A potato cDNA encoding a homologue of mammalian multidrug resistant P-glycoprotein

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with³⁵S-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170–180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.     

Hydroxycinnamoylputrescines are not causally involved in the tuberization process in potato plants

The possible role of hydroxycinnamoylputrescines in the tuberization process of potato plants was studied using in vitro tuberization systems. Minitubers in shoot cultures of Solanum tuberosum ssp. andigena and S. tuberosum ssp. tuberosum were obtained in vitro within 3 weeks of dark incubation after increasing the sucrose concentration in the Murashige-Skoog (T. Murashige and F. Skoog. 1962. Physiol. Plant. 15: 473–497.) medium (without hormones) from 60 to 240 m M . both in the presence and absence of benzylaminopurine (BAP). Feruloylputrescine (FP) and caffeoylputrescine (CP) increased with tuberization, with a sharp maximum at day 9 in the shoot, but only when the medium contained BAP. When inhibitors of phenylalanine ammonia-lyase (PAL) and of polyamine biosynthesis were added to the medium containing BAP, the levels of FP and CP were reduced to values lower than those observed in the absence of BAP, but there was no significant effect on the number and dry weight of tubers formed. Addition of BAP without increasing the sucrose content also resulted in CP and FP accumulation. but failed to induce tuberization of the cultures. Experiments with in vitro stolon cultures and leaf cuttings also supported the conclusion that CP and FP accumulated as a response to the application of BAP, without having any effect on optimal tuberization. These results indicate that the increase of hydroxycinnamoylputrescines during tuber formation is unlikely to be causally involved in the tuberization process in potato plants.     

Theobroxide Triggers Jasmonic Acid Production to Induce Potato Tuberization in vitro

Theobroxide, a novel compound isolated from fungus Lasiodiplodia theobromae culture, stimulates potato (Solanum tuberosum L.) tuber formation in vitro and in vivo, and induces flowering of Japanese morning glory (Pharbitis nil) under non-inductive long day conditions. To assess the mode of action of theobroxide in the tuberization process we measured endogenous levels of jasmonic acid and lipoxygenase activity in the cultures after the treatment with theobroxide. The results showed that theobroxide not only stimulated microtuber formation alone, but also enhanced the inductive effect of jasmonic acid when they were used in combination. The endogenous JA content increased in response to theobroxide in both old and new tissues. Moreover, theobroxide increased lipoxygenase activity in the cultures, at 2 and 3 week after culture initiation. Additionally, histological observations indicated that theobroxide might play a role in the swelling of micro-tubers formed in vitro in a similar manner as that of jasmonic acid. These results suggest that the inductive effect of theobroxide on potato micro-tuber formation might be achieved through triggering jasmonic acid production.     

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.     

Carboxylic acids affect induction,development and quality of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) microtubers grown <Emphasis Type="Italic">in vitro</Emphasis> from single-node explants

The role of three carboxylic acids with increasing alkyl-chain length, viz., formic, acetic and propionic acids in microtuberization was investigated in three potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of these carboxylic acids (0.0, 1.5, 3.0, 4.5 and 6.0 mM) were supplemented in microtuber induction medium, which was based on MS medium containing 8% sucrose, and their efficacy for induction, development and quality of microtubers was studied using single-node explants under continuous darkness at 20 °C. The carboxylic acids exhibited a strong stolon- and root-inhibiting effect on single-node explants with their increasing concentrations as well as alkyl-chain length (i.e., formic < acetic < propionic acids), and their mode of action was synonymous with antigibberellin substances. However, they did not have any significant inductive effect on microtuberization as compared to that under 8% sucrose medium. Rather they did show a detrimental effect on microtuber development in terms of average microtuber fresh weight with increasing concentrations as well as alkyl-chain length; both acetic and propionic acids at 6.0 mM induced the smallest microtubers in vitro. The carboxylic acids could, however, significantly increase the harvest indices suggesting their possible role in the regulation of source-sink co-ordination during microtuberization from single-node explants. But the most favourable effect of carboxylic acids on microtubers was apparent in terms of dry matter concomitant with higher starch synthesis and enhanced accumulation of reducing and total sugars. Acetic acid was the most effective in increasing the percentage of microtuber dry matter. The higher percentage of dry matter with higher carbohydrate reserves in microtubers induced by the carboxylic acids could be assumed to affect the quality of microtubers for subsequent storage, dormancy release and sprout growth.     

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the <Emphasis Type="Italic">PHYB</Emphasis> Gene from <Emphasis Type="Italic">Arabidopsis</Emphasis>

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.     

Growth pattern, tuber formation and hormonal balance in in vitro potato plants carrying ipt gene

Nodal cuttings of in vitro grown potato plants (Solanum tuberosum, cv. Miranda) were transformed by a vector plasmid carrying ipt gene of Agrobacterium tumefaciens. From the initial teratoma stage 5 clones of transgenic plants (1, 2, 11, 13 and 15) were obtained, which displayed in varying degree shortening of the internodes, decrease of the leaf size, decrease of apical dominance and poor rooting. In addition, two of the clones (11 and 13) showed increased stolon and tuber formation. In all these clones the endogenous level of free cytokinins (CKs) was increased: from 40% in clone 11 to almost 300% in clone 1. Also free indole-3-acetic acid (IAA) level was increased, but to a lower degree; the maximal increase was 160% (clone 13). Applied kinetin or IAA (1 mg·l^-1) strongly suppressed root and tuber formation in clones 11 and 13, although they did not affect or even stimulated these processes in control plants. For control plants the minimal medium sucrose concentration necessary for tuber initiation was 6% whereas in clone 11 plants 2% was sufficient. Different distribution of endogenous CKs and IAA was observed in clone 11 and control plants. The highest CK content was found in transgenic plants in stems and in controls in leaves. In clone 11 plants abscisic acid (ABA) level was significantly increased in comparison to the control throughout the cultivation period. Ethylene formation was strongly increased the first week after the subcultivation and later on the difference between transgenic and control plants rapidly diminished. Reactions of clone 11 plants to red (RL) and blue light (BL) were similar to reactions of control plants. In RL clone 11 plants were tall and thin with stunted leaves; in BL they had a teratoma-like appearance and formed a very high number of tubers. The role of hormones in these changes in growth and tuber formation is discussed.     

Comparison of tuber and shoot formation from in vitro cultured potato explants

The development of axillary buds of potato (Solanum tuberosum L.) plants, cultured in vitro, was analyzed. Depending on the composition of the culture medium, the buds developed into either tubers (medium with 8% sucrose), shoots (1% sucrose), or stolons (8% sucrose and 0.5 μM gibberellin). Endogenous sugar and starch levels, and key-enzymes involved in the conversion of sucrose to starch were determined at different stages of development. Moreover, the spatial distribution of sugar levels and enzyme activities were determined within the developing structures. Glucose and fructose decreased upon tuber formation, most noticeably in the swelling parts, where also starch accumulated. The activities of sucrose synthase, fructokinase and ADP-glucose pyrophosphorylase were highest under tuber-inducing conditions, the increase being confined to the tubers, and absent in the subtending stolons. It is concluded that changes in the measured parameters, observed under tuberizing conditions, are specifically related to the formation of the tuber, and are confined to the swelling part only. This revised version was published online in June 2006 with corrections to the Cover Date.