SyPro POPLAR: IMPROVING POPLAR BIOMASS PRODUCTION UNDER ABIOTIC STRESS CONDITIONS: AN INTEGRATED OMICS, BIOINFORMATICS, SYNTHETIC BIOLOGY AND GENETIC ENGINEERING APPROACH.
The objective is to produce a suite of promoters that are short, precise and strong for induction. These synthetic promoters will be used to drive the expression of known and novel stress-responsive genes in trasnsgenic poplar trees under controlled and field conditions. Our objectives are: (1) Study the function of selected stress-responsive genes; (2) Discover novel motifs and construct stress-responsive synthetic promoters; (3) Use these promoters to drive the expression of genes known to confer abiotic stress tolerance in a variety of crops and develop abiotic-stress tolerant poplar seedlings; (4) Evaluate stress tolerance and yield potential of the transgenic poplar trees in the field.
- Amir Ahkami, Pacific Northwest National Laboratory.
- C. Neal Stewart, University of Tennessee.
- Stephen DiFazio, West Virginia University.
THE EFFECTS OF MODIFICATIONS OF SOURCE-SINK RELATIONSHIPS ON CROP STRESS TOLERANCE
Abiotic stress is the primary cause of crop plant yield losses worldwide. Improving yield production and stability under stressful environments is needed to fulfill the food demand of the ever-growing world population, and an estimated increase of 50% in grain yield of major crops swill be needed by 2050. Drought, the most prominent threat to agricultural production worldwide, accelerates leaf senescence, leading to a decrease in canopy size, loss in photosynthesis and reduced yields. We hypothesized that it may be possible to enhance drought tolerance by altering sink/source relationships in the plant by promoting the stress-induced synthesis of cytokinins. The regulated expression of IPT (isopentenyltransferase) under the control of PSARK significantly improved drought tolerance in both laboratory and field conditions. Transgenic plants produced higher yields than wild-type plants in the field and the seeds from PSARK::IPTplants were normal, indicating that the nutritional value of the transgenic seeds was not altered. We used a multidisciplinary approach that combined genomics, proteomics, metabolomics and enzyme function analysis to identify and characterize cellular/biochemical components that regulate Carbon and Nitrogen metabolism in plants grown under water deficient conditions. Stress-induced cytokinin production had a positive effect on nitrate uptake as well as on the expression of genes associated with primary N assimilation and N re-assimilation, enhanced higher protein synthesis and the strengthening of the transgenic plants sink capacity. A System Biology approach was applied to identify genes and gene networks mediating the stress-response of crops to abiotic stress. A number of genes have been identified, and their expression has been modified in a number of crop species. The role of these genes and their effects on survival to stress is under study.
CELLULAR ION HOMEOSTASIS: EMERGING ROLES OF INTRACELLULAR NHX-TYPE Na+/H+ ANTIPORTERS IN PLANT GROWTH AND DEVELOPMENT:
Na+/H+ antiporters (NHX-type) are important regulators of intracellular pH and K+(Na+) homeostasis in plant cells. In Arabidopsis the NHX gene family includes six intracellular Na+/H+ members (AtNX1-6), which localize to either the vacuole (AtNHX1-4), or to trafficking endosomes (AtNHX5-6). Previously we showed that the double knockout nhx1nhx2 had reduced growth rates, smaller cells, shorter hypocotyls in etiolated seedlings and abnormalities in flower development. Measurements of intravacuolar pH and K+ concentrations indicated that nhx1nhx2 vacuoles were more acidic and accumulated less K+.nhx1nhx2 plants also displayed severe sensitivity to added K+ (10mM) but not to Na+, and the addition of 50mM NaCl to the growth medium partially rescued the growth phenotype. These results demonstrated that NHX1 and NHX2 play significant roles in vacuolar K+/H+ exchange and are key determinants in establishing vacuolar K+ homeostasis and raised questions regarding the cellular ‘compatibility’ of K+ ions, the transport of Na+ into the vacuole, the role of both ions in generating the vacuolar turgor needed to drive cell expansion, as well as the cellular response to high salinity. We extended these studies to a larger comparative analysis of additional multiple knockout lines lacking all possible combinations of NHX1, NHX2, NHX3 and NHX4. Triple and tetra knockouts exhibited complex and unique growth phenotypes suggesting that individual NHX isoforms contribute differentially to cell growth. We measured and compared vacuolar pH, K+ and Na+ concentrations, under varying conditions, in different knockout lines with the goal of quantifying the contribution of each vacuolar isoform to cell pH and ion homeostasis. Furthermore, other results showed that the Golgi and trans-Golgi network-localized NHX members, NHX5 and NHX6 are required for vesicular trafficking, cell growth and salt-stress responses. To investigate the roles of NHX5 and NHX6 in maintaining vesicular pH homeostasis, we developed genetically encoded pH sensors and targeted these to the distinct endomembrane compartments, along the secretory pathway. Measurements of endosomal pH, indicated that a gradual acidification of compartments from the ER to the vacuole exists. Using nhx5nhx6 double knockouts, we further investigated the role of intravesicular pH and ion homeostasis on vesicular trafficking and protein processing. Using a combination of approaches including in vivo protein-protein interactions, biochemical characterization of protein complexes, and in vivo endomembrane pH measurements, we provide evidence indicating that pH and/or ion homeostasis, controlled by NHX5 and NHX6, is a requisite for receptor mediated processing of storage proteins and trafficking of storage proteins to protein storage vacuoles.
CELLULAR AND MOLECULAR DETERMINANTS FOR CLIMACTERIC AND NON-CLIMACTERIC BEHAVIOR IN PLUM FRUITS
Japanese plums represent the most abundant and variable group among tree species and include most of the fresh-market plums commercialized worldwide. We characterized and compared two Japanese plum cultivars, “Santa Rosa” (SR) and its bud-sport mutant “Sweet Miriam” (SM). These cultivars share the same genetic background but display contrasting ripening behaviors (SR, climacteric and SM, non-climacteric). Both cultivars differ in their sugar metabolism conferring the SM fruits with unusual quality properties (lower glucose and fructose, higher sorbitol and galactose-metabolism related sugars, etc). The main objective of this research is to characterize the differences in the complex sugar metabolic pathways between the cultivars and their possible crosstalk with ethylene biosynthesis-related enzymes, underlying their climacteric and non-climacteric fruit ripening behaviors. Fruits from each each cultivar were harvested at an early (S2: pit hardening) and late (S4: fully-ripe) stages of fruit development and assessed using a Systems Biology approach. Transcriptomics, proteomics and metabolomics methodologies, together with targeted gene expression and enzymatic activity assays were analyzed to reveal complex sugar metabolic interrelations and identify differences between the cultivars that could be associated to the observed changes in sugar homeostasis as well as ethylene biosynthesis and ethylene signaling. This experimental system provides a unique tool to study metabolic pathways underlying climacteric and non-climacteric fruit ripening behaviors and offers several practical applications. Understanding mechanisms that allow fruits to ‘switch’ to a sorbitol-based metabolism would have a great industry impact, since sorbitol is an alternative and healthier natural sweetener to sucrose. In addition, it could also allow the identification of candidate genes for breeding programs focused on fruit quality improvement.