Identification and expression pattern of GhCHS, GhANR and GhLAR
In the genome of G. hirsutum, the 13 GhCHS and GhCHS-like genes in the CHS family, 2 GhANR genes and 3 GhLAR genes were scanned, the gene and protein sequences of GhCHS, GhANR and GhLAR were highly conserved, but the genes of GhCHS, GhANR and GhLAR had the expression specificity in cotton plant, GhCHS2 gene was predominantly expressed in colored cotton fibers, GhCHS1 and GhCHS3 expressed weakly in the developing fibers, the other GhCHS and GhCHS-like transcripts in the developing fibers were not measured. GhLAR1, GhLAR2 and GhLAR3 were all expressed in the developing fibers, but differentially expressed in the different cotton species with different colors or color-depth, the 3 GhLAR genes represented the high expressive abundance in the deeply colored fibers of HZ, and perhaps the GhLAR genes could improve the fiber color depth. The 2 GhANR genes were expressed in the developing fibers and obviously increased their transcripts in the colored cotton species, and also showed high expression abundance in the deeply colored fibers of HZ. Among the three types of genes for anthocyanidin biosynthesis and transport, the GhANR genes always maintained high expression level and as well represented the main flow way for anthocyanidins into fiber cell [11] and played the major role for anthocyanidins transport.
The expression levels of GhCHS, GhANR and GhLAR closely related to fiber color
The 5 cotton species were used to measure the influence of GhCHS, GhANR and GhLAR gene expression on the fiber color formation. The expression levels of GhCHSs and GhANRs, GhLAR1 and GhLAR3 were all predominantly expressed early in developing fibers of colored fibers, especially in the dark brown fiber of HZ (Fig. 5). The expression levels of GhCHS, GhANR and GhLAR positively influenced the color formation of fiber in colored cotton. Therefore, for improving the color of cotton fiber, firstly the GhCHS gene expression would be increased to enhance the anthocyanin biosynthesis, then the GhANR and GhLAR increased their expression for transporting anthocyanidins into fiber cell. In the GhANRi and GhLARi cotton lines, the GhCHS gene was upregulated by the suppression of GhANRi and GhLARi, perhaps in natural colored cotton, the PA formation in the fiber cell could feedback the anthocyanidins biosynthesis, PA formation in fiber cell was mainly resulted from the anthocyanidin transport and accumulation through GhANR and GhLAR. Correspondingly, the suppression of GhCHS in GhCHSi cotton lines, the GhANR was downregulated, perhaps no more anthocyanidins could be transported into fiber cell through GhANR. The content of anthocaynidins in cotton kernels, fiber and seedcoat of GhANRi, GhLARi and GhCHSi plants decreased and increased pattern in leaves could confirm this hypothesis.
The suppression of GhCHS, GhANR and GhLAR had negative effect on fiber color
The GhANR, GhLAR and GhCHS genes in natural colored cotton ZX1 was silenced, and the fiber color in the transgenic RNAi ZX1 plants was significantly different from the WT and CK. In the transgenic ZX1 plants, the endogenous genes of GhANR, GhLAR and GhCHS were suppressed, especially in the fiber of 5 DPA and 10 DPA (Fig 8), the fiber color in the transgenic ZX1 plants faded to lighter and even more lighter. The down-regulation levels of the 3 genes emerged as negative correlation with fiber color. In the general phenylpropanoid pathway, chalcone synthase was the first committed enzyme of flavonoid biosynthesis, among the 3 genes, the conserved sequence of GhCHS1, GhCHS2 and GhCHS3 silenced has little significant effect on cotton fiber color. Firstly, it may be multiple members of CHS family in G. hirsutum, although GhCHS2 predominantly expressed early in developing fiber in colored cotton, other members existed functional complementarity after GhCHS2, even GhCHS1 and GhCHS3 suppressed; Secondly, GhCHS genes were in the upstream location of anthocyanidin biosynthesis, suppression of GhCHS had little effect on downstream synthesis and metabolism of anthocyanins. Early biosynthetic genes (EBGs)—CHS, CHI, and F3H are the common flavonoid pathway genes which are involved in the biosynthesis of all downstream flavonoids. In general, the reported expression profile of EBGs varies and there is no consistent correlation between their expression levels and anthocyanin content in Solanaceous vegetables [51]. In eggplant, the expression level of SmCHS was reported to be significantly upregulated in black or violet fruits compared to the green or white mutants [52, 53]. In potato tubers, the association of expression of CHS and anthocyanin accumulation is more consistent. CHS were highly expressed in red and purple tubers and correlated with anthocyanin content [54-57].
The GhANR and GhLAR worked for anthocyanidins transport in the anthocyanin metabolic pathway, the GhANR played the main role for colored anthocyanidins into fiber cell, the GhLAR worked for transporting leucoanthocyanidin in fiber cell and also could enhance the fiber color perhaps by polymerization and oxidation to form anthocyanin derivatives [11]. The GhLARs were preferentially expressed in the deep colored fiber of HZ plant, the fiber color became lighter in the GhLAR suppressed plants.
PAs (also called condensed tannins) are synthesized via a branch of anthocyanin biosynthesis pathway under the catalyzation of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). LAR catalyzes the conversion of leucoanthocyanidin (flavan-3, 4-diol) to catechin, while ANR catalyzes the synthesis of epicatechin from anthocyanidin [36, 38, 58]. Ectopic expression of the tea CsLAR gene in tobacco results in the accumulation of higher level of epicatechin than that of catechin, suggesting LAR maybe involved in the biosynthesis of epicatechin [37]. ANRs from grapevine and tea are proven to have epimerase activity and thus can convert anthocyanidin to a mixture of epicatechin and catechin [37, 59]. Further, previous engineering experiments in soybean, Arabidopsis, and petunia have redirected metabolic flux from anthocyanin biosynthesis into the isoflavone pathway, from lignin biosynthesis into the flavonoid pathway, and from flavonol biosynthesis into the anthocyanin pathway, by suppressing anthocyanin, lignin, and flavonol branchpoint genes respectively [60-62]. The overexpression of the ANR gene from Medicago truncatula in tobacco resulted in reduced anthocyanin pigmentation in the flower and elevated PA levels [58]. These results suggested the potential for ANR to compete with the anthocyanin biosynthesis enzyme UDP-glycose: flavonoid-3-O-glycosyltransferase (UF3GT) for the substrate anthocyanidin, suppression of ANR genes results in increased anthocyanin accumulations.
The Arabidopsis ANR (or BAN) knockout mutant displayed precocious accumulation of cyanic pigments in the seed coat during early seed development [63]. The accumulations were only temporary, and resulted in a transparent testa (tt) phenotype with black pigmentation confined to the raphe of the dried grain [63]. This contrasts the phenotype in soybean, where high-level suppression of ANR genes gives a red-brown grain [41]. There may exist underlying mechanistic and metabolite differences that could explain the differences in grain phenotypes between these species. In Arabidopsis, the UF3GT gene (UGT78D2) and the ANR gene are regulated reciprocally, with UGT78D2 expressed with anthocyanins in the seedling, and ANR expressed with PAs in the seed coat [64]. By contrast, soybean UF3GT genes (UGT78K1 and UGT78K2) and ANR genes (ANR1 and ANR2) are both expressed in the seed coat [41]. Thus, it is possible that the difference in phenotype between the soybean grain undergoing high-level ANR gene suppressions and the Arabidopsis ANR knockout grain, may be attributed to the presence and absence of UF3GT expressions, respectively, that stabilize anthocyanins in soybean allowing their accumulations to provide the red-brown grain phenotype.
The biosynthesis of flavan-3-ols has been well characterized in many plant species, both genetically and biochemically. Two distinct enzymes, LAR and ANR, are involved in catalyzing the last steps of the pathway to flavan-3-ol monomers in PA-producing plants [37, 65, 66]. Genes encoding LAR and ANR can occur as single gene, for example in Arabidopsis [38], or as multigene families, for example in grapevine [65] and tea [37]. Analysis of the P. trichocarpa genome revealed three loci encoding LAR proteins and two loci encoding ANR proteins [67, 68], the enzymatic activity of the proteins encoded by all loci by heterologous expression and in vitro enzyme assays and showed that they are likely involved in the catalysis of the last steps of flavan-3-ol biosynthesis in native black poplar. ANRs and LARs are two distinct classes of enzymes and that DFR is more related to ANRs than LARs. Similar evolutionary relationships for ANR and LAR proteins were reported [37, 68]. Transcript levels of all three PnLAR and two PnANR genes increased in rust-infected black poplar leaves over the course of infection. Monomeric catechin synthesized from the LAR branch is freely available and accumulated in black poplar, while free ANR-dependent epicatechin was observed only at very low concentrations. The recovery of epicatechin after hydrolysis of PAs indicates that epicatechin might contribute to the extension of PA chains. Similar mechanisms also were observed in grape and Norway spruce [65, 69]. LARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression of NtLAR and NtANR s in CsMYB5b transgenic tobacco [70]. So the transport of anthocyanidins through GhANR, GhLAR into fiber cell will be the important link for genetic engineering of colored fiber molecular improvement.
The anthocyanidins content in the fiber directly influenced fiber color
In the transgenic RNAi cotton plants, the content of anthocyanidins was reduced by suppression of the endogenous GhANR, GhLAR and GhCHS genes, which resulted in the fiber color fading. CHS plays an important role in the phenylalanine metabolic pathway, plant growth and development, such as stress response, plant fertility and plant color [71]. LAR is a key enzyme in the synthetic pathway of plant flavonoids from phenylalanine, which catalyzes the conversion of colorless anthocyanins to catechins [58, 65, 66]. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin to regulate PA synthesis, respectively. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers [66].
The anthocyanidins content in the fiber and seedcoat of GhLARi plants was higher than GhANRi plants, and the fiber color was also deeper than that of GhANRi plant, although LAR transported colorless anthocyanins into fiber cell. From our previous research, the transcription level of GhLAR in the fibers of brown cotton was higher than that in white cotton, during the fiber development, the fiber color of GhLARi plants lightly faded here. Compared with white cotton fibers, the expression level of GhANR in brown cotton fibers was significantly higher. The gene expression of GhANR was active in brown cotton fibers and reached its peak at 12 DPA, when the expression level of GhANR in brown cotton fibers was >7 times higher than that in white cotton fibers [11]. During the fiber development, the GhLAR expression level in brown cotton was much lower than that of GhANR, so effect of suppression of GhLAR on the fiber color change was lower than that of GhANR, the suppression of GhANR in ZX1 could cause the fiber color to be significantly lighter. Our work of NMR analyses demonstrated that the flavan-3-ols in brown and white cotton fibers were in the 2, 3-cis form, but part of the proanthocyanidins in the white cotton fibers were modified by acylation. The prodelphidin (PD) relative percentage was similar to that of procyanidin (PC) in white cotton fibers, and proanthocyanidins with 90.1% PD were found in brown cotton fibers. The proanthocyanidin monomeric composition was consistent with the expression profiles of proanthocyanidin synthase genes, suggesting that ANR represented the major flow of the proanthocyanidin biosynthesis pathway in brown cotton fibers. Compared with white fibers, all of the proanthocyanidin synthase genes were expressed at a higher level in brown fibers [11]. The cis-form and trans-form of flavan-3-ols were synthesized via leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) branches, respectively [11, 38, 58, 72]. Biochemical analyses by mass spectrometry (MS) revealed that the main PA monomers in brown cotton fibers contained three hydroxyls on the B ring (gallocatechin or epigallocatechin) [11,21,73], PA accumulation in brown fibers starts at an early stage (5 DPA) and peaks at 30 DPA, whereas in mature brown fibers, PAs are converted to oxidized derivatives (quinones). Because developing brown fibers do not exhibit distinct coloration until maturation, the condensed quinones were proposed instead of their PA precursors, directly contribute to brown pigmentation in cotton fibers [11]. Therefore, the three key genes in the anthocyanin metabolic pathways played the very important role in the coloration of cotton fibers, and became the target genes for genetic manipulation to improve cotton fiber color.