Immunocytochemical localization of patatin,the major glycoprotein in potato (Solanum tuberosum L.) tubers

Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanum tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers.Abbreviations PHA phytohemagglutinin - TFMS trifluoromethanesulfonic acid     

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.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum) tubers. This protein has been reported not only to serve as a storage protein, but also to exhibit enzymic activity. By using a baculovirus system to express protein from the patatin cDNA clone pGM01, it was unambiguously shown that the patatin coded by this DNA has lipid acyl hydrolase and acyltransferase activities. The enzyme is active with phospholipids, monoacylglycerols and p-nitrophenyl esters, moderately active with galactolipids, but is apparently inactive with di- and tri-acylglycerols.     

Resistance to Phytophthora infestans in somatic hybrids of Solanum nigrum L. and diploid potato

In breeding for resistance to late blight, ( Phytophthora infestans Mont. de Bary), an economically important disease affecting potatoes, the search for new sources of durable resistance includes the non-host wild Solanum species. The aim of this work was to evaluate the resistance to P. infestans in the somatic hybrids between S. nigrum L. and diploid potato clone ZEL-1136. Sixteen somatic hybrids, their fusion parents, and three standard potato cultivars were screened for resistance to P. infestans in two types of tests-on whole plants and detached leaves-with two highly aggressive and virulent isolates of P. infestans, US8 and MP322. In the whole plant assay, the foliage of the somatic hybrids showed no symptoms of infection, while the foliage of the potato fusion parent and the standard cultivars was infected with P. infestans. In the detached leaflet assay, the breaking-down of resistance of the S. nigrum L. parent and the variable response of individual hybrid clones were noted. Nine S. nigrum L. (+) ZEL-1136 hybrids showed a resistance that was significantly higher than that of S. nigrum, while six clones expressed a resistance to P. infestans similar to that of S. nigrum. The results confirm the effective transfer of late blight resistance of S. nigrum into its somatic hybrids with potato.     

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae.

The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 microg/ml (agronomic dose), 10.62 microg/ml and 10 microg/ml. G. mosseae was able to germinate in the presence of 2.12 microg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 microg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi.     

Expression of a tuber-specific storage protein in transgenic tobacco plants: demonstration of an esterase activity

A chimaeric gene composed of the 5' upstream region of STLS1, a leaf/stem specifically expressed gene from Solanum tuberosum, and the RNA-coding as well as the 3' downstream region of patatin, the major storage protein of potato tubers, has been transferred into tobacco plants using the Agrobacterium system. The introduction of this gene led to a leaf/stem specific expression of a 42-kd large protein which immunocrossreacts with patatin antiserum. Only low amounts of immunoreacting protein of smaller size could be detected in transgenic tobacco leaves indicating that the patatin protein is fairly stable in this heterologous environment. The size of the protein as well as the size of the RNA detected in transgenic tobacco leaves using a patatin-specific probe indicates that the patatin RNA was accurately processed in both leaf and stem tissue of tobacco. The expression of the patatin gene led to the appearance of a new esterase activity in the transformed tobacco which co-migrated with a protein immunoreacting with patatin antiserum. These data therefore demonstrate that patatin in addition to serving as a storage protein displays an enzymatic activity.     

Expression and Characterization of Hepatitis B Surface Antigen in Transgenic Potato Plants

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.     

Induction and accumulation of major tuber proteins of potato in stems and petioles

A family of immunologically identical glycoproteins with apparent molecular weights of approximately 40,000 are among the major tuber proteins of potato (Solanum tuberosum L.). These proteins, as purified by ion-exchange and affinity chromatography, have been given the trivial name `patatin.' To determine if patatin can be used as a biochemical marker to study the process of tuberization, its amount was measured in a variety of tissues by rocket immunoelectrophoresis and by enzyme-linked immunosorbent assay (ELISA).

Patatin comprises 40 to 45% of the soluble protein in tubers regardless of whether they are formed on underground stolons or from axillary buds of stem cuttings. Under normal conditions, patatin is present in only trace amounts, if at all, in leaves, stems, or roots of plants which are either actively forming tubers or which have been grown under long days to prevent tuberization. However, if tubers and axillary buds are removed, patatin can accumulate in stems and petioles. This accumulation occurred without any obvious tuber-like swelling and would occur even under long days. In all tissues containing large amounts of patatin, the other tuber proteins were also found as well as large amounts of starch.

     

DRY-ROT DISEASE OF THE POTATO

A laboratory method is described in which a modified hypodermic syringe is used to inoculate potato tubers with Fusarium caeruleum. This injection method gives consistent results and permits reliable assessments of factors such as varietal susceptibility.
The occurrence of arrested lesions is noted.
Suspensions of very high spore concentration or of spores already germinated did not greatly increase the amount of dry rot above that given by the standard too spores per inoculation, provided that spore germination was adequate.
The size of tuber and site of inoculation were found to have a considerable effect upon the results of inoculation experiments, large tubers being more susceptible than small ones and the heel end being more susceptible than the rose end. The necessity for uniformity of material used in inoculation experiments is emphasized.     

Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber-specific and sucrose-inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B-box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA-binding protein designated Storekeeper (STK) that specifically recognises the B-box motif in vitro. Gel shift experiments with an STK-specific antibody suggest that STK is the B-box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B-box motif.     

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.     

Characterization of patatin esterase activity in AOT-isooctane reverse micelles

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT-isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, w(o) (= H(2)O/AOT), and the percentage of H(2)O, theta, were investigated. The results obtained show that patatin esterase activity varies with w(o) but remains constant throughout the range of theta values studied. The variation with w(o) showed that the activity follows an S-shaped behavior pattern, reaching a maximum at about w(o) = 20 for 2% H(2)O. Patatin esterase activity was compared with p-nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP-caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 degrees C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated (K(m) = 10 mM, V(m) = 158 microM/min, V(m)/K(m) = 15.8 x 10(-3) min(-1)). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.     

人乳铁蛋白在转基因马铃薯块茎中的表达

人乳铁蛋白(human lactoferrin,hLF)是人体非特异性免疫系统的重要成员之一,具有抗细菌、真菌和抗病毒活性及其他多种功能.报道将hLF基因的cDNA与马铃薯(Solarium tuberosum L.)块茎专一性表达patain基因启动子融合后通过农杆菌介导导入马铃薯,PCR检测证实获得了多个转基因株系,RT-PCR阳性结果说明hLF mRNA在马铃薯植株中得到了表达.同时,经过ELISA及Western blot检测证实,转基因马铃薯表达了hLF并具有人乳铁蛋白的活性.     

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.     

Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco

Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)⁶⁰, and Asn⁹⁰, but the third glycosylation site (Asn²⁰²) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.Abbreviations Asn asparagine - ConA concanavalin A - EndoH endoglycosidase H - Fuc fucose - GlcNAc N-acetylglucosamine - HPLC high-performance liquid chromatography - Man mannose - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl-sulfate - Ser serine - TFMS trifluoromethanesulfonic acid - Thr threonine - Xyl xylose     

Regulation of potato tuber protein accumulation by gibberellic Acid

Many studies have shown that gibberellic acid (GA₃) inhibits tuberization in potato (Solanum tuberosum L.). In this study, we have utilized the 40 kilodalton glycoprotein, patatin, as a marker for biochemical events associated with the process of tuberization. To determine the effects of exogenous applications of GA₃ on the induction of the accumulation of this major tuber protein, we measured patatin levels in tubers from treated whole plants, petioles from a single-node cutting system with GA₃ applied in a lanolin paste, and stolon tips cultured in vitro on an inductive medium supplemented with GA₃. In all three systems, GA₃ inhibited the accumulation of patatin and the major 15 and 22 kilodalton tuber proteins. This effect appeared to be selective since most of the other proteins were not affected and, in tubers, at least one protein was stimulated by GA₃. These results suggest that GA₃ can reverse biochemical events of tuberization in tubers as well as prevent the accumulation of the major tuber proteins in other inducible tissues.     

Evaluation of somatic hybrids of potato with Solanum stenotomum after a long-term in vitro conservation.

Somatic hybrids of potato with a cultivated relative, Solanum stenotomum also called Solanum tuberosum Stenotomum group, were evaluated for their physiological and agronomical characteristics as well as the stability of the introgressed resistance to bacterial wilt, caused by Ralstonia solanacearum, after a long-term in vitro conservation for more than 5 years. Analysis of photosynthesis showed that the PEPC/Rubisco ratio remained lower than 0.5 for all vitroplants of potato and the somatic hybrids, except for the relative species. This indicates that the carbon metabolism is heterotrophic (ratio>1) for S. stenotomum, and autotrophic for potato and the somatic hybrids (ratio<1). In both in vitro and greenhouse conditions, potato and the somatic hybrids produced few bigger tubers, while many small tubers were obtained from the relative. The hybrid tubers were morphologically intermediate. The starch content of hybrid tubers was much lower than that of potato, but similar to that of the relative species. Interestingly, the level of bacterial resistance, introgressed from S. stenotomum into potato, was shown to be very stable and remained as high as that of the relative after a long-term period of in vitro conservation.