Ling Min

Sugar and auxin signaling pathways respond to high temperature stress during anther development as revealed by transcript profiling analysis in cotton

Anther indehiscence at high temperatures is coordinately regulated by sugar and auxin. Male reproduction in flowering plants is highly sensitive to high temperature (HT). To investigate molecular mechanisms of the response of cotton (Gossypium hirsutum) anthers to HT, a relatively complete comparative transcriptome analysis was performed during anther development of cotton lines 84021 and H05 under normal temperature and HT conditions. In total, 4,599 differentially expressed genes were screened; the differentially expressed genes were mainly related to epigenetic modifications, carbohydrate metabolism, and plant hormone signaling. Detailed studies showed that the deficiency in S-ADENOSYL-l-HOMOCYSTEINE HYDROLASE1 and the inhibition of methyltransferases contributed to genome-wide hypomethylation in H05, and the increased expression of histone constitution genes contributed to DNA stability in 84021. Furthermore, HT induced the expression of CASEIN KINASEI (GhCKI) in H05, coupled with the suppression of starch synthase activity, decreases in glucose level during anther development, and increases in indole-3-acetic acid (IAA) level in late-stage anthers. The same changes also were observed in Arabidopsis (Arabidopsis thaliana) GhCKI overexpression lines. These results suggest that GhCKI, sugar, and auxin may be key regulators of the anther response to HT stress. Moreover, PHYTOCHROME-INTERACTING FACTOR genes (PIFs), which are involved in linking sugar and auxin and are regulated by sugar, might positively regulate IAA biosynthesis in the cotton anther response to HT. Additionally, exogenous IAA application revealed that high background IAA may be a disadvantage for late-stage cotton anthers during HT stress. Overall, the linking of HT, sugar, PIFs, and IAA, together with our previously reported data on GhCKI, may provide dynamic coordination of plant anther responses to HT stress.

Cotton WRKY1 Mediates the Plant Defense-to-Development Transition during Infection of Cotton by Verticillium dahliae by Activating JASMONATE ZIM-DOMAIN1 Expression

A transcription factor regulates plant development during pathogen infection by attenuating jasmonate signaling. Plants have evolved an elaborate signaling network to ensure an appropriate level of immune response to meet the differing demands of developmental processes. Previous research has demonstrated that DELLA proteins physically interact with JASMONATE ZIM-DOMAIN1 (JAZ1) and dynamically regulate the interaction of the gibberellin (GA) and jasmonate (JA) signaling pathways. However, whether and how the JAZ1-DELLA regulatory node is regulated at the transcriptional level in plants under normal growth conditions or during pathogen infection is not known. Here, we demonstrate multiple functions of cotton (Gossypium barbadense) GbWRKY1 in the plant defense response and during development. Although GbWRKY1 expression is induced rapidly by methyl jasmonate and infection by Verticillium dahliae, our results show that GbWRKY1 is a negative regulator of the JA-mediated defense response and plant resistance to the pathogens Botrytis cinerea and V. dahliae. Under normal growth conditions, GbWRKY1-overexpressing lines displayed GA-associated phenotypes, including organ elongation and early flowering, coupled with the down-regulation of the putative targets of DELLA. We show that the GA-related phenotypes of GbWRKY1-overexpressing plants depend on the constitutive expression of Gossypium hirsutum GhJAZ1. We also show that GhJAZ1 can be transactivated by GbWRKY1 through TGAC core sequences, and the adjacent sequences of this binding site are essential for binding specificity and affinity to GbWRKY1, as revealed by dual-luciferase reporter assays and electrophoretic mobility shift assays. In summary, our data suggest that GbWRKY1 is a critical regulator mediating the plant defense-to-development transition during V. dahliae infection by activating JAZ1 expression.

Asymmetric subgenome selection and cis -regulatory divergence during cotton domestication

Comparative population genomics offers an excellent opportunity for unraveling the genetic history of crop domestication. Upland cotton (Gossypium hirsutum) has long been an important economic crop, but a genome-wide and evolutionary understanding of the effects of human selection is lacking. Here, we describe a variation map for 352 wild and domesticated cotton accessions. We scanned 93 domestication sweeps occupying 74 Mb of the A subgenome and 104 Mb of the D subgenome, and identified 19 candidate loci for fiber-quality-related traits through a genome-wide association study. We provide evidence showing asymmetric subgenome domestication for directional selection of long fibers. Global analyses of DNase I–hypersensitive sites and 3D genome architecture, linking functional variants to gene transcription, demonstrate the effects of domestication on cis-regulatory divergence. This study provides new insights into the evolution of gene organization, regulation and adaptation in a major crop, and should serve as a rich resource for genome-based cotton improvement.

Expression profile analysis of genes involved in cell wall regeneration during protoplast culture in cotton by suppression subtractive hybridization and macroarray

The molecular mechanisms underlying cell wall biosynthesis are poorly understood. In this study, microscopic analysis showed that protoplasts generated a new cell wall within 48 h after transfer to a wall-regeneration medium. To identify genes related to cell wall biosynthesis in cotton, suppression subtractive hybridization was used to visualize differential gene expression at seven time points within the first 48 h. In total, 412 differentially expressed sequence tags (ESTs; >3-fold) were identified, and 210 unigenes were sequenced successfully. As confirmed by reverse-transcription PCR (RT-PCR) and real-time quantitative reverse-transcription PCR (QRT-PCR) analysis, the selected genes displayed complex expression patterns during cell wall regeneration from protoplasts and included most previously published cell-wall-associated genes. ESTs similar to cell-wall-protein genes, such as proline-rich protein (PRPL), glycine-rich protein (GRP), extension (EPR1), fasciclin-like arabinogalactan protein (FLA2), and expensing-like protein (EXLA and EXLB), which might participate in primary cell wall or secondary cell wall construction and modification, were up-regulated during cell wall regeneration from protoplasts. Sucrose synthase, an important enzyme in the sugar signalling pathway, played important roles in cellulose biosynthesis. Our findings also highlighted the function of some transcription factors during cell wall regeneration from protoplasts, including the squamosa promoter binding protein-like 14 (SPL14), NAC, Gbiaa-re, MYB, WRKY, swellmap 1 (SMP1), RAD5, and zinc finger family protein, as well as the enrichment of Ca2+-calmodulin signal molecules. On the basis of the gene expression profiles, a model of cell wall regeneration from protoplasts derived from cotton suspension cultures is proposed.

Cotton cytochrome P450 CYP82D regulates systemic cell death by modulating the octadecanoid pathway

Plant oxylipins are derived from unsaturated fatty acids and play roles in plant growth and development as well as defence. Although recent studies have revealed that fatty acid metabolism is involved in systemic acquired resistance, the precise function of oxylipins in plant defence remains unknown. Here we report a cotton P450 gene SILENCE-INDUCED STEM NECROSIS (SSN), RNAi suppression of which causes a lesion mimic phenotype. SSN is also involved in jasmonate metabolism and the response to wounding. Fatty acid and oxylipin metabolite analysis showed that SSN overexpression causes hyperaccumulation of hydroxide and ketodiene fatty acids and reduced levels of 18:2 fatty acids, whereas silencing causes an imbalance in LOX (lipoxygenase) expression and excessive hydroperoxide fatty acid accumulation. We also show that an unknown oxylipin-derived factor is a putative mobile signal required for systemic cell death and hypothesize that SSN acts as a valve to regulate HR on pathogen infection.

Defective pollen wall contributes to male sterility in the male sterile line 1355A of cotton.

To understand the mechanisms of male sterility in cotton (Gossypium spp.), combined histological, biochemical and transcription analysis using RNA-Seq was carried out in the anther of the single-gene recessive genic male sterility system of male sterile line 1355A and male fertile line 1355B, which are near-isogenic lines (NILs) differing only in the fertility trait. A total of 2,446 differentially expressed genes were identified between the anthers of 1355AB lines, at three different stages of development. Cluster analysis and functional assignment of differentially expressed genes revealed differences in transcription associated with pollen wall and anther development, including the metabolism of fatty acids, glucose, pectin and cellulose. Histological and biochemical analysis revealed that a major cellular defect in the 1355A was a thicker nexine, consistent with the RNA-seq data, and further gene expression studies implicated differences in fatty acids synthesis and metabolism. This study provides insight into the phenotypic characteristics and gene regulatory network of the genic male sterile line 1355A in upland cotton.

LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 network regulates somatic embryogenesis by regulating auxin homeostasis

A gene network regulates auxin homeostasis, thereby affecting cell proliferation and the transition from nonembryogenic callus to somatic embryos during somatic embryogenesis. Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network.

Multiple shoots induction in wild cotton (Gossypium bickii) through organogenesis and the analysis of genetic homogeneity of the regenerated plants

Fertile plants were regenerated via organogenesis from cultures of Gossypium bickii Prokhanov cotyledonary nodes devoid of cotyledons. Cotyledonary nodes from 7-day-old seedlings yielded the maximum number of shoots (9.12 shoots per explant) using MSB medium [MS medium (Murashige & Skoog 1962) and B5 (Gamborg et al. 1968) vitamins] supplemented with the combination of 4 mg/L BAP (N6-benzyladinine) and 0.1 mg/L TDZ (thidiazouron), and the subculture was made on a medium containing 2 mg/L BAP. Elongation of multiple shoots was obtained on MSB medium containing 0.05 mg/L GA3 (gibberellic acid). For rooting, a modified half-strength MS medium was utilized. Some regenerants were transferred to a greenhouse, and most of them were morphologically normal and fertile. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants. Flow cytometric analyses and chromosome counting suggested that the regenerants and their offsprings were diploid. DNA content of regenerants and their offsprings was homogeneous and similar to that of seed-derived plants with unchanged chromosome number (2n = 26). Random amplified polymorphic DNA (RAPD) analysis confirmed the genetic homogeneity of the regenerants and their offsprings with the diploid parent.

Laccase GhLac1 Modulates Broad-Spectrum Biotic Stress Tolerance via Manipulating Phenylpropanoid Pathway and Jasmonic Acid Synthesis

Modification of GhLac1 expression leads to redirection of phenylpropanoid metabolism and alteration of JA synthesis to confer broad spectrum resistance to both pathogens and pests. Plants are constantly challenged by a multitude of pathogens and pests, which causes massive yield and quality losses annually. A promising approach to reduce such losses is to enhance the immune system of plants through genetic engineering. Previous work has shown that laccases (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) function as lignin polymerization enzymes. Here we demonstrate that transgenic manipulation of the expression of the laccase gene GhLac1 in cotton (Gossypium hirsutum) can confer an enhanced defense response to both pathogens and pests. Overexpression of GhLac1 leads to increased lignification, associated with increased tolerance to the fungal pathogen Verticillium dahliae and to the insect pests cotton bollworm (Helicoverpa armigera) and cotton aphid (Aphis gosypii). Suppression of GhLac1 expression leads to a redirection of metabolic flux in the phenylpropanoid pathway, causing the accumulation of JA and secondary metabolites that confer resistance to V. dahliae and cotton bollworm; it also leads to increased susceptibility to cotton aphid. Plant laccases therefore provide a new molecular tool to engineer pest and pathogen resistance in crops.

Disrupted Genome Methylation in Response to High Temperature Has Distinct Affects on Microspore Abortion and Anther Indehiscence

Integrated multi-omics data analysis reveals the role of genome methylation in male fertility, shedding light on the mechanism underlying male sterility in response to high temperature. High-temperature (HT) stress induces male sterility, leading to yield reductions in crops. DNA methylation regulates a range of processes involved in plant development and stress responses, but its role in male sterility under HT remains unknown. Here, we investigated DNA methylation levels in cotton (Gossypium hirsutum) anthers under HT and normal temperature (NT) conditions by performing whole-genome bisulfite sequencing to investigate the regulatory roles of DNA methylation in male fertility under HT. Global disruption of DNA methylation, especially CHH methylation (where H = A, C, or T), was detected in an HT-sensitive line. Changes in the levels of 24-nucleotide small-interfering RNAs were significantly associated with DNA methylation levels. Experimental suppression of DNA methylation led to pollen sterility in the HT-sensitive line under NT conditions but did not affect the normal dehiscence of anther walls. Further transcriptome analysis showed that the expression of genes in sugar and reactive oxygen species (ROS) metabolic pathways were significantly modulated in anthers under HT, but auxin biosynthesis and signaling pathways were only slightly altered, indicating that HT disturbs sugar and ROS metabolism via disrupting DNA methylation, leading to microspore sterility. This study opens up a pathway for creating HT-tolerant cultivars using epigenetic techniques.