Longfu Zhu

Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry

The incompatible pathosystem between resistant cotton (Gossypium barbadense cv. 7124) and Verticillium dahliae strain V991 was used to study the cotton transcriptome changes after pathogen inoculation by RNA-Seq. Of 32 774 genes detected by mapping the tags to assembly cotton contigs, 3442 defence-responsive genes were identified. Gene cluster analyses and functional assignments of differentially expressed genes indicated a significant transcriptional complexity. Quantitative real-time PCR (qPCR) was performed on selected genes with different expression levels and functional assignments to demonstrate the utility of RNA-Seq for gene expression profiles during the cotton defence response. Detailed elucidation of responses of leucine-rich repeat receptor-like kinases (LRR-RLKs), phytohormone signalling-related genes, and transcription factors described the interplay of signals that allowed the plant to fine-tune defence responses. On the basis of global gene regulation of phenylpropanoid metabolism-related genes, phenylpropanoid metabolism was deduced to be involved in the cotton defence response. A closer look at the expression of these genes, enzyme activity, and lignin levels revealed differences between resistant and susceptible cotton plants. Both types of plants showed an increased level of expression of lignin synthesis-related genes and increased phenylalanine-ammonia lyase (PAL) and peroxidase (POD) enzyme activity after inoculation with V. dahliae, but the increase was greater and faster in the resistant line. Histochemical analysis of lignin revealed that the resistant cotton not only retains its vascular structure, but also accumulates high levels of lignin. Furthermore, quantitative analysis demonstrated increased lignification and cross-linking of lignin in resistant cotton stems. Overall, a critical role for lignin was believed to contribute to the resistance of cotton to disease.

Isolation and Characterization of Genes Associated to Cotton Somatic Embryogenesis by Suppression Subtractive Hybridization and Macroarray

Somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and preglobular somatic embryos, as the tester, and cDNA from nonembryogenic callus, as the driver. After differential screening and subsequent confirmation by reverse Northern blot analysis, a total of 671 differentially expressed cDNA fragments were identified, and 242 unigenes significantly up-regulated during cotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCR analysis of representative cases, including most previously published SE-related genes in plants. In total, more than half had not been identified previously as SE-related genes, including dominant crucial genes involved in transcription, posttranscription, and transportation, and about one-third had not been reported previously to GenBank or were expected to be unknown, or newly identified genes. We used cDNA arrays to further investigate the expression patterns of these genes in differentiating gradient culture, ranging from proembryogenic masses to somatic embryos at every stage. The cDNA collection is composed of a broad repertoire of SE genes which is an important resource for understanding the genetic interactions underlying SE signaling and regulation. Our results suggested that a complicated and concerted mechanism involving multiple cellular pathways is responsible for cotton SE. This report represents a systematic and comprehensive analysis of genes involved in the process of somatic embryogenesis.

The genome sequence of Sea-Island cotton (Gossypium barbadense) provides insights into the allopolyploidization and development of superior spinnable fibres

Gossypium hirsutum contributes the most production of cotton fibre, but G. barbadense is valued for its better comprehensive resistance and superior fibre properties. However, the allotetraploid genome of G. barbadense has not been comprehensively analysed. Here we present a high-quality assembly of the 2.57 gigabase genome of G. barbadense, including 80,876 protein-coding genes. The double-sized genome of the A (or At) (1.50 Gb) against D (or Dt) (853 Mb) primarily resulted from the expansion of Gypsy elements, including Peabody and Retrosat2 subclades in the Del clade, and the Athila subclade in the Athila/Tat clade. Substantial gene expansion and contraction were observed and rich homoeologous gene pairs with biased expression patterns were identified, suggesting abundant gene sub-functionalization occurred by allopolyploidization. More specifically, the CesA gene family has adapted differentially temporal expression patterns, suggesting an integrated regulatory mechanism of CesA genes from At and Dt subgenomes for the primary and secondary cellulose biosynthesis of cotton fibre in a “relay race”-like fashion. We anticipate that the G. barbadense genome sequence will advance our understanding the mechanism of genome polyploidization and underpin genome-wide comparison research in this genus.

Proteomic and virus-induced gene silencing (VIGS) analyses reveal that Gossypol, Brassinosteroids and Jasmonic acid contribute to the resistance of cotton to Verticillium dahliae

Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

Overexpression of Rice NAC Gene SNAC1 Improves Drought and Salt Tolerance by Enhancing Root Development and Reducing Transpiration Rate in Transgenic Cotton

The SNAC1 gene belongs to the stress-related NAC superfamily of transcription factors. It was identified from rice and overexpressed in cotton cultivar YZ1 by Agrobacterium tumefaciens-mediated transformation. SNAC1-overexpressing cotton plants showed more vigorous growth, especially in terms of root development, than the wild-type plants in the presence of 250 mM NaCl under hydroponic growth conditions. The content of proline was enhanced but the MDA content was decreased in the transgenic cotton seedlings under drought and salt treatments compared to the wild-type. Furthermore, SNAC1-overexpressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in the greenhouse. The performances of the SNAC1-overexpressing lines under drought and salt stress were significantly better than those of the wild-type in terms of the boll number. During the drought and salt treatments, the transpiration rate of transgenic plants significantly decreased in comparison to the wild-type, but the photosynthesis rate maintained the same at the flowering stage in the transgenic plants. These results suggested that overexpression of SNAC1 improve more tolerance to drought and salt in cotton through enhanced root development and reduced transpiration rates.

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.

Suppression of GhAGP4 gene expression repressed the initiation and elongation of cotton fiber.

Cotton fibers, important natural raw materials for the textile industry, are trichomes elongated from epidermal cells of cotton ovules. To date, a number of genes have been shown to be critical for fiber development. In this study, the roles of genes encoding fasciclin-like arabinoglactan proteins (FLAs) in cotton fiber were examined by transforming RNA interfering (RNAi) construct. The RNAi according to the sequence of GhAGP4 caused a significant reduction of its mRNA level, and the expression of other three FLAs (GhAGP2, GhAGP3, GhFLA1) were also partially suppressed. The fiber initiation and fiber elongation were inhibited in the transgenic plants. As for the mature fibers of transgenic cotton, the fiber length became significantly shorter and the fiber quality became worse. In addition, the RNAi of GhAGP4 also affected the cytoskeleton network and the cellulose deposition of fiber cells. Through ovule culture, it was found that the expression of cotton FLA genes were upregulated by GA3, especially for GhAGP2 and GhAGP4. These results indicate that the FLAs are essential for the initiation and elongation of cotton fiber development.

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.