various environmental stresses. Understanding photosynthetic responses of plants exposed to various stresses will help plant breeders to improve genotype, as well as crop physiologists and agronomists to develop the best management practice, to deal with environmentally-induced abiotic stresses. Salinity effects on photosynthesis in plants are complex. Slightly elevated salinity levels sometimes even increase photosynthetic performance (Greenway and Munns, 1980; Heuer and Plaut 1981; Marschner, 1995). In contrast, medium or high salinity can inhibit leaf photosynthesis (Singh and Dubey, 1995; Tan and Blake, 1997; Abadia et al., 1999; Delfine et al., 1999; Netondo et al., 2004). Two major adverse effects of salinity in non-tolerant species are osmotic stress, and Na + and Cl - toxicity (Greenway and Munns, 1980; Serrano et al., 1999; Shabala, 2000). Due to complexity and multiplicity of pathways of salt effect on photosynthesis, there is a problem of how to interpret changes in photosynthetic parameters. The effect from osmotic stress of salinity implies that stomata is involved in regulation of plant

31 photosynthetic performance. A significant decrease in stomatal conductance has always been considered an important factor of the overall decrease in net CO 2 assimilation in affected plants (Seemann and Critchley, 1985; Fedina and Tsonev, 1997; Khan et al., 1997; Delfine et al., 1998; Morales et al., 1998; Belkhodja et al., 1999; Chatrath et al., 2000; Redondo-Gomez et al., 2007). Significant reduction in net CO 2 assimilation under saline conditions was reported for wheat (Kasai et al., 1998), rice (Welfare et al., 1996; Khan et al., 1997), oat (Chatrath et al., 2000), barley (Belkhodja et al., 1999), spinach (Delfine et al., 1998), pea (Fedina and Tsonev, 1997), tomato (Xu et al., 1997), guava (Ali-Dinar et al., 1999), citrus (Banuls et al., 1997), alfalfa (Anand et al., 2000), pepino (Chen et al., 1999), sorghum (Netondo et al., 2004) and other species (Redondo-Gomez et al., 2007; Wang et al., 2007b). Salinity stress results in a decrease in plant photosynthesis through stomatal and nonstomatal factors, with the latter not yet fully understood (Sharma and Hall, 1992; Dionisio-Sese and Tobita, 2000; Zhao et al., 2007). The relative contribution of stomatal and non-stomatal effects on photosynthesis is highly dependent on the level of the salt stress. At medium salinities, the major mechanism is the limitation from stomatal component, whereas under more severe salt stress the non-stomatal limitation at the biochemical level dominates (Seemann and Critchley, 1985; Plaut et al., 1989; Bethke and Drew, 1992; Robertson et al., 1993; Everard et al., 1994). The latter makes chlorophyll fluorescence, especially F v /F m (the variable to maximum fluorescence ratio, indicating photochemical efficiency of leaf), a useful tool for screening plants for salt tolerance (Marler and Mickelbart, 1993; Tiwari et al., 1997; Keiper et al., 1998; Chen et al., 1999; Dionisio-Sese and Tobita, 2000). Salinity decreased chlorophyll content in affected leaves and changed chlorophyll a-to-b ratio (Singh and Dubey, 1995; Lutts et al., 1996; Kasai et al., 1998; Rawat and Banerjee, 1998; Abadia et al., 1999; Chen et al., 1999; Delfine et al., 1999; Netondo et al., 2004; Zhao et al., 2007). Hence, decreased chlorophyll content (usually associated with leaf chlorosis) could be one possible reason for the observed salt-induced decrease in photosynthetic rates. However, the Chl a/b ratio remained unchanged by salt treatment up to 200 mm NaCl in spinach (Robinson et al., 1983). Only a 14% decline in chlorophyll content was measured under 150 mm NaCl in capsicum (Bethke and Drew, 1992); however, the chlorophyll content increased at 100 mm during the first days of

32 the experiments. There are also controversial reports on the total chlorophyll content being significantly higher in mung bean and brassica grown under saline conditions (Misra et al., 1995). One of the reasons of such controversy might be due to the fact that a significant decrease in the water content of leaf tissue may cause the apparent increase in total chlorophyll content in affected leaves due to an absence of dilution (Wang and Nii, 2000). These results indicated that loss of chlorophyll is not the main cause of decreased photosynthesis under salinity and cannot be the primary biochemical factor of the observed growth decrease (Everard et al., 1994). Therefore, for screening purpose, pigment analysis is not a sensitive enough parameter to be used unless results are expressed on a dry weight basis (Shabala et al., 1998). However, some studies have shown that there was a progressive decrease in both the fluorescence yield (Y) and the coefficient of photochemical quenching (qp) in pre-illuminated leaf samples of maize (Shabala et al., 1998) when NaCl concentration was higher than 50 mm. There was an increase in the coefficient of non-photochemical quenching (qn) and high correlation (about 0.9) between each of these parameters (y, qp, qn) and Na + accumulation in leaves. A similar conclusion was obtained for two salinized poplar genotypes (Wang et al., 2007b),sweet almond (Ranjbarfordoei et al., 2006) and sorghum (Netondo et al., 2004). A significant decrease in qp of naked oat (Zhao et al., 2007) was observed at 200 and 250 mm NaCl. Therefore, we can conclude that, in spite of the variability, fluorescence parameters, particularly fluorescence quenching, in pre-illuminated leaves are the most sensitive photosynthetic factor to be used for detecting salinity sensitivity in maize (Shabala et al., 1998), spinach (Robinson et al., 1983; Delfine et al., 1999), barley (Abadia et al., 1999), pepino (Chen et al., 1999), rice (Dionisio-Sese and Tobita, 2000), oat (Zhao et al., 2007) and some other species Another chlorophyll fluorescence parameter, F v /F m ratio, is widely used in measuring the effect of stress on PSII activity (Larcher et al., 1990; Krause and Weis, 1991; Belkhodja et al., 1994; Everard et al., 1994; Marler and Zozor, 1996; Jimenez et al., 1997; Chen et al., 2004; Broetto et al., 2007; Lin et al., 2007; Redondo-Gomez et al., 2007; Zhao et al., 2007). This ratio is a measure of the maximal photochemical efficiency of PSII, and it is convenient to measure. A significant change in the F v /F m ratio in celery was observed when 300 mm NaCl was applied for 5 weeks (Everard et

33 al., 1994). F v /F m ratio decreased in rice (Tiwari et al., 1997), pea (Velitchkova and Fedina, 1998), pepino (Chen et al., 1999) in radish (Jamil et al., 2007) and in sweet almond (Ranjbarfordoei et al., 2006) Although in some species F v /F m is not a sensitive parameter for estimation of plant physiological conditions at low and/or intermediate salinity levels (Brugnoli and Bjorkman, 1992), it might be attributed to the low level of salinity, short time of exposure to salt stress and plant ability to resist salt stress. Salt concentration and time of exposure should be always considered simultaneously to characterize the level of salt stress. On the other hand, salt-tolerant plants are not sensitive to low concentrations of NaCl. Therefore, no reduction in Fv/Fm estimated in some studies on halophyte Suaeda salsa or salt cress is rather due to a low dose of NaCl (Lu et al., 2003). Hence, together with other measurements, such as ion (Na +, K + and Ca 2+ ) content changes in plant tissues, F v /F m can be one option to detect the salt stress-induced damage in plants. In summary, salt stress severity and stress-induced damage in plants can be measured by the traditional ecophysiological methods such as measuring rates of photo assimilation, stomatal conductance, leaf water potential, etc. (Han et al., 1994; Johnson and Asay, 1995; Kleinhenz et al., 1995; Lichtenthaler, 1996; Tan and Blake, 1997). However, these changes are known to undergo significant variations during the day. For example, changes in weather situation might cause significant changes in rates of transpiration, net CO 2 assimilation and stomatal conductance (Cardon et al., 1994; Steduto and Hsiao, 1998). Therefore, these changes cannot be used to detect plant stress responses. Moreover, these changes are subject to diurnal (Marler and Zozor, 1996; Steduto and Hsiao, 1998; Kumar et al., 1999) or circadian (Snaith and Mansfield, 1986; Hennessey and Field, 1991; Luttge et al., 1996) rhythmicity of most ecophysiological characteristics. Even under constant temperature, light and humidity conditions, net CO 2 assimilation and stomatal conductance could undergo 3-fold circadian variations (Hennessey and Field, 1991; Luttge et al., 1996). The net CO 2 fixation in canopy was significantly higher in the morning than in the afternoon although the photosynthetically active photon flux density (PPFD, μmol m -2 s -1 ) remained at a constant level. Therefore, the absolute values of such ecophysiological characteristics may not be suitable to be used for screening plants for environmental fitness. To solve this problem, the best option is to measure the physiological parameters as relative values, eg. some ratio

34 values. One answer to this question is chlorophyll fluorescence. Recent technical improvements in fluorescence measurement have made this method a convenient and important tool in plant physiology research. Chlorophyll fluorescence, especially Fv/Fm ratio, has been widely used in detecting crop responses to salt, heat, chilling and drought stresses (Daniel, 1997; During, 1998; Janda et al., 1999; Tezara et al., 2003; Lin et al., 2007). This ratio is normally very close to 0.83 for every species under unstressed conditions (Bjorkman and R, 1987; Johnson et al., 1993). When exposed to stress, Fv/Fm ratio decreased significantly if the activity of PSII is affected by stress, suggesting that the stress factor had a severe effect on plants (Warner and Burke, 1993; Dolstra et al., 1994; Zotz and Tyree, 1996; Gillies and Binder, 1997). 2.5 Summary Salinity is a complex phenomenon and its complete understanding requires joint efforts of agronomists, biochemists, geneticists, molecular biologists, plant physiologists and soil scientists. Plant salt tolerance involves a set of complex parameters. Membrane transport activity can be one component, but not all, of a salt tolerance mechanism. Several mechanisms may be operating together to resist salt stress in plants under saline conditions because processes in many different plant parts are affected, and more than one of these processes can occur concurrently within a specific plant. These tolerance mechanisms can occur from the cell to the whole-plant level. They can occur in all cells or just in specific cell types. To further understand complexity of the whole plant adaptations to salinity, more knowledge is required of cell-specific transport processes and the consequences of manipulating transporters, channels, and signaling factors in specific cell types. The most common solution to solve the salinity problem is to enhance the salt resistance of conventional crop plants, but the improvement in yield by this method is not obivous (Tester and Davenport, 2003). In addition, salt tolerance at the cell level can contribute to the whole-plant salt tolerance and vice versa. Studying the specific genes from closely related halophytic species can also be an effective way to improve salt tolerance by genetic manipulation technologies. Nowadays, more and more attention has been paid to development of salt-tolerant crops through breeding and genetic engineering. Many genes encoding transporters and/or channels associated with plant salt tolerance

35 have been identified and localized in cell, with the complete genome sequences being available for some of them. However, there is a lack of knowledge on (i) the functional analysis for most of these genes directly in plants, (ii) functional characterization of new mutants with altered pathways and processes that might be involved in salt tolerance. 2.6 Outline of the study General The purpose of this study was to (i) investigate the involvement of Arabidopsis cyclic-nucleotide-gated channel 10 (AtCNGC10) in plant salt tolerance, (ii) to provide evidence for the activity and the role of this channel in plants, and (iii) to test the Na + /H + antiport activity of SOS1 gene directly in plants, thus making a contribution to the functional characterization of AtCNGC10 and SOS1. The general approach was to carry out a series of experiments on plant growth and development under salt stress conditions, and measure various parameters. Electrophysiological measurements were also taken to test the Na + /H + antiport activity of SOS1 gene directly in plants Aims To test the hypothesis that Arabidopsis cyclic neucleotide-gated channel 10 (AtCNGC10) is involved in plant salt tolerance; To investigate the response of the AtCNGC10 antisense lines to salt stress in different developmental stage; To test the activity of Na + /H + antiporter SOS1 directly in plants Structure of the thesis A general introduction and literature review that embraces all aspects of the study have been given in Chapter 1 and Chapter 2. General details of plant materials, plant growth conditions, experimental procedures, analytical instruments and techniques and principles, and statistical analyses are contained in Chapter 3. The experimental results are given in Chapters 4-6. The cyclic nucleotide-gated channel, AtCNGC10,

36 influences salt tolerance in Arabidopsis (Chapter 4). Chapter 5 deals with involvement of the cyclic nucleotide-gated channel, AtCNGC10, in transporting Ca 2+ and Mg 2+ as assessed by Fluorescence Lifetime Imaging and ion fluxes analysis. Chapter 6 deals with the Na + /H + antiporter activity of SOS1 Gene: Fluorescence Lifetime Imaging analysis and electrophysiological studies on Arabidopsis seedlings. General conclusions and discussions, suggestions for future study are given in Chapter

37 CHAPTER 3 GENERAL MATERIALS AND METHODS 3.1 General This chapter describes common plant materials, plant growth conditions, and analytical and experimental procedures employed in this study. Further details of materials and methods specific to individual experiments are indicated in individual chapters. 3.2 Materials and Methods Plant material and growth conditions Wild type (WT) Arabidopsis plants (Arabidopsis thaliana L. [Heynh]) ecotype Col-0 and AtCNGC10 antisense lines, A2 and A3, in Columbia background were from Department of Molecular Biosciences and Bioengineering, University of Hawaii, USA. The Arabidopsis wild type was originally obtained from the Arabidopsis Biological Resource Center. In this study, we use the term seedlings for plants less than 2 weeks old. Older plants in the flowering stage (start of budding) were also used. For all experiments, seeds were surfaced sterilized using CaCl 2 O 2 for 10 minutes, followed by several rinses with sterile water. For biomass, root length and ion content measurements surface sterilised seeds were placed into Petri dishes on agar media [¼ Hoagland solution (Table 1) and 0.8% w/v agar). For ion fluxes and intracellular ph and cytosolic Ca 2+ measurements, seeds were surface sterilised and placed into Petri dishes on agar media [basic salt medium (BSM): 1 mm KCl mm CaCl 2 and 0.8% w/v agar). Petri dishes were placed vertically in a growth chamber (25 o C/20 o C, 14/10 h day/night cycle, 120 μmol m -2 s -1 )

38 For biomass and chlorophyll fluorescence studies on plants in the flowering stage, wild type (WT) Arabidopsis (Arabidopsis thaliana L. [Heynh]) ecotype Columbia-0 (Col-0) and two Arabidopsis cyclic nucleotide-gated channel 10 (AtCNGC10) antisense lines in Columbia background (A2 and A3) were grown in a temperature-controlled chamber with a cycle of 16 h light (150 μmol m -2 s -1 ) and 8 h dark at 20 ± 1 o C. Seeds were surface sterilised and placed on a thin (1-2 mm) rock-wool base in ¼ Hoagland nutrient solution that was changed every 3 days. Based on a preliminary experiment, WT seeds were germinated 10 days earlier than A2 and A3 to synchronise the growth stage for measurements. Treatments with different concentration of NaCl solutions were applied on day 26 after germination for A2 and A3, and on day 36 for WT. Table 1 Hoagland s solution Salt MW Stock solution (St) (g/l) Stock solution conc. Full strength Hoagland concontration 1 KH 2 PO M M KNO M M Ca(NO 3 ) 2.4H 2 O M M MgSO 4.7H 2 O M M 1 5 EDTA-Na % w/v 5% w/v 0.25 FeSO 4.7H 2 O H 3 BO MnCl 2.4H 2 O 1.81 ZnCl CuCl 2.2H 2 O 0.05 Na 2 MoO 2.2H 2 O ¼ Strength of Hoagland solution (ml St/L) To normalise the assessment of growth of WT and antisense lines, biomass accumulation in plants in flowering stage was expressed as a percentage of their own controls: biomass of plants grown in ¼ Hoagland solution without NaCl was defined as 100%

39 WT A2 A3 Fig. 1 Arabidopsis genotypes. WT - wild type and AtCNGC10 antisense lines A2 and A3 grown in 1/4 Hoagland solution which was changed every two days, the pictures were taken at flowering.stage. Ion content Plants in flowering stage were harvested and weighed after 4 days of salt treatment (in preliminary experiments, the highest level of NaCl caused wilting in mutant plants on the 5 th day after salt application, making the fluorescence measurements impossible, in order to keep all the treatments identical for ion content measurement, plants under all the levels of salt are harvested on day 5). In contrast, seedlings were harvested after 7 days of salt exposure seedlings from three Petri dishes were pooled for each replicate, with three replicates for a treatment. Plant samples were dried in an oven at 70 o C for 72 h. Plant materials were digested with the mixture of 70% (v/v) HNO 3 and 70% (v/v) HClO 4 (3:1 v/v). For plants in the flowering stage, ion content was determined separately in roots and shoots, whereas for seedlings the whole plants were used. The contents of K, Na, Ca, and Mg were determined by a Perkin Elmer Analyst 300 Atomic Absorption Spectrometer (AAS). Ion concentration was calculated on a dry weight basis. Microelectrode Ion Flux Estimation (MIFE) measurements

40 MIFE theory The MIFE TM system was developed at the University of Tasmania by Newman (2001). Ions in solution are carried to or from the cell surface by diffusion or mass flow. If the ion is taken up by living cells, its concentration will be lower around the cell surface than further away. By the same token, if ion is extruded across the cytoplasmic membrane, its concentration will be higher close to the cell surface, and there will be an obvious concentration gradient away from the cell surface. The magnitude of this gradient is positively correlated to the rate of ion movement crossing the cytoplasmic membrane. Thus, the net fluxes (the resultant sum of influx and efflux) of the specific ion can be measured if an ion-selective micro-electrode measures ion concentration at two positions with variable distance to the cell surface. Therefore, the principle of the MIFE is the slow, square-wave movement of ion-selective microelectrode probes between two positions: close to and distant from the sample surface (Shabala et al., 1997). The recorded voltage gradients (dv) between these two positions are converted into concentration differences using the calibrated Nernst slopes of the electrodes. Different equations are used to calculate the net ion fluxes depending on diffusion geometry. In my study, cylindrical diffusion geometry typical of Arabidopsis root surface was used to calculate the net fluxes of ions. One of advantages of this technique is that up to four ions can be measured at the same time in a relatively small volume of solution. Another advantage is that net ion fluxes can be measured with high temporal and spatial resolution, thus providing useful information on ion flux kinetics (Babourina et al., 2000). Micro-electrode fabrication Micro-electrodes for the MIFE TM system were pulled (Sachs flaming micropipette puller PC-84, Sutter Instrument Co.) from 1.5 mm (external diameter) borosilicate glass capillaries (final micro-electrode diameter < 1 μm) (Babourina et al., 2001). Micro-electrodes were subsequently oven dried (220 o C) overnight and silanised with tributylchlorosilane (Fluka #90796) for 10 min. The micro-electrode tips were then broken back, increasing the diameter to approximately 2 μm whilst removing imperfections prior to back filling with solutions. Immediately after back-filling, the

41 electrode tips were front-filled with commercially available ionophore cocktails and calibrated against a range of standards. The specific details about the type of liquid ion exchanger (LIX), composition of back-filling solutions and range of standards used for this study are given in Table 2. Electrodes with slope less than 50 mv per decade for monovalent ions and 25 mv per decade for divalent, and with correlation coefficients less than were discarded. A reference electrode was prepared in a similar way from a glass microcapillary and filled with 100 mm KCl in 2% (w/v) agar. Table 2. Details of ion-selective microelectrodes used in experiments Ion Liquid Ion Exchanger Back-filling solution Calibration solutions (Fluka Catalogue No.) (mm) Hydrogen NaCl + 40 KH 2 PO (ph) Potassium KCl (mm) Sodium NaCl (mm) Calcium CaCl (µm) Magnesium MgCl (mm) Measurements of ion fluxes under salt stress Seedlings were placed in a 90-mm diameter Petri dish containing 30 ml of an unbuffered basal solution (0.01 mm CaCl 2, 0.1 mm KCl, ph 5.5). Electrodes were positioned at the meristematic zone (in root zoning experiments, we found, the fluxes of all the ions measued in our experiments have the most obvious differences in meristematic zone, so meristematic zone was chosen for the measuements of ion fluxes under salt stress). Ion fluxes were measured for 10 min prior to salt application (50 mm NaCl). Salt application occurred by pipetting the required volume of 1 M NaCl stock into the Petri dish. After addition, the bathing solution was thoroughly mixed by sucking and expelling it from a pipette ~5 times. The saline solution was allowed to

42 equilibrate for one minute prior to recording ion fluxes under saline conditions; hence, flux measurements during the first minute after salt stress application were discarded from the analysis and appear as gaps in the figures. Ion fluxes were subsequently measured for 30 minutes. Fluorescence lifetime imaging (FLIM) FLIM theory Fluorescence microscopy techniques have become a useful tool for research in biosciences because they can be used for measuring living cells under native, physiological conditions (Day, 2005; Lichtman and Conchello, 2005). Confocal and two-photon laser scanning techniques represent a great progress in fluorescence microscopy (White et al., 1987; Denk et al., 1990). In particular, multi-dimensional features such as the optical sectioning capability and the large penetration depth of two-photon excitation and the multi-wavelength capability (Dickinson et al., 2002) have led to a new quality of biological imaging. However, the fluorescence light emitted by organic molecules does not only depend on its emission intensity and emission spectrum, but it also has a specific lifetime. The fluorescence lifetime is an inherent characteristic of a chromophore, and thus is independent of chromophore concentration, photobleaching and excitation intensity. However, it depends on ph, ion concentrations, and the local environmental factors that affect the non-radiative rate of a chromophore (Lakowicz, 1999). Because the local environment determines the fluorescence lifetime, this fluorescence lifetime is used to calculate an image that is independent of chromophore concentration (Lakowicz et al., 1992). The fluorescence lifetime can be used to measure cell parameters such as ph, ion concentrations or oxygen saturation (Lakowicz and Szmacinski, 1993), aggregation effects (Kelbauskas and Dietel, 2002) and can be used for investigating protein or DNA structures by lifetime-sensitive dyes (Knemeyer et al., 2000). This makes fluorescence lifetime imaging (FLIM) a very useful and powerful tool for quantitative imaging at cellular levels. FLIM can also be used to discriminate the fluorescence components in measurements of tissue autofluorescence (Konig and Riemann, 2003). FLIM measurements

43 For dye loading, Fluorescent dyes 2,7 -bis-(2-carboxyethyl)-5-(and-6) carboxy -fluorescein acetoxymethyl ester (BCECF-AM) and Calcium Green TM (Ca-G-AM) (both from Molecular Probes, Eugene, OR, USA) were dissolved in dimethylsulphoxide (DMSO) (Sigma, Castle Hill, Australia) and diluted by loading solution (0.2 mm CaCl 2 and 50 mm mannitol, ph 4.2) to the final concentration of 20 μm. Final concentration of DMSO in loading solution was 1% v/v. After 2-h treatment in the loading solution on ice, plants were placed into the BSM solution for 30 min to recover. For two-photon-flim measurements, a seedling was placed in the chamber on the stage of an inverted confocal microscope (Leica TCS SP2 AOBS, Leica Microsystems GmbH, Wetzlar, Germany). Light pulses were generated at a frequency of 80 MHz by the Mai Tai Laser (Spectra Physics, Mountain View, CA, USA). Fluorescence was recorded by photo multipliers, and FLIM analysis was performed using electronics (SPC-730; Becker & Hickl, Berlin, Germany) and software (SPC7.22; Becker & Hickl) for time-correlated two-photon counting (O Connor and Desmond, 1994). Lifetime images were analysed using SPCImage 2.6 (Becker & Hickl). BCECF potassium salt dissolved in different buffers was used for calibration based on the median lifetime assuming a single exponential decay for BCECF (Fig. 2) (Nakabayashi et al., 2007). However, we found a shift (~0.45 ns) between in vitro and in vivo calibrations, similar to other studies where a shift of 0.4 ns was also found (Nakabayashi et al., 2007; Wang et al., 2007a). For in vivo calibrations we used carbonyl cyanide m-chlorophenylhydrazone (CCCP) as a protonophore to eliminate gradient between external and internal H + concentrations. The calibration curve was calculated after CCCP calibration (by subtracting the 0.45 ns shift)

44 τ m (ns) y = x R 2 = E ph BCECF ph BCECF λ Ex =940nm Fig. 2 The ph-dependence of mean lifetimes (τ m ) of the fluorescence intensity decay of 2,7 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfl uorescein (BCECF). The graph summarises the lifetime of the BCECF molecules loaded into the root and exposed to different buffers and treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (in vivo calibration which was used in calculations of intracellular ph) (E). The left upper panel (A) demonstrates the lifetime distribution of fluorescent molecules, which naturally reside in plant tissue, excited by 940 nm ( autofluorescence ). The mean lifetime of these molecules is around ns (panel B). Panel C displays the lifetime distribution of BCECF molecules loaded into an Arabidopsis seedling at the beginning of the root hair zone: the black arrowheads indicate nuclear zone of the cell; the white arrowhead is pointed at the area of the emerging root hair tip. The nuclear area is more alkaline (blue colour) and the cytoplasm close to the surface of the cell with the emerging root hair is more acidic (yellow/brown) (C). The mean lifetime of the BCECF molecules for the whole picture is 2.58 ns, as seen in the panel D

45 For FLIM calibration of cytosolic Ca 2+ activity ([Ca 2+ ] cyt ), stock solution of Ca-Green potassium salt was prepared in 0 mm Ca 2+ buffer (Calcium Calibration Kit #2, Molecular Probes, Eugene, OR, USA). Immediately after preparation, stock solution was diluted to 2 μm in different Ca 2+ buffers (free Ca 2+ concentration range from 0 to 1 mm). For calibration purposes, we intensified the first component assuming a double exponential decay for Ca-Green (Fig. 3) (Sanders et al., 1994)

46 A B lifetime ( ps ) a 1 B E τm τ Intenc ity coefficient of the fast component, a 1 (%) Ca 2+ concen tration (nm) C D 80 F y = x R 2 = α Ca 2+ co n cen tratio n (nm ) Fig. 3 Ca-Green calibration using fluorescence lifetime imaging (FLIM) and colour coding for different parameters calculated for a double exponential decay. The left upper panel demonstrates the distribution of Ca-Green molecules according to their median lifetime (A). Colour coding of the image is based on the median lifetime distribution as indicated in panel B: the peak of the faster lifetime component (a1, unbound Ca-Green molecules) is around 1.2 ns, the slower lifetime component is around 2450 ns. The yellow/brown colour indicates relatively low Ca 2+ concentration within cells, whereas the blue colour indicates relatively high Ca 2+ concentration. Panel C demonstrates the same image with intensity of the first (faster) component a1 used for colour coding. Because this parameter decreases when intracellular Ca 2+ concentration is higher (E, F), i.e. there are fewer free Ca-Green molecules, yellow/brown colour indicates higher Ca 2+ concentration and blue colour shows lower Ca 2+ concentrations. As seen in graph E, with more Ca-Green molecules bound to Ca 2+, the lifetime of the mean of the second (slower) component (τ 2 ) and, correspondingly, the mean lifetime (τm) increase. Although Ca-Green was calibrated over a broad concentration range (graph E), we used linear equations within a narrow concentration range (graph F)

47 Seedlings were attached to the cover glass polarized with the drop of poly-l-lysine solution (Sigma, Castle Hill, Australia) for about 30 min before rinsing with de-ionized water. To localize solution in a certain area, an Aqua-Hold Barrier Pap Pen was used to draw a line along the edges of the cover slip. Seedlings were scanned in BSM for 5 min before 50 mm NaCl application and immediately after NaCl application for 30 min. Each lifetime image of the plant was taken for 5 min. Emission wavelength was 940 nm (preliminary experiment demonstrated that this wavelength minimized contribution from autofluorescence). Intracellular [Ca 2+ ] cyt and ph were calculated according to calibration curves with Data Analysis Software for Fluorescence Lifetime Imaging Microscopy Systems (SPCImage Version 2.6, MP-FLIM and D-FLIM). Statistical analysis All growth experiments were repeated at least three times. Significant differences between means were assessed by MS Excel Software for the t-test and Genstat 8 th edition (VSN International Ltd, Hemel Hempstead, UK) for two-way analysis of variance (ANOVA)

48 CHAPTER 4 THE CYCLIC NUCLEOTIDE-GATED CHANNEL, AtCNGC10, INFLUENCES SALT TOLERANCE IN ARABIDOPSIS Kun Mei Guo 1, Olga Babourina 1, David A. Christopher 2, Tamas Borsics 2, Zed Rengel 1 1 School of Earth and Environment, University of Western Australia, Crawley WA 6009, Australia. 2 Department of Molecular Biosciences and Bioengineering, University of Hawaii 1955 East-West Road, Agsciences 218, Honolulu, HI 96822, USA. Abstract Cyclic nucleotide-gated channels (CNGCs) in the plasma membrane were suggested to transport K + and other cations, but their roles in the response and adaptation of plants to environmental salinity are unclear. Growth, cation contents, salt tolerance and K + fluxes were assessed in wild type and two AtCNGC10 antisense lines (A2 and A3) of Arabidopsis thaliana (L.) Heynh. Compared to the wild type, plants in the flowering stage of both antisense lines had higher K + and Na + concentrations in roots and shoots and were more sensitive to salt stress, as assessed by biomass and chlorophyll fluorescence. The shoots of A2 and A3 plants had higher Na + concentrations and significantly higher Na + /K + ratios compared to wild type, whereas roots had higher K + concentrations and lower Na + /K + ratios. Four-day-old seedlings of both antisense lines exposed to salt stress had lower Na + /K + ratios and longer roots than the wild type. Immediately after the application of salt stress, the Na + influx was smaller and the K + efflux was bigger in the antisense lines, indicating that AtCNGC10 might function as a channel providing Na + influx and K + efflux at the root/soil interface. We conclude that the AtCNGC10 channel is involved in Na + and K + transport including cation uptake in roots as well as long-distance transport, such as phloem loading and/or

49 xylem retrieval. A2 and A3 plants with impaired AtCNGC10 activity became more salt sensitive than wild-type plants due to impaired photosynthesis induced by a higher Na + concentration in the leaves. Key words: Arabidopsis thaliana, AtCNGC10, chlorophyll fluorescence, ion fluxes, K +, Na +, salt stress

50 4.1 Introduction Ion transport across cellular membranes plays a critical role in plant salt tolerance (Tester and Davenport, 2003). In order to survive salt stress, plants need to maintain low Na + and Cl - concentrations and a low Na + /K + ratio in the cytoplasm by regulating ion transport via the plasma membrane and endomembranes, including the tonoplast (Fox and Guerinot, 1998; Amtmann and Sanders, 1999). A family of cyclic nucleotide-gated channels (CNGCs) was identified in plants, with 20 members of this family in the Arabidopsis genome (Maser et al., 2001; Talke et al., 2003). Even though CNGCs exist in many species (Talke et al., 2003), their functions in plants are not clearly understood. The CNGC genes have been suggested to encode non-selective channels that regulate cation currents (Very and Sentenac, 2002; Demidchik et al., 2002b; Demidchik and Maathuis, 2007), and may be involved in ion uptake and translocation (Chan et al., 2003; Li et al., 2005). Moreover, they might provide a possible pathway for the entry of toxic ions such as Na + and heavy metals into plant cells (Sunkar et al., 2000; Gobert et al., 2006; Demidchik and Maathuis, 2007). Several CNGCs (AtCNGC1, AtCNGC3 and AtCNGC13) were up-regulated, whereas AtCNGC7 and AtCNGC16 were down-regulated during exposure to 150 mm NaCl (Maathuis et al., 2003), further suggesting a role for CNGCs during adaptation to salt in the environment. Functional analysis shows that heterologously expressed plant CNGCs act as cation transporters (Li et al., 2005; Gobert et al., 2006). In particular, AtCNGC1 and AtCNGC4 can transport cations such as Na + and K + (Leng et al., 2002; Balague et al., 2003). AtCNGC10 was shown to rescue potassium channel mutants of Escherichia coli (LB650) and yeast (CY162) and partially rescue Arabidopsis akt1 (Li et al., 2005), indicating its involvement in K + transport. Recently, heterologously expressed AtCNGC10 demonstrated substantial inward K + and outward cation currents (Christopher et al., 2007). Generally, it is difficult to predict whether a plant will become more salt tolerant or more salt sensitive if one of the CNGC channels is suppressed. CNGC suppression might limit Na + uptake into the plant, and thereby increase plant salt tolerance. Also, it

51 might alter K + transport, causing plants to become more salt tolerant if outward K + fluxes are suppressed, leading to higher K + concentrations in the tissue. Alternatively, plants may become more salt sensitive if inward K + fluxes decrease, which will lead to lower internal K + concentration. In addition, these channels might be involved in long distance cation transport (xylem/phloem uploading/retrieving), a process in which the effects on plant salt tolerance are unclear. AtCNGC10 is located in the plasma membrane in diverse types of plant cells, and is expressed at higher levels in roots relative to shoots (Christopher et al., 2007). Molecular genetic studies have shown that AtCNGC10 is an essential gene for plant development (Borsics et al., 2007). In this report, we tested the hypothesis that AtCNGC10 functions in plant salt tolerance. We tested this by examining the effects of salt treatments on various responses of the AtCNGC10 antisense knockdown lines. We found that root growth in the seedling stage was less inhibited in the antisense lines than WT at high external NaCl, whereas plants in the flowering stage were more salt sensitive compared with WT as assessed by biomass and chlorophyll fluorescence. We concluded that changes in ion transport in the root causing a lower Na + /K + ratio were essential for the higher salt tolerance of AtCNGC10 antisense seedlings compared with WT, whereas antisense plants in the flowering stage were more sensitive to salt than wild type plants due to higher Na + /K + ratios in the leaves, which, in turn, affected photosynthesis. 4.2 Materials and Methods Plant materials and growth conditions for mature and seedling plants, determination of ion content in tissue, MIFE theory, microelectrode fabrication and ion fluxes measurements, and statistical analysis were described in Chapter 3. Root length measurement Four-day-old seedlings from agar plates (1/4 Hoagland solution and 0.8% w/v agar) were transferred to fresh media supplemented with 50 mm NaCl. Seedlings were allowed to grow vertically for additional 7 days. During harvesting, plants were scanned

52 and weighed. The root length of seedlings per treatment was measured using the ImageJ 1.31v program (developed at the United States National Institutes of Health and available at Chlorophyll fluorescence The chlorophyll fluorescence parameter F v /F m was chosen because it is an established tool for detecting early stages of stress (Lichtenthaler, 1988; Bjoerkman and Demmig-Adams, 1994; Maxwell and Johnson, 2000). Chlorophyll fluorescence from the upper surface of the leaves was measured at 20 o C using a pulse-amplitude modulation portable fluorometer (Hansatech Instrument LTD, Norfolk, UK). All measurements were performed in the saturation pulse mode (Shabala et al., 1998). Dark adaptation of leaves for minimum (F o ) and maximum (F m ) fluorescence yields lasted 20 minutes. Three measurements were taken for each treatment. During the measurements, the leaf chamber was clamped over the central portion of the youngest fully expanded leaf of 25- to 30-day-old plants. The ratio of variable to maximum fluorescence was calculated as F v /F m = (F m -F o )/F m. Because chlorophyll fluorescence measurements can damage the leaves, these plants were not used for biomass and ion uptake assessments. 4.3 Results Salt stress in plants in flowering stage One of plant s quick responses to environmental stresses, including salt stress, is inhibition of photosynthesis. In our experiments, plants in the flowering stage (the onset of budding) were exposed to different levels of NaCl in solution. No significant difference was found for the F v /F m ratio between A2, A3 and WT plants in control conditions (Fig. 1). When plants were exposed to mild salt stress (<50 mm NaCl), the F v /F m values for WT, A2 and A3 plants were not changed during the first 6 days; however, the F v /F m of A2 exposed to 50 mm NaCl started to decrease 6 days after the onset of salt stress. At high concentrations of NaCl, the decrease in F v /F m for the A2 and A3 lines started earlier: after 4 days at 75 and 100 mm NaCl and after 2 days at 125 mm NaCl. At 100 mm NaCl, the F v /F m for A2 and A3 decreased after 4 days to

53 and 0.825, respectively (a 3% and 4% decrease compared with their respective controls, which was not significantly different). The F v /F m values for WT plants remained relatively constant during the whole experiment regardless of whether plants were exposed to 25, 50 or 75 mm NaCl mm NaCl mm NaCl 0.85 LSD 0.85 WT A2 A mm NaCl mm NaCl F v /F m mm NaCl mm NaCl Days under salt stress Fig. 1 The effect of salt stress on Fv/F m (maximal quantum yield) in leaves of wild type Arabidopsis plants (WT) and AtCNGC10 antisense lines, A2 and A3. The experiment was started when plants were in the flowering stage. Values are means ± SE (n = 9)

54 Exposure of A2, A3 and WT plants to NaCl (25-75 mm) for 4 days did not significantly affect biomass accumulation. Higher levels of salt stress (100 and 125 mm) resulted in insignificant changes in the biomass of WT plants in comparison to plants grown in control conditions. However, at 100 and 125 mm NaCl, A2 and A3 plants had significantly lower biomass than their own controls (Fig. 2). 120 WT A2 A3 FW (% of control) * ** ** *** NaCl (mm) Fig. 2 The effect of salt stress on the biomass (FW) of Arabidopsis wild type (WT) and AtCNGC10 antisense lines, A2 and A3. Treatments were applied at the flowering stage; plants were harvested on day 4 after the onset of treatment. Values are means ± SE (n = 9). (*, P 0.05, **, P 0.01, *** P 0.001, treated plants compared with their own controls as estimated by the paired t-test). In shoots, A2 and A3 had higher K + concentrations in the control treatment than WT, and this difference was not observed in the presence of increasing salinity concentrations. At the same time, Na + concentrations in the shoots were significantly higher in A2 and A3 than in WT plants at all salinity levels (Fig. 3)

55 Shoot Root 1.6 K + WT A2 A3 1.0 K LSD LSD Ion concentration (mmol g -1 DW) Na Na NaCl (mm) 0.0 NaCl (mm) Fig. 3 The effect of salt stress on ion content in shoots and roots of wild type Arabidopsis plants (WT) and AtCNGC10 antisense lines, A2 and A3, from one representative experiment. Similar patterns of ion accumulation were obtained in three independent experiments. Salt stress was applied at the flowering stage. Plants were harvested on day 4 after the onset of salt stress. Values are means (n = 9), LSD bars are shown. Changes in ion concentrations in the roots during exposure to salt stress were different from those observed in shoots. The K + concentrations in the roots of A2 and A3 plants were higher than in WT plants at all levels of salinity, although they gradually declined in response to an increase in external NaCl concentration. In contrast, no significant differences were found with regard to Na + concentrations in the roots between WT and the antisense lines (Fig. 3). These changes in Na + and K + concentrations in the shoots and roots of plants in the flowering stage in the five salinity treatments caused the

56 Na + /K + ratio in WT to be significantly lower in shoots and higher in roots (Fig. 4) (P 0.05 for 100 mm NaCl) compared to A2 and A3 plants A WT A2 A3 Na + /K + in shoots B Na + /K + in roots NaCl (mm) Fig. 4 Na + /K + ratio in shoots and roots of wild type Arabidopsis plants (WT) and AtCNGC10 antisense lines, A2 and A3, exposed to different levels of salt stress. Salt stress was applied at the flowering stage. Plants were harvested on day 4 after the onset of salt stress

57 Seedling response to salt stress treatment A reduction in root elongation is one of the first and quickest plant responses to salt stress (Munns, 2002). Seedlings of A2 and A3 lines performed better than WT plants under salt stress conditions: they had longer roots (P 0.05) (Fig. 5). The A2 and A3 seedlings had a higher initial K + concentration and lower Na + concentration, resulting in a significantly lower Na + /K + ratio before and after salinity treatments (P 0.01 and P 0.05, respectively). 150 Root length (% to control) WT A2 A3 control ** * * 50 mm NaCl * Fig. 5 Relative response of root growth in seedlings of wild type Arabidopsis plants (WT) and AtCNGC10 antisense lines, A2 and A3, exposed to 50 mm NaCl for 7 days. (*) indicates a significant difference at P 0.05 for treated plants compared with their own controls as estimated by the paired t-test (n = 30-50). Absolute values for the control treatment are 5.0 ± 0.1, 2.4 ± 0.1, 3.5 ± 0.2 cm for WT, A2, and A3 seedlings, respectively. Generally, the K + and Na + concentrations in A2 and A3 seedlings were similar to those found in the roots in the plant flowering stage. However, A2 and A3 seedlings had (i) significantly lower Na + concentrations (P 0.05), and (ii) higher K + concentration compared with WT under control and salt treatment conditions, with a decrease in the concentration of K + in all lines studied after 7 days of salt exposure (Fig. 6)

58 4.0 WT A2 A3 Na + (mmol g -1 DW) * * K + (mmol g -1 DW) * * Na + /K + ratio 4 ** ** 2 0 ** control ** 50 mm NaCl Fig. 6 Changes in Na +, K + and Na + /K + in seedlings of WT, A2 and A3 exposed to 50 mm NaCl for 7 days. Thirty to 50 seedlings from three Petri dishes were pooled for each replicate in a treatment. Values are means ± SE (n = 3). (*, P 0.05, **, P 0.01, paired t-test between WT and antisense lines, A2 or A3)

59 Ion fluxes during sudden NaCl exposure Ion fluxes were measured at the root meristem zone in response to salt stress. A large Na + influx was observed during the first few minutes after 50 mm NaCl addition (Fig. 7). This net Na + influx gradually reversed to efflux within the first 5 min in A2 and A3 seedlings. The reduced Na + influx in WT was observed to occur at a slower rate, reaching zero values 6-8 min after NaCl addition. However, Na + fluxes in WT did not demonstrate negative values, i.e. Na + efflux into the medium did not dominate over Na + influx into the root. Ten minutes after the onset of salt stress, Na + flux in the A2 and A3 lines was significantly shifted towards efflux compared with WT (Table 1) (paired t-test, P 0.05). This difference in Na + fluxes between the antisense lines and WT continued for the next 20 min. Immediately after application of 50 mm NaCl, a significant K + efflux from the root was observed in all Arabidopsis lines studied (Fig. 7). Within min, this initial K + efflux gradually decreased and stabilized, but at different levels for the different lines. Thirty minutes after application of NaCl, K + efflux from the roots of WT seedlings was significantly higher than in the A2 and A3 lines (Table 1)

60 Table 1. Na + and K + fluxes measured at the meristem zone of 4-day-old seedlings of wild type Arabidopsis (WT) and AtCNGC10 antisense lines A2 and A3 after application of 50 mm NaCl. Values are mean ±SE (n = 8) WT A2 A3 Na + fluxes (nmol m -2 s -1 ) before NaCl application 40 ± ± ± min after NaCl application 1376 ± ± 1218* ± 1309* 30 min after NaCl application -129 ± ± ± 850 K + fluxes (nmol m -2 s -1 ) before NaCl application -155 ± 67 3 ± 38-4 ± min after NaCl application -371 ± ± ± min after NaCl application -197 ± ± 29* 17 ± 36* Paired t-test, A2 and A3 compared with WT. *, P<

61 mm NaCl Na + WT A2 A3 Influx 0 Efflux Ion fluxes (nmol m -2 s -1 ) Influx K Efflux time (min) Fig. 7 Net Na + and K + fluxes (influx positive) measured near the root meristem zone of WT, A2 and A3 seedlings before and after addition of 50 mm NaCl. Similar patterns of ion fluxes were obtained in three independent experiments (only one representative experiment is shown). (Similar results were obtained for WT, A2 and A3 before NaCl addition, causing partial covering of the data points for WT). Timing of NaCl addition is indicated by the arrows. Summarized statistics for several time intervals are presented in Table

62 4.4 Discussion Differences in salt tolerance in seedlings and plants in flowering stage have been well documented. However, plants in flowering stage are usually more salt tolerant. In many halophytes, seed germination and seedling growth at earlier stages are similar to glycophytes (Weisel, 1973; Ungar et al., 1991). The salt tolerance of many halophytes is usually linked to better Na + translocation/sequestration to special vascular tissues or glands that plants develop later on in their growth cycle. Under the control conditions of our experiments, A2 and A3 lines had higher K + concentrations in seedlings as well as in the shoots and roots of plants in the flowering stage. This can be explained by lower activity of the channels that transport K + from the roots to the medium, i.e. the outward rectifying K + channel, such as NORC (Wegner and De Boer, 1997), is suppressed. This suggestion is consistent with the K + fluxes measured at the meristem zone of seedlings: there is a trend toward lower K + efflux in A2 and A3 seedlings compared to WT. This trend was also observed in another set of experiments designed to establish fluxes for different zones of the root (data not shown). The ability of AtCNGC10 channels to produce both outward and inward cation currents has recently been shown using heterologous expression in HEK293 cells (Christopher et al., 2007). Therefore, for plants grown under control conditions in the present study we suggest that K + efflux from plants with suppressed functioning of AtCNGC10 is inhibited: a higher K + concentration in both leaves and roots and smaller K + efflux from the roots was observed (Fig. 3, Table 1). Exposure of plants in the flowering stage to different levels of salt revealed that AtCNGC10 might have diverse functions in different plant organs. These channels may also be involved in long-distance transport such as K + loading into the phloem or retrieval from the xylem, in addition to K + uptake/release in the roots. This type of transport activity has been suggested for SOS1 and AtHKT1 transporters (Shi et al., 2002; Davenport et al., 2007) and for AKT2 subfamily of Shaker-type K + channels in higher plants (Gambale and Uozumi, 2006). If AtCNGC10 is permeable to both K + and Na +, a higher concentration of Na + in leaves under salt stress can be explained by

63 decreased Na + recycling in A2 and A3 plants in the flowering stage, where K + (Na + ) phloem loading or xylem retrieval in leaves is suppressed. It is known that after being taken up from the soil, loaded into the xylem, and transported to the leaves and other organs, up to 85% of K + is transported back to the phloem, and routed again into the xylem stream (Jeschke and Pate, 1991). Similarly, plants can re-distribute Na + accumulated during salt stress, and there is minimal discrimination against Na + during phloem loading (Pate and Jeschke, 1993). Therefore, it is feasible to suggest that plants employ non-selective pathways/transporters (e.g. AtCNGC10) that are used in unstressed conditions as well for ion transport when under environmental stress. The inability of A2 and A3 plants in the flowering stage to redistribute Na + within the plant leads to higher Na + /K + ratio in the leaves (Fig. 4). This is critical for maintaining a certain ionic environment in the leaves, and for functioning of the photosynthetic apparatus. Indeed, A2 and A3 plants had lower F v /F m (a major chlorophyll fluorescence parameter) under salt stress than WT (Fig. 1). The impaired photosynthesis coincided with lower biomass, which is generally one of several indicators of plant tolerance to an environmental factor. Therefore, we suggest that A2 and A3 plants in the flowering stage were more salt-sensitive than WT plants due to impaired photosynthesis, which was affected by high Na + /K + ratio in leaves. From our results, it can be concluded that A2 and A3 plants are more salt tolerant at the seedling stage because their root lengths in the presence of 50 mm NaCl are significantly longer compared to their non-salt controls (P 0.05), whereas WT seedlings had a significant reduction in root growth at the same level of salt stress (P 0.05) (Fig. 5). Compared with WT, the superior performance of A2 and A3 seedlings in the presence of high external salinity could be explained by the higher K + and lower Na + concentrations found in tissues for both control and salinity-treatment conditions, leading to a significantly lower Na + /K + ratios (P 0.01) (Fig. 6). Different concentrations of K + and Na + in A2, A3 and WT seedlings under control and salt stress condition are consistent with the flux results obtained before salt stress onset. The flux measurements demonstrate that suppression of AtCNGC10 channel activity in A2 and A3 plants led to a dual effect: a lower K + efflux and higher Na + efflux (Fig. 7, Table 1). The difference in ion fluxes between the antisense lines and WT was observed within 5-10 min, when A2 and A3 lines began to exclude Na + from the roots and WT seedling

64 were still taking up Na +, although at a much lower rate than during the first few minutes (Fig. 7). Given that we measured net Na + fluxes, it is difficult to ascertain whether Na + uptake or release transport systems were affected in A2 and A3 plants. However, we can assume a scenario in which reduced CNGC10 activity may decrease net Na + influx into the cell and hence cause smaller membrane depolarization. This in turn will cause smaller activation of the outward-rectifying depolarization-activated K + (GORK) channels; hence, smaller NaCl-induced K + efflux in A2/A3 lines was observed (Fig. 7, Table 1). Recent results regarding AtCNGC10 activity (Christopher et al., 2007) indicate that this channel belongs to the family of voltage-independent non-selective cation channels (VI-NSCC) as defined by Demidchik and Maathuis (2007). These channels are suggested to provide a main pathway for Na + entry into the plant cell, and this is the only function of these channels included in the diagram of a recent review (Demidchik and Maathuis, 2007). However, based on our current results, we would add K + efflux to the functions of this channel under both salt stress and control conditions. Future experiments should be designed to decipher the selectivity and pharmacology of this channel, as well as its tissue specificity and whether AtCNGC10 is involved in xylem loading and retrieval and/or phloem loading in Arabidopsis plants

65 CHAPTER 5 THE CYCLIC NUCLEOTIDE-GATED CHANNEL AtCNGC10 TRANSPORTS Ca 2+ AND Mg 2+ IN ARABIDOPSIS Kun Mei Guo 1, Olga Babourina 1, David A. Christopher 2, Tamas Borsics 2, Zed Rengel 1 1 School of Earth and Environment, University of Western Australia, Crawley WA 6009, Australia. 2 Department of Molecular Biosciences and Bioengineering, University of Hawaii 1955 East-West Road, Agsciences 218, Honolulu, HI 96822, USA. Abstract Suppression of cyclic nucleotide-gated channel AtCNGC10 alters K + uptake by seedlings and mature Arabidopsis plants. However, other cyclic nucleotide-gated channels (CNGCs) have been shown to transport Ca 2+, K +, Li +, Cs + and Rb + across the plasma membrane when expressed in heterologous systems, but there is no knowledge about their capacity to transport such ions in plants. Also, the ability of AtCNGC10 channel to transport nutrients other than K + in plants has not been tested so far. Growth and cation contents were assessed in Arabidopsis thaliana seedlings and plants (flowering stage) of the wild type (WT) and two AtCNGC10 antisense lines (A2 and A3) exposed to different concentrations of Ca 2+, Mg 2+ and K + in media. Exposing plants in flowering stage to various K +, Ca 2+ and Mg 2+ concentrations in the solution led to altered K +, Ca 2+ and Mg 2+ concentrations in shoots of antisense lines in comparison with WT, indicating disturbed long-distance ion transport of these cations, possibly due to changes in xylem loading/retrieval and/or phloem loading. Exposure to different levels of salt stress indicated that AtCNGC10 is involved in long-distance transport of Ca 2+ and Mg

66 Ion fluxes along different zones of the seedling roots, estimated by the non-invasive ion-specific microelectrode (MIFE) technique, were significantly different in antisense A2 and A3 lines in comparison to WT. Most notably, the influx of H +, Ca 2+ and Mg 2+ in the meristem of antisense A2 and A3 lines was significantly lower than that in WT. The lower Ca 2+ influx from the external media corresponded to the lower intracellular Ca 2+ activity ([Ca 2+ ] cyt ) estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, intracellular ph in the meristem zone of the roots of A2 and A3 seedlings was significantly lower (more acidic) than that of WT, which might indicate a feedback block of H + influx into meristematic cells caused by low intracellular ph. This study demonstrates that AtCNGC10 mutation affects H + and Ca 2+ fluxes at the root surface and long-distance transport of Ca 2+, Mg 2+ and K +. Key words: Arabidopsis thaliana, cyclic nucleotide-gated channels, CNGC10, H +, K +, Ca 2+, Mg 2+, ion fluxes, root zone, fluorescence lifetime imaging (FLIM), salinity

67 5.1 Introduction Arabidopsis genome contains 20 members of cyclic nucleotide-gated channels (CNGCs) family (Maser et al., 2001; Talke et al., 2003). Despite intensive work on heterologous expression and knockout studies, the function of these channels in plants is still unclear (Leng et al., 1999; Leng et al., 2002; Chan et al., 2003; Li et al., 2005; Gobert et al., 2006). Electrophysiologically characterised voltage-independent non-selective cation channels (VI-NSCC) in plants were suggested to represent CNGC channels (Very and Sentenac, 2002; Demidchik et al., 2002b); this suggestion was recently confirmed at least for AtCNGC10 (Christopher et al., 2007). In addition, some Arabidopsis CNGC mutants demonstrated altered cation content in plants, indicating involvement of CNGCs in ion uptake and translocation (Chan et al., 2003; Li et al., 2005; Guo et al., 2008). Moreover, CNGCs might provide a pathway for the entry of toxic ions, such as Pb 2+ (Sunkar et al., 2000; Gobert et al., 2006) and Na + into plant cells (Sunkar et al., 2000; Gobert et al., 2006). Although the channels from the Arabidopsis CNGC family have sequence similarity ranging between 55% and 83%, they have different ion selectivity. AtCNGC10 rescued potassium channel mutants of Escherichia coli (LB650) and yeast (CY162) and partially rescued Arabidopsis akt1 (Li et al., 2005), indicating its involvement in K + transport. For AtCNGC1 and AtCNGC4, electrophysiological studies have revealed that they can transport cations such as Na + and K + (Leng et al., 2002; Balague et al., 2003). AtCNGC3 forms a non-selective ion transporter of Na + and K + involved in seed germination (Gobert et al., 2006). In contrast, AtCNGC2 transports Ca 2+, K + and other monovalent cations (Li +, Cs + and Rb + ), but the influx of Na + is low (Leng et al., 1999; Leng et al., 2002). Because CNGCs can transport Ca 2+, it has been proposed that they could be involved in Ca 2+ signalling pathways (Talke et al., 2003). Heterologous expression of CNGC18 in E. coli demonstrated time- and concentration-dependent accumulation of Ca 2+ (Frietsch et al., 2007). Complementation analysis using a Ca 2+ -uptake-deficient yeast mutant demonstrated that the AtCNGC11/12 channel is permeable to Ca 2+ (Urquhart et al., 2007)

68 The variation in cation transport among members of the highly conserved CNGC family has caused different phenotypes in CNGC mutants. The AtCNGC1 knockout has improved tolerance to Pb 2+ (Sunkar et al., 2000) and lower Ca 2+ concentration in its tissues (Ma et al., 2006). AtCNGC3 mutants are sensitive to Na + toxicity during germination and have altered monovalent cation content (Gobert et al., 2006). Knockout AtCNGC2 plants are hypersensitive to Ca 2+, but not to Na + or K + (Chan et al., 2003). Analysis of AtCNGC2 mutants indicates the function of this channel in Ca 2+ -mediated plant development as well as in disease resistance and programmed cell death (Clough et al., 2000). Likewise, AtCNGC4 mutants have altered responses to hypersensitive pathogen resistance signalling (Balague et al., 2003) as do AtCNGC11 and AtCNCG12 (Yoshioka et al., 2006). In our previous study we demonstrated altered K + and Na + uptake by AtCNGC10 mutants (Guo et al., 2008) and varied salt tolerance at different developmental stages, in order to elucidate the relationship between the functions of the AtCNGC10 channel and cation uptake, we tested the ablility of the AtCNGC10 channel to transporter Ca 2+, Mg 2+ and K + to the roots and shoots and changes of ion fluxes and concentrations in shoots and roots under salt stress. In this study, we used AtCNGC10 antisense knockdown lines to non-invasively measure ion fluxes along the roots, intracellular concentrations of Ca 2+ and H + and ion content changes in shoot and root under salt stress. 5.2 Materials and Methods Plant material and growth conditions Plant materials and growth conditions for seedlings and mature plants, determination of ion content in plant tissues, MIFE theory, microelectrode fabrication, ion flux measurements, and statistical analysis were described in Chapter 3. Treatments with low K or high Ca 2+ or Mg 2+ Low K or high Ca 2+ or Mg 2+ treatment was applied at the same developmental stage (the start of budding, which is easy to identify). Instead of having the K +, Ca 2+ and Mg 2+ ion concentrations in the control (1/4 Hoagland solution), There was 0.05 mm K + in the

69 low-k treatment, 5 mm Ca 2+ in the high-ca treatment and 5 mm Mg 2+ in the high-mg treatment. Ion fluxes measurement in different root zone The seedling root length and diameter were measured in a Petri dish placed on an inverted microscope. The seedling was fastened to a glass slide by a Parafilm M strip. The bathing solution contained (in mm): KCl 1, CaCl and MgCl 2 1 at ph 5.5. Measurements of ion fluxes commenced 30 min after placing a seedling into a bathing solution at the distance of μm from the root surface. To cope with the oscillation pattern of ion fluxes in a given zone, we measured fluxes over the same portion of each root twice, going back and forth, and the final value of ion fluxes for each root zone was averaged from these two-way measurements on different plants. Experiments were performed at ºC under standard laboratory lighting (the light intensity close to plant was 20 μmol m -2 s -1 ). Fluxes were measured using three ion-specific electrodes simultaneously in different combinations, but keeping the H + electrode in all measurements. 5.3 Results H +, Ca 2+, Mg 2+ and K + fluxes along different root zones Fluxes of H +, Ca 2+, Mg 2+ and K + were measured at three positions (the meristem, elongation and mature zones) along the seedling roots of WT and antisense AtCNGC10 lines A2 and A3 (Fig. 1). In all genotypes studied, the H +, Ca 2+ and Mg 2+ fluxes demonstrated the highest positive values (influx) in the meristematic zone compared with other root zones. Wheares K + fluxes in the meristematic zone are lowest in all genotypes compared with other root zones. However, A2 and A3 lines had significantly lower H +, Ca 2+ and Mg 2+ influx in meristematic zone and elongation zone than WT (P 0.05), and there was no significant difference in H +, Ca 2+ and Mg 2+ fluxes in mature zone between WT and A2, A3. There was a smaller (statistically non-significant) K + efflux in the meristematic zone of A2 and A3 roots compared with WT, there was no significant difference in K + fluxes in three zones between WT and A2, A

70 H + WT A2 100 ** ** A3 0 * * Ca Ion fluxes (nmol m -2 s -1 ) ** ** Mg 2+ * * * * ** ** K MERISTEM ZONE ELONGATION ZONE MATURE ZO NE Fig. 1 Ion flux distribution along the root axis of 4-day-old seedlings of Arabidopsis wild type (WT) and AtCNGC10 antisense lines A2 and A3. Paired t-test, A2 or A3 compared with WT. *, P 0.05; **, P<

71 Intracellular Ca 2+ and ph Intracellular ph of the root meristematic cells was more acidic in A2 and A3 seedlings than WT (P 0.05). No significant difference among genotypes was found in elongation and mature zones (Fig. 2). The root meristematic cells of A2 and A3 seedlings had lower [Ca 2+ ] cyt than it was found for WT, whereas the root elongation and mature zones of A2 and A3 lines were not different from WT (Fig. 3)

72 WT 8 a Meristematic zone 7 b b ph A2 ph 6.9 Intracellular ph a Elongation zone a a 5 A3 8 7 Mature zone a a a ph WT A2 A3 Fig. 2 Intracellular ph in different root zones of 4-day-old seedlings of Arabidopsis wild type (WT) and AtCNGC10 antisense lines A2 and A3 assessed by the fluorescence life-time (FLIM) analysis (right panes). Values are means ± SE (n=5). Different lower case letters indicate significant difference at P The left and central panes demonstrate ph distribution in representative roots of WT, A2 and A3 seedlings: colour distribution in microphotographs in the left panes is based on 2,7 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF) lifetime distribution which is measured in meristematic zone (framed), calculated and presented in the central panes. The mean ph is more acidic in the root meristematic zones of A2 and A3 than WT (central panes)

73 WT a Meris tematic zone 120 b b A2 [Ca 2+ ] intradellular (nm) a Elongation zone a a 0 A a Mature zone a a WT A2 A3 Fig. 3 Intracellular Ca 2+ concentration in different root zones of 4-day-old seedlings of Arabidopsis wild type (WT) and AtCNGC10 antisense lines A2 and A3 assessed by the fluorescence life-time (FLIM) analysis (right panes). Values are means ± SE (n=5-7). Different lower case letters indicate significant difference at P The left panes show representative roots of the three lines with the selected regions of interest (framed)