Transcriptome profiling, physiological, and biochemical analyses provide new insights towards drought stress response in sugar maple (Acer saccharum Marshall) saplings

Sugar maple (Acer saccharum Marshall) is a temperate tree species in the northeastern parts of the United States and is economically important for its hardwood and syrup production. Sugar maple trees are highly vulnerable to changing climatic conditions, especially drought, so understanding the physiological, biochemical, and molecular responses is critical. The sugar maple saplings were subjected to drought stress for 7, 14, and 21 days and physiological data collected at 7, 14, and 21 days after stress (DAS) showed significantly reduced chlorophyll and Normalized Difference Vegetation Index with increasing drought stress time. The drought stress-induced biochemical changes revealed a higher accumulation of malondialdehyde, proline, and peroxidase activity in response to drought stress. Transcriptome analysis identified a total of 14,099 differentially expressed genes (DEGs); 328 were common among all stress periods. Among the DEGs, transcription factors (including NAC, HSF, ZFPs, GRFs, and ERF), chloroplast-related and stress-responsive genes such as peroxidases, membrane transporters, kinases, and protein detoxifiers were predominant. GO enrichment and KEGG pathway analysis revealed significantly enriched processes related to protein phosphorylation, transmembrane transport, nucleic acids, and metabolic, secondary metabolite biosynthesis pathways, circadian rhythm-plant, and carotenoid biosynthesis in response to drought stress. Time-series transcriptomic analysis revealed changes in gene regulation patterns in eight different clusters, and pathway analysis by individual clusters revealed a hub of stress-responsive pathways. In addition, qRT-PCR validation of selected DEGs revealed that the expression patterns were consistent with transcriptome analysis. The results from this study provide insights into the dynamics of physiological, biochemical, and gene responses to progressive drought stress and reveal the important stress-adaptive mechanisms of sugar maple saplings in response to drought stress

DataSheet_1_Co-expression of stress-responsive regulatory genes, MuNAC4, MuWRKY3 and MuMYB96 associated with resistant-traits improves drought adaptation in transgenic groundnut (Arachis hypogaea l.) plants.doc

Groundnut, cultivated under rain-fed conditions is prone to yield losses due to intermittent drought stress. Drought tolerance is a complex phenomenon and multiple gene expression required to maintain the cellular tolerance. Transcription factors (TFs) regulate many functional genes involved in tolerance mechanisms. In this study, three stress-responsive regulatory TFs cloned from horse gram, (Macrotyloma uniflorum (Lam) Verdc.), MuMYB96, involved in cuticular wax biosynthesis; MuWRKY3, associated with anti-oxidant defense mechanism and MuNAC4, tangled with lateral root development were simultaneously expressed to enhance drought stress resistance in groundnut (Arachis hypogaea L.). The multigene transgenic groundnut lines showed reduced ROS production, membrane damage, and increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzyme activity, evidencing improved antioxidative defense mechanisms under drought stress. Multigene transgenic plants showed lower proline content, increased soluble sugars, epicuticular wax content and higher relative water content suggesting higher maintenance of tissue water status compared to wildype and mock plants. The scanning electron microscopy (SEM) analysis showed a substantial increase in deposition of cuticular waxes and variation in stomatal number in multigene transgenic lines compared to wild type and mock plants. The multigene transgenic plants showed increased growth of lateral roots, chlorophyll content, and stay-green nature in drought stress compared to wild type and mock plants. Expression analysis of transgenes, MuMYB96, MuWRKY3, and MuNAC4 and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16 and DBP using qRT-PCR showed a two- to four-fold increase in transcript levels in multigene transgenic groundnut plants over wild type and mock plants under drought stress. Our study demonstrate that introducing multiple genes with simultaneous expression of genes is a viable option to improve stress tolerance and productivity under drought stress.</p

DataSheet_2_Co-expression of stress-responsive regulatory genes, MuNAC4, MuWRKY3 and MuMYB96 associated with resistant-traits improves drought adaptation in transgenic groundnut (Arachis hypogaea l.) plants.doc

Groundnut, cultivated under rain-fed conditions is prone to yield losses due to intermittent drought stress. Drought tolerance is a complex phenomenon and multiple gene expression required to maintain the cellular tolerance. Transcription factors (TFs) regulate many functional genes involved in tolerance mechanisms. In this study, three stress-responsive regulatory TFs cloned from horse gram, (Macrotyloma uniflorum (Lam) Verdc.), MuMYB96, involved in cuticular wax biosynthesis; MuWRKY3, associated with anti-oxidant defense mechanism and MuNAC4, tangled with lateral root development were simultaneously expressed to enhance drought stress resistance in groundnut (Arachis hypogaea L.). The multigene transgenic groundnut lines showed reduced ROS production, membrane damage, and increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzyme activity, evidencing improved antioxidative defense mechanisms under drought stress. Multigene transgenic plants showed lower proline content, increased soluble sugars, epicuticular wax content and higher relative water content suggesting higher maintenance of tissue water status compared to wildype and mock plants. The scanning electron microscopy (SEM) analysis showed a substantial increase in deposition of cuticular waxes and variation in stomatal number in multigene transgenic lines compared to wild type and mock plants. The multigene transgenic plants showed increased growth of lateral roots, chlorophyll content, and stay-green nature in drought stress compared to wild type and mock plants. Expression analysis of transgenes, MuMYB96, MuWRKY3, and MuNAC4 and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16 and DBP using qRT-PCR showed a two- to four-fold increase in transcript levels in multigene transgenic groundnut plants over wild type and mock plants under drought stress. Our study demonstrate that introducing multiple genes with simultaneous expression of genes is a viable option to improve stress tolerance and productivity under drought stress.</p