Drought stress is a most critical limitation to plant growth and productivity. Plants have complex adaptation mechanisms that include Ca²⁺ signaling as a focal secondary messenger. Calmodulin (CaM) is presumed to be one of the primary Ca²⁺ signature-decoding molecules. Genome-wide screening of expression libraries using labeled recombinant CaM has revealed that AtGT-2 (GT elements-binding proteins) family are potential CaM binding transcription factors. AtGTL1, one of the AtGT-2 family, encodes a putative Ca²⁺/CaM-binding transcriptional activator. gtl1 T-DNA insertional mutations (gtl1-1, gtl1-2 and gtl1-3) substantially enhance the capacity of plants to survive in response to severe water deficit stress by which maintain leaf relative water content through reduced transpiration. gtl1 plants exhibit reduced stomatal density in abaxial leaves and increased trichome density/size in adaxial leaves, which may reduce transpiration. AtGTL1 expression is down-regulated by dehydration stress, which is consistent with the notion that the transcription factor is a negative regulator of drought adaptation response, which is important to maintain homeostasis for adaptation processes. Gene expression analysis by RT-PCR revealed that GTL1 regulates DREB2A expression in ABA-independent pathway, not ABA-dependent gene expression, suggesting that GTL1 mediates dehydration signal necessary for DREB2A expression. We hypothesize that Ca²⁺/CaM-mediated GTL1 regulates drought stress adaptation through mechanism by which is linked to efficient water usage process. This research will provide functional understanding about how plants decode Ca²⁺/CaM signals to initiate stress adaptation processes that could enhance crop yield stability under water deficit conditions.

In view of the anticipated severe global shortage of water and desertification, much needs to be done to improve the efficiency of water acquisition by plants, and to improve plant tolerance to extreme water-deficient conditions. It is well accepted that future developments in these directions must be based on comprehensive understanding of the molecular and cellular processes that occur in the plant in stress situations. The proposed research seeks to reveal the changes in carbon metabolism and partitioning under drought stress, and to elucidate the metabolic and gene networks underlying these changes in the model plant Arabidopsis thaliana. The focus of the proposed research is a family of transcription factors (designated GTLs) that bind calcium/calmodulin, whose function is important for drought tolerance (preliminary unpublished results). Their known downstream target genes encode proteins functioning in the chloroplast and mitochondria and are involved in regulating carbon metabolism and energy balance. The proposed research includes the following approaches and methodologies: (1) Identifying all the downstream target genes of GTLs by chromatin immuno-precipitation and hybridization of immuno-enriched DNA fragments to genomic DNA chips (ChIP on chip). This approach will be complemented by in vitro DNA-protein binding assays [Israel] and investigation of cell-specific expression of GTLs under control and stress conditions [USA]; (2) Studies of GTL transcription activity in vivo and effects of cellular signals and calmodulin on transcription [Israel]; (3) physiological, transcriptome and metabolome investigation of genetically engineered plants and mutants under dehydration and rehydration conditions [USA]; (4) Bioinformatic studies of GTL DNA-target sites [Israel], and the topology of metabolic and expressed gene networks [USA]. The proposed research is expected to provide novel information on carbon metabolism and partitioning under stress, and the cellular factors that underlie these metabolic activities. These cellular processes and their physiological consequences must be taken into consideration in future strategies of crop improvement for harsh environments.

Our studies have determined that the SUMO (small ubiquitin-related modifier) E3 ligase AtSIZ1 facilitates cold-induced CBF3 expression and cold acclimation in Arabidopsis. SIZ1-dependent sumoylation of ICE1 (CBF activator) is necessary for CBF activation and freezing tolerance. The project objectives are to determine the mechanisms by which SIZ1 mediated, SUMO-conjugated ICE1 activates CBF expression and represses expression of MYB15 (CBF3 repressor), and establish that, like CBF, SIZ1 and ICE1 orthologs function in freezing tolerance of crops such as rice and tomato. Specifically, Objective 1 will determine if sumoylated ICE1 activates CBF3 and represses MYB15 expression through remodeling of CBF3 and/or MYB15 chromatin. Alternatively, sumoylation of ICE1 may affect DNA-binding activity or protein-protein interaction or subnuclear compartmentalization of the transcription factor, which will be assessed by ChIP analysis, proteomics, and fluorescence imaging. Objective 2 is to determine if sumoylation activates ICE1 or if other post-translational modification processes are linked to SUMO conjugation. Previously, we determined that K393 is the target residue for sumoylation of ICE and recent results implicate S403 as a possible phosphorylation or O-linked β-N-acetylglucosamine (O-GlcNAc) conjugation site, which will be resolved by co-immunopreciptation assays. Objective 3 is to provide molecular genetic evidence that the SIZ1-ICE1 mediated freezing tolerance process and the post-translational regulatory mechanisms (sumoylation and phosphorylation or O-GlcNAc conjugation) of ICE1 are conserved in rice and tomato. The proposed research is focused to provide novel discovery information about cold signaling and gene expression regulatory mechanisms that mediate freezing tolerance that are conserved in plants and applicable to crops.