Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.Abbreviation ssRubisco small subunit of ribulose-1,5-bis-phosphate carboxylase     

Abscisic acid-deficient plants do not accumulate proteinase inhibitor II following systemin treatment

The role of systemin inPin2 gene expression was analyzed in wild-type plants of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.), as well as in abscisic acid (ABA)-deficient tomato (sitiens) and potato (droopy) plants. The results showed that systemin initiates Pin2 mRNA accumulation only in wildtype tomato and potato plants. As in the situation after mechanical wounding,Pin2 gene expression in ABA-deficient plants was not activated by systemin. Increased endogenous levels of jasmonic acid (JA) and accumulation of Pin2 mRNA were observed following treatment with α-linolenic acid, the precursor of JA biosynthesis, suggesting that these ABA mutants still have the capability to synthesize de novo JA. Measurement of endogenous levels of ABA and JA showed that systemin leads to an increase of both phytohormones (ABA and JA) only in wild-type but not in ABA-deficient plants.     

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.).     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants

The introduction of an antisense DNA into transgenic potato (Solanum tuberosum L.) plants decreased the expression of the chloroplast triose-phosphate translocator and lowered its activity by 20–30%. With plants propagated from tubers, the effect of the transformation on photosynthetic metabolism was analysed by measuring photosynthesis, the formation of leaf starch, and the total and subcellular metabolite contents in leaves. Although the transformants, in contrast to those propagated from cell cultures, did not differ from the wild-type plants in respect to rates of photosynthesis, plant appearance, growth and tuber production, their photosynthetic metabolism was found to be severely affected. The results show that the decrease in activity of the triose-phosphate translocator in the transformants caused a fourfold increase in the level of 3-phosphoglycerate and a corresponding decrease in inorganic phosphate in the stromal compartment, resulting in a large increase in the synthesis of starch. Whereas during a 12-h day period wild-type plants deposited 43% of their CO₂ assimilate into starch, this value rose to 61–89% in the transformants. In contrast to the wild-type plants, where the rate of assimilate export from the leaves during the night period was about 75% of that during the day, the export rate from leaves of transformants appeared to be much higher during the night than during the day. As the mobilisation of starch occurs in part hydrolytically, resulting in the formation of glucose, the triose-phosphate translocator loses its exclusive function in the export of carbohydrates from the chloroplasts when the photoassimilates are temporarily deposited as starch. It appears that by directing the CO₂ assimilates mainly into starch, the transformants compensate for the deficiency in triose-phosphate translocator activity in such a way that the productivity of the plants is not affected by the transformation.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - 3-PGA 3-phosphoglycerate - Rubisco ribulose,1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - trioseP triose phosphate - WT wild type The able technical assistance of Mrs. K. Wildenberger and Mrs. A. Großpietsch is gratefully acknowledged. This work has been supported by the Bundesminister für Forschung und Technologie.     

The use and limits of AFLP data in the taxonomy of polyploid wild potato species in Solanum series Conicibaccata

Solanum sect. Petota (tuber-bearing wild and cultivated potatoes) are a group of approximately 190 wild species distributed throughout the Americas from the southwestern United States south to Argentina, Chile, and Uruguay. Solanum series Conicibaccata are a group of approximately 40 species within sect. Petota, distributed from central Mexico to central Bolivia, composed of diploids (2n = 2x = 24), tetraploids (2n = 4x = 48) and hexaploids (2n = 6x = 64); the polyploids are thought to be polysomic polyploids. This study initially was designed to address species boundaries of the four Mexican and Central American species of series Conicibaccata with AFLP data with the addition of first germplasm collections of one of these four species, Solanum woodsonii, as a follow-up to prior morphological, chloroplast DNA, and RAPD studies; and additional species of series Conicibaccata from South America. AFLP data from 12 primer combinations (1722 polymorphic bands) are unable to distinguish polyploid species long thought to be distinct. The data suggest a complex reticulate history of the tetraploids or the need for a broad downward reevaluation of the number of species in series Conicibaccata, a trend seen in other series of sect. Petota. Separately, through flow cytometry, we report the first ploidy level of S. woodsonii, as tetraploid (2n = 48). The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Interaction between photorespiration and respiration in transgenic potato plants with antisense reduction in glycine decarboxylase

Potato (Solanum tuberosum L. cv. Désirée) plants with an antisense reduction in the P-protein of the glycine decarboxylase complex (GDC) were used to study the interaction between respiration and photorespiration. Mitochondria isolated from transgenic plants had a decreased capacity for glycine oxidation and glycine accumulated in the leaves. Malate consumption increased in leaves of GDC deficient plants and the capacity for malate and NADH oxidation increased in isolated mitochondria. A lower level of alternative oxidase protein and decreased partitioning of electrons to the alternative pathway was found in these plants. The adenylate status was altered in protoplasts from transgenic plants, most notably the chloroplastic ATP/ADP ratio increased. The lower capacity for photorespiration in leaves of GDC deficient plants was compensated for by increased respiratory decarboxylations in the light. This is interpreted as a decreased light suppression of the tricarboxylic acid cycle in GDC deficient plants in comparison to wild-type plants. The results support the view that respiratory decarboxylations in the light are restricted at the level of the pyruvate dehydrogenase complex and/or isocitrate dehydrogenase and that this effect is likely to be mediated by mitochondrial photorespiratory products.     

Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein

The expression patterns of plant defense genes encoding osmotin and osmotin-like proteins imply a dual function in osmotic stress and plant pathogen defense. We have produced transgenic potato (Solanum commersonii Dun.) plants constitutively expressing sense or antisense RNAs from chimeric gene constructs consisting of the cauliflower mosaic virus 35S promoter and a cDNA (pA13) for an osmotin-like protein. Transgenic potato plants expressing high levels of the pA13 osmotin-like protein showed an increased tolerance to the late-blight fungus Phytophthora infestans at various phases of infection, with a greater resistance at an early phase of fungal infection. There was a decrease in the accumulation of osmotin-like mRNAs and proteins when antisense transformants were challenged by fungal infection, although the antisense transformants did not exhibit any alterations in disease susceptibility. Expression of pA13 sense and antisense RNAs had no effect on the development of freezing tolerance in transgenic plants when assayed under a variety of conditions including treatments with abscisic acid or low temperature. These results provide evidence of antifungal activity for a potato osmotin-like protein against the fungus P. infestans, but do not indicate that pA13 osmotin-like protein is a major determinant of freezing tolerance.     

Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding

The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.Abbreviations BA 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog (1962) - 2,4-d dichlorophenoxyacetic acid     

Proteinase Inhibitors in the Nonvenomous Defensive Secretion of Grasshoppers: Antiproteolytic Range and Possible Significance

The defensive secretion produced in the metathoracic tracheal glands of the grasshoppers, Romalea guttata and Taeniopoda eques, contains a diversity of proteinase inhibitors. The exudate contains inhibitors of trypsin, chymotrypsin, cathepsin G, papain and pancreatic and leukocyte elastases. The inhibitory profile of the secretion differs markedly from that of the hemolymph both, in terms of antiproteolytic activities and isoinhibitor composition. We suggest that these proteinase inhibitors may constitute a complementary defense system, for example, directed against diverse entomopathogenic fungi that characteristically attack grasshoppers.     

Jasmonic acid spraying does not induce tuberisation in short-day-requiring potato species kept in non-inducing conditions

The potato species Solanum andigena (Juz. and Buk.) and Solanum demissum (Lindl.) that both require short days for tuberisation were kept in either long days (16 h light), or short days (8 h light) with a 30-min night break mid-way through the dark period. Tuberisation of these species was inhibited under both conditions. Repeated spraying of these plants with up to 100 μM jasmonic acid did not induce them to tuberise even though jasmonic acid was shown to be taken up and transported within the plant. This result argues against jasmonic acid itself being the transported tuber-inducing signal, although it does not exclude a role for jasmonic acid later in tuber formation and development once induction has taken place.     

Protocol for DNA extraction from potato tubers

A method to extract high-quality DNA from potato tubers was developed and tested on 3 wild potato species (Solanum raphanifolium, S. megistracrolobum, S. bukasovii) and on the tetraploid B3 bred population, (population number 393228, derived fromS. tuberosum subsp.tuberosum). The average yield of extracted DNA varied from 10–30 μg of DNA per gram of processed tissue. The DNA was pure and suitable for ligation-mediated polymerase chain reaction (LM-PCR) amplification, producing clear, distinctive, and reproducible banding patterns in polyacrylamide gels.     

Heterologous expression of a<Emphasis Type="Italic"> keto</Emphasis>hexokinase in potato plants leads to inhibited rates of photosynthesis,severe growth retardation and abnormal leaf development

In the present paper we investigated the effect of heterologous expression of a rat liver ketohexokinase in potato (Solanum tuberosum L.) plants with the aim of investigating the role of fructose 1-phosphate in plant metabolism. Plants were generated that contained appreciable activity of ketohexokinase but did not accumulate fructose 1-phosphate. They were, however, characterised by a severe growth retardation and abnormal leaf development. Studies of ¹⁴CO₂ assimilation and metabolism, and of the levels of photosynthetic pigments, revealed that these lines exhibited restricted photosynthesis. Despite this fact, the levels of starch and soluble sugars remained relatively constant. Analysis of intermediates of starch and sucrose biosynthesis revealed large increases in the triose phosphate and fructose 1,6-bisphosphate pools but relatively unaltered levels of inorganic phosphate and 3-phosphoglycerate, and these lines were also characterised by an accumulation of glyceraldehyde. The transformants neither displayed consistent changes in the activities of Calvin cycle enzymes nor in enzymes of sucrose synthesis but displayed a metabolic profile partially reminiscent of that brought about by end-product limitation, but most likely caused by an inhibition of photosynthesis brought about by the accumulation of glyceraldehyde. Analysis of the metabolite contents in lamina and vein fractions of the leaf, and of the enzymes of carbohydrate oxidation indicate that the phloem-enriched veins of ketohexokinase-expressing leaves tend toward hypoxia and indicate a problem of phloem transport.Abbreviations CaMV Cauliflower mosaic virus - DHAP Dihydroxyacetone phosphate - F1P Fructose 1-phosphate - FBP Fructose 1,6-bisphosphate - KHK Ketohexokinase - NADP-GAPdH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFP Pyrophosphate: fructose 6-phosphate 1-phosphotransferase - 3PGA 3-Phosphoglycerate - PEP Phosphoenolpyruvate - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase - SPS Sucrose phosphate synthase - SuSy Sucrose synthase     

Allelism of endosperm balance number (EBN) in Solanum acaule Bitt. and other wild potato species

The inheritance of endosperm balance number (EBN), a genetic, dose-dependent crossability system functioning in tuber-bearing Solanum (potato) species, was investigated for certain wild potato species having an EBN equal to one half of their ploidy. The EBN of Solanum acaule, a disomic 4(2EBN) South American species, was investigated by producing F₁ and F₂ hybrids with artificial 4x(2EBN) S. commersonii. This allowed assessment of recombination among the two genomes of disomic S. acaule and that of S. commersonii. When crossability of the hybrids with 1EBN, 2EBN and 4EBN standards was tested, no variation for EBN was detected. The apparent lack of recombination and segregation for EBN in these hybrids indicates that the genomes of S. acaule and S. commersonii carry EBN in a genetically-similar way. Combined with previous reports, these data indicate that the inheritance of EBN is similar in widely-separated taxa from South America and Mexico.     

Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers

The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose K _m=64, 90 and 100 (M) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (K _m=26, 25 and 240 M) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (K _i=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the K _i (ADP) was about threefold below the K _m (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (K _i=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (K _m=41, 130 and 35 M) and mannose but a poor affinity for fructose (K _m=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (K _m=90, 280 and 560 M) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (K _i=40 and 108 M) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (K _i=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.Abbreviations FK fructokinase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - HK hexokinase - NTP nucleoside triphosphate - P_i inorganic phosphate - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Froschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Olfaction in the Colorado potato beetle: Ultrastructure of antennal sensilla inLeptinotarsa sp

Sensillae on the antennae of the Colorado potato beetle,Leptinotarsa decemlineata are described using scanning (SEM) and transmission (TEM) electron microscopy and compared with SEM observations of antennal sensilla inL. haldemani andL. texana. In all the three species, 13 distinct sensillar types were identified with a higher density of sensilla in the more polyphagous species,L. decemlineata than in the moderately host specificL. haldemani and the highly host specificL. texana. Cuticular specializations and the predominance of olfactory sensilla are discussed in relation to host specificity in the three species.     

Injury to potato leaves exposed to subzero temperatures in the absence of freezing

Electrolyte leakage was measured in hardened and nonhardened leaves of three potato species, Solanum tuberosum L., S. acaule Bitt. and S. commersonii Dun., and one interspecific cross, Alaska Frostless (S. acaule x S. tuberosum) when exposed to various subzero temperatures. The leaves were undercooled (no ice present) from 0°C to -12.5°C for 45 min and to-4°C for up to 10 d. Regardless of the degree of undercooling no injury was observed in any of the potatoes, hardened or nonhardened, for up to 12 h. After 5 d, however, electrolyte leakage was observed in hardened S. tuberosum, S. acaule and S. commersonii, and in nonhardened Alaska Frostless. After 10 d exposure all potatoes, hardened and nonhardened, showed a significant amount of electrolyte leakage as compared to their controls kept at 0°C for 10 d.Scientific Journal Series Paper No. 13842 of the Minnesota Agricultural Experiment Station, St. Paul, Minn     

Characterisation of a gene that is expressed in leaves at higher levels upon tuberisation in potato and upon flowering in tobacco

A partial cDNA clone has been isolated which represents a gene that is induced in potato (Solanum tuberosum L.) leaves upon tuber initiation. The gene is also present in tobacco (Nicotiana tobacum L.) where it is induced upon flowering, and in tomato (Lycopericon esculentum, L.) where it is induced when the plant starts to form fruit. This provides further evidence for the similarity of the floral-induction and tuber-induction pathways, and raises the possibility that this gene may be involved in a common step in both of these pathways. Alternatively, it could be involved in a secondary process that is induced during tuberisation, flowering and fruit development.