Global Phosphoproteomic Analysis Reveals the Defense and Response Mechanisms of Jatropha Curcas Seedling under Chilling Stress

Abstract

As a promising energy plant for biodiesel, Jatropha curcas is a tropical and subtropical shrub and its growth is affected by one of major abiotic stress, chilling. Therefore, we adopt the phosphoproteomic analysis, physiological measurement and ultrastructure observation to illustrate the responsive mechanism of J. curcas seedling under chilling (4 °C) stress. After chilling for 6 h, 308 significantly changed phosphoproteins were detected. Prolonged the chilling treatment for 24 h, obvious physiological injury can be observed and a total of 332 phosphoproteins were examined to be significantly changed. After recovery (28 °C) for 24 h, 291 phosphoproteins were varied at the phosphorylation level. GO analysis showed that significantly changed phosphoproteins were mainly responsible for cellular protein modification process, transport, cellular component organization and signal transduction at the chilling and recovery periods. On the basis of protein-protein interaction network analysis, phosphorylation of several protein kinases, such as SnRK2, MEKK1, EDR1, CDPK, EIN2, EIN4, PI4K and 14-3-3 were possibly responsible for cross-talk between ABA, Ca²⁺, ethylene and phosphoinositide mediated signaling pathways. We also highlighted the phosphorylation of HOS1, APX and PIP2 might be associated with response to chilling stress in J. curcas seedling. These results will be valuable for further study from the molecular breeding perspective.

Keywords: Jatropha curcas; chilling stress; phosphoproteomics; regulated mechanism; seedling.

Figure 1

Morphological and physiological responses of J. curcas seedling to different treatment condition. Four-leaf stage seedling were treated at 4 °C for 0, 6 and 24 h (C0 h, C6 h and C24 h) and then allowed to recover for 24 h (R24 h) (A). The black bar equals 6 cm. The Pn, Cond, Ci and Trmmol were showed in (B). The values of relative % for each column are means ±S.D. of three biological replicates. The different lowercase letters labeled above columns indicate significant changes according to one-way ANOVA (p < 0.05).

Figure 2

The ultrastructures of leaves at different treatment condition (C0 h, C6 h, C24 h, R24 h) from J. curcas seedling. The bar equals 2 μm. C, chloroplast; M, Mitochondria; S, starch granules; V, vacuole.

Figure 3

Statistical analysis of phosphoproteomics of J. curcas seedling under different treatment. The significantly changed phosphopeptides in each sample (C6 h, C24 h and R24 h) when compared to C0 h (A); Venn diagram of significantly changed phosphorylation sites and phosphoproteins distributed in each sample (C6 h, C24 h and R24 h) when compared to C0 h (B).

Figure 3

Statistical analysis of phosphoproteomics of J. curcas seedling under different treatment. The significantly changed phosphopeptides in each sample (C6 h, C24 h and R24 h) when compared to C0 h (A); Venn diagram of significantly changed phosphorylation sites and phosphoproteins distributed in each sample (C6 h, C24 h and R24 h) when compared to C0 h (B).

Figure 4

Functional classification of the significantly changed phosphoproteins from J. curcas seedling under different treatment by GO analysis. Over-represented GO terms were displayed graphically as pie charts for three GO vocabularies: (A) biological process; (B) cellular component; (C) molecular function. The number in brackets represents the phosphoprotein number within the group and the color of the pie chart represents the significance of enrichment.

Figure 4

Functional classification of the significantly changed phosphoproteins from J. curcas seedling under different treatment by GO analysis. Over-represented GO terms were displayed graphically as pie charts for three GO vocabularies: (A) biological process; (B) cellular component; (C) molecular function. The number in brackets represents the phosphoprotein number within the group and the color of the pie chart represents the significance of enrichment.

Figure 5

Conservation analysis of significantly changed phosphoproteins in J. curcas seedling under different treatment.

Figure 6

Phosphorylation motifs extracted from the phosphopeptides with the significantly changes by Motif-X from J. curcas seedling under different treatment.

Figure 7

Protein-protein interaction (PPI) network of signal transduction, posttranslational modification and intracellular trafficking, transport related phosphoproteins by STRING. Nodes with orange, red and blue background color represent the KOGs of differential phosphoproteins related with signal transduction, posttranslational modification and intracellular trafficking, transport, respectively.

Figure 8

Schematic presentation of systematic chilling response and defense mechanisms in J. curcas seedling at phosphorylation level. The phosphorylation regulatory sites were shown in Table 1. Black solid lines with arrows represent catalytic reaction, while dotted lines with arrows represent material transport. Blue solid lines with arrows represent direct relationship, while magenta lines with arrows represent ubiquination. Black solid lines with stubs represent negative regulation. Green dotted lines with arrows in chloroplast represent transport between PSII and PSI. The proteins labeled with red colors represent significantly changed phosphoproteins among C0 h, C6 h, C24 h and R24 h. The area outlined with dotted square represents ER associated degradation (ERAD).

Figure 9

Sequence alignment and predicted structural composition of TRAB1 in J. curcas. (A) JcTRAB1 (KDP38330.1) sequence alignment with OsTRAB1 (BAA83740.1). The red line with arrow indicated the phosphorylation site of TRAB1 in J. curcas while black line with arrow indicated the phosphorylation site Ser 102 in O. sativa. The pink rectangle showed the conserved region II of TRAB1. (B) Predicted functional domain distribution of JcTRAB1.