G. N. Mohan Kumar

Characterization of Solanum Tuberosum Multicystatin and its Structural Comparison with Other Cystatins.

Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO4−-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, α-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.

Characterization of Solanum tuberosum Multicystatin and the Significance of Core Domains

This study characterizes the structure and significance of the core of potato multicystatin (PMC), a multidomain cysteine protease inhibitor found in the cortical parenchyma tissue of potato tubers. Papain inhibitory properties of native and recombinant PMC containing core domains are affected by pH. It is likely that pH-mediated regulation imparts unique properties to PMC that modulate proteolysis upon wounding and/or infection via inhibiting cysteine proteases. Potato (Solanum tuberosum) multicystatin (PMC) is a unique cystatin composed of eight repeating units, each capable of inhibiting cysteine proteases. PMC is a composite of several cystatins linked by trypsin-sensitive (serine protease) domains and undergoes transitions between soluble and crystalline forms. However, the significance and the regulatory mechanism or mechanisms governing these transitions are not clearly established. Here, we report the 2.2-Å crystal structure of the trypsin-resistant PMC core consisting of the fifth, sixth, and seventh domains. The observed interdomain interaction explains PMC’s resistance to trypsin and pH-dependent solubility/aggregation. Under acidic pH, weakening of the interdomain interactions exposes individual domains, resulting in not only depolymerization of the crystalline form but also exposure of cystatin domains for inhibition of cysteine proteases. This in turn allows serine protease–mediated fragmentation of PMC, producing ∼10-kD domains with intact inhibitory capacity and faster diffusion, thus enhancing PMC’s inhibitory ability toward cysteine proteases. The crystal structure, light-scattering experiments, isothermal titration calorimetry, and site-directed mutagenesis confirmed the critical role of pH and N-terminal residues in these dynamic transitions between monomer/polymer of PMC. Our data support a notion that the pH-dependent structural regulation of PMC has defense-related implications in tuber physiology via its ability to regulate protein catabolism.

Correlative changes in proteases and protease inhibitors during mobilisation of protein from potato (Solanum tuberosum) seed tubers

Potato tubers (Solanum tuberosum L.) contain protease inhibitors that function in plant defence and as storage proteins. A multi-domain cysteine protease inhibitor, potato multicystatin (PMC), has also been implicated in regulating protein accumulation in developing tubers by inhibiting proteases. Unlike developing tubers, sprouting tubers mobilise protein reserves to support growth of developing plants and, therefore, show an increase in protease activity. Using single-eye containing cores (seedcores) from seed tubers, we characterised the relative changes in patatin, PMC, proteases and serine (Ser) protease inhibitors, as a prerequisite to further research on their potential roles in protein mobilisation from tubers during plant establishment. Approximately 63% of seedcore dry matter was mobilised over a 29-day period of plant establishment (1.7 mg seedcore dry matter mobilised for every mg increase in plant dry matter). The gelatinolytic protease isoforms induced in seedcores during plant establishment differed from those characterised previously in developing tubers. Total protease activity increased progressively in seedcores and reached a maximum 23 days after planting. Conversely, seedcore soluble protein content declined, with patatin accounting for the greatest decrease in the soluble protein fraction during plant establishment. PMC also decreased 44% and Ser (trypsin) protease inhibitors decreased to levels barely detectable in seedcores over the 29-day growth interval. Moreover, the temporal changes in PMC, protease activity and patatin content were highly correlated. As PMC decreased from 6 to 4 ng core–1, protease activity increased 9-fold, patatin decreased 2.6-fold and total soluble protein decreased by 58%. These results suggest that catabolism of protease inhibitors may facilitate protein mobilisation from seed tubers. Further work to define unequivocally the role of protease inhibitors in modulating the activity of proteases during protein mobilisation from tubers is warranted.

Protein Mobilization from Potato Tubers during Long-Term Storage and Daughter Tuber Formation

The role of protease inhibitors in modulating changes in protein content of potato (Solanum tuberosum L.) tubers was investigated using a mother/daughter tuber model system. Changes in patatin, potato multicystatin (PMC), proteases, serine (Ser) protease inhibitors, and their gene expression were temporally coordinated over a 22-mo storage interval in genotypes with short (cv. Ranger Russet) and long (cv. Russet Burbank) dormancy. Daughter tubers were initiated on Ranger Russet tubers at ∼15 mo. PMC (Cys protease inhibitor) declined linearly (∼4.2-fold) in Ranger Russet mother tubers from 4 to 15 mo and then maintained low levels through 22 mo. Conversely, protease activity was low and constant from 4 to 15 mo before increasing 7.4-fold through 22 mo. This increase coincided with the most rapid decline (54%) in patatin and the formation of daughter tubers. The proteases induced during aging of mother tubers were inhibited by PMC. Ser protease inhibitors were maintained in mother tubers throughout storage. In contrast, as daughter tubers developed, PMC and Ser protease inhibitors increased, protease activity declined to 17% of initial levels, and patatin increased threefold. These results implicate a role for protease inhibitors in regulating protein content during mobilization from mother tubers and deposition in daughter tubers.

Zebra chip disease enhances respiration and oxidative stress of potato tubers (Solanum tuberosum L.)

AbstractMain conclusionThe physiological phenotype of potato tubers afflicted by zebra chip disease is characterized by increased oxidative stress metabolism and upregulation of systems for its mitigation. Starch catabolism and extensive buildup of reducing sugars render potatoes infected with zebra chip (ZC) pathogen (Candidatus Liberibacter solanacearum) unsuitable for fresh market and processing into chips/fries. Here we show that the disease inflicts considerable oxidative stress, which likely constitutes a substantial sink for metabolic energy, resulting in increased respiration rate of afflicted tubers. In contrast to healthy tubers, tissue from diseased tubers had greater ability to reduce 2,3,5-triphenyl-tetrazolium chloride to formazan, indicating enhanced dehydrogenase activity, probable disease-induced changes in cellular redox potential, and increased respiratory activity. The respiration rate of diseased tubers (cv. Atlantic) was 2.4-fold higher than healthy tubers and this correlated with increased activities of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, key enzymes responsible for synthesis of cytosolic reducing equivalents. Wound-induced NADPH oxidase activity was greater for ZC than healthy tubers, but the resulting superoxide was rapidly catabolized by higher superoxide dismutase activity in ZC tubers. Peroxidase, catalase, glutathione reductase and ascorbate free radical reductase activities were also higher in diseased tubers, as was malondialdehyde, a biomarker of peroxidative damage and oxidative stress. Upregulation of the glutathione–ascorbate pathway is a direct response to (and indicator of) oxidative stress, which consumes reducing equivalents (NADPH) to catabolize reactive oxygen species and maintain cellular redox homeostasis. ZC disease substantially altered the oxidative metabolism of tubers, resulting in a physiological phenotype defined by metabolic changes directed toward mitigating oxidative stress. Paradoxically, the increased respiration rate of ZC tubers, which fuels the metabolic pathways responsible for attenuating oxidative stress, likely also contributes to oxidative stress.