Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology

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

Sarah M. Weeda A , G. N. Mohan Kumar A and N. Richard Knowles A B

A Postharvest Physiology and Biochemistry Laboratory, Department of Horticulture and Landscape Architecture, PO Box 646414, Washington State University, Pullman, WA 99164-6414, USA.

B Corresponding author. Email:

Functional Plant Biology 37(1) 32-42
Submitted: 22 July 2009  Accepted: 9 September 2009   Published: 5 January 2010


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.

Additional keywords: plant establishment, potato multicystatin, protein mobilisation, proteolysis.


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. The Biochemical Journal 252 199 206

Bewley JD , Black M (1982) ‘Physiology and biochemistry of seeds in relation to germination. 2. Viability, dormancy and environmental control.’ (Springer-Verlag: Berlin)

Boulter D Harvey PJ 1985 Accumulation, structure and utilization of tuber storage proteins, with particular reference to Dioscora rotundata. Physiologia Vegetalis 23 61 74

Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248 254

Davies HV Ross HA 1984 The pattern of starch and protein degradation in tubers. Potato Research 27 209 218 doi:10.1007/BF02357468

Dunaevsky YE Belozersky MA 1989 The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds. Planta 179 316 322 doi:10.1007/BF00391076

Green TR Ryan CA 1972 Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science, USA 175 776 777

Hopkins M , McNamara L , Taylor C , Wang TW , Thompson J (2007) Membrane dynamics and regulation of subcellular changes during senescence. In ‘Senescence processes in plants’. (Ed. S Gan) pp. 39–68. (Blackwell Publishing Ltd: Oxford)

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 Science 169 423 431

Kitamura N Maruyama Y 1986 Purification and properties of cysteine proteinase in sprouting potato tubers. Agricultural and Biological Chemistry 50 381 390

Kumar GNM Houtz RL Knowles NR 1999 Age-induced protein modifications and increased proteolysis in potato seed tubers. Plant Physiology 119 89 100

Kumar GNM Iyer S Knowles NR 2007 Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues. Journal of Agricultural and Food Chemistry 55 1674 1678 doi:10.1021/jf062941m

Laemmli UK 1970 Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227 680 685 doi:10.1038/227680a0

Łojkowska E Hołubowska M 1989 Changes of the lipid catabolism in potato tubers from cultivars differing in susceptibility to autolysis during the storage. Potato Research 32 463 470 doi:10.1007/BF02358502

Lulai EC Sowokinos JR Knoper JA 1986 Translucent tissue defects in Solanum tuberosum L. Plant Physiology 80 424 428 doi:10.1104/pp.80.2.424

Macrae AR Visicchio JE Lanot A 1998 Application of potato lipid acyl hydrolase for the synthesis of monoacylglycerols. Journal of the American Oil Chemists’ Society 75 1489 1494 doi:10.1007/s11746-998-0083-y

Madureira HC Da Cunh M Jacinto T 2006 Immunolocalization of a defense-related 87 kDa cystatinin leaf blade of tomato plants. Environmental and Experimental Botany 55 201 208 doi:10.1016/j.envexpbot.2004.10.013

Michaud D Nguyen-Quoc B Bernier-Vadnais N Faye L Yelle S 1994 Cysteine proteinase forms in sprouting potato tuber. Physiologia Plantarum 90 497 503 doi:10.1111/j.1399-3054.1994.tb08807.x

Müntz K 1996 Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds. Journal of Experimental Botany 47 605 622 doi:10.1093/jxb/47.5.605

Müntz K Belozersky MA Dunaevsky YE Schlereth A Tiedemann J 2001 Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth. Journal of Experimental Botany 52 1741 1752 doi:10.1093/jexbot/52.362.1741

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. The Plant Cell 21 861 875 doi:10.1105/tpc.108.064717

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. Analytical Biochemistry 244 161 166 doi:10.1006/abio.1996.9895

Orr GL Strickland JA Walsh TA 1994 Inhibition of Diabrotica larval growth by a multicystatin from potato tubers. Journal of Insect Physiology 40 893 900 doi:10.1016/0022-1910(94)90023-X

Park WD , Hannapel DJ , Mignery GA , Pikaard CS (1985) Molecular approaches to the study of the major tuber proteins. In ‘Potato physiology’. (Ed. PH Li) pp. 261–278 (Academic Press Inc.: Orlando, FL)

Popovič T Brzin J 2007 Purification and characterization of two cysteine proteinases from potato leaves and the mode of their inhibition with endogenous inhibitors. Croatica Chemica Acta 80 45 52

Pots AM Gruppen H van Diepenbeek R van der Lee JJ van Boekel MAJS Wijngaards G Voragen AGJ 1999 The effect of storage of whole potatoes of three cultivars on the patatin and protease inhibitor content; a study using capillary electrophoresis and MALDI-TOF mass spectrometry. Journal of the Science of Food and Agriculture 79 1557 1564

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. Journal of Agricultural and Food Chemistry 49 2864 2874 doi:10.1021/jf010126v

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. Journal of Agricultural and Food Chemistry 51 5001 5005 doi:10.1021/jf0212486

Rodis P Hoff JE 1984 Naturally occurring protein crystals in the potato. Plant Physiology 74 907 911 doi:10.1104/pp.74.4.907

Ryan CA 1990 Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28 425 449 doi:10.1146/

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. Molecular Genetics and Genomics 203 15 20

Schlereth A Standhardt D Mock HP Muntz K 2001 Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination. Planta 212 718 727

Shutov A Vaintraub IA 1987 Degradation of storage proteins in germinating seeds. Phytochemistry 26 1557 1566 doi:10.1016/S0031-9422(00)82245-1

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 1275 doi:10.1534/genetics.105.051219

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 Molecular Biology 26 1013 1018 doi:10.1007/BF00028869

Tiedemann 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 legume seeds. Planta 212 728 738 doi:10.1007/s004250000435

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. Journal of Agricultural and Food Chemistry 52 4928 4934 doi:10.1021/jf049832x

Visal-Shah S Vrain TC Yelle S Nguyen-Quoc B Michaud D 2001 An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels. Electrophoresis 22 2646 2652 doi:10.1002/1522-2683(200108)22:13<2646::AID-ELPS2646>3.0.CO;2-8

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 Molecular Biology 23 801 812 doi:10.1007/BF00021535

Walsh TA Strickland JA 1993 Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains. Plant Physiology 103 1227 1234 doi:10.1104/pp.103.4.1227

Weeda SM Kumar GNM Knowles NR 2009 Developmentally linked changes in proteases and protease inhibitors suggest a role for potato multicystatin in regulating protein content of potato tubers. Planta 230 73 84 doi:10.1007/s00425-009-0928-0

Wrobel R Jones BJ 1992 Appearance of endoproteolytic enzymes during the germination of barley. Plant Physiology 100 1508 1516 doi:10.1104/pp.100.3.1508

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 Physiology 41 185 191

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 Physiology 42 710 716

Yamagishi K Mitsumori C Kikuta Y 1991 Nucleotide sequence of a cDNA encoding the putative trypsin inhibitor in potato tuber. Plant Molecular Biology 17 287 288 doi:10.1007/BF00039507

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. Physiologia Plantarum 116 172 185 doi:10.1034/j.1399-3054.2002.1160206.x

Rent Article Export Citation Cited By (5)