2007 Annual Report 1a.Objectives (from AD-416) The goal is to identify mechanisms by which polyamines enhance the accumulation of choline and N-transport amino acids in tomato fruit. Further, we will determine if increased production and quality attributes in tomato plants grown under leguminous cover crop (hairy vetch) involve polyamine-responsive component(s). 1b.Approach (from AD-416) We will use transgenic technology to develop genotypes enriched in nutrients beneficial for human health. These genotypes will be analyzed for gene expression, particularly to elucidate mechanisms responding to higher polyamines. The interaction of methyl jasmonate with polyamines in regulating fruit metabolism will be analyzed in methyl jasmonate deficient transgenic tomato genotype. Influence of cover crop mulches on gene expression and metabolite profiles will be conducted on field grown transgenic and non-transgenic plants. 3.Progress Report Previously we developed novel tomato genotypes engineered to accumulate higher polyamines, spermidine (Spd) and spermine (Spm), and which have higher nutritional and processing quality. The fruit from these genotypes provide a model system to define alterations at the transcriptome level and effects of Spd/Spm on the metabolome. To bring an insight into the metabolic processes that Spd/Spm impact in modifying the nutrient levels in tomato fruit, we carried out metabolite profiling analyses of the fruit at different stages of ripeness from two transgenic and one azygous control line using NMR spectroscopic analysis. Spd/Spm were found to elevate the levels of amino acids glutamine and asparagine, organic acids citrate, fumarate and malate, and the micronutrient choline while the levels of valine, aspartate, sucrose and glucose were significantly lowered as compared to the control (wild-type and azygous) red fruit. The levels of isoleucine, glutamae, GABA, phenylalanine and fructose remained similar in the non-transgenic and transgenic fruits. Statistical treatment of the metabolite variables distinguished the control fruits from the transgenic fruit and provided credence to the pronounced, differential metabolite profiles seen during ripening of the transgenic fruits. The pathways involved in the nitrogen sensing/signaling and carbon metabolism were preferentially activated in the high Spd/Spm transgenics. The metabolite profiling analysis revealed that Spd/Spm are perceived as nitrogenous metabolites by the fruit cells, which in turn results in the stimulation of carbon sequestration. This is manifested in higher respiratory activity and up-regulation of PEP carboxylase and NADP-dependent isocitrate dehydrogenase transcripts in the transgenic fruit compared to controls indicating high metabolic status of the transgenics even late in fruit ripening. The increased respiratory activity in the transgenic tomatoes as compared to wild-type/azygous tomatoes reveals an in vivo role of polyamines in mitochondrial metabolic regulation, consistent with a previous proposal for spermine function in rat liver mitochondria. For the effects on gene expression, subtractive cloning of total RNA of transgenic from wild type ripening fruits resulted in isolation of several genes that were up-regulated and represented a wild range of functional classifications. The differentially expressed genes represented functional categories including transcription, translation, signal transduction, chaperone family, stress related, amino acid biosynthesis, ethylene biosynthesis and action, polyamine biosynthesis, isoprenoid pathway, and flavonoid biosynthesis. About 44% of the differentially regulated cDNAs included genes encoding products not yet classified for the functional attributes. Based on these results on the limited transcriptome in conjunction with metabolite profiles showing significant enhancement of anabolic pathways in transgenic fruits, we have proposed that Spd/Spm act as anabolic growth regulators in plants. 4.Accomplishments Higher Biogenic Amines, Spermidine and Spermine, Act as Anabolic Plant Growth Regulators and Enhance Accumulation of Phytonutrients. Pharmacological doses of higher polyamines, spermidine (Spd) and spermine (Spm), elicit a number of effects in living cells but their precise biological function(s)/role(s) remained unknown until recently. Using tomato as a plant model, we successfully enhanced the accumulation of Spd/Spm specifically in ripening tomato fruits by introduction of a yeast S-adenosylmethionine decarboxylase gene engineered to be driven a fruit-specific promoter. Such a molecular engineering technology enabled development of nutritionally enhanced tomato lines that have become a unique genetic resource to study the function of polyamines in plants. Employing the techniques of DNA arrays for analysis of global gene expression, immunoblots for protein expression profiling, and nuclear magnetic resonance-based metabolite profiling of these transgenic fruits have revealed previously unknown facets of Spd/Spm function in living cells. Some of these facets include initiation of anabolic pathways, nitrogen-carbon interactions, and positive impact on nutrient composition including effects on aspartate family of amino acids, antioxidant lycopene, and the micronutrient ‘vital amine’ choline. Thus, these studies conducted by scientist at the ARS’s Sustainable Agricultural Systems Laboratory, Beltsville, Maryland in collaboration with researchers at the Purdue University and at CNR, Rome, Italy show for the first time that polyamines (Spd/Spm) act as anabolic growth regulators and impact processes of transcription, translation and metabolic networks. Aspects particularly related to the fruit metabolism and signaling include: presence of sensing mechanisms for Spd/Spm late in the ripening process, revival of metabolic memory by Spd/Spm, and cross talk of Spd/Spm with other plant hormones to regulate cell metabolism. These findings provide new basic, fundamental knowledge on the role of polyamines, which is extendable to other biological systems, and likely critical in understanding pathways by which nutrient levels are regulated in fruits and vegetables. NP 302: Plant Biological and Molecular Processes. Program Component: Biological Processes that Improve Crop Productivity and Quality. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 7 Number of newspaper articles and other presentations for non-science audiences 4 Review Publications Mattoo, A.K., Sobolev, A.P., Neelam, A., Goyal, R.K., Handa, A.K., Segre, A.L. 2006. Nmr spectroscopy based metabolite profiles of transgenic tomato fruit engineered to accumulate polyamines spermidine and spermine reveal enhanced anabolic nitrogen-carbon interactions. Plant Physiology. 142:1759-1770. Mattoo, A.K., Srivastava, A., Chung, S.H., Fatima, T., Datsenka, T., Handa, A. 2007. Polyamines as anabolic growth regulators revealed by transcriptome analysis and metabolite profiles of tomato fruits engineered to accumulate spermidine and spermine. Plant Biotechnology Journal. 24:57-70.