Co-Suppression of NbClpC1 and NbClpC2, Encoding Clp Protease Chaperons, Elicits Significant Changes in the Metabolic Profile of Nicotiana benthamiana

Abstract

Metabolites in plants are the products of cellular metabolic processes, and their differential amount can be regarded as the final responses of plants to genetic, epigenetic, or environmental stresses. The Clp protease complex, composed of the chaperonic parts and degradation proteases, is the major degradation system for proteins in plastids. ClpC1 and ClpC2 are the two chaperonic proteins for the Clp protease complex and share more than 90% nucleotide and amino acid sequence similarities. In this study, we employed virus-induced gene silencing to simultaneously suppress the expression of ClpC1 and ClpC2 in Nicotiana benthamiana (NbClpC1/C2). The co-suppression of NbClpC1/C2 in N. benthamiana resulted in aberrant development, with severely chlorotic leaves and stunted growth. A comparison of the control and NbClpC1/C2 co-suppressed N. benthamiana metabolomes revealed a total of 152 metabolites identified by capillary electrophoresis time-of-flight mass spectrometry. The co-suppression of NbClpC1/C2 significantly altered the levels of metabolites in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, and the purine biosynthetic pathway, as well as polyamine and antioxidant metabolites. Our results show that the simultaneous suppression of ClpC1 and ClpC2 leads to aberrant morphological changes in chloroplasts and that these changes are related to changes in the contents of major metabolites acting in cellular metabolism and biosynthetic pathways.

Keywords: Clp protease; ClpC1; ClpC2; co-suppression; metabolites; virus-induced gene silencing.

Figure 1

Heat map of the results of the hierarchical cluster analysis of metabolites in the leaves of NbClpC1/C2 co-suppressed Nicotiana benthamiana. The horizontal and vertical axes show sample names and peaks, respectively. The distances between samples and between peaks are displayed in three diagrams. NBC-1, 2, 3: Control 1, 2, and 3; NBT-1, 2, 3: NbClpC1/C2 co-suppressed N. benthamiana 1, 2 and 3.

Figure 2

Comparison of glycolysis metabolite contents between control and NbClpC1/C2 co-suppressed Nicotiana benthamiana plants. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6P, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 3PG, 3-phosphoglyceric acid; 2PG, 2-phosphoglyceric acid; PEP, phosphoenolpyruvate. The error bars indicate the standard deviation (SD) of triplicate samples. The different letters on the bars indicate statistically significant differences between groups (p < 0.05).

Figure 3

Changes in TCA cycle-related metabolite contents with co-suppression of NbClpC1/C2. ClpC: NbClpC1/C2 co-suppressed N. benthamiana. The error bars indicate the SD of triplicate samples. The different letters on the bars indicate statistically significant differences between groups (p < 0.05).

Figure 4

Comparison of pentose phosphate pathway-related (oxidative and nonoxidative) metabolite contents in Nicotiana benthamiana plants with co-suppression of NbClpC1/C2. ClpC: NbClpC1/C2 co-suppressed plants. The error bars indicate the SD of triplicate samples. The different letters on the bars indicate statistically significant differences between groups (p < 0.05).

Figure 5

Changes in polyamine and antioxidant metabolite contents with co-suppression of NbClpC1/C2 in Nicotiana benthamiana plants. ClpC: NbClpC1/C2 co-suppressed plants. SAM, S-adenosyl methionine; GSH, reduced glutathione; GSSG, oxidised glutathione. The error bars indicate the SD of triplicate samples. The different letters on the bars indicate statistically significant differences between groups (p < 0.05).

Figure 6

Changes in purine nucleotide biosynthetic pathway-related metabolite contents in Nicotiana benthamiana plants with co-suppression of NbClpC1/C2. ClpC: NbClpC1/C2 co-suppressed plants. The error bars indicate the SD of triplicate samples. The different letters on the bars indicate statistically significant differences between groups (p < 0.05).