Trehalose-6-phosphate phosphatase (TPP) is a key enzyme involved in trehalose synthesis in higher plants. Previous studies have shown that TPP family genes increase yields without affecting plant growth under drought conditions, but their functions in cotton have not been reported. In this study, 17, 12, 26 and 24 TPP family genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. The 79 TPP family genes were divided into three subgroups by phylogenetic analysis. Virus-induced gene silencing (VIGS) of GhTPP22 produced TRV::GhTPP22 plants that were more sensitive to drought stress than the control plants, and the relative expression of GhTPP22 was decreased, as shown by qRT–PCR. Moreover, we analysed the gene structure, targeted small RNAs, and gene expression patterns of TPP family members and the physicochemical properties of their encoded proteins. Overall, members of the TPP gene family in cotton were systematically identified, and the function of GhTPP22 under drought stress conditions was preliminarily verified. These findings provide new information for improving drought resistance for cotton breeding in the future.

Glycerol-3-phosphate dehydrogenase (GPDH) is a key enzyme in plant glycerol synthesis and metabolism, and plays an important role in plant resistance to abiotic stress. Here, we identified 6, 7, 14 and 14 GPDH genes derived from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. Phylogenetic analysis assigned these genes into three classes, and most of the genes within the family were expanded by whole-genome duplication (WGD) and segmental duplications. Moreover, determination of the nonsynonymous substitution rate/synonymous substitution rate (Ka/Ks) ratio showed that the GPDH had an evolutionary preference for purifying selection. Transcriptome data revealed that GPDH genes were more active in the early stages of fiber development. Additionally, numerous stress-related cis-elements were identified in the potential promoter region. Then, a protein–protein-interaction (PPI) network of GPDH5 in G. hirsutum was constructed. In addition, we predicted 30 underlying miRNAs in G. hirsutum. Functional validation results indicated that silencing GhGPDH5 diminished drought tolerance in the upland cotton TM-1 line. In summary, this study provides a fundamental understanding of the GPDH gene family in cotton, GhGPDH5 exerts a positive effect during drought stress and is potentially involved in stomatal closure movements.

YTH domain-containing proteins are one kind of RNA-binding protein involved in post-transcriptional regulation and play multiple roles in regulating the growth, development, and abiotic stress responses of plants. However, the YTH domain-containing RNA-binding protein family has not been previously studied in cotton. In this study, a total of 10, 11, 22, and 21 YTH genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium barbadense, and Gossypium hirsutum, respectively. These Gossypium YTH genes were categorized into three subgroups by phylogenetic analysis. The chromosomal distribution, synteny analysis, structures of Gossypium YTH genes, and the motifs of YTH proteins were analyzed. Furthermore, the cis-element of GhYTH genes promoter, miRNA targets of GhYTH genes, and subcellular localization of GhYTH8 and GhYTH16 were characterized. Expression patterns of GhYTH genes in different tissues, organs, and in response to different stresses were also analyzed. Moreover, functional verifications revealed that silencing GhYTH8 attenuated the drought tolerance in the upland cotton TM-1 line. These findings provide useful clues for the functional and evolutionary analysis of YTH genes in cotton.

Chilling stress seriously affects the growth and yield of tomato. Anthocyanin is a typical chilling-induced metabolite with strong antioxidant activity and photoprotective capacity. Here, we found that anthocyanin was also involved in ascorbic acid biosynthesis and glycolysis under chilling stress. SlAN2 is an important positive gene in anthocyanin biosynthesis. The results of physiological indicators showed that SlAN2 overexpression lines (A189) had a greater ability to tolerate cold stress than wild-type (WT) plants. Conjoint analysis of transcriptomics and metabonomics of A189 lines and WT plants was used to analyze the metabolic difference and the cold resistance mechanisms caused by anthocyanin under chilling stress. The anthocyanin accumulated more in A189 than that in WT under chilling stress at 4 °C for 24 h, which led to hexoses and ascorbic acid increased significantly. Results indicate that SlAN2 overexpression reduces the expression of key enzyme genes in glycolytic pathway such as phosphofructokinase (PFK) and pyruvate kinase (PK) genes, weakens glycolysis ability, and promotes accumulation of hexoses in A189 lines at 4 °C for 24 h compared with wild lines. Additionally, ascorbic acid content is increased by up-regulated the genes of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR). The increased hexose content can reduce cell osmotic potential, freezing point and synthesize more ascorbic acid, while the increased ascorbic acid content can enhance the ability to scavenge reactive oxygen species, so improves the cold resistance of tomato. The glycolysis and ascorbic acid metabolism pathway mediated by SlAN2 provides a new insight for the molecular mechanism of anthocyanins in improving the cold resistance of tomato and provides a new theoretical basis for cultivating new cold-tolerant tomato varieties.