Changes in Levels of Butanol- and Water-Soluble Cytokinins during the Life Cycle of Potato Tubers

The nature of the water-soluble cytokinin in potato tubers (Solanumtuberosum L.) and the changes in the levels of both butanol-and water-soluble cytokinins during their life cycle were examined.The main cytokinin detected in the water-soluble fraction hadthe same chromatographic behavior and susceptibility to enzymesas zeatin ribotide. The level of butanol-soluble cytokinin inelongating stolon tips was low, while that of water-solublecytokinin was extremely high. Upon swelling of the stolon tips,the former increased greatly as the latter decreased. The resultssuggest that the increased butanol-soluble cytokinin is responsiblefor the subsequent vigorous thickening growth of the stolonsto form tubers and that the water-soluble cytokinin is a temporarystorage form. (Received February 6, 1982; Accepted May 13, 1982)     

The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, ¹⁴C-glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch-degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U-[¹⁴C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3-fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m^?3 U-[¹⁴C]glucose, the rate of labelling of sucrose in slices from 6 d cold-stored material was higher than in slices from warm-stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose-phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5-fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the ¹⁴C-labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose-phosphate are accompanied by a 6–8-fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold-stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose-phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.     

The ascorbate system and lipid peroxidation in stored potato (Solanum tuberosum L.) tubers

The changes in the components of the ascorbateglutathione systemduring the storage of potato (Solanum tuberosum L. cv. Spunta)tubers for 40 weeks at both 3C and 9C were studied in relationto lipid peroxidation. The level of malondialdehyde was foundto be higher at 3C than at 9C throughout storage. Thus, lipidperoxidation, which is the main cause of membrane deterioration,was accelerated at the lower temperature. Catalase activityincreased throughout storage independently of temperature. Theascorbate content of tubers decreased during storage both at3C and at 9C, as in other ageing processes. However, ascorbateperoxidase activity reached a maximum after about 8 weeks ofstorage, then declined at 9C, but held a higher level at 3C.The dehydroascorbic content also reached a maximum after about8 weeks and was significantly higher in tubers stored at 3C.These findings indicate a greater utilization of ascorbate byascorbate peroxidase at the lower temperature. Ascorbate freeradical reductase, dehydroascorbate reductase and glutathionereductase, the enzymes involved in the regeneration of ascorbate,were not affected by temperature and remained quite unchangedthroughout storage. It can be concluded that the ascorbate systemis involved in the scavenging of the free-radicals responsiblefor lipid peroxidation in stored potato tubers, at least atlow temperatures and in the first period of storage. Key words: Ascorbate, lipid peroxidation, potato tubers, Solanum tuberosum L     

Free Sulfhydryl Groups in Ripening Fruits

Ripening was found to be accompanied by an increase in sulfhydryl (SH) group content in several fruits. In ripening, climacteric,tomato fruits, this increase was due mostly to an increase inthe glutathione level. In orange, a non-climacteric fruit, therelatively high SH levels did not change during developmentand maturation. Non-ripening tomato mutants, e.g., rin and nor,were characterized by low and constant SH levels during fruitgrowth and senescence. Ripening-inducing storage conditions,such as high temperature (30?C) and ethylene treatment, concomitantlyhastened the increase in SH level in fast-ripening tomato varieties.Storage conditions that slowed down the ripening process, suchas low temperatures (2, 12?C) and low oxygen levels (5%), sloweddown the increase in SH content. Storage of red tomatoes at30?C caused an increase in the SH content in comparison withlower temperature treatments (2, 12, 20?C). SH compounds (reducedglutathione, cysteine), dithiothreitol and an SH-binding compound(n-ethylmaleimide), did not affect the ripening process of greentomatoes. The results suggest that the increase in SH groupsaccompanies the ripening processes rather than regulating them. (Received April 26, 1982; Accepted September 6, 1982)     

The Distribution of Cytokinins in Tuberizing Potatoes

Potato tubers (Solanum tuberosum L.) stored at low temperaturein the dark for a prolonged period of time develop a disorderknown as ‘little potato’. This phenomenon involvesthe formation of stolons and new tubers, the reserves for thelatter being obtained from the parent tubers. A study of thedistribution of endogenous cytokinins in the various regionsof the system showed it to be very complex. Zeatin ribosideappeared to be the major cytokinin throughout the whole system.It was present in the highest levels at the stolon tips andin the smallest little potatoes. The internodal sprout tissue,which can be regarded as a link between the parent tubers andthe bulking little potatoes, contained the highest total levelof cytokinin. The total activity was greater due to the presenceof higher levels of compounds which co-eluted with zeatin andzeatin glucoside. It is possible that the cytokinin glucosideis either a translocatoly or storage form in the present system.The decreased levels of the glucoside at the stolon tips andin the bulking little potatoes could well be the result of hydrolysisor perhaps its active participation in the tuberizing process.     

Changes in cis-zeatin and cis-zeatin riboside levels and biological activity during potato tuber dormancy

The effects of postharvest storage duration and temperature on endogenous cis -zeatin ( cis -Z) and cis -zeatin riboside ( cis -ZR) levels in potato ( Solanum tuberosum L.) tubers were determined in relation to tuber bud dormancy. The tubers used in these studies were completely dormant for at least 81 days of storage. Thereafter, tuber bud dormancy diminished gradually and after 165 days of postharvest storage, the tubers were completely non-dormant. Immediately after harvest, endogenous levels of cis- Z and cis -ZR were approximately 25 pmol (g fresh weight)⁻¹ and 8 pmol (g fresh weight)⁻¹, respectively. In tubers exiting dormancy but stored at a growth-inhibiting temperature (3°C), endogenous levels of cis -Z rose over threefold after 25 days of storage and remained elevated for the duration of the study. Levels of cis -ZR remained essentially constant during this same period. In tubers transferred to a growth permissive temperature (20°C) prior to use, the rise in endogenous cis -Z was less dramatic and more protracted; increasing twofold after 53 days of storage. No change in cis -Z riboside content was observed in these tubers during this period. Dose-response studies using either cis -Z or trans -Z demonstrated a time-dependent increase in cytokinin sensitivity during postharvest storage. Immediately after harvest, dormant tubers were insensitive to both zeatin isomers. Thereafter, tubers exhibited a dose-dependent increase in premature sprouting following injection with either cytokinin isomer. After injection into dormant tubers, cis -[8-¹⁴C]-zeatin was metabolized primarily to adenine/adenosine and cis -Z riboside. Seven days after injection, less than 10% of the recovered radioactivity was associated with trans -ZR. These results are consistent with a role for endogenous cis -Z (and its derivatives) in the regulation of potato tuber dormancy.     

Relation between Nitrogen Nutrition, Cytokinin Activity and Tuberization in Solanum tuberosum

In water-culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), the influence of tuberization initiated by a 7-day period of nitrogen withdrawal (discont. N) on the cytokinin activity in shoots, roots and exudate was studied. Plants with a constant supply of nitrogen (cont. N) were used as control. — Whereas no tuberization could be observed with cont. N, discont. N led to tuberization already 2 days after nitrogen withdrawal and all plants had been induced after another 4 days. In the roots of plants with discont. N, there was a temporary increase in cytokinin activity, whereas the activity decreased steadily with cont. N. In the exudate, cytokinin activity was greatly reduced during nitrogen withdrawal, whereas this activity in the exudate increased steadily with cont. N. — In the shoot with cont. N cytokinin activity decreased steadily, but with discont. N, after an initial decrease, the activity increased steeply. This increase is mainly or exclusively caused by a shift between the water-soluble and butanol-soluble fractions of the cytokinins in favour of the latter. The shift in cytokinin activity of the shoot is assumed to be in causal connection with an increased photosynthetic activity after the onset of tuber growth as ‘sink’ for assimilates.     

Changes in Endogenous Cytokinin Levels During Abscission and Senescence of Streptocarpus Leaves

The cytokinin levels of butanol and aqueous extracts of proximalportions of Streptocarpus leaves showed very little change duringthe summer and early autumn months, whereas those of the distalportions changed markedly. These changes appear to be closelyrelated to the formation of abscission layers and the subsequentsenescence of the distal part of the leaves. It is postulatedthat the increase in butanol-soluble cytokinins during Januarymay induce abscission, while the decrease in February appearsto be directly related to senescence of the distal portionsof the leaves.     

The Effect of Photoperiod on Levels of Endogenous Cytokinins in Xanthium strumarium

The cytokinin content of Xanthium strumarium L. plants decreased markedly when they were exposed to short days (SD). There was a significant decrease in the content of the butanol-soluble cytokinins of the mature leaves after only 5 SD cycles, and after 10 SD there was no significant cytokinin activity in butanol extracts; the changes in the young leaves were less marked. Most of the cytokinin activity in mature leaves appears to be present in the aqueous fraction, whereas in young leaves most activity occurs in the butanol-soluble fraction. SD treated plants produced less root exudate than LD plants, but there were no significant differences in the amounts of cytokinin in the root exudates from LD and SD plants collected over an equivalent time period. The cytokinin levels of SD-induced leaves remained low even when transferred back to LD. The observed differences in cytokinin levels did not appear to be the result of photosynthetic differences. Exposure of detached leaves to LD or SD did not result in differences in cytokinin content. It is not clear whether the observed changes in cytokinin levels in the leaves under SD are involved in the flowering response, but they may be causally related to a reduced chlorophyll content observed in SD-induced leaves.     

A Comparative Auxin and Cytokinin Study in Normal and to-2 Mutant Tomato Plants

Torosa-2 (to-2), a tomato mutant with strong apical dominance,was studied in order to determine the mechanism of shoot outgrowthcontrol. In decapitated or defoliated to-2 plants only a fewshoots grew and IAA or morphactin application had little oronly short term effects. No differences were found in auxinand cytokinin activities between normal and mutant plants upto 20 days after sowing. In the period from 40 to 90 days theIAA content increased equally in both genotypes. During thesame period, however, cytokinin increased only in normal plants.The results obtained with the to-2 mutant indicate that budsdo not shoot, probably because bud differentiation does notoccur. The ratio of auxin to cytokinin clearly was affectedby the low level of cytokinin in all tissues of the to-2 plants.This led us to the conclusion that insufficient quantities ofcytokinin for lateral bud differentiation is the cause of thestrong apical dominance in to-2. (Received January 20, 1982; Accepted April 26, 1982)     

Role of Aminocyclopropane-1-carboxylic Acid (ACC) in Control of Ethylene Production in Fresh and Cold-stored Rose Flowers

The relationships between ethylene production, aminocyclopropane-1-carboxylicacid (ACC) content and ethylene-forming-enzyme (EFE) activityduring ageing and cold storage of rose flower petals (Rose hybridaL. cv. Gabriella) were investigated. During flower ageing at20 °C there was a climacteric rise in petal ethylene production,a parallel increase in ACC content, but a continuous decreasein EFE activity. Applied ACC increased petal ethylene productionc. 200-fold. During cold storage of flowers at 1 °C therewere parallel increases in petal ethylene production and ACCcontent, to levels greater than those reached in fresh flowersheld at 20 °C. EFE activity decreased during storage. Immediatelyafter cold-stored flowers were transferred to 20 °C ethyleneproduction and ACC levels were c. four times greater than infreshly cut flowers. These levels increased to maximum valuesof two to four times the maximum values reached during ageingof fresh, unstored, flowers. It was concluded that in rose petalsethylene synthesis is probably regulated by ACC levels and thatcold storage stimulates ethylene synthesis because it increasesthe levels of ACC in the petals. Key words: Rose flower, senescence, ethylene     

Identification of cytokinins associated with mitosis in synchronously cultured tobacco cells

The amount of endogenous cytokinins in the butanol-soluble fractionincreased about 5 times during the mitosis of synchronouslydividing cultured tobacco cells (Nicotiana tabacum cv. Xanthi).The free cytokinins were identified as trans-zeatin and zeatinribonucleoside by paper and thin layer chromatography and byhigh-performance liquid chromatography combined with mass spectrometryand bioassay. The water-soluble fraction contained zeatin ribonucleotide,isopentenyladenosine monophosphate, phosphoglucosylzeatin andphosphoglucosylzeatin ribonucleoside. Changes in the cytokininsin the water-soluble and transfer RNA fractions during the cellcycle did not support the hypothesis that they might be thesource of the butanol-soluble cytokinins which increased duringmitosis. These seem to be synthesized de novo during mitosis. (Received February 1, 1980; )     

Seed Dormancy in Acer: CHANGES IN ENDOGENOUS CYTOKININS, GIBBERELLINS AND GERMINATION INHIBITORS DURING THE BREAKING OF DORMANCY IN ACER SACCHARUM MARSH

The present studies on changes in the levels of cytokinins,gibberellins, and germination inhibitors during stratificationof Acer saccharum sceds suggest that the breaking of dormancyis a phasic process. Stratification at 5°C led to a markedincrease in the butanol-soluble cytokinins after 20 d. Furtherchilling resulted in a loss of cytokinin. The majority of thedetectable cytokinin activity co-chromatographed with zoatin.Similarly, at day 40, acidic gibberellin-like substances increasedand declined with further chilling. A loss of acidic germinationinhibitors was also detected during stratification. The acidicfraction was shown to contain abscisic acid (ABA) by its behaviouron GLC and isomerization with ultra-violet light. Determinationsof endogenous levels of ABA by combined GLC and isotope dilutionmethods indicated a loss of 97.9 per cent ABA during stratification.The data suggest that the breaking of seed dormancy at low temperatureinvolves the initiation of an ordered sequence of events thatinclude the changes in growth hormones necessary to determinethe course of development for the removal of dormancy.     

Enzyme Formation in Higher Plant Tissue:1

Discs cut from Jerusalem artichoke tubers stored at 20 °Cproduced considerably less invertase than discs cut from tubersstored at 4 °C. Gibberellic acid stimulated synthesis ofinvertase in tissue previously stored at 20 °C, restoringit to the level of cold-stored tubers.     

Identification of Free Cytokinins and the Changes in Endogenous Levels during Tuber Development of Sweet Potato (Ipomoea batatas Lam.)

Trans-zeatin, trans-zeatin riboside and N⁶-(²-isopentenyl)adenosine(i⁶Ado) were found to be the major cytokinins by high performanceliquid chromatographic separation and gas chromatography-massspectrometric analysis in the small tubers of sweet potato (Ipomoeabatatas Lam. cv. Minamiyutaka) with a diameter of about 5 mm.During tuber development cytokinin levels were high in tubershaving a diameter below 12 mm, minimal in tubers with a diameterof 22.5 mm, and then gradually increased as the tuber developed. The role of cytokinins in tuber development of the sweet potatois discussed. (Received May 20, 1982; Accepted August 10, 1983)     

A [beta]-Amylase in Potato Tubers Is Induced by Storage at Low Temperature

A new starch-degrading enzyme activity is induced by storage of potato (Solanum tuberosum L.) tubers at low temperatures (L. Hill, R. Reimholz, R. Schroder, T.H. Nielsen, M. Stitt [1996] Plant Cell Environ 14: 1223-1237). The cold-induced activity was separated from other amylolytic activities in zymograms based on iodine staining of polyacrylamide gels containing amylopectin. A similar band of activity was detected at normal growth temperatures in leaves, stems, and growing tubers but was present only at low activity in warm-stored tubers. The cold-induced enzyme was separated by ion-exchange chromatography from other amylolytic activities. It has a broad neutral pH optimum. Characterization of its hydrolytic activity with different substrates showed that the cold-induced activity is a [beta]-amylase present at low activity in tubers stored at 20[deg]C and induced progressively when temperatures are decreased to 5 and 3[deg]C. The first clear induction of [beta]-amylase activity was observed within 3 d of storage at 3[deg]C, and the activity increased 4- to 5-fold within 10 d. The possible involvement of the cold-induced [beta]-amylase in sugar accumulation during cold storage is discussed.     

Comparative proteomic analysis of cold-induced sweetening in potato (Solanum tuberosum L.) tuber

Cold-induced sweetening is one of the major factors limiting the quality of fried potato products. To understand the mechanisms of protein regulation for cold-induced sweetening in potato tubers, a comparative proteomic approach was used to analyse the differentially expressed proteins both during control (25 °C, 30 days) and cold treatment (4 °C, 30 days) using two-dimensional gel electrophoresis. Quantitative image analyses indicated that there were 25 protein spots with their intensities significantly altered more than twofold. Of these proteins, 9 were up-regulated, 13 were down-regulated, 2 were absent, and 1 was induced in the cold-stored tubers. The MALDI-TOF/TOF MS analyses led to the identification of differentially expressed proteins that are involved in several processes and might work cooperatively to maintain metabolic homeostasis in tubers during low-temperature storage. The preponderance of metabolic proteins reflects the inhibition of starch re-synthesis and the accumulation of sugars in carbon fluxes, linking starch–sugar conversion. The respiration-related proteins suggest the transfer of respiratory activity from aerobic respiration to anaerobic respiration in the cold-stored tubers. The proteins associated with defence appear to protect the tuber cells from low-temperature stress. Some heat shock proteins that act as chaperones also displayed a differential expression pattern, suggesting a potentially important role in cold-stored tubers, although their exact contribution remains to be investigated. The proposed hypothetical model might explain the interaction of these differentially expressed proteins that are associated with cold-induced sweetening in tubers.     

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.     

Decreased amount of reducing sugars in transgenic potato tubers and its influence on yield characteristics

This work focuses on the comparison of field characteristics and amounts of reducing sugars in cold-stored tubers of transgenic plants derived from two potato cultivars. The bacterial gene coding for phosphofructokinase under the tuber-specific promoter was used to support the glycolysis in stored tubers. While the tubers from untransformed control plants steadily accumulated reducing sugars during cold storage, the tubers from transformed plants regardless the genotype were characterized by subsequent decrease in the sugar content. After long period of cold storage the greatest reduction in the reducing sugar content was by more than 60 % compared to control. Before the storage, however, the content of reducing sugars was in 80 % of transgenic lines higher than in control ones. The plants evaluated in field trials for their appearance showed any changes in growth characteristics in about 25 % of the transgenic lines. Despite the introduced modification of sugar metabolism the yield of transgenic plants with normal appearance did not differ significantly from the yield of control plants.