Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.     

Expression of full-length bioactive antimicrobial human lactoferrin in potato plants

A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEKDEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods. The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter. The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods. Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis. The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF. Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein. Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues. This is the first report of synthesis of full length, biologically active hLF in edible plants.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

Ectopic Expression of StERF94 Transcription Factor in Potato Plants Improved Resistance to Fusarium solani Infection

ERF proteins (ethylene-responsive factors), which belong to the AP2/ERF superfamily, play essential roles in plant development, growth, and response to abiotic and biotic constraints. In a previous study, we cloned a cDNA encoding the StERF94 factor from potato plants and the phylogenetic analyses showed that it belongs to group IX of the ERF family. Genes of this group are known to be involved in plant response to biotic stress. The StERF94 cDNA was overexpressed in transgenic potato plants and the resulting transgenic plants showed a high tolerance to salinity. In this study, we investigated the response of StERF94 transgenic plants to biotic stress by evaluating their resistance to Fusarium solani infection. A significant enhanced resistance to the fungus was noticed in the transgenic plants which displayed limited malondialdehyde and H₂O₂ production and increasing antioxidant enzyme activities. Our findings also revealed that overexpression of StERF94 in potato enhanced expression of relevant defense genes like those encoding PR proteins (pathogenesis related) which led to a protection against disease propagation and reduction of fungus development in plant tissues.

     

Increased accumulation of anthocyanins in transgenic potato tubers by overexpressing the 3GT gene

In order to explore a biotechnological method for improving potato tuber color and creating plants with increased anthocyanin contents, a potato UDP-glucose: flavonoid-3-O-glucosyltransferase (3GT) gene was inserted behind the GBSSI promoter of pBin19, and this construct was introduced into Solanum tuberosum L. cultivar Désirée plants by Agrobacterium-mediated transformation. Six independent transgenic lines overexpressing the 3GT gene were identified by PCR and Southern blot analysis from 18 kanamycin-resistant plants. Due to the expression of 3GT gene, the tuber color and the anthocyanin content were enhanced noticeably in the transgenic plants compared to the wild-type control plants. This result suggests that the 3GT gene can potentially be used to improve potato color and enhance levels of antioxidants in the diet.     

cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)

Using an Arabidopsis thaliana expressed sequence tag with sequence similarity to human lysosomal α-glucosidase as a probe, a potato cDNA was isolated. The cDNA encodes a polypeptide with an Mr value of 105,400 and the most significant matches of the deduced amino acid sequence are with members of family 31 of glucosyl transferase. The potato cDNA was expressed in a strain of Saccharomyces cerevisiae that is deficient in maltase activity and unable to grow using maltose as a carbon source (ABYSMAL81). Expression of the potato cDNA in the mutant yeast strain restores its ability to use maltose as a carbon source for growth. Additionally, α-glucosidase activity could be measured in extracts of the yeast cells following complementation. A range of maltodextrins were substrates for this activity. The steady-state expression level of the potato α-glucosidase gene was low in most tissues examined, the highest levels occurring in sprouting tubers and source leaves.     

Cloning and characterization of the Rubisco activase gene from <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam

A full-length cDNA of Rubisco activase (IBrcaI) was cloned from sweet potato (Ipomoea batatas (L.) Lam) using Rapid-Amplification of cDNA Ends (RACE). IBrcaI contains a 1,347 bp open reading frame encoding a protein of 439 amino acids. The sequence alignment of multiple Rubisco activase genes from sweet potato and other plants showed high homology at two previously described ATP-binding sites. Western blot analysis indicated that there are two Rubisco activase proteins in sweet potato. Expression of IBrcaI was only detected in leaves. In the 14 h light and 10 h dark photoperiods, maximal and minimal IBrcaI mRNA expression levels were detected at 8:00 in the morning and at midnight, respectively.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Functional characterisation of urease accessory protein G (ureG) from potato

The activation of the nickel metalloenzyme urease is a complex process. In bacteria, several urease accessory proteins are essential for incorporation of nickel into the active centre of urease. Comparatively little is known about the activation process and the proteins involved in plants. We cloned five different cDNAs encoding isoforms of urease accessory protein G (ureG) in potato. The 5-coding region of these cDNAs is highly polymorphic within Solanum tuberosum ssp. tuberosum, containing mainly a simple sequence repeat encoding histidine and aspartate. Mapping on an ultrahigh-density map of the potato genome and Southern blot analysis showed that the isoforms arise from allelic differences of a single-copy gene which was located on chromosome 2. Expression analysis at the mRNA and protein levels indicated the presence of ureG in almost all tissues examined, consistent with the ubiquitous expression of urease. An attempt to correlate urease activity with ureG expression levels in different tissues was made. Allelic copies of ureG were expressed in a tissue-specific manner. UreG from potato and the Klebsiella aerogenes urease operon defective in bacterial ureG were co-expressed in Escherichia coli. The plant gene complements the K. aerogenes ureG mutation, demonstrating that it encodes a urease accessory protein and indicating a structural conservation between the plant and the bacterial urease activation complexes.     

Changes in translatable poly(A) RNA from differentiated potato tissues transformed with shoot-inducing Ti TL-DNA of Agrobacterium tumefaciens

Summary Two dimensional gel electrophoresis was used to examine differences in steady state total poly(A) RNA from untransformed potato (Solanum tuberosum cv. Maris Bard) and potato transformed with shoot-inducing T_L-DNA from A. tumefaciens. RNA was compared from phenotypically very distinct in vitro cultured shoots, more similar grafted plants and tubers. In each case between 200–400 translation products were identified representing the more abundant poly(A) mRNA's. In general, poly(A) RNA from the transformed tissues gave more high molecular weight products. This increase was most evident in poly(A) RNA from shoot cultures. Depending on the tissue examined, 1–5% of the translation products with a molecular weight <43 KD were observed to increase or decrease in abundance. The influence of T-DNA on cellular gene expression in the different transformed potato tissues is discussed in relation to previously determined changes in T-DNA gene expression (particularly of the T-DNA cytokinin gene) and the corresponding changes in endogenous hormone concentrations. It is concluded that some of the specific changes in low molecular weight products are either directly caused by the increased cytokinin levels or are indirectly involved in maintaining the transformed phenotype. re]19850530 rv]19851206 ac]19851210     

Molecular analysis of a homogentisate phytyltransferase gene from <Emphasis Type="Italic">Lactuca sativa</Emphasis> L.

Tocochromanols, usually known as vitamin E, play a crucial role in human and animal nutrition. The enzyme homogentisate phytyltransferase (HPT) performs the first committed step of the vitamin E biosynthetic pathway. The full-length cDNA encoding HPT was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPT, was 1,670 bp long containing an open reading frame (ORF) of 1,185 bp which encoded a protein of 395 amino acids. Sequence analysis indicated that the deduced protein, named as LsHPT, shared high identity with other dicotyledonous HPTs. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPT was preferentially expressed in mature leaves compared with other tissues. When lettuce plants were subjected to drought and high-light stress treatments, LsHPT expression was markedly increased. Expression of LsHPT in Arabidopsis showed that LsHPT could enhance the α-tocopherol biosynthesis in Arabidopsis. Transient expression of LsHPT via agroinfiltration resulted in 9-fold increase in LsHPT mRNA level and nearly 18-fold enhancement in α-tocopherol content compared with the negative controls.     

Synthesis and Assembly of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Fimbrial Protein in Potato Tissues

Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266–337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266–337) protein. Further, a protein band of ~160 kDa was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against periodontal disease caused by P. gingivalis.     

TILLING to detect induced mutations in soybean

Background

Potato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants.

Results

In higher plants, cystathionine γ-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of theArabidopsis CgS (CgS _Δ90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein theCgS _Δ90was co-transformed with the methionine-rich15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgS_Δ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased.

Conclusion

Ectopic expression of CgS_Δ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgS_Δ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgS_Δ90 and can not significantly improve the nutritional value of tubers. The level ofPAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgS_Δ90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.