Genetic basis for glandular trichome formation in cotton

Abstract

Trichomes originate from epidermal cells and can be classified as either glandular or non-glandular. Gossypium species are characterized by the presence of small and darkly pigmented lysigenous glands that contain large amounts of gossypol. Here, using a dominant glandless mutant, we characterize GoPGF, which encodes a basic helix-loop-helix domain-containing transcription factor, that we propose is a positive regulator of gland formation. Silencing GoPGF leads to a completely glandless phenotype. A single nucleotide insertion in GoPGF, introducing a premature stop codon is found in the duplicate recessive glandless mutant (gl2gl3). The characterization of GoPGF helps to unravel the regulatory network of glandular structure biogenesis, and has implications for understanding the production of secondary metabolites in glands. It also provides a potential molecular basis to generate glandless seed and glanded cotton to not only supply fibre and oil but also provide a source of protein for human consumption.

Figure 1. Origins of the genotypes and phenotypes of glandless tetraploid cottons.

The genotypes include a dominant mutation, Gl₂^eGl₃; two recessive mutations, gl₂Gl₃ and Gl₂gl₃; and a duplicated recessive cotton, gl₂gl₃. Black points in each graph show the glands on the surface of seeds.

Figure 2. Fine mapping of dominant glandless gene Gl and the expression pattern of GoPGF.

(a) Gland traits of seeds, leaves and stems of TM-1 (glanded) and Hai-1 (glandless). (b) Fine mapping of the dominant glandless gene, Gl. Seven genes (ORF1–7) in the mapping region are indicated by boxes. (c) Real-time RT–PCR expression analysis of ORF2 in the root, stem and leaf of Hai7124, TM-1, Hai-1 and Hai-1 × TM-1. Error bars represent the s.d. of the mean values of three biological replicates. (d) Sequence diversity of PGF. An amino acid change from alanine to valine was observed in the dominant glandless gene (Gl). The premature translation termination resulted in the production of fewer glands (Gl₂gl₃ and gl₂Gl₃ mutant) or completely glandless (gl₂gl₃ mutant) phenotypes. (e) Pyrosequencing analysis of the relative expression levels of GoPGF_A12 and GoPGF_D12 homoeologous alleles in the root, stem and leaf of TM-1, Hai-1, 2(Gl₂gl₃), 2(gl₂Gl₃) and 2(gl₂gl₃).

Figure 3. Functional characterization of GoPGF by VIGS.

(a) Phenotypes of TM-1 before and after GoPGF silencing by VIGS, showing the presence and the absence of the glands. (b) Transcript level of GoPGF in GoPGF-silenced leaves. (c) Cavity observed in the leaves of TM-1 but disappeared in the leaves emerged after VIGS. Scale bar, 10 μm. (d) Hemigossypol and gossypol content in control (CK), empty vector (TRV:00) and in the GoPGF-silenced (#1 and #2) leaves of TM-1, determined by high-performance liquid chromatography. **P<0.01; Student's t-test, n=3. Error bars are s.d. of three biological repeats.

Figure 4. Functional characterization of GoPGF by VIGS in G. barbadense cv. Giza 45.

(a) Transcript level of GoPGF in normal Giza 45 and corresponding GoPGF-silenced leaves. Error bars are s.d. of three biological repeats. **P<0.01; Student's t-test, n=3. (b,c) Phenotypes of Giza 45 before and after GoPGF silencing by VIGS. Yellow and blue arrows indicate the cotyledonary node and the first vegetative branch, respectively.

Figure 5. A proposed model of gossypol biosynthesis and gland formation.

(a) Among 111 bHLH members (Supplementary Data 2), 19 bHLHs from the distinct clade II may be responsible for glandular trichome formation. Numbers on branch nodes indicate percentage support in 1,000 bootstrap trials. (b) A proposed model of gossypol biosynthesis and gland formation. Blue text indicates genes downregulated in GoPGF-silenced TM-1. Heat map shows gene expression level with FPKM value in VIGS-free TM-1 (AT), PGF-silenced TM-1 (P) and Hai-1 (H), each block indicates one gene. First, the expression of GoPGF is induced by exogenous signal including JA. Then, GoPGF protein, as a regulator, controls the specification and differentiation of gland cells through regulating the expression of JAZ, WRKYs or other genes. On the other hand, GoPGF can specifically interact with the G-box motif, which is commonly found in TPSs and WRKYs promoter regions to feedback on gossypol biosynthesis pathway.