A comparative proteomic analysis of Pinellia ternata leaves exposed to heat stress

Abstract

Pinellia ternata is an important traditional Chinese medicinal plant. The growth of P. ternata is sensitive to high temperatures. To gain a better understanding of heat stress responses in P. ternata, we performed a comparative proteomic analysis. P. ternata seedlings were subjected to a temperature of 38 °C and samples were collected 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress was frequently generated in rice leaves exposed to high temperature. Two-dimensional electrophoresis (2-DE) was used to analyze heat-responsive proteins. More than 600 protein spots were reproducibly detected on each gel; of these spots, 20 were up-regulated, and 7 were down-regulated. A total of 24 proteins and protein species were successfully identified by MALDI-TOF/TOF MS. These proteins and protein species were found to be primarily small heat shock proteins (58%) as well as proteins involved in RNA processing (17%), photosynthesis (13%), chlorophyll biosynthetic processes (4%), protein degradation (4%) and defense (4%). Using 2-DE Western blot analysis, we confirmed the identities of the cytosolic class II small heat shock protein (sHSPs-CII) identified by MS. The expression levels of four different proteins [cytosolic class I small heat shock protein (sHSPs-CI), sHSPs-CII, mitochondrial small heat shock protein (sHSPs-MIT), glycine-rich RNA-binding protein (GRP)] were analyzed at the transcriptional level by quantitative real-time PCR. The mRNA levels of three sHSPs correlated with the corresponding protein levels. However, GRP was down-regulated at the beginning of heat stress but then increased substantially to reach a peak after 24 h of heat stress. Our study provides valuable new insight into the responses of P. ternata to heat stress.

Figure 1

Physiological responses of P. ternata leaf under heat stress. (A) Relative electrolyte leakage; (B) Malondialdehyde (MDA) content of P. ternata leaves after heat treatments. Each point represents the average of three individual identical experiments (±SD). Letters above the bars (a, b, c, d, ab and bc) indicate a statistically significant difference (p < 0.05) according to Duncan’s multiple range test.

Figure 2

Representative 2-DE gels of P. ternata leaf proteins. 2-DE was performed using 1200 μg of total protein and 24 cm immobiline dry strips with linear pH gradients from pH 4–7. SDS-PAGE was performed with 12.5% gels. The gels were stained with CBB R-250. (CK):2-DE gel of the control sample. The down-regulated spots are numbered; (H24): 2-DE gel of sample treated at 38 °C for 24 h. The up-regulated spots are numbered. The framed regions a, b, c, d, e and f are enlarged in Figure 2.

Figure 3

Magnified regions of differentially expressed proteins in P. ternata leaves.

Figure 3

Magnified regions of differentially expressed proteins in P. ternata leaves.

Figure 4

Functional groups of differentially expressed proteins identified in the control and heat-treated P. ternata leaves. This classification is based on their homologs and the literature. A total of six functional categories and their percentages are shown.

Figure 5

Relative gene expression levels of selected proteins in P. ternata leaves exposed to heat stress for 0, 0.5, 2, 8, 12, 24 and 72 h. The GAPDH gene was used as an internal control. The gene expression levels relative to those in the unstressed state are represented by relative quantification (RQ) values (fold changes).

Figure 6

Protein level changes for sHSPs-CI and sHSPs-CII in P. ternata leaves exposed to heat stress. (A) 2-DE Western blot analysis of sHSPs-CII proteins in P. ternata leaves. The total proteins extracted from the unstressed (CK) or stressed samples at 24 h (H24) were separated by 2-DE. The portion of the gel corresponding to 12–18 kDa and pI 5.2–5.8 was excised; (B) Accumulation of sHSP-CI and sHSPs-CII proteins in the leaves of P. ternata induced by exposure to 38 °C; and (C) Accumulation of sHSP-CI and sHSP-CII proteins in the leaves of P. ternata maintained at 25 °C. Equal amounts (15 mg) of total protein were loaded onto sodium dodecylsulfate-polyacrylamide gel electrophoresis gels. The levels of sHSPs-CI, sHSPs-CII and actin proteins were estimated by immunoblotting, as described in the Materials and Methods Section.