The effect of an elevated cytokinin level using the ipt gene and N 6-benzyladenine on single node and intact potato plant tuberization in vitro

Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L^-1 N ⁶-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.Abbreviations BA N ⁶-benzyladenine - PCR polymerase chain reaction - HPLC high performance liquid chromatography - ELISA enzyme-linked immunosorbent assay - NAA -naphthylacetic acid     

Phospholipid and phospholipase changes by jasmonic acid during stolon to tuber transition of potato

Potato tuber formation starts with the stolon swelling and is regulated by jasmonates. The cascade of events leading to tuber formation is not completely understood. The aim of this study was to evaluate phospholipid composition and phospholipase activities during four stages of stolon-to-tuber transition of Solanum tuberosum L., cv. Spunta, and involvement of phosphatidic acid (PA) in stolon cell expansion during early stages. Effects of jasmonic acid (JA) treatment on phospholipid content and activation of phospholipase D (PLD) (EC 3.1.4.4) and phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP₂-PLC) (EC 3.1.4.3) were studied in the early stages (first stage, hooked apex stolon; second stage, initial swelling stolon) of tuberization. All the phospholipid species identified, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), PA, and cardiolipin (CL), decreased as tuber formation progressed. PLD and PLC were activated in control tissues at an early stage. JA treatment caused a decrease of PC and PS in first stage stolons, accumulation of PA in second stage stolons, and modification of PLD and PLC activities. PA increased stolon cell area in the first and second stages. These findings indicate that phospholipid catabolism is activated from the early stages of tuber formation, and that JA treatment modifies the pattern of phospholipid (PC, PS, and PA) composition and phospholipase (PLD and PLC) activity. These phospholipids therefore may play a role in activation of an intracellular mechanism that switches the developmental fate of stolon meristem cells, causing differentiation into a tuber.     

Use of the growth retardant tetcyclacis for potato tuber formation in vitro

The effects of the plant growth retardant tetcyclacis on in vitro tuber formation in potatoes was studied, using two different approaches: 1. tuber formation in various lines that did not or hardly form tubers under control conditions, and 2. tuber formation by the variety Bintje, which readily forms tubers. The ABA-deficient (droopy) lines of S. phureja hardly formed tubers without the addition of tetcyclacis. In the presence of this growth retardant tuberization was nearly 100%, within three weeks of in vitro culture, even in the absence of cytokinin. A series of somatic hybrids between S. tuberosum and S. brevidens, that did not form tubers in field and pot experiments, were tested. They all formed tubers in vitro in the presence of tetcyclacis. Stoloniferous shoots formed on single-node cuttings from in vitro grown Solanum tuberosum var Bintje plantlets were transferred to media containing a high level of sucrose. In the presence of tetcyclacis, tuber formation started after 4 days, reaching a maximum level of 80% at day 7. Tubers formed in the presence of tetcyclacis, accumulated starch and expressed several tuber-specific genes. These effects were fully antagonized by gibberellic acid. It is concluded that the growth retardant tetcyclacis is a potent tool in the study of tuber formation in potatoes.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid - STS silver thiosulphate - TET tetcyclacis     

Sucrose application causes hormonal changes associated with potato tuber induction

Stems of potato plants (Solanum tuberosum L. cv. Dianella) were immersed in solutions containing water (control), sucrose, glucose, paclobutrazol, and gibberellic acid. The effects of these treatments on the ethylene release, levels of endogenous gibberellins, glucose, sucrose, and starch were correlated with tuberization of nodal cuttings, excised from potato stems. Paclobutrazol and sucrose improved the percent of tuberization and/or increased tuber weight. In contrast, GA₃ inhibited tuber formation compared with the control. The level of endogenous free GAs was negatively correlated with percent tuberization. However, the level of conjugated GAs was positively correlated with both percent tuberization and tuber weight. The effect of sucrose on potato tuber induction in relation to the possible role of sucrose in GA-conjugate formation is discussed.     

The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation in Vitro

The effects of plant hormones and sucrose (Suc) on potato (Solanum tuberosum L.) tuberization were studied using in vitro cultured single-node cuttings. Tuber-inducing (high Suc) and -noninducing (low Suc or high Suc plus gibberellin [GA]) media were tested. Tuberization frequencies, tuber widths, and stolon lengths were measured during successive stages of development. Endogenous GAs and abscisic acid (ABA) were identified and quantified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Exogenous GA_4/7 promoted stolon elongation and inhibited tuber formation, whereas exogenous ABA stimulated tuberization and reduced stolon length. Indoleacetic acid-containing media severely inhibited elongation of stolons and smaller sessile tubers were formed. Exogenous cytokinins did not affect stolon elongation and tuber formation. Endogenous GA₁ level was high during stolon elongation and decreased when stolon tips started to swell under inducing conditions, whereas it remained high under noninducing conditions. GA₁ levels were negatively correlated with Suc concentration in the medium. We conclude that GA₁ is likely to be the active GA during tuber formation. Endogenous ABA levels decreased during stolon and tuber development, and ABA levels were similar under inducing and noninducing conditions. Our results indicate that GA is a dominant regulator in tuber formation: ABA stimulates tuberization by counteracting GA, and Suc regulates tuber formation by influencing GA levels.     

Sulfate activation in Desulfotomaculum

Enzyme activities conceivably involved in the activation of sulfate were studied with Desulfotomaculum ruminis, D. acetoxidans, D. nigrificans, D. orientis, and Desulfovibrio vulgaris. Cell lysates of these species revealed activities of at least 8 nkat/mg protein (i.e., 480 nmol per min and mg protein) of ATP sulfurylase, acetate kinase, phosphotransacetylase and adenylate kinase. ADP sulfurylase was not detected. Pyrophosphatase activity was high (73 to 97 nkat/mg protein) in Desulfotomaculum orientis and Desulfovibrio vulgaris. In these strains pyrophosphatase was activated by addition of a reductant (dithionite). In Desulfotomaculum ruminis, D. acetoxidans, and D. nigrificans, only low pyrophosphatase activity (2.5 to 6.3 nkat/mg protein) was measured, which was not reductant-activated. Some hints indicated a membrane association of the pyrophosphatase in D. ruminis, and possibly also in D. acetoxidans and D. nigrificans. Activities of a pyrophosphate-dependent acetate kinase (PP_i:acetate kinase), a PP_i:AMP kinase or a polyphosphate:AMP kinase were not detected or negligible. The results are not in favour of the assumption that pyrophosphate formed by ATP sulfurylase during sulfate activation might be utilized to form acetyl phosphate in Desulfotomaculum species. Contrary results of other authors were shown to be artefacts caused by chemical hydrolysis of acetyl phosphate in the molybdate-sulfuric acid reagent used for phosphate determination.Abbreviations P_i orthophosphate - PP_i pyrophosphate - APS adenosine phosphosulfate - AP₅A, P¹ P⁵-di(adenosine-5-)pentaphosphate - CTAB cetyltrimethylammonium bromide - MOPS 3-(N-morpholino)propanesulfonic acid - HEPES N(-2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid     

Relative importance of maltose and sucrose supplied during a 2-step potato microtuberization process

A 2-stage in vitro tuberization process comprising first micropropagation via nodal explants and then tuber induction in the resultant in vitro plantlets was studied using 2 cultivars of potato, Iwa and Daeji. In particular, the effects on both plantlet growth and subsequent in vitro tuberization of Murashige and Skoog (1962) basal medium containing either sucrose or maltose, each at 3 % (w/v), used for micropropagation were investigated. Sucrose and maltose were found to be equally effective in supporting development of vigorous plantlets from the nodal explants of both potato cultivars. Upon transfer to a medium with an optimised level of sucrose (i.e. 8 %, w/v) for in vitro tuberization, only the plantlets previously grown in the sucrose-containing medium were capable of forming more microtubers of the larger size category (greater than 0.5 g). The relative importance of sucrose supply at the mircropropagation stage was further confirmed when the resultant plantlets grown in the 3 % sucrose-containing medium were transferred to an in vitro tuberization medium containing either sucrose or maltose, each at 8 % (w/v). In this experiment, maltose and sucrose had indistingushable effects on in vitro tuberization.     

Regulation of potato tuber protein accumulation by gibberellic Acid

Many studies have shown that gibberellic acid (GA₃) inhibits tuberization in potato (Solanum tuberosum L.). In this study, we have utilized the 40 kilodalton glycoprotein, patatin, as a marker for biochemical events associated with the process of tuberization. To determine the effects of exogenous applications of GA₃ on the induction of the accumulation of this major tuber protein, we measured patatin levels in tubers from treated whole plants, petioles from a single-node cutting system with GA₃ applied in a lanolin paste, and stolon tips cultured in vitro on an inductive medium supplemented with GA₃. In all three systems, GA₃ inhibited the accumulation of patatin and the major 15 and 22 kilodalton tuber proteins. This effect appeared to be selective since most of the other proteins were not affected and, in tubers, at least one protein was stimulated by GA₃. These results suggest that GA₃ can reverse biochemical events of tuberization in tubers as well as prevent the accumulation of the major tuber proteins in other inducible tissues.     

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 μmol·dcm⁻³ of CdCl₂ solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm⁻³ concentration of CdCl₂ rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of K_I for cadmium and K_M for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic 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.     

High K<Superscript>+</Superscript> does not affect potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) tuber induction,but represses its development <Emphasis Type="Italic">in vitro</Emphasis>

The role of K⁺ in potato (Solanum tuberosum L.) tuberization, based on the effects of K fertilizer and soil exchangeable K⁺, appears to be mostly contradictory. Here, we provide evidence that K⁺ at high concentrations is detrimental to tuber development in vitro once induction has taken place. An experimental system using in vitro-cultured single-node cuttings showed that K⁺ at ≥30.0 mM significantly reduced tuber fresh mass concomitant with a corresponding decline in starch content. However, high K⁺ did not affect tuber induction in terms of number of tubers developed per cutting. High K⁺-induced inhibitory effect on tuber development was attributed to a reduced rate of assimilate partitioning. ⁸⁶Rb(K) transport to stolons, and tubers that acted as strong sinks in vitro were proportional to exogenous K⁺ levels; however, ⁸⁶Rb accumulation and K⁺ deposition were markedly reduced in tubers as compared with that in stolons, especially at higher K⁺ levels. The results indicated a diminishing sink strength developed by tubers with increasing K nutrition. Highly significant negative correlations between ⁸⁶Rb accumulation/K⁺ deposition in both the sink organs and tuber fresh mass reinforced the inhibitory effect of high K⁺ on tuber development. The rate of tuber K removal in vitro was similar to that of crop K removal reported in vivo, suggesting highly conserved K uptake and transport mechanisms during tuberization process. The results have been discussed in the context of possible effects of high K⁺ on impairing sucrose uptake and metabolism.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum)

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA+Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA₃) was the most abundant in all the organs. While GA₁and GA₃ were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.     

Starch Synthesis in Developing Potato Tubers

The activities of enzymes involved in starch metabolism were measured at intervals during tuberization and the early stages of tuber growth in Solanum tubersum grown in water culture under controlled environmental conditions. Starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase activities all increased during tuber development, the most pronounced increases occurring in the activities of ADP-glucose pyrophosphorylase and phosphorylase. The activity ratio ADPglucose pyrophosphorylase/phosphorylase was lowest in slow growing tubers and hightest in fast growing tubers. In addition, high sugar concentrations in fast growing tubers and low sugar concentrations in slow growing tubers suggested that enzyme levels might be influenced by sugar concentration. The activities of starch synthase, phosphorylase and ADPglucose pyrophosphorylase were increased 2–2.5 fold by the presence of 100 mM K⁺. It is concluded that the major enzyme changes occur as a consequence of tuber initiation and that starch accumulation is controlled, at least in part, by the activities of ADPglucose pyrophosphorylase and phosphorylase.     

The control of bud dormancy in potato tubers

Potato (Solanum tuberosum L.) tuber buds normally remain dormant through the growing season until several weeks after harvest. In the cultivar Majestic, this innate dormancy persisted for 9 to 12 weeks in storage at 10° C, but only 3 to 4 weeks when the tubers were stored at 2° C. At certain stages, supplying cytokinins to tubers with innately dormant buds induced sprout growth within 2 d. The growth rate was comparable to that of buds whose innate dormancy had been lost naturally. Cytokinin-treatment did not accelerate the rates of cell division and cell expansion in buds whose innate dormancy had already broken naturally. Gibberellic acid did not induce sprout growth in buds with innate dormancy. We conclude that cytokinins may well be the primary factor in the switch from innate dormancy to the non-dormant state in potato tuber buds, but probably do not control the subsequent sprout growth.Abbreviations tio ⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)purine, zeatin - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)-9--D-ribofuranosyl purine, zeatin riboside     

Enzymic activities of carbohydrate,purine, and pyrimidine metabolism in the Anaeroplasmataceae (class Mollicutes)

Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161^T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PP_i-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PP_i-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P ₂). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P ₂), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PP_i-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.Abbreviations EMP Embden-Meyerhof-Parnas - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PEP phosphoenolpyruvate - PFK phosphofructokinase - PPDK pyruvate, orthophosphate dikinase - TCA cycle tricarboxylic acid cycle Note: Other abbreviations used are as per the instruction to authors, or the reference cited therein (Eur J Biochem 1:259), or Biochem J 120:449 (which supercedes a portion of the first reference)