The miR164-GhCUC2-GhBRC1 module regulates plant architecture through abscisic acid in cotton

Abstract

Branching determines cotton architecture and production, but the underlying regulatory mechanisms remain unclear. Here, we report that the miR164-GhCUC2 (CUP-SHAPED COTYLEDON2) module regulates lateral shoot development in cotton and Arabidopsis. We generated OE-GhCUC2m (overexpression GhCUC2m) and STTM164 (short tandem target mimic RNA of miR164) lines in cotton and heterologous expression lines for gh-miR164, GhCUC2 and GhCUC2m in Arabidopsis to study the mechanisms controlling lateral branching. GhCUC2m overexpression resulted in a short-branch phenotype similar to STTM164. In addition, heterologous expression of GhCUC2m led to decreased number and length of branches compared with wild type, opposite to the effects of the OE-gh-pre164 line in Arabidopsis. GhCUC2 interacted with GhBRC1 and exhibited similar negative regulation of branching. Overexpression of GhBRC1 in the brc1-2 mutant partially rescued the mutant phenotype and decreased branch number. GhBRC1 directly bound to the NCED1 promoter and activated its transcription, leading to local abscisic acid (ABA) accumulation and response. Mutation of the NCED1 promoter disrupted activation by GhBRC1. This finding demonstrates a direct relationship between BRC1 and ABA signalling and places ABA downstream of BRC1 in the control of branching development. The miR164-GhCUC2-GhBRC1-GhNCED1 module provides a clear regulatory axis for ABA signalling to control plant architecture.

Keywords: GhBRC1; GhCUC2; ABA; lateral branch; miR164.

Figure 1

RNA‐seq and cluster analysis show differential GhCUC2 up‐regulation in short‐branch cotton. (a) Relative expression correlation analysis and cluster analysis between short‐ and long‐branch cotton cultivars. (b) MA plot of differentially expressed genes (DEGs) for long‐branch and short‐branch cotton plants. (c) Unrooted phylogenetic tree of NAC transcription factors. Numbers between branches indicate bootstrap values based on 1000 replications. The red box indicates the CUC2 clade. Names and references for other NACs: Arabidopsis thaliana—ATAF1, ATAF2 (Aida et al., 1997), AtNAC2 (He et al., 2005), AtNAC3 (Takada et al., 2001), AtNAM (Duval et al., 2002), CUC1, CUC2 (Takada et al., 2001), CUC3 (Vroemen et al., 2003), NAC1 (Xie et al., 2000), NAC2, NAP (Sablowski and Meyerowitz, 1998), TIP (Ren et al., 2000); Oryza sativa—OsNAC6 (Kusano et al., ; Ohnishi et al., 2005), OsNAC2 (Mao et al., 2007); Petunia—NAM (Souer et al., 1996). (d) GhCUC2 expression levels among long‐branch and short‐branch cotton plants based on RNA‐seq. (e) qRT‐PCR detection of relative expression of GhCUC2 among long‐branch and short‐branch cotton plants. (f) Fluorescence in situ hybridization (FISH) detection of GhCUC2 in different tissues. Blue, DAPI; Red, GhCUC2; Scale bars = 100 μm.

Figure 2

miR164 directly cleaves GhCUC2 transcripts. (a) Cleavage positions predicted from degradome data. Red peak indicates the miR164 cleavage site position. (b) Sequence alignment of miR164, GhCUC2 and GhCUC2m. Red letters indicate nonsynonymous substituted nucleotides in the miR164 target site that disrupt miR164‐mediated cleavage without altering translated amino acid residues. Blue lines indicate Watson‐Crick base pairing between the GhCUC2 mRNA and miR164. Mismatches are indicated by stars. (c) Tobacco leaf cells transiently expressing eGFP::CUC2 (OD600 nm = 0.6) or (e) eGFP::CUC2m (OD600 nm = 0.6) alone or co‐expressed with miR164 (OD600 nm = 0.3/0.6/0.9). eGFP::CUC2 protein accumulation decreases with increasing miR164 concentration. (d) and (f) Change in eGFP intensity with increasing miR164 concentration. Results represent the mean ± SD of three independent experiments. Significance was determined by t‐test; ** indicates P < 0.01).

Figure 3

GhCUC2 and miR164 regulate branch development in Arabidopsis. (a) Phenotypic analysis of 35S::GhCUC2, 35S::GhCUC2m and 35S::pre164 transgenic Arabidopsis overexpression lines at 30 days after emergence (DAE). Scale bar, 2 cm. (b) Statistical analysis of branch number in wild‐type (WT), OE‐GhCUC2, OE‐GhCUC2m and OE‐gh‐pre164 Arabidopsis at 40 DAE. (c) Statistical analysis of branch length phenotype between OE‐GhCUC2 and OE‐GhCUC2m Arabidopsis overexpression lines. (d) Semi‐quantitative PCR detection of GhCUC2 mRNA levels in WT, OE‐GhCUC2 and OE‐GhCUC2m Arabidopsis lines. The Actin gene served as an internal reference. (e) Semi‐quantitative PCR detection of miR164 transcripts in OE‐gh‐pre164 Arabidopsis. The U6 gene served as an internal reference.

Figure 4

miR164 and GhCUC2 regulate branch length in cotton. (a) Representative images showing morphological and growth phenotypes of STTM164 cotton plants silenced for miR164 (right) and empty vector controls (left). Scale bar, 10 cm. (b) Representative images showing phenotypes of empty vector control, CUC2‐silenced cotton and miR164 overexpression cotton. (c) Distribution of branch lengths of CUC2‐silenced, WT, miR164‐silenced and miR164 overexpression cotton lines. (d) Phenotypic analysis of GhCUC2m transgenic cotton plants. Scale bar, 20 cm. (e) Image of branch phenotype in WT and OE‐GhCUC2m cotton plants. Scale bar, 2 cm. (f) Statistical analysis of branch length in WT and OE‐GhCUC2m cotton plants.

Figure 5

GhCUC2 physically interacts with GhBRC1 (a) Representative images of transgenic brc1‐2 Arabidopsis mutant (right) and brc1‐2 mutant carrying a 35S‐GhBRC1 complementation vector (left). (b) Quantitative analysis of rosette‐leaf branches in 45‐day‐old brc1‐2 and transgenic plant. (c) Fluorescence in situ hybridization (FISH) detection of GhBRC1 expression patterns in different tissues. Blue, DAPI; green, GhBRC1; Scale bars = 100 μm. (d) Yeast two‐hybrid (Y2H) detection of interaction between GhCUC2 and GhBRC1. AD, pGADT7; BD, pGBDT7; SD, medium. (e) Bimolecular fluorescence complementation (BiFC) assay showing putative interaction between GhBRC1 and GhCUC2. The N‐terminus of yellow fluorescent protein (YFP) was fused to GhBRC1, while the C‐terminus of YFP was fused to GhCUC2. EV, empty vector. Scale bars = 50 μm. (f) In vivo co‐immunoprecipitation (CoIP) assay. After the co‐transformation of Flag‐GhBRC1 and HA‐GhCUC2 in Nicotiana benthamiana leaf, total proteins of N. benthamiana leaf were immunoprecipitated using an anti‐HA antibody and were detected with anti‐HA and anti‐Flag antibodies. IB, immunoblot; IP, immunoprecipitation. (g) In vitro pull‐down assay. Recombinant GST‐GhBRC1 and His‐GhCUC2 proteins were used for the pull‐down assay. IB, immunoblot.

Figure 6

GhBRC1 binds to the promoter of NCED1 and induces its transcription. (a) Alignment of the reporter constructs used in yeast one‐hybrid (Y1H) analysis. Red stars indicate three tandem copies of the NCED1 promoter core sequence in NCED1 mutagenized promoter variant and WT, both of which were inserted into pAbAi as reporter constructs. (b) Y1H analysis showing that GhBRC1 binds the core sequence of NCED1, but not the NCED1 mutated motif. SD/−Leu, SD medium without Leu; SD/−Leu/AbA¹⁰⁰, SD medium without Leu supplemented with 100 ng/mL AbA. (c) EMSA of GhBRC1 binding to the promoter of GhNCED1 in vitro. A GhBRC1‐His protein expressed in Escherichia coli was purified; its binding to biotin‐labelled promoters was detected in the absence or presence of unlabelled wild‐type probes (competitor). No specific binding was observed with the labelled mutant probes. (d) Luminescence imaging of dual‐LUC assays showing interaction between GhCUC2 and GhBRC1 activates GhNCED1 transcription in Nicotiana benthamiana leaf cells. PCambia2300 is empty vector; Gh BRC1+proNCED1‐LUC is PCambia2300+Gh; BRC1+proNCED1‐LUC proNCED1‐LUC is ‘PCambia2300+PCambia2300+proNCED1‐LUC; and GhCUC2+GhBRC1+proNCED1‐LUC is GhCUC2+GhBRC1+proNCED1‐LUC GhCUC2+proNCED1‐LUC is ‘PCambia2300+GhCUC2+proNCED1‐LUC. (e) Quantitative comparison of luciferase signals in (d). The error bars indicate mean ± SD of three independent experiments. **P < 0.01, (Student’s t‐test).

Figure 7

Proposed model of how miR164‐GhCUC2‐GhBRC1 module regulates plant architecture through ABA in cotton. The number of dots indicates the content of ABA.