Shuxun Yu

Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton

BackgroundGenetic male sterility (GMS) in cotton (Gossypium hirsutum) plays an important role in the utilization of hybrid vigor. However, the molecular mechanism of the GMS is still unclear. While numerous studies have demonstrated that microRNAs (miRNA) regulate flower and anther development, whether different small RNA regulations exist in GMS and its wild type is unclear. A deep sequencing approach was used to investigate the global expression and complexity of small RNAs during cotton anther development in this study.ResultsThree small RNA libraries were constructed from the anthers of three development stages each from fertile wild type (WT) and its GMS mutant cotton, resulting in nearly 80 million sequence reads. The total number of miRNAs and short interfering RNAs in the three WT libraries was significantly greater than that in the corresponding three mutant libraries. Sixteen conserved miRNA families were identified, four of which comprised the vast majority of the expressed miRNAs during anther development. In addition, six conserved miRNA families were significantly differentially expressed during anther development between the GMS mutant and its WT.ConclusionsThe present study is the first to deep sequence the small RNA population in G. hirsutum GMS mutant and its WT anthers. Our results reveal that the small RNA regulations in cotton GMS mutant anther development are distinct from those of the WT. Further results indicated that the differently expressed miRNAs regulated transcripts that were distinctly involved in anther development. Identification of a different set of miRNAs between the cotton GMS mutant and its WT will facilitate our understanding of the molecular mechanisms for male sterility.

Genome-wide analysis of the WRKY gene family in cotton.

WRKY proteins are major transcription factors involved in regulating plant growth and development. Although many studies have focused on the functional identification of WRKY genes, our knowledge concerning many areas of WRKY gene biology is limited. For example, in cotton, the phylogenetic characteristics, global expression patterns, molecular mechanisms regulating expression, and target genes/pathways of WRKY genes are poorly characterized. Therefore, in this study, we present a genome-wide analysis of the WRKY gene family in cotton (Gossypium raimondii and Gossypium hirsutum). We identified 116 WRKY genes in G. raimondii from the completed genome sequence, and we cloned 102 WRKY genes in G. hirsutum. Chromosomal location analysis indicated that WRKY genes in G. raimondii evolved mainly from segmental duplication followed by tandem amplifications. Phylogenetic analysis of alga, bryophyte, lycophyta, monocot and eudicot WRKY domains revealed family member expansion with increasing complexity of the plant body. Microarray, expression profiling and qRT-PCR data revealed that WRKY genes in G. hirsutum may regulate the development of fibers, anthers, tissues (roots, stems, leaves and embryos), and are involved in the response to stresses. Expression analysis showed that most group II and III GhWRKY genes are highly expressed under diverse stresses. Group I members, representing the ancestral form, seem to be insensitive to abiotic stress, with low expression divergence. Our results indicate that cotton WRKY genes might have evolved by adaptive duplication, leading to sensitivity to diverse stresses. This study provides fundamental information to inform further analysis and understanding of WRKY gene functions in cotton species.

Transcriptomic analysis of differentially expressed genes during anther development in genetic male sterile and wild type cotton by digital gene-expression profiling

BackgroundCotton (Gossypium hirsutum) anther development involves a diverse range of gene interactions between sporophytic and gametophytic tissues. However, only a small number of genes are known to be specifically involved in this developmental process and the molecular mechanism of the genetic male sterility (GMS) is still poorly understand. To fully explore the global gene expression during cotton anther development and identify genes related to male sterility, a digital gene expression (DGE) analysis was adopted.ResultsSix DGE libraries were constructed from the cotton anthers of the wild type (WT) and GMS mutant (in the WT background) in three stages of anther development, resulting in 21,503 to 37,352 genes detected in WT and GMS mutant anthers. Compared with the fertile isogenic WT, 9,595 (30% of the expressed genes), 10,407 (25%), and 3,139 (10%) genes were differentially expressed at the meiosis, tetrad, and uninucleate microspore stages of GMS mutant anthers, respectively. Using both DGE experiments and real-time quantitative RT-PCR, the expression of many key genes required for anther development were suppressed in the meiosis stage and the uninucleate microspore stage in anthers of the mutant, but these genes were activated in the tetrad stage of anthers in the mutant. These genes were associated predominantly with hormone synthesis, sucrose and starch metabolism, the pentose phosphate pathway, glycolysis, flavonoid metabolism, and histone protein synthesis. In addition, several genes that participate in DNA methylation, cell wall loosening, programmed cell death, and reactive oxygen species generation/scavenging were activated during the three anther developmental stages in the mutant.ConclusionsCompared to the same anther developmental stage of the WT, many key genes involved in various aspects of anther development show a reverse gene expression pattern in the GMS mutant, which indicates that diverse gene regulation pathways are involved in the GMS mutant anther development. These findings provide the first insights into the mechanism that leads to genetic male sterility in cotton and contributes to a better understanding of the regulatory network involved in anther development in cotton.

Genomic organization, differential expression, and functional analysis of the SPL gene family in Gossypium hirsutum

SQUAMOSA promoter binding protein-like (SPL) genes encode plant-specific transcription factors that are involved in many fundamental developmental processes. Certain SPL genes contain sequences complementary to miR156, a microRNA (miRNA) that plays a role in modulating plant gene expression. In this study, 30 SPL genes were identified in the reference genome of Gossypium raimondii and 24 GhSPLs were cloned from Gossypium hirsutum. G. raimondii is regarded as the putative contributor of the D-subgenome of G. hirsutum. Comparative analysis demonstrated sequence conservation between GhSPLs and other plant species. GhSPL genes could be classified into seven subclades based on phylogenetic analysis, diverse intron–exon structure, and motif prediction. Within each subclade, genes shared a similar structure. Sequence and experimental analysis predicted that 18 GhSPL genes are putative targets of GhmiR156. Additionally, tissue-specific expression analysis of GhSPL genes showed that their spatiotemporal expression patterns during development progressed differently, with most genes having high transcript levels in leaves, stems, and flowers. Finally, overexpression of GhSPL3 and GhSPL18 in Arabidopsis plants demonstrated that these two genes are involved in the development of leaves and second shoots and play an integral role in promoting flowering. The flowering integrator GhSOC1 may bind to the promoter of GhSPL3 but not GhSPL18 to regulate flowering. In conclusion, our analysis of GhSPL genes will provide some gene resources and a further understanding of GhSPL3 and GhSPL18 function in flowering promotion. Furthermore, the comparative genomics and functional analysis deepened our understanding of GhSPL genes during upland cotton vegetative and reproductive growth.

Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton.

BackgroundThe cotton (Gossypium spp.) fiber cell is an important unicellular model for studying cell differentiation. There is evidence suggesting that phosphorylation is a critical post-translational modification involved in regulation of a wide range of cell activities. Nevertheless, the sites of phosphorylation in G. hirsutum and their regulatory roles in fiber cell initiation are largely unknown. In this study, we employed a mass spectrometry-based phosphoproteomics to conduct a global and site-specific phosphoproteome profiling between ovules of a fuzzless-lintless (fl) Upland cotton (G. hirsutum) mutant and its isogenic parental wild type (WT) at -3 and 0xa0days post-anthesis (DPA).ResultsA total of 830 phosphopeptides and 1,592 phosphorylation sites from 619 phosphoproteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation). Of these, 76 phosphoproteins and 1,100 phosphorylation sites were identified for the first time after searching the P3DB public database using the BLAST program. Among the detected phosphopeptides, 69 were differentially expressed between the fl mutant and its WT in ovules at -3 and 0 DPA. An analysis using the Motif-X program uncovered 19 phosphorylation motifs, 8 of which were unique to cotton. A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction, protein modification, carbohydrate metabolic processes, and cell cycle and cell proliferation.ConclusionsOur phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation, and also offers a helpful dataset for elucidation of signaling networks in fiber development of G. hirsutum.

Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana

Extensive studies on floral transition in model species have revealed a network of regulatory interactions between proteins that transduce and integrate developmental and environmental signals to promote or inhibit the transition to flowering. Previous studies indicated FLOWERING PROMOTING FACTOR 1 (FPF1) gene was involved in the promotion of flowering, but the molecular mechanism was still unclear. Here, FPF1 homologous sequences were screened from diploid Gossypium raimondii L. (D-genome, nu200a=u200a13) and Gossypium arboreum L. genome (A-genome, nu200a=u200a13) databases. Orthologous genes from the two species were compared, suggesting that distinctions at nucleic acid and amino acid levels were not equivalent because of codon degeneracy. Six FPF1 homologous genes were identified from the cultivated allotetraploid Gossypium hirsutum L. (AD-genome, nu200a=u200a26). Analysis of relative transcripts of the six genes in different tissues revealed that this gene family displayed strong tissue-specific expression. GhFPF1, encoding a 12.0-kDa protein (Accession No: KC832319) exerted more transcripts in floral apices of short-season cotton, hinting that it could be involved in floral regulation. Significantly activated APETALA 1 and suppressed FLOWERING LOCUS C expression were induced by over-expression of GhFPF1 in the Arabidopsis Columbia-0 ecotype. In addition, transgenic Arabidopsis displayed a constitutive shade-avoiding phenotype that is characterized by long hypocotyls and petioles, reduced chlorophyll content, and early flowering. We propose that GhFPF1 may be involved in flowering time control and shade-avoidance responses.

Molecular Cloning and Function Analysis of Two SQUAMOSA-Like MADS-Box Genes From Gossypium hirsutum L.†

The MADS-box genes encode a large family of transcription factors having diverse roles in plant development. The SQUAMOSA (SQUA)/APETALA1 (AP1)/FRUITFULL (FUL) subfamily genes are essential regulators of floral transition and floral organ identity. Here we cloned two MADS-box genes, GhMADS22 and GhMADS23, belonging to the SQUA/AP1/FUL subgroup from Gossypium hirsutum L. Phylogenetic analysis and sequence alignment showed that GhMADS22 and GhMADS23 belonged to the euFUL and euAP1 subclades, respectively. The two genes both had eight exons and seven introns from the start codon to the stop codon according to the alignment between the obtained cDNA sequence and the Gossypium raimondii L. genome sequence. Expression profile analysis showed that GhMADS22 and GhMADS23 were highly expressed in developing shoot apices, bracts, and sepals. Gibberellic acid promoted GhMADS22 and GhMADS23 expression in the shoot apex. Transgenic Arabidopsis lines overexpressing 35S::GhMADS22 had abnormal flowers and bolted earlier than wild type under long-day conditions (16u2009h light/8u2009h dark). Moreover, GhMADS22 overexpression delayed floral organ senescence and abscission and it could also respond to abscisic acid. In summary, GhMADS22 may have functions in promoting flowering, improving resistance and delaying senescence for cotton and thus it may be a candidate target for promoting early-maturation in cotton breeding.

Genome-wide analysis of the family 1 glycosyltransferases in cotton.

Family 1 GT, designated as UGT, is the largest and most functionally important multigene family in the plant kingdom. In this study, we carried out a genome-wide identification, analysis, and comparison of 142, 146, and 196 putative UGTs from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively. All members present the 44 amino-acid conserved consensus sequence termed the plant secondary product glycosyltransferase motif. According to the phylogenetic relationship among the cotton UGT proteins and those from other species, GrUGTs and GaUGTs could be classified into 16 major phylogenetic groups (A–P), whereas GhUGTs are classified into 15 major phylogenetic groups with a lack of group C. All cotton UGTs are dispersed throughout the chromosomes and are displayed in clusters with the same open reading frame orientation. The expansion of them appears to result from genome duplication and rearrangement. Two conserved introns, A and B, are detected in most of the intron-containing-UGTs in G. raimondii and G. arboreum, whereas only intron A is detected in the intron-containing-UGTs in G. hirsutum. Furthermore, expression patterns of the UGT genes in G. hirsutum wild type and its near isogenic fuzzless–lintless mutant at the stage of fiber initiation were analyzed using the RNA-seq data. Overall, this study not only deepens our understanding of the structure, phylogeny, evolution, and expression of cotton UGT genes, but also provides a solid foundation for further cloning and functional studies of the UGT family genes.

Selection and Characterization of a Novel Photoperiod‐Sensitive Male Sterile Line in Upland Cotton

Upland cotton (Gossypium hirsutum L.) shows strong heterosis. However, heterosis is not widely utilized owing to the high cost of hybrid seed production. Creation of a photoperiod-sensitive genetic male sterile line could substantially reduce the cost of hybrid seed production in upland cotton. Such a mutant with virescent marker was found by space mutation in near-earth orbit and its traits had been stable after 4 years of selection in Anyang and Sanya, China. This mutant was fertile with an 11-12.5u2009h photoperiod when the temperature was higher than 21.5u2009°C and was sterile with a 13-14.5u2009h photoperiod. Genetic analysis indicated that both traits were controlled by a single recessive gene or two closely linked genes. Also, the cytological observations and transcriptome profiling analysis showed that the degradation of pollen grain cytoplasm should be the primary reason why the mutant line were male sterile under long-day conditions.

Generation and analysis of a large-scale expressed sequence Tag database from a full-length enriched cDNA library of developing leaves of Gossypium hirsutum L.

Background Cotton (Gossypium hirsutum L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. Methodology/Principal Findings In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. Conclusions/Significance These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in G. hirsutum and comparative genomics among Gossypium species.