Effects of Elicitors on the Accumulation of Proteinase Inhibitors in Injured Potato Tubers

The time course of accumulation and the composition of proteinase-inhibiting proteins in diffusates from potato tubers treated with elicitors such as salicylic, jasmonic, and arachidonic acids were studied. The 40-kDa reserve protein patatin and the chymotrypsin inhibitors, among which proteins of 24.6, 22.0, and 16.0 kDa were prevalent, accumulated in diffusates from potato tubers. Jasmonic and arachidonic acids activated the accumulation of the chymotrypsin inhibitors in tubers in response to the injury stress, whereas salicylic acid inhibited this process. The effects of jasmonic and arachidonic acids increased when their concentrations decreased to 10^–6M. Salicylic acid inhibited this process. The data suggest an important role of the lipoxygenase metabolism in signal transduction of the anti-injury defense system in dormant potato tubers.     

Role of proteinase inhibitors in potato protection

Mechanical damage or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

Role of Protease Inhibitors in Potato Protection

Mechanical wounding or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

Wound healing and induced resistance in potato tubers

It was demonstrated that biogenic elicitors, arachidonic acid and chitosan, locally and systemically stimulated wound healing in potato tuber tissues by increasing the number of wound periderm layers, accelerating the development of cork cambium (phellogen), and inducing proteinase inhibitors. The signal molecules, jasmonic and salicylic acids, had different effects on the development of wound periderm: jasmonic acid locally and systemically stimulated potato wound healing and elevated the level of proteinase inhibitors, whereas salicylic acid did not have any effect on wound healing and even blocked the formation of proteinase inhibitors.     

Effect of systemic signaling molecules on the rate of spread of the immunizing effect of elicitors over potato tissues

Mobile systemic signaling molecules (salicylic and jasmonic acids) enhance and accelerate the spread of systemic immunizing effect of elicitors (arachidonic acid and chitosan) over potato tuber tissues (Solanum tuberosum L.).     

Effect of methyljasmononate on induction of late-blight resistance of potatoes using arachidonic acid

Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused by Phytophthora infestans. Treatment of potato tuber disk surface with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10(-6)-10(-5) M) increased the rate of rishitin biosynthesis induced by arachidonic acid or P. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.     

Influence of Systemic Signal Molecules on the Rate of Spread of the Immunizing Effect of Elicitors over Potato Tissues

Mobile systemic signal molecules (salicylic and jasmonic acids) enhance and accelerate the spread of the systemic immunizing effect of elicitors (arachidonic acid and chitosan) over potato tuber tissues (Solanum tuberosum L.).     

Effect of methyl jasmonate on arachidonic acid-induced resistance of potato to late blight

Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused byPhytophthora infestans. Treatment of potato tuber disk surfaces with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10⁻⁶–10⁻⁵ M) increased the rate of rishitin biosynthesis induced by arachidonic acid orP. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Activation of elicitor defensive properties by systemic signal molecules during the interaction between potato and Phytophthora

The elicitor arachidonic acid in combination with jasmonic acid (JA) induced a higher level of defense against the late blight agent in potato (Solanum tuberosum L.) tissues than in combination with salicylic acid (SA). On the contrary, the elicitor chitosan displayed a higher inductive effect in combination with SA as compared with JA. The optimal concentrations of tested compounds were selected for designing the compositions activating wound repair, induction of proteinase inhibitors, and resistance to the biotrophic pathogen Phytophthora infestans (Mont.) de Bary. It was demonstrated that the compositions of elicitor and systemic signal molecules provided a faster spreading of an inducing effect in the potato tissues.     

Activation of elicitor defensive properties by systemic signal molecules during the interaction between potato and the late blight agent

The elicitor arachidonic acid in combination with jasmonic acid (JA) induced a higher level of defense against the late blight agent in potato (Solanum tuberosum L.) tissues than in combination with salicylic acid (SA). On the contrary, the elicitor chitosan displayed a higher inductive effect in combination with SA as compared with JA. The optimal concentrations of tested compounds were selected for designing the compositions activating wound repair, induction of proteinase inhibitors, and resistance to the biotrophic pathogen Phytophthora infestans (Mont.) de Bary. It was demonstrated that the compositions of elicitor and systemic signal molecules provided a faster spreading of an inducing effect in the potato tissues.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Jasmonic acid-inducible gene expression of a Kunitz-type proteinase inhibitor in potato tuber disks

Messenger RNAs of a potato (Solanum tuberosum L.) Kunitz-type proteinase inhibitor(s) (PKPI) were present in potato disks excised from tubers stored for 14 months (old tubers) or 2 months (young tubers) after harvest, and disappeared during the aseptic culture. The PKPI mRNA accumulation was found to be induced in potato disks from the old tubers by the addition of jasmonic acid (JA) [3-oxo-2-(2-cis-pentenyl)-cyclopentane-1-acetic acid].     

Effect of jasmonic acid on Ca<Superscript>+2</Superscript> transport through the plasmalemma of potato tuber cells

The rate of Ca²⁺ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10^?5–10^?10 M jasmonic acid on the accumulation of Ca⁺² in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca⁺²,Mg⁺²-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca⁺²,Mg⁺²-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.     

Extensive post-translational processing of potato tuber storage proteins and vacuolar targeting

Potato tuber storage proteins were obtained from vacuoles isolated from field-grown starch potato tubers cv. Kuras. Vacuole sap proteins fractionated by gel filtration were studied by mass spectrometric analyses of trypsin and chymotrypsin digestions. The tuber vacuole appears to be a typical protein storage vacuole absent of proteolytic and glycolytic enzymes. The major soluble storage proteins included 28 Kunitz protease inhibitors, nine protease inhibitors 1, eight protease inhibitors 2, two carboxypeptidase inhibitors, eight patatins and five lipoxygenases (lox), which all showed cultivar-specific sequence variations. These proteins, except for lox, have typical endoplasmic reticulum (ER) signal peptides and putative vacuolar sorting determinants of either the sequence or structure specific type or the C-terminal type, or both. Unexpectedly, sap protein variants imported via the ER showed multiple molecular forms because of extensive and unspecific proteolytic cleavage of exposed N- and C-terminal propeptides and surface loops, in spite of the abundance of protease inhibitors. Some propeptides are potential novel vacuolar targeting peptides. In the insoluble vacuole fraction two variants of phytepsin (aspartate protease) were identified. These are most probably the processing enzymes of potato tuber vacuolar proteins. Database Proteome data have been submitted to the PRIDE database under accession number 17707.     

Free and conjugated forms of salicylic acid: content and role in potato

Hydrolysis of conjugated forms of salicylic acid and accumulation of its free form was observed after infection of potato tubers (Solanum tuberosum L.) with an incompatible race of phytophthora or treatment with an elicitor (chitosan). Infection of tubers with a compatible race of the pathogen or treatment with a suppressor (laminarin) decreased both the degree of hydrolysis of conjugated forms of salicylic acid and the accumulation of its free form.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds.

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

马铃薯POTHR-1 cDNA的克隆及晚疫病菌和其他非生物因子诱导表达分析

利RACE和重叠延伸相结合的方法,从经晚疫病菌接种诱导的马铃薯水平抗性材料叶片中克隆了一个POTHE 1基因(potato Phytophthora infestans induced hypersensitive response related protein gene)的全长cDNA.序列分析表明,该基因编码225个氨基酸,与烟草harpin诱导蛋白基因hinl有很高的同源性(编码区核苷酸和氨基酸序列分别为83%和81%).Southern杂交结果显示在马铃薯基因组中有2～3个拷贝.对其诱导表达模式研究表明:晚疫病病原菌接种36 h后,该基因表达迅速增加;机械伤害及茉莉酸(JA)处理能够诱导表达;渗透胁迫(NaCl浸泡)能够诱导其微弱表达;但水杨酸(SA)不能诱导表达.该基因可能和病原与寄主互作时寄主产生过敏反应及细胞生理性死亡有关.