Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.)

Abstract

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Keywords: floral buds; iTRAQ; pistil and stamen development; proteomics; walnut (Juglans regia L.).

Figure 1

The number of up-regulated and down-regulated DEPs identified in each contrast.

Figure 2

The distribution of DEPs in S2/S1, S3/S2, S4/S2, and S4/S3. A Venn diagram illustrating the specific and common proteins between the different contrasts.

Figure 3

The most significant enriched GO terms of the differentially expressed proteins at each contrast. (A), S2/S1; (B), S3/S1; (C), S4/S2; (D), S4/S3. Note: The full name with ellipsis in (A) are generation of precursor metabolites and energy, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor and oxidoreductase activity, acting on the CH-CH group of donors respectively. The full name with ellipsis in (B) are generation of precursor metabolites and energy, oxidoreductase activity, acting on the CH-CH group of donors, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor and protein binding involved in protein folding respectively. The full name with ellipsis in (C) are generation of precursor metabolites and energy, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor, oxidoreductase activity, acting on the CH-CH group of donors and peptidase activity, acting on L-amino acid peptides respectively. The full name with ellipsis in (D) is protein binding involved in protein folding.

Figure 4

The most significant associated KEGG pathways of differentially expressed proteins at each contrast. (A), S2/S1; (B), S3/S1; (C), S4/S2; (D), S4/S3.

Figure 5

The KEGG classification of the identified DEPs. p values below the 0.05 threshold. (A), S2/S1; (B), S3/S1; (C), S4/S2; (D), S4/S3. Note: The full names with ellipses in (A,C) are both Amino sugar and nucleotide sugar metabolism.

Figure 6

The string networks of the DEPs of XP_018841899.1.

Figure 7

The string networks of the DEPs of XP_018830780.1.

Figure 8

Photographs of the pistils and stamens of the walnut plant cultivar ‘Zhuxing’. Unopened pistils (S1); opened pistils (S2); unopened stamens (S3); opened stamens (S4).