Transgenic Poplar Expressing Pine GS1a Show Alterations in Nitrogen Homeostasis During Drought

Juan Jesús Molina-Rueda and Edward G. Kirby, Rutgers University and the University of Málaga

               Transgenic hybrid poplars engineered to express ectopically the heterologous pine cytosolic GS1a display significant pleiotropic phenotypes including enhanced growth, enhanced nitrogen use efficiency, and resistance to drought stress.  The present study was undertaken in order to assess mechanisms whereby ectopic expression of pine GS1a in transgenic poplars results in enhanced agronomic phenotypes.  Microarray analysis using the Agilent Populus whole genome array allowed identification of genes differentially expressed between wild type (WT) and GS transgenics in four tissues (sink leaves, source leaves, stems, and roots) under three growth conditions (well-watered, drought, and recovery).  Analysis revealed that differentially expressed genes in functional categories related to nitrogen metabolism show a trend of significant down-regulation in GS poplars compared to the WT, including genes encoding nitrate and nitrite reductases.  Down-regulation of these genes was verified using qPCR, and downstream effects were tested using NR activity assays.  Results suggest that higher glutamine levels in GS transgenics regulate nitrate uptake and reduction.  Transcript levels of nitrogen-related genes in leaves, including GS/GOGAT cycle enzymes, aspartate aminotransferase, GABA shunt enzymes, photorespiration enzymes, asparagine synthetase, phenylalanine ammonia lyase, isocitrate dehydrogenase, and PII, were also assessed using qPCR revealing significant differences between GS poplars and the WT.  Moreover, metabolites related to these differentially expressed genes showed alterations, including higher levels of GABA, hydroxyproline, and putrescine in the GS transgenic.  These alterations in nitrogen homeostasis offer insights into mechanisms accounting for drought tolerance observed in GS poplars.