Long noncoding RNA lncRNA354 functions as a competing endogenous RNA of miR160b to regulate ARF genes in response to salt stress in upland cotton.

Long non-coding RNAs (lncRNAs) play important roles in response to biotic and abiotic stress through acting as competing endogenous RNAs (ceRNAs) to decoy mature miRNAs. However, whether this mechanism is involved in cotton salt stress response remains unknown. We report the characterization of an endogenous lncRNA, lncRNA354, whose expression was reduced in salt-treated cotton and was localized at the nucleus and cytoplasm. Using endogenous target mimic (eTM) analysis, we predicted that lncRNA354 had a potential binding site for miR160b. Transient expression in tobacco demonstrated that lncRNA354 was a miR160b eTM and attenuated miR160b suppression of its target genes, including auxin response factors (ARFs). Silencing or overexpressing lncRNA354 affected the expression of miR160b and target ARFs. Silencing lncRNA354 and targets GhARF17/18 resulted in taller cotton plants and enhanced the resistant to salt stress. Overexpression of lncRNA354 and targets GhARF17/18 in Arabidopsis led to dwarf plants, decreased root dry weight and reduced salt tolerance. Our results show that the lncRNA354-miR160b effect on GhARF17/18 expression may modulate auxin signalling and thus affect growth. These results also shed new light on a mechanism of lncRNA-associated responses to salt stress.

Mitigation of salt stress response in upland cotton (Gossypium hirsutum) by exogenous melatonin.

As a pleiotropic signal molecule, melatonin is ubiquitous throughout the animal and plant kingdoms and plays important roles in the regulation of plant growth, development, and responses to environmental stresses. In this study, we quantified the endogenous melatonin levels in upland cotton (Gossypium hirsutum L.), using high-performance liquid chromatography-tandem mass spectrometry. The melatonin concentrations in root, stem, and leaf were 150.60, 37.92, and 40.58 ng g fresh weight- 1, respectively. The effects of exogenous melatonin (1 µM) on plant growth, photosynthesis, antioxidant enzyme activity, and ion homeostasis in upland cotton seedlings exposed to 100 mM NaCl treatment were determined. Pretreatment (prior to exposure to salt stress) of seedlings with exogenous melatonin significantly alleviated plant growth inhibition by salt stress and maintained an improved photosynthetic capacity. The application of melatonin also significantly reduced the salt-induced oxidative damage, possibly through the accumulation of osmotic regulatory substances and the activation of antioxidant enzymes. We also showed that exogenous melatonin regulated the expression of stress-responsive and ion-channel genes under salinity, which could contribute to improved salt tolerance in cotton. Taken together, our study provides evidence that cotton contains endogenous melatonin, and it may have unraveled crucial evidence of the role of melatonin in cotton against salt stress.

Comprehensive analysis of the Gossypium hirsutum L. respiratory burst oxidase homolog (Ghrboh) gene family.

BACKGROUND: Plant NADPH oxidase (NOX), also known as respiratory burst oxidase homolog (rboh), encoded by the rboh gene, is a key enzyme in the reactive oxygen species (ROS) metabolic network. It catalyzes the formation of the superoxide anion (O2•-), a type of ROS. In recent years, various studies had shown that members of the plant rboh gene family were involved in plant growth and developmental processes as well as in biotic and abiotic stress responses, but little is known about its functional role in upland cotton. RESULTS: In the present study, 26 putative Ghrboh genes were identified and characterized. They were phylogenetically classified into six subfamilies and distributed at different densities across 18 of the 26 chromosomes or scaffolds. Their exon-intron structures, conserved domains, synteny and collinearity, gene family evolution, regulation mediated by cis-acting elements and microRNAs (miRNAs) were predicted and analyzed. Additionally, expression profiles of Ghrboh gene family were analyzed in different tissues/organs and at different developmental stages and under different abiotic stresses, using RNA-Seq data and real-time PCR. These profiling studies indicated that the Ghrboh genes exhibited temporal and spatial specificity with respect to expression, and might play important roles in cotton development and in stress tolerance through modulating NOX-dependent ROS induction and other signaling pathways. CONCLUSIONS: This comprehensive analysis of the characteristics of the Ghrboh gene family determined features such as sequence, synteny and collinearity, phylogenetic and evolutionary relationship, expression patterns, and cis-element- and miRNA-mediated regulation of gene expression. Our results will provide valuable information to help with further gene cloning, evolutionary analysis, and biological function analysis of cotton rbohs.

Mechanisms and Functions of Long Non-Coding RNAs at Multiple Regulatory Levels.

Long non-coding (lnc) RNAs are non-coding RNAs longer than 200 nt. lncRNAs primarily interact with mRNA, DNA, protein, and miRNA and consequently regulate gene expression at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in a variety of ways. They play important roles in biological processes such as chromatin remodeling, transcriptional activation, transcriptional interference, RNA processing, and mRNA translation. lncRNAs have important functions in plant growth and development; biotic and abiotic stress responses; and in regulation of cell differentiation, the cell cycle, and the occurrence of many diseases in humans and animals. In this review, we summarize the functions and mechanisms of lncRNAs in plants, humans, and animals at different regulatory levels.

The long non-coding RNA lncRNA973 is involved in cotton response to salt stress.

BACKGROUND: Long non-coding (lnc) RNAs are a class of functional RNA molecules greater than 200 nucleotides in length, and lncRNAs play important roles in various biological regulatory processes and response to the biotic and abiotic stresses. LncRNAs associated with salt stress in cotton have been identified through RNA sequencing, but the function of lncRNAs has not been reported. We previously identified salt stress-related lncRNAs in cotton (Gossypium spp.), and discovered the salt-related lncRNA-lncRNA973. RESULTS: In this study, we identified the expression level, localization, function, and preliminary mechanism of action of lncRNA973. LncRNA973, which was localized in the nucleus, was expressed at a low level under nonstress conditions but can be significantly increased by salt treatments. Here lncRNA973 was transformed into Arabidopsis and overexpressed. Along with the increased expression compared with wild type under salt stress conditions in transgenic plants, the seed germination rate, fresh weights and root lengths of the transgenic plants increased. We also knocked down the expression of lncRNA973 using virus-induced gene silencing technology. The lncRNA973 knockdown plants wilted, and the leaves became yellowed and dropped under salt-stress conditions, indicating that the tolerance to salt stress had decreased compared with wild type. LncRNA973 may be involved in the regulation of reactive oxygen species-scavenging genes, transcription factors and genes involved in salt stress-related processes in response to cotton salt stress. CONCLUSIONS: LncRNA973 was localized in the nucleus and its expression was increased by salt treatment. The lncRNA973-overexpression lines had increased salt tolerance compared with the wild type, while the lncRNA973 knockdown plants had reduced salt tolerance. LncRNA973 regulated cotton responses to salt stress by modulating the expression of a series of salt stress-related genes. The data provides a basis for further studies on the mechanisms of lncRNA973-associated responses to salt stress in cotton.

The Catalase Gene Family in Cotton: Genome-Wide Characterization and Bioinformatics Analysis.

Catalases (CATs), which were coded by the catalase gene family, were a type notably distinguished ROS-metabolizing proteins implicated to perform various physiological functions in plant growth, development and stress responses. However, no systematical study has been performed in cotton. In the present study, we identified 7 and 7 CAT genes in the genome of Gossypium hirsutum L. Additionally, G. barbadense L., respectively. The results of the phylogenetic and synteny analysis showed that the CAT genes were divided into two groups, and whole-genome duplication (WGD) or polyploidy events contributed to the expansion of the GossypiumCAT gene family. Expression patterns analysis showed that the CAT gene family possessed temporal and spatial specificity and was induced by the Verticillium dahliae infection. In addition, we predicted the putative molecular regulatory mechanisms of the CAT gene family. Based on the analysis and preliminary verification results, we hypothesized that the CAT gene family, which might be regulated by transcription factors (TFs), alternative splicing (AS) events and miRNAs at different levels, played roles in cotton development and stress tolerance through modulating the reactive oxygen species (ROS) metabolism. This is the first report on the genome-scale analysis of the cotton CAT gene family, and these data will help further study the roles of CAT genes during stress responses, leading to crop improvement.

MicroRNA414c affects salt tolerance of cotton by regulating reactive oxygen species metabolism under salinity stress.

Salinity stress is a major abiotic stress affecting the productivity and fiber quality of cotton. Although reactive oxygen species (ROS) play critical roles in plant stress responses, their complex molecular regulatory mechanism under salinity stress is largely unknown in cotton, especially microRNA (miRNA)-mediated regulation of superoxide dismutase gene expression. Here, we report that a cotton iron superoxide dismutase gene GhFSD1 and the cotton miRNA ghr-miR414c work together in response to salinity stress. The miRNA ghr-miR414c targets the coding sequence region of GhFSD1, inhibiting expression of transcripts of this antioxidase gene, which represents the first line of defense against stress-induced ROS. Expression of GhFSD1 was induced by salinity stress. Under salinity stress, ghr-miR414c showed expression patterns opposite to those of GhFSD1. Ectopic expression of GhFSD1 in Arabidopsis conferred salinity stress tolerance by improving primary root growth and biomass, whereas Arabidopsis constitutively expressing ghr-miR414c showed hypersensitivity to salinity stress. Silencing GhFSD1 in cotton caused an excessive hypersensitive phenotype to salinity stress, whereas overexpressing miR414c decreased the expression of GhFSD1 and increased sensitivity to salinity stress, yielding a phenotype similar to that of GhFSD1-silenced cotton. Taken together, our results demonstrated that ghr-miR414c was involved in regulation of plant response to salinity stress by targeting GhFSD1 transcripts. This study provides a new strategy and method for plant breeding in order to improve plant salinity tolerance.

Plant MicroRNAs in Cross-Kingdom Regulation of Gene Expression.

MicroRNAs (miRNAs) are a class of noncoding small RNAs, which play a crucial role in post-transcriptional gene regulation. Recently, various reports revealed that miRNAs could be transmitted between species to mediate cross-kingdom regulation by integrating into a specific target gene-mediated regulatory pathway to exert relevant biological functions. Some scholars and researchers have observed this as an attractive hypothesis that may provide a foundation for novel approaches in the diagnosis, prognosis, and treatment of disease. Meanwhile, others deem the mentioned results were obtained from a "false positive effect" of performed experiments. Here, we focus on several current studies concerning plant miRNA-mediated cross-kingdom regulation (from both fronts) and discuss the existing issues that need further consideration. We also discuss possible miRNA horizontal transfer mechanisms from one species to another and analyze the relationship between miRNA-mediated cross-kingdom regulation and coevolution during a long-term specific host⁻pathogen interaction.

Role of plant respiratory burst oxidase homologs in stress responses.

Plant respiratory burst oxidase homologs (Rbohs), which are also named nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), are the homologs of mammalian phagocyte gp91phox. As a unique among other reactive oxygen species (ROS) production mechanisms in plants, NADPH oxidases can integrate different signal transduction pathways, such as calcium, protein phosphorylation catalysed by protein kinases, nitric oxide, and lipid messengers. Coupling with genetic studies, the ability of plant NADPH oxidases to integrate different signal transduction pathways with ROS production demonstrates their involvement in many important biological processes in cells, such as morphogenesis and development, and stress responses. Here, we focus on several current studies concerning the role of plant NADPH oxidases in stress responses.

Identification of Gossypium hirsutum long non-coding RNAs (lncRNAs) under salt stress.

BACKGROUND: Long non-coding RNAs (lncRNAs) represent a class of riboregulators that either directly act in long form or are processed into shorter microRNAs (miRNAs) and small interfering RNAs. Long noncoding RNAs (lncRNAs) are arbitrarily defined as RNA genes larger than 200 nt in length that have no apparent coding potential. lncRNAs have emerged as playing important roles in various biological regulatory processes and are expressed in a more tissue-specific manner than mRNA. Emerging evidence shows that lncRNAs participate in stress-responsive regulation. RESULTS: In this study, in order to develop a comprehensive catalogue of lncRNAs in upland cotton under salt stress, we performed whole-transcriptome strand-specific RNA sequencing for three-leaf stage cotton seedlings treated with salt stress (S_NaCl) and controls (S_CK). In total we identified 1117 unique lncRNAs in this study and 44 differentially expressed RNAs were identified as potential non-coding RNAs. For the differentially expressed lncRNAs that were identified as intergenic lncRNAs (lincRNA), we analysed the gene ontology enrichment of cis targets and found that cis target protein-coding genes were mainly enriched in stress-related categories. Real-time quantitative PCR confirmed that all selected lincRNAs responsive to salt stress. We found lnc_388 was likely as regulator of Gh_A09G1182. And lnc_883 may participate in regulating tolerance to salt stress by modulating the expression of Gh_D03G0339 MS_channel. We then predicted the target mimics for miRNA in Gossypium. six miRNAs were identified, and the result of RT-qPCR with lncRNA and miRNA suggested that lnc_973 and lnc_253 may regulate the expression of ghr-miR399 and ghr-156e as a target mimic under salt stress. CONCLUSIONS: We identified 44 lincRNAs that were differentially expressed under salt stress. These lincRNAs may target protein-coding genes via cis-acting regulation. We also discovered that specifically-expressed lincRNAs under salt stress may act as endogenous target mimics for conserved miRNAs. These findings extend the current view on lincRNAs as ubiquitous regulators under stress stress.

Genome-wide characterization and expression analyses of superoxide dismutase (SOD) genes in Gossypium hirsutum.

BACKGROUND: Superoxide dismutases (SODs) are a key antioxidant enzyme family, which have been implicated in protecting plants against the toxic effects of reactive oxygen species. Despite current studies have shown that the gene family are involved in plant growth and developmental processes and biotic and abiotic stress responses, little is known about its functional role in upland cotton. RESULTS: In the present study, we comprehensively analyzed the characteristics of the SOD gene family in upland cotton (Gossypium hirsutum). Based on their conserved motifs, 18 GhSOD genes were identified and phylogenetically classified into five subgroups which corroborated their classifications based on gene-structure patterns and subcellular localizations. The GhSOD sequences were distributed at different densities across 12 of the 26 chromosomes. The conserved domains, gene family evolution cis-acting elements of promoter regions and miRNA-mediated posttranscriptional regulation were predicted and analyzed. In addition, the expression pattern of 18 GhSOD genes were tested in different tissues/organs and developmental stages, and different abiotic stresses and abscisic acid, which indicated that the SOD gene family possessed temporal and spatial specificity expression specificity and may play important roles in reactive oxygen species scavenging caused by various stresses in upland cotton. CONCLUSIONS: This study describes the first genome-wide analysis of the upland cotton SOD gene family, and the results will help establish a foundation for the further cloning and functional verification of the GhSOD gene family during stress responses, leading to crop improvement.

Data set for phylogenetic tree and RAMPAGE Ramachandran plot analysis of SODs in Gossypium raimondii and G. arboreum.

The data presented in this paper is supporting the research article "Genome-Wide Analysis of Superoxide Dismutase Gene Family in Gossypium raimondii and G. arboreum" [1]. In this data article, we present phylogenetic tree showing dichotomy with two different clusters of SODs inferred by the Bayesian method of MrBayes (version 3.2.4), "Bayesian phylogenetic inference under mixed models" [2], Ramachandran plots of G. raimondii and G. arboreum SODs, the protein sequence used to generate 3D sructure of proteins and the template accession via SWISS-MODEL server, "SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information." [3] and motif sequences of SODs identified by InterProScan (version 4.8) with the Pfam database, "Pfam: the protein families database" [4].

Corrigendum: Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton Confers Salinity Tolerance and Increases Yield in a Saline Field.

[This corrects the article on p. 1227 in vol. 6, PMID: 26779246.].

Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L.) were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular.

Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton Confers Salinity Tolerance and Increases Yield in a Saline Field.

Soil salinity is a major environmental stress limiting plant growth and productivity. We have reported previously the isolation of an Apocynum venetum DEAD-box helicase 1 (AvDH1) that is expressed in response to salt exposure. Here, we report that the overexpression of AvDH1 driven by a constitutive cauliflower mosaic virus-35S promoter in cotton plants confers salinity tolerance. Southern and Northern blotting analyses showed that the AvDH1 gene was integrated into the cotton genome and expressed. In this study, the growth of transgenic cotton expressing AvDH1 was evaluated under saline conditions in a growth chamber and in a saline field trial. Transgenic cotton overexpressing AvDH1 was much more resistant to salt than the wild-type plants when grown in a growth chamber. The lower membrane ion leakage, along with increased activity of superoxide dismutase, in AvDH1 transgenic lines suggested that these characteristics may prevent membrane damage, which increases plant survival rates. In a saline field, the transgenic cotton lines expressing AvDH1 showed increased boll numbers, boll weights and seed cotton yields compared with wild-type plants, especially at high soil salinity levels. This study indicates that transgenic cotton expressing AvDH1 is a promising option for increasing crop productivity in saline fields.

Genome-wide analysis of the RNA helicase gene family in Gossypium raimondii.

The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes), DEAH-box (52 genes), or DExD/H-box (58 genes) in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5%) are within the identified syntenic blocks. Sixty-six (40.99%) helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

Genome-wide profiling of miRNAs and other small non-coding RNAs in the Verticillium dahliae-inoculated cotton roots.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are short (19-25 nucleotides) non-coding RNA molecules that have large-scale regulatory effects on development and stress responses in plants. Verticillium wilt is a vascular disease in plants caused by the fungal pathogen Verticillium dahliae. The objective of this study is to investigate the transcriptional profile of miRNAs and other small non-coding RNAs in Verticillium-inoculated cotton roots. Four small RNA libraries were constructed from mocked and infected roots of two cotton cultured species which are with different Verticillium wilt tolerance ('Hai-7124', Gossypium barbadense L., a Verticillium-tolerant cultivar, and 'Yi-11', Gossypium hirsutum L. a Verticillium-sensitive cultivar). The length distribution of obtained small RNAs was significantly different between libraries. There were a total of 215 miRNA families identified in the two cotton species. Of them 14 were novel miRNAs. There were >65 families with different expression between libraries. We also identified two trans-acting siRNAs and thousands of endogenous siRNA candidates, and hundred of them exhibited altered expression after inoculation of Verticillium. Interesting, many siRNAs were found with a perfect match with retrotransposon sequences, suggested that retrotransposons maybe one of sources for the generation of plant endogenous siRNAs. The profiling of these miRNAs and other small non-coding RNAs lay the foundation for further understanding of small RNAs function in the regulation of Verticillium defence responses in cotton roots.

[Cloning and functional analysis of chitinase gene GbCHI from sea-island cotton (Gossypium barbadense)].

Chitinase is one of the important pathogenesis-related (PR) proteins in plants. By comparative proteomics study, a novel pathogen-responsive chitinase (known as GbCHI) has been identified from sea-island cotton (Gossypium barbadense). The GbCHI cDNA was cloned from wilt-resistant sea-island cotton and the anti-fungal activity of the gene product was investigated. qRT-PCR analysis indicated that GbCHI was expressed constitutively in root, stem, leaf, flower, and ovule of cotton plant, and the expression could be induced by Verticillium dahliae and plant hormone SA, ACC, and JA. Subcellular localization analysis using GFP-tagged proteins showed that GbCHI-GFP fusion proteins were targeted mainly to the plasma membrane. Anti-fungal assay demonstrated that GbCHI could inhibit spore germination and hyphae growth of V. dahliae significantly. These results provide important information for understanding the cellular function of GbCHI and for exploring the application potential of this gene in molecular breeding of wilt-tolerant cotton plants.

Difference in miRNA expression profiles between two cotton cultivars with distinct salt sensitivity.

MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that play important roles in many developmental processes and stress responses in plants and animals. Cotton (Gossypium hirsutum L.) is considered a relatively salt-tolerant non-halophytic plant species. To study the role of miRNAs in salt adaptation, a salt-tolerant cotton cultivar SN-011 and a salt-sensitive cultivar LM-6 were used to detect differentially expressed miRNAs. Using miRNA microarray analysis and a computational approach, 17 cotton miRNAs belonging to eight families were identified. Although they are conserved, 12 of them showed a genotype-specific expression model in both the cultivars. Under salt stress treatment, miR156a/d/e, miR169, miR535a/b and miR827b were dramatically down-regulated in SN-011, while miR167a, miR397a/b and miR399a were up-regulated. Only miR159 was found to be down-regulated in LM-6 under salt stress. To gain insight into their functional significance, 26 target genes were predicted and their functional similarity was further analyzed. Quantitative real-time PCR showed that the expression of seven target genes showed a significant inverse correlation with corresponding miRNAs. These differentially expressed miRNAs can help in further study into the role of transcriptome homeostasis in the adaptation responses of cotton to salt.

Identification of conserved microRNAs and their target genes in tomato (Lycopersicon esculentum).

MicroRNAs (miRNAs) are a class of non-coding RNAs that have important gene regulation roles in various organisms. To date, a total of 1279 plant miRNAs have been deposited in the miRNA miRBase database (Release 10.1). Many of them are conserved during the evolution of land plants suggesting that the well-conserved miRNAs may also retain homologous target interactions. Recently, little is known about the experimental or computational identification of conserved miRNAs and their target genes in tomato. Here, using a computational homology search approach, 21 conserved miRNAs were detected in the Expressed Sequence Tags (EST) and Genomic Survey Sequence (GSS) databases. Following this, 57 potential target genes were predicted by searching the mRNA database. Most of the target mRNAs appeared to be involved in plant growth and development. Our findings verified that the well-conserved tomato miRNAs have retained homologous target interactions amongst divergent plant species. Some miRNAs express diverse combinations in different cell types and have been shown to regulate cell-specific target genes coordinately. We believe that the targeting propensity for genes in different biological processes can be explained largely by their protein connectivity.