Transcriptome Analysis of a New Peanut Seed Coat Mutant for the Physiological Regulatory Mechanism Involved in Seed Coat Cracking and Pigmentation

Abstract

Seed-coat cracking and undesirable color of seed coat highly affects external appearance and commercial value of peanuts (Arachis hypogaea L.). With an objective to find genetic solution to the above problems, a peanut mutant with cracking and brown colored seed coat (testa) was identified from an EMS treated mutant population and designated as "peanut seed coat crack and brown color mutant line (pscb)." The seed coat weight of the mutant was almost twice of the wild type, and the germination time was significantly shorter than wild type. Further, the mutant had lower level of lignin, anthocyanin, proanthocyanidin content, and highly increased level of melanin content as compared to wild type. Using RNA-Seq, we examined the seed coat transcriptome in three stages of seed development in the wild type and the pscb mutant. The RNA-Seq analysis revealed presence of highly differentially expressed phenylpropanoid and flavonoid pathway genes in all the three seed development stages, especially at 40 days after flowering (DAF40). Also, the expression of polyphenol oxidases and peroxidase were found to be activated significantly especially in the late seed developmental stage. The genome-wide comparative study of the expression profiles revealed 62 differentially expressed genes common across all the three stages. By analyzing the expression patterns and the sequences of the common differentially expressed genes of the three stages, three candidate genes namely c36498_g1 (CCoAOMT1), c40902_g2 (kinesin), and c33560_g1 (MYB3) were identified responsible for seed-coat cracking and brown color phenotype. Therefore, this study not only provided candidate genes but also provided greater insights and molecular genetic control of peanut seed-coat cracking and color variation. The information generated in this study will facilitate further identification of causal gene and diagnostic markers for breeding improved peanut varieties with smooth and desirable seed coat color.

Keywords: RNA-seq; flavonoid pathway; peanut (Arachis hypogaea); pigmentation; seed-coat cracking.

Figure 1

Phenotypic characterization of peanut seed coat development in pscb mutant and WT. (A) Phenotypic characterization of peanut seed coat development in pscb mutant and WT. (B) Fresh and dry weight of matured seed coat of pscb mutant and WT. ^***Means significant differences.

Figure 2

Water uptake efficiency and germination in pscb mutant and WT. (A) Water uptake efficiency of pscb mutant and WT. (B) Germination time of pscb mutant and WT. (C) The radicals elongated faster in WT when compared with pscb mutant.

Figure 3

The pscb mutant has decreased lignin, anthocyanin, and Proanthocyanidin content. (A) Lignin content of matured seed coat in pscb mutant and WT. (B) Anthocyanin levels from pscb mutant and WT matured seed coat. (C) Soluble PA levels of pscb mutant and WT seed coat. (D) In-soluble PA content from pscb mutant and WT matured seed coat. FW, Fresh weight. (E,F) Changes of proanthocyanidins and phenolic compounds during seed coat development of pscb mutant and WT. (e1–e5) Detection and localization of proanthocyanidin and phenolic compounds in seed coat development in pscb mutant. Bars = 500 μm. (f1–f5) Detection and localization of proanthocyanidin and phenolic compounds in seed coat development in WT. Bars = 500 μm. Black arrows, accumulation site for polymeric phenolic compounds. ^***Means significant differences.

Figure 4

Number of DEGs between pscb mutant and WT at DAF20, DAF40, and DAF60.

Figure 5

qRT-PCR validation of differential expression. (A) Transcript levels of 17 genes, which were involved in plant cell wall organization (B) Comparison between the gene expression ratios obtained from RNA-seq data and qRT-PCR. The RNA-seq log₂ value of the expression ratio (y-axis) has been plotted against the developmental stages (x-axis).

Figure 6

The pscb mutant testa accumulates phytomelanin through higher level of peroxidase and polyphenol oxidase expression. (A) Phytomelanin contents in pscb mutant and WT matured seed coat. The phytomelanin content was expressed as the mg g-1 fresh weight of seed coat. (B) Heatmaps represent the expression level of nine peroxidases in pscb mutant and WT seed coat of DAF20, DAF40, and DAF60. (C) Heatmaps represent the expression level of 24 polyphenol oxidase in pscb mutant and WT seed coat of DAF20, DAF40, and DAF60.The gene expression was scaled using Z-score of FPKM in the heatmap.

Figure 7

Phenylalanine metabolism was down-regulated in pscb mutant seed coat. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; C3H, 4-coumarate 3-hydroxylase; COMT, caffeic acid o-methyltransferase; CCoAOMT, caffeoyl-CoA o-methyltransferase; F5H, ferulate-5-hydroxylase. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; UGTs, UDP sugar glycosyltransferases; DFR, dihydroflavonol reductase.

Figure 8

Summary of some biological pathways involved in peanut seed coat pigmentation and crack formation. Red boxes indicate genes/proteins that were upregulated in pscb mutant compared with WT; green boxes indicate genes/proteins that were downregulated in pscb mutant compared with WT, 1-aminocyclopropane-1-carboxylate oxidase; ETR, ethylene receptor; EBF, EIN3-binding F-box protein; ERF1, ethylene-responsive transcription factor; PYR/PYL, abscisic acid receptor; PP2C, protein phosphatase 2C; SnRK2, serine/threonine-protein kinase SRK2; POD, peroxidase; PPOD, polyphenol oxidase; IR, interaction.