The Comparatively Proteomic Analysis in Response to Cold Stress in Cassava Plantlets

Abstract

Cassava (Manihot esculenta Crantz) is a tropical root crop and sensitive to low temperature. However, it is poorly to know how cassava can modify its metabolism and growth to adapt to cold stress. An investigation aimed at a better understanding of cold-tolerant mechanism of cassava plantlets was carried out with the approaches of physiology and proteomics in the present study. The principal component analysis of seven physiological characteristics showed that electrolyte leakage (EL), chlorophyll content, and malondialdehyde (MDA) may be the most important physiological indexes for determining cold-resistant abilities of cassava. The genome-wide proteomic analysis showed that 20 differential proteins had the same patterns in the apical expanded leaves of cassava SC8 and Col1046. They were mainly related to photosynthesis, carbon metabolism and energy metabolism, defense, protein synthesis, amino acid metabolism, signal transduction, structure, detoxifying and antioxidant, chaperones, and DNA-binding proteins, in which 40 % were related with photosynthesis. The remarkable variation in photosynthetic activity and expression level of peroxiredoxin is closely linked with expression levels of proteomic profiles. Moreover, analysis of differentially expressed proteins under cold stress is an important step toward further elucidation of mechanisms of cold stress resistance.

Keywords: Cassava plantlets; Cold stress; Comparative proteome; Differential proteins.

Fig. 1

Phenotypic changes in cold-stressed cassava and recovery after cold stress. a1–5, 40-day-old SC8 subjected to 5 °C for 0, 3, 7, 10, and 15 days in a chamber under light showing phenotypic changes, respectively; b1–5, 40-day-old Col1046 subjected to 5 °C for 0, 3, 7, 10, and 15 days in a chamber under light showing phenotypic changes, respectively. c Cassava SC8 plantlets exposed to 5 °C for 15 days were moved to room temperature for 2 months. d Cassava Col1046 plantlets exposed to 5 °C for 15 days were moved to room temperature for 2 months

Fig. 2

Contents of chlorophyll, EL, and MDA in cold-stressed cassava apical expanded leaves. Chlorophyll a contents (a), chlorophyll b contents (b), electrolyte leakage (c), and MDA (d) in SC8 and Col1046 apical leaves exposed to 5 °C for 0, 3, 7, 10, and 15 days. The mean values are calculated from three biological replicates; the error bars represent the standard error of the mean

Fig. 3

Content proline and soluble sugar and activities of SOD and POD in cold-stressed cassava apical expanded leaves. Proline contents (a), soluble sugar contents (b), SOD activities (c), and POD activities (d) in SC8 and Col1046 apical leaves exposed to 5 °C for 0, 3, 7, 10, and 15 days. The mean values are calculated from three biological replicates; the error bars represent the standard error of the mean

Fig. 4

Coomassie-stained 2-D gel protein profiles of SC8 apical expanded leaves: a 5 °C for 0 day, b 5 °C for 10 days, and c wrapped 2-DE map from 5 °C for 0 day and 5 °C for 10 days. The white and black arrows in c indicate proteins that showed detectable changes (>2.0-fold the normalized volume) in abundance compared with those seen in 5 °C for 0 day; white arrows indicate a down-regulated match, and black arrows indicate an up-regulated match

Fig. 5

Coomassie-stained 2-D gel protein profiles of Col1046 apical expanded leaves: a 5 °C for 0 day, b 5 °C for 10 days, and c wrapped 2-DE map from 5 °C for 0 day and 5 °C for 10 days. The white and black arrows in c indicate proteins that showed detectable changes (>2.0-fold the normalized volume) in abundance compared with those seen in 5 °C for 0 day; white arrows indicate a down-regulated match, and black arrows indicate an up-regulated match

Fig. 6

Functional categories of differential proteins. a Functional categories of differential proteins in SC8 apical expanded leaves exposed to 5 °C for 10 days compared with 5 °C for 0 day. b Functional categories of differential proteins in Col1046 apical expanded leaves exposed to 5 °C for 10 days compared with 5 °C for 0 day. Functional categorization was performed according to MIPS database (http://mips.gsf.de)

Fig. 7

Functional categories of the common differential proteins identified in both SC8 and Col1046 apical expanded leaves exposed to 5 °C for 10 days compared with 5 °C for 0 day. a Number of spots altered in expression of SC8 and Col1046. b Functional categories of 20 common differential proteins in SC8 and Col1046

Fig. 8

Western blotting of Rubisco and peroxiredoxin. The expressions of Rubisco (a) and peroxiredoxin (b) in apical expanded leaves of SC8and Col1046 were detected by Western blotting using anti-Rubisco polyclonal antibody (AS07218) and anti-peroxiredoxin antibody (AS05093) from Agrisera, respectively. Mr, protein marker; lines a and b, the expression of Rubisco and peroxiredoxin of SC8 in 5 °C for 0 and 10 days, respectively; lines c and d, the expression of Rubisco and peroxiredoxin of Co11046 in 5 °C for 0 and 10 days, respectively

Fig. 9

Imaging pulse amplitude modulation of SC8 and Col1046 collected from apical expanded leaves exposed to 5 °C for 0 and 10 days. Parameters were Fv/Fm (maximal photosystem II (PSII) quantum yield), ΦPSII (effective PSII quantum yield), and NPQ/4 (non photochemical quenching). The color gradient provided a scale from 0 to 100 % for assessing the magnitude of the parameters

Fig. 10

Biological networks generated for combination of 11 differential proteins. Differential proteins including ATP synthase subunit beta, RCA, Rubisco, phosphoglycerate kinase, APX, CDSP32, peroxiredoxin, chaperone, heat shock protein, glutathione transferase, and 14-3-3 were used to generate a protein-protein interaction network through Pathway Studio analysis. Regulation is marked as an arrow with R, chemical reaction as an arrow with C, and binding as an arrow without any marks. Blue arrows indicate down-regulated expression, and read indicates up-regulated expression (Color figure online)