Gene expression profiling during gland morphogenesis of a mutant and a glandless upland cotton.

To identify genes involved in pigment gland morphogenesis in cotton, gene expression was profiled using genechip (Affymetrix) during pigment gland morphogenesis in cotton variety Xiangmian-18, which has glandless seeds but glanded plants, and a glandless line, N5. The results showed that 303 genes were differentially expressed by a factor greater than two during gland morphogenesis; 59% (180) of these genes shared similarity with known genes in GenBank. These genes play roles in defense response, response to oxidative stress, peroxidase activity, and other metabolic pathways. KOBAS (KEGG Orthology-Based Annotation System) indicate that these genes are involved in 68 biochemical pathways. These findings suggest that the related defense response, gossypol biosynthesis pathway and other complex regulation may be associated with pigment gland morphogenesis in cotton. The results may provide a basis for further study and serve as a guide for related research.

Functional analysis of the GbDWARF14 gene associated with branching development in cotton.

Plant architecture, including branching pattern, is an important agronomic trait of cotton crops. In recent years, strigolactones (SLs) have been considered important plant hormones that regulate branch development. In some species such as Arabidopsis, DWARF14 is an unconventional receptor that plays an important role in the SL signaling pathway. However, studies on SL receptors in cotton are still lacking. Here, we cloned and analysed the structure of the GbD14 gene in Gossypium barbadense and found that it contains the domains necessary for a SL receptor. The GbD14 gene was expressed primarily in the roots, leaves and vascular bundles, and the GbD14 protein was determined via GFP to localize to the cytoplasm and nucleus. Gene expression analysis revealed that the GbD14 gene not only responded to SL signals but also was differentially expressed between cotton plants whose types of branching differed. In particular, GbD14 was expressed mainly in the axillary buds of normal-branching cotton, while it was expressed the most in the leaves of nulliplex-branch cotton. In cotton, the GbD14 gene can be induced by SL and other plant hormones, such as indoleacetic acid, abscisic acid, and jasmonic acid. Compared with wild-type Arabidopsis, GbD14-overexpressing Arabidopsis responded more rapidly to SL signals. Moreover, we also found that GbD14 can rescue the multi-branched phenotype of Arabidopsis Atd14 mutants. Our results indicate that the function of GbD14 is similar to that of AtD14, and GbD14 may be a receptor for SL in cotton and involved in regulating branch development. This research provides a theoretical basis for a profound understanding of the molecular mechanism of branch development and ideal plant architecture for cotton breeding improvements.

To Be a Flower or Fruiting Branch: Insights Revealed by mRNA and Small RNA Transcriptomes from Different Cotton Developmental Stages.

The architecture of the cotton plant, including fruit branch formation and flowering pattern, is the most important characteristic that directly influences light exploitation, yield and cost of planting. Nulliplex branch is a useful phenotype to study cotton architecture. We used RNA sequencing to obtain mRNA and miRNA profiles from nulliplex- and normal-branch cotton at three developmental stages. The differentially expressed genes (DEGs) and miRNAs were identified that preferentially/specifically expressed in the pre-squaring stage, which is a key stage controlling the transition from vegetative to reproductive growth. The DEGs identified were primarily enriched in RNA, protein, and signalling categories in Gossypium barbadense and Gossypium hirsutum. Interestingly, during the pre-squaring stage, the DEGs were predominantly enriched in transcription factors in both G. barbadense and G. hirsutum, and these transcription factors were mainly involved in branching and flowering. Related miRNAs were also identified. The results showed that fruit branching in cotton is controlled by molecular pathways similar to those in Arabidopsis and that multiple regulated pathways may affect the development of floral buds. Our study showed that the development of fruit branches is closely related to flowering induction and provides insight into the molecular mechanisms of branch and flower development in cotton.

An optimized gossypol high-performance liquid chromatography assay and its application in evaluation of different gland genotypes of cotton.

A comparative study on gossypol content of various genetic types of pigment glands of cotton varieties was conducted through an optimized high-performance liquid chromatography (HPLC) on a C18 column (4.6 mm x 250 mm, 5 microm particle) with methanol-0.5% acetic acid aqueous solution, 90 : 10 (v/v), as mobile phase, at a flow rate of 0.8 ml/min and UV detection at 254 nm. The method was shown to be highly reproducible, with precision [as relative standard deviation (RSD)] and accuracy [as relative mean error (RME)] < 10%, both intra-day and inter-day. Absolute recoveries were > 94%. The results revealed major differences among the different gland varieties or species of cotton, including the special and ordinary glandless and glanded Gossypium hirsutum, G. barbadense, and displayed the precious resources of different glands of extraordinary cotton.

Identification of novel microRNAs in the Verticillium wilt-resistant upland cotton variety KV-1 by high-throughput sequencing.

Plant microRNAs (miRNAs) play essential roles in the post-transcriptional regulation of gene expression during development, flowering, plant growth, metabolism, and stress responses. Verticillium wilt is one of the vascular disease in plants, which is caused by the Verticillium dahlia and leads to yellowing, wilting, lodging, damage to the vascular tissue, and death in cotton plants. Upland cotton varieties KV-1 have shown resistance to Verticillium wilt in multiple levels. However, the knowledge regarding the post-transcriptional regulation of the resistance is limited. Here two novel small RNA (sRNA) libraries were constructed from the seedlings of upland cotton variety KV-1, which is highly resistant to Verticillium wilts and inoculated with the V991 and D07038 Verticillium dahliae (V. dahliae) of different virulence strains. Thirty-seven novel miRNAs were identified after sequencing these two libraries by the Illumina Solexa system. According to sequence homology analysis, potential target genes of these miRNAs were predicted. With no more than three sequence mismatches between the novel miRNAs and the potential target mRNAs, we predicted 49 target mRNAs for 24 of the novel miRNAs. These target mRNAs corresponded to genes were found to be involved in plant-pathogen interactions, endocytosis, the mitogen-activated protein kinase (MAPK) signaling pathway, and the biosynthesis of isoquinoline alkaloid, terpenoid backbone, primary bile acid and secondary metabolites. Our results showed that some of these miRNAs and their relative gene are involved in resistance to Verticillium wilts. The identification and characterization of miRNAs from upland cotton could help further studies on the miRNA regulatory mechanisms of resistance to Verticillium wilt.