Analysis of flavonoids and the flavonoid structural genes in brown fiber of upland cotton

Abstract

Background: As a result of changing consumer preferences, cotton (Gossypium Hirsutum L.) from varieties with naturally colored fibers is becoming increasingly sought after in the textile industry. The molecular mechanisms leading to colored fiber development are still largely unknown, although it is expected that the color is derived from flavanoids.

Experimental design: Firstly, four key genes of the flavonoid biosynthetic pathway in cotton (GhC4H, GhCHS, GhF3'H, and GhF3'5'H) were cloned and studied their expression profiles during the development of brown- and white cotton fibers by QRT-PCR. And then, the concentrations of four components of the flavonoid biosynthetic pathway, naringenin, quercetin, kaempferol and myricetin in brown- and white fibers were analyzed at different developmental stages by HPLC.

Result: The predicted proteins of the four flavonoid structural genes corresponding to these genes exhibit strong sequence similarity to their counterparts in various plant species. Transcript levels for all four genes were considerably higher in developing brown fibers than in white fibers from a near isogenic line (NIL). The contents of four flavonoids (naringenin, quercetin, kaempferol and myricetin) were significantly higher in brown than in white fibers and corresponding to the biosynthetic gene expression levels.

Conclusions: Flavonoid structural gene expression and flavonoid metabolism are important in the development of pigmentation in brown cotton fibers.

Figure 1. Schematic representation of the flavonoid biosynthetic pathway modified from –.

C4H: Cinnamate-4-hydroxylase; 4CL: 4-Coumarate acyl-COA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3′H: flavonoid-3′-hydroxylase; F3′5′H: flavonoid-3′5′-hydroxylase; FLS: flavonol synthase; DFR: dihydroflavonol reductase; ANS: Anthocyanidin synthase; 3GT: 3-O-glucosyltransferase.

Figure 2. Multiple alignment of deduced amino acid sequences of plant chalcone synthase.

Abbreviations of sequences RhCHS: Rosa hybrid cultivar 'Kardinal' (BAC66467); PcCHS: Pyrus communis (AAX16494); VvCHS:Vitis vinifera (BAB84111); AmCHS: Abelmoschus manihot(ACE60221); RsCHS: Rhododendron simsii (CAC88858).

Figure 3. Phylogenetic analyses of chalcone synthase and cytochrome P450 superfamily proteins from various species.

(A) Phylogenetic analyses of chalcone syntheses, (B) Phylogenetic analyses of cytochrome P450 superfamily proteins. VvCHS: Vitis vinifera (ABM67586), IpCHS: Ipomoeapurpurea (BAA20387), RhCHS: Rud-beckia hirta (ABN79673), ZmCHS: Zea mays (CAA42763), OsCHS: Oryza sativa (japonica cultivar-group) (BAA19186), AsCHS: Aquilaria sinensis (ABM73434), AtCHS; Arabidopsis thaliana (CAI30418), ThCHS: Torenia hybrid cultivar (BAB20074), AhCHS: Arachis hypogaea (AAO32821), AmCHS: Abelmoschus manihot (ACE60221); PhCHS: Petunia x hybrida (BAM17286); GhCHS: Gossypium hirsutum (ABS52573); PtC4H: Populus trichocarpa (ACC63873); HlC4H: Humulus lupulus (ACM69364); Ntc4h: Nicotiana tabacum (ABC69412); PhC4H: Petunia x hybrid (ADX33332); VvC4H: Vitis vinifera (XP_002266238); MdC4H: Malus x domestica (AAY87450); PaC3′H: Prunus avium (ADZ54783); VvC3′H: Vitis vinifera (XP_002284151); MdC3′H: Malus x domestica (ACR14867); ArC3′H: Arabidopsis lyrata subsp. Lyrata (XP_002871298); CsC3′H: Camellia sinensis (ACV74415); CiC3′H: Cichorium intybus (ACN65825); PtC3′5′H: Populus trichocarpa (XP_002314004); VvC3′5′H: Vitis vinifera (BAE47007); CsC3′5′H: Camellia sinensis (AAY23287); VwC3′5′H: Viola x wittrockiana (BAF93855); PiC3′5′H: Petunia integrifolia subsp. integrifolia (BAF34563); MtC3′5′H: Medicago truncatula (XP_003638760).

Figure 4. Multiple alignment of deduced amino acid sequences of Cinnamate-4-hydroxylase (C4H), flavanone-3-hydroxylase (F3′H), flavonoid-3'5'-hydroxylase (F3′5′H) from the brown cotton fiber.

Figure 5. QRT-PCR analysis of four flavonoid structural genes in the brown and white cotton.

A: GhC4H1, B: GhCHS, C: GhF3′H, D: GhF3′5′H. In the horizontal axis, 1: roots; 2: hypocotyls; 3: leaves; 4: flowers, 5–14 represent 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 DPA fiber cells, respectively.

Figure 6. The contents of four flavonoids during the development of brown and white cotton fiber.

A: naringenin, B: quercetin, C: myricetin, D: kaempferol. The numbers in horizontal axis represent 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 DPA fiber cells, respectively.