Construction and use of cDNA arrays in studying barley seed development and foxtail millet salt tolerance

Single-pass sequencing of randomly chosen cDNA clones is currently the most efficient method for the discovery of many genes from cereals with large genomes. Management and analysis of the enormous amount of low-quality sequence data requires great care and powerful computational methods for annotation. In order to study the network of gene expression underlying seed development during the pre-storage (0 to 4 days after flowering, DAF) and the initial storage phase (6 to 12 DAF) in barley, we employed EST-based macroarrays. Radioactive labelled cDNA probes were prepared from pericarp and postfertilization embryo sac preparations of developing caryopses (0 –12 DAF) in two-day intervals and hybridized to cDNA macroarrays containing 1412 cDNA inserts, which represent 1184 unique sequences. Grouping of genes by K-mean cluster analysis according to gene expression patterns resulted in 16 gene sets, which can be arranged into 6 cluster groups. Most of the genes up regulated in the pericarp encode proteases, hormonal regulated proteins, and proteins involved in energy production and carbohydrate and lipid metabolism (cluster group1). In the embryo sac probe unique developmental stage specific transcript profiles were identified. During the pre-storage phase, 25% of genes up regulated in embryo sac probe are related to cell division and cell elongation (cluster group 2). In the intermediate phase, photosynthetic genes are up regulated in embryo sac (cluster group3). During the onset of storage phase the embryo sac fraction mainly represents genes belonging to specific metabolic pathways, for instance, the starch (cluster group 4) and storage protein (cluster group 5) biosynthetic pathways including several protease and amylase/trypsin inhibitor genes. On the basis of annotated ESTs and global expression analysis an attempt was made to unravel the complex metabolic and regulatory networks involved during barley seed development. A detailed examination of gene expression patterns related to sucrose to starch and sucrose to pyruvate metabolism pathways provides interesting results of gene networking. Some of the differentially regulated genes detected by expression analysis were studied and further characterized by northern analysis and in situ hybridizations. seg8, a barley mutant defective in seed development, provides a unique opportunity to study the influence of the maternal tissue on endosperm development and storage product accumulation. In order to gain deeper insight into the complex regulatory and metabolic control of maternal and filial tissues and their interaction we analysed seg8 mutant by expression analysis and metabolite profiling. During pre-storage phase of early caryopsis development no obvious difference were found in seed fresh weight between wild type and 1 mutant; mutant seeds weighed approximately 43% of normal ‘Bowman’ wild type during storage phase. Microscopic studies revealed that seg8 mutant shows massive growth of nucellar projection tissue (maternal) with abnormal shrunken endosperm at 4 days after flowering. The failure of proper endosperm development in seg8 that was evident already during 4 DAF became a prominent event at 10 DAF onwards with two lobes of endosperm with professed nucellar projection touching the dorsal crease. In the present study we used the 1412 cDNA array to analyse expression of genes involved in different metabolic pathways during early stages of development (2-14 DAF, days after flowering) between ‘seg8’ mutant and its corresponding wild type ‘Bowman’. A comparison of seg8 versus Bowman during 0-14 DAF at whole caryopsis level as well as in maternal and filial fractions hinted that key genes of carbohydrate metabolism from sugar to starch pathway are down regulated in seg8 mutant. The results provide evidence that genes encoding the UDP-glucose metabolising enzymes are specifically down regulated in seg8. As expected a decrease in the ADP-glucose content was registered in the filial fraction containing endosperm. On the other hand transcripts coding for storage proteins did not yield any considerable differences between mutant and wild type. The reason for maternal inheritance of the abnormal endosperm mutant is not clear, since there is no differences found among transcripts expressed in maternal tissue in the very early stages except at 0 DAF. Characteristic irregularities occur in the endosperm tissue itself with lower expression of carbohydrate metabolic genes immediately before storage activity starts in endosperm. The observed major changes in the expression of starch biosynthetic pathway genes in seg8 mutant result in less starch accumulation. In addition, we observed decreased transcript levels of some transporters in filial fraction of mutant during 4 to 14 DAF. Using cDNA macroarray encoding stress genes selected from a barley EST library, we identified transcripts differentially expressed in salt (NaCl)-treated tolerant and sensitive seedlings of foxtail millet. Transcripts of unknown genes and hydrogen peroxide scavenging enzymes such as phospholipid hydroperoxide glutathione peroxidase (PHGPX) and, additionally, ascorbate peroxidase (APX) and catalase 1 (CAT1) were found to be up regulated during salinity treatment in five-day-old salt-tolerant foxtail millet seedlings (Cv. Prasad). In order to understand the protection mechanism induced in salt-treated tolerant seedlings at the molecular level, we cloned and characterized a foxtail millet cDNA encoding a PHGPX homologue, which shows 85% and 95% homology to one stress-induced member of the small barley PHGPX gene family coding for non-selenium glutathione peroxidases at 2 the DNA and protein level, respectively. As shown by Southern blot analysis, a small family of PHGPX genes exists in foxtail millet, too. The expression of the PHGPX gene is markedly induced in tolerant seedlings by high salt concentrations, suggesting that its product plays a role in defence against salt-induced oxidative damage. To analyse this process further at the protein level we examined protein expression patterns under various stress conditions. A 25 kD protein was found to be induced prominently under high salt concentrations (250 mM). The salt-induced 25 kD protein has been purified and identified as PHGPX protein based on its peptide sequence. The increase of the PHGPX protein level induced under salt stress conditions only in tolerant seedlings parallels the result found for the PHGPX mRNA in the comparative expression analysis (see above). Most likely, this non-selenium glutathione peroxidase is one of the components conferring resistance against salt to the tolerant foxtail millet cultivar. The tolerant five-day-old seedlings grown during high salinity treatment (200 mM NaCl) contained a lower amount of Na ions and showed a lower electrolyte leakage than sensitive seedlings. In conclusion, our comparative studies indicate that at least in part, saltinduced oxidative tolerance is conferred by an enhanced compartment specific activity of antioxidant enzymes (Sreenivasulu et al., 2000).