Identification of G-Box Sequence as an Essential Element for Methyl Jasmonate Response of Potato Proteinase Inhibitor II Promoter

The potato proteinase inhibitor II promoter was studied to identify cis-acting regulatory sequences involved in methyl jasmonate (MJ) response using transgenic tobacco plants carrying various lengths of the promoter fused to a chloramphenicol acetyltransferase reporter gene. An internal fragment between −625 and −520 was sufficient to confer a response to MJ, wounding, or sucrose when it was placed upstream of the nos promoter −101, which contains the CAAT-TATA region. Deletion of the proteinase inhibitor II promoter sequence upstream of −611 did not affect the MJ response, but a further deletion to −573 eliminated the response. The 3′-deletion study showed that the DNA sequence downstream from −520 is dispensable. However, 3′-deletion mutant −574 did not respond to the MJ treatment. These results indicated that an element essential for the MJ response is located at the −574/−573 region where the G-box sequence (CACGTGG) is located. The G-box sequence was not required for the sucrose enhancer effect, suggesting that the MJ response mechanism is different from that of sucrose.     

Cloning of a Sequence Encoding the SKTI Family Proteinase Inhibitor from Potato

Seven structurally similar clones from potato (Solanum tuberosum L.), cv. Istrinskii genomic DNA were isolated by cloning of the PCR products. It was suggested that five of these clones were the amplified copies of the same gene. Based on comparative and structural analysis of these clones, initial nucleotide structure of the gene was reconstructed. It appeared to be highly homologous (98%) to the already published sequences encoding the proteins belonging to the soybean Kunitz trypsin inhibitor family (SKTI). Comparison of the results with the previously published data on the SKTI-type proteinase inhibitors from potato of cv. Istrinskii suggests that the gene examined encodes both chains of the earlier described PSPI-21-6.3 protein [9].     

Localized Wounding by Heat Initiates the Accumulation of Proteinase Inhibitor II in Abscisic Acid-Deficient Plants by Triggering Jasmonic Acid Biosynthesis

To test whether the response to electrical current and heat treatment is due to the same signaling pathway that mediates mechanical wounding, we analyzed the effect of electric-current application and localized burning on proteinase inhibitor II (Pin2) gene expression in both wild-type and abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) and potato (Solanum phureja) plants. Electric-current application and localized burning led to the accumulation of Pin2 mRNA in potato and tomato wild-type plants. Among the treatments tested, only localized burning of the leaves led to an accumulation of Pin2 mRNA in the ABA-deficient plants. Electric-current application, like mechanical injury, was able to initiate ABA and jasmonic acid (JA) accumulation in wild-type but not in ABA-deficient plants. In contrast, heat treatment led to an accumulation of JA in both wild-type and ABA-deficient plants. Inhibition of JA biosynthesis by aspirin blocked the heat-induced Pin2 gene expression in tomato wild-type leaves. These results suggest that electric current, similar to mechanical wounding, requires the presence of ABA to induce Pin2 gene expression. Conversely, burning of the leaves activates Pin2 gene expression by directly triggering the biosynthesis of JA by an alternative pathway that is independent of endogenous ABA levels.     

Abscisic Acid Mediates Wound Induction but Not Developmental-Specific Expression of the Proteinase Inhibitor II Gene Family

The expression of the potato and tomato proteinase inhibitor II (pin2) gene family is subject to both developmental and environmental control, being constitutively expressed in potato tubers while only being present in the foliage of the potato or tomato plants after mechanical damage. There is evidence that the phytohormone abscisic acid (ABA) is involved in this wound induction of pin2 gene expression. This paper describes experiments that demonstrate that ABA is able to induce the expression of the pin2 gene family, both locally and systemically, at physiological concentrations. The significance of the ABA involvement in the pin2 induction upon wounding has been further strengthened by analyzing the expression of a pin2 promoter-[beta]-glucuronidase gene fusion in transgenic ABA-deficient mutant potato plants. We have analyzed the developmental regulation of pin2 gene expression in wild-type and ABA-deficient potato and tomato plants. The pin2 mRNA level is identical in mutant and wild-type parental Solanum phureja tubers. In addition, evidence is presented for pin2 also being constitutively expressed at certain stages in the development of both tomato and potato flowers. Again, the ABA deficiency appears to have little influence in this tissue-specific expression in the mutants. These results suggest the action of separate pathways for the developmental and environmental regulation of pin2 gene expression.     

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp³²–Asp²¹⁵ diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.     

Modulation of Sucrose and Starch Metabolism by Salicylic Acid Induces Thermotolerance in Spring Maize

Russian Journal of Plant Physiology - Two inbred lines of spring maize (Zea mays L.), CML 32 (stress tolerant) and LM 11 (stress susceptible) were taken to study the effect of salicylic acid (SA)...     

Modification of Proteinase Inhibitor II in Adzuki Beans during Germination

We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.     

水杨酸对陈化马铃薯切片乙烯产生的促进作用 (英)

外源水杨酸（talicylicacid,SA）处理可明显促进陈化马铃薯切片的乙烯产生。SA促进乙烯产生的作用强度与SA浓度。pH值、陈化温度以及块茎的生理状态有关：在90μmol－1内,作用强度与SA浓度呈正相关;30℃下的作用强于20℃下的作用;pH6．4的SA溶液作用最强;对休眠块茎切片的作用强于破除休眠块茎切片。     

Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.

S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^?10M, Rh. glutinis acid protease; 1.8×10^?7M, Cladosporium acid protease; 3.0×10^?6M.

These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.

Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form.     

Inhibition of Cathepsin B1 by E-64, a Thiol Proteinase Inhibitor,and Its Derivatives

Four formaldehyde-resistant yeasts were isolated from soil. Three were tentatively identified as Debaryomyces vanriji and one as Trichosporon penicillatum. These yeasts almost completely consumed formaldehyde at 0.15 to 0.55% in growth medium containing glucose as carbon source, but the carbon of formaldehyde was not incorporated into the cell constituents. In formaldehyde-containing medium, yeast growth occurred after formaldehyde consumption. The yeasts showed relatively high activities of formaldehyde dehydrogenase, S-formylglutathione hydrolase and formate dehydrogenase. The resistance to formaldehyde is attributed to detoxification by oxidation.     

Inhibition of Soybean and Potato Lipoxygenases by Bhilawanols from Bhilawan (Semecarpus anacardium) Nut Shell Liquid and Some Synthetic Salicylic Acid Analogues

Bhilawanol diene (3) isolated from bhilawan nut shell liquid was found to be a potent inhibitor of both soybean and potato lipoxygenases with IC 50 values of 0.85 μM and 1.1 μM, respectively. However, the monoene (2) and saturated (1) bhilawanols exhibited relatively lower inhibitory activity. In addition, inhibition studies with synthetic analogues of salicylic acid (4 - 8) suggested that the unsaturated lipophilic side chain may be an absolute requirement for inhibitory activity.     

水杨酸对锌胁迫下小麦幼苗生长抑制的缓解效应

以小麦品种‘新麦18’为材料,采用室内水培实验研究了不同浓度水杨酸(SA)处理对300 mg.L-1锌胁迫下小麦种子萌发和幼苗生长的影响。结果表明:在Zn2+胁迫下,小麦种子的发芽势和发芽率、幼苗根长、芽长以及幼苗叶片的可溶性蛋白含量、根系活力显著降低,而脯氨酸和丙二醛(MDA)含量显著增加(P<0.05);外施SA显著提高了Zn2+胁迫下小麦种子的发芽势和发芽率,同时也使Zn2+胁迫7 d后的小麦幼苗的根长、芽长,幼苗叶片的脯氨酸和可溶性蛋白含量以及根系活力显著升高,膜脂过氧化产物MDA含量却显著降低(P<0.05)。由此可见,外施SA可通过提高小麦幼苗根长和芽长来增加幼苗根系活力,通过提高小麦幼苗可溶性蛋白含量、脯氨酸含量来维持细胞膜的稳定性,降低膜脂过氧化伤害程度,从而缓解了Zn2+胁迫对幼苗生长的抑制,并以14 mg.L-1外源水杨酸缓解效果最好。     

The Effect of Gamma-Irradiation on the Sucrose Content in Sweet Potato Roots and Potato Tubers

The sucrose content in both potato tubers and sweet potato roots was considerably increased by gamma-irradiation. The maximum increase was achieved by an irradiation dose of 3 to 4 kGy for potatoes and 0.8 to 2 kGy for sweet potatoes. Cooling treatment (15°C, 2 weeks) for sweet potato roots also enhanced the sucrose content (almost 2 times) but was not additive to the irradiation treatment; the maximum sucrose content in irradiated sweet potato roots was in the range of 7 to 12% irrespective of the cooling treatment, depending on the variety of sweet potatoes. Irradiation made the sucrose content in the roots 2 to 4 times higher.     

Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)