Yim Lab of Plant Genomics

University of Nevada, Reno

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Crassulacean Acid Metabolism Abiotic Stress-Responsive Transcription Factors: a Potential Genetic Engineering Approach for Improving Crop Tolerance to Abiotic Stress

This perspective paper explores the utilization of abiotic stress-responsive transcription factors (TFs) from crassulacean acid metabolism (CAM) plants to improve abiotic stress tolerance in crop plants. CAM is a specialized type of photosynthetic adaptation that enhances water-use efficiency (WUE) by shifting CO2uptake to all or part of the nighttime when evaporative water losses are minimal. Recent studies have shown that TF-based genetic engineering could be a useful approach for improving plant abiotic stress tolerance because of the role of TFs as master regulators of clusters of stress-responsive genes. Here, we explore the use of abiotic stress-responsive TFs from CAM plants to improve abiotic stress tolerance and WUE in crops by controlling the expression of gene cohorts that mediate drought-responsive adaptations. Recent research has revealed several TF families including AP2/ERF, MYB, WRKY, NAC, NF-Y, and bZIPthat might regulate water-deficit stress responses and CAM in the inducible CAM plant Mesembryanthemum crystallinum under water-deficit stress-induced CAM and in the obligate CAM plant Kalanchoe fedtschenkoi. Overexpression of genes from these families in Arabidopsis thaliana can improve abiotic stress tolerance in A. thaliana in some instances. Therefore, we propose that TF-based genetic engineering with a small number of CAM abiotic stress-responsive TFs will be a promising strategy for improving abiotic stress tolerance and WUE in crop plants in a projected hotter and drier landscape in the 21st-century and beyond.

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. Introducing the CAM photosynthetic machinery into C3 (or C4) photosynthesis plants (CAM Biodesign) represents a potentially breakthrough strategy for improving WUE while maintaining high productivity. To optimize the success of CAM Biodesign approaches, the functional analysis of individual C4 metabolism cycle genes is necessary to identify the essential genes for robust CAM pathway introduction. Here, we isolated and analyzed the subcellular localizations of 13 enzymes and regulatory proteins of the C4 metabolism cycle of CAM from the common ice plant in stably transformed Arabidopsis thaliana. Six components of the carboxylation module were analyzed including beta-carbonic anhydrase (McBCA2), phosphoenolpyruvate carboxylase (McPEPC1), phosphoenolpyruvate carboxylase kinase (McPPCK1), NAD-dependent malate dehydrogenase (McNAD-MDH1, McNAD-MDH2), and NADP-dependent malate dehydrogenase (McNADP-MDH1). In addition, seven components of the decarboxylation module were analyzed including NAD-dependent malic enzyme (McNAD-ME1, McNAD-ME2), NADP-dependent malic enzyme (McNADP-ME1, NADP-ME2), pyruvate, orthophosphate dikinase (McPPDK), pyruvate, orthophosphate dikinase-regulatory protein (McPPDK-RP), and phosphoenolpyruvate carboxykinase (McPEPCK). Ectopic overexpression of most C4-metabolism cycle components resulted in increased rosette diameter, leaf area, and leaf fresh weight of A. thaliana except for McNADP-MDH1, McPPDK-RP, and McPEPCK.Overexpression of most carboxylation module components resulted in increased stomatal conductance and dawn/dusk titratable acidity (TA) as an indirect measure of organic acid (mainly malate) accumulation in A. thaliana. In contrast, overexpression of the decarboxylating malic enzymes reduced stomatal conductance and TA. This comprehensive study provides fundamental insights into the relative functional contributions of each of the individual components of the core C4-metabolism cycle of CAM and represents a critical first step in laying the foundation for CAM Biodesign.

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Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resulting in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules.