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.

Monodehydroasorbate reductase (MDHAR) (EC1.6.5.4), a key enzyme in ascorbate-glutathione recycling, plays important roles in cell growth, plant development and physiological response to environmental stress via control of ascorbic acid (AsA)-mediated reduction/oxidation (redox) regulation. Until now, information regarding MDHAR function and regulatory mechanism in Gossypium have been limited. Herein, a genome-wide identification and comprehensive bioinformatic analysis of 36 MDHAR family genes in four Gossypium species, Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, were performed, indicating their close evolutionary relationship. Expression analysis of GhMDHARs in different cotton tissues and under abiotic stress and phytohormone treatment revealed diverse expression features. Fiber-specific expression analysis showed that GhMDHAR1A/D, 3A/D and 4A/D were preferentially expressed in fiber fast elongating stages to reach peak values in 15-DPA fibers, with corresponding coincident observances of MDHAR enzyme activity, AsA content and ascorbic acid/dehydroascorbic acid (AsA/DHA) ratio. Meanwhile, there was a close positive correlation between the increase of AsA content and AsA/DHA ratio catalyzed by MDHAR and fiber elongation development in different fiber-length cotton cultivars, suggesting the potential important function of MDHAR for fiber growth. Following H₂O₂ stimulation, GhMDHAR demonstrated immediate responses at the levels of mRNA, enzyme, the product of AsA and corresponding AsA/DHA value, and antioxidative activity. These results for the first time provide a comprehensive systemic analysis of the MDHAR gene family in plants and the four cotton species and demonstrate the contribution of MDHAR to fiber elongation development by controlling AsA-recycling-mediated cellular redox homeostasis.

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.

Brown cotton is a major cultivar of naturally colored cotton, and brown cotton fibers (BCFs) are widely utilized as raw materials for textile industry production due to their advantages of being green and dyeing-pollution-free. However, the mechanisms underlying the pigmentation in fibers are still poorly understood, which significantly limits their extensive applications in related fields. In this study, we conducted a multidimensional comparative analysis of the transcriptomes and metabolomes between brown and white fibers at different developmental periods to identify the key genes and pathways regulating the pigment deposition. The transcriptomic results indicated that the pathways of flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched regulatory pathways, especially in the late development periods of fiber pigmentation; furthermore, the genes distributed in the pathways of PAL, CHS, F3H, DFR, ANR, and UFGT were identified as significantly up-regulated genes. The metabolic results showed that six metabolites, namely (−)-Epigallocatechin, Apiin, Cyanidin-3-O-glucoside, Gallocatechin, Myricetin, and Poncirin, were significantly accumulated in brown fibers but not in white fibers. Integrative analysis of the transcriptomic and metabolomic data demonstrated a possible regulatory network potentially regulating the pigment deposition, in which three MYB transcription factors promote the expression levels of flavonoid biosynthesis genes, thereby inducing the content increase in (−)-Epigallocatechin, Cyanidin-3-O-glucoside, Gallocatechin, and Myricetin in BCFs. Our findings provide new insights into the pigment deposition mechanism in BCFs and offer references for genetic engineering and breeding of colored cotton materials.

Phenylalanine ammonia-lyase (PAL) serves as a pivotal regulatory enzyme at the initial branching point of the phenylpropanoid pathway, exerting a profound influence on downstream reactions essential for flavonoid biosynthesis. Glycyrrhiza species are important medicinal plants and provide plenty of roots as raw materials for further utilization, with the components of glycyrrhizic acid and flavonoids as two major active ingredients. However, functional studies of the PAL genes in the medicinal Glycyrrhiza species remain limited. In this study, we identified seven PAL family genes from each of the three medicinal Glycyrrhiza species, Glycyrrhiza uralensis Fisch., G. inflata Bat., and G. glabra L., and comprehensively analyzed their phylogenetic relationships, gene structures, motif distributions, and promoter cis-elements. Gene expression profiling revealed that PAL1 is highly expressed in roots and significantly induced by drought and salt stresses. We further selected G. uralensis GuPAL1 for functional investigation in Arabidopsis. GuPAL1-overexpression lines (GuPAL1-OE) demonstrated significant enhancements in plant growth, flavonoid accumulation, and hormone levels in Arabidopsis thaliana. Conversely, the Atpal1 mutant plants displayed marked reductions in these traits, while the transgenic lines of GuPAL1-OE in the Atpal1 mutant (Atpal1/GuPAL1) recovered to the normal phenotypes similar to wild type (WT). Transcriptomic analysis of the GuPAL1-OE plants compared to WT demonstrated that several key genes in the phenylpropanoid and flavonoid metabolic pathways (4CL, CCoAOMT, CAD, POD, F3H, FLS) were significantly enriched, suggesting that GuPAL1 may promote plant growth and flavonoid biosynthesis by regulating diverse cellular functions, metabolic pathways, and associated gene expressions. These findings highlight the functional importance of GuPAL1 in flavonoid biosynthesis, and provide valuable insights into the molecular mechanisms underlying the medicinal properties of Glycyrrhiza species.