Comparative proteomic analysis provides insight into a complex regulatory network of taproot formation in radish (Raphanus sativus L.)

Abstract

The fleshy taproot of radish is an important storage organ determining its yield and quality. Taproot thickening is a complex developmental process in radish. However, the molecular mechanisms governing this process remain unclear at the proteome level. In this study, a comparative proteomic analysis was performed to analyze the proteome changes at three developmental stages of taproot thickening using iTRAQ approach. In total, 1862 differentially expressed proteins (DEPs) were identified from 6342 high-confidence proteins, among which 256 up-regulated proteins displayed overlapped accumulation in S1 (pre-cortex splitting stage) vs. S2 (cortex splitting stage) and S1 vs. S3 (expanding stage) pairs, whereas 122 up-regulated proteins displayed overlapped accumulation in S1 vs. S3 and S2 vs. S3 pairs. Gene Ontology (GO) and pathway enrichment analysis showed that these DEPs were mainly involved in several processes such as "starch and sucrose metabolism", "plant hormone signal transduction", and "biosynthesis of secondary metabolites". A high concordance existed between iTRAQ and RT-qPCR at the mRNA expression levels. Furthermore, association analysis showed that 187, 181, and 96 DEPs were matched with their corresponding differentially expressed genes (DEGs) in S1 vs. S2, S1 vs. S3, and S2 vs. S3 comparison, respectively. Notably, several functional proteins including cell division cycle 5-like protein (CDC5), expansin B1 (EXPB1), and xyloglucan endotransglucosylase/hydrolase protein 24 (XTH24) were responsible for cell division and expansion during radish taproot thickening process. These results could facilitate a better understanding of the molecular mechanism underlying taproot thickening, and provide valuable information for the identification of critical genes/proteins responsible for taproot thickening in root vegetable crops.

Fig. 1. GO and KEGG pathway functional classification of the identified proteins.

a The cell component category of GO classification. b The molecular function category of GO classification. c The biological function category of GO classification. d KEGG pathway functional classification and annotation

Fig. 2. The characteristic of DEPs distribution in three libraries.

a The number of DEPs in any two different stages. b Venn diagrams of DEPs mutual relationship among three libraries. c Venn diagrams of up-regulated proteins mutual relationship among three libraries

Fig. 3. Association results of proteome and transcriptome in three libraries.

a–c Venn diagram of associated DEPs/DEGs. d–f R value of DEPs and DEGs association. g–i R value of the same trends DEPs/DEGs. a, d, g S1 vs. S2 pair. b, e, h S1 vs. S3 pair. c, f, i S2 vs. S3 pair. R is the Pearson correlation coefficient

Fig. 4. Cluster diagram for the concordance between proteome and transcriptome based on pairwise comparison among the three libraries.

a S1 vs. S2 pair. b S1 vs. S3 pair. c S2 vs. S3 pair

Fig. 5. Validation of the mRNA expression levels of proteins from iTRAQ results by RT-qPCR.

a S1 vs. S2 pair. b S1 vs. S3 pair. c S2 vs. S3 pair

Fig. 6. An overview of proteome changes involved in radish taproot thickening at three developmental stages.

The blue font represents overlap proteins between DEPs and DEGs, and the normal font represents unique proteins in this study. MAPK4 mitogen-activated protein kinase 4, CAM putative calmodulin, SRP54 signal recognition particle subunit, PP2C58 probable protein phosphatase 2C 58, SPP1 sucrose-phosphatase 1, PFK3 6-phosphofructokinase 3, PFK5 6-phosphofructokinase 5, bglL beta-glucosidase L, UGD1 UDP-glucose 1-dehydrogenase, SUS1 sucrose synthase 1, PME51 phosphoglucomutase 1, bZIP59 bZIP transcription factor 59, EF1BB elongation factor 1B alpha-subunit 2, PP2C59 protein phosphatase 2C 59, Gns7 glucan endo-1,3-beta-glucosidase 7, MAPK6 mitogen-activated protein kinase 6, BSK8 brassionsteroid-signaling kinase 8, BSL1 serine/threonine-protein phosphatase, EXPL2 expansin-like A2, EXPB1 expansion B1, XTH24/XTH32 xyloglucan endotransglucosylase 24/32, FRUCT5 beta-fructofuranosidase, bglH beta-glucosidase H, PGM1 phosphoglucomutase 1, APL3 glucose-1-phosphate adenylyltransferase, bglI beta-glucosidase I, CDC5 cell division cycle 5-like protein