Identification of Early Nitrate Stress Response Genes in Spinach Roots by Suppression Subtractive Hybridization

To better understand the molecular basis of plant responses to nitrate stress, suppression subtractive hybridization (SSH) was used to identify the potential important or novel genes involved in the early stage of spinach responses to nitrate stress. Complementary DNAs (cDNAs) of 15 min, 1 h, 2 h, 6 h and 24 h of 160 mM nitrate stress treatment seedlings were used as tester, and cDNAs of normal nutrient solution seedlings of the same time were used as driver. The SSH analysis identified 189 non-redundant putative nitrate stress responsive cDNAs out of 798 clones. These ESTs were categorized into 12 functional groups, with the largest group of genes involved in metabolism, followed by the group of genes related to cell rescue, defense and virulence. Reverse transcriptase (RT)-PCR was conducted for several genes, confirming the induction by nitrate stress. The results indicated that osmolyte biosynthesis genes, reactive oxygen scavengers, transporters, signaling components and transcription factor played important roles in fighting against nitrate stress. The diversity of the putative functions of these genes indicated that nitrate stress resulted in a complex response in spinach plants.