Although the significance of some plant WRKYs in response to cold stress have been identified, the molecular mechanisms of most WRKYs remain unclear in grapevine. In this study, we demonstrate that cold-induced expression of VaBAM3 in Vitis amurensis executes a beneficial role in enhancing resistance by the regulating starch decomposition. VaWRKY65 was identified as an upstream transcriptional activator of VaBAM3 through yeast one-hybrid library screening and validated to directly interact with the W-box region inside the VaBAM3 promoter. Transgenic Arabidopsis thaliana plants and grapevine roots overexpression VaWRKY65 exhibited improved cold tolerance along with higher BAM activity and soluble sugar levels, whereas opposite changes were observed in VaWRKY65 knockdown lines created by virus-induced gene silencing (VIGS) in grapevine plants and in the knockout wrky65 mutants generated by CRISPR/Cas9 technology in grapevine roots. The transcriptome data show that overexpression of VaWRKY65 led to significant alteration of a diverse set of stress-related genes at the transcriptional level. One of the genes, Peroxidase 36 (VaPOD36), was further verified as a direct target of VaWRKY65. Consistently, VaWRKY65-overexpressing plants had higher VaPOD36 transcript levels and POD activity but a reduced ROS level, while silencing VaWRKY65 results in contrary changes. Collectively, these results reveal that VaWRKY65 enhanced cold tolerance through modulating soluble sugars produced from starch breakdown and ROS scavenging.

Xyloglucan endotransglycosylase/hydrolase (XTH) is a cell-wall-modifying enzyme participating in diverse cell morphogenetic processes and adaptation to stress. In this study, 48 XTH genes were identified from two pineapple (Ananas comosus) cultivars (‘F153’ and ‘MD2’) and designated Ac(F153)XTH1 to -24 and Ac(MD2)XTH1 to -24 based on their orthology with Arabidopsis thaliana genes. Endoglucanase family 16 members were identified in addition to XTHs of glycoside hydrolase family 16. Phylogenetic analysis clustered the XTHs into three major groups (Group I/II, III and Ancestral Group) and Group III was subdivided into Group IIIA and Group IIIB. Similar gene structure and motif number were observed within a group. Two highly conserved domains, glycosyl hydrolase family 16 (GH16-XET) and xyloglucan endotransglycosylase C-terminus (C-XET), were detected by multiple sequences alignment of all XTHs. Segmental replication were detected in the two cultivars, with only the paralogous pair Ac(F153)XTH7-Ac(F153)XTH18 presented in ‘F153’ prior to genomic expansion. Transcriptomic analysis indicated that XTHs were involved in the regulation of fruit ripening and crassulacean acid metabolism with tissue specificity and quantitative real-time PCR analysis suggested that Ac(MD2)XTH18 was involved in root growth. The results enhance our understanding of XTHs in the plant kingdom and provide a basis for further studies of functional diversity in A. comosus.

Cissus rotundifolia Lam. is an evergreen climber that mainly distributed in the tropical savannas of eastern Africa with excellent drought resistance. While the mechanism underlying drought stress response in C. rotundifolia remains unknown. In this study, we investigated the transcriptomics and flavonoid metabolomics responses of C. rotundifolia leaves under different drought conditions. We identified a total of 2401 differentially expressed genes (DEGs) in drought-treated leaves. The continuously increasing DEGs were significantly enriched in pathways related to phenylpropanoid biosynthesis, flavonoid biosynthesis, and galactose metabolism. The total flavonoids content were also found increased significantly during drought treatment in the leaves of C. rotundifolia. A total of 57 distinct flavonoids were identified using UPLC-MS/MS. Among them, nine out of ten differentially accumulated metabolites (DAMs) displayed notable accumulation profiles under drought conditions. These DAMs included two flavanols [(-)-Catechin gallate and (-)-gallocatechin gallate], two flavonols (myricetin and astragalin), four flavones (orientin, cynaroside, isoorientin, and vitexin), and one flavanone (naringenin-7-glucoside), indicating their pivotal roles in drought response. Additionally, a DEG with continuously high-expression, annotated flavone synthase (FLS) synthesises, and synthetizing myricetin under drought stress was tightly related to additional genes belonging to ABA-signaling genes, stomata movement genes, transcription factors, and protein kinases, these genes were contained Top 10 hub-genes of network constructed all DEGs. The results reflect the significance of FLS and potentially regulatory genes under drought conditions. These findings suggest that flavonoids play a pivotal role in the drought stress response of C. rotundifolia, advancing our understanding of flavonoids accumulation and the transcriptional regulation involved in this process.

Cissus rotundifolia Lam. is used as a medicinal herb and vegetable. Flavonoids are the major components for the therapeutic effects. However, flavonoids constituents and expression profiles of related genes in C. rotundifolia organs are unknown. Colorimetric assay showed the highest flavonoid concentration in roots compared to the stem and leaf. Widely target-based metabolome analysis allowed tentative identification of 199 compounds in three organs. Flavonols and flavones were the dominant flavonoids subclasses. Among the metabolites, 171 were common in the three organs. Unique accumulation profile was observed in the root while the stem and leaf exhibited relatively similar patterns. In the root, six unique compounds (jaceosidin, licoagrochalcone D, 8-prenylkaempferol, hesperetin 7-O-(6″malonyl) glucoside, aureusidin, apigenin-4′-O-rhamnoside) that are used for medicinal purposes were detected. In total, 18,427 expressed genes were identified from transcriptome of the three organs covering about 60% of annotated genes in C. rotundifolia genome. Fourteen gene families, including 52 members involved in the main pathway of flavonoids biosynthesis, were identified. Their expression could be found in at least one organ. Most of the genes were highly expressed in roots compared to other organs, coinciding with the metabolites profile. The findings provide fundamental data for exploration of metabolites biosynthesis in C. rotundifolia and diversification of parts used for medicinal purposes.

Cissus quadrangularis is a tetraploid species belonging to the Vitaceae family and is known for the Crassulacean acid metabolism (CAM) pathway in the succulent stem, while the leaves perform C₃ photosynthesis. Here, we report a high-quality genome of C. quadrangularis comprising a total size of 679.2Mb which was phased into two subgenomes. Genome annotation identified 51,857 protein-coding genes, while approximately 47.75% of the genome was composed of repetitive sequences. Gene expression ratios of two subgenomes demonstrated that the sub-A genome as the dominant subgenome played the vital role during the drought tolerance. Genome divergence analysis suggests that the tetraploidization event occurred around 8.9 million years ago. Transcriptome data revealed that pathways related to cutin, suberine, and wax metabolism were enriched in the stem during drought treatment, suggesting that these genes contributed to the drought adaption. Additionally, a subset of CAM-related genes displayed diurnal expression patterns in the succulent stems but not in leaves, indicating that stem-biased expression of existing genes contributed to the CAM evolution. Our findings provide insights into the mechanisms of drought adaptation and photosynthesis transition in plants.