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.
Highlights
Plant proteases control a broad range of functions, including differentiation, development, cell death via gene expression, protein targeting, protein sorting, protein folding, protein quality control, and protein degradation [1]
With co-suppression of NbClpC1/C2, the levels of most intermediates of the tricarboxylic acid (TCA) cycle were significantly increased at the beginning of the cycle, and decreased gradually compared to the control (Figure 3)
The high levels of citrate in NbClpC1/C2 co-suppressed plants indicate that it is not being consumed by the TCA cycle and glucose breakdown might be slowed due to the regulatory effect of citrate on PFK-1
Summary
Plant proteases control a broad range of functions, including differentiation, development, cell death via gene expression, protein targeting, protein sorting, protein folding, protein quality control, and protein degradation [1]. In addition to a role in Clp proteolytic activity, ClpC proteins import cytosolic preproteins into the chloroplast in association with the integral membrane proteins Tic110, Tic, Tic, and the intermembrane space protein Tic, as well as other stromal chaperones (cpHsp, Hsp90C) [7,23,24,25,26]. Several studies have reported the functional roles of ClpC1 and ClpC2 proteins [7,23,24,25,26], but the chaperonic roles of the Clp protease complex in relation to cellular metabolism remain unknown. Employing virus-induced gene silencing (VIGS), we simultaneously suppressed NbClpC1/C2 in N. benthamiana plants using only one silencing vector, and subsequently identified their roles in metabolite pool changes
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