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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.

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.

Cotton fi ber development at the stages of elongation and secondary wall synthesis determines the traits of fi ber length and strength. To date, the mechanisms controlling the progression of these two phases remain elusive. In this work, the function of a fi ber-preferential actin-binding protein (GhPFN2) was characterized by cytological and molecular studies on the fi bers 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 fi bers. Cytoskeleton staining and quantitative assay revealed that thicker and more abundant F-actin bundles formed during the elongation stage in GhPFN2 -overexpressing fi bers. 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 fi bers, 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.

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.

Using rapid amplification of cDNA ends (RACE)-PCR, two full-length cDNAs encoding putative seven-transmembrane receptors (designated Gh7TMpR1 and Gh7TMpR2) were cloned from cotton plants. Southern blot and an ApaL1 restriction site polymorphism analyses revealed that Gh7TMpR1 was derived from the ancestral A diploid genome, while Gh7TMpR2 was from the D subgenome. Northern blot hybridization indicated that both Gh7TMpR1 and Gh7TMpR2 were expressed preferentially in the elongation phase of fiber development. Majority of the Gh7TMpR1 proteins were located within the membrane structure and displayed a punctuate pattern of distribution. Overexpression of Gh7TMpR1 in fission yeast disrupted the polar growth and caused the formation of rounded cells. These results suggest that GhT7MpR1 may play a critical role in cotton fiber development, perhaps as a signaling receptor that is involved in controlling fiber elongation.

Cotton fiber is an extremely elongated single cell that is considered as an ideal system for studying the mechanisms controlling plant cell elongation. In this study, suppression subtractive hybridization (SSH) between RNA from leaves and fibers of cotton plant was conducted to identify genes that are specifically or preferentially expressed in fiber cells. Screening the resulting SSH library by reverse Northern analysis identified a total of 180 differentially expressed cDNA fragments. Sequencing determination and database analysis revealed 64 non-redundant cDNA clones, of which, 25 code for unknown-function proteins. Among these cDNAs, four were found to encode for proteins that showed high homology to calcium signaling components including calmodulin (CaM), glutamate decarboxylase (GAD) and calcineurin B-like (CBL) protein-interacting protein kinases (CIPKs). RT-PCR analysis indicated that the transcripts of these genes were accumulated predominantly in elongating fiber cells. Moreover, the expression level of these genes was significantly reduced in the li (ligon-lintless) mutant fibers as compared to the wild-type control. One of the two CIPKs, designated as GhCIPK1, was further characterized in this study. Structural analysis showed that GhCIPK1 contained the characteristic domains of CIPK proteins and was highly expressed in the elongating phase in developing fiber, and in vitro assay demonstrated that the gene product was a functional protein kinase. Our results suggest that calcium-mediated signal transduction may play an important role in cotton fiber elongation.

Summary 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.