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Gene revolution during genomic era: Its impact in dissecting the mechanisms of abiotic stress tolerance in plants N. Sreenivasulu* Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466, Gatersleben, Germany *corresponding author e-mail: [email protected] Tel: 0049-39482-5172 Fax: 0049-39482-5595 Summary Abiotic stress is one of the major limiting factors for agricultural productivity. This review surveys how plants deal with abiotic stress conditions and how tolerance mechanisms can be revealed by genomic tools. Large scale sequencing of complete genomes from Arabidopsis and rice as well as largely available Expressed Sequence Tags (ESTs) from abiotic stress treated tissues has provided overwhelming information concerning the discovery of stress-related genes. Especially, cDNA based expression profiling as well as EST data mining tools are used to define extended sets of genes and pathways involved in abiotic stress responses. Among the identified components, accumulation of osmolytes, expression of antioxidant components as well as stress proteins plays a pivotal role in tolerance. Exclusion or compartmentation of Na+ ions into vacuoles provides another efficient mechanism to avert deleterious effects of Na+ in the cytosol. In order to explore the putative function of unknown abiotic stress responsive genes set, forward and reverse genetic approaches has been employed. Many stressinducible genes have been used to improve stress tolerance of plants by gene transfer and genetic engineering. A variety of crop plants have been engineered with respect to the synthesis of osmoprotectants and ion-compartmentation, but there are other cellular pathways involved in abiotic stress response that are still not completely explored. Key words: abiotic stress tolerance, osmoregulation, EST mining, genomic approaches, transgenics.

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Introduction Plant productivity is severely affected by abiotic stress. Abiotic stresses such as salinity, drought, high temperature, cold and heavy metals induce various physiological and biochemical responses in plants. As a result, hundreds of genes and its corresponding gene products have been known to respond to these stresses at transcriptional and translational level (for reviews see Sreenivasulu et al., 2004a; Cushman and Bohnert, 2000; Bohnert et al., 2001). Understanding the putative functions of these stressinducible genes helps to understand the possible mechanisms of stress tolerance. Recently established functional genomic approaches have triggered a major paradigm from single gene discovery to thousands of genes by using multi-parallel high throughput techniques. Generation of expressed sequence tags (ESTs) from abiotic stress-treated libraries of various crop plants, complete genome sequence information for rice and Arabidopsis provide a valuable resource for gene discovery. Furthermore, employment of multiparallel techniques such as expression profiling by microarrays, random and targeted mutagenesis, complementation and promoter-trapping strategies allow us to identify the key stress-responsive gene-pools and in turn provide important clues for functional characterization of stress responsive genes and stress tolerance mechanisms (Bohnert et al., 2001). Recent genomic studies show considerable overlap of plant responses to cold, drought, and salinity stresses (Kreps et al., 2002; Chen et al., 2002). Dehydration, salinity, low as well as high-temperature stresses leads to metabolic toxicity, membrane disorganization, generation of reactive oxygen species (ROS), inhibition of photosynthesis and altered nutrient acquisition (Hasegawa et al., 2000). At the molecular level, abiotic stress tolerance can be achieved by gene transfer by altering the accumulation of osmoprotectants, production of chaperones, superoxide radical scavenging mechanisms, exclusion or compartmentation of ions by efficient transporter and symporter systems (for review see Ingram and Bartels, 1996; Hasegawa et al., 2000; Zhu, 2001; Apse and Blumwald, 2002). In the following sections, I will provide an account on the recent development of functional genomic approaches of abiotic stress tolerance. Each of the components of tolerance mechanism is regulated and coordinated by multiple genes, whose expressions were controlled by signaling networks and transcriptional regulators. Based on the available high-throughput expression results of

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abiotic stress studies, recently revealed signaling cascades as well as regulatory mechanisms will be discussed in the following chapters. Functional genomic approaches for elucidating abiotic stress tolerance mechanisms The greater power of genomics held in the factor of integrating large data obtained at global scale of genome in order to address challenges occur at molecular, metabolic, physiological, cellular and morphological level during abiotic stress treatments. This would not have been possible without technical advancement in the platform of (a) gene discovery, (b) high-throughput gene expression, (c) functional characterisation of genes of interest through high-throughput gene inactivation techniques and (d) altering gene expression via transformation technologies. The role of different disciplines of functional genomic approaches in crop improvement for stress tolerance shown in Fig. 1 and details will be discussed in the following sections.

Fig. 1 Functional genomics approaches employed to dissect abiotic stress tolerance. The schematic representation reveals interesting networks of techniques employed in functional genomic approaches of abiotic stress tolerance.

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Discovery of abiotic stress responsive genes An important genomic approach to identify abiotic stress related genes is based on ESTs generated from different cDNA libraries representing abiotic stress treated tissues collected at various stages of development. Detailed information on the type of libraries and number of ESTs generated from each library of various abiotic stress tolerant species is

indexed

at

NCBI

dbEST

database

(http://www.ncbi.nlm.nih.gov/

dbEST/dbEST_summary.html). In order to enrich plant EST datasets with stressresponsive genes, specific sequencing programmes based on cDNA libraries from stresstreated plant tissues and organs of diverse species at more developmental time points are necessary. Since EST data set generated from control as well as stress-treated tissues were derived mostly from non-normalized cDNA libraries, counting the abundance of a particular gene provide information on relative expression levels of stress-responsive genes. In addition, the clustering of EST sequences generated from abiotic stress-treated cDNA libraries provided information on gene number, gene content and possible number of gene families involved in stress responses. Putative functions were assigned to the stress-responsive genes by BLASTX comparison to the Swissprot database. This type of analysis provides a valuable resource of information regarding a gene index associated with stress responsive genes among various species. Further, the clustering data yielded consensus sequences that provide a much cleaner data set than typical EST-data. For functional classification and analysis, the consensus sequence of clusters and sequence information

from

all

singletons

was

considered

(as

per

the

MIPS

[http://mips.gsf.de/proj/thal/] and MapMan functional catalogues). The majority of ESTs belong to functional categories of metabolism, cell rescue/defense, protein biosynthesis and no hit/unknown genes. Unknown genes still represent a very high percentage (20 - 30 %) in all cDNA libraries of stress-treated plants. They need to be annotated in order to find possible functions and to get a comprehensive picture of the tolerance mechanisms. Recently, we made an attempt to define a unigene set by clustering programs and to further identify abundantly expressed ESTs in libraries of a salt-treated halophyte (ice plant) as well as glycophytes like barley, wheat, maize, rice, Arabidopsis and soybean (Sreenivasulislu et al., unpublished data). Analysing the various EST collections helped

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to find salt-stress regulated genes. Further, these data should also assist in unravelling the underlying regulatory and metabolic networks. Identification of a stress-responsive gene by transcript profiling In contrast to digital in silico quantification of expression levels based on EST counts, approaches such as SAGE, MPSS, array-based transcript profiling technologies allow us to perform assessment of high-throughput expression of thousands of genes during control and stress-treated tissue during various developmental stages. Insights into gene expression patterns and functions coupled to stress tolerance can be explored by EST based cDNA arrays. Gene expression profiling using cDNA macroarrays (Sreenivasulu et al., 2004b, 2002a) or cDNA microarrays (Kawasaki et al., 2001) is a useful approach to identify higher numbers of transcripts and pathways related to stress-tolerance mechanisms than before. Kawasaki et al. (2001) reported large-scale gene expression profiling in the salt-tolerant rice variety Pokkali as well as in the salt-sensitive variety IR29 at 15-minutes to 7-day time intervals under control and high salinity conditions. These authors concluded that the tolerant cultivar responded at the level of transcription already after 15 min of salt stress and displayed up regulation of many genes encoding glycine-rich proteins, ABA and stress induced proteins, metallothionein-like proteins, glutathione S-transferase, ascorbate peroxidase, water channel protein isoforms, subtilisin inhibitor, tyrosine inhibitor and others. Many transcripts that were up regulated in the tolerant cultivar were generally down regulated in the sensitive one during salt treatment. Ozturk et al. (2002) carried out transcript profiling in 3-week old, salt-stressed barley seedlings and identified upregulated transcripts such as metallothionein-like protein, heat shock protein (70 kDa), lipid transfer protein, glutathione S-transferase, LEA protein, pathogenesis related protein, ribosomal protein (60S) etc. Recently, the ability of barley cDNA arrays was explored to examine the gene expression patterns in tolerant and sensitive seedlings of foxtail millet (Setaria italica L.) exposed to 250 mM NaCl (Sreenivasulu et al., 2004c). Further, 14 unique ESTs up-regulated in the salt-tolerant line were identified under prolonged salt stress and found to be similar to that identified in rice and barley. In conclusion, among cereal plants during long-term abiotic stress

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treatments protease inhibitors, stress proteins, aquaporins and antioxidant components were induced and expected to impart tolerance during various abiotic stress treatments. Several microorganisms have been used as models to characterize the properties of salt stress-induced genes. Multiparallel expression analysis of 6144 open reading frames (ORFs) of yeast to salinity response (10 to 30 min) revealed up-regulation of genes of nucleotide and amino acid metabolism, protein synthesis and protein destination, as well as of intracellular transport genes. Under long-term salt exposure (90 min), genes associated with detoxification, lipid and fatty acid biosynthesis and energy metabolism were up regulated (Yale & Bohnert, 2001). Bohnert et al. (2001) made an attempt to identify plant homologues (from ice plant, rice and corn) for yeast ORFs regulated during salinity stress. The time pattern of up regulation of genes under salinity treatment in yeast seems to be similar in plants. Genes belonging to protein synthesis and turn over were expressed as part of the early response to salinity. Under prolonged salt-exposure, detoxification pathway genes were expressed both in yeast and rice. Similarly, another peace of work carried out on expression-profiling of halophyte plant confirmed that ESTs highly expressed in salt-treated epidermal bladder cells of ice plant belong to antimicrobial, pathogenesis-related and antifungal proteins (Bohnert et al., 2001). Putative functional relevance of these stress-induced genes in halophyte needs to be explored by functional genomic approaches. Another important aspect that needs to be investigated by functional genomics analysis is identification of gene expression patterns related to multistress interactions and signal transduction pathways involved in pathway cross talks. Along that line, Chen et al. (2002) identified groups of transcription factors regulated (a) specifically by abiotic stress (class I) and (b) both by biotic and abiotic stresses (class II) in Arabidopsis. Among the class I group, approximately 20 genes were preferentially induced by abiotic stresses such as salinity, osmotic, cold and jasmonic acid treatment. These transcription factors include DRE/CRT binding factors activated by cold stress, CCA1 and Athb-8 (regulated by hormones, Baima et al., 2001), Myb proteins as well as bZIP/HD-ZIPs and AP2/EREBP domain proteins (Kizis et al., 2001). Further, Seki et al. (2002) employed a full-length cDNA microarray containing 7000 independent Arabidopsis cDNAs to identify cold, drought and salinity induced target genes and stress-related transcription

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factor family members such as DREB, ERF, WRKY, MYB, bZIP, helix-loop-helix and NAC. These results indicated that there is a greater crosstalk between salt and drought stress signaling process in comparison to salt and cold stress. As large-scale gene expression data sets become available from the analyses of stress responses of various plant systems, it is important to set up effective clustering algorithms and bioinformatic tools for identification of the stress regulons (for technical details see the review of Sreenivasulu et al., 2002b). This approach enables the identification of new promoter elements/transcription factor binding sites in co-expressed gene sets and further helps to explore regulatory networks controlling abiotic stress response (Aarts et al., 2003). Further, such clustering data also provide an opportunity to identify organism specific gene sets that are regulated under abiotic stress treatments. However, to achieve a more holistic picture of stress tolerance mechanisms, it is important to combine different functional genomic approaches such as transcriptome, proteome, and metabolome studies (for technical details see the review of Colebatch et al. 2002). A final test of the validity of the developed concepts is either testing the putative function of the genes by forward genetics or by the transfer of several of the stress-responsive genes into crops to enhance the tolerance levels. Such experiments will be discussed in the following section. Implementation of high-throughput gene inactivation techniques to identify puataive function of unknown genes involved in abiotic stress responses As an increasing array of genomic assisted programs continued to be developed, the identification of abiotic stress responsive candidate genes is at large number. Interestingly, more than 40-50% of identified stress-responsive gene functions are not known. In order to reveal its putative functions involved in abiotic stress tolerance various high throughput methods developed for the confirmation and validation of gene function by targeted gene inactivation. There are two main complementary approaches developed for identifying mutations in target genes, namely TILLING (Targeted Induced Local Lesions In Genomes) and T-DNA insertion mutant lines. Using these techniques various attempts were made in order to show the putative functions of abiotic stress-responsive genes in Arabidopsis, Rice, maize and barlely. As an example we dealt

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with report on Arabidopsis SOS mutants identified by Zhu group in a genetic screen showing hypersensitive to growth inhibition by NaCl stress (Zhu, 2001). Zhu et al. (1998) identified sos mutants, where mutant growth is impaired on media that are deficient in K+ and particularly hypersensitive to Na+ and Li+ ions. Based on recent biochemical and physiological data the role of sos1 in K+ acquisition is indirect and SOS1 gene is identified as Na+/H+ antiporter, which maintains low concentration of Na+ in cytoplasm by pumping Na+ ions into acidic vacuoles (Shi et al., 2000). Subsequently, another locus, SOS2, and a third salt tolerance locus, SOS3, were identified in Arabidopsis by Zhu et al. (1998). SOS2 locus has also been found to be necessary for K+ nutrition since sos2 mutants could not be grown in a medium with low potassium and recently been identified as a serine/threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase (Liu et al., 2000). High concentration of Na+ elicits a cytoplasmic calcium signal, which will be perceived by SOS3, a calcium binding protein. SOS3 interacts with SOS2, a protein kinase, the regulatory domain is located in SOS2 gene. The calcium-dependent kinase pathway of SOS3-SOS2 activates the Na+/H+ antiporter gene under excess of Na+. It, therefore, appears that SOS1, SOS2, SOS3 encode regulatory components controlling K+ nutrition essential for salt tolerance in plants. Recently, Shi et al. (2002) isolated sos4 mutant from Arabidopsis (hypersensitive to Na+, K+, and Li+ ions) and demonstrated that SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. Functional aspects of stress tolerance mechanisms identified through transgenic approaches Genetic engineering for stress tolerance was limited in the pre-genomics era by the limited availability of genes and specific promoters (Zhu et al., 1997). Now, it is possible to study many genes simultaneously on a genome wide-scale with respect to their structure and function. Thus, a current trend in stress-biology is to use large-scale genomics data to scrutinize and revalidate the protective mechanisms which have been described based on transgenic approaches (osmolyte biosynthesis, stress proteins, antioxidants, aquaporins, ion homeostasis and signaling pathways). In conclusion, there is a good correlation among the gene sets identified by traditional and genomic studies.

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Moreover, many important components identified for abiotic stress tolerarnce were tested by transgenic technologies and confirmed the inferred role of osmolyte genes, stress proteins, antioxidants, aquaporins and ion homeostasis genes in conferring abiotic stress tolerance across various crop species. In this review we have demonstrated and discussed the role of antioxidant protection in abiotic stress tolerance identified through genomic and transgenic technologies. Ion toxicity and water deficiency impair photosynthesis and produce reactive oxygen species (ROS). ROS generation leads to phytotoxic reactions like lipid peroxidation, protein degradation and DNA mutation (Leprince et al., 2000). The degree of oxidative cellular damage in plants exposed to salt stress is controlled by the antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) (Sreenivasulu et al., 2000). Over expression of Mn-SOD (Tanaka et al., 1999), FeSOD (Van-Camp et al., 1996;) and chloroplastic Cu/Zn-SOD (Gupta et al., 1993) in transgenics leads to higher tolerance against various stress conditions including salt. In transgenic rice, over expression of SOD leads to an increase in total SOD as well as APX activity upon salt stress (Tanaka et al., 1999). The transgenic rice plants showed better growth with higher PS II activity during salinity treatments. Transgenic plants over expressing APX (Wang et al., 1999), GPX and glutathione reductase displayed better resistance to oxidative as well as salt stress conditions (Roxas et al., 1997). Over expression of GST and GPX in transgenic tobacco plants resulted in higher levels of glutathione and ascorbate than in wild type seedlings. Oxidative damage was also reduced and salt tolerance increased (Roxas et al., 1997). The potential role of catalase in the protection against osmotic stress was investigated by expression of Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) in transgenic tomato (Hsieh et al., 2002). These plants displayed higher catalase activity and increased resistance to osmotic stress. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in the generated tomato plants and catalase1 was characterized. Overexpression of CBF1 in Arabidopsis induced cold responsive gene expression, which in turn promoted freezing tolerance (Jaglo-Ottosen et al., 1998).

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Outlook In recent years an enormous increase in our knowledge on plant's responses to stress including salt stress has been achieved. Several genes have been isolated from microorganisms and plants and transferred into crop plants to improve tolerance against various abiotic stress conditions. Most reports state an increased stress tolerance of the respective transgenic lines. Since engineered higher accumulation of osmolytes and stress proteins also increased tolerance for water and cold stresses the gain in agricultural productivity with such plants would be even more dramatic. However, no scientific reports on extensive field tests have been published yet. Only such trials can show which approaches are promising for further development of varieties able to meet the economical demands. It is evident from the published research that more extensive work is necessary in several areas. In most of the reviewed experiments the foreign gene was constitutively expressed and thus is likely to cause unwanted effects. Therefore, it is highly desirable to achieve organ-specific and stress-responsive expression of the introduced genes. Especially genomics approaches have identified a set of salt-inducible genes. Their promoters can be isolated and thoroughly tested for specificity. Several different such promoters are advisable to prevent gene silencing when gene pyramiding is tried as a promising technology to achieve higher tolerance levels. Genomic approaches are also highly suitable to identify whole pathways involved in abiotic stress response and their relationships to the whole metabolic network. At the same time general pathway regulators can be identified. However, unwanted side effects of manipulating such regulatory genes are even more likely than with single gene members of a pathways and, therefore, need intensive research and breeding efforts to use their great potential. Beside genetic engineering approaches, other more classical approaches, not reviewed here, are of no less importance. Convinsingly, we are optimistic that the high expectations raised by the recent reports on functional genomic of abiotic stress tolerance could be a valuable information for generation of highly tolerant plants to abiotic stresses, which can be met in the not too distant future. Acknowledgements I gratefully acknowledge Prof. U. Wobus, IPK for expert comments on the manuscript. This work was supported by IPK internal funds.

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