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.     

Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression

The proteinase inhibitor II (pin2) gene family exhibits two different modes of expression. It is, on the one hand, constitutively expressed in flowers of potato and tomato plants. and in potato tubers. On the other hand, its expression is induced in the plant foliage by mechanical wounding. To define cis-regulatory elements involved in pin2 promoter activity, deletion analysis of a potato pin2 promoter has been performed in stably and transiently transformed potato and tobacco plants. Two different elements, a quantitative enhancer and a regulatory element, are required for promoter activity. While functional promoter elements required for pin2 activity in tubers and wounded leaves could not be separated, its expression in flowers is mediated by different cis-acting sequences. Induction of pin2 expression in leaves by treatment with the plant growth regulators abscisic acid and jasmonic acid, and the general metabolite sucrose, depends on the presence of the regulatory element involved in expression in tubers and wounded leaves. Thus, pin2 expression in tubers and wounded leaves apparently results from the action of similar hormonal signals on closely linked promoter elements, while a different signal pathway leads to its constitutive expression in flowers.     

<Emphasis Type="Italic">Strboh A</Emphasis> homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers

During 30-months of storage at 4°C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18–24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca⁺² declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25–30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.     

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression

Summary Two cDNA clones containing the complete coding region of a developmentally controlled (tuber-specific) as well as environmentally inducible (wound-inducible) gene from potato (Solanum tuberosum) have been sequenced. The open reading frame codes for 154 amino acids. Its sequence is highly homologous to the proteinase inhibitor II from tomato, indicating that the cDNA's encode the corresponding proteinase inhibitor II of potato. In addition the putative potato proteinase inhibitor II contains a sequence which is completely homologous with that of another small peptide proteinase inhibitor from potato, called PCI-I. Evidence is presented that this small peptide is probably derived from the proteinase inhibitor II by posttranslational processing.Northern type experiments using RNA from wounded and nonwounded leaves demonstrate that RNA homologous to the putative proteinase inhibitor II cDNA's accumulates in leaves as a consequence of wounding, whereas normally the expression of this gene is under strict developmental control, since it is detected only in tubers of potato (Rosahl et al. 1986). In addition the induction of this gene in leaves can also be achieved by the addition of different polysaccharides such as poly galacturonic acid or chitosan. In contrast to the induction of its expression by wounding in leaves, wounding of tubers results in a disappearance of the proteinase II inhibitor m-RNA from these organs.     

Biochemical reactions of different tissues of potato (Solanum tuberosum) to zoospores or elicitors from Phytophthora infestans

The time courses of sesquiterpenoid phytoalexin accumulation were examined in compatible and incompatible interactions of leaves and tubers from five different R genotypes of potato (Solanum tuberosum) with corresponding pathotypes of Phytophthora infestans, as well as in non-host interactions of all five potato cultivars with Phytophthora megasperma f. sp. glycinea and in elicitor-treated tubers from five, and cell suspension cultures from two, of the cultivars. In tubers, rishitin and several structurally related sesquiterpene derivatives accumulated rapidly in non-host incompatible interactions, less rapidly in host incompatible interactions, and more slowly in compatible interactions. Treatment of tubers or cell cultures with fungal culture filtrate or arachidonic acid elicited in most cases a transient accumulation of the sesquiterpenoid phytoalexins. None of these compounds was detectable under any of the applied conditions either in infected or in elicitortreated leaves. Sesquiterpenoid phytoalexins might therefore be helpful, but appear not to be essential, in disease resistance of potato.Abbreviations CF concentrated culture filtrate of Pi - cv. cultivar - Pi Phytophthora infestans (numbering indicates pathotypes corresponding to R genes in potato) - Pmg Phytophthora megasperma f. sp. glycinea     

Expression of the Phytophthora infestans ipiB and ipi0 genes in planta and in vitro

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

5'' upstream sequences from the wun1 gene are responsible for gene activation by wounding in transgenic plants.

A 1.2-kilobase pair fragment of the 5' upstream region of a potato wound-inducible gene (wun1) was fused to different marker genes (wun1-CAT, wun1-NPTII). Stable integration of a wun1-CAT chimeric gene into the tobacco genome led to a high wound-inducible chloramphenicol acetyltransferase activity in leaves. Transient expression experiments in potato protoplasts showed that wun1 carries a strong promoter sequence similar in strength to the 35S promoter. The same intensity of expression was also observed using wun1 constructs in transient experiments with rice protoplasts. wun1 mRNA was shown to accumulate to high levels in potato leaves collapsing as a result of infection with the phytopathogen Phytophthora infestans. The wun1 product might, therefore, play a role in a general physiological reaction to stress correlated with cell death.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Expression of the ipomoelin gene from sweet potato is regulated by dephosphorylated proteins, calcium ion and ethylene

A wound‐inducible cDNA, ipomoelin (IPO) was isolated from the subtraction library of sweet potato (Ipomoea batatas cv. Tainung 57) and used as a molecular probe to investigate the transduction pathway of wounding signal within plant cells. Following mechanical wounding of the leaves of sweet potato, IPO mRNA accumulation peaked at 6 h and then continuously declined. However, IPO gene expression in the apical unwounded leaves began at 6 h after wounding and continued for a further 10 h. Besides mechanical wounding, methyl jasmonate (MeJA) was identified as a signal transducer leading to the accumulation of IPO mRNA. Treatment with salicylic acid reduced the production of IPO mRNA, further supporting the involvement of the octadecanoid pathway in the signal transduction of wounding in sweet potato. In addition, ethylene was involved in the signal pathway and induced the expression of the IPO gene. Furthermore, the application of okadaic acid, a protein phosphatase inhibitor, blocked the accumulation of IPO mRNA induced by MeJA or ethylene, indicating that activation of the IPO gene by both MeJA and ethylene was via dephosphorylated proteins. The presence of a calcium ion chelator or channel blockers also inhibited the expression of the IPO gene after wounding. However, investigation by confocal scanning microscopy further pointed out that mechanical wounding rather than the application of MeJA induced the accumulation of the calcium ion. These results may indicate that the calcium ion is also involved in the activation of IPO mRNA. In addition, wounding signals the accumulation of calcium ion first and then stimulates the biosynthesis of MeJA in sweet potato. Hence, the reaction sequence of signal transducers, including the calcium ion, MeJA and protein kinase/phosphatase, in the wounding signalling pathway of sweet potato is suggested in this report.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.