MICROARRAY ANALYSIS TO IDENTIFY DROUGHT RESPONSIVE GENES INVOLVED IN CARBOHYDRATE METABOLISM IN Medicago sativa LEAVES

¹ New Mexico State University, Dept. Agronomy and Horticulture, Las Cruces, NM, 88003, USA

² Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, OK, 73402 USA

Microarray technology was utilized to study the expression of several genes, that encode for enzymes involved in carbon metabolism, under drought-stress. Leaf tissue of a drought tolerant alfalfa genotype was collected for microarray and glycosyl composition analysis when leaf relative water content of the drought-stressed and well-watered (control) treatments reached 0.53 ± 0.04 and 0.95 ± 0.01, respectively. cDNA was prepared from leaf total RNA and hybridized to microarrays derived from Medicago truncatula cDNAs and Arabidopsis thaliana 70-mer oligonucleotides. Statistically significant changes (p equals 0.05) in expression for those genes examined were determined by analysis of variance across replicated biological samples and arrays. Glycosyl composition analysis of the plants under drought treatment showed increased levels of pinitol, mannose and galactose, and a decrease of glucose when compared to the well-watered treatment. Expression of genes involved in the synthesis of pinitol (i.e. myo-inositol-1 phosphate synthase, phospholipase C, phosphatidylinositol-4-phosphate 5-kinase, and myo-inositol monophosphatase), mannose (i.e. xylose isomerase) and sucrose (sucrose synthase) increased almost two-fold in response to drought. Constitutive levels of expression were found for the genes involved in galactose synthesis. These results suggest that observed decreases in glucose content in drought-stressed leaves may reflect conversion of glucose to mannose, sucrose and galactose. Several genes encoding for enzymes involved in Gluconeogenesis (i.e. phosphoglyceromutase, glyceraldhehyde-3 phosphate dehydrogenase and fructose-biphosphate aldolase) were up regulated under drought-stress. Such a response could partially compensate for the loss of glucose to other metabolic pathways. Transcript levels of the genes examined in this study were confirmed by Northern blotting.