Comparative Metabolome and Transcriptome Analysis of Anthocyanin Biosynthesis in White and Pink Petals of Cotton (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) is one of the important fiber crops. Cotton flowers usually appear white (or cream-colored) without colored spots at the petal base, and turn pink on the next day after flowering. In this study, using a mutant showing pink petals with crimson spots at their base, we conducted comparative metabolome and transcriptome analyses to investigate the molecular mechanism of coloration in cotton flowers. Metabolic profiling showed that cyanidin-3-O-glucoside and glycosidic derivatives of pelargonidins and peonidins are the main pigments responsible for the coloration of the pink petals of the mutant. A total of 2443 genes differentially expressed (DEGs) between the white and pink petals were identified by RNA-sequencing. Many DEGs are structural genes and regulatory genes of the anthocyanin biosynthesis pathway. Among them, MYB21, UGT88F3, GSTF12, and VPS32.3 showed significant association with the accumulation of cyanidin-3-O-glucoside in the pink petals. Taken together, our study preliminarily revealed the metabolites responsible for the pink petals and the key genes regulating the biosynthesis and accumulation of anthocyanins in the pink petals. The results provide new insights into the biochemical and molecular mechanism underlying anthocyanin biosynthesis in upland cotton.

Transcriptome Analysis Reveals Differences in Anthocyanin Accumulation in Cotton (Gossypium hirsutum L.) Induced by Red and Blue Light.

Many factors, including illumination, affect anthocyanin biosynthesis and accumulation in plants. light quality is the key factor affecting the process of photoinduced anthocyanin biosynthesis and accumulation. We observed that the red color of the Upland cotton accession Huiyuan with the R1 mutation turned to normal green color under light-emitting diodes (LEDs), which inspired us to investigate the effect of red and blue lights on the biosynthesis and accumulation of anthocyanins. We found that both red and blue lights elevated accumulation of anthocyanins. Comparative transcriptomic analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA, revealed that genes differentially expressed under different light conditions were enriched with the pathways of circadian rhythm, phenylpropanoid biosynthesis, anthocyanin biosynthesis, and flavone and flavonol biosynthesis. Not surprisingly, all the major structural genes related to biosynthesis of anthocyanins, including the key regulatory MYB transcription factor (GhPAP1D) and anthocyanin transporter (GhGSTF12), were induced by red or blue light treatment. However, LARs and MATEs related to biosynthesis of proanthocyanidins were more significantly up-regulated by red light radiation than by blue light radiation. Vice versa, the accumulation of anthocyanins under red light was not as high as that under blue light. In addition, we demonstrated a potential role of GhHY5, a key regulator in plant circadian rhythms, in regulation of anthocyanin accumulation, which could be achieved via interaction with GhPAP1D. Together, these results indicate different effect of red and blue lights on biosynthesis and accumulation of anthocyanins and a potential module including GhHY5 and GhPAP1D in regulation of anthocyanin accumulation in cotton. These results also suggest that the substrates responsible the synthesis of anthocyanins under blue light is diverted to biosynthesis of proanthocyanidin under red light.

GhGSTF12, a glutathione S-transferase gene, is essential for anthocyanin accumulation in cotton (Gossypium hirsutum L.).

Anthocyanins are flavonoid pigments providing plants a range of colors from red, pink, orange to blue. Anthocyanins are synthesized in the cytosol but accumulate predominantly in the vacuoles through vacuolar sequestration involving glutathione S-transferases (GSTs) and multidrug and toxic compound extrusion (MATE) and the ATP binding cassette (ABC) transporters. However, little is known about anthocyanin-related GSTs in Upland cotton (Gossypium hirsutum L.). In this study, we performed genome-wide identification of GST genes in Upland cotton and identified GST genes functioning in accumulation of anthocyanins. We demonstrated that GhGSTF12 was able to complement the defective leaf color phenotypes of the Arabidopsis tt19 mutant caused by mutation in a GSTF gene. Virus-induced silencing of GhGSTF12 in the red leaf cultivar turned its red color to green and transient overexpression of GhGSTF12 accelerated anthocyanin accumulation in the red leaf cultivar but not in the green leaf cultivar. Collectively, GhGSTF12 may be involved in transport of anthocyanins from cytosol to vacuoles in cotton. These results also demonstrated a conserved function of plant GSTF genes in anthocyanin accumulation and provide a candidate gene for manipulating pigmentation in cotton tissues.

Screening the Reference Genes for Quantitative Gene Expression by RT-qPCR During SE Initial Dedifferentiation in Four Gossypium hirsutum Cultivars that Have Different SE Capability.

RNA sequencing (RNA-Seq)-based gene expression analysis is applicable to a wide range of biological purposes in various species. Reverse transcription quantitative PCR (RT-qPCR) is also used to assess target gene expression utilizing stably expressed reference genes as internal control under a given set of conditions. However, investigations of the reference genes for RT-qPCR normalization in the process of somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum are rarely reported. In this study, on the basis of our previous transcriptome data of three different induction stages during SE initial dedifferentiation process in four G. hirsutum cultivars that have different SE capability, 15 candidate genes were selected during SE initial dedifferentiation process, and their expression stability was evaluated by geNorm, NormFinder, and BestKeeper. The results indicated that the two genes of endonuclease 4 (ENDO4) and 18S ribosomal RNA (18S rRNA) showed stable expression in the four different G. hirsutum cultivars, endowing them to be appropriate reference genes during three induction stages in the four cotton cultivars. In addition, the stability and reliability of the two reference genes of ENDO4 and 18S rRNA were further verified by comparing the expressions of auxin-responsive protein 22 (AUX22) and ethylene-responsive transcription factor 17 (ERF17) between RT-qPCR results and the RNA-seq data, which showed strong positive correlation coefficient (R2 = 0.8396-0.9984), validating again the steady expression of ENDO4 and 18S rRNA as the reliable reference genes. Our results provide effective reference genes for RT-qPCR normalization during SE process in different G. hirsutum cultivars.

Comparative Transcriptome Analysis of SE initial dedifferentiation in cotton of different SE capability.

Somatic embryogenesis (SE) is a critical transition from vegetative to embryogenic growth in higher plants; however, few studies have investigated the mechanism that regulates SE initial differentiation. Most cotton varieties have not undergone regeneration by SE, so only a few varieties can be used in genetic engineering. Here, two varieties of cotton with different SE capabilities (HD, higher differentiation and LD, lower differentiation) were analyzed by high throughout RNA-Seq at the pre-induction stage (0h) and two induction stages (3h and 3d) under callus-induction medium (CIM). About 1150 million clean reads were obtained from 98.21% raw data. Transcriptomic analysis revealed that "protein kinase activity" and "oxidoreductase activity" were highly represented GO terms during the same and different treatment stages among HD and LD. Moreover, several stress-related transcription factors might play important roles in SE initiation. The SE-related regulation genes (SERKs) showed different expression patterns between HD and LD. Furthermore, the complex auxin and ethylene signaling pathway contributes to initiation of differentiation in SE. Thus, our RNA-sequencing of comparative transcriptome analysis will lay a foundation for future studies to better define early somatic formation in cotton with different SE capabilities.