The GhTT2_A07 gene is linked to the brown colour and natural flame retardancy phenotypes of Lc1 cotton (Gossypium hirsutum L.) fibres

Abstract

Some naturally coloured brown cotton fibres from accessions of Gossypium hirsutum L. can be used to make textiles with enhanced flame retardancy (FR). Several independent brown fibre loci have been identified and mapped to chromosomes, but the underlying genes have not yet been identified, and the mechanism of lint fibre FR is not yet fully understood. In this study, we show that both the brown colour and enhanced FR of the Lc1 lint colour locus are linked to a 1.4Mb inversion on chromosome A07 that is immediately upstream of a gene with similarity to Arabidopsis TRANSPARENT TESTA 2 (TT2). As a result of the alternative upstream sequence, the transcription factor GhTT2_A07 is highly up-regulated in developing fibres. In turn, genes in the phenylpropanoid metabolic pathway are activated, leading to biosynthesis of proanthocyanidins and accumulation of inorganic elements. We show that enhanced FR and anthocyanin precursors appear in developing brown fibres well before the brown colour is detectible, demonstrating for the first time that the polymerized proanthocyanidins that constitute the brown colour are not the source of enhanced FR. Identifying the particular colourless metabolite that provides Lc1 cotton with enhanced FR could help minimize the use of synthetic chemical flame retardant additives in textiles.

Keywords: Lc1 locus; brown fibre; cotton; fame retardant; flavonoid; proanthocyanidin; textiles; transparent testa..

Fig. 1.

Time lapse depiction in 10s increments of the ASTM D1230-10 (45-degree) flammability test with nonwoven fabrics produced by hydroentanglement of cotton fibres harvested from Lc1 and white cotton lines. (A) Following a 10s exposure to the ignition source, nonwoven fabrics produced from MC-BL fibres immediately self-extinguished with afterglow visible in the remaining panels. (B) Nonwoven fabrics produced from MC-WL fibres ignited and stopped the test timer at ~20s followed by complete consumption of the material by ~50s.

Fig. 2.

Accumulation of flavonoid PA precursor molecules demonstrated by DMACA staining is correlated with enhanced flame retardancy (lower heat release capacity) and elevated levels of inorganic material after pyrolysis (% char yield). (A) Bolls from the cotton lines MC-BL and MC-WL with the carpels removed and fibres stained with DMACA and mature fibres. (B) Comparative analysis of fibre flammability (heat release capacity) and inorganic material remaining after pyrolysis (% char yield) from developing and mature fibre of the cotton lines MC-BL and MC-WL. Developmental stages are indicated by days post-anthesis (DPA) on the x-axis. Error bars represent standard deviation of three biological replicates.

Fig. 3.

Linkage and physical maps of the Lc1 genetic locus in Gossypium hirsutum. Genetic distances on G. hirsutum chromosome A07 are shown in centimorgans (cM) and physical distance along the orthologous G. arboreum reference sequence is shown in base pairs (bp). See also Supplementary Fig. S2 for sequencing-based evidence for the 1.4Mb genomic inversion.

Fig. 4.

Correlations between CIE L*a*b* colour space and fibre flammability (heat release capacity) in a subset of the F₂ segregating population. A lower HRC indicated a more flame retardant material. Whisker bars represent the maximum and minimum of three measurements. (A) Heat release capacity increased in order from homozygous Lc1 fibres (A genotype) to heterozygous Lc1 fibres (H phenotype) to homozygous wild-type (B genotype). (B) The CIE L*a*b* colour space coordinated with increased HRC starting with dark brown fibres (A genotype) to light brown fibres (H genotype) to white wild-type fibres (B genotype). (C) Percentage of sample not consumed (% char yield), which indicates the level of inorganic material remaining after pyrolysis, decreased in order from homozygous Lc1 fibres (A genotype) to heterozygous Lc1 fibres (H phenotype) to homozygous wild-type (B genotype). (D) Scanned images of single locules representative of the indicated Lc1 inversion genotype. In CIE L*a*b* colour space, L* indicates whiteness (0=black; 100=white); a* indicates colours from greenish (negative values) to reddish (positive values); and b* indicates colours from bluish (negative values) to yellowish (positive values).

Fig. 5.

Differential expression of structural and regulatory genes in the committed phenylpropanoid and flavonoid pathways, and in the upstream phenylalanine and shikimate pathways. Genes that were statistically significant and upregulated more than 2-fold based on the mRNA-seq analysis are indicated along with the accession numbers based on the Gossypium hirsutum cv. TM-1 draft genome. The fold differences in expression between MC-BL and MC-WL are indicated in the boxes below the accession numbers, with 8 days post anthesis (DPA) in the left box and 20 DPA in the right. The boxes are colour-coded brown for upregulation in MC-BL and white for upregulation in MC-WL. No statistically significant difference in gene expression is indicated by ns. The genes known to be directly regulated by the TT2-TT8-TTG1 regulon in Arabidopsis are indicated within the bracket next to the regulatory complex in the illustration. The GhTT2_A07 gene (Gh_A07G2341) activated by the upstream genomic inversion is indicated within the red box.