A single-nucleotide mutation of G301A in GaIAA14 confers leaf curling in Gossypium arboreum

Abstract

Cotton is a crucial fiber and oil crop, playing a significant role in the textile and food industries. Its yield heavily relies on photosynthesis, a process that primarily occurs in the leaves. Consequently, leaf morphology stands as a vital agronomic trait in cotton breeding. However, research on the molecular mechanisms underlying cotton leaf morphogenesis remains relatively limited. Here we identified a curly leaf mutant (CU) in Gossypium arboreum by ethyl methylsulfonate (EMS) mutagenesis. The genetic analysis revealed that the curly leaf trait in this mutant is a semi-dominant characteristic controlled by a single gene. The map-based cloning of the CU locus showed a single-nucleotide mutation from G to A at the 301st positions in AUX/IAA14 protein (GaIAA14), which resulted in an amino acid substitution from valine (V) to isoleucine (I). After silencing GaIAA14 through virus-induced gene silencing (VIGS) technology in CU mutant, the leaves exhibited a flattened phenotype, indicating that GaIAA14 is a key gene regulating leaf curling in cotton. Comparative transcriptomic RNA-Seq analysis revealed significant changes in the expression levels of most auxin-related genes, suggesting that the mutation disrupts auxin signaling transduction. These findings establish a foundation for further functional studies of this gene and provide research strategies for leaf morphology improvement.

Keywords: CU; G301A; GaIAA14; Gossypium arboreum; curly leaf.

Figure 1

Phenotype characterization of the CU mutant. (a, b) Plant architecture of the wild-type Shixiya 1 and the CU mutant (scale bar = 10 cm). (c) Plant leaf morphology of the wild-type Shixiya 1 and the CU mutant (scale bar = 5 cm); (d, e) Longisection analysis of the third to the last leaf in the wild-type Shixiya 1 and the CU mutant. The red arrows mark the palisade tissue, and the blue boxes mark the spongy tissue (scale bar = 200 µm).

Figure 2

The CU locus is located on chromosome A03. (a) The CU locus was mapped on chromosome A03 by QTL-Seq analysis. The red arrow indicates the only window with a ΔSNP value exceeding the 95% significance threshold confidence interval across the whole genome. (b) Enlarged view of chromosome A03. The red arrow indicates the candidate region on the chromosome.

Figure 3

Fine mapping and cloning of CU. (a) Fine mapping of the CU locus in a population with 91 BML × CU F₂ dominant individual plants by InDel primers, and the number of recombinants is shown below the black line. (b) Genetic mapping of the CU locus in a population with 211 DQJ × CU F₂ recessive individual plants by KASP, and six ORFs located in the 48.2-kb candidate region are indicated by arrows. The number of recombinants is shown below the black line. (c) Single-nucleotide mutation from G to A in the second exon of GaIAA14 in the CU mutant. (d) 3D protein structure of GaIAA14 in the wild-type Shixiya 1 and the CU mutant. The white arrows indicate the absence of a segment of the α-helix in the CU mutant.

Figure 4

Silencing of GaIAA14 in CU mutant results in leaf flattening. (a, b) The level of GaIAA14 transcript in the leaves of GaIAA14-silenced (TRV: GaIAA14) plants and the negative control (TRV: 00). Two-tailed Student’s t-test was used for paired comparison of the GaIAA14 gene in TRV: GaIAA14 and TRV: 00 leaves (***P < 0.001 or **P < 0.01 or *P < 0.05).

Figure 5

Transcriptomic comparison of the CU mutant versus Shixiya 1. (a) Venn diagram showing the overlaps between the different stages of the CU mutant and Shixiya 1. The number above each stage designation is the total differentially expressed genes (DEGs) detected in that stage(s). (b) Volcano plot of differentially expressed genes between the CU mutant and Shixiya 1 at T₃ stage (after the curling degree of the first true leaf stabilized). (c) Top 20 KEGG enriched pathways of upregulated genes. (d) Top 20 KEGG enriched pathways of downregulated genes. The top 20 KEGG enriched pathways are selected by p. adjust value sorting. Count, the bubble size, represents the number of enriched genes. Rich factor represents the multiple of enrichment level of differentially expressed genes in a target pathway relative to the entire genomic background.