No abstract

β-1,3-glucanase functions in plant physiological and developmental processes. However, how β-1,3-glucanase participates in cell wall development remains largely unknown. Here, we answered this question by examining the role of GhGLU18, a β-1,3-glucanase, in cotton (Gossypium hirsutum) fibers, in which the content of β-1,3-glucan changes dynamically from 10% of the cell wall mass at the onset of secondary wall deposition to < 1% at maturation. GhGLU18 was specifically expressed in cotton fiber with higher expression in late fiber elongation and secondary cell wall (SCW) synthesis stages. GhGLU18 largely localized to the cell wall and was able to hydrolyze β-1, 3-glucan in vitro. Overexpression of GhGLU18 promoted polysaccharide accumulation, cell wall reconstruction and cellulose synthesis, which led to increased fiber length and strength with thicker cell walls and shorter pitch of the fiber helix. However, GhGLU18-suppressed cotton resulted in opposite phenotypes. Additionally, GhGLU18 was directly activated by GhFSN1 (fiber secondary cell wall-related NAC1), a NAC transcription factor reported previously as the master regulator in SCW formation during fiber development. Our results demonstrate that cell wall-localized GhGLU18 promotes fiber elongation and SCW thickening by degrading callose and enhancing polysaccharide metabolism and cell wall synthesis.

Cotton fiber is the most important natural textile material in the world. Identification and functional characterization of genes regulating fiber development are fundamental for improving fiber quality and yield. However, stable cotton transformation is time-consuming, low in efficiency, and technically complex. Moreover, heterologous systems, such as Arabidopsis and tobacco, did not always work to elucidate the function of cotton fiber specifically expressed genes or their promoters. For these reasons, constructing a rapid transformation system using cotton fibers is necessary to study fiber’s specifically expressed genes. In this study, we developed an easy and rapid Agrobacterium-mediated method for the transient transformation of genes and promoters in cotton fibers. First, we found that exogenous genes could be expressed in cotton fibers via using β-glucuronidase (GUS) and green fluorescence protein (GFP) as reporters. Second, parameters affecting transformation efficiency, including LBA4404 Agrobacterium strain, 3 h infection time, and 2-day incubation time, were determined. Third, four different cotton genes that are specifically expressed in fibers were transiently transformed in cotton fibers, and the transcripts of these genes were detected ten to thousand times increase over the control. Fourth, GUS staining and activity analysis demonstrated that the activity profiles of GhMYB212 and GhFSN1 promoters in transformed fibers are similar to their native activity in developmental fibers. Furthermore, the transient transformation method was confirmed to be suitable for subcellular localization studies. In summary, the presented Agrobacterium-mediated transient transformation method is a fast, simple, and effective system for promoter characterization and protein expression in cotton fibers.

Plant cell expands via a tip growth or diffuse growth mode. In plants, RabA is the largest group of Rab GTPases that regulate vesicle trafficking. The functions of RabA protein in modulating polarized expansion in tip growth cells have been demonstrated. However, whether and how RabA protein functions in diffuse growth plant cells have never been explored. Here, we addressed this question by examining the role of GhRabA4c in cotton fibers. GhRabA4c was preferentially expressed in elongating fibers with its protein localized to endoplasmic reticulum and Golgi apparatus. Over- and down-expression of GhRabA4c in cotton lead to longer and shorter fibers, respectively. GhRabA4c interacted with GhACT4 to promote the assembly of actin filament to facilitate vesicle transport for cell wall synthesis. Consistently, GhRabA4c-overexpressed fibers exhibited increased content of wall components and the transcript levels of the genes responsible for the synthesis of cell wall materials. We further identified two MYB proteins that directly regulate the transcription of GhRabA4c. Collectively, our data showed that GhRabA4c promotes diffused cell expansion by supporting vesicle trafficking and cell wall synthesis.