As calcium signal sensors, calcium-dependent protein kinases (CPKs) play vital roles in stimulating the production of secondary metabolites to participate in plant development and response to environmental stress. However, investigations of the Glycyrrhiza uralensis CPK family genes and their multiple functions are rarely reported. In this study, a total of 23 GuCPK genes in G. uralensis were identified, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, and promoter cis-acting elements were analyzed. Ten GuCPKs showed root-specific preferential expressions, and GuCPKs indicated different expression patterns under treatments of CaCl₂ and NaCl. In addition, under 2.5 mM of CaCl₂ and 30 mM of NaCl treatments, the diverse, induced expression of GuCPKs and significant accumulations of glycyrrhizic acid and flavonoids suggested the possible important function of GuCPKs in regulating the production of glycyrrhizic acid and flavonoids. Our results provide a genome-wide characterization of CPK family genes in G. uralensis, and serve as a foundation for understanding the potential function and regulatory mechanism of GuCPKs in promoting the biosynthesis of glycyrrhizic acid and flavonoids under salt stress.

L-Ascorbate (Asc) plays important roles in cell growth and plant development, and its de novo biosynthesis was catalyzed by the first rate-limiting enzyme VTC1. However, the function and regulatory mechanism of VTC1 involved in cell development is obscure in Gossypium hirsutum. Herein, the Asc content and AsA/DHA ratio were accumulated and closely linked with fiber development. The GhVTC1 encoded a typical VTC1 protein with functional conserved domains and expressed preferentially during fiber fast elongation stages. Functional complementary analysis of GhVTC1 in the loss-of-function Arabidopsis vtc1-1 mutants indicated that GhVTC1 is genetically functional to rescue the defects of mutants to normal or wild type (WT). The significant shortened primary root in vtc1-1 mutants was promoted to the regular length of WT by the ectopic expression of GhVTC1 in the mutants. Additionally, GhVTC1 expression was induced by ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the GhVTC1 promoter showed high activity and included two ethylene-responsive elements (ERE). Moreover, the 5′-truncted promoters containing the ERE exhibited increased activity by ACC treatment. Our results firstly report the cotton GhVTC1 function in promoting cell elongation at the cellular level, and serve as a foundation for further understanding the regulatory mechanism of Asc-mediated cell growth via the ethylene signaling pathway.

Ascorbate oxidase (AO) plays important roles in plant growth and development. Previously, we reported a cotton AO gene that acts as a positive factor in cell growth. Investigations on Gossypium hirsutum AO (GhAO) family genes and their multiple functions are limited. The present study identified eight GhAO family genes and performed bioinformatic analyses. Expression analyses of the tissue specificity and developmental feature of GhAOs displayed their diverse expression patterns. Interestingly, GhAO1A demonstrated the most rapid significant increase in expression after 1 h of light recovery from the dark. Additionally, the transgenic ao1-1/GhAO1A Arabidopsis lines overexpressing GhAO1A in the Arabidopsis ao1-1 late-flowering mutant displayed a recovery to the normal phenotype of wild-type plants. Moreover, compared to the ao1-1 mutant, the ao1-1/GhAO1A transgenic Arabidopsis presented delayed leaf senescence that was induced by the dark, indicating increased sensitivity to hydrogen peroxide (H₂O₂) under normal conditions that might be caused by a reduction in ascorbic acid (AsA) and ascorbic acid/dehydroascorbate (AsA/DHA) ratio. The results suggested that GhAOs are functionally diverse in plant development and play a critical role in light responsiveness. Our study serves as a foundation for understanding the AO gene family in cotton and elucidating the regulatory mechanism of GhAO1A in delaying dark-induced leaf senescence.

Myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) plays important roles in plant growth and development, stress responses, and cellular signal transduction. MIPS genes were found preferably expressed during fiber cell initiation and early fast elongation in upland cotton (Gossypium hirsutum), however, current understanding of the function and regulatory mechanism of MIPS genes to involve in cotton fiber cell growth is limited. Here, by genome-wide analysis, we identified four GhMIPS genes anchoring onto four chromosomes in G. hirsutum and analyzed their phylogenetic relationship, evolutionary dynamics, gene structure and motif distribution, which indicates that MIPS genes are highly conserved from prokaryotes to green plants, with further exon-intron structure analysis showing more diverse in Brassicales plants. Of the four GhMIPS members, based on the significant accumulated expression of GhMIPS1D at the early stage of fiber fast elongating development, thereby, the GhMIPS1D was selected to investigate the function of participating in plant development and cell growth, with ectopic expression in the loss-of-function Arabidopsis mips1 mutants. The results showed that GhMIPS1D is a functional gene to fully compensate the abnormal phenotypes of the deformed cotyledon, dwarfed plants, increased inflorescence branches, and reduced primary root lengths in Arabidopsis mips1 mutants. Furthermore, shortened root cells were recovered and normal root cells were significantly promoted by ectopic expression of GhMIPS1D in Arabidopsis mips1 mutant and wild-type plants respectively. These results serve as a foundation for understanding the MIPS family genes in cotton, and suggest that GhMIPS1D may function as a positive regulator for plant cell elongation.