Patatin Gene Research Articles

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.

Presence of a transposon-like element in the promoter region of an inactive patatin gene in Solanum tuberosum L.

The promoter of the PGT3 patatin gene belonging to the class II subfamily is highly homologous to other class II patatin genes except for a 736 bp insertion in front of the putative transcription start site. The insertion is characterized by structural features resembling a transposable element such as an 11 bp inverted repeat at the termini and an 8 bp duplication flanking the insertion site. Despite the high homology to active patatin genes, fusion of its promoter to the beta-glucuronidase reporter gene does not lead to detectable beta-glucuronidase (GUS) activity in transgenic potato or tobacco plants, suggesting that the inactivation of this gene might be caused by the insertion of the transposon like element.

Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato ( Solanum tuberosum)

Patatin, a group of glycoproteins in potato ( Solanum tuberosum), is encoded by a multigene family. The protein is found in mass amounts in potato tubers and considered to represent the main storage protein. However, unlike other storage proteins patatin displays enzymatic activity and furthermore, related proteins are found in lower amounts in organs of potato plants which do not serve as storage organs, i.e. stolons, roots and flowers. Evidence is presented showing differences in the enzymatic activities of patatin purified from these organs. The possible implications of these results are discussed. In a genetical approach two single isoforms derived from a class I resp. a class II patatin gene were expressed in transgenic tobacco plants under control of heterologous promoters in order to reduce the complexity of the gene family. The enzymatic activity of these single isoforms was compared to that of patatin of different organs. A class I specific isoform showed exactly the same activity profile as tuber derived patatin. A class II specific isoform showed a pattern different from both tuber and root patatin.

Translatability of a plant-mRNA strongly influences its accumulation in transgenic plants

Current knowledge of parameters affecting RNA stability is very restricted in plants. Here we investigated factors which might contribute to the stability of a particular plant messenger RNA. To this end, insertion and deletion mutants were made in two different exons and an intron of the transcribed region of a well characterised patatin gene (pgT5). Mutant genes were expressed under the control of a strong leaf-stem specific promoter (ST-LS1) and analysed in vivo in transgenic tobacco plants. Northern analysis revealed the importance of the translatability of the mature messenger RNA with respect to its accumulation in transgenic plants. Enlargement of the 3' non-translated region by several hundred base-pairs reduced the steady state mRNA level slightly; the introduction of a stop codon leading to premature termination of translation of the RNA led to a dramatic decrease of the steady state mRNA level.

A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants.

A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the beta-glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.

Sucrose-regulated expression of a chimeric potato tuber gene in leaves of transgenic tobacco plants

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato tubers. Class-I patatin genes encode 98 to 99% of the patatin mRNA in tubers, but are not normally expressed in other tissues. They are not totally 'tuber-specific'; however, since they can be induced to express at high levels in other tissues under conditions of sink limitation or in explants cultured on medium containing elevated levels of sucrose. To examine the evolution of the mechanisms that regulate patatin gene expression, we introduced a chimeric patatin-beta-glucuronidase (GUS) gene containing 2.5 kb of 5' flanking sequence from the Class-I potato patatin gene PS20 into tobacco plants. The construct was not expressed at significant levels in leaves of juvenile plants or plantlets cultured in vitro, but was expressed at high levels in explants cultured on medium containing 0.3 to 0.4 M sucrose. While there were differences in the expression of the chimeric gene between transgenic tobacco and potato plants, the pattern of sucrose induction was very similar. These results suggest that the mechanism that controls patatin gene expression in potato tubers evolved from a widely distributed mechanism in which gene expression is regulated by the level of available photosynthate.

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 Asn(90), but the third glycosylation site (Asn(202)) 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.

Both developmental and metabolic signals activate the promoter of a class I patatin gene

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5'-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the beta-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.

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.

A rapid and efficient transformation method for the production of large numbers of transgenic potato plants

A procedure is described for the rapid and efficient production of transgenic potato ( Solanum tuberosum L.) plants using Agrobacterium-based vectors. The key factors are the genotype and the level of kanamycin sulfate used for selection. Optimized culture conditions easily and routinely permitted the isolation of hundreds of individual regenerates expressing β-glucuronidase (GUS) transcriptionally fused to promoters from cauliflower mosaic virus (CaMV 35S) or a class-I patatin gene.

Structural diversity of the patatin gene family in potato cv. Desiree.

We have used a combined genetical and molecular approach to study the structural diversity of the patatin gene family in tetraploid Solanum tuberosum L. cv. Desiree (2n = 4x = 48). Nine dihaploid derivatives (2n = 2x = 24) of cv. Desiree were isolated by gynogenesis through prickle pollination with S. phureja Juz et Buk. Patatin DNA sequences in Desiree and in the dihaploids were examined by probing Southern blots of restriction endonucleases HindIII and XbaI digested DNA with patatin cDNA region-specific gene probes and by more detailed examination (restriction endonuclease mapping and partial DNA sequencing) of 10 patatin genomic clones in bacteriophage lambda replacement vector EMBL4. This provided positive identification for most individual patatin gene family members and some estimate of their organisation and diversity. Most of the 64-72 patatin DNA copies showed little allelic variation based on HindIII and XbaI restriction fragment length polymorphism (RFLP) mapping and did not appear to be very tightly clustered. Four of the 6-8 class I patatin genes (without a characteristic 22 bp insert in their untranslated leader DNA), showed apparent allelic homogeneity, whilst the remaining class I genes comigrated with a single class II patatin gene RFLP subclass. Of the isolated clones, 4 contained apparent pseudogenes lacking 5' control sequences and exon-1 DNA while another clone contained a patatin gene truncated at the 5' region due to the cloning event. The remaining 5 all contained class II genes (with the 22 bp insert) and these showed varying degrees of sequence homology for 400 bp of conserved 5' coding and non-coding DNA (from 77%-95%). In one case the extent of homology differed, with complete sequence divergence upstream of position -80 from the start of transcription. The structural diversity of the patatin gene family is discussed in relation to expression of individual patatin genes and the use of cv. Desiree as a host for potato transformation experiments.

Molecular characterization of the patatin multigene family of potato

Patatin is a family of glycoproteins that accounts for up to 40% of the total soluble protein in potato tubers. We isolated and characterized 25 patatin genomic clones. All of these exhibit different restriction patterns, but can be divided into two classes based on the presence (class II) or absence (class I) of a 22-bp sequence within the 5'-untranslated region. We determined the complete nucleotide sequence of the class-I clone PS20 and the 5'-flanking sequence of seven additional clones. The transcribed region of PS20 spans 3197 bp and is divided by six introns. The seven exons encode a transcript which is identical to the cDNA, cloned in pGM203 [Mignery et al., Nucl. Acids Res. 12 (1984) 7987–8000]. The 5' -flanking sequences of both class-I and class-II patatin genes are highly homologous up to bp position −87 and then diverge. This conserved region contains the CAAT and TATA homologies as well as a homology to the core enhancer sequence. Within a class there are additional large regions of homology, but differences exist that allow the patatin genes to be further divided into several discrete subclasses. S1 nuclease protection experiments with both class-I and class-II probes showed that class-I transcripts are the predominant species present in tubers. Class-II transcripts are present in tubers, but are 50–100-fold less abundant. In potato roots, class-II transcripts are the predominant species and few, if any, class-I transcripts are present.

Differential expression of genes in potato tubers after wounding

Tubers of a common tetraploid species of Solanum tuberosum (Granola) were mechanically wounded by slicing. After 18 hr only small differences were detectable in the pattern of the steady-state protein extracted from wounded versus unwounded tubers. In contrast the protein pattern obtained by in vitro translation of mRNA isolated from wounded and unwounded tubers differed significantly. A cDNA library was established and screened for wound-induced cDNA clones by differential hybridization. Two clones, wun-1 and wun-2, were found that corresponded to genes that were highly expressed in wounded potato tubers but were not expressed in unwounded tubers. The expression of the gene corresponding to wun-1 is detectable 30 min after wounding; the expression of the gene corresponding to wun-2 is detectable 4 hr after wounding. The expression of both genes (hereafter referred to as wun-1 and wun-2) remains constant for up to 24 hr after wounding. Interestingly the RNA corresponding to patatin, a major storage protein of potato tubers, behaves in the opposite way; it decreases dramatically in tubers within 30 min after wounding. The low level of patatin mRNA observed in unwounded roots and stems also disappears after wounding. Run-off transcription experiments, performed with isolated nuclei, indicate that the activation of the wound-induced genes as well as the inhibition of the patatin gene are controlled at the transcriptional level.

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.

The 5? flanking DNA of a patatin gene directs tuber specific expression of a chimaeric gene in potato

A member of the patatin multigene family which encodes the major soluble tuber protein was isolated from potato cultivar Désirée. Analysis by strategic nucleotide sequencing demonstrated close homology to analogous regions of previously isolated patatin genomic clones from different cultivars. A 3.8-kb fragment containing the promoter and 5' flanking DNA of the patatin gene was used to construct a transcriptional fusion gene with the coding DNA of the bacterial chloramphenicol acetyltransferase (CAT) gene and the polyadenylation/termination sequences of the nopaline synthase gene (nos). The chimaeric gene was reintroduced into potato cultivar Désirée by agrobacterial transformation of tissue slices. Regenerated transformed plants showed expression of the chimaeric gene (as determined by CAT activity) in tubers, but not in leaves, stems or roots of in vitro grown plants. Independent transformants did not show substantial variation in the level of induced tuber-specific CAT activity. Thus, information contained within 3.8 kb of the 5' flanking DNA of the patatin gene analysed is sufficient to direct tuber-specific expression, largely independent of position effects.

Isolation and characterization of a gene from Solanum tuberosum encoding patatin, the major storage protein of potato tubers

The isolation and analysis is described of several cDNA- as well as one genomic clone encoding patatin, the major storage protein from tubers of Solanum tuberosum. The structure of the patatin gene was determined by sequencing a near full-length cDNA clone as well as a genomic fragment of 5.3 kb containing the entire RNA coding part plus about 0.8 kb 5′ upstream sequences. The transcription start site was determined by S1-mapping experiments. An open reading frame of 1,158 nucleotides is found in both the cDNA and the genomic clone. Six introns interrupt the ORF in the genomic sequence. This open reading frame allows for a protein of 42 kd which is 2.5 kd larger than the size of the mature protein. The patatin gene has all the characteristic sequences normally found in eukaryotic genes. A TATA box is found 25 nucleotides upstream of the transcription initiation site and the sequence CAAACT at position -60. In the 3′ region the consensus sequence AATAAA is found 13–18 nucleotides in front of the poly-A-addition site.

The structure and transcription start site of a major potato tuber protein gene.

We have isolated recombinant lambda clones containing intact major tuber protein (patatin) genes and flanking sequences from the commercial tetraploid variety Maris Piper. The gene is composed of seven exons and six introns, spread over 4 kb of DNA. Nuclease mapping defined the 5' end of the mRNA approximately 45 bp upstream of the initiation codon. The 5' end of the gene is preceeded by a canonical TATA box sequence. The three known patatin genes encode proteins of nearly identical Mr but very different isoelectric points. The sequence of the gene does not indicate a role for patatin as one of the globulin class of plant storage proteins.