, Volume 230, Issue 1, pp 73–84 | Cite as

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

  • Sarah M. Weeda
  • G. N. Mohan Kumar
  • N. Richard Knowles
Original Article


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 µg 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.


Potato multicystatin Protein Protease Tuber development Solanumtuberosum Patatin Tuberization 





Days after planting


Enzyme ImmunoAssay


Fluorescein isothiocyanate


Lipolytic acyl hydrolase


p-Nitrophenyl myristate


Potato cysteine protease inhibitor


Potato protease inhibitor I


Potato protease inhibitor II


Potato Kunitz-type protease inhibitor


Potato multicystatin


p-Nitrophenyl phosphate





Financial support provided by grants from the USDA/ARS, USDA/CSREES, Washington State Potato Commission and WSU Agricultural Research Center is gratefully acknowledged.


  1. Andrews DL, Beames B, Summers MD, Park WD (1988) Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector. Biochem J 252:199–206PubMedGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  3. Brunelle F, Girard C, Cloutier C, Michaud D (2005) A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases. Arch Insect Biochem Physiol 60:20–31PubMedCrossRefGoogle Scholar
  4. Dunaevsky YE, Belozersky MA (1989) The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds. Planta 179:316–322CrossRefGoogle Scholar
  5. Etienne P, Desclos M, Le Gou L, Gombert J, Bonnefoy J, Maurel K, Le Dily F, Ourry A, Avice JC (2007) N-protein mobilization associated with the leaf senescence process in oilseed rape is concomitant with the disappearance of trypsin inhibitor activity. Funct Plant Biol 34:895–906CrossRefGoogle Scholar
  6. Goulet MC, Dallaire C, Vaillancourt LP, Khalf M, Badri AM, Preradov A, Duceppe MC, Goulet C, Cloutier C, Michaud D (2008) Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites. Plant Physiol 146:1010–1019PubMedCrossRefGoogle Scholar
  7. Green TR, Ryan CA (1972) Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Sci USA 175:776–777Google Scholar
  8. Grudkowska M, Zagdańska B (2004) Multifunctional role of plant cysteine proteinases. Acta Biochim Pol 51:609–624PubMedGoogle Scholar
  9. Huang DJ, Chen HJ, Hou WC, Chen TE, Hsu WY, Lin YH (2005) Expression and function of a cysteine proteinase cDNA from sweet potato (Ipomoea batatas [L.] Lamm ‘Tainong 57’) storage roots. Plant Sci 169:423–431CrossRefGoogle Scholar
  10. Knowles NR, Pavek MJ, Knowles LO, Holden Z (2008) Developmental profiles and postharvest behavior of long-season processing cultivars. In: Proceedings of the 47th annual Washington State potato conference, Feb. 5–7, Moses Lake, WA, pp 45–65Google Scholar
  11. Kumar GNM, Houtz RL, Knowles NR (1999) Age-induced protein modifications and increased proteolysis in potato seed-tubers. Plant Physiol 119:89–99PubMedCrossRefGoogle Scholar
  12. Kumar GNM, Iyer S, Knowles NR (2007) Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues. J Agric Food Chem 55:1674–1678PubMedCrossRefGoogle Scholar
  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  14. Lecardonnel A, Chauvin L, Jouanin L, Beaujean A, Prévost G, Sangwan-Norreel B (1999) Effects of rice cystatin I expression in transgenic potato on Colorado potato beetle larvae. Plant Sci 140:71–79CrossRefGoogle Scholar
  15. Lenfesty CM (1967) Soil survey: Adams County, Washington. Washington DCGoogle Scholar
  16. Lojkowska E, Holubowska M (1989) Changes of the lipid catabolism in potato tubers from cultivars differing in susceptibility to autolysis during the storage. Potato Res 32:463–470CrossRefGoogle Scholar
  17. Lulai EC, Sowokinos JR, Knoper JA (1986) Translucent tissue defects in Solanum tuberosum L. Plant Physiol 80:424–428PubMedCrossRefGoogle Scholar
  18. Macrae AR, Visicchio JE, Lanot A (1998) Application of potato lipid acyl hydrolase for the synthesis of monoacylglycerols. J Am Oil Chem Soc 75:1489–1494CrossRefGoogle Scholar
  19. Michaud D, Faye L, Yelle S (1993) Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. Electrophoresis 14:94–98PubMedCrossRefGoogle Scholar
  20. Michaud D, Nguyen-Quoc B, Bernier-Vadnais N, Faye L, Yelle S (1994) Cysteine proteinase forms in sprouting potato tuber. Physiol Plant 90:97–503CrossRefGoogle Scholar
  21. Nissen MS, Kumar GNM, Youn B, Knowles DB, Lam KS, Ballinger WJ, Knowles NR, Kang C (2009) Characterization of potato multicystatin and its structural comparison with other cystatins. Plant Cell (in press).
  22. Oliver GW, Leferson JD, Stetler-Stevenson WG, Kleiner DE (1997) Quantitative reverse zymography: analysis of pictogram amounts of metalloproteinase inhibitors using gelatinase A and B reverse zymograms. Anal Biochem 244:161–166PubMedCrossRefGoogle Scholar
  23. Orr GL, Strickland JA, Walsh TA (1994) Inhibition of Diabrotica larval growth by a multicystatin from potato tubers. J Insect Physiol 40:893–900CrossRefGoogle Scholar
  24. Palma JM, Sandalio LM, Corpas FJ, Romero-Puertas MC, McCarthy I, del Río LA (2002) Plant proteases, protein degradation, and oxidative stress: role of peroxisomes. Plant Physiol Biochem 40:521–530CrossRefGoogle Scholar
  25. Popovič T, Brzin J (2007) Purification and characterization of two cysteine proteinases from potato leaves and the mode of their inhibition with endogenous inhibitors. Croat Chem Acta 80:45–52Google Scholar
  26. Pouvreau L, Gruppen H, Piersma SR, van den Broek LAM, van Koningsveld GA, Voragen AGJ (2001) Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana. J Agric Food Chem 49:2864–2874PubMedCrossRefGoogle Scholar
  27. Pouvreau L, Gruppen H, van Koningsveld GA, van den Broek LAM, Voragen AGJ (2003) The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family. J Agric Food Chem 51:5001–5005PubMedCrossRefGoogle Scholar
  28. Prins A, van Heerden PDR, Olmos E, Kunert KJ, Foyer CH (2008) Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) veisicular bodies. J Exp Bot 59:1935–1950PubMedCrossRefGoogle Scholar
  29. Rivard D, Girard C, Anguenot R, Vézina LP, Trépanier S, Michaud D (2007) MsCYS1, a developmentally-regulated cystatin from alfalfa. Plant Physiol Biochem 45:508–514PubMedCrossRefGoogle Scholar
  30. Rodis P, Hoff JE (1984) Naturally occurring protein crystals in the potato. Plant Physiol 74:907–911PubMedCrossRefGoogle Scholar
  31. Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28:425–449CrossRefGoogle Scholar
  32. Sanchez-Serrano J, Schmidt R, Schell J, Willmitzer L (1986) Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mol Gen Genet 203:15–20CrossRefGoogle Scholar
  33. Sheokand S, Dahiya P, Vincent JL, Brewin NJ (2005) Modified expression of cysteine protease affects seed germination, vegetative growth and nodule development in transgenic lines of Medicago truncatula. Plant Sci 169:966–975CrossRefGoogle Scholar
  34. Sin SF, Yeung EC, Chye ML (2006) Downregulation of Solanum americanum genes encoding proteinase inhibitor II causes defective seed development. Plant J 45:58–70PubMedCrossRefGoogle Scholar
  35. Siqueira-Júnior CL, Fernandes KVS, Machado OLT, da Cunha M, Gomes VM, Moura D, Jacinto T (2002) 87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants. Plant Physiol Biochem 40:247–254CrossRefGoogle Scholar
  36. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–443PubMedCrossRefGoogle Scholar
  37. Stupar RM, Beaubien KA, Jin W, Song J, Lee MK, Wu C, Zhang HB, Han B, Jing J (2006) Structural diversity and differential transcription of the patatin multicopy gene family during potato tuber development. Genetics 172:1263–1275PubMedCrossRefGoogle Scholar
  38. Taylor MA, Wright F, Davies HV (1994) Characterization of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.). Plant Mol Bio 26:1013–1018CrossRefGoogle Scholar
  39. Thoenen M, Herrmann B, Feller U (2007) Senescence in wheat leaves: is a cysteine endopeptidase involved in the degradation of the large subunit of Rubisco? Acta Physiol Plant 29:339–350CrossRefGoogle Scholar
  40. Tiedmann J, Schlereth A, Muntz K (2001) Differential tissue-specific expression of cysteine proteinases forms the basis for fine tuned mobilization of storage globulin during and after germination in legumes seeds. Planta 212:728–738CrossRefGoogle Scholar
  41. Valdés-Rodríguez S, Guerrero-Rangel A, Melgoza-Villagómez C, Chagolla-López A, Delgado-Vargas F, Martínez-Gallardo N, Sánchez-Hernández C, Délano-Frier J (2007) Cloning of a cDNA encoding a cystatin from grain amaranth (Amaranthus hypochondriacus) showing a tissue-specific expression that is modified by germination and abiotic stress. Plant Physiol Biochem 45:790–798PubMedCrossRefGoogle Scholar
  42. van den Broek LAM, Pouvreau L, Lommerse G, Schipper B, van Koningsveld GA, Gruppen H (2004) Structural characterization of potato protease inhibitor I (cv. Bintje) after expression in Pichia pastoris. J Agric Food Chem 52:4928–4934PubMedCrossRefGoogle Scholar
  43. Van der Hoorn RAL (2008) Plant proteases: from phenotypes to molecular mechanisms. Annu Rev Plant Biol 59:191–223PubMedCrossRefGoogle Scholar
  44. Waldron C, Wegrich LM, Merlo PAO, Walsh TA (1993) Characterization of a genomic sequence coding for potato multicystatin, an 8-domain cysteine proteinase-inhibitor. Plant Mol Bio 23:801–812CrossRefGoogle Scholar
  45. Walsh TA, Strickland JA (1993) Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains. Plant Physiol 103:1227–1234PubMedCrossRefGoogle Scholar
  46. Wang J, Li Y, Lo SW, Hillmer S, Sun SSM, Robinson DG, Jiang L (2007) Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies. Plant Physiol 143:1628–1639PubMedCrossRefGoogle Scholar
  47. Weeda SM, Kumar GNM, Knowles NR (2009) Changes in protease inhibitors during protein mobilization from seed-tubers. In: Proceedings of 92nd Annual Meeting Potato Association of America. Am J Potato Res (in press)Google Scholar
  48. Yamada T, Ohta H, Shinohara A, Iwamatsu A, Shimada H, Tuschiya T, Masuda T, Takamiya K (2000) A cysteine protease from maize isolated in complex with cystatin. Plant Cell Physiol 41:185–191PubMedGoogle Scholar
  49. Yamada T, Kondo A, Ohta H, Masuda T, Shimada H, Takamiya K (2001) Isolation of the protease component of maize cysteine protease–cystatin complex: release of cystatin is not crucial for the activation of the cysteine protease. Plant Cell Physiol 42:710–716PubMedCrossRefGoogle Scholar
  50. Yamagishi K, Mitsumori C, Kikuta Y (1991) Nucleotide sequence of a cDNA encoding the putative trypsin inhibitor in potato tuber. Plant Mol Bol 17:287–288CrossRefGoogle Scholar
  51. Zabrouskov V, Kumar GNM, Spychalla JP, Knowles NR (2002) Oxidative metabolism and the physiological age of seed potatoes are affected by increased α-linolenate content. Physiol Plant 116:172–185PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Sarah M. Weeda
    • 1
  • G. N. Mohan Kumar
    • 1
  • N. Richard Knowles
    • 1
  1. 1.Postharvest Physiology and Biochemistry Laboratory, Department of Horticulture and Landscape ArchitectureWashington State UniversityPullmanUSA

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