Kinetic model for carbon partitioning in Solanum tuberosum tubers stored at 2 degrees C and the mechanism for low temperature stress-induced accumulation of reducing sugars

Exposure to low but nonfreezing temperatures induces the breakdown of starch and the accumulation of sucrose, glucose and fructose in potato tubers, a complex phenomenon known as low-temperature sweetening (LTS). A kinetic model for the degradation of starch to sucrose, fructose, glucose, hexose phosphates and carbon dioxide in 2 degrees C-stored mature Solanum tuberosum cv. Norchip (LTS-sensitive) and Solanum tuberosum seedlling ND860-2 (LTS-tolerant) tubers is presented in this work. Analysis of sugar accumulation data in tubers grown in 1993 and 1994 showed no significant differences in the rates of conversion of starch to hexose phosphates and hexose phosphates to sucrose for both cultivars (P > 0.05). The rate constant corresponding to invertase activity was 2.3 day(-1) for Norchip tubers and 1.1 day(-1) for ND860-2 tubers grown in 1993 (P < or = 0.05); however, no significant differences were observed in invertase activity for 1994-grown tubers (P > 0.05). The accumulation of the reducing sugars fructose and glucose was found to be dependent on the relative difference in rate constants corresponding to invertase activity and glycolytic/respiratory capacity. This difference was 3-4 fold greater for Norchip in 1993, and 4-6 fold greater for Norchip in 1994, than for ND860-2 (P < or = 0.05). Results from the analysis also suggest that the amount of available starch for degradation was greater in Norchip tubers than ND860-2 tubers (P < or = 0.05). Our analysis suggests that tubers with decreased invertase activity coupled to increased glycolytic/respiratory capacity should be more tolerant to low-temperature stress.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Tecia solanivora infestation increases tuber starch accumulation in Pastusa Suprema potatoes

In response to infestation with larvae of the Guatemalan tuber moth(Tecia solanivora), some Solanum tuberosum(potato) varieties exhibit an overcompensation response, whereby the total dry mass of uninfested tubers is increased. Here, we describe early responses,within the first few days, of T. solanivora feeding, in the Colombian potato variety Pastusa Suprema. Nontargeted metabolite profiling showed significant secondary metabolism changes in T. solanivora-infested tubers,but not in uninfested systemic tubers. In contrast,changes in primary metabolism were greater in uninfested systemic tubers than in the infested tubers, with a notable 80% decline in systemic tuber sucrose levels within 1 d of T. solanivora infestation. This suggested either decreased sucrose transport from the leaves orincreased sink strength, i.e., more rapid sucrose to starch conversion in the tubers. Increased sucrose synthesis was indicated by higher rubisco activase and lower starch synthase gene expression in the leaves of infested plants.Elevated sink strength was demonstrated by 45% more total starch deposition in systemic tubers of T. solanivorainfested plants compared to uninfested control plants.Thus, rather than investing in increased defense of uninfested tubers, Pastusa Suprema promotes deposition of photoassimilates in the form of starch as a response to T. solanivora infestation.     

A major QTL and an SSR marker associated with glycoalkaloid content in potato tubers from <Emphasis Type="Italic">Solanum tuberosum </Emphasis>× <Emphasis Type="Italic">S. sparsipilum</Emphasis> located on chromosome I

New potato (Solanum tuberosum) varieties are required to contain low levels of the toxic glycoalkaloids and a potential approach to obtain this is through marker-assisted selection (MAS). Before applying MAS it is necessary to map quantitative trait loci (QTLs) for glycoalkaloid content in potato tubers and identify markers that link tightly to this trait. In this study, tubers of a dihaploid BC(1) population, originating from a cross between 90-HAF-01 (S. tuberosum(1)) and 90-HAG-15 (S. tuberosum(2) x S. sparsipilum), were evaluated for content of alpha-solanine and alpha-chaconine (total glycoalkaloid, TGA) after field trials. In addition, tubers were assayed for TGA content after exposure to light. A detailed analysis of segregation patterns indicated that a major QTL is responsible for the TGA content in tubers of this potato population. One highly significant QTL was mapped to chromosome I of the HAG and the HAF parent. Quantitative trait loci for glycoalkaloid production in foliage of different Solanum species have previously been mapped to this chromosome. In the present research, QTLs for alpha-solanine and alpha-chaconine content were mapped to the same location as for TGA content. Similar results were observed for tubers exposed to light. The simple sequence repeat marker STM5136 was closely linked to the identified QTL.     

Polyphenol oxidase in potato. A multigene family that exhibits differential expression patterns.

Polyphenol oxidase (PPO) activity in potato (Solanum tuberosum) plants was high in stolons, tubers, roots, and flowers but low in leaves and stems. PPO activity per tuber continued to increase throughout tuber development but was highest on a fresh weight basis in developing tubers. PPO activity was greatest at the tuber exterior, including the skin and cortex tissue 1 to 2 mm beneath the skin. Flowers had high PPO activity throughout development, particularly in the anthers and ovary. Five distinct cDNA clones encoding PPO were isolated from developing tuber RNA. POT32 was the major form expressed in tubers and was found in all parts of the tuber and at all stages of tuber development. It was also expressed in roots but not in photosynthetic tissues. POT33 was expressed in tubers but mainly in the tissue near the skin. POT72 was detected in roots and at low levels in developing tubers. NOR333 was identical with the P2 PPO clone previously isolated from potato leaves (M.D. Hunt, N.T. Eannetta, Y. Haifeng, S.M. Newman, J.C. Steffens [1993] Plant Mol Biol 21: 59-68) and was detected in young leaves and in tissue near the tuber skin but was highly expressed in flowers. The results indicate that PPO is present as a small multigene family in potato and that each gene has a specific temporal and spatial pattern of expression.     

Factors Affecting the Formation of In vitro Tubers of Potato (Solanum tuberosum L.)

In vitro (mini) tubers were induced within 6–8 weeks inserially propagated potato shoot cultures by subculturing tomedium containing 2.0 mg 1^–1 benzylaminopurine (BAP) and6 per cent sucrose in 8- and 24-h days. The effect of BAP inpromoting tubering was greater in short than in long days. Inshort days most of the tubers were formed above the agar, inlong days within the agar. Tubering was promoted less effectivelyby the addition of (2-chloroethyl)-trimethylammonium chloride(CCC) to the medium, but CCC reinforced the effect of BAP leadingto earlier tubering above the agar. Tubering eventually tookplace after 4—5 months on medium without hormones, soonerin short than in long days. Periods of short days and low temperaturesgiven to long-day cultures did not accelerate tubering. Abscisicacid had little effect on, and GA2 strongly inhibited, tubering.Tubering was also inhibited by sealing the culture vessels butnot if ethylene-absorbing agents were included. Solanum tuberosum L, potato, tissue culture, tubers, cytokinin, ethylene, daylength, propagation     

Identification and quantification of catecholamines in potato plants (Solanum tuberosum) by GC-MS

Dopamine, norepinephrine, and normetanephrine were identified by GC-MS in potato (Solanum tuberosum L.) plants, the latter was new for plants. The highest amount of catecholamines was found in leaves. A developmental stage dependent variation in potato leaf catecholamines accumulation was also observed with highest level in third leaves. Catecholamine contents decrease during cold storage of tubers to undetectable levels. Mechanical wounding of leaves led to a small increase in the level of catecholamines investigated.     

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.     

Light-induced changes in the lipid composition and ultrastructure of plastids from potato tubers

Sandelius, A. S. and Liljenberg, C. 1982. Light-induced changes in the lipid composition and ultrastructure of plastids from potato tubers. – Physiol. Plant. 56: 266–272.
Amyloplasts and starch containing plastids from green tissue – amylochloroplasts – from potato tubers ( Solanum tuberosum L., var. King Edward) were separated from other cell organelles by sedimentation in a discontinuous sucrose gradient. Their lipid composition was analysed with emphasis on galactolipids and phospholipids and the fatty acid compositions of these lipids. Irradiation of the tubers caused increased ratios of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and of total galactolipids to total phospholipids in the plastid membranes. Furthermore, the degree of unsaturation of the fatty acids increased in all lipid classes analysed, this effect being most prominent in the galactolipids. The ultrastructural studies made on tuber tissue revealed that irradiation caused a change in starch grain size distribution concomitant with formation of membrane structures resembling grana within the envelope. In many cases prolamellar bodies and plastoglobuli were present.     

Antisense Repression of Hexokinase 1 Leads to an Overaccumulation of Starch in Leaves of Transgenic Potato Plants But Not to Significant Changes in Tuber Carbohydrate Metabolism

Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.     

Expression of a bacterial xylose isomerase in potato tubers results in an altered hexose composition and a consequent induction of metabolism

Here we investigate the role of hexoses in the metabolism of the developing potato (Solanum tuberosum) tuber by the expression of a bacterial xylose isomerase which catalyzes the interconversion of glucose and fructose. Previously, we found that glycolysis was induced in transgenic tubers expressing a yeast invertase in the cytosol and postulated that this was due either to the decreased levels of sucrose or to effects downstream of the sucrose cleavage. In the present study xylose isomerase was expressed under the control of the tuber-specific patatin promoter. Selected transformants exhibited minor changes in the levels of tuber glucose and fructose but not in sucrose. Analysis of the enzyme activities of the glycolytic pathway revealed minor yet significant increases in the maximal catalytic activities of aldolase and glyceraldehyde 3-phosphate dehydrogenase but no increase in the activities of other enzymes of glycolysis. These lines were also characterized by an elevated tuber number, glycolytic and sucrose synthetic fluxes and in some metabolite levels downstream of glycolysis. When considered together these data suggest that the perturbation of hexose levels can result in increased glycolytic and sucrose (re)synthetic fluxes in the potato tuber even in the absence of changes in the level of sucrose. The consequences of altering hexose levels in the tuber are, however, not as severe as those observed following perturbation of the level of tuber sucrose.     

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.     

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.     

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1

Transgenic potato ( Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene ( SnRK1 ) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%−167% compared with that in the wild-type. Glucose levels were decreased, at 17%−56% of the levels of the wild-type, and the starch content showed an increase of 23%−30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%–60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.     

An integrated view of the hormonal regulation of tuber formation in potato (Solanum tuberosum)

The hormonal regulation of the consecutive steps in the formation of tubers on the potato plant ( Solanum tuberosum L.) is described and discussed. An integrated view of the complex regulation of the initiation and growth of stolons and tubers is presented, with special emphasis on the commonly observed lack of synchronization of the various steps in tuber formation within a plant.     

Roles of alcohol dehydrogenase, lactate dehydrogenase and pyruvate decarboxylase in low-temperature sweetening in tolerant and susceptible varieties of potato (Solanum tuberosum)

Low-temperature sweetening (LTS) results when tubers of potato ( Solanum tuberosum ) are stored at temperatures below 9–10°C with the accumulation of sucrose and reducing sugars glucose and fructose. Our earlier study showed that the LTS-tolerant varieties have higher ethanol and lactate tissue levels compared with the LTS-susceptible variety Monona ( Blenkinsop et al. 2003 ), which led us to investigate the role of the anaerobic respiratory pathway in LTS tolerance. The anaerobic respiratory enzymes alcohol dehydrogenase (ADH), l -lactate dehydrogenase (LDH) and pyruvate decarboxylase (PDC) were, therefore, investigated in LTS-tolerant and -susceptible potato varieties. A positive correlation ( P ≤ 0.05) was observed between reducing sugar concentration and the K _M of PDC, with the LTS-tolerant ND 860-2 possessing a lower K _M and reducing sugar content than the LTS-susceptible Monona variety. The moderately LTS-tolerant variety, Snowden, exhibited intermediate behavior between the two aforementioned cultivars at 4°C. The isozyme profile of the tolerant varieties differed from the susceptible variety. Two groups of LDH isozyme families were observed in all varieties with the exception of ND 860-2, where the second group appeared only during low-temperature exposure. Moreover, the tolerant variety possessed one additional ADH isozyme. Gene expression levels of these enzymes were higher in ND 860-2 as compared with Monona at 4°C. The above results suggest that the anaerobic respiratory enzymes contribute to LTS-tolerance.     

Lipid and oxylipin profiles during aging and sprout development in potato tubers (Solanum tuberosum L.)

Potato tubers (Solanum tuberosum L. cv Bintje) were stored at 20 degrees C for 210 days without desprouting to study the lipoxygenase pathway during aging. After 15 days of storage, potato tubers sprouted, while after 45-60 days, apical dominance was lost and multiple sprouts developed. Analysis of the fatty acid hydroperoxides (HPOs) revealed that 9-S-hydroperoxide of linoleic acid (9-HPOD) was the main oxylipin formed. Between 45 and 60 days of storage, increases in the levels of 9-HPOD and colneleic acid were observed. Analysis of phospholipids and galactolipids by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that a decrease in the levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), digalactosyldiacylglycerol (DGDG), and monogalactosyldiacylglycerol (MGDG) occurred between 0 and 45 days of aging. The decrease in the amount of linoleic acid in complex lipids correlates well with the amount of 9-HPOD and colneleic acid produced.     

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.