The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation.

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells.

Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae.

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.

Overexpression of a profilin (GhPFN2) promotes the progression of developmental phases in cotton fibers.

Cotton fiber development at the stages of elongation and secondary wall synthesis determines the traits of fiber length and strength. To date, the mechanisms controlling the progression of these two phases remain elusive. In this work, the function of a fiber-preferential actin-binding protein (GhPFN2) was characterized by cytological and molecular studies on the fibers of transgenic green-colored cotton (Gossypium hirsutum) through three successive generations. Overexpression of GhPFN2 caused pre-terminated cell elongation, resulting in a marked decrease in the length of mature fibers. Cytoskeleton staining and quantitative assay revealed that thicker and more abundant F-actin bundles formed during the elongation stage in GhPFN2-overexpressing fibers. Accompanying this alteration, the developmental reorientation of transverse microtubules to the oblique direction was advanced by 2 d at the period of transition from elongation to secondary wall deposition. Birefringence and reverse transcription-PCR analyses showed that earlier onset of secondary wall synthesis occurred in parallel. These data demonstrate that formation of the higher actin structure plays a determinant role in the progression of developmental phases in cotton fibers, and that GhPFN2 acts as a critical modulator in this process. Such a function of the actin cytoskeleton in cell phase conversion may be common to other secondary wall-containing plant cells.

Proteomic identification of differentially expressed proteins in the Ligon lintless mutant of upland cotton (Gossypium hirsutum L.).

Cotton fiber is an ideal model for studying plant cell elongation. To date, the underlying mechanisms controlling fiber elongation remain unclear due to their high complexity. In this study, a comparative proteomic analysis between a short-lint fiber mutant (Ligon lintless, Li(1)) and its wild-type was performed to identify fiber elongation-related proteins. By 2-DE combined with local EST database-assisted MS/MS analysis, 81 differentially expressed proteins assigned to different functional categories were identified from Li(1) fibers, of which 54 were down-regulated and 27 were up-regulated. Several novel aspects regarding cotton fiber elongation can be illustrated from our data. First, over half of the down-regulated proteins were newly identified at the protein level, which is mainly involved in protein folding and stabilization, nucleocytoplasmic transport, signal transduction, and vesicular-mediated transport. Second, a number of cytoskeleton-related proteins showed a remarkable decrease in protein abundance in the Li(1) fibers. Accordingly, the architecture of actin cytoskeleton was severely deformed and the microtubule organization was moderately altered, accompanied with dramatic disruption of vesicle trafficking. Third, the expression of several proteins involved in unfolded protein response (UPR) was activated in Li(1) fibers, indicating that the deficiency of fiber cell elongation was related to ER stress. Collectively, these findings significantly advanced our understanding of the mechanisms associated with cotton fiber elongation.

Down-regulation of GhADF1 gene expression affects cotton fibre properties.

Cotton fibre is the most important natural fibres for textile industry. To date, the mechanism that governs the development of fibre traits is largely unknown. In this study, we have characterized the function of a member of the actin depolymerizing factor (ADF) family in Gossypium hirsutum by down-regulation of the gene (designated as GhADF1) expression in the transgenic cotton plants. We observed that both the fibre length and strength of the GhADF1-underexpressing plants increased as compared to the wild-type fibre, and transgenic fibres contained more abundant F-actin filaments in the cortical region of the cells. Moreover, the secondary cell wall of the transgenic fibre appeared thicker and the cellulose content was higher than that of the control fibre. Our results suggest that organization of actin cytoskeleton regulated by actin-associated proteins such as GhADF1 plays a critical role in the processes of elongation and secondary cell wall formation during fibre development. Additionally, our study provided a candidate intrinsic gene for the improvement of fibre traits via genetic engineering.

Co-expression and preferential interaction between two calcineurin B-like proteins and a CBL-interacting protein kinase from cotton.

The CBL/CIPK signaling system mediates a variety of responses to environmental stimuli in plants. In this work, we identified four CBL genes from Gossypium hirsutum, two of which (designated GhCBL2 and GhCBL3) showed preferential expression in the elongating fiber cells. Moreover, the expression patterns of these two CBL genes coincided with that of a putative CBL-interacting protein kinase gene (GhCIPK1) that we isolated in a previous study. Yeast two-hybrid assay indicated that among the four CBLs, GhCIPK1 interacted selectively with GhCBL2 and GhCBL3. The co-expression and interactions of these proteins suggest that they are components of the same signaling pathway. These findings strengthen our previous prediction that CBL/CIPK signaling plays a critical role in the regulation of cotton fiber elongation.