The cotton GhWIN2 gene activates the cuticle biosynthesis pathway and influences the salicylic and jasmonic acid biosynthesis pathways

Abstract

Background: Metabolic pathways are interconnected and yet relatively independent. Genes involved in metabolic modules are required for the modules to run. Study of the relationships between genes and metabolic modules improves the understanding of metabolic pathways in plants. The WIN transcription factor activates the cuticle biosynthesis pathway and promotes cuticle biosynthesis. The relationship between the WIN transcription factor and other metabolic pathways is unknown. Our aim was to determine the relationships between the main genes involved in cuticle biosynthesis and those involved in other metabolic pathways. We did this by cloning a cotton WIN gene, GhWIN2, and studying its influence on other pathways.

Results: As with other WIN genes, GhWIN2 regulated expression of cuticle biosynthesis-related genes, and promoted cuticle formation. Silencing of GhWIN2 resulted in enhanced resistance to Verticillium dahliae, caused by increased content of salicylic acid (SA). Moreover, silencing of GhWIN2 suppressed expression of jasmonic acid (JA) biosynthesis-related genes and content. GhWIN2 positively regulated the fatty acid biosynthesis pathway upstream of the JA biosynthesis pathway. Silencing of GhWIN2 reduced the content of stearic acid, a JA biosynthesis precursor.

Conclusions: GhWIN2 not only regulated the cuticle biosynthesis pathway, but also positively influenced JA biosynthesis and negatively influenced SA biosynthesis.

Keywords: Cuticle; GhWIN2; Jasmonic acid; Salicylic acid; Systems biology; VIGS; Verticillium dahliae.

Fig. 1

Cloning and characterization of GhWIN2. (a) Phylogenetic analysis of WIN proteins from cotton (Gossypium hirsutum, Gh), rice (Oryza sativa, Os), barley (Hordeum vulgare, Hv), tomato (Solanum lycopersicum, Sl), and Arabidopsis (At). Accession numbers: OsWR1(Os02g0202000); OsWR2(AK061163); HvHUD (KP245804.1); AtWIN1/SHN1(AT1g15360); AtWIN2/SHN2(At5g11190); AtWIN3/SHN3(At5g25390); SlSHN1(XP_004235965.1); SlSHN2(XP_004251719.1); SlSHN3(NM_001319202). The GhWIN2 is indicated by a red box. The rooted neighbor-joining tree was based on multiple sequence alignment using the MEGA5.1 software. (b) Subcellular localization of GhWIN2. GFP and DAPI fluorescence in the cotyledons of five-day-old plants of line 4 (scale bars: 10 μm). (C, D) The expression of GhWIN2 in two-week-old cotton seedlings after treatment with 100 μM ABA (c) and 100 mM NaCl (d). The mock samples were treated with double distilled water. Data were from three independent replicates and the results are means ± SD. The relative transcription levels were normalized using GhUBQ7. Asterisks indicate significant differences between treated and mock plants according to Student’s t-test (* P < 0.05; ** P < 0.01)

Fig. 2

GhWIN2 regulation of cuticle development. (a) Expression of genes involved in cuticle biosynthesis in four-week-old WT and transgenic Arabidopsis plants. Arabidopsis elongation factor-1-α gene (EF-1-α) was the endogenous reference for normalization. Data represent means ± SD for three biological replicates. Student’s t-test; * P < 0.05, ** P < 0.01. (b) Expression of the putative cuticle biosynthesis-related genes in cotton plants 14 days after infiltration. (c) SEM images of the stems of the WT and transgenic Arabidopsis plants. (Scale bars in main image, 50 μm; scale bars in insets, 10 μm) (d) TEM images of the cuticle of the upper leaf surface (scale bar: 500 nm). CW, cell wall; white arrows mark the cuticle. Images were taken at 50,000× magnification. (e) The thickness of the cuticle layer from the leaf upper surface (d). Data are presented as means ± SD from at least four independent biological replicates. Asterisks denote Student’s t test significance compared with WT plants (* P < 0.05; ** P < 0.01)

Fig. 3

GhWIN2 activation of the promoter of the GhCYP86A4 gene. (a) Luminescence signal of transient co-expression of GhWIN2/GhCYP86A4_pro:LUC or GhWIN2^V-A/GhCYP86A4_pro:LUC on Nicotiana benthamiana leaves. The photos were taken 2 days after infiltration with the same amount of Agrobacterium tumefaciens cultures containing the specified constructs. Two sites were injected for every treatment. (b) Sequence alignments of AP2/EREBP proteins. The conserved valine is marked in red. GenBank accession numbers: Oryza sativa OsDREB1A (AAN02486), OsDREB2A (AAN02487), AtDREB1A (Q9M0L0), Limonium bicolor LbDREB (ACP18975), Populus euphratica PeDREB2 (ABL86587), Hordeum vulgare HvNUD (AKF40403). (c, d) Expression of LUC transcripts 2 days after co-infiltration of Agrobacterium cells harboring GhWIN2/GhCYP86A4_pro:LUC (c) and GhWIN2^V-A/GhCYP86A4_pro:LUC (d). Data represent means ± SD from three independent biological replicates; Student’s t-test; * P < 0.05, ** P < 0.01

Fig. 4

GhWIN2 involvement in the ABA-cuticle pathway. (a) Expression levels of GhWIN2 were detected in TRV:GhPYL1 and TRV:GhNCED1 plants 14 days after infiltration. Values represent the means ± SD from at least three independent biological replicates. Student’s t-test; * P < 0.05. (B–D) Expression levels of GhCYP86A4 (b), GhCYP86A7 (c), and GhLACS2 (d) in TRV:00 and TRV:GhWIN2 plants after treatment with 100 μM ABA. The numbers represent the inducible multiples of plants treated with ABA compared to untreated plants

Fig. 5

GhWIN2 negative regulation of plant immune response to V. dahliae. (a) Disease symptoms after inoculation with V. dahliae. (b) Expression of JA biosynthesis-related genes in TRV:00 and TRV:GhWIN2 plants 14 days after agroinfiltration. Values are shown as means ± SD from at least three independent biological replicates. Student’s t-test; * P < 0.05. (C, D) Content of JA (c) and SA (d). Values are the means ± SD from six independent biological replicates. Student’s t-test; * P < 0.05, ** P < 0.01, *** P < 0.001. (e, f) Expression of marker genes involved in SA response

Fig. 6

Regulation of 16:0-ACP metabolic flux. (a) GhWIN2 positively regulates the expression of 16:0-ACP metabolic flux-related genes. Values are shown as means ± SD from three independent biological replicates. Student’s t-test; * P < 0.05, ** P < 0.01. (b) Content of stearic acid in TRV:00 and TRV:GhWIN2 plants

Fig. 7

Model of GhWIN2-related metabolic pathways. GhWIN2 positively regulated cuticle biosynthesis and content of JA, and negatively affected SA biosynthesis