A genomics approach towards salt stress tolerance

https://doi.org/10.1016/S0981-9428(00)01237-7Get rights and content

Abstract

Abiotic stresses reduce plant productivity. We focus on gene expression analysis following exposure of plants to high salinity, using salt-shock experiments to mimic stresses that affect hydration and ion homeostasis. The approach includes parallel molecular and genetic experimentation. Comparative analysis is employed to identify functional isoforms and genetic orthologs of stress-regulated genes common to cyanobacteria, fungi, algae and higher plants. We analyze global gene expression profiles monitored under salt stress conditions through abundance profiles in several species: in the cyanobacterium Synechocystis PCC6803, in unicellular (Saccharomyces cerevisiae) and multicellular (Aspergillus nidulans) fungi, the eukaryotic alga Dunaliella salina, the halophytic land plant Mesembryanthemum crystallinum, the glycophytic Oryza sativa and the genetic model Arabidopsis thaliana. Expanding the gene count, stress brings about a significant increase of transcripts for which no function is known. Also, we generate insertional mutants that affect stress tolerance in several organisms. More than 400 000 T-DNA tagged lines of A. thaliana have been generated, and lines with altered salt stress responses have been obtained. Integration of these approaches defines stress phenotypes, catalogs of transcripts and a global representation of gene expression induced by salt stress. Determining evolutionary relationships among these genes, mutants and transcription profiles will provide categories and gene clusters, which reveal ubiquitous cellular aspects of salinity tolerance and unique solutions in multicellular species.

Introduction

For at least 8 000 years, mankind has manipulated plant species from subtropical regions to grow under diverse climatic conditions. The results of this activity provide all our food, feed and fiber. Domestication extended to just a few hundred species, with maybe ten of these providing the majority of all foods. Selection, increasingly based on scientific criteria, has allowed us to keep pace with population growth 〚9〛. Population growth, changes in lifestyle, competition for fresh water between farmers and cities, and possible global environmental changes have led to alarmist projections that seem to argue for additional strategies by which food supply can be guaranteed 〚32〛. Areas where food production is problematic coincide largely with places where water is a precious commodity, prolonged droughts are frequent, where saline and marginal soils are found and where temperature extremes compromise production. Moreover, the increased productivity achieved in irrigated agriculture has become a double-edged sword, because salinization following prolonged irrigation is unavoidable 〚13〛, 〚37〛. These considerations have galvanized strong interest in studying plant abiotic stress responses and understanding the meaning of stress tolerance as a biological phenomenon.

Understanding abiotic stress tolerance, not to mention breeding for stress tolerance, proved difficult because of the trait’s multigenicity. As a consequence no traditional crop lines exist that combine tolerance to high salinity or drought with high yield, which represents yet another complex trait. Many physiological studies have analyzed the problem. In the search for tolerance mechanisms, plant biologists have generated hypotheses by interpreting correlative evidence from many species based on biochemical and biophysical principles that govern stress tolerance or resistance 〚27〛 These insights provided guiding principles for moving from physiology to protein and enzyme analysis, genetic structure, gene function and gene expression studies, finally culminating in transgenic and mutant generation and analyses.

The last decade has brought a sea change in our views about stress sensitivity fueled by results from molecular genetics. Changes in the expression of individual genes and proteins induced by stress have been monitored under different conditions. As of the year 2000, the sequence of the Arabidopsis thaliana genome is nearly completed, and soon we will have a catalog of plant gene expression exceeding a million transcripts (http://www.ncbi.nlm.nih.gov/dbEST/index.html) 〚28〛, 〚44〛, 〚45〛. While numbers are important, more important is that from the molecular analysis of these transcripts, collected as ESTs (expressed sequence tags), the frequency and type of transcribed genes are obtained for organs, tissues and cells during development and under various perturbations. Such expression profiles are augmented by the analysis of EST-derived microarrays or oligonucleotide-based DNA-chips both providing information on changes in the expression levels over time or during experimental perturbations of the plants 〚7〛, 〚39〛, 〚50〛. Accompanying these activities has been yet another breakthrough, whose impact on stress biology and crop breeding will be, we think, even more profound. In A. thaliana, rice and corn saturation mutagenesis seems to have been achieved 〚4〛, 〚31〛, 〚36〛. The resulting mutants make possible proof of concept experiments on a large scale.

Here we present an initial set of data from an ongoing project that is focussing on defining the complete gene set associated with salinity stress. In addition, we report on Arabidopsis mutants with altered stress sensing and signal transduction characteristics. New technologies are employed to advance understanding: large-scale EST preparation and expression profiling, microarray analysis identifying target genes whose expression is stress-regulated, and the generation of mutants in stress-relevant signal transduction and response pathways. As the first results emerge, additional strategies can be employed that target the analysis of the many functionally unknown genes or transcripts, which are regulated when plants experience abiotic stress.

Section snippets

Results and discussion

For continued vegetative growth and the development of reproductive organs under osmotic and ionic stress conditions, plants must, above all, obtain water. To satisfy this essential need, each of many adaptive mechanisms that have been detected as plant defenses must be subordinate to this goal. When stomata are closed to limit water loss, a series of events adjusts photosynthesis, carbon fixation and carbohydrate transport, initiating processes that maintain the integrity of the photosynthetic

Conclusion

With the instrumentation in place that allows large-scale DNA sequencing, genome-wide analysis of the transcriptome, and large-scale analysis of all proteins in a cell, we must learn to utilize the bioinformatics tools that are essential if we wish to sort and understand the data avalanche. The extent to which biology is presently driven by these new technologies and by the need for sophisticated bioinformatics tools requires a new mindset. We have arrived at a threshold that permits the

Libraries from stressed plantsand EST sequencing

Our main objective was to obtain cDNA libraries from different tissues of salt-stressed plants to be used for the generation of EST sequences. Up to now, approximately fifty cDNA libraries have been generated, listed at stress-genomics.org (see also table I). They include cDNA libraries from the non-plant models Synechocystis sp. PCC6803, Saccharomyces cerevisiae, Aspergillus nidulans and Dunaliella salina. The plant models for which cDNA libraries have been established are Arabidopsis thaliana

Acknowledgements

We thank the technical staff and students in our laboratories, who have been essential in making high-throughput DNA preparation, analysis of ESTs, and mutant generation possible, especially Rashid Abduraham, Inna Akselrod, Adam Barb, Alison Berne, Susan Brazille, Amanda Breen, Davi Bufford, Juang-Hong Chong, Michael Dellinger, Monica Dennis, Andrea Hanlon, Xiaojing Jin, Melissa Knue, Julianne Lazarr, Saul Leite, Fang Li, Xianghui Liu, Heather McLaughlin, Jeson Martajaja, Jessica Martinez,

References (50)

  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • M.P. Apse et al.

    Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis

    Science

    (1999)
  • H.J. Bohnert et al.

    Molecular mechanisms of salinity tolerance

  • R.L. Burnap et al.

    The highly abundant chlorophyll-protein complex of iron-deficient Synechococcus sp. PCC7942 (CP43') is encoded by the isiA gene

    Plant Physiol.

    (1993)
  • M.B. Eisen et al.

    Cluster analysis and display of genome-wide expression patterns

    Proc. Natl. Acad. Sci. USA

    (1998)
  • L.T. Evans

    Feeding the Ten Billion

    (1998)
  • B. Ewing et al.

    Base-calling of automated sequencer traces using Phred. II. Error probabilities

    Genome Res.

    (1998)
  • B. Ewing et al.

    Base-calling of automated sequencer traces using Phred. I. accuracy assessment

    Genome Res.

    (1998)
  • T.L. Ferea et al.

    Systematic changes in gene expression patterns following adaptive evolution in yeast

    Proc. Natl. Acad. Sci. USA

    (1999)
  • T.J. Flowers et al.

    Breeding for salinity tolerance in crop plants: Where next?

    Aust. J. Plant Physiol.

    (1995)
  • C. Frova et al.

    Mapping quantitative trait loci for tolerance to abiotic stresses in maize

    J. Exp. Zool.

    (1999)
  • U. Halfter et al.

    The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

    Proc. Natl. Acad. Sci. USA

    (2000)
  • P.D. Hare et al.

    The involvement of cytokinins in plant responses to environmental stress

    Plant Growth Regul.

    (1997)
  • P.M. Hasegawa et al.

    Plant cellular and molecular responses to high salinity

    Annu. Rev. Plant Physiol. Plant Mol. Biol.

    (2000)
  • P. Horton et al.

    Regulation of light harvesting in green plants

    Annu. Rev. Plant Physiol. Plant Mol. Biol.

    (1996)
  • Cited by (0)

    1

    Contributed experimental data from ongoing sub-projects within the stress genomics consortium.

    View full text