Nitrogen metabolism in woody plants; forest biotechnology
We have developed a molecular approach to increase glutamine production in hybrid poplar (Populus tremula L. X P. alba L.) by overexpression of a conifer glutamine synthetase (GS) gene (Gallardo et al., 1999). Biochemical analysis of GS transgenic poplar lines has revealed that ectopic expression of GS leads not only to increased GS activity, but also to enhanced levels of chlorophyll and protein, and total amino acids in leaves (Gallardo et al., 1999; Fu et al., 2003). Greenhouse and field studies of GS transgenic poplar (Figure 1) showed that enhanced GS activity in leaves is correlated with significant increases in growth (Fu et al., 2003). 15N-enrichment experiments showed that significantly more15N was incorporated into structural compounds in leaves of transgenic lines than in leaves of non-transgenic controls (Man et al., 2005). Taken together, these studies provide strong evidence that GS poplar are characterized by enhanced nitrogen assimilation efficiency.
Although the role of cytosolic GS is not well defined in leaves of angiosperms, its expression in photosynthetic tissues has been associated with responses to biotic and abiotic stresses. Therefore, modification of GS levels has important implications, not only for nitrogen partitioning, but also for stress tolerance. We have shown that when water stress was applied to GS transgenic and control poplar lines, GS lines showed resistance to stress. We have investigated possible mechanisms for enhanced water stress resistance in GS transgenic lines in ecophysiological studies (El-Khatib et al, 2004). Taken as a whole, our work suggests that overexpression of cytosolic GS in leaves of transgenic poplars under water stress conditions results in engagement of antennae-level, energy-dependent mechanisms that protect the reaction centers during water stress.
Figure 2: cDNA microarray analysis of gene expression in leaves of control and GS transgenic poplar exposed to water stress conditions and during recovery. A microarray image. B Representation of results: T/C_NS: transgenic vs. controls non-stress conditions; T/C_WS transgenic vs. controls water stress conditions; WS/NS_C water stress vs. non-stress in controls; WS/NS_T water stress vs. non-stress in transgenics. Ned Kirby
We have recently begun analysis of changes in gene expression in GS poplar and non-transgenic controls during water stress and recovery. Preliminary results (Figure 2) show that under non-stress conditions a number of genes are significantly up-regulated in GS transgenics in comparison to controls. Assessment and annotation of the data from this preliminary experiment show that changes in gene expression reflect global changes associated with overall decreases in growth and metabolic activity accompanying water stress in both transgenics and controls.
We have proposed to utilize the new Agilent poplar array, representing 43,000 transcripts (60-mer probes), to assess global gene expression profiles in control and GS transgenic poplar. These analyses will provide insight into mechanisms of accelerated poplar growth due to ecotopic expression of GS. Such assessment will lead to a comprehensive profile of gene expression during water stress and recovery in poplar and will lead to the identification of genes involved in stress resistance and recovery. We will pay special attention to transcription factors during array data-mining in order to identify candidate master regulators associated with stress responses. Additional datasets such as protein–protein interaction data, genetic interactions, and metabolic data will provide additional support for networks of co-regulated genes and their interactions. Results from these studies will lead to the identification of particular genes and markers for water stress resistance that could be important in selecting stress-resistant genotypes.
Recently we have begun work in the general field of molecular ecology, specifically identifying molecular markers useful in restoration of native upland salt marsh communities in the New Jersey Meadowlands.
B.S. in Biology, University of Michigan.
Ph.D. in Botany, University of Florida, 1977.
Molina-Rueda JJ, Tsai CJ, Kirby EG (2013) The Populus Superoxide Dismutase Gene Family and Its Responses to Drought Stress in Transgenic Poplar Overexpressing a Pine Cytosolic Glutamine Synthetase (GS1a). PLoS ONE 8(2): e56421. doi:10.1371/journal.pone.0056421.
Coleman, HD, FM Cánovas, H Man, EG Kirby, SD Mansfield. Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in filed grown hybrid poplar (Populus tremula X alba) (717-1B4). Plant Biotechnology Journal DOI: 10.1111/j.1467-7652.2012.00714.x, (2012).
Castro-Rodriguez, V, A Garcia-Gutierrez, J Canales, C Avila, EG Kirby, FM Cánovas. The glutamine synthetase gene family in Populus. BMC Plant Biology 11, 119 (2011).
Man, H, S Pollmann, EW Weiler, EG Kirby. Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase alpha-subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics? Journal of Experimental Botany 62, 4423-4431 (2011).
Pascual, MP, PJ Zhong, EG Kirby, FM Cánovas, F Gallardo. Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin. Phytochemistry 62, 382-389 (2008).
Kirby, EG, F Gallardo, H Man, R El-Khatib. The overexpresion of glutamine synthetase in transgenic poplar: A review. Silvae Genetica (2006).
Man, H, R Boriel, R El-Khatib, EG Kirby. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytologist 167: 31–39 (2005).
El-Khatib, R, EP Hamerlynck, F Gallardo, EG Kirby. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiology 24, 729–736 (2004).
Fu, J, R Sampalo, F Gallardo, FM Cánovas, EG Kirby. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant, Cell & Environment 26, 411–418 (2003).
Gallardo, F, J Fu, JP Jing, EG Kirby, FM Cánovas. Genetic modification of amino acid metabolism in woody plants. Plant Physiology & Biochemistry. 41, 587-594 (2003).