Identification and analysis of genes involved in cotton fibre initiation.

Abstract

Cotton fibres are single-celled hairs, arising from the epidermal surface of the cotton ovule. One factor in determining the length of the mature cotton fibre is the timing of fibre initiation, which is therefore a crucial step in obtaining commercial cotton fibres. To achieve a greater understanding of the regulation of cotton fibre differentiation, more fundamental information is needed on the signals and mechanisms associated with fibre initiation. The extensive genetic knowledge of Arabidopsis leaf trichomes could aid in the elucidation of the genetic mechanisms controlling cotton fibre differentiation. Trichomes are small hairs on the plant surface, originating from single epidermal cells in a developmental process that appears very similar to that of cotton fibres. Arabidopsis trichome development has been extensively investigated, and several genes that control the process have been characterised. One gene essential for trichome initiation is TRANSPARENT TESTA GLABRAI (TTGI), and loss-of-function mutations in TTGI result in an almost complete absence of leaf trichomes. TTG 1 plays additional roles in numerous pathways in Arabidopsis, including root hair initiation, anthocyanin production and seed coat mucilage production. In order to isolate genes required for fibre initiation in cotton, functional homologues of Arabidopsis TTG 1 in cotton have been sought. Four putative homologues of Arabidopsis TTG 1 have previously been isolated in this laboratory by RT-PCR of mRNA prepared from cotton fibres, and are termed GhTTG 1-4. Sequence comparisons between the four cotton deduced proteins and Arabidopsis TTG 1 showed that they form two groups, with GhTTG 1 and GhTTG3 being closely related to each other (87% identical and 93% similar) and to TTG 1 (79% and 80% amino acid identity respectively). GhTTG2 and GhTTG4 formed the second group, with 95% amino acid identity to each other and lower (approximately 62%) identity to TTG 1. An analysis of the genomic origins of the GhTTG genes demonstrated that each is derived from the same ancestral diploid genome. Cross-species complementation experiments were performed to test for functional homology of these cotton TTG I-like genes to AtTTG 1, by introducing the cotton genes into Arabidopis ttgI-I mutants via Agrobacterium-mediated transformation. This experiment showed that two of the four genes, GhTTGl and GhTTG3 were able to restore trichome initiation in the Arabidopsis mutant plants, and a further investigation of GhTTG3 transgenic plants demonstrated complementation of the full range of ttgl mutant phenotypes. An analysis of the temporal and spatial expression of the GhTTG genes in cotton is also described. It was shown that each of the genes is expressed ubiquitously throughout the cotton plant, in common with many plant WD-repeat genes. A closer examination of transcript abundance in the developing cotton ovule utilising in situ hybridisation revealed predominant expression of GhTTG lIGhTTG3 in the epidermal cells destined to become cotton fibres. A yeast two-hybrid assay was utilised to identify transcription factors that may interact with GhTTG3 during .fibre development. This experiment identified three cotton fibre cDNAs encoding putative interacting proteins, including one with a similar secondary structure to several TTG I-interacting proteins in Arabidopsis, raising the possibility of similar regulat01;y-complexes controlling trichome initiation in Arabidopsis and cotton.