Functional Genomics

Redox homeostasis is important for plants to be able to maintain cellular metabolism, and disrupting cellular redox homeostasis will cause oxidative damage to cells and adversely affect plant growth. In this study, a cotton CCCH-type tandem zinc finger gene defined as GhTZF1, which was isolated from a cotton cell wall regeneration SSH library in our previous research, was characterized. GhTZF1 was predominantly expressed during early cell wall regeneration, and it was expressed in various vegetative and reproductive tissues. The expression of GhTZF1 was substantially up-regulated by a variety of abiotic stresses, such as PEG and salt. GhTZF1 also responds to MeJA and H2O2 treatment. Overexpression of GhTZF1 enhanced drought tolerance and delayed drought-induced leaf senescence in transgenic Arabidopsis. Subsequent experiments indicated that dark- and MeJA-induced leaf senescence was also attenuated in transgenic plants. The amount of H2O2 in transgenic plants was attenuated under both drought conditions and with MeJA-treatment. The activity of SOD and POD was higher in transgenic plants than in wild type plants under drought conditions. qRT-PCR analysis revealed that overexpression of GhTZF1 reduced the expression of oxidative-related senescence-associated genes (SAGs) under drought conditions. Overexpression of GhTZF1 also enhanced oxidative stress tolerance, which was determined by measuring the expression of a set of antioxidant genes and SAGs that were altered in transgenic plants during H2O2 treatment. Hence, we conclude that GhTZF1 may serve as a regulator in mediating drought stress tolerance and subsequent leaf senescence by modulating the reactive oxygen species homeostasis.

Cotton fibers, the single-celled trichomes derived from the ovule epidermis, provide the most important natural material for the global textile industry. A number of studies have demonstrated that regulating endogenous hormone levels through transgenic approaches can improve cotton fiber qualities. Phytosulfokine-α (PSK-α) is a novel peptide hormone in plants that is involved in regulating cell proliferation and elongation. However, its potential applications in crop genetic improvement have not been evaluated. In this study, we describe how exogenous PSK-α application promotes cotton fiber cell elongation in vitro. Chlorate, an effective inhibitor of peptide sulfation, suppressed fiber elongation in ovule culture. Exogenously applied PSK-α partly restored the chlorate-induced suppression. A putative PSK gene (GhPSK) was cloned from Gossypium hirsutum. Expression pattern analysis revealed that GhPSK is preferentially expressed in rapidly elongating fiber cells (5-20 DPA). Overexpression of GhPSK in cotton increased the endogenous PSK-α level and promoted cotton fiber cell elongation, resulting in longer and finer fibers. Further results from electrophysiological and physiological analyses suggest that GhPSK affects fiber development through regulation of K+ efflux. Digital Gene Expression (DGE) profile analysis of GhPSK overexpression lines indicates that PSK signaling may regulate the respiratory electron-transport chain and ROS to affect cotton fiber development. These results imply that peptide hormones are involved in cotton fiber growth, and suggest a new strategy for the biotechnological improvement of cotton fiber quality.

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 and maturation. We constructed 7 fiber RNA libraries representing the initiation, elongation and second cell wall stages. A total of 47 conserved miRNA families and 7 novel miRNAs were profiled using small RNA sequencing. Northern blot and real-time PCR analyses revealed the dynamic expression of miRNAs during fiber development. In addition, 140 targets of 30 conserved miRNAs and 38 targets of 5 novel miRNAs were identified through degradome sequencing. Analysis of correlated expression between miRNAs and their targets demonstrated that specific miRNAs regulate transcription factors, SBP and MYB, a leucine-rich receptor-like protein kinase, a pectate lyase, α-tubulin, a UDP-glucuronic acid decarboxylase and cytochrome c oxidase subunit 1 to affect fiber development. Histochemical analyses showed that the biological activity of miRNA156/157 was specific 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. This study is the first to verify miRNA function in the cotton fiber.

Cotton fiber is a single cell that differentiates from the ovule epidermis and undergoes synchronous elongation with high secretion and growth rate. Apart from economic importance, cotton fiber provides an excellent single-celled model for studying mechanisms of cell growth. Annexins are Ca2+- and phospholipid-binding proteins that have been reported to be localized in multiple cellular compartments and involved in control of vesicle secretions. Although several annexins have been found to be highly expressed in elongating cotton fibers, their functional roles in fiber development remain unknown. Here, 14 annexin family members were identified from the fully sequenced diploid G. raimondii (D5 genome), and half of which were expressed in fibers of the cultivated tetraploid species G. hirsutum (cv YZ1). Among them, GhAnn2 (DT genome) displayed high expression level in elongating fiber. The expression of GhAnn2 could be induced by some phytohormones that played important roles in fiber elongation, such as IAA and GA3. RNAi-mediated down-regulation of GhAnn2 inhibited fiber elongation and secondary cell wall synthesis, resulting in shorter and thinner mature fibers in the transgenic plants. Measurement with non-invasive scanning ion-selective electrode revealed that the rate of Ca2+ influx from extracellular to intracellular was decreased at the fiber cell apex of GhAnn2 silencing lines, in comparison to that in the wild type. These results indicate that GhAnn2 may regulate fiber development through modulating Ca2+ fluxes and signaling. A gene encoding a calcium sensor, GhCaM7, was also isolated based on its high expression level relative to other GhCaMs in fiber cells at the fast elongation stage. The level of expression of GhCaM7 in wild-type and the fuzzless/lintless mutant corresponds to the presence and absence respectively of fiber initials. Overexpressing GhCaM7 promotes early fiber elongation, whereas GhCaM7 suppression by RNAi delays fiber initiation and inhibits fiber elongation. Reactive oxygen species (ROS) play important roles in fiber early development. ROS induced by exogenous H2O2 and Ca2+ starvation promotes early fiber elongation. GhCaM7 overexpression (OE) fiber cells show increased ROS levels compared to wildtype, while GhCaM7 RNAi fiber cells have reduced levels. Furthermore, we show that H2O2 enhances Ca2+ influx into the fiber and feedback regulates the expression of GhCaM7.

Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during cotton leafroll dwarf polerovirus (CLRDV) infection Elisson Romanel1,2, Tatiane F. Silva2, Régis L. Corrêa1, Laurent Farinelli3, Jennifer S. Hawkins4, Carlos E. G. Schrago1 & Maite F.S. Vaslin2 1- Depto. Genética, I. Biologia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil, 2- Depto. Virologia, I. Microbiologia, UFRJ, Rio de Janeiro, RJ, Brazil, 3- Fasteris SA, Plan-les-Ouates, Switzerland, 4- Department of Biology, West Virginia University, Morgantown, WV 26506, USA maite@micro.ufrj.br Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new conserved miRNAs. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks. Financial support: CAPES, CNPq and FAPERJ

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique largely used in the investigation of gene expression. However, for correct analysis and interpretation of their results, the choice of a suitable gene to normalize the data is a crucial factor. The genes used to normalize expression data, called as reference genes, most have constant expression levels in different tissues and across different conditions. However find genes stable in all situations and that work at the same form in different plant species and conditions is not an accessible goal. The technique of second generation sequencing has contributed to the identification a number of new genes, including microRNAs (miRNAs). miRNAs have important regulatory roles in eukaryotic acting in at post-trasncription level at different biological functions. In the present study, we evaluated, for the first time cotton, micro RNAs as candidates as reference genes for cotton gene expression studies. The stability of miRNAs in Gossypium hirsutum in different tissues (root, stem, leaf and flower), different cultivars (FiberMax FM966, Delta Opal and Cedro) and under biotic stress caused by infection of Cotton leaf roll dwarf virus (CLRDV) was analyzed. mRNAs already described as reference genes in cotton were also included in these analyzes. Cotton samples were organized in 12 sets where the expression stabilities of the six miRNAs and five mRNA reference genes were evaluated. Four algorithms, geNorm, NormFinder, BestKeeper and ΔCt were used to identify the stability of these genes and therefore provide an accurate selection of reference genes. In 8 of the 12 sets tested, the micro RNA (miRNAs) were the best housekeeping or reference genes. After found the best reference genes between the micro RNAs and the mRNAs for these twelve experimental conditions, we validated them in expression assays performed in study cases. For that, we studied the expression of miRNAs and mRNA already know to show distinct expression levels in the analyzed experimental conditions. Study cases used leaves of “cv FM966” under stress biotic caused by Cotton blue disease and flower x leaves from FM966 and Cedro. miR2910, miR164, miR2118, miR2111, miR3476, miR159, GhDCL1, GhDCL2, GhDCL3 and GhDCL4 were evaluated under biotic stress and miR164, GhPGFS7 and GhXTH expressions levels were evaluated in leaves and flowers. Ours results show that the reference microRNA/mRNA genes previously selected work as excellent reference genes in all of the study cases. Statistical teste p sustained the expression levels of the differentially expressed miRNA and mRNAeven under biotic stress as in flower x leaves. These analyzis also showed that to normalize target mRNA the origin, miRNA or mRNA, of the reference gene is not important, but to normalize miRNA expression levels, miRNA reference genes seems to be better than mRNAs. The indication that for miRNA expressions analyzes we should use miRNA as reference genes instead of mRNAs was already well supported in human miRNAs studies, however this is the first study were this is shown in plants. Financial Support: CAPES and FAPERJ

Brown fiber cotton is currently the major raw material for naturally colored cotton textiles. Brown pigmentation in cotton fiber is related to the biosynthesis and accumulation of proanthocyanidins. To clarify the proanthocyanidin pathway in brown cotton fiber, we compared the gene expression profiles in brown and white fibers. Digital gene expression profiling and real-time RT-PCR analyses indicated that, the structural genes encoding most of proanthocyanidin biosynthesis enzymes (including flavonoid 3’, 5’-hydroxylase and leucoanthocyanidin reductase) were significantly up-regulated in brown fibers. Thin layer chromatography and liquid chromatography electrospray ionization mass spectrometry analyses showed that, in brown cotton fiber, the majority of free favan-3-ols were in 2,3-trans form and the proanthocyanidins were mainly composed of trihydroxylated flavan-3-ol units. Our data revealed a detailed proanthocyanidin pathway and indicated that flavonoid 3’, 5’-hydroxylase and leucoanthocyanidin reductase represented the major flow of proanthocyanidin pathway in brown cotton fiber.

ABSTRACT: Genetic diversity/phylogenetic study in six cotton cultivars were carried out realeased by Central Cotton Research Institute Sakrand. The study was carried out by using RAPD markers. In this study 15 universal RAPD primers were used. The RAPD profiles obtained were successfully used to differentiate the genotypes. Based on the pair-wise comparison of amplification products, the genetic diversity/similarity was estimated. The four cotton cultivars showed variation at the DNA level. The genetic similarity among all cotton cultivars ranged from 70 to 85%. CRIS-467 was quite distant from CRIS-134, CRIS-121, CRIS-342 and CRIS-5A (Marvi) cultivars. A dendrogram was constructed using NTSYSpc programme. On the basis of this analysis, the populations were clustered into two clusters: cluster I contained CRIS-121 and CRIS-134 cultivars and cluster II contained CRIS-467, CRIS-342, CRIS-5A and CRIS-9 cultivars. Therefore, the polymorphism detected and its reproducibility suggest that RAPD markers are reliable for identification of cotton cultivars.

Very long-chain fatty acids (VLCFAs) are predominantly present in the form of sphingolipids, which are essential components of plasma membrane microdomains (lipid rafts) that mediate signal transduction pathways in a wide range of eukaryotic organisms, we speculate that sphingolipid may exert an important function during G. hirsutum fiber development. Five groups of cotton sphingolipids including ceramides, hydroxyceramides, inositolphosphoceramides, glucosylceramides, and glycosylinositolphosphoceramides were quantified by using 5500 QTRAP mass spectrometer. Ceramides and inositolphosphoceramides containing VLCFAs accumulated in fast elongating fibers. Ceramides are bioactive lipids and precursors to sphingolipids and have been shown to take part in a wide variety of different physiological processes in eukaryotic organisms and are thought to be toxic at high concentrations. Ceramides are synthesized by condensation of the sphingoid base sphinganine and a fatty acyl CoA by ceramide synthases, a family of enzymes that differ in their specificity for the length of the acyl CoA substrate. A yeast strain in which the endogenous ceramide synthase was replaced by a cotton gene GhLag1, resulting in yeast cells producing C18 rather than C26 dihydroceramides indicating that GhLag1 is a bona fide ceramide synthase with specificity towards C18 acyl CoA. Further, the yeast cells expressing GhLag1 after deletion of the normally essential AUR1 encoding inositol phosphorylceramide synthase is viable, and are delayed in cytokinesis. The data suggest the essential roles of complex sphingolipids in controlling cytokninesis in eukaryotic cells.

Using the recent assembled G. raimondii D5 genome, we performed genome-wide mapping at single base-pair resolution using about 10 Gb RNA-seq data and 0.8 Gb non-coding RNA-seq data from Illumina sequencing platform. About sixty percent of intron-containing genes were alternative spliced in G. raimondii and intron retention was the most frequent (~39%) alternative slicing (AS) event while exon skipping, most commonly found in vertebrates, constituted only 6%. Comparative genomics across nine eukaryote species revealed that AS events profiles were conserved between G. raimondii and Arabidopsis thaliana, but varied from eudicots to monocots and other species. In some KNOX genes previously reported to be involved in leaf development, AS events result in the loss of homeodomain that is essential for the biochemical function of the encoded protein as a transcription factor. Combined with a significantly different expression level during plant leaf development, our data indicates that AS could be a potential regulatory mechanism of morphogenesis in plant. In addition, we identified 562 potential miRNA gene loci on G. raimondii genome, all of which were supported by non-coding RNA-seq data. Using cotton EST and microarray data, we found 463 putative miRNA-target genes and most significant up/down-regulation occurred in 10-20 days post anthesis (DPA), indicating that miRNAs played an important role during the elongation and secondary cell wall synthesis stages of cotton fiber development. Our study have enhanced the accuracy and diversity of the assembled G. raimondii genome and also provided better understanding of plant ribogenome.