Li-Bo Han

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.

The Dual Functions of WLIM1a in Cell Elongation and Secondary Wall Formation in Developing Cotton Fibers

This work examines the role of a cotton LIM-domain protein, WLIM1a, finding that this protein has dual functions in actin bundling and transcriptional regulation of the genes involved in phenylpropanoid biosynthesis. WLIM1a translocates from the cytoplasm to the nucleus in response to H2O2 and plays important roles in cell elongation and secondary wall formation during fiber development. LIN-11, Isl1 and MEC-3 (LIM)-domain proteins play pivotal roles in a variety of cellular processes in animals, but plant LIM functions remain largely unexplored. Here, we demonstrate dual roles of the WLIM1a gene in fiber development in upland cotton (Gossypium hirsutum). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages in developing fibers. Overexpression of WLIM1a in cotton led to significant changes in fiber length and secondary wall structure. Compared with the wild type, fibers of WLIM1a-overexpressing plants grew longer and formed a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness. Functional studies demonstrated that (1) WLIM1a acts as an actin bundler to facilitate elongation of fiber cells and (2) WLIM1a also functions as a transcription factor to activate expression of Phe ammonia lyase–box genes involved in phenylpropanoid biosynthesis to build up the secondary cell wall. WLIM1a localizes in the cytosol and nucleus and moves into the nucleus in response to hydrogen peroxide. Taken together, these results demonstrate that WLIM1a has dual roles in cotton fiber development, elongation, and secondary wall formation. Moreover, our study shows that lignin/lignin-like phenolics may substantially affect cotton fiber quality; this finding may guide cotton breeding for improved fiber traits.

The Cotton Transcription Factor TCP14 Functions in Auxin-Mediated Epidermal Cell Differentiation and Elongation

GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, and it may act as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells. 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.

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.

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton

The root apoplast secretome of Gossypium barbadense actively changes upon infection, and a thioredoxin plays an important role in apoplastic ROS balance. Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.

Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae

In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants.

Orchestration of microtubules and the actin cytoskeleton in trichome cell shape determination by a plant-unique kinesin

Microtubules (MTs) and actin filaments (F-actin) function cooperatively to regulate plant cell morphogenesis. However, the mechanisms underlying the crosstalk between these two cytoskeletal systems, particularly in cell shape control, remain largely unknown. In this study, we show that introduction of the MyTH4-FERM tandem into KCBP (kinesin-like calmodulin-binding protein) during evolution conferred novel functions. The MyTH4 domain and the FERM domain in the N-terminal tail of KCBP physically bind to MTs and F-actin, respectively. During trichome morphogenesis, KCBP distributes in a specific cortical gradient and concentrates at the branching sites and the apexes of elongating branches, which lack MTs but have cortical F-actin. Further, live-cell imaging and genetic analyses revealed that KCBP acts as a hub integrating MTs and actin filaments to assemble the required cytoskeletal configuration for the unique, polarized diffuse growth pattern during trichome cell morphogenesis. Our findings provide significant insights into the mechanisms underlying cytoskeletal regulation of cell shape determination. DOI: http://dx.doi.org/10.7554/eLife.09351.001

The Tobacco BLADE-ON-PETIOLE2 Gene Mediates Differentiation of the Corolla Abscission Zone by Controlling Longitudinal Cell Expansion

The BLADE-ON-PETIOLE (BOP) genes of Arabidopsis (Arabidopsis thaliana) have been shown to play an essential role in floral abscission by specializing the abscission zone (AZ) anatomy. However, the molecular and cellular mechanisms that underlie differentiation of the AZ are largely unknown. In this study, we identified a tobacco (Nicotiana tabacum) homolog of BOP (designated NtBOP2) and characterized its cellular function. In tobacco plants, the NtBOP2 gene is predominantly expressed at the base of the corolla in an ethylene-independent manner. Both antisense suppression of NtBOP genes and overexpression of NtBOP2 in tobacco plants caused a failure in corolla shedding. Histological analysis revealed that the differentiation of the corolla AZ was blocked in the transgenic flowers. This blockage was due to uncontrolled cell elongation at the region corresponding to wild-type AZ. The role of NtBOP2 in regulating cell elongation was further demonstrated in Bright Yellow 2 single cells: perturbation of NtBOP2 function by a dominant negative strategy led to the formation of abnormally elongated cells. Subcellular localization analysis showed that NtBOP2-green fluorescent protein fusion proteins were targeted to both the nucleus and cytoplasm. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that NtBOP2 proteins interacted with TGA transcription factors. Taken together, these results indicated that NtBOP2 mediated the differentiation of AZ architecture by controlling longitudinal cell growth. Furthermore, NtBOP2 may achieve this outcome through interaction with the TGA transcription factors and via an ethylene-independent signaling pathway.

The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection

Highlight Cotton MYB108 interacts with CML11 and acts as a positive regulator in defense against V. dahliae infection.

GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation

Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.