3.1. Fiber phenotypic characterization of brown and green cottons
We collected four colored cotton accessions, including three accessions of green colored cottons (G1, G2, G3) and one accession of brown colored cotton (T586/T). YZ1, one accession of white colored cotton was used as control. The fiber color and length of these materials are shown in Fig. 1. The fiber color of the three green cotton accessions is yellow-green, and similar to each other. Another feature of these three green cotton fibers is the uneven coloration. The regions of the fibers near the base of the seed coat, which were wrapped inside, are dark green, but fibers exposed to the outside exhibit a light green or even white. The brown cotton T586 (T) fiber colored uniformly (Fig. 1A-B).
The fiber lengths of these three different colored cottons were measured, and white cotton YZ1 has the longest fiber, with an average length of 27.9 mm. The length of three accessions of green cotton (G1, G2, G3) was similar to each other but shorter than white cotton YZ1, with a length of about 24 mm. The brown cotton T586 (T) had significantly shorter fibers than white and green cottons, at a mean of 15.8 mm (Fig. 1B-C).
3.2. Expression analysis of flavonoid biosynthesis genes in brown and green cotton fibers.
Flavonoids are thought to be involved in the formation of NCC fiber pigments (Feng et al. 2014; Hua et al. 2007; Liu et al. 2018; Tan et al. 2013; Yan et al. 2018). To investigate expression patterns, annotated flavonoid biosynthesis genes in G. hirsutum genome were selected, and a heatmap for these genes in white cotton fiber was constructed to illustrate transcriptional changes during fiber development (Fig. 2). All genes in the flavonoid biosynthesis pathway had only one or two copies in each subgenome except CHS, which has 9 copies in the Dt subgenome and 8 copies in the At subgenome. Most genes of flavonoid biosynthesis pathway had a similar expression pattern, being highly expressed at the fiber initiation stage (0 DPA, 5 DPA), and the expression levels decreased during fiber development.
To reveal the relationship between endogenous flavonoid biosynthesis gene expression and fiber colors, the expression levels of flavonoid biosynthesis pathway genes were analysed by qRT-PCR in NCC accessions (Fig. 3). F3H was the most abundantly expressed gene in brown cotton fiber, and its expression gradually increased during fiber development, with the highest expression level at 15 DPA. The stage with the highest F3H expression in green cottons was 10 DPA and in white cotton was 5 DPA. Moreover, the expression of F3H in brown cotton fibers was 10 times higher than in white and green cotton fibers.
As the first key enzyme in the flavonoid biosynthesis pathway, CHS plays an extremely important role in flavonoid metabolism. The expression of CHS in brown cotton fibers was the highest in 10 DPA fiber. Like F3H, the expression level peak in green cottons was in 10 DPA and white cotton was 5 DPA. ANR can catalyze the synthesis of PAs, and the corresponding gene was also found to be highly expressed in brown cotton fiber. ANR shows the highest expression at 10 DPA in the brown fibers, and its expression level was 4-5 times higher than that in white and green fibers.
ANS is the downstream gene of flavonoid metabolism, catalyzing the synthesis of anthocyanins. ANS was highly expressed in brown cotton fibers to a level of 4-5 times higher than that of white and green cotton fibers. Whether anthocyanins are involved in brown fiber pigmentation remains to be explored. Nevertheless, the expression level of flavonoid biosynthetic genes in brown cotton fibers was significantly higher than in green and white cotton fibers.
3.3. Endogenous flavonoid contents in brown and green cotton fibers
We measured the total flavonoid contents (TFCs) in the ovules and fibers from 5 DPA to 20 DPA to determine whether they changed during the development of different colored fibers (Fig. 4A). White cotton and green cotton fibers had similar trends in TFCs during different developmental stages. In these accessions, the TFCs accumulated to the highest levels in 5 DPA ovule samples, about 8mg/g. In fibers, TFCs were the highest in 15 DPA fibers. TFCs of all fiber samples were significantly lower than in 5 DPA ovule samples. In contrast for brown fibers, the total flavonoid contents in 10 DPA, 15 DPA and 20 DPA fibers were markedly higher than in 5 DPA ovule samples. The TFCs concnetrations in 10 DPA fibers were the highest (35mg/g), and at 15 DPA fiber the levels decreased sharply, but then increased again in 20 DPA fibers. Overall, the TFC contents in brown fibers were significantly higher than those of white and green fibers.
3.4. Proathocyanidin (PA) contents in brown and green cotton fibers.
To further investigate whether PA plays the same role in the pigmentation of green and brown cotton fibers, the PA contents were measured. 4-dimethylaminocinnamaldehyde (DMACA) staining method, which gives a blue coloration in the presence of PA, was employed to visualize PA in mature fibers. Brown cotton fibers showed the presence of PA while white and green fibers showed no difference with controls (Fig. 5). These results suggested that PA accumulated in mature brown cotton fibers, but not detectably in mature fibers of white and green cottons.
We also checked the PA contents in immature fibers (Fig. 4B-C). Like the results found for the total flavonoids, the PA contents at different developmental stages in white and green cotton fibers were similar, but significantly lower than those in brown fiber samples. The highest PA content in brown cotton fibers was at 10 DPA, and PA contents decreased slightly at 15 DPA and 20 DPA. In summary, significantly higher level of PA was accumulated in brown cotton than in green and white cottons.
3.5. Expression analysis of lignin and lignan biosynthesis pathway genes in brown and green cotton fibers.
The presence of caffeoyl and caffeoyl glycerides in extracts of green cotton fibers have been studied (Feng et al. 2017; Ma et al. 2015). Caffeic acid and caffeoyl-CoA are intermediate metabolites of lignin and lignan metabolism (Davin and Lewis, 2000). Therefore, qRT-PCR was used to detect the expression levels of lignin and lignan biosynthetic pathway genes in these three types of cotton fibers (Fig. 6).
PAL, C4H and 4CL are the most upstream genes in phenylpropanoid metabolism, and are involved in the synthesis of not only flavonoids, but also lignin and lignan. The expression levels of PAL and C4H in brown fibers were significantly higher than those in white and green fibers. PAL and C4H transcripts accumulated to the highest levels in 5 DPA samples of white cotton (Fig. 6, Table S4), but in 10 DPA fibers of green cottons. The expression levels of these two genes in green fibers were slightly higher than in white fibers. The expression of 4CL in brown fibers was higher than in ovules at 0 DPA and 5 DPA and than in white and green cotton fibers, but lower than in green fibers at 10 DPA and 15 DPA.
HCT is the first key enzyme in the lignin synthesis pathway. The expression of this gene in white and green fibers was higher than in brown fibers, and the expression levels in G1 fibers were significantly higher than in white fibers. CCoAOMT and COMT are downstream genes in lignin metabolism, and influence the biosynthesis of monolignols, which are further used to synthesize lignin or lignan. The green fibers showed a slightly higher expression of this gene than brown or white fibers at 10 DPA and 15 DPA (Fig. 6). PCBER encodes a key enzyme in the metabolism of lignan, and is a novel candidate gene that potentially is responsible for pigmentation in green cotton fibers (Li et al. 2018). Analysis of its expression in fibers showed that, similar to HCT, the expression levels of this gene were higher in white cotton and green cotton fibers than in brown fibers (Fig. 7).