4.1. Flavonoid accumulation is not a key determinant in green cotton fiber pigmentation
Green and brown cotton are the major two colored cotton types grown around the world. Determining the pigment components of colored fibers is a key first step in breeding improved quality of colored cotton. Most research has been carried out on the pigmentation of brown cotton fiber, with little known about green fiber pigments. We therefore chose 3 green cotton accessions, 1 brown cotton accession and 1 white cotton accession to study the differences between these 3 types. Unlike brown cotton, green cotton fibers colored unevenly (Fig. 1). Green but not white or brown cotton fiber color changed when treated with 6 M HCl/95% ethanol solution (Fig. 5), likely due to the instability of the green pigments. Previous studies have shown the color of green cotton fiber was easily changed by oxidants, reductants, metallic ions, alkalis, UV exposure and high temperature (Günaydin et al., 2019; Zhang and Hu, 2003). All these results suggested that the fiber pigment components of brown and green cotton are different.
Flavonoids are one of the three major plant pigments, include six major subgroups such as chalcones, anthocyanins and proanthocyanins. Intensive biochemical and transcriptomics analysis has indicated that flavonoid biosynthesis, and especially PAs biosynthesis and accumulation, plays a key role in the coloration of brown cotton fibers (Feng et al., 2014; Gong et al., 2014; Li et al., 2013; Yan et al., 2018). In agreement with previous studies, we found that flavonoid metabolism was transcriptionally activated in brown cotton fibers, and high levels of flavonoids were synthesized during fiber development (Fig. 2 - Fig. 5).
The relationship between green fiber pigmentation and flavonoids is still controversial. Flavonoids have been found to be the dominant pigment in green cotton fibers by measuring the flavonoids content during fiber development in previous work (Hua et al., 2007; Yuan et al., 2016). Further study found PAs were not to be the pigments in green cotton fibers based on DMACA staining (Li et al., 2018). But a recent study about transcriptomic and transgenic analysis of green and brown cotton suggested that the flavonoid biosynthetic pathway controls green fiber pigmentation (Liu et al., 2018).
Our results found that differences in the flavonoid metabolism between green and white fibers were not as significant as between brown and white fibers (Fig. 2 - Fig. 5). The expression levels of flavonoid metabolism genes in green fibers were similar to those in white fibers and significantly lower than in brown fibers (Fig. 3), which was consistent with the measurement of flavonoid contents (Fig. 4 - Fig. 5). The measurement of PA contents and DMACA staining of green fibers also indicated that PA was not the accumulated pigment in green fibers. These results suggest that flavonoids are not the key determinant of pigmentation in green cotton fibers.
4.2. Lignin and lignan biosynthesis pathways were slightly activated at the transcriptional level during the development and coloration of green cotton fibers.
Caffeic acid is a key intermediate in the biosynthesis of lignin and lignan (Davin and Lewis, 2000),and caffeic-acid derivatives have been detected in green cotton fibers (Feng et al., 2017; Ma et al., 2015; Schmutz et al., 1993; Schmutz et al., 1994). Furthermore, colored cotton fibers have been found to contain more lignin and lignan than white cotton fibers (Ioelovich and Leykin, 2008; Li et al., 2018). However, the comparison of lignin contents in green cotton and brown cotton fibers depends on the variety tested. Some brown cotton fibers contained higher total lignin contents than green cotton fibers (Morais Teixeira et al., 2010), but some are exactly the opposite (Ioelovich and Leykin, 2008).
We checked the expression levels of six key genes involved in caffeic acid and lignin biosynthesis to gain insights into whether this pathway participates in green fiber development. The phenylpropanoid pathway was significantly up regulated in brown fibers compared with white and green fibers (Fig. 6), consistent with previous reports. The expression levels of PAL and C4H in brown fibers were markedly higher than in white and green fibers. However, the expression of genes for the metabolic flux to lignin biosynthesis was similar or slightly lower than in white and green fibers, implying that a large amount of phenylpropanoid metabolism is directed to flavonoids in brown fibers.
Although most of the caffeic-acid and lignin and lignan biosynthesis genes in green fibers did not exhibit noticeably increased expression compared with white and brown fibers, they did have a slightly higher expressions during some stages of fiber development. C4H, 4CL, HCT are the enzymes directly responsible for caffeic acid and caffeoyl-CoA synthesis (Vanholme et al., 2012). At 10 DPA and 15 DPA, the expression levels of C4H, 4CL in green fibers were higher than in white fibers, and the green accession G1 had a significantly higher expression of HCT at 10 DPA and 15 DPA (Fig. 6). A similar situation also was seen for lignan metabolism. 15 DPA is the point of secondary cell wall biosynthesis, and also an important stage for the initiation of pigmentation in colored cotton fibers (Kim, 2015; Yuan et al., 2012). Our results indicate that caffeic acid derivatives, and lignin and lignan biosynthesis pathways are activated during the development and coloration of green fibers, which may explain why green fibers have a higher lignan and caffeic acid derivatives contents than white fibers. A detailed biochemical and transcriptional systems biology analysis should be carried out to investigate the precise roles of the caffeic acid derivatives, lignin and lignan in the pigmentation of green cotton fibers.
4.3. Suberin may be a key structure for green cotton fiber pigmentation
Suberin is an analogous biopolymer of cutin found in some specialized plant cell walls, and serves as a lipid-based barrier to protect cells from various environmental stresses (Cohen et al., 2017; Graca, 2015). It is composed of very long chain aliphatic acid derivatives, glycerol, and linked with phenolics and embedded waxes. Typically, the phenolic components are ferulic acid, caffeic acid, coumaric acid and monolignol derivatives (Cohen et al., 2017; Vishwanath et al., 2015).
Interestingly, electron microscope thin sections of cotton fibers revealed that suberin lamellae are found in the cell wall of green cotton fibers (Ryser et al., 1983). Caffeic acid and glycerol have been detected in extracts of green fibers, and presence of these two chemicals in the suberin of green fiber has been confirmed in subsequent studies, leading to the proposal that they may be the pigments of green fibers (Schmutz et al., 1996). However, no studies have been conducted to compare the location of fiber pigments and suberin lamellae in green cotton fibers. We compared the previous studies on the location of pigments and suberin lamellae in green cotton fibers and surprisingly, we found both are deposited in alternating layers with cellulose in the secondary cell walls of fibers (Ryser et al., 1983; Zhang et al., 2011). Suberin lamellae must therefore be a key feature of green cotton fibers that is involved in fiber coloration.
Since some caffeic acid derivatives have a yellow-green color and have been detected in extracts of green fibers (Feng et al., 2017), caffeic acid derivatives are likely to be some of the pigments in green fibers. Monolignol derivatives and lignan might act as structural components of suberin. So far, few studies have focused on this particular cell wall structure as compared with other components in plant cell walls. Green cotton fibers, with available genetic and molecular resources, are therefore an excellent system for studying the synthesis and composition of suberin. More effort is needed in this area and on the relationship between the suberin lamellae and lignin and lignan. A comprehensive research effort on suberin lamellae will greatly assist in understanding the control of green cotton pigmentation and inform fiber quality breeding in green cotton cultivars.