Background: The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. Results: The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. Conclusion: The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.

Summary: Naturally coloured cotton (NCC) fibres need little or no dyeing process in textile industry to low‐carbon emission and are environment‐friendly. Proanthocyanidins (PAs) and their derivatives were considered as the main components causing fibre coloration and made NCCs very popular and healthy, but the monotonous fibre colours greatly limit the wide application of NCCs. Here a G. hirsutum empurpled mutant (HS2) caused by T‐DNA insertion is found to enhance the anthocyanidins biosynthesis and accumulate anthocyanidins in the whole plant. HPLC and LC/MS‐ESI analysis confirmed the anthocyanidins methylation and peonidin, petunidin and malvidin formation are blocked. The deficiency of GhOMT1 in HS2 was associated with the activation of the anthocyanidin biosynthesis and the altered components of anthocyanidins. The transcripts of key genes in anthocyanidin biosynthesis pathway are significantly up‐regulated in HS2, while transcripts of the genes for transport and decoration were at similar levels as in WT. To investigate the potential mechanism of GhOMT1 deficiency in cotton fibre coloration, HS2 mutant was crossed with NCCs. Surprisingly, offsprings of HS2 and NCCs enhanced PAs biosynthesis and increased PAs levels in their fibres from the accumulated anthocyanidins through up‐regulated GhANR and GhLAR. As expected, multiple novel lines with improved fibre colours including orange red and navy blue were produced in their generations. Based on this work, a new strategy for breeding diversified NCCs was brought out by promoting PA biosynthesis. This work will help shed light on mechanisms of PA biosynthesis and bring out potential molecular breeding strategy to increase PA levels in NCCs.

Cotton Verticillium wilt, caused by the notorious fungal phytopathogen Verticillium dahliae (V. dahliae), is a destructive soil-borne vascular disease and severely decreases cotton yield and quality worldwide. Transcriptional and post-transcriptional regulation of genes responsive to V. dahliae are crucial for V. dahliae tolerance in plants. However, the specific microRNAs (miRNAs) and the miRNA/target gene crosstalk involved in cotton resistance to Verticillium wilt remain largely limited. To investigate the roles of regulatory RNAs under V. dahliae induction in upland cotton, mRNA and small RNA libraries were constructed from mocked and infected roots of two upland cotton cultivars with the V. dahliae-sensitive cultivar Jimian 11 (J11) and the V. dahliae-tolerant cultivar Zhongzhimian 2 (Z2). A comparative transcriptome analysis revealed 8330 transcripts were differentially expressed under V. dahliae stress and associated with several specific biological processes. Moreover, small RNA sequencing identified a total of 383 miRNAs, including 330 unique conserved miRNAs and 53 novel miRNAs. Analysis of the regulatory network involved in the response to V. dahliae stress revealed 31 differentially expressed miRNA–mRNA pairs, and the up-regulation of GhmiR395 and down-regulation of GhmiR165 were possibly involved in the response to V. dahliae by regulating sulfur assimilation through the GhmiR395-APS1/3 module and the establishment of the vascular pattern and secondary cell wall formation through GhmiR165-REV module, respectively. The integrative analysis of mRNA and miRNA expression profiles from upland cotton lays the foundation for further investigation of regulatory mechanisms of resistance to Verticillium wilt in cotton and other crops.

The wild cotton species Gossypium stocksii produces a brown fiber that provides a valuable resource for the color improvement of naturally colored cotton (NCC) fiber. However, the biochemical basis and molecular mechanism of its fiber pigmentation remain unclear. Herein, we analyzed the dynamics of proanthocyanidins (PAs) accumulation in developing the fiber of G. stocksii, which suggested a similar role of PAs and/or their derivatives in the fiber coloration of G. stocksii. In addition, comparative transcriptomics analyses revealed that the PA biosynthetic genes were expressed at higher levels and for a longer period in developing fibers of G. stocksii than G. arboreum (white fiber), and the transcription factors, such as TT8, possibly played crucial regulatory roles in regulating the PA branch genes. Moreover, we found that the anthocyanidin reductase (ANR) was expressed at a higher level than the leucoanthocyanidin reductases (LARs) and significantly upregulated during fiber elongation, suggesting a major role of ANR in PA synthesis in G. stocksii fiber. In summary, this work revealed the accumulation of PAs and the expression enhancement of PA biosynthetic genes in developing fibers of G. stocksii. We believe this work will help our understanding of the molecular mechanisms of cotton fiber coloration and further promote the future breeding of novel NCCs.