Subtilisin Protein Inhibitor from Potato Tubers

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.     

中药郁金中姜黄素类成分及微量元素分析

本文报道了用双波长薄层扫描法对5种郁金中姜黄素类化合物的含量测定结果,同时采用原子吸收光谱法、等离子发射光谱法测定和比较了郁金生药中20种微量元素的含量。     

Cyclic AMP phosphodiesterase from dormant tubers of Jerusalem artichoke

An enzyme, which hydrolyzes 3′,5′-cyclic AMP to 3′-AMP and 5′-AMP, has been isolated from dormant tubers of Jerusalem artichoke and purified 850 × with a recovery of 15% of total activity. The partially purified enzyme differs greatly from both animal and bacterial phosphodiesterases in terms of pH optimum, substrate specificity, cation dependence and sensitivity to methylxanthines. The plant hormones are without effect, whereas ATP, 5′-AMP, 3′-AMP, inorganic phosphate and pyrophophosphate are inhibitors. The enzyme seems to be greatly inhibited in vivo by inorganic phosphate during dormancy.     

On the ecological and evolutionary significance of storage in clonal plants

Environmental heterogeneity has received wide attention in clonal plant research over the last decade. Most studies have focussed on the effects of spatial variation in environmental conditions on the performance of ramets and genets, while the effects of temporal heterogeneity have only occasionally been studied. As a consequence, our current knowledge about functional responses of clonal plants to habitat patchiness is biased towards spatial aspects of environmental heterogeneity. Nevertheless, temporal changes in biotic and abiotic conditions do occur in most natural habitats, and they are very likely to affect plant growth and performance, and to create positive selection pressures on traits that can buffer plants against unfavorable consequences of this variability. Storage of resources is a widespread phenomenon in clonal plant species. Typical clonal structures such as stolons, rhizomes and hibernacles serve as storage organs. However, the ecologic significance of storage in clonal plant structures remains partly unclear. We suggest that the lack of understanding with respect to resource storage in clonal plants be related to our poor knowledge of ecologic implications of temporal habitat heterogeneity in natural environments. Resource storage can be understood as a safety measure against temporal changes in the growing conditions of plants. This paper summarizes existing information about the ecologic relevance of storage in clonal plants and it tries to develop a framework for further investigation of resource storage as a strategy to enhance the performance of clonal plants in temporally variable environments.     

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.     

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect‐resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad‐spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI‐expressing and control (comparator) lines, followed by a two‐dimensional gel electrophoresis (2‐DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI‐expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2‐DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2‐DE showed unaltered deposition patterns in SlCDI‐expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI‐expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel‐generation transgenic plants.     

A novel hypersensitive resistance response against potato virus A in cultivar ’Shepody’

The potato cultivar ’Shepody’ is susceptible to a number of potato viruses including potato virus Y (PVY, potyvirus) but was found to possess extreme resistance to another potyvirus, potato virus A (PVA). ’Shepody’ plants were resistant to PVA infection in manual and graft inoculations. PVA replication was not detected in any of the inoculated plants by ELISA, an infectivity assay and RT-PCR. However, ’Shepody’ plants grafted with shoots containing PVA developed a novel symptomology which resembled a virus infection in appearance and in rate of translocation to the entire plant. Efforts to transmit the symptom-inducing agent manually failed. Graft-inoculation to potato virus indicator plants and PVA-susceptible potato plants showed that the symptom inducer was PVA at an extremely low concentration, detected using RT-PCR followed by Southern blot assay. Tubers from grafted but resistant ’Shepody’ plants had necrotic surfaces and internal spots. PVA was detected from necrotic areas but not from the non-necrotic ones. However, plants resulting from necrotic tubers were free from aerial leaf symptoms observed in grafted plants and produced non-necrotic normal tubers. A trace-back of the parental lineage of ’Shepody’ indicated that the resistance had been introgressed from the cultivar ’Bake King’. Analysis of progeny of a cross of resistant ’Shepody’ to the susceptible ’Goldrus’ indicated that this resistance is controlled by two independent dominant complementary genes in contrast to monogenic resistance reported for other potato viruses. Received: 6 April 1999 / Accepted: 28 July 1999     

The biosynthesis of lubimin from [1-14C]isopentenyl pyrophosphate by cell-free extracts of potato tuber tissue inoculated with an elicitor preparation from Phytophthora infestans

A cell-free enzyme system, which catalyses the incorporation of radiolabel from [$\text{1}\text{2}$-¹⁴C]isopentenyl pyrophosphate into the sesquiterpenoid phytoalexin lubimin, has been prepared from tuber tissue of Solanum tuberosum inoculated with an elicitor preparation from Phytophthora infestans. Biosynthesis of lubimin is optimum at pH 7.$\text{3}\text{2}$-7.5 and is dependent upon Mg²⁺ and NADPH. Lubimin labelling by cell-free enzyme system prepared from tissue 48 hr after treatment with elicitor rises rapidly to a maximum over the first 30 min of incubation and does not decline for a further 150 min. The biosynthetic capacity for lubimin in cell free extracts can be observed as early as 3 hr after inoculation of tuber tissue, and rises to a maximum at about 48 hr after treatment, declining thereafter. Lubimin labelling is inhibited by iodoacetamide, the effect of which is reversed by 3,3-dimethylallylpyrophosphate. Preliminary observations on the cell-free system show that it will also catalyse the incorporation of [2-¹⁴C]mevalonic acid into lubimin in the presence ofan ATP-generating system.     

Sucrose uptake and partitioning in discs derived from source versus sink potato tubers

The uptake of sucrose into isolated discs cut from sink (growing) and source (sprouting) potato (Solanum tuberosum L.) tuber tissue was studied. The uptake of sucrose into sink-tuber discs demonstrated biphasic kinetics. The large saturable component was inhibited by incubation of the discs with p-chloromercuribenzene sulfonic acid (PCMBS) whilst both the saturable and linear components were inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP). By contrast, in source-tuber discs, the linear component represented the majority of sucrose taken up, the saturable component playing only a minor role. In source discs, only the saturable component of uptake was inhibited by either PCMBS or CCCP. A large proportion (up to 25%) of sucrose taken up into sink-tuber discs was converted to starch but as the tubers aged the proportion of sucrose converted to starch decreased to the level found in source-tuber discs (approx. 3%). By contrast with sink-tuber discs (see Oparka and Wright, 1988b, Planta 175, 520–526) sucrose uptake into source discs was insensitive to turgor and demonstrated an uptake pattern similar to that of CCCP-treated sink tissue. It is proposed that exogenous sucrose is taken into the storage parenchyma of sink-tuber discs by both a carrier-mediated and a diffusional process. By contrast, uptake into the storage parenchyma of source-tuber discs appears to be essentially diffusional. The turgor sensitivity of sucrose uptake into sink-tissue discs may be mediated via the plasmalemma H⁺-ATPase. As the tuber ages the sucrose-uptake activity decreases and the capacity of the storage parenchyma to synthesise starch is lost. The data are discussed in relation to the in-vivo mechanisms of sucrose transport in storage tissues.     

Effect of gibberellic acid on growth and carbohydrate metabolism of developing tubers of potato (Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Ostara) were grown in aerated water culture in a controlled environment. When the tubers had reached a diameter of 1–3 cm. ¹⁴C-labelled or unlabelled gibberellic acid (GA₃) was applied to the surface of the stolons at points approximately 1 crn from the developing tubers, and treatment continued for 10 days. - Significant quantities of GA₃ moved into tuber tissue within 2–4 days of hormone application. This influx of GA₃ was accompanied by a marked reduction in both the activity of ADPG-pyrophospharylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase and an increase in the activity of UDPG-pyrophosphorylase. Starch phosphorylase activity initially increased slightly but then fell, whereas the activity of starch synthase remained constant throughout the experiment. The soluble sugar composition of the tubers changed qualitatively towards a pattern characteristic of growing stolon tips prior to tuber initiation, but there was no clear evidence of net starch degradation. Changes in the activities of the enzymes were observed prior to noticeable effects of the hormone on tuber growth rate or the development of new stolons at the tuber eyes. - GA₃- treated tubers imported more ¹⁴C from labelled photosynthate than expected on the basis of growth rate. However, the capacity to convert solub#e-¹⁴C to ethaTiol-insoluble-¹⁴C (predominantly starch) was reduced in comparison with non-treated tubers. - The observed changes in carbohydrate composition and enzyme activities indicate that GA₃ induces a drastic change in potato tuber metabolism towards a pattern characteristic for the termination of the storage process.