Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.     

Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative Proteomics

Stable Isotope Labelling by Amino acids in Cell culture (SILAC) is a powerful technique for comparative quantitative proteomics, which has recently been applied to a number of different eukaryotic organisms. Inefficient incorporation of labelled amino acids in cell cultures of Arabidopsis thaliana has led to very limited use of SILAC in plant systems. We present a method allowing, for the first time, efficient labelling with stable isotope-containing arginine and lysine of whole Arabidopsis seedlings. To illustrate the utility of this method, we have combined the high labelling efficiency (>95%) with quantitative proteomics analyses of seedlings exposed to increased salt concentration. In plants treated for 7 days with 80 mM NaCl, a relatively mild salt stress, 215 proteins were identified whose expression levels changed significantly compared to untreated seedling controls. The 92 up-regulated proteins included proteins involved in abiotic stress responses and photosynthesis, while the 123 down-regulated proteins were enriched in proteins involved in reduction of oxidative stress and other stress responses, respectively. Efficient labelling of whole Arabidopsis seedlings by this modified SILAC method opens new opportunities to exploit the genetic resources of Arabidopsis and analyse the impact of mutations on quantitative protein dynamics in vivo.     

Uptake of ant-derived nitrogen in the myrmecophytic orchid Caularthron bilamellatum

Background and Aims Mutualistic ant-plant associations are common in a variety of plant families. Some myrmecophytic plants, such as the epiphytic orchid Caularthron bilamellatum, actively form hollow structures that provide nesting space for ants (myrmecodomatia), despite a substantial loss of water-storage tissue. This study aimed at assessing the ability of the orchid to take up nitrogen from ant-inhabited domatia as possible trade-off for the sacrifice of potential water storage capacity. Methods Nitrogen uptake capabilities and uptake kinetics of (15)N-labelled compounds (NH(4)(+), urea and l -glutamine) were studied in field-grown Caularthron bilamellatum plants in a tropical moist forest in Panama. Plants were either labelled directly, by injecting substrates into the hollow pseudobulbs or indirectly, by labelling of the associated ants in situ. Key Results Caularthron bilamellatum plants were able to take up all tested inorganic and organic nitrogen forms through the inner surface of the pseudobulbs. Uptake of NH(4)(+) and glutamine followed Michaelis-Menten kinetics, but urea uptake was not saturable up to 2 mm. (15)N-labelled compounds were rapidly translocated and incorporated into vegetative and reproductive structures. By labelling ants with (15)N in situ, we were able to prove that ants transfer N to the plants under field conditions. Conclusions Based on (15)N labelling experiments we were able to demonstrate, for the first time, that a myrmecophytic orchid is capable of actively acquiring different forms of nitrogen from its domatia and that nutrient flux from ants to plants does indeed occur under natural conditions. This suggests that beyond anti-herbivore protection host plants benefit from ants by taking up nitrogen derived from ant debris.     

Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase

Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m^–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.     

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect‐resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad‐spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI‐expressing and control (comparator) lines, followed by a two‐dimensional gel electrophoresis (2‐DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI‐expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2‐DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2‐DE showed unaltered deposition patterns in SlCDI‐expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI‐expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel‐generation transgenic plants.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Treatment of potato tubers with a growth promotingPseudomonas sp.: Plant growth responses and bacterium distribution in the rhizosphere

Rifampicin-nalidixic acid resistant mutants of a plant growth promotingPseudomonas sp., strain PsJN, were evaluated for their ability to stimulate in vitro growth of potato. Two mutant strains, MFE (a consistent growth promoter), and IIM15 (an inconsistent growth promoter), were selected for root colonization study. Root colonization of potato plants was consistently greater with MFE than with IIM15. The population density of indigenous bacteria on the root surface of potato plants inoculated with strain MFE was significantly lower as compared to non-bacterized controls and to the plants bacterized with strain IIM15. Soil sterilization did not affect plant growth in any of the treatments. Bacterization of seed tubers with strain MFE stimulated plant emergence and root development in the field, during the first two weeks after planting. Bacterized plants also formed stolons and tubers earlier and had increased yields of commercial size tubers (55 mm) as compared to non-bacterized controls. Root colonization by strain MFE was positively correlated with plant growth stimulation.     

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology,for the production of human-therapeutic glycoproteins

Background  

Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.     

Association of Bactericera cockerelli (Homoptera: Psyllidae) with "zebra chip," a new potato disease in southwestern United States and Mexico

A new defect of potato, Solanum tuberosum L., "zebra chip," so named for the characteristic symptoms that develop in fried chips from infected potato tubers, has recently been documented in several southwestern states of the United States, in Mexico, and in Central America. This defect is causing millions of dollars in losses to both potato producers and processors. Zebra chip plant symptoms resemble those caused by potato purple top and psyllid yellows diseases. Experiments were conducted to elucidate the association between the psyllid Bactericera cockerelli (Sulc) (Homoptera: Psyllidae) and zebra chip by exposing clean potato plants to this insect under greenhouse and field conditions. Potato plants and tubers exhibiting zebra chip symptoms were tested for phytoplasmas by polymerase chain reaction. Potato psyllids collected from infected potato fields also were tested. Results indicated that there was an association between the potato psyllid and zebra chip. Plants exposed to psyllids in the greenhouse and field developed zebra chip. In the greenhouse, 25.8 and 59.2% of tubers exhibited zebra chip symptoms in the raw tubers and fried chips, respectively. In the field, 15 and 57% of tubers showed symptoms in raw tubers and chips, respectively. No zebra chip was observed in tubers from plants that had not been exposed to psyllids, either in the greenhouse or field. No phytoplasmas were detected from potato plants or tubers with zebra chip symptoms, suggesting that these pathogens are not involved in zebra chip. Of the 47 samples of potato psyllids tested, only two tested positive for the Columbia Basin potato purple top phytoplasma.     

High-throughput screening of structural proteomics targets using NMR

We applied a high-throughput strategy for the screening of targets for structural proteomics of Xanthomonas axonopodis pv citri. This strategy is based on the rapid (1)H-(15)N HSQC NMR analysis of bacterial lysates containing selectively (15)N-labelled heterologous proteins. Our analysis permitted us to classify the 19 soluble candidates in terms of 'foldedness', that is, the extent to which they present a well-folded solution structure, as reflected by the quality of their NMR spectra. This classification allowed us to define a priority list to be used as a guide to select protein candidates for further structural studies.     

DNA repair in plant mitochondria – a complete base excision repair pathway in potato tuber mitochondria

Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg²⁺ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.     

Evaluation of different multidimensional LC-MS/MS pipelines for isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of potato tubers in response to cold storage

Cold-induced sweetening in potato tubers is a costly problem for the food industry. To systematically identify the proteins associated with this process, we employed a comparative proteomics approach using isobaric, stable isotope coded labels to compare the proteomes of potato tubers after 0 and 5 months of storage at 5 °C. We evaluated both high pH reverse phase (hpRP) liquid chromatography (LC) and off-gel electrophoresis (OGE) as first dimension fractionation methods followed by nanoLC-MS/MS, using two high performance mass spectrometry platforms (Q-TOF and Orbitrap). We found that hpRP-LC consistently offered better resolution, reduced expression ratio compression, and a more MS-compatible workflow than OGE and consistently yielded more unique peptide/protein identifications and higher sequence coverage with better quantification. In this study, a total of 4463 potato proteins were identified, of which 46 showed differential expressions during potato tuber cold storage. Several key proteins important in controlling starch-sugar conversion, which leads to cold-induced sweetening, as well as other proteins that are potentially involved in this process, were identified. Our results suggest that the hpRP-RP shotgun approach is a feasible and practical workflow for discovering potential protein candidates in plant proteomic analysis.     

A Low Temperature Therapy and Meristem-Tip Culture for Eliminating four Viroids from Infected Plants

Viroid-free potato and chrysanthemum plants were obtained from meristem-tips cut from potato spindle tuber viroid-infected potato plants and from chrysanthemum plants infected with chrysanthemum stunt, chrysanthemum chlorotic mottle or cucumber palefruit viroids after 6 months therapy in a growth chamber at 5 °C and 16 hours daily light of 5.000 lx intensity. Chrysanthemum plants survived quite well the conditions of therapy while potato plants grown from stem cuttings survived these conditions much worse and potato plants grown from tubers did not survive these conditions. PSTV-free plants were obtained from meristem-tips cut from sprouts grown from potato tubers infected with severe (s-PSTV) or mild (m-PSTV) strains of potato spindle tuber viroid after 6 months therapy at 6–7 °C in the dark. The tubers survived these conditions quite well. The 3 months therapy period was found too short for any plant material. The efficiency of 6 months therapy in viroid elimination varied for different viroids and different plant material from 18.5 to 80.0 %.     

Accumulation of vitamin E in potato (Solanum tuberosum) tubers

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). α-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in α-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less α-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.     

Zebra chip disease incidence on potato is influenced by timing of potato psyllid infestation, but not by the host plants on which they were reared

Abstract  The Zebra chip (ZC) syndrome is an emerging disease of potato and a major threat to the potato industry. The potato psyllid, Bactericerca cockerelli (Sulc) is believed to be a vector of the ZC pathogen, which is now thought to be Candidatus Liberibacter, a bacterium. To further understand the relationship between potato psyllid infestation and ZC disease expression, healthy potato plants at different growth stages (4, 6 and 10 weeks after germination) were exposed separately to potato psyllids that were separately reared on four solanaceous hosts plants (potato, tomato, eggplant or bell pepper) for more than 1 year. ZC symptoms, leaf rates and total nonstructural carbohydrate accumulation in leaves and tubers of healthy and psyllid-infested plants were monitored and recorded. Typical ZC symptoms were observed in leaves and tubers of all plants exposed to potato psyllids regardless of the host plant on which they were reared. This was also accompanied by significant reductions in net photosynthetic rate. Caged potato plants without exposure to potato psyllids (uninfested controls) did not show any ZC symptom in both foliage and in harvested tubers. Foliage damage and ZC expression were most severe in the potato plants that were exposed to potato psyllids 4 weeks after germination compared to plants infested at later growth stages. Tubers from potato psyllid-infested plants had significantly higher levels of reducing sugars (glucose) and lower levels of starch than those in healthy plants, indicating that potato psyllid infestation interfered with carbohydrate metabolism in either leaves or tubers, resulting in ZC expression.     

Recent developments in solid-state magic-angle spinning, nuclear magnetic resonance of fully and significantly isotopically labelled peptides and proteins

In recent years, a large number of solid-state nuclear magnetic resonance (NMR) techniques have been developed and applied to the study of fully or significantly isotopically labelled ((13)C, (15)N or (13)C/(15)N) biomolecules. In the past few years, the first structures of (13)C/(15)N-labelled peptides, Gly-Ile and Met-Leu-Phe, and a protein, Src-homology 3 domain, were solved using magic-angle spinning NMR, without recourse to any structural information obtained from other methods. This progress has been made possible by the development of NMR experiments to assign solid-state spectra and experiments to extract distance and orientational information. Another key aspect to the success of solid-state NMR is the advances made in sample preparation. These improvements will be reviewed in this contribution. Future prospects for the application of solid-state NMR to interesting biological questions will also briefly be discussed.     

Role of proteinase inhibitors in potato protection

Mechanical damage or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.     

Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot

Potato is the world's largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora. Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.