Genome-Wide Identification, Expression, and Protein Analysis of CKX and IPT Gene Families in Radish (Raphanus sativus L.) Reveal Their Involvement in Clubroot Resistance

Abstract

Cytokinins (CKs) are a group of phytohormones that are involved in plant growth, development, and disease resistance. The isopentenyl transferase (IPT) and cytokinin oxidase/dehydrogenase (CKX) families comprise key enzymes controlling CK biosynthesis and degradation. However, an integrated analysis of these two gene families in radish has not yet been explored. In this study, 13 RsIPT and 12 RsCKX genes were identified and characterized, most of which had four copies in Brassica napus and two copies in radish and other diploid Brassica species. Promoter analysis indicated that the genes contained at least one phytohormone or defense and stress responsiveness cis-acting element. RsIPTs and RsCKXs were expanded through segmental duplication. Moreover, strong purifying selection drove the evolution of the two gene families. The expression of the RsIPT and RsCKX genes distinctly showed diversity in different tissues and developmental stages of the root. Expression profiling showed that RsCKX1-1/1-2/1-3 was significantly upregulated in club-resistant materials during primary infection, suggesting their vital function in clubroot resistance. The interaction network of CKX proteins with similar 3D structures also reflected the important role of RsCKX genes in disease resistance. This study provides a foundation for further functional study on the IPT and CKX genes for clubroot resistance improvement in Raphanus.

Keywords: 3D structure; Raphanus sativus; RsCKX; RsIPT; clubroot resistance; expression profile; interaction network.

Figure 1

Chromosomal mapping of the RsIPT and RsCKX genes in radish. The green bars represent chromosomes. Values corresponding to the RsIPT and RsCKX genes represent the physical distance (Mb).

Figure 2

Phylogenetic tree of IPT genes from Arabidopsis thaliana, Brassica rapa, B. oleracea, B. nigra, B. napus, and Raphanus sativus. IPT genes are divided into four groups according to phylogenetic analysis. Different groups are indicated with corresponding colors. The gene RsIPT10 which not belong to any group was presented by gray color. The IPT genes of R. sativus are highlighted by black stars, and the IPT genes of A. thaliana are highlighted by black stars and squares. The A. thaliana, B. rapa, B. oleracea, B. nigra, B. napus, and R. sativus genes are marked with “At”, “Bra”, “Bol”, “Bni”, “Bna”, and “Rs”, respectively.

Figure 3

Phylogenetic tree of CKX genes from Arabidopsis thaliana, Brassica rapa, B. oleracea, B. nigra, B. napus, and Raphanus sativus. CKX genes are divided into four groups according to phylogenetic analysis. Different groups are indicated with corresponding colors. The IPT genes of R. sativus are highlighted by black stars, and the IPT genes of A. thaliana are highlighted by black stars and squares. The A. thaliana, B. rapa, B. oleracea, B. nigra, B. napus, and R. sativus genes are marked with “At”, “Bra”, “Bol”, “Bni”, “Bna”, and “Rs”, respectively.

Figure 4

Phylogenetic relationship (a), gene structure (b), conserved motifs (c), and cis-acting elements in promoters (d) of IPT genes in radish. The yellow rectangles and black lines represent exons and introns of genes, respectively, and their lengths are presented proportionally. The 10 conserved motifs and 21 cis-acting elements are indicated in different colors.

Figure 5

Phylogenetic relationship (a), gene structure (b), conserved motifs (c), and cis-acting elements in promoters (d) of CKX genes in radish. The green rectangles and black lines represent exons and introns of genes, respectively, and their lengths are presented proportionally. The 10 conserved motifs and 25 cis-acting elements are indicated in different colors.

Figure 6

Collinearity analysis of RsIPT genes among radish chromosomes. Different chromosomes are indicated with corresponding number and colors in different regions of circle. The genes connected by the red line are segmental duplication pairs.

Figure 7

Collinearity analysis of RsCKX genes among radish chromosomes. Different chromosomes are indicated with corresponding number and colors in different regions of circle. The genes connected by the red line are segmental duplication pairs.

Figure 8

Expression profiles of (a) RsIPT and (b) RsCKX genes in different tissues and root growth stages in XYB36-2 and Xinlimei radish. ESS: seedling stage; SS: splitting stage; EES: early expanding stage; RES: rapid expanding stage; MS: mature stage. Color scale indicates the relative expression of genes by log₂ transformation compared with the controls.

Figure 9

Expression profiles of (a) RsIPT and (b) RsCKX genes during different infection periods after inoculation with Plasmodiophora brassicae in susceptible (P1, YS-472) and resistant varieties (P2, YR-456). P1d7: susceptible variety 7 days after inoculation/corresponding control with no infection; P2d7: resistant variety 7 days after inoculation/corresponding control with no infection; P1d34: susceptible variety 34 days after inoculation/corresponding control with no infection; P2d34: resistant variety 34 days after inoculation/corresponding control with no infection. Color scale indicates the relative expression of genes by log₂ transformation compared with the controls.

Figure 10

Expression levels of selected RsIPT and RsCKX genes in eight treatments of susceptible and resistant materials under Plasmodiophora brassicae stress by qRT-PCR analysis. P1d7: susceptible variety 7 days after inoculation; P1d7CK: susceptible variety 7 days with no infection; P2d7: resistant variety 7 days after inoculation; P2d7CK: resistant variety after 7 days with no infection; P1d34: susceptible variety 34 days after inoculation; P1d34CK: susceptible variety after 34 days with no infection; P2d34: resistant variety 34 days after inoculation; P2d34CK: resistant variety after 34 days with no infection. Different lowercase letters indicate significant differences (p < 0.05).

Figure 11

3D structure of 12 RsCKX proteins. The secondary structure in green represents an alpha helix, purple represents an extended strand, and pink represents a random coil.

Figure 12

Protein-protein interaction networks of RsCKX proteins with other proteins predicted using STRING. The red circles represent RsCKXs, and the inner circles represent proteins interacting with RsCKXs. The different colors indicate that the proteins belong to different families. Different colors including brown, lime, aqua, turquoise, purple, blue, yellow, pink, spring green, orange, and light green represent cytokinin synthase, beta-glucosidase, wuschel related homeobox 1, peptidoglycan-binding LysM domain-containing protein, F-box/LRR-repeat protein 15, ATP synthase, Polyprenyltransferase 1, cytokinin riboside 5′-monophosphate phosphoribohydrolase, histidine-containing phosphotransfer protein, histone acetyltransferase, and cytokinin receptors. The gray lines indicate interactions.