Characterization of the early events of potato tuber development

The early events of potato ( Solanum tuberosum L. cv. Superior) tuberization were examined by using a model system of axillary bud tuber development from petiole-leaf single-node cuttings. Both fresh weight and starch accumulation were monitored to establish a developmental framework for morphological changes. Fresh weight and starch content began to increase in axillary buds after 2 days. Visible changes in bud morphology could be detected 4 days after the start of incubation. Substantial increases in both total protein and total RNA were observed at the onset of tuber morphology. Immunoblot analysis showed that the major tuber protein, patatin, could be initially detected in day 4 buds and that a 22-kDa proteinase inhibitor could be initially detected at day 8. Northern blot analysis corroborated this pattern of accumulation at the RNA level for both protein types. Substantial accumulation of the two proteinase inhibitor mRNAs occurred later than patatin mRNA accumulation. The results of this study showed that there is considerable accumulation of both protein and mRNA occurring during the early stages of tuber development prior to the substantial accumulation of the major tuber storage proteins.     

Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Effect of cadmium and Phytophthora infestans on polyamine levels in potato leaves

Changes in putrescine, spermidine and spermine concentrations in potato ( Solanum tuberosum L.) leaves stressed either with cadmium, with the fungal pathogen Phytophthora infestans Mont. (de Bary) or with both simultaneously were investigated. The leaves of two cultivars, Bintje and Bzura, respectively susceptible and resistant to P. infestans were examined. The level of of putrescine did not change under any stress conditions. Cadmium stress caused at least a 2-fold increase in spermidine and spermine concentrations in susceptible leaves. In resistant leaves there was a 4-fold increase in spermidine and a decrease in spermine. The pathogenic stress produced similar changes in polyamine concentrations, i. e. with differences between the cultivars. The double stress induced antagonistic alterations in the concentrations of spermidine and spermine.     

Tuber surface microorganisms influence the susceptibility of potato tubers to late blight

Potato tubers (cvs Cara and Bintje) were grown in compost in a glasshouse and immature tubers harvested 57, 68 and 78 days after planting. Two moisture levels were imposed after the first harvest by disconnecting the water supply to one of the treatments and allowing the soil in that treatment to dry naturally. Tubers from wetter compost (59.4% moisture holding capacity) were more resistant to Phytophthora infestans than those from drier compost (14.7% moisture holding capacity) 78 days after planting. The potential causes of this difference were investigated. Aqueous extracts of wet compost did not inhibit the growth of P. infestans. The susceptibility of the internal tuber tissue, from which the periderm had been removed, was different to whole tuber susceptibility. The internal tissue of tubers from wet compost was more susceptible (cv. Cara), or as susceptible (cv. Bintje) as that of tubers from dry compost 78 days after planting. Fungi were isolated from the surface of whole tubers and there were no differences between the populations of potentially antagonistic fungal genera on tubers from wet and dry compost. As the experiment progressed, the number of bacteria per gram fresh weight on tubers grown in wet compost increased, whereas that on tubers from drier compost decreased (cv. Bintje) or remained similar (cv. Cara). There were significantly (P= 0.008) more bacteria on the surface of tubers from wet compost 78 days after planting. When P. infestans was co-cultured in Petri dishes with randomly selected tuber surface bacteria, some isolates (≤ 16.7%) inhibited the growth of the fungus. The percentage of the total bacterial population that was antagonistic to P. infestans was not significantly affected by soil moisture level (P= 0.368). The greater numbers of bacteria, of which a proportion are antagonistic to P. infestans, on the surface of tubers grown in wet compost may account for the greater resistance to tuber blight in that instance.     

Jasmonic acid-inducible gene expression of a Kunitz-type proteinase inhibitor in potato tuber disks

Messenger RNAs of a potato (Solanum tuberosum L.) Kunitz-type proteinase inhibitor(s) (PKPI) were present in potato disks excised from tubers stored for 14 months (old tubers) or 2 months (young tubers) after harvest, and disappeared during the aseptic culture. The PKPI mRNA accumulation was found to be induced in potato disks from the old tubers by the addition of jasmonic acid (JA) [3-oxo-2-(2-cis-pentenyl)-cyclopentane-1-acetic acid].     

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Draga?evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.     

Change in phosphorylase isoenzymes during dedifferentiation of potato tuber cells

The phosphorylase isoenzyme composition of soluble preparations isolated from potato ( Solanum tuberosum L. cv. Spunta) tuber-derived callus has been studied by polyacrylamide gel electrophoresis and affinity electrophoresis. Native electrophoretic profiles indicate that dedifferentiated callus tissue contains a single form of phosphorylase that differs in primer requirement, charge and affinity towards branched α-1,4/1,6-glucans from the major phosphorylase form (phosphorylase II) in potato tuber. This latter molecular form is missing in dedifferentiated callus. However, callus phosphorylase appears to be closely related to tuber phosphorylase I, a minor form found in the original explant tissue.     

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.     

Memristors: Memory elements in potato tubers

A memristor is a nonlinear element because its current-voltage characteristic is similar to that of a Lissajous pattern for nonlinear systems. This element was postulated recently and researchers are looking for it in different biosystems. We investigated electrical circuitry of red Irish potato tubers (Solanum tuberosum L.). The goal was to discover if potato tubers might have a new electrical component - a resistor with memory. The analysis was based on a cyclic current-voltage characteristic where the resistor with memory should manifest itself. We found that the electrostimulation by bipolar sinusoidal or triangle periodic waves induces electrical responses in the potato tubers with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K⁺ channels, transforms a memristor to a resistor in potato tubers. Our results demonstrate that a voltage gated K⁺ channel in the excitable tissue of potato tubers has properties of a memristor. Uncoupler carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decreases the amplitude of electrical responses at low and high frequencies of bipolar periodic sinusoidal or triangle electrostimulating waves. The discovery of memristors in plants creates a new direction in the understanding of electrical phenomena in plants.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.     

Aspects of tuber resistance in hybrid potatoes to potato tuber worm

Tubers produced from crosses between the wild potato, Solanum berthaultii Hawkes (Solanaceae), and the cultivated species Solanum tuberosum L. (Solanaceae) are resistant to potato tuber worm (PTW), Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), infestation compared to those of the popular commercial North American cultivars Allegany, Atlantic, Chieftain, Katahdin, MaineChip, NorDonna, Norwis, Russet Norkotah, Snowden, and Yukon Gold. Given a choice between Atlantic and hybrid tubers, female PTW deposited ca. 50% fewer eggs on hybrid tubers than on those of Atlantic; larval survival and production of prepupae on hybrid tubers were reduced similarly. Time needed for neonates to penetrate eye buds was ca. 100 min greater on hybrid tubers compared to that on cv. Atlantic. Periderm of hybrid tubers is thicker than that of cv. Atlantic and may contribute to the delay in larval penetration of tubers and the success of initial establishment.     

Exogenously applied jasmonic acid induces changes in apical meristem morphology of potato stolons

Hooked apex stolons and initial swelling stolons of potato plants were treated with 3 x 10-8 mol l-1 jasmonic acid (JA) to study the effect of this compound on histology, cell expansion and tissue differentiation. In hooked apex stolons, JA application increased the meristem thickness and reduced the length of the leaf primordia, whereas in initial swelling stolons narrowing of the apical region, absence of leaf primordia and swelling of the subapical meristem were evident. Early vascular tissue differentiation was observed in response to JA treatment, especially of xylem elements from regions proximal to the tunic. Protoxylem elements, such as tracheal elements, were present with thin primary cell walls. The cell area was measured in two zones: zone I, central mother cells situated immediately under the tunic; and zone II, rib meristem cells. JA caused a four- and six-fold increase in cell area in both zones in hooked apex stolons and initial swelling stolons, respectively. Thus, tuber formation is concluded to occur as a consequence of increased cell expansion, a reduction in the length of leaf primordia, enlargement of meristems, and early vascular tissue differentiation.     

Comparative analysis of Phytophthora infestans induced gene expression in potato cultivars with different levels of resistance

Differential gene expression was analyzed after infection with Phytophthora infestans in six potato cultivars with different levels of resistance to late blight. To verify the infection of the potato leaflets, the amount of phytopathogen mRNA within the plant material was quantified by real-time quantitative PCR. The expression of 182 genes selected from two subtracted cDNA libraries was studied with cDNA array hybridization using RNA from non-infected and infected potato leaflets. Gene up- and down-regulation were clearly detectable in all cultivars 72 h post inoculation. Gene expression patterns in susceptible cultivars differed from those in potato varieties with a higher level of resistance. In general, a stronger gene induction was observed in the susceptible cultivars compared to the moderately to highly resistant potato varieties. Five genes with the highest homology to stress and/or defence-related genes were induced specifically in the susceptible cultivars. Four genes responded to pathogen attack independently of the level of resistance of the cultivar used, and three genes were repressed in infected tissue of most cultivars. Even in the absence of P. infestans infection, six genes showed higher expression levels in the somewhat resistant cultivars Bettina and Matilda. Possible reasons for the different levels of gene expression are discussed.     

Characterization of potato plants with reduced StSYR1 expression

Vesicle fusion processes in plants are important for both development and stress responses. Transgenic potato plants with reduced expression of SYNTAXIN-RELATED1 (StSYR1), a gene encoding the potato homolog of Arabidopsis PENETRATION1 (AtPEN1), display spontaneous necrosis and chlorosis at later stages of development. In accordance with this developmental defect, tuber number, weight and overall yield are significantly reduced in StSYR1-RNAi lines. Enhanced resistance of StSYR1-RNAi plants to Phytophthora infestans, the causal agent of late blight disease of potato, correlates with enhanced levels of salicylic acid, whereas levels of 12-oxophytodienoic acid and jasmonic acid are unaltered. Cultured cells of StSYR1-RNAi lines secrete at least two compounds which are not detectable in the supernatant of control cells, suggesting an involvement of StSYR1 in secretion processes to the apoplast.     

Postharvest changes in endogenous cytokinins and cytokinin efficacy in potato tubers in relation to bud endodormancy

Using an indirect enzyme‐linked immunosorbent assay (ELISA), the effects of postharvest storage duration and temperature on endogenous cytokinins in potato ( Solanum tuberosum L. cv. Russet Burbank) tuber apical bud tissues in relation to endodormancy status were determined. Following fractionation by HPLC, a total of eight cytokinins were detected and these were: zeatin riboside‐5'‐monophosphate (ZRMP), zeatin‐ O ‐glucoside (ZOG), zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine‐5'‐monophosphate (IPMP), isopentenyl adenine‐9‐glucoside (IP‐9‐G), isopentenyl adenine (IP) and isopentenyl adenosine (IPA). Regardless of postharvest storage temperature or endodormancy status, IP‐9‐G was the most abundant cytokinin detected while ZRMP and ZOG were the least abundant ones. In tubers preincubated at a growth‐permissive temperature (20°C) prior to extraction, the loss of endodormancy was preceded by significant increases in the endogenous levels of Z, ZR, IPMP and IP‐9‐G. When stored continuously at a growth‐inhibiting temperature (3°C), significant increases in ZR, IP‐9‐G and IP + IPA were observed. The total content of cytokinins increased by over 7‐fold during postharvest storage and this increase was a result of de novo biosynthesis. Dose‐response studies using IPA and ZR demonstrated a time‐dependent increase in apparent cytokinin sensitivity during postharvest storage. With the exception of IP‐9‐G, injection of any of these endogenous cytokinins resulted in the rapid and complete termination of tuber endodormancy. The significance of these results with respect to endodormancy regulation and the possible mechanisms controlling cytokinin levels in potato tubers are discussed.     

Temporal dynamics of tuber formation and related processes in a crossing population of potato (Solanum tuberosum)

The chronological relationships between stolon formation, stolon tip swelling, tuber initiation, flowering, senescence, growth and resorption of tubers were studied under field conditions in a diploid population of potato with 238 genotypes, the parental clones and seven tetraploid cultivars. Timing of tuber initiation was not closely related to the timing of stolon formation, flowering and duration of the plant cycle. Tuber initiation very often preceded stolon branching. The number and size distribution of tubers were largely influenced by the degree of stolon branching, the length of the stolon swelling period and tuber resorption. The peak production of stolons and swollen stolon tips largely took place within the flowering period, although in most genotypes, some stolon tip swelling took place until the end of the plant cycle. More information on the general temporal relationships between events related to tuber formation and plant development will contribute to a better understanding of the physiological and genetic basis of the processes leading to the production of harvestable tubers.     

Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S . tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes ( AtCBF1-3 ) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 °C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.