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. Molecular & Cellular

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.

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 latestage 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.

Summary Long noncoding RNAs (lncRNAs) are transcripts of at least 200 bp in length, possess no apparent coding capacity and are involved in various biological regulatory processes. Until now, no systematic identification of lncRNAs has been reported in cotton (Gossypium spp.). Here, we describe the identification of 30 550 long intergenic noncoding RNA (lincRNA) loci (50 566 transcripts) and 4718 long noncoding natural antisense transcript (lncNAT) loci (5826 transcripts). LncRNAs are rich in repetitive sequences and preferentially expressed in a tissue‐specific manner. The detection of abundant genome‐specific and/or lineage‐specific lncRNAs indicated their weak evolutionary conservation. Approximately 76% of homoeologous lncRNAs exhibit biased expression patterns towards the At or Dt subgenomes. Compared with protein‐coding genes, lncRNAs showed overall higher methylation levels and their expression was less affected by gene body methylation. Expression validation in different cotton accessions and coexpression network construction helped to identify several functional lncRNA candidates involved in cotton fibre initiation and elongation. Analysis of integrated expression from the subgenomes of lncRNAs generating miR397 and its targets as a result of genome polyploidization indicated their pivotal functions in regulating lignin metabolism in domesticated tetraploid cotton fibres. This study provides the first comprehensive identification of lncRNAs in Gossypium.

SummaryAs a product of asexual reproduction in plants, the somatic embryo (SE) differentiates into a new plantlet via a zygotic embryogenesis-like process. Here, we present the phenotypic and cellular differences between SEs and zygotic embryos (ZEs) revealed by histological section scanning using three parallel development stages of the two types of embryos of cotton (Gossypium hirsutum cv. YZ1), including globular, torpedo and cotyledonary-stages. To identify the molecular characteristics of SE development in cotton, the digital gene expression system was used to profile the genes active during SE and ZE development. A total of 4242 differentially expressed genes (DEGs) were identified in at least one developmental stage. Expression pattern and functional classification analysis based on these DEGs reveals that SE development exhibits a transcriptional activation of stress responses. RT-PCR analysis further confirmed enhanced expression levels of stress-related genes in SEs than in ZEs. Experimental stress treatment, induced by NaCl and ABA, accelerated SE development and increased the transcription of genes related to stress response, in parallel with decelerated proliferation of embryogenic calluses under stress treatment. Our data reveal that SE development involves the activation of stress responses, which we suggest may regulate the balance between cell proliferation and differentiation. These results provide new insight into the molecular mechanisms of SE development and suggest strategies that can be used for regulating the developmental processes of somatic embryogenesis.

Pectins are fundamental polysaccharides in the plant primary cell wall. Pectins are synthesized and secreted to cell walls as highly methyl-esterified polymers and then demethyl-esterified by pectin methylesterases (PMEs), which are spatially regulated by pectin methylesterase inhibitors (PMEIs). Although PME and PMEI genes are pivotal in plant cell wall formation, few studies have focused on the evolutionary patterns of the PME and PMEI gene families. In this study, the gene origin, evolution, and expression diversity of these two families were systematically analyzed using 11 representative species, including algae, bryophytes, lycophytes and flowering land plants. The results show that 1) for the two subfamilies (PME and proPME) of PME, the origin of the PME subfamily is consistent with the appearance of pectins in early charophyte cell walls, 2) Whole genome duplication (WGD) and tandem duplication contribute to the expansion of proPME and PMEI families in land plants, 3) Evidence of selection pressure shows that the proPME and PMEI families have rapidly evolved, particularly the PMEI family in vascular plants, and 4) Comparative expression profile analysis of the two families indicates that the eudicot Arabidopsis and monocot rice have different expression patterns. In addition, the gene structure and sequence analyses show that the origin of the PMEI domain may be derived from the neofunctionalization of the pro domain after WGD. This study will advance the evolutionary understanding of the PME and PMEI families and plant cell wall development.

Nanozymes with superoxide dismutase (SOD)-like activity have attracted increasing interest due to their ability to scavenge superoxide anion, the origin of most reactive oxygen species in vivo. However, SOD nanozymes reported thus far have yet to approach the activity of natural enzymes. Here, we report a carbon dot (C-dot) SOD nanozyme with a catalytic activity of over 10,000 U/mg, comparable to that of natural enzymes. Through selected chemical modifications and theoretical calculations, we show that the SOD-like activity of C-dots relies on the hydroxyl and carboxyl groups for binding superoxide anions and the carbonyl groups conjugated with the π-system for electron transfer. Moreover, C-dot SOD nanozymes exhibit intrinsic targeting ability to oxidation-damaged cells and effectively protect neuron cells in the ischemic stroke male mice model. Together, our study sheds light on the structure-activity relationship of C-dot SOD nanozymes, and demonstrates their potential for treating of oxidation stress related diseases.

SUMMARY microRNAs (miRNAs) are 20-24 nucleotide non-coding small RNAs that play important roles in plant development. The stages of cotton fiber development include initiation, elongation, secondary wall thickening (SWT) and maturation. We constructed seven fiber RNA libraries representing the initiation, elongation and SWT stages. In total, 47 conserved miRNA families and seven candidate miRNAs were profiled using small RNA sequencing. Northern blotting and real-time polymerase chain reaction (PCR) analyses revealed the dynamic expression of miRNAs during fiber development. In addition, 140 targets of 30 conserved miRNAs and 38 targets of five candidate miRNAs were identified through degradome sequencing. Analysis of correlated expression between miRNAs and their targets demonstrated that specific miRNAs suppressed the expression of transcription factors, SBP and MYB, a leucine-rich receptor-like protein kinase, a pectate lyase, a-tubulin, a UDP-glucuronic acid decarboxylase and cytochrome C oxidase subunit 1 to affect fiber development. Histochemical analyses detected the biological activity of miRNA156/157 in ovule and fiber development. Suppressing miRNA156/157 function resulted in the reduction of mature fiber length, illustrating that miRNA156/157 plays an essential role in fiber elongation.