Xueying Guan

Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.)

BackgroundCotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression.ResultsHere we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers.ConclusionsEnrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

miR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development

Although polyploidy is common in plants and some animals, mechanisms for functional divergence between homoeologous genes are poorly understood. MYB2 gene promotes cotton fibre development and is functionally homologous to Arabidopsis GLABROUS1 (GL1) in trichome formation. The most widely cultivated cotton is an allotetraploid (Gossypium hirsutum, AADD) that contains GhMYB2A and GhMYB2D homoeologs. Here we show that GhMYB2D mRNA accumulates more than GhMYB2A during fibre initiation and is targeted by miR828 and miR858, generating trans-acting siRNAs (ta-siRNAs) in the TAS4 family. Overexpressing GhMYB2A but not GhMYB2D complements the gl1 phenotype. Mutating the miR828-binding site or replacing its downstream sequence in GhMYB2D abolishes ta-siRNA production and restores trichome development in gl1 mutants. Moreover, disrupting Dicer-like protein 4 or RDR6, the biogenesis genes for ta-siRNAs, in the gl1 GhMYB2D overexpressors restores trichome development. These data support a unique role for microRNAs in functional divergence between target homoeologous genes that are important for evolution and selection of morphological traits.

The HD-Zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2

Most of the plant homeodomain-containing proteins play important roles in organ patterning and development, and Arabidopsis GLABRA2 (GL2), a member of the class IV homeodomain-leucine zipper (HD-ZIP) proteins, is a trichome and non-root hair cell regulator. Here we report the analysis of two cotton homeodomain-containing proteins, GaHOX1 and GaHOX2, isolated from the diploid cotton Gossypium arboreum. Both GaHOX1 and GaHOX2 belong to the class IV HD-ZIP family. When expressed under the control of the GL2 promoter, GaHOX1 rescued trichome development of an Arabidopsis glabrous mutant of gl2-2 (SALK_130213), whereas GaHOX2 did not. On the other hand, expression of GaHOX1 with a Cauliflower mosaic virus (CaMV) 35S promoter in the wild-type Arabidopsis plants suppressed the trichome development just as the GL2 ectopic expression. Expression analysis by Northern, RT-PCR and in situ hybridization indicated that GaHOX1 is predominantly expressed in cotton fiber cells at early developmental stages, consistent with its putative role in regulating cotton fiber development, while GaHOX2 is expressed in both fiber and other ovular tissues, including outer and inner integuments. Our results suggest that GaHOX1 is a functional homolog of GL2 in plant trichome development.

Apyrase (Nucleoside Triphosphate-Diphosphohydrolase) and Extracellular Nucleotides Regulate Cotton Fiber Elongation in Cultured Ovules

Ectoapyrase enzymes remove the terminal phosphate from extracellular nucleoside tri- and diphosphates. In Arabidopsis (Arabidopsis thaliana), two ectoapyrases, AtAPY1 and AtAPY2, have been implicated as key modulators of growth. In fibers of cotton (Gossypium hirsutum), transcript levels for GhAPY1 and GhAPY2, two closely related ectoapyrases that have high sequence similarity to AtAPY1 and AtAPY2, are up-regulated when fibers enter their rapid growth phase. In an ovule culture system, fibers release ATP as they grow, and when their ectoapyrase activity is blocked by the addition of polyclonal anti-apyrase antibodies or by two different small molecule inhibitors, the medium ATP level rises and fiber growth is suppressed. High concentrations of the poorly hydrolyzable nucleotides ATPγS and ADPβS applied to the medium inhibit fiber growth, and low concentrations of them stimulate growth, but treatment with adenosine 5′-O-thiomonophosphate causes no change in the growth rate. Both the inhibition and stimulation of growth by applied nucleotides can be blocked by an antagonist that blocks purinoceptors in animal cells, and by adenosine. Treatment of cotton ovule cultures with ATPγS induces increased levels of ethylene, and two ethylene antagonists, aminovinylglycine and silver nitrate, block both the growth stimulatory and growth inhibitory effects of applied nucleotides. In addition, the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, lowers the concentration of nucleotide needed to promote fiber growth. These data indicate that ectoapyrases and extracellular nucleotides play a significant role in regulating cotton fiber growth and that ethylene is a likely downstream component of the signaling pathway.

Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3

Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3–GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation.

Activation of Arabidopsis Seed Hair Development by Cotton Fiber-Related Genes

Each cotton fiber is a single-celled seed trichome or hair, and over 20,000 fibers may develop semi-synchronously on each seed. The molecular basis for seed hair development is unknown but is likely to share many similarities with leaf trichome development in Arabidopsis. Leaf trichome initiation in Arabidopsis thaliana is activated by GLABROUS1 (GL1) that is negatively regulated by TRIPTYCHON (TRY). Using laser capture microdissection and microarray analysis, we found that many putative MYB transcription factor and structural protein genes were differentially expressed in fiber and non-fiber tissues. Gossypium hirsutum MYB2 (GhMYB2), a putative GL1 homolog, and its downstream gene, GhRDL1, were highly expressed during fiber cell initiation. GhRDL1, a fiber-related gene with unknown function, was predominately localized around cell walls in stems, sepals, seed coats, and pollen grains. GFP:GhRDL1 and GhMYB2:YFP were co-localized in the nuclei of ectopic trichomes in siliques. Overexpressing GhRDL1 or GhMYB2 in A. thaliana Columbia-0 (Col-0) activated fiber-like hair production in 4–6% of seeds and had on obvious effects on trichome development in leaves or siliques. Co-overexpressing GhRDL1 and GhMYB2 in A. thaliana Col-0 plants increased hair formation in ∼8% of seeds. Overexpressing both GhRDL1 and GhMYB2 in A. thaliana Col-0 try mutant plants produced seed hair in ∼10% of seeds as well as dense trichomes inside and outside siliques, suggesting synergistic effects of GhRDL1 and GhMYB2 with try on development of trichomes inside and outside of siliques and seed hair in A. thaliana. These data suggest that a different combination of factors is required for the full development of trichomes (hairs) in leaves, siliques, and seeds. A. thaliana can be developed as a model a system for discovering additional genes that control seed hair development in general and cotton fiber in particular.

Polyploidy and small RNA regulation of cotton fiber development

Cotton is not only the most important source of renewal textile fibers, but also an excellent model for studying cell fate determination and polyploidy effects on gene expression and evolution of domestication traits. The combination of A and D-progenitor genomes into allotetraploid cotton induces intergenomic interactions and epigenetic effects, leading to the unequal expression of homoeologous genes. Small RNAs regulate the expression of transcription and signaling factors related to cellular growth, development and adaptation. An example is miRNA-mediated preferential degradation of homoeologous mRNAs encoding MYB-domain transcription factors that are required for the initiation of leaf trichomes in Arabidopsis and of seed fibers in cotton. This example of coevolution between small RNAs and their homoeologous targets could shape morphological traits such as fibers during the selection and domestication of polyploid crops.

Genetic basis for glandular trichome formation in cotton

Trichomes originate from epidermal cells and can be classified as either glandular or non-glandular. Gossypium species are characterized by the presence of small and darkly pigmented lysigenous glands that contain large amounts of gossypol. Here, using a dominant glandless mutant, we characterize GoPGF, which encodes a basic helix-loop-helix domain-containing transcription factor, that we propose is a positive regulator of gland formation. Silencing GoPGF leads to a completely glandless phenotype. A single nucleotide insertion in GoPGF, introducing a premature stop codon is found in the duplicate recessive glandless mutant (gl2gl3). The characterization of GoPGF helps to unravel the regulatory network of glandular structure biogenesis, and has implications for understanding the production of secondary metabolites in glands. It also provides a potential molecular basis to generate glandless seed and glanded cotton to not only supply fibre and oil but also provide a source of protein for human consumption.

Auxin boost for cotton

Increasing auxin levels at the right time and place during ovule and fiber development improves the yield and quality of cotton fibers.

A Long-Read Transcriptome Assembly of Cotton ( Gossypium hirsutum L.) and Intraspecific Single Nucleotide Polymorphism Discovery

Upland cotton (Gossypium hirsutum L.) has a narrow germplasm base, which constrains marker development and hampers intraspecific breeding. A pressing need exists for high‐throughput single nucleotide polymorphism (SNP) markers that can be readily applied to germplasm in breeding and breeding‐related research programs. Despite progress made in developing new sequencing technologies during the past decade, the cost of sequencing remains substantial when one is dealing with numerous samples and large genomes. Several strategies have been proposed to lower the cost of sequencing for multiple genotypes of large‐genome species like cotton, such as transcriptome sequencing and reduced‐representation DNA sequencing. This paper reports the development of a transcriptome assembly of the inbred line Texas Marker‐1 (TM‐1), a genetic standard for cotton, its usefulness as a reference for RNA sequencing (RNA‐seq)‐based SNP identification, and the availability of transcriptome sequences of four other cotton cultivars. An assembly of TM‐1 was made using Roche 454 transcriptome reads combined with an assembly of all available public expressed sequence tag (EST) sequences of TM‐1. The TM‐1 assembly consists of 72,450 contigs with a total of 70 million bp. Functional predictions of the transcripts were estimated by alignment to selected protein databases. Transcriptome sequences of the five lines, including TM‐1, were obtained using an Illumina Genome Analyzer‐II, and the short reads were mapped to the TM‐1 assembly to discover SNPs among the five lines. We identified >14,000 unfiltered allelic SNPs, of which ∼3,700 SNPs were retained for assay development after applying several rigorous filters. This paper reports availability of the reference transcriptome assembly and shows its utility in developing intraspecific SNP markers in upland cotton.