POTATO LEAF EXTRACT AND ITS COMPONENT, α‐SOLANINE,EXERT SIMILAR IMPACTS ON DEVELOPMENT AND OXIDATIVE STRESS IN Galleria mellonella L.

Plants synthesize a broad range of secondary metabolites that act as natural defenses against plant pathogens and herbivores. Among these, potato plants produce glycoalkaloids (GAs). In this study, we analyzed the effects of the dried extract of fresh potato leaves (EPL) on the biological parameters of the lepidopteran, Galleria mellonella (L.) and compared its activity to one of the main EPL components, the GA α‐solanine. Wax moth larvae were reared from first instar on a diet supplemented with three concentrations of EPL or α‐solanine. Both EPL and α‐solanine affected survivorship, fecundity, and fertility of G. mellonella to approximately the same extent. We evaluated the effect of EPL and α‐solanine on oxidative stress in midgut and fat body by measuring malondialdehyde (MDA) and protein carbonyl (PCO) contents, both biomarkers of oxidative damage. We evaluated glutathione S‐transferase (GST) activity, a detoxifying enzyme acting in prevention of oxidative damage. EPL and α‐solanine altered MDA and PCO concentrations and GST activity in fat body and midgut. We infer that the influence of EPL on G. mellonella is not enhanced by synergistic effects of the totality of potato leaf components compared to α‐solanine alone.     

Synergistic interaction between the potato glycoalkaloids α-solanine and α-chaconine in relation to lysis of phospholipid/sterol liposomes

At pH 7.2, the steroidal glycoalkaloid α-chaconine disrupted phosphatidylcholine/cholesterol liposomes whereas α-solanine was virtually without effect. A glycoalkaloid mixture extracted from potato sprouts and comprising approximately equal amounts of solanine and chaconine had, at 150 μM, a lytic effect the same as a 150 μM solution of chaconine only. The apparent synergistic interaction between the two compounds was confirmed using 1:1 mixtures of authentic solanine and chaconine from different sources and of different batches. Combinations (1:1) of solanine or chaconine and tomatine or digitonin (both of which lysed liposomes) or β₂-chaconine (which is non-lytic) did not produce synergistic effects. The synergism between solanine and chaconine was observed only when the two compounds were present together, although the order of addition into the test system did not appear crucial. Pretreatment of liposomes with one glycoalkaloid and its subsequent removal did not permanently sensitize the membranes to the second glycoalkaloid. The magnitude of the synergism was dependent on the relative amounts of solanine and chaconine with maximal effects where chaconine comprised 40% or more of the mixture.     

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.     

Phytochrome B mediates the photoperiodic control of tuber formation in potato

To determine whether phytochrome B is involved in the response of potato plants to photoperiod, a potato PHYB cDNA fragment was inserted in the antisense orientation behind the 35S CaMV promoter in Bin19 and this construct was transformed into Solanum tuberosum ssp. andigena plants which normally require short days for tuberization. Two independent transformants were obtained that had much lower levels of PHYB mRNA and protein, and which exhibited phenotypes characteristic of phyB mutants, for example, elongated stems and decreased chlorophyll content. The level of phyA, and of several phytochrome A-controlled responses, was unaffected in these plants. The photoperiodic control of tuberization in these antisense PHYB plants was abolished, the plants tuberizing in short day, long day, or short day plus night break conditions. This result shows that phytochrome B is required for the photoperiodic control of tuberization in potato ( Solanum tuberosum ssp. andigena ) and that it regulates this developmental process by preventing tuber formation in non-inductive photoperiods rather than by promoting tuberization in inductive photoperiods.     

Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics

GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA₁ and GA₄. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato.     

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the ??cytokinin deficiency syndrome?? were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.     

Possible involvement of jasmonates in various morphogenic events

Jasmonates (jasmonic acid and related compounds) seem to be involved in various morphogenic events of plants, such as tuberization (potato, yam and Jerusalem artichoke), tuberous root formation (sweet potato), bulb formation (onion and garlic), determination of plant structure (soybean) and thigmomorphogenesis (coiling of tendrils of Bryonia dioica ). The involvement of jasmonates in tuberization in these plants was inferred from their ability to induce tubers in vitro, and from changes in the levels of endogenous jasmonates during the growth of the plants, which can account for the initiation of tuberization. As to potato tuberization, jasmonic acid (JA) and its methyl ester (JA-Me) have strong tuber-inducing activity. These compounds seem to exert their tuber-inducing effects by elicting the expansion of cells, because JA and JA-Me are capable of causing the expansion of cells in potato tubers. The JA-induced expansion of cells is attributable to both an increase in osmotic pressure due to the accumulation of sucrose and changes in cell wall architecture that appear to affect the extensibility of the wall. And, moreover, the synthesis of cellulose might be indispensable for the JA-induced expansion. The tuberization and the expansion of cells induced by JA always involve the reorientation of cortical microtubules (MTs), suggesting that JA controls the direction of cell expansion by changing the arrangement of MTs. However, the reorientation of MTs itself seems to be insufficient for the induction of expansion of cells.
Involvement of jasmonates in bulb formation and tuberous root formation is presumed from the fact that JA is able to induce these in vitro. The exact nature of the control that the jasmonates exert on morphogenesis remains to be elucidated.     

Blue Light Inhibition of Tuberization in a Day-Neutral Potato

In tests on the effects of light quality on potato tuberization, continuous blue light was found to consistently inhibit tuberization of tissue-cultured plantlets of Solanum tuberosum ssp. tuberosum cv. ??Norland??. Other tested cultivars, including sports of ??Norland??, formed tubers under continuous blue light. Microarrays identified BL, GA7ox, and Nudix genes as exhibiting altered expression in response to blue light treatment. Quantitative RT-PCR (qRT-PCR) showed that GA7ox RNA increased in ??Norland?? but not in ??Sangre?? plantlets in blue light compared to darkness. RNA levels of genes identified in the literature as having roles in potato tuberization were also measured using qRT-PCR. Levels of GA20o1x, but not GA2ox, RNA increased in response to blue light in ??Norland?? plantlets. BEL5 RNA content was greater under blue light compared to darkness for both ??Norland?? and ??Sangre?? plants. Levels of FT were not significantly different in blue light compared to dark-treated ??Norland?? plants, but were low in blue light-treated compared to dark-treated ??Sangre?? plants. Addition of ancymidol to ??Norland?? plants exposed to blue light overcame blue light inhibition of tuberization. Ancymidol prevents the oxidation of ent-kaurene to ent-kaurenoic acid, thus inhibiting gibberellin biosynthesis. These data suggest that blue light may increase GA accumulation in ??Norland?? plants, as has been shown to occur in Arabidopsis plants. The novel effect of blue light in inhibiting tuberization of ??Norland?? plants suggests that this system could be a useful tool in further elucidating the mechanisms of day-neutral potato tuberization.