Genome-wide identification of multifunctional laccase gene family in cotton (Gossypium spp.); expression and biochemical analysis during fiber development

Abstract

The single-celled cotton fibers, produced from seed coat epidermal cells are the largest natural source of textile fibers. The economic value of cotton fiber lies in its length and quality. The multifunctional laccase enzymes play important roles in cell elongation, lignification and pigmentation in plants and could play crucial role in cotton fiber quality. Genome-wide analysis of cultivated allotetraploid (G. hirsutum) and its progenitor diploid (G. arboreum and G. raimondii) cotton species identified 84, 44 and 46 laccase genes, respectively. Analysis of chromosomal location, phylogeny, conserved domain and physical properties showed highly conserved nature of laccases across three cotton species. Gene expression, enzymatic activity and biochemical analysis of developing cotton fibers was performed using G. arboreum species. Of the total 44, 40 laccases showed expression during different stages of fiber development. The higher enzymatic activity of laccases correlated with higher lignin content at 25 DPA (Days Post Anthesis). Further, analysis of cotton fiber phenolic compounds showed an overall decrease at 25 DPA indicating possible incorporation of these substrates into lignin polymer during secondary cell wall biosynthesis. Overall data indicate significant roles of laccases in cotton fiber development, and presents an excellent opportunity for manipulation of fiber development and quality.

Figure 1. Phylogenetic, conserved domain and gene architecture analysis of G. arboreum laccases.

(A) The phylogenetic tree of 44 G. arboreum laccase proteins. (B) Characteristic conserved domains present in the laccase proteins [CuRO_1_LCC_plant (cd13849), CuRO_2_LCC_plant (cd13875) and CuRO_3_LCC_plant (13897)] (C) Exon-intron architecture of G. arboreum laccase genes. Green boxes represent exons whereas introns are represented with black lines. Big introns were compressed in order to fit within space where one double slash (\\) represents reduction of 1 Kb.

Figure 2. Phylogenetic analysis of cotton laccase gene family.

Phylogenetic tree was constructed using 240 protein sequences from Gossypium arboreum (44), G. raimondii (46), G. hirsutum A-subgenome (42), G. hirsutum D-subgenome (42), Arabidopsis thaliana (17) and Populus trichocarpa (49). MEGA v6.2. Neighbor-joining method was used with boot strap replication of 1000 times to create the phylogenetic tree. Arabidopsis laccases are highlighted with red colored text.

Figure 3. Chromosomal localization and synteny mapping of cotton laccase gene family.

(A) G. arboreum (A-genome) (B) G. raimondii (D-genome) (C) G. hirsutum A-subgenome (D) G. hirsutum D-subgenome. Colored lines represent the syntenic relationships of laccase genes within genome. Chromosomes containing laccase gene(s) were represented in the figure.

Figure 4. Relative mRNA expression of G. arboreum laccase genes during fiber development.

(A) A total of 11 out of 40 laccases are expressed during active phase of fiber elongation (5 DPA). (B) A total of 15 out of 40 laccases are expressed during transition/onset of SCW development of fiber (10 and 15 DPA). (C) A total of 14 out of 40 laccases are expressed during active phase of SCW development of fiber (20 and 25 DPA). GaHistone-3b was used as internal control to normalize the expression data. Error bars represent difference in expression pattern between technical replicates.

Figure 5. Laccase enzyme activity assay in developing G. arboreum fibers.

Left panel: Laccase enzyme activity was assayed using 1-D Zymography. 50 μg of total proteins from 10, 15, 20 and 25 DPA fibers along with control (stem), were run on non-denaturing SDS-PAGE gel, incubated in Sodium tartarate buffer and later in ABTS solution to visualize laccase activity. Right panel: Loading control gel ran with same amounts of total proteins and stained with Coomassie Brilliant Blue. M: represents protein marker.

Figure 6. Lignin content of different developmental stages of G. arboreum fiber.

Lignin was extracted from different stages of cotton fiber development (10, 15, 20 and 25 DPA) and estimated using TGA method (UV absorbance at 280 nm). Industrial lignin was used for standard curve preparation to calculate lignin content in cotton fibers. Asterisk indicates significant difference in lignin quantity (t-test; p < 0.05).

Figure 7. Soluble and wall bound phenolic acid content during G. arboreum fiber development.

(A) Soluble phenolic acid content was represented as μg/gram of fresh fiber weight. (B) Wall bound phenolic acid content was represented as μg/gram of cell wall weight. Soluble and wall bound phenolic acids were extracted from different developmental stages (10, 15, 20 and 25 DPA) and analyzed using HPLC with C18 column. Mobile phases composed of 33% A (100% acetonitrile) and 67% B (ultra-pure water pH: 2.1) at a flow rate of 1.5 ml/minute. Standards for ferulic acid, p-coumaric acid, caffeic acid, sinapic acid and vanillic acid were used at 1 mg/ml concentration. 1-Napthalene acetic acid (1 mg/ml) was used as an internal control to calculate the phenolic acid content of cotton fibers. Asterisk indicates significant difference in phenolic acid levels (t-test; p < 0.05).

Figure 8. Potential roles of laccases in cotton fiber development.

Phenolic compounds are found to have important roles in auxin regulation, cell elongation, lignin biosynthesis, cellular signaling and pigmentation in various plant species. The roles of laccases in cotton fiber development have not been characterized however the numbers and expression levels of laccases at different stages of fiber development indicate their potential roles in cotton fiber development and quality.