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. In the early stage of anthocyanidin biosynthetic, CHS, CHI, and F3H are the common flavonoid pathway genes (also called early biosynthesis genes, EBGs), and are responsible for the biosynthesis of all downstream flavonoids. The varied expression profile of EBGs was not directly resulted in the change of anthocyanin content in Solanaceous vegetables [51]. The transcript of SmCHS was significantly increased in black or violet eggplant fruits compared to the light colored mutants of green or white [52, 53]. In potato tubers, CHS were highly expressed in red and purple tubers and more 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 LAR and 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]. The tea CsLAR gene ectopically expressed in tobacco caused the accumulation of higher level of epicatechin than that of catechin, indicating LAR may be response for the biosynthesis of epicatechin [37]. ANRs in grapevine and tea had the epimerase activity and thus could convert anthocyanidin to a mixture of epicatechin and catechin [37, 59]. The metabolic fluxes were successfully genetically modified in soybean, Arabidopsis, and petunia to redirect the biosynthesis of the isoflavone, the flavonoid, and the anthocyanin, by suppressing the corresponding branchpoint genes [60-62]. The overexpressed Medicago truncatula ANR gene in tobacco reduced anthocyanin pigmentation in the flower and elevated PA levels [58]. Perhaps ANR competes with UF3GT for the substrate anthocyanidin, suppression of ANR genes results in increasing anthocyanin accumulations.
During early seed development, the seed coat was precociously accumulated cyanic pigments in the Arabidopsis ANR (or BAN) knockout mutant [63]. The pigments in seed coat was transitorily accumulated as a transparent testa (tt) phenotype and black pigmentation confined to the raphe of the dried grain [63]. This was different with the phenotype in soybean with red-brown grain by strongly suppressed ANR genes [41]. The underlying mechanistic and metabolite resulted in the different grain phenotypes of different species. In Arabidopsis, the UF3GT (UGT78D2) for anthocyanins in the seedling and the ANR for PAs in the seed coat were regulated reciprocally [64]. In soybean, UF3GT genes including UGT78K1 and UGT78K2, ANR genes including ANR1 and ANR2 were expressed in the seed coat concurrently [41]. The difference of soybean grains phenotype perhaps resulted from ANR gene significant suppression. The presence and absence of UF3GT expression gave the Arabidopsis ANR knockout grain. Thus the red-brown grain phenotype in soybean was resulted from the accumulated of stable anthocyanins.
Two distinct enzymes of LAR and ANR were responsible for catalyzing the last step of the biosynthesis of flavan-3-ol monomers in PA-producing plants [37, 65, 66]. LAR and ANR gene occurs as single gene in Arabidopsis [38], or as multigene families, in grapevine [65] and tea [37]. Analysis of the P. trichocarpa genome revealed three loci encoding LAR and two loci encoding ANR [67, 68], which were likely involved in the catalysis of the last steps of flavan-3-ol biosynthesis in native black poplar from the enzymatic activity and in vitro enzyme assays. ANRs and LARs belong to two distinct classes of enzymes even with the similar evolutionary relationships, DFR was more related to ANRs than LARs [37, 68]. Freely available monomeric catechin was synthesized from the LAR branch and accumulated in black poplar, the concentration of free ANR-dependent epicatechin was very low. The epicatechin might contribute to the extension of PA chains in poplar, 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 GhANR was actively expressed in brown cotton fibers and predominantly expressed at 12 DPA, when the transcript level of GhANR in brown cotton fibers was higher than that in white cotton fibers with 7-fold [11]. During the fiber development, the transcript level of GhLAR 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. The flavan-3-ols exist in brown and white cotton fibers as the 2, 3-cis form. The most of proanthocyanidins in brown fibers were prodelphidin (PD), while in white cotton fibers, PD content was similar to that of procyanidin (PC). The proanthocyanidin monomeric composition conformed with the expression profiles of proanthocyanidin synthase genes, and ANR played the key role in the proanthocyanidin biosynthesis in brown fibers. The proanthocyanidin synthase genes were expressed at a higher level in brown fibers than in white fibers [11]. The cis-form and trans-form of flavan-3-ols were synthesized in LAR and ANR branches, respectively [11, 38, 58, 72]. Mass spectrometry (MS) analyses 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, PAs are gradually converted into oxidized derivatives (quinones) in mature brown fibers. Because developing brown fibers do not exhibit distinct coloration until maturation, the condensed quinones maybe directly contribute to brown pigmentation in cotton fibers instead of their PA precursors [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.