New Molecular Approaches to Improving Salt Tolerance in Crop Plants

Annals of Botany 82 : 703–710, 1998 Article No. bo980731

BOTANICAL BRIEFING

New Molecular Approaches to Improving Salt Tolerance in Crop Plants I. W I N I C O V* Departments of Microbiology and Biochemistry, UniŠersity of NeŠada Reno, Reno, NV 89557, USA Received : 5 May 1998

Returned for revision : 6 June 1998

Accepted : 21 July 1998

The last century has seen enormous gains in plant productivity and in resistance to a variety of pests and diseases through much innovative plant breeding and more recently molecular engineering to prevent plant damage by insects. In contrast, improvements to salt and drought tolerance in crop and ornamental plants has been elusive, partially because they are quantitative traits and part of the multigenic responses detectable under salt\drought stress conditions. However, the rapidly expanding base of information on molecular strategies in plant adaptation to stress is likely to improve experimental strategies to achieve improved tolerance. Recently studies of salinity tolerance in crop plants have ranged from genetic mapping to molecular characterization of salt\drought induced gene products. With our increasing understanding of biochemical pathways and mechanisms that participate in plant stress responses it has also become apparent that many of these responses are common protective mechanisms that can be activated by salt, drought and cold, albeit sometimes through different signalling pathways. This review focuses on recent progress in molecular engineering to improve salt tolerance in plants in context of our current knowledge of metabolic changes elicited by salt\drought stress and the known plant characteristics useful for salt tolerance. While it is instructive to draw parallels between molecular mechanisms responsive to salt-stress with accumulating evidence from studies of related abiotic stress-responses, more data are needed to delineate those mechanisms specific for salt tolerance. Also discussed is the alternative genetic strategy that combines single-step selection of salt tolerant cells in culture, followed by regeneration of salt tolerant plants and identification of genes important in the acquired salt tolerance. Currently, transgenic plants have been used to test the effect of overexpression of specific prokaryotic or plant genes, known to be up-regulated by salt\drought stress. The incremental success of these experiments indicates a potentially useful role for these stress-induced genes in achieving long term tolerance. In addition, it is possible that enhanced expression of gene products that function in physiological systems especially sensitive to disruption by salt, could incrementally improve salt tolerance. Current knowledge points towards a need to reconcile our findings that many genes are induced by stress with the practical limitations of overexpressing all of them in a plant in a tissue specific manner that would maintain developmental control as needed. New approaches are being developed towards being able to manipulate expression of functionally related classes of genes by characterization of signalling pathways in salt\drought stress and characterization and cloning of transcription factors that regulate the expression of many genes that could contribute to salt\drought tolerance. Transcription factors that regulate functionally related genes could be particularly attractive targets for such investigations, since they may also function in regulating quantitative traits. Transgenic manipulation of such transcription factors should help us understand more about multigene regulation and its relationship to tolerance. # 1998 Annals of Botany Company Key words : Salt tolerance, salt stress, molecular engineering, cellular selection, transgenic plants, gene regulation, transcription factors.

INTRODUCTION Improving salinity and drought tolerance of crop plants by genetic means has been an important but largely unfulfilled aim of modern agricultural development. As more land becomes salinized through poor local irrigation practice, the regional impact of salinity on crop production is becoming increasingly important world-wide (Tanji, 1990 ; Flowers and Yeo, 1995) creating a pressing need for improved salt tolerant plants. At the genetic level, salinity tolerance has been considered to be a quantitative trait (Foolad and Jones, 1993) and has been generally resistant to improvements by plant breeding. Since quantitative traits influence maximal plant yield and productivity, introducing a trait that improves tolerance to saline growth conditions may * Fax (702) 784-1620, e-mail winicov!unr.edu

0305-7364\98\120703j08 $30.00\0

actually lower the potential yield under normal conditions. Thus, the need to balance productivity with salinity\drought tolerance has become a contested point of discussion that is likely to be resolved in favour of ‘ relative ’ yield only in areas of limited arable land. However, rapid progress in understanding biochemical mechanisms that may participate in plant stress responses and salt tolerance, as well as the molecular cloning of genes involved in the various metabolic pathways that respond to salt stress, offer new approaches to solving this persistent problem (Bohnert and Jensen, 1996 ; Winicov and Bastola, 1997). Accordingly, this review will focus on recent progress in molecular engineering and cellular selection to improve salt-tolerance in plants. The rapid expansion of our knowledge of the diverse genes that are induced and repressed by dehydration (Ingram and Bartels, 1996 ; Bray, 1997 ; Shinozaki and YamaguchiShinozaki, 1997) includes the concept that most of these # 1998 Annals of Botany Company

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WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants T     1. Examples of differential actiŠation of NaCl inducible genes by dehydration, cold and ABA Function*

Gene Alfin1 ARSK1 ATCDPK1 ATCDPK2 Atmyb2 AtP5CS AtPLC1 cor6.6 kin1 mlip15 MsPRP2 OsBZ8 PKABA1 rd22 rd29A (COR78) rd29B

Alfalfa Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Maize Alfalfa Rice Wheat Arabidopsis Arabidopsis Arabidopsis

DNA-binding Prot. kinase Prot. kinase Prot. kinase DNA-binding Proline biosyn. Phospholipase C Antifreeze prot. Antifreeze prot. DNA-binding Cell wall prot. DNA-binding Protein kinase Seed protein

NaCl

Dehdr.

Cold

j j j j j j j j j j j j j j j p

nd j j j j j j j j k nd j j j j p

nd j k k k j j j j j nd p j nd j k

ABA Reference k j k k j j j j j j k j j j p j

Bastola et al., 1998 a Hwang and Goodman, 1995 Urao et al., 1994 Ibid Urao et al., 1993 Yoshiba et al., 1995 Hirayama et al., 1995 Wang et al., 1995 Ibid Kussano et al., 1995 Deutsch and Winicov, 1995 Nakagawa, Ohmiya and Hattori, 1996 Holappa and Walker-Simmons, 1995 Iwasaki, Yamaguchi-Shinozaki and Shinozaki, 1995 Yamaguchi-Shinozaki and Shinozaki, 1994 Ibid

nd, Not determined. p, Weak or delayed response. * Function shown or implied by sequence similarity with proteins of known function.

genes also respond to salt stress. In addition, it is becoming increasingly apparent that among the genes that respond to salinity stress and drought, some, but not all, respond to cold stress (Shinozaki and Yamaguchi-Shinozaki, 1996). Table 1 gives selected examples of a variety of genes that are induced by salt stress, but differ in their responses to dehydration, cold and the plant hormone abscisic acid (ABA), which accumulates under these same abiotic stress conditions. The commonality of responses may indicate similar functions of these gene products in detoxification and cellular maintenance for plants under stress conditions involving water deficit. They also suggest interacting signal perception and transduction pathways. In contrast, genes that are induced in a stress specific manner emphasize the likely existence of several signalling pathways and increase the complexity of adjustments that may need to be made for engineering tolerance traits in plants. At present, we do not know to what extent acute stress responses can best be utilized at the molecular level to achieve significant long-term salt tolerance, nor which responses hold the key to tolerance. Adaptive mechanisms leading to increased long-term salt tolerance may utilize either the gene products accumulated under short-term salt stress or use other means for increased resilience. It is possible that improved salt tolerance may be achieved by the maintenance, activation or enhanced function of physiological systems that are especially sensitive to disruption by increased levels of salt. Overexpression or activation of the limiting components of such systems would overcome the system failure under salt stress. A number of such physiological systems could contribute individually to a specific aspect of salt tolerance and so provide both incremental and additive improvements in salt tolerance (Winicov, 1994). Recently, it has been reported that overexpression of the cold regulated gene (COR) Binding Factor 1 (CBF1), which normally regulates COR genes turned on by the cold acclimation response, leads to

enhanced freezing tolerance (Jaglo-Ottosen et al., 1998). This example indicates that some adaptive responses can provide increased tolerance by activation of a subset of genes. Other data on temporal analysis of gene activation in both wheat and barley have shown that while coordinated induction of genes responsive to salt occurs within 2 h, many transcripts decline in abundance within 24 h and others disappear after about 6 d (Robinson, Tanaka and Hurkman, 1990 ; Gulick and Dvor) a! k, 1992). These results emphasize the transient nature of the early response to salt stress, but do not address the potential of their use long term. Our comparisons of salt inducible polypeptides and mRNAs between salt-tolerant and salt-sensitive cells within the same alfalfa genotype demonstrated different salt inducible classes of genes for tolerant and sensitive cell lines (Winicov et al., 1989). Since the tolerant cells were selected at mutational frequencies from the salt-sensitive cells, these results support the concept that different short-term and long-term responses each contribute to salt tolerance. As we become increasingly able to evaluate the results from experimentally constructed transgenic plants harbouring different trans-genes, the efficacy of these temporal responses will become clarified in terms of long-term salt tolerance. SALT TOLERANCE—THE GENERAL AIM Optimistic discussions of future salt tolerance in plants have sometimes invoked the unrealistic vision of crop plants growing as halophytes on severely salt affected land or being irrigated with sea water. This scenario appears unlikely, since centuries of plant breeding have developed desirable agronomic traits for most crop and ornamental plants. Also, there are major morphological and physiological differences between halophytes and most cultivated plants. For the purposes of this discussion, the qualitative term of ‘ improved salt tolerance ’ will imply continued survival and productive growth of a salt-tolerant plant under conditions

WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants where a similar salt-sensitive genotype shows severely inhibited growth or dies. The varietal utilization of genetic information has long been recognized as a potential source of beneficial traits for salt tolerance. Native varietal halotolerance was exploited to characterize differences between salt-sensitive and salttolerant barley varieties (Hurkman, Fornari and Tanaka, 1989). Examples from other plant species that show varietal differences in salt tolerance are tomato (Tal, Heikin and Dehan, 1978), rice (Flowers and Yeo, 1981) and alfalfa (Smith and McComb, 1981), demonstrating that the genetic repertoire within each species can provide enhanced salt tolerance characteristics for improving general salt tolerance of plants. The potential for augmenting this repertoire with exogenous genetic information lacking in salt-sensitive plants, or enhancing the utilization of endogenous genes remains intriguing and is currently being explored in a number of laboratories. An alternate strategy for salt tolerance improvement adopted by our laboratory is to utilize the regeneration of alfalfa and rice with heritable improved salt tolerance after selection of salt tolerant cells in culture (Winicov, 1991, 1996). The cell culture selection and regeneration protocol has been undertaken in a number of other laboratories with limited success. Many of the selected variants cannot be evaluated, since we lack information about the heritability of the trait. For others, the tolerance trait has been epigenetic in nature and has yielded many albino plants as in the case of rice (KrishnaRaj and SreeRangasamy, 1993) or produced dwarf plants with limited fertility as shown in rice (Yano, Ogawa and Yamada, 1982) or no fertility as in alfalfa (McCoy, 1997). It is likely, however, that the early problems encountered with this method were due to prolonged selection on NaCl in culture and lack of secondary screening of an adequate number of the regenerated plants (Winicov, 1996). When successful, the cellular selection and regeneration approach relies on identification of mutants optimized for continued survival and productive growth under saline conditions, but does not provide ready identification of the affected genes without further study of changes in regulation of the endogenous genetic information (Winicov and Krishnan, 1996 ; Winicov and Bastola, 1997). However, since the tolerant cells\plants continue productive growth under saline conditions for months, we assume that the changes in gene regulation are associated with the ability to survive otherwise lethal conditions.

CHARACTERISTICS IMPORTANT IN SALT TOLERANCE Salt tolerance of plants depends primarily on characteristics that can be broadly grouped in three categories : (1) physical uptake or exclusion of salt followed by transport and compartmentation of salt ; (2) morphological features and biomass distribution of plant shoots and roots, which would include rates of transpiration and stomatal closure ; (3) physiological and metabolic events that counteract the presence of salt at the cellular level. These characteristics

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could be the primary targets for manipulation in engineering of salt\drought tolerance. Plant morphology and salt transport in the xylem depend on a complex pattern of developmental regulation and have so far received little attention with current molecular techniques. Similarly, guard cell responses to environmental stimuli have been documented (Kearns and Assmann, 1993), but currently these responses cannot be manipulated in a heritable fashion. General inhibition of shoot growth with continued root growth has been considered as a morphological adaptation to salt stress or water stress (Creelman et al., 1990 ; Saab et al., 1990). While enhanced root development could be beneficial in salt\drought tolerance as indicated from studies on adaptation, molecular techniques for effectively manipulating root mass have not been developed (Aeschbacher, Schiefelbein and Benfey, 1994). Ion uptake and transfer across membranes has been investigated as an integral metabolic change in salt stress and adaptation (Niu et al., 1995). Membrane components of ion pumps could thus be encoded by a group of genes which, when activated, would counteract acute salt stress. This mechanism is suggested by increased Na+ tolerance in yeast with a mutated transmembrane domain of the high affinity K+-transporter, HKT1 (Rubio, Gassman and Schroeder, 1996). However, much more needs to be understood about the role different members of these multigene families play in tissue specific function. Most progress to date has been made in understanding biochemical mechanisms in physiological or metabolic adaptation to salt\drought stress at the cellular level as a means of providing potential candidate genes for engineering improved tolerance.

ENGINEERING PHYSIOLOGIC OR METABOLIC ADAPTATION Physiologic or metabolic adaptations to salt stress at the cellular level are the main responses amenable to molecular analysis and have led to the identification of a large number of genes induced by salt (Ingram and Bartels, 1996 ; Bray, 1997 ; Shinozaki and Yamaguchi-Shinozaki, 1997). These genes can be classified in groups related to their physiologic or metabolic function predicted from sequence homology with known proteins and are summarized in Table 2. Most of the genes in the functional groups have been identified as salt inducible under stress conditions. Other genes have

T     2. Functional groups of genes\proteins actiŠated in salt stress with potential for proŠiding tolerance 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Carbon metabolism and energy production\photosynthesis Cell wall\membrane structural components Osmoprotectants and molecular chaperons Water channel proteins Ion transport Oxidative stress defences Detoxifying enzymes Proteinases Proteins involved in signalling Transcription factors

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WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants

been detected by a salt-hypersensitivity assay in Arabidopsis, which led to the identification of mutants in potassium uptake as being critical in salt sensitivity through the manifestation of increased salt sensitivity (Wu, Ding and Zhu, 1996). However, other physiological systems may be equally limiting under stress conditions and mutants in these physiological pathways could lead to increased salt toxicity and would affect survival in a negative manner. Our approach has been to clone genes that are differentially induced by salt in salt-tolerant alfalfa cells (Winicov and Bastola, 1997) and are also regulated by salt at the whole plant level (Winicov, 1993 ; Winicov and Deutch, 1994 ; Deutch and Winicov, 1995 ; Winicov and Shirzadegan, 1997) with the aim of testing their relevance to the improved salt tolerance of the selected plants. For example, one of these genes Alfin1 encodes a transcription factor that has been found to induce enhanced expression of the saltinducible MsPRP2 gene in callus and in plants (Bastola, Pethe and Winicov, 1998 b). It will be interesting to determine if Alfin1 can also influence the expression of other salt inducible genes and if it functions in concert with other regulatory factors under salt stress conditions. SINGLE GENE TRANSFER TO IMPROVE SALT TOLERANCE While salt induced gene activation has been demonstrated for genes belonging to all the functional groups listed in Table 2, only a limited number of genes have been tested in transgenic plants for their effect on stress tolerance. Primarily these genes encode enzymes involved in osmoprotectant synthesis, molecular chaperons and detoxifying enzymes involved in oxidative stress responses. Increased osmoprotectant synthesis has been manipulated in plants by overexpression of enzymes leading to increased mannitol synthesis in tobacco (Tarczynski, Jensen and Bohnert, 1993) and Arabidopsis (Thomas et al., 1995), ononitol production in tobacco (Sheveleva et al., 1997), fructan synthesis in tobacco (Pilon-Smits et al., 1995), trehalose synthesis in tobacco (Holmstrom et al., 1996) and the amino acid proline in tobacco (Kishor et al., 1995). In each case incremental improvements in salt\drought tolerance were measured under laboratory conditions that correlated with increased constitutive accumulation of the manipulated solutes. The applicability of this approach for all plant species has been questioned (Flowers et al., 1997). In addition, the results with transgenics accumulating mannitol and proline provided some unexpected information. While transgenic plants accumulating proline demonstrated that degradative as well as synthetic pathways may need to be manipulated if constitutively higher than normal proline levels were to be attained, elevated basal levels of proline provided immediate protection against stress. These results are consistent with observations in maize, where the osmoprotectant glycinebetaine accumulation has been shown to correlate with Bet1 gene copy number and improved salt tolerance (Saneoka et al., 1995) and with our findings that salt-tolerant alfalfa plants rapidly double their proline concentration in the roots, while salt-sensitive plants had a delayed response (Petrusa and Winicov, 1997). In the case of mannitol, it has

been proposed that this solute may provide protection by reducing oxidative damage in the chloroplast (Shen, Jensen and Bohnert, 1997) since cytoplasmic mannitol concentrations were considered to be too low to provide sufficient osmotic adjustment. Late embryogenesis abundant (LEA) proteins are thought to play a role in dessication tolerance in seed development and in response to dehydration, salinity and cold stress (Close, 1997). Rice plants transformed with the barley LEA gene, HVA1, have shown increased tolerance to water deficit and salt stress (Xu et al., 1996) while improved salt and freezing tolerance has been observed in yeast transformed with a tomato LEA-class gene (Imai et al., 1996). At present these proteins are thought to preserve the structural integrity of the cell, but the extent of their utility in improving salt tolerance needs to be further explored. Oxidative stress has been considered to be a common component of both biotic and abiotic stress in plants. Many of the genes for antioxidant enzymes have been cloned and expressed in transgenic plants to test their role in plant protection against oxidative and other stresses (Foyer, Descourvieres and Kunert, 1994 ; Allen, 1995). Transgenic alfalfa overexpressing superoxide dismutase has been reported to show reduced injury from water deficit stress along with field performance (McKersie et al., 1996) and oxidative stress tolerance was shown in Fe-superoxide dismutase overproducing chloroplasts (Van Camp et al., 1996). Increased tolerance to oxidative stress has also been reported in tobacco plants that overexpress cytosolic ascorbate peroxidase (APX) but not those that express a chloroplast targeted isoform (Torsethaugen et al., 1997). Overexpression of glutathione S-transferase\glutathione peroxidase provided some protection against cold and salt stress in tobacco (Roxas et al., 1997). The complexity of the interacting pathways, cellular compartmentalization and differential responses of antioxidant gene expression (Conklin and Last, 1995) make it somewhat difficult to compare results obtained from different systems at the present time and will require further elucidation of the extent of tolerance that can be obtained with single gene transfer. In addition, most expression of the transferred genes has been ubiquitous under the direction of the CAMV 35 S promoter, which may overlook the necessary tissue specificity for regulation in order to achieve optimal protection.

MECHANISMS OF COORDINATE REGULATION OF STRESS RELATED GENES Signalling pathways Multigenic cellular adaptation to increases in the ionic environment implies integrated changes in regulation of gene expression for groups of functionally related genes. The phenomenon of specific and coordinate mRNA accumulation in response to salt and water stress has been documented in a variety of plants (Bohnert, Nelson and Jensen, 1995 ; Ingram and Bartels, 1996 ; Bray, 1997) indicating that creating transgenic plants overexpressing a

WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants gene encoding a single function may not be sufficient to lead to optimal adaptation to saline environments. The signalling pathways and molecular mechanisms responsible for this coordinate transcript accumulation have been reviewed recently (Bray, 1997 ; Shinozaki and Yamaguchi-Shinozaki, 1997). Although an increasing number of kinases and phosphatases have been identified that respond to both salt and drought stress (see Table 1), the pathways themselves have remained generally unresolved. Since the level of the plant hormone abscisic acid (ABA) increases with salt, drought and cold stress, it has been postulated to play a central role in signalling for these stress responses besides playing an important role in seed production. Exogenous ABA can activate transcription of many of the genes induced by salt\drought stress, while other salt\drought inducible genes are not activated by ABA, suggesting both ABA-dependent and ABA-independent signalling pathways (Bray, 1997 ; Shinozaki and Yamaguchi-Shinozaki, 1997). Signal perception and transduction pathways certainly make attractive targets for signal manipulation to coordinate gene regulation, but it is currently difficult to design definitive experiments in this area because of the cascade characteristics of signalling systems and the likely multiplicity of intersecting signalling pathways (Ishitani et al., 1997).

Transcriptional regulators Another attractive target category for manipulation and coordinate gene regulation is the small group of transcription factors that have been identified to bind to promoter regulatory elements in genes regulated by salt\ drought stress Shinozaki and Yamaguchi-Shinozaki, 1997 ; Winicov and Bastola, 1997). As recently shown for the cold acclimation response (Jaglo-Ottoson et al., 1998), overexpression of the transcription factor CBF1 that regulates numerous COR genes was able to enhance demonstrable freezing tolerance in Arabidopsis, while previous overexpression of just one of the COR genes gave much more subtle results. This suggests that transcriptional activation of salt\drought induced genes might be possible in transgenic plants overexpressing one or more transcription factors that recognize promoter regulatory elements of these genes. Many of the salt\drought stress regulated genes may depend on a combinatorial activation by several transcription factors as suggested by the requirement of a coupling element for stress regulation of the barley HVA22 gene containing the ABRE (PyACGTGGC) element (Shen et al., 1996). It will be a challenge to identify those factors that may be limiting in the overall response and to manipulate their expression in a tissue targeted manner. Information to date on transcriptional regulation in response to salt\drought stress is relatively gene specific. Much of the current information focuses on cis-acting elements of the genome involved in ABA induced gene expression and the trans-acting bZIP proteins that recognize these elements (Bray, 1997 ; Shinozaki and YamaguchiShinozaki, 1997). The broader function of ABRE elements in plant gene regulation has been recently investigated by overexpression in tobacco of the EmBP-1 gene (ABRE

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binding protein from wheat embryo) and its truncated form containing the DNA binding and dimerization domains. While overexpression of EmBP did not alter the plant response to water stress, the truncated form acted as a dominant negative inhibitor, revealing an important developmental function of this protein in vegetative tissues (Eckardt, McHenry and Guiltinan, 1998). ABA-mediated inducible expression of genes that are also induced by salt and drought stress has been recently linked to several myband myc-related transcriptional activators, two classes of transcription factors associated with cellular proliferation. Atmyb2 from Arabidopsis (Urao et al., 1993) and cpm10 and cpm7, which have been cloned from Cereterostigma (Iturriaga et al., 1996), encode MYB type transcription factors and are induced by dehydration stress. The rd22BP1 gene from Arabidopsis has been shown to encode a myc type transcription factor (Abe et al., 1997) expressed in seeds, but not induced under stress conditions. Both myc- and mybtranscriptional activators belong to multigene families and the myc (CANNTG) and myb (PyAACPyN) recognition sites can be found in many salt\drought stress activated gene promoters, including rd22 (Abe et al., 1997) and the alfalfa MsPRP2 promoter (Bastola, Pethe and Winicov 1998 a). Recent transient transactivation experiments with Arabidopsis leaf protoplasts have shown that the Arabidopsis MYC (rd22BP1 ) and MYB (ATMYB2 ) proteins function as transcriptional activators in ABA and dehydration inducible expression using a 67 bp region of the rd22 gene promoter containing the myc and myb DNA recognition elements (Abe et al., 1997). These results further show the multiplicity of factors likely to be involved in salt\drought stress regulation. The ABA-independent salt\drought inducible DRE element (TACCGACAT) was initially identified in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki, 1994) and is recognized by the transcription factor CBF1 (Stockinger, Gilmour and Thomashow, 1997). While CBF1 overexpression has been shown to increase COR gene transcription and provide increased cold tolerance (JagloOttosen et al., 1998), no data are currently available on the salt\drought tolerance of these transgenic plants. We have documented coordinate gene regulation in longterm acquired salt tolerance in alfalfa and rice (Winicov, 1991, 1996) and have focused on the role for a putative transcriptional regulator Alfin1 in altered gene expression in salt tolerant alfalfa (Winicov, 1993 ; Winicov and Bastola, 1997). Alfin1 cDNA encodes a novel member of zinc finger family of proteins. It contains sequence information for one Cys and one His\Cys zinc finger domain, and an acidic % $ region characteristic of DNA binding proteins that are likely to interact with other proteins. Alfin1 appears to be a unique or a low copy gene in the alfalfa genome and shows conservation among diverse plants, such as rice and Arabidopsis as demonstrated by southern analysis (Winicov and Bastola, 1997). Alfin1 is expressed primarily in roots and binds DNA in a sequence specific manner (Winicov and Bastola, 1997 ; Bastola et al., 1998 a). Elements of this GC rich binding sequence are found in promoter fragments of the salt inducible gene MsPRP2 that is also primarily expressed in roots (Winicov and Deutch, 1994 ; Deutch and Winicov, 1995). We have transformed alfalfa with Alfin1

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WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants

cDNA and obtained expression with the 35S CAMV promoter in callus and regenerated plants. The regenerated plants appear normal despite the ubiquitous expression of Alfin1. Interestingly, we have found that Alfin1 overexpression in transgenic alfalfa leads to enhanced levels of MsPRP2 transcript accumulation in callus and in roots (Bastola et al., 1998 b), indicating that Alfin1 can act as transcriptional regulator on endogenous genes when transformed into alfalfa. Particularly interesting is our finding that Alfin1 overexpression can induce the MsPRP2 gene, which is also induced by salt. These results suggest that Alfin1 may be an important transcription factor involved in gene regulation in our salt tolerant alfalfa and an excellent target of gene manipulation for improved salt tolerance.

CONCLUSIONS Salt tolerance is a complex trait in plants, but molecular and genetic approaches are beginning to characterize the diverse biochemical events that occur in response to salt stress. In the short term, it will remain a challenge to manipulate the essential protective mechanisms in plants and to utilize our biochemical knowledge for optimal molecular engineering of salt tolerance in plants. A major unresolved question is the extent and importance of both short-term and long-term stress responses for sustained tolerance and their effects on agriculturally desirable traits in crop plants. With the recognition that the enhanced expression of a number of functionally related genes may be required for optimal improvements in salt tolerance, molecular engineering has been expanded to include proposals for multiple gene transfers to enhance salt tolerance (Bohnert and Jensen, 1996). An equally promising approach to manipulating many genes may emerge as we learn more about the specificity of signalling pathways that turn on transcription of related genes that counteract salt stress at the cellular level. Redundancy of the intersecting signalling pathways and communication between the different pathways, however, is likely to create difficulties in using this information in a directed approach at improving salinity tolerance in the near future. Transcriptional regulation is another new area with potential for coordinate regulation of genes relevant to tolerance, but will require identification of factors limiting the sustained response so that their expression may be manipulated in a tissue targeted manner. Overall, we are likely to see continued significant progress in our understanding and ability to modify salt tolerance by molecular engineering using both model and crop plants based on knowledge of how salinity affects plant biochemistry and physiology through gene expression.

A C K N O W L E D G E M E N TS My thanks for the helpful comments of the reviewers and to Dr M. J. Guiltinan for providing the EmBP results, in press. This work was supported in part by a Hatch grant from NAES, NSF EPSCoR WISE Program and NRICGP 9401235 grant to I.W.

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