Patatin Protein Research Articles

Variation of nitrogenous components in potato (Solanum tuberosum L.) tubers produced under organic and conventional crop management

The information related to protein and other nitrogenous components in organically produced potato tubers can be interesting for consumers with specific diets. Such information is still limited. Here we report results on the effects of organic and conventional production systems on accumulation of nitrogenous substances including total tuber protein and the nutritiously most important tuber protein, patatin, observed in five cultivars on two localities during three consecutive years. Organically produced potato tubers contained significantly lower content of total nitrogen, crude protein, nitrates and glycoalkaloids (α-solanine and α-chaconine). Protein content, patatin relative abundance in total tuber protein and patatin content in organically produced potato tubers were slightly lower than in conventional ones but the differences were not statistically significant. The cultivar and interactions between cultivars and other production factors affected considerably contents of crude protein, protein, glycoalkaloids and patatin relative abundance. The effect of cultivar on these nitrogenous characteristics was stronger for potatoes produced under conventional farming indicating the sensitive reaction of organically produced potatoes on less favourable agro-ecological production conditions. The results indicate that cultivar-specific genetic factors play the predominant role in the reciprocal interaction with other cultivation factors affecting the nitrogenous qualitative parameters of organically produced potato tubers. This should be considered to include primarily the cultivar in quality standards of organic farming. The late maturing cultivar Bionta exhibited high instability in tubers yield, total protein and patatin protein characteristics when produced under organic farming system, while the early maturing cultivar Karin reacted on the conditions of organic farming by an increase of the patatin content.

A deep gold mine metagenome as a source of novel esterases

New sources of enzymes for biotechnological applications are continually being sought for. While diverse microbial ecosysyems have been demonstrated in the deep subsurfaces, deep mines provide easy access to these specialist communities. Therefore, the aim of this study was to assess a deep mine biofilm as a source of novel esterase enzymes. Biofilm was collected from the Beatrix Mine in South Africa, at a depth of 808 m. Assessment of the diversity revealed a group of previously uncultured bacteria and archaea. A metagenome library was screened for esterolytic activity, producing two esterolytic clones: a phospholipase patatin protein and an isochorismatase family protein. The isochorismatase family protein contained the catalytic Asp and Cys but not the Arg, which is considered as important for catalysis. The patatin showed 55% similarity to its closest relative; the patatin family protein from Plesiocystis pacifica . The expressed patatin displayed a preference for the C6 ester and was maximally active at pH 8 and 30°C. This study reported that screening of a relatively small metagenome library from the deep mine biofilm provided two esterolytic clones, which differed from their known counterparts. This indicates that the deep mine ecosystems contain an untapped resource of novel and potentially useful enzymes which may have applications in chemical syntheses. Key words: Metagenome library, functional screening, lipolytic activity, patatin, isochorismatase

Analysis of 176 expressed sequence tags generated from cDNA clones of hot pepper by single-pass sequencing

As a part of the project to identify novel genes from the hot pepper (Capsicum annuum L. cv. Happy Dry), we have constructed several cDNA libraries and 176 randomly selected cDNA clones were partially sequenced. This expressed sequence tag (EST) analysis identified 95 clones (54.0%) that had a significant homology to a known protein sequence in the NCBI database. Of these clones, eighteen of them are related to genes not from the plant kingdom, indicating that 10.2% of the ESTs were newly identified in plants. Eunctional categorization of these clones revealed that the genes involved in metabolic pathways such as glycolysis and photosynthesis are most abundant, and genes in translational apparatus ranked next in abundance. Expression patterns of four ESTs were examined by RNA blot analysis. The CAN14 clone, which has a 58% identity to the potato patatin protein over a 117 amino acid overlap, was highly expressed in anther tissue but not in fruit tissues. The CFR2 clone, which is 88% identical to apospory-associated protein, showed relatively higher expression levels in seedlings and roots compared to other tissues. The transcript of the CFR11 clone, which shows a homology to γ-thionin, was abundant in every organ that was examined except roots. The CFR 29 clone which is 92% identical to the putative osmoprotectant from tomato root showed a root-preferential expression pattern.

Transgenic potato plants depleted for the major tuber protein patatin via expression of antisense RNA

Patatin, one of the major potato tuber proteins, is a glycoprotein of approx. 40 kDa and contributes 20% of the water soluble protein fraction of tubers of the potato cultivar Désirée. Despite a detailed molecular and biochemical analysis, no definitive physiological role has been assigned to the protein. In order to approach this question, we reduced the amount of patatin in potato tubers by Agrobacterium-mediated introduction of a chimeric gene containing a class I patatin cDNA cloned in reverse orientation to the 35S CaMV promoter into potato plants, thus resulting in formation of patatin antisense RNA. In several transformants the amount of patatin protein and RNA is drastically reduced, the maximum level being about 90% reduction in case of the protein and 99% in case of the RNA. This reduction of patatin protein in tubers of transgenic potato plants does not lead to readily visible changes in phenotype and development, rather the plants are indistinguishable from controls. Whereas no antisense RNA and only trace amounts of sense RNA were detected in tubers of inhibited plants, both sense and antisense RNA could be detected in leaf tissue of these plants. Induction of expression of the endogenous patatin genes in leaves by incubation of detached leaves in sucrose leads to a reduction of the level of antisense RNA. This can be interpreted as evidence for a direct interaction between sense and antisense RNA.

Expression of mutant patatin protein in transgenic tobacco plants: role of glycans and intracellular location.

The influence of N-glycosylation and subcellular compartmentation on various characteristics of a vacuolar glycoprotein is described. One member of the patatin gene family was investigated as a model system. Different glycosylation mutants obtained by destroying the consensus site Asn-X-Ser/Thr by oligonucleotide-directed mutagenesis were expressed in leaves of transgenic tobacco plants under the control of a light-inducible promoter. The various patatin glycomutants retained their properties in comparison with the wild-type protein with respect to protein stability, subcellular compartmentation, enzymatic activity, and various physicochemical properties studied showing the N-glycosylation not to be essential for any of these characteristics. To test the importance of the cotranslational transport and the subcellular (vacuolar) location for the properties of the patatin protein, another mutant was constructed in which the signal peptide was deleted, leading to its synthesis and accumulation in the cytosol. Biochemical analysis of this protein in comparison with its vacuolar form again revealed no significant differences with respect to its enzymatic activity or its stability in normal vegetative cells. During seed development, however, the cytoplasmic form was more stable than the vacuolar form, indicating the appearance of proteases specific for the protein bodies of developing seeds.

Expression of Mutant Patatin Protein in Transgenic Tobacco Plants: Role of Glycans and Intracellular Location

The influence of N-glycosylation and subcellular compartmentation on various characteristics of a vacuolar glycoprotein is described. One member of the patatin gene family was investigated as a model system. Different glycosylation mutants obtained by destroying the consensus site Asn-X-Ser/Thr by oligonucleotide-directed mutagenesis were expressed in leaves of transgenic tobacco plants under the control of a light-inducible promoter. The various patatin glycomutants retained their properties in comparison with the wild-type protein with respect to protein stability, subcellular compartmentation, enzymatic activity, and various physicochemical properties studied showing the N-glycosylation not to be essential for any of these characteristics. To test the importance of the cotranslational transport and the subcellular (vacuolar) location for the properties of the patatin protein, another mutant was constructed in which the signal peptide was deleted, leading to its synthesis and accumulation in the cytosol. Biochemical analysis of this protein in comparison with its vacuolar form again revealed no significant differences with respect to its enzymatic activity or its stability in normal vegetative cells. During seed development, however, the cytoplasmic form was more stable than the vacuolar form, indicating the appearance of proteases specific for the protein bodies of developing seeds.

Analysis of a chimeric class-I patatin-GUS gene in transgenic potato plants: High-level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. To examine the regulation of the patatin genes, we constructed a chimeric gene containing 2.5 kb of 5' flanking sequence from the class I patatin genomic clone PS20 transcriptionally fused to β-glucuronidase (GUS) and introduced it into potato plants using an Agrobacterium tumefaciens Tiplasmid vector. While the chimeric gene was expressed at high levels in tubers and in stolons attached to developing tubers, it was not normally expressed in leaves, stems, roots, or in stolons before tuberizatization. However, the expression of the class I patatin-GUS construct was not "tuber-specific" since leaf and stem explants cultured on medium containing 300 to 400 mM sucrose showed GUS activity equal or greater than that of tubers. The sucrose induction of GUS activity in leaf and stem explants was accompanied by the accumulation of patatin protein and large amounts of starch, but not by the morphological changes that normally are associated with tuberization. In contrast, the GUS reporter gene under the control of the 35S promoter of cauliflower mosaic virus showed an essentially uniform pattern of expression in transgenic potato plants and was not induced by sucrose.

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.

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.