Integrative transcriptome, proteome, phosphoproteome and genetic mapping reveals new aspects in a fiberless mutant of cotton.

To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at -3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins, and 131 phosphoproteins were differentially expressed (DE) between WT and fl, and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting, and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation.

[Genome-wide analysis of the LPAAT gene family in Gossypium raimondii and G. arboreum, and expression analysis of its orthologs in G. hirsutum].

Lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid into phosphatidic acid is a key enzyme in biosynthesis pathway of lipid in plants. In this study, we identified 17 members of the LPAAT gene family from genomic data of G. raimondii-D5 and G. arboreum-A2. Analysis of gene structure, chromosome distribution and phylogenetic evolution of LPAAT genes in diploid Gossypium using bioinformatics approaches showed that these genes can be divided into distinct subfamilies based on the distance of their genetic relationship. Moreover, the gene structures were similar within LPAAT subfamily members. The amino acid sequences encoded by LPAAT family genes contained three conserved motifs, including ΦFPEGTR-G binding site and Φ-NHQS- ΦDΦΦ catalytic site. Phylogenetic analysis of LPAAT gene family demonstrated significant differences in evolution of LPAAT in different species. Finally, expression analysis of G. hirsutum ovules in different stages from RNA-seq and qRT-PCR data indicated that LPAAT gene may play a positive role in oil accumulation. Our studies facilitate understanding of the function of LPAAT gene family in Gossypium and selecting better LPAAT genes for further functional validation.

Cloning and characterization of a FLO/LFY ortholog in Gossypium hirsutum L.

KEY MESSAGE: GhLFY was cloned from G. hirsutum L. Its expression, subcellular localization, and function were analyzed, as well as the in vivo regulation of GhLFY by the MADS-box protein SOC1 (GhSOC1). ABSTRACT: Flowering is a very important phase during which plants produce the organs for sexual reproduction. The FLORICAULA/LEAFY (FLO/LFY) homologs play a major role in the initiation of flowering. To understand the mechanism of the transition from the vegetative to reproductive phases in Upland cotton (Gossypium hirsutum L.), we isolated a candidate LFY gene from G. hirsutum L. (GhLFY) that showed a high degree of similarity to other plant homologs of FLO/LFY. qPCR analysis showed that GhLFY was highly expressed in the shoot apex, with substantial upregulation at the third true leaf expansion stage during floral bud differentiation. Subcellular localization studies revealed GhLFY localization in the nucleus. Ectopic expression of the GhLFY coding region in Arabidopsis resulted in early flowering. The expression of the GhLFY coding region under the control of the 35S promoter complemented the lfy-5 mutation in transgenic Arabidopsis lfy-5 mutant plants. Furthermore, a chromatin immunoprecipitation assay revealed that GhLFY may function downstream of GhSOC1 during the initiation of flowering in G. hirsutum L. GhLFY was likely to be regulated by GhSOC1, which binds to the LFY promoter in Arabidopsis. These results suggest that GhLFY is a FLO/LFY ortholog that may be involved in controlling flowering time and floral development.

Comparative proteomics indicates that biosynthesis of pectic precursors is important for cotton fiber and Arabidopsis root hair elongation.

The quality of cotton fiber is determined by its final length and strength, which is a function of primary and secondary cell wall deposition. Using a comparative proteomics approach, we identified 104 proteins from cotton ovules 10 days postanthesis with 93 preferentially accumulated in the wild type and 11 accumulated in the fuzzless-lintless mutant. Bioinformatics analysis indicated that nucleotide sugar metabolism was the most significantly up-regulated biochemical process during fiber elongation. Seven protein spots potentially involved in pectic cell wall polysaccharide biosynthesis were specifically accumulated in wild-type samples at both the protein and transcript levels. Protein and mRNA expression of these genes increased when either ethylene or lignoceric acid (C24:0) was added to the culture medium, suggesting that these compounds may promote fiber elongation by modulating the production of cell wall polymers. Quantitative analysis revealed that fiber primary cell walls contained significantly higher amounts of pectin, whereas more hemicellulose was found in ovule samples. Significant fiber growth was observed when UDP-L-rhamnose, UDP-D-galacturonic acid, or UDP-D-glucuronic acid, all of which were readily incorporated into the pectin fraction of cell wall preparations, was added to the ovule culture medium. The short root hairs of Arabidopsis uer1-1 and gae6-1 mutants were complemented either by genetic transformation of the respective cotton cDNA or by adding a specific pectin precursor to the growth medium. When two pectin precursors, produced by either UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase 4-reductase or by UDP-D-glucose dehydrogenase and UDP-D-glucuronic acid 4-epimerase successively, were used in the chemical complementation assay, wild-type root hair lengths were observed in both cut1 and ein2-5 Arabidopsis seedlings, which showed defects in C24:0 biosynthesis or ethylene signaling, respectively. Our results suggest that ethylene and C24:0 may promote cotton fiber and Arabidopsis root hair growth by activating the pectin biosynthesis network, especially UDP-L-rhamnose and UDP-D-galacturonic acid synthesis.

[Development and appraisement of functional molecular marker: intron sequence amplified polymorphism (ISAP)].

Molecular markers are playing an increasingly important role in map construction, QTL analysis, gene mapping and marker-assisted selection. Researchers hope the target gene and locus are as close as possible, one locus can present one gene, or linked with some important trait, then, individuals with useful trait can be selected through molecular markers selecting, and it's the functional molecular marker. PCR-based molecular markers such as RAPD, SSR, AFLP amplified non-coding regions, or the whole genome randomly, the locus is far away from the gene of targeted trait, this limit the ap-plication of these molecular markers. This study established a kind of functional molecular markers based on intron of gene sequence, trying to link loci with gene sequence to achieve the purpose of its function. It used the conservative consistent sequence of intron splicing sites as its core sequence of amplification. ISAP is a PCR-based marker system, it has two kinds of primers: forward primer and reverse primer, both primers are 18 bases. Any of the primers can be used to construct a primer combination with the other kind of primers. Seventeen primers, 9 forward and 8 reverse, were used to construct 72 primer combinations, 67 of them showed polymorphism in a G. hirsutum cv. CCRI36 x G. barbadense cv. H7124 F2 population and a total of 212 loci were obtained. Together with 164 SRAP loci, these 212 loci were used to construct a genetic linkage map. ISAP markers distributed evenly in the entire linkage group, part of the region had a high saturation, might be the coding sequence-rich region. Sequencing results of 20 fragments showed that 85% of the sequences announced homology with published EST sequence stored in the NCBI which indicated that they were amplified adjacent to expressed sequences. These results showed that ISAP marker system was simple, efficient, reliable, and had a relatively high polymorphism, furthermore, it directly targeted gene sequence, was a functional molecular marker system. ISAP was also used to amplify other plants and good results were achieved.

Cloning and expression of the chloroplast copper/zinc-superoxide dismutase gene in upland cotton (Gossypium hirsutum L.).

A full-length 1,043-base-pair cDNA clone encoding a chloroplast copper/zinc superoxide dismutase (Cu/Zn-SOD) of upland cotton was first isolated by rapid amplification of cDNA ends (RACE) from the leaves of the cotton (Gossypium hirsutum L.) variety 'CRI36'. Nucleotide sequence analysis of the clone revealed that it contained the complete coding sequence of the mature SOD isozyme subunit, along with a 60-amino acid transit peptide at N-terminal. The amino acid sequence predicted from the full-length clone showed 66%-74% homology with the amino acid sequences of Cu/Zn-SOD from several other plants. This gene was found to be expressed in the leaves and stems, but not in roots, flowers, and hypocotyls, indicating that the gene was expressed only in green tissues. Also, its expression was found to be most active at seedling stage and declined gradually in later development stages. Expression of this cotton Cu/Zn-SOD gene by using the pET-21a (+) expression vector in E. coli BL21 (DE3) led to the production of a novel 29 kD polypeptide with SOD enzyme activity, confirming that the cloned cotton Cu/Zn-SOD cDNA was indeed encoding a functioning SOD enzyme.

Relationship between gene differential expression of leaves in full opening flower stages of hybrids & their parents and heterosis in pest-resistant cotton.

cDNAs of leaves of 24 pest-resistant cotton (authorization) crosses and their parents in full opening flower stages were amplified and different bands were displayed by DDRT-PCR, with 45 primer combinations. The mean percentage of bands duplicated was 70.1%, which implied that there was a high proportion of pseudopositive fragments in the amplified cDNA. These pseudopositive bands can be reduced using duplicate PCR. Correlation analysis between differential gene expression and hybrid performance and heterosis showed that M4 (bands detected in one parent and F1) was positively correlated with all yield traits, and significantly correlated with boll number; M2 (bands observed in one parent but not in F1 and another parent) was negatively correlated with heterosis of boll number; and M3 (bands observed only in F1) was positively correlated with heterosis of lint yield. These results showed that genes that had dominant and specific expression in top leaves of full opening flower stages were beneficial to yield formation and heterosis occurrence.