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.

Cell elongation is a fundamental process for plant growth and development. Studies have shown lipid metabolism plays important roles in cell elongation; however, the related functional mechanisms remain largely unknown. Here, we report that cotton (Gossypium hirsutum) LIPID TRANSFER PROTEIN4 (GhLTP4) promotes fiber cell elongation via elevating ceramides (Cers) content and activating auxin-responsive pathways. GhLTP4 was preferentially expressed in elongating fibers. Over-expression and down-regulation of GhLTP4 led to longer and shorter fiber cells, respectively. Cers were greatly enriched in GhLTP4-overexpressing lines and decreased dramatically in GhLTP4-down-regulating lines. Moreover, auxin content and transcript levels of IAA-responsive genes were significantly increased in GhLTP4-overexpressing cotton fibers. Exogenous application of Cers promoted fiber elongation, while NPA (N-1-naphthalic acid, a polar auxin transport inhibitor) counteracted the promoting effect, suggesting that IAA functions downstream of Cers in regulating fiber elongation. Furthermore, we identified a bHLH transcription factor, GhbHLH105, that binds to the E-box element in the GhLTP4 promoter region and promotes the expression of GhLTP4. Suppression of GhbHLH105 in cotton reduced the transcripts level of GhLTP4, resulting in smaller cotton bolls and decreased fiber length. These results provide insights into the complex interactions between lipids and auxin signaling pathways to promote plant cell elongation.

Cotton (Gossypium hirsutum) fiber is a highly elongated single cell with a thickened cell wall. MYB transcription factors are important regulators of plant cell elongation; however, the molecular mechanism involved in regulating fiber elongation remains to be explored. Here, we present evidence that the R2R3-MYB transcription factor GhMYB4 negatively regulates cotton fiber cell elongation by suppressing the expression of two crucial genes previously reported to affect fiber development: lipid transfer protein 4 (GhLTP4) and sucrose transporter 12 (GhSWEET12). GhMYB4 is preferentially expressed in elongating fiber cells. Knockdown of GhMYB4 in cotton results in longer fiber cells, whereas overexpression of GhMYB4 in Arabidopsis leads to reduced plant height and root length. Transcriptomic and lipidomic analyses revealed that GhMYB4 is involved in coordinating three interconnected biological processes, namely lipid content regulation, auxin signaling, and sugar metabolism. Additionally, we showed that GhMYB4 inhibits the expression of GhLTP4 and GhSWEET12 by binding to the MYB cis-element (TTTAGTG) in their respective promoters. Interestingly, bHLH transcription factor 105 (GhbHLH105) and MYB transcription factor 212 (GhMYB212) counteract the inhibitory effects of GhMYB4 on the expression of GhLTP4 and GhSWEET12, respectively. These findings provide insights into the complex molecular mechanisms regulating plant cell elongation.

Plant cell walls are complex and dynamic cellular structures critical for plant growth, development, physiology, and adaptation. Cellulose is one of the most important components of the cell wall. However, how cellulose microfibrils deposit and assemble into crystalline cellulose remains elusive. The COBRA-LIKE plant-specific protein family plays a vital role in modulating the deposition and orientation of cellulose microfibril in plant cell walls. Here, we investigate the role of GhCOBL9 in cotton (Gossypium hirsutum) fiber development, an ideal model for studying cell elongation and cell wall thickening. The expression period of GhCOBL9 is consistent with the thickening stage of the secondary wall of cotton fibers. Overexpression of GhCOBL9 results in increased cellulose content in the cell wall and produces shorter, thicker, and stronger fibers, while RNA interference (RNAi)-mediated downregulation of GhCOBL9 leads to the opposite phenotypes, indicating its crucial role in cell wall development. Subcellular localization and binding activity assays reveal that GhCOBL9 targets the cell wall and binds to crystalline cellulose with high affinity. Transcriptomic analysis of GhCOBL9 transgenic lines uncovers expression alterations in genes related to cellulose and monosaccharide biosynthesis. Furthermore, we identify a fasciclin-like arabinogalactan protein 9 (GhFLA9) as an interacting partner of GhCOBL9 to modulate cell wall development. Additionally, the R2R3-MYB transcription factor GhMYB46-5 activates GhCOBL9 expression by binding to the MYB46-responsive cis-regulatory element in the GhCOBL9 promoter. These findings broaden our knowledge of COBL function in modulating plant cell wall development.