Developmentally linked changes in proteases and protease inhibitors suggest a role for potato multicystatin in regulating protein content of potato tubers

Abstract

The soluble protein fraction of fully developed potato (Solanum tuberosum L.) tubers is dominated by patatin, a 40 kD storage glycoprotein, and protease inhibitors. Potato multicystatin (PMC) is a multidomain Cys-type protease inhibitor. PMC effectively inhibits degradation of patatin by tuber proteases in vitro. Herein we show that changes in PMC, patatin concentration, activities of various proteases, and their gene expression are temporally linked during tuber development, providing evidence that PMC has a role in regulating tuber protein content in vivo. PMC was barely detectable in non-tuberized stolons. PMC transcript levels increased progressively during tuberization, concomitant with a 40-fold increase in PMC concentration (protein basis) as tubers developed to 10 g fresh wt. Further increases in PMC were comparatively modest (3.7-fold) as tubers developed to full maturity (250 g). Protease activity declined precipitously as PMC levels increased during tuberization. Proteolytic activity was highest in non-tuberized stolons and fell substantially through the 10-g fresh wt stage. Cys-type proteases dominated the pre-tuberization and earliest stages of tuber development. Increases in patatin transcript levels during tuberization were accompanied by a notable lag in patatin accumulation. Patatin did not begin to accumulate substantially on a protein basis until tubers had reached the 10-g stage, wherein protease activity had been inhibited by approximately 60%. These results indicate that a threshold level of PMC (ca. 3 microg tuber(-1), 144 ng mg(-1) protein) is needed to favor patatin accumulation. Collectively, these results are consistent with a role for PMC in facilitating the accumulation of proteins in developing tubers by inhibiting Cys-type proteases.

Characterization of Solanum tuberosum Multicystatin and Its Structural comparison with Other Cystatins

“…It is also tempting to speculate that PMC likely has more than one target protease in vivo, thus enabling PMC to regulate myriad physiological events central to tuber ontogeny. As previously indicated, PMC appears to be involved in regulating protein deposition in tubers during development (Weeda et al, 2008) and protein mobilization from seed tubers during sprouting by regulating protease activity (Weeda et al, 2009). For example, during tuber development, active monomeric PMC accumulation precedes deposition of patatin, a major storage protein in the potato tuber (Weeda et al, 2008).…”

“…A relatively low level of cytosolic Pi favors the monomeric (active) form of PMC, which in turn would inhibit Cys protease activity and thus facilitate protein synthesis and deposition in the developing tubers. Indeed, PMC accumulation precedes the deposition of patatin, a major storage glycoprotein (Weeda et al, 2008). Tissue probed with anti-PMC has demonstrated the uniform distribution of monomeric PMC throughout the tuber during early development (Kumar et al, 1999).…”

“…PMC accumulation in tubers precedes patatin (45-kD storage glycoprotein) deposition during the initial stages of tuber development (Weeda et al, 2008). In storage, the PMC content of tubers declines over time, concomitant with a loss of patatin.…”

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

“…Until now, PhyCys have been shown to have a role in plant growth and development, senescence, and programmed cell death (Solomon et al, 1999;Corre-Menguy et al, 2002;Belenghi et al, 2003;Kiyosaki et al, 2007;Weeda et al, 2009). However, these studies involved individual cystatin members from multiple plant species.…”

“…C1A Cys-proteases from plants are synthesized as preproteins, processed either automatically or with the aid of processing enzymes, transported via the trans-Golgi network, and finally either are stored in the vacuoles and lysosomes or externally secreted (Grudkowska and Zagdanska, 2004). These plant proteases participate in protein degradation during senescence and abscission processes, programmed cell death, and the accumulation and mobilization of storage proteins in seeds and tubers (Grudkowska and Zagdanska, 2004;van der Hoorn, 2008;Shi and Xu, 2009;Weeda et al, 2009). Moreover, they also play an essential role in local and systemic defense responses against pathogens and pests (van der Hoorn, 2008;McLellan et al, 2009).…”