Genome-wide survey indicates diverse physiological roles of the turnip (Brassica rapa var. rapa) calcium-dependent protein kinase genes

Abstract

Calcium-dependent protein kinases (CDPKs) as crucial sensors of calcium concentration changes play important roles in responding to abiotic and biotic stresses. In this study, 55 BrrCDPK genes, which were phylogenetically clustered into four subfamilies, were identified. Chromosome locations indicated that the CDPK family in turnip expanded by segmental duplication and genome rearrangement. Moreover, gene expression profiles showed that different BrrCDPKs were expressed in specific tissues or stages. Transcript levels of BrrCDPKs indicated that they were involved in abiotic and biotic stresses and that paralogs exhibited functional divergence. Additionally, we identified 15 Rboh genes in turnip; the results of yeast two-hybrid analysis suggested that BrrRbohD1 interacted only with BrrCDPK10 and that BrrRbohD2 interacted with BrrCDPK4/7/9/10/17/22/23. Most of the genes play an important role in pst DC3000 defense by regulating the accumulation of H₂O₂ and stomatal closure. Our study may provide an important foundation for future functional analysis of BrrCDPKs and reveal further biological roles.

Figure 1

Phylogenetic relationship and gene structure of turnip CDPKs. A neighbor-joining tree was created for 55 turnip CDPK proteins using the MEGA7.0 program with 1000 bootstrap replicates. Four subfamilies were labeled Groups 1–4 with vertical bars in specific colors. Exons and introns are represented by yellow boxes and black lines, respectively.

Figure 2

BrrCDPKs’ chromosome distributions, synteny blocks, and the turnip genome duplication event caused paralogous relationships. Chromosomes are shown in different colors and in the outer circle, where the numbers represent the chromosome length in 100 Kb. The BrrCDPK genes are marked at their approximate positions with specific colored lines on the circle. Filled blocks in different colors denote the syntenic relationships of turnip CDPK genes.

Figure 3

Heat maps showing the expression profiles of turnip CDPK genes across different tissues and developmental stages of tuberous roots. Quantitative RT-PCR was used to assess BrrCDPK transcript levels in total RNA samples extracted from mature plants, root, stem, leaf, and flower tissue. The developmental stages were based on transcriptional data generated by Jingjuan Li. Samples were collected on day 18 (the early stage before cortex splitting, ES), day 28 (the cortex splitting stage, CSS), and day 64 (the stage of root thickening, RTS) after sowing. The relative expression was log transformed and visualized as heat maps.

Figure 4

Differential expression of turnip CDPK genes under different stresses. Quantitative RT-PCR analyses were performed and expression values were calculated using the 2^−△△CT method. Data are mean values ± standard error obtained from three replicates. Red indicates upregulated genes and green downregulated genes. Asterisks denote statistically significant differences (t-test, p < 0.05).

Figure 5

Interaction of BrrCDPKs with BrrRboh proteins. (A) Phylogenetic relationship of turnip Rbohs. A neighbor-joining tree was created for 15 turnip Rboh proteins using the MEGA7.0 program with 1000 bootstrap replicates. (B) Yeast two-hybrid analysis of interactions between CDPK and Rboh proteins in turnip. The yeast cells of strain AH109 containing the indicated plasmid combinations were grown on either the nonselective (SD-LW) or selective (SD-LWHA) media. AD is the empty pGADT7 vector. (C) Expression profiles of BrrRbohD1 and BrrRbohD2 under ACC, JA, SA, and pst DC3000 stress. Quantitative RT-PCR analyses were performed and expression values were calculated using the 2^−△△CT method. Data are mean values ± standard error obtained from three replicates. Red indicates upregulated genes and green indicates downregulated genes. Asterisks denote statistically significant differences (t-test, p < 0.05).

Figure 6

H₂O₂ in guard cells and stomatal closure. (A) H₂O₂ content in leaves of turnip treated with pst DC3000 for 0, 1, and 3 h. Data are mean values ± standard error (SE) obtained from three replicates. Different letters within a column indicate a significant difference (p < 0.05; Tukey’s test). (B) DC3000-induced production of H₂O₂ by turnip leaf guard cells. Epidermal pieces of turnip leaves without (1 and 4) or with 1 h (2 and 5) or 3 h (3 and 6) of pst DC3000 treatment were loaded with 50 µmol/L H2DCF-DA for 10 min. Photographs were taken using a laser-scanning confocal microscope. Pictures 1–3 are fluorescent images, 4–6 are overlapped images. (C) Changes in stomatal aperture of turnip leaves treated with pst DC3000 for 0, 1, and 3 h. Stomatal aperture size (mean values ± SE, n = 90 stomata from three leaves) of leaves treated with pst DC3000 for 0, 1, and 3 h. Different letters within a column indicate a significant difference (p < 0.05; Tukey’s test).