SUMMARYLittle is still known about the developmental control of the long seed coat trichomes of cotton (Gossypium hirsutum L.). In Arabidopsis, leaf trichome initiation is regulated by a group of well-defined transcription factors that includes MYB and homeodomain types. Many MYBs are expressed in fibres, but their roles in fibre development remain unclear. We analysed the function of one MYB transcription factor, GhMYB25, identified from transcriptome comparisons between wild-type and fibreless cotton mutants. A GhMYB25 promoter-GUS construct in transgenic cotton was expressed in the epidermis of ovules, developing fibre initials and fibres, in the trichomes of a number of tissues including leaves, stems and petals, as well as in the anthers, pollen and the epidermal layers of roots and root initials, but not in root hairs. Cotton is an allotetraploid with two very similar GhMYB25 genes that were silenced by a single RNAi construct. GhMYB25-silenced cotton showed alterations in the timing of rapid fibre elongation, resulting in short fibres, dramatic reductions in trichomes on other parts of the plant, and reductions in seed production. Reciprocal crosses between transgenic and nontransgenic plants indicated that pollen and ovule viability per se were not disrupted. Ectopic over-expression of GhMYB25 had more subtle impacts, with increases in cotton fibre initiation and leaf trichome number. High expression appeared to adversely affect fertility. Our results provide convincing evidence for a role of GhMYB25, like other MIXTA-like MYBS, in regulating specialized outgrowths of epidermal cells, including, in this case, cotton fibres.

Cotton fibres are a subset of single epidermal cells that elongate from the seed coat to produce the long cellulose strands or lint used for spinning into yarn. To identify genes that might regulate lint fibre initiation, expression profiles of 0 days post-anthesis (dpa) whole ovules from six reduced fibre or fibreless mutants were compared with wild-type linted cotton using cDNA microarrays. Numerous clones were differentially expressed, but when only those genes that are normally expressed in the ovule outer integument (where fibres develop) were considered, just 13 different cDNA clones were down-regulated in some or all of the mutants. These included: a Myb transcription factor (GhMyb25) similar to the Antirrhinum Myb AmMIXTA, a putative homeodomain protein (related to Arabidopsis ATML1), a cyclin D gene, some previously identified fibre-expressed structural and metabolic genes, such as lipid transfer protein, alpha-expansin and sucrose synthase, as well as some unknown genes. Laser capture microdissection and reverse transcription-PCR were used to show that both the GhMyb25 and the homeodomain gene were predominantly ovule specific and were up-regulated on the day of anthesis in fibre initials relative to adjacent non-fibre ovule epidermal cells. Their spatial and temporal expression pattern therefore coincided with the time and location of fibre initiation. Constitutive overexpression of GhMyb25 in transgenic tobacco resulted in an increase in branched long-stalked leaf trichomes. The involvement of cell cycle genes prompted DNA content measurements that indicated that fibre initials, like leaf trichomes, undergo DNA endoreduplication. Cotton fibre initiation therefore has some parallels with leaf trichome development, although the detailed molecular mechanisms are clearly different.

Here we present a quick and low-cost method to separate the different layers of tissue from the ovules and young seeds of cotton (Gossypium hirsutum L.) for use in high- and low-throughput molecular applications. This method is performed at room temperature using standard laboratory equipment and does not require embedding of the samples, time-consuming fixation, or micro-sectioning procedures. We show that the three main tissues can be efficiently separated from isolated ovules collected on the day of anthesis. RNA and genomic DNA extracted from tissues separated by this method are of good quality and suitable for a variety of molecular applications to study the early stages of cotton seed and fiber development.