BACKGROUND Foxtail millet grain has higher folate content than other cereal crops. However, the folate metabolite content and the expression patterns of folate metabolite‐related genes are unknown. RESULTS Liquid chromatography–mass spectrometry was used to investigate 12 folate metabolites in a foxtail millet panicle. The content of total folate and derivatives gradually decreased during panicle development. Polyglutamate 5‐formyl‐tetrahydrofolate was the major form. Twenty‐eight genes involved in the folate metabolic pathway were identified through bioinformatic analysis. These genes in Setaria italica, S. viridis and Zea mays showed genomic collinearity. Phylogenetic analysis revealed that the folate‐related genes were closely related among the C4 plants compared to C3 plants. The gene expressions were then studied at three panicle development stages. The gene expression patterns were classified into two groups, namely SiADCL1 and SiGGH as two key enzymes, which are responsible for folate synthesis and degradation; their expression levels were highest at the early panicle development stage, up to 179.11‐ and 163.88‐fold, respectively. Their expression levels had a similar downward trend during panicle development and were significantly positively correlated with the concentration of total folate and folate derivatives. However, SiSHMT3 expression levels were significantly negatively correlated with total folate concentration. CONCLUSION Besides being the major determinants of folate and folate derivatives accumulation, SiADCL1 and SiGGH expression levels are key limiting factors in the foxtail millet panicle. Therefore, SiADCL1 and SiGGH expression levels can be targeted in genetic modification studies to improve folate content in foxtail millet seeds in the future. © 2021 Society of Chemical Industry

The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT–PCR (qRT–PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT–PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.

Soil salinization conditions seriously restrict cotton yield and quality. Related studies have shown that the DUF4228 proteins are pivotal in plant resistance to abiotic stress. However, there has been no systematic identification and analysis of the DUF4228 gene family in cotton and their role in abiotic stress. In this study, a total of 308 DUF4228 genes were identified in four Gossypium species, which were divided into five subfamilies. Gene structure and protein motifs analysis showed that the GhDUF4228 proteins were conserved in each subfamily. In addition, whole genome duplication (WGD) events and allopolyploidization might play an essential role in the expansion of the DUF4228 genes. Besides, many stress-responsive (MYB, MYC) and hormone-responsive (ABA, MeJA) related cis-elements were detected in the promoters of the DUF4228 genes. The qRT-PCR results showed that GhDUF4228 genes might be involved in the response to abiotic stress. VIGS assays and the measurement of relative water content (RWC), Proline content, POD activity, and malondialdehyde (MDA) content indicated that GhDUF4228-67 might be a positive regulator of cotton response to salt stress. The results in this study systematically characterized the DUF4228s in Gossypium species and will provide helpful information to further research the role of DUF4228s in salt tolerance.