DELLA-NAC Interactions Mediate GA Signaling to Promote Secondary Cell Wall Formation in Cotton Stem

Abstract

Gibberellins (GAs) promote secondary cell wall (SCW) development in plants, but the underlying molecular mechanism is still to be elucidated. Here, we employed a new system, the first internode of cotton, and the virus-induced gene silencing method to address this problem. We found that knocking down major DELLA genes via VIGS phenocopied GA treatment and significantly enhanced SCW formation in the xylem and phloem of cotton stems. Cotton DELLA proteins were found to interact with a wide range of SCW-related NAC proteins, and virus-induced gene silencing of these NAC genes inhibited SCW development with downregulated biosynthesis and deposition of lignin. The findings indicated a framework for the GA regulation of SCW formation; that is, the interactions between DELLA and NAC proteins mediated GA signaling to regulate SCW formation in cotton stems.

Keywords: DELLA proteins; NAC proteins; cotton stem; gibberellin; secondary cell wall formation.

Figure 1

Effects of exogenous GA and PAC on stem elongation and lignification, and DELLA protein level in cotton. Ten-day-old seedlings sprayed with water (Mock), 100 μM GA (GA), 50 μM PAC (PAC), and mixed GA and PAC (GA + PAC) were grown at 25°C under 16 h/8 h (light/dark) conditions for 8 days. (A) The phenotype of cotton plants. Bar = 10 mm. (B) Time courses of first internode elongation. (C) Phloroglucinol staining of cross-sections of the first internode, aligned with the enlarged view at the lower panel. Bar = 1 mm. (D) Cotton DELLA protein level detected with anti-GhGAI1 in the first internodes. The amounts of total proteins transferred onto membrane are indicated by Ponceau S staining.

Figure 2

Functional characterization of GhGAIs by VIGS analysis. Agrobacterium cultures carrying pTRV2-GhGAI1&2 (TRV:GhGAI1&2), pTRV2-GhGAI3&4 (TRV:GhGAI3&4) or both (TRV:GhGAI1&2&3&4), mixed with pTRV1 Agrobacterium, were infiltrated into two fully expanded cotyledons of 7-day-old cotton seedlings. Empty vector pTRV2-00 (TRV:00) was used as control. (A) Transcript levels of 8 GhGAIs in the first internode were detected 15 days post infiltration (DPI) by qRT-PCR. (B) DELLA protein levels detected with anti-GhGAI1 in immunoblot analysis. The amounts of total proteins transferred onto the membrane are indicated by Ponceau S staining. (C) Fifteen DPI seedlings. Bar = 10mm. (D) The first internode length measured 15 DPI. ** indicates a significant difference compared with control (t-test, P < 0.01, n ≥ 10). Error bars indicate SD.

Figure 3

Silencing of GhGAI1 and GhGAI 2 enhances SCW development in the first internodes. Agrobacterium cultures carrying empty vector pTRV2-00 (TRV:00) or pTRV2-GhGAI1&2 (TRV:GAI1&2), mixed with pTRV1 Agrobacterium, were infiltrated into two fully expanded cotyledons of 7-day-old cotton seedlings. (A) The first internode diameters at the top, middle and bottom parts measured 20 DPI. * indicates a significant difference compared with control (t-test, P < 0.05, n ≥ 10). Error bars indicate SD. (B) Phloroglucinol staining of the cross-sections of 20 DPI first internode in the middle region. Bar = 1 mm or 500 μm (amplification). (C) Microscopic observation of cross-sections of 20 DPI first internode in the middle region under UV conditions. (D) Lignin and (E) cellulose contents in the first internodes, respectively. The internodes were sampled 10, 15, and 20 DPI. Lignin content is shown relative to the control level 10 days post infiltration. * and ** indicate a significant difference compared with the control, with P-values of 0.05 and 0.01, respectively (t-test, n = 3). Error bars indicate SD. P, phloem; PF, phloem fibers; X, xylem; and IF, interfascicular region.

Figure 4

Quantitative RT-PCR analysis of genes related to SCW formation. Total RNAs were isolated from the first internodes. Mock, GA, and PAC represent 10-day-old seedlings treated with water, 100 μM GA, and 50 μM PAC for 8 days. VIGS-treated materials are indicated with pTRV2 vectors (TRV:00 and TRV:GhGAI1&2) plus sampling time (15 or 20 DPI). The relative expression levels in GA/PAC-treated and GhGAI1&2-silenced seedlings are then normalized to Mock and TRV:00, respectively. Gene accession numbers in cotton FGD: CesA4 (Gh_D08G0509); CesA7 (Gh_D05G0079); CesA8 (Gh_D10G0333); IRX9 (Gh_A09G1418); IRX14 (Gh_D11G1966); PAL (Gh_D10G2528); 4CL (Gh_D10G0473); CCoAOMT (Gh_A04G1032); F5H (Gh_Sca004990G01); LAC4 (Gh_A11G2936); PER4 (Gh_A03G0199); and PER21 (Gh_A09G1415).

Figure 5

BiFC analyses of interactions between GhGAI1D and SCW-related NACs. NACs fused to N-terminal fragment of YFP (NAC-nYFP) were co-infiltrated with GhGAI1D fused to C-terminal fragment of YFP (GhGAI1D-cYFP) or unfused cYFP into N. benthamiana leaves. Unfused cYFP and nYFP were used as negative controls. The nuclei were indicated by DAPI staining.

Figure 6

VIGS analysis of GhSND2s. Agrobacterium cultures that carry pTRV1 and pTRV2-GhSND2s (TRV:GhSND2s) were infiltrated into two fully expanded cotyledons of 7-day-old cotton seedlings. Empty vector pTRV2-00 (TRV:00) was used as control. (A) Transcript levels of six GhSND2 genes in the first internodes 15 days post infiltration (DPI) by qRT-PCR analysis. (B) Phloroglucinol staining of the cross-sections of 15 DPI first internodes. Bar = 1 mm or 500 μm (amplification). (C) Quantitative RT-PCR analysis of the genes related to SCW formation in the first internodes.