Differential cold tolerance, starch, sugar, protein, and lipid of yellow and purple nutsedge tubers

From measurements of viability after exposure of tubers to natural overwintering in the soil and 6 weeks exposure at 2 C, species cold tolerance of the tubers was ranked in decreasing order: yellow nutsedge `I' (Cyperus esculentus L.), an ecotype originating in Illinois; yellow nutsedge `G', an ecotype originating in Georgia; and purple nutsedge (Cyperus rotundus L.). The ratios of unsaturated-saturated fatty acids in tuber triglycerides, tuber polar lipids, and leaf polar lipids followed the same order as the cold tolerance rankings, with the most cold-hardy species having the highest ratios. Lipid content was less than 1% of dry weight in purple nutsedge tubers, but was from 5 to 7% in both yellow nutsedge tubers. Starch, sugar, and lipid contents increased significantly in the hardy yellow nutsedge `I' tubers during a 6-week exposure to 2 C, but did not change in the susceptible purple nutsedge tubers; only sugar increased in yellow nutsedge `G' tubers after this treatment. Protein content was not altered by the 2 C treatment in any of the tubers. Apparently, several factors involving starch, sugar, lipids, and fatty acids are related to the differences in tolerance to cold in these species.     

HRE-type genes are regulated by growth-related changes in internal oxygen concentrations during the normal development of potato (Solanum tuberosum) tubers

The occurrence of hypoxic conditions in plants not only represents a stress condition but is also associated with the normal development and growth of many organs, leading to adaptive changes in metabolism and growth to prevent internal anoxia. Internal oxygen concentrations decrease inside growing potato tubers, due to their active metabolism and increased resistance to gas diffusion as tubers grow. In the present work, we identified three hypoxia-responsive ERF (StHRE) genes whose expression is regulated by the gradual decrease in oxygen tensions that occur when potato tubers grow larger. Increasing the external oxygen concentration counteracted the modification of StHRE expression during tuber growth, supporting the idea that the actual oxygen levels inside the organs, rather than development itself, are responsible for the regulation of StHRE genes. We identified several sugar metabolism-related genes co-regulated with StHRE genes during tuber development and possibly involved in starch accumulation. All together, our data suggest a possible role for low oxygen in the regulation of sugar metabolism in the potato tuber, similar to what happens in storage tissues during seed development.     

Studies on the biochemical basis of physiological processes in the potato tuber

Summary The breakdown of starch in potato tubers which starts when buds begin to grow, stops if the sprouts are removed. The sprout controls the utilization and translocation of food reserves from the tuber. Movement of reserves can occur over the whole cross section of the tuber and is not restricted to the vascular shell. The presence of a growing sprout does not affect the permeability of the tuber tissue to sugar or amino acid.Previous part: Edelman and Singh (1968).     

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.     

Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes.

Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch biosynthesis in plants. Instead up to 30% of the dry weight of the transgenic potato tubers was represented by sucrose and up to 8% by glucose. The process of tuber formation also changed, resulting in significantly more tubers both per plant and per stolon. The accumulation of soluble sugars in tubers of antisense plants resulted in a significant increase of the total tuber fresh weight, but a decrease in dry weight of tubers. There was no significant change in the RNA levels of several other starch biosynthetic enzymes, but there was a great increase in the RNA level of the major sucrose synthesizing enzyme sucrose phosphate synthase. In addition, the inhibition of starch biosynthesis was accompanied by a massive reduction in the expression of the major storage protein species of potato tubers, supporting the idea that the expression of storage protein genes is in some way connected to carbohydrate formation in sink storage tissues.     

Fatty acids, membrane permeability, and sugars of stored potato tubers

The relationships of potato (Solanum tuberosum L.) tuber membrane permeability and membrane lipid composition to sugar accumulation were examined. Tubers from four potato cultivars were stored for 40 weeks at 3°C and 9°C. Rates of tuber membrane electrolyte leakage, total fatty acid composition, free fatty acid composition, and sugar content were measured throughout the storage period. Storage of tubers at 3°C caused dramatic increases in total fatty acid unsaturation, membrane permeability, and sugar content compared to tubers stored at 9°C. Cultivars with higher levels of fatty acid unsaturation had lower rates of membrane electrolyte leakage and lower sugar contents. We propose that high initial levels or high induced levels of membrane lipid unsaturation mitigate increases in tuber membrane permeability during storage, thus positively influencing the processing quality of stored potato tubers.     

Effect of gibberellic acid on growth and carbohydrate metabolism of developing tubers of potato (Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Ostara) were grown in aerated water culture in a controlled environment. When the tubers had reached a diameter of 1–3 cm. ¹⁴C-labelled or unlabelled gibberellic acid (GA₃) was applied to the surface of the stolons at points approximately 1 crn from the developing tubers, and treatment continued for 10 days. - Significant quantities of GA₃ moved into tuber tissue within 2–4 days of hormone application. This influx of GA₃ was accompanied by a marked reduction in both the activity of ADPG-pyrophospharylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase and an increase in the activity of UDPG-pyrophosphorylase. Starch phosphorylase activity initially increased slightly but then fell, whereas the activity of starch synthase remained constant throughout the experiment. The soluble sugar composition of the tubers changed qualitatively towards a pattern characteristic of growing stolon tips prior to tuber initiation, but there was no clear evidence of net starch degradation. Changes in the activities of the enzymes were observed prior to noticeable effects of the hormone on tuber growth rate or the development of new stolons at the tuber eyes. - GA₃- treated tubers imported more ¹⁴C from labelled photosynthate than expected on the basis of growth rate. However, the capacity to convert solub#e-¹⁴C to ethaTiol-insoluble-¹⁴C (predominantly starch) was reduced in comparison with non-treated tubers. - The observed changes in carbohydrate composition and enzyme activities indicate that GA₃ induces a drastic change in potato tuber metabolism towards a pattern characteristic for the termination of the storage process.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

太子参微块根发育的解剖学与组织化学定位

采用植物组织培养、解剖学及组织化学定位方法研究太子参试管微块根发育的形态结构与营养物质积累特征的结果表明：太子参微块根由组培苗膨大的腋芽基部长出的不定根发育而成,经历了初生结构与次生结构发育,其膨大加粗是由于不定根的次生生长。维管形成层向内形成大量的次生木质部构成微块根的主要部分。淀粉粒是太子参微块根的主要营养存储方式。随着微块根的次生生长,淀粉粒先在次生木质部薄壁细胞中形成,随后在次生韧皮薄壁细胞中也大量积累。膨大的微块根可以合成太子参皂苷,成熟微块根中次生韧皮部的皂苷含量略高于次生木质部。离体太子参微块根的生长发育和营养物质的积累与块根中的相同。     

Acid phosphatase activity may affect the tuber swelling by partially regulating sucrose-mediated sugar resorption in potato

APase activity is involved in regulating many physiological and developmental events by affecting the resorption process.In this study, we investigate the role of APase activity in tuber development in potato. APase activities were mainly localized in cytoplasm, gaps among cells and stroma of amyloplasts of parenchyma cells at the stage of tuber swelling. AP1, encoding a putative APase, was also highly expressed in swelling tubers and a low level of expression was observed in elongated stolons and matured tubers. Inhibition of APase activity by applying Brefeldin A, an inhibitor of APase production and secretion, significantly suppressed the tuber swelling and moderately affected the stolon elongation and the tuberization frequency. During tuber development, sucrose serves as the main soluble sugar for long-distance transportation and resorption. Moreover, Inhibition of APase activity by Brefeldin A markedly reduced the sucrose content in tubers and further decreased the starch accumulation, suggesting that the function of APase in regulating the tuber swelling might be at least artially mediated by the sugar resorption. Exogenous sucrose treatments further indicate the important role of sucrose-mediated sugar resorption in tuber swelling. These results suggest that the APase activity might affect the tuber swelling by partially regulating the sucrose-mediated sugar resorption.     

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.     

Biogenesis and Degradation of Starch: I. The Fate of the Amyloplast Membranes during Maturation and Storage of Potato Tubers

Storage of mature or developing potato tubers (Solanum tuberosum “Up-to-Date” variety) at 4 C causes a reduction in the starch content and the elevation in the level of free sugars. This phenomenon is not observed when the tubers are stored at 25 C. Changes in the morphology of cells from developing or mature tubers after storage at 4 or 25 C have been followed by electron microscopy. During all stages of the tuber development the starch granules are surrounded by a membrane derived from the plastid envelope. Storage in the cold induces disintegration of this membrane. A membrane fraction isolated from starch granules of tubers stored at 4 C has a lower buoyant density, and the electrophoretic pattern of its proteins is different from that of a similar membrane fraction obtained from tubers stored at 25 C. It is suggested that the cold-induced changes in the starch and sugar content during storage of potato tubers might be correlated with damage to the membranes surrounding the starch granules and changes in their permeability to degradative enzymes and substrates.     

Long-term Metabolism of [14C]Sugars in Stored Potatoes

[¹⁴C]Sucrose, [¹⁴C]glucose and [¹⁴C]fructose were introducedinto potato tubers held at 10 °C and the redistributionof label chased over a 65 d period in storage. Respiratory losseswere identical in all treatments, as was the partitioning of¹⁴C between soluble and insoluble forms. Sucrose was the predominantlabelled sugar in the tubers after 20 h, regardless of the original[¹⁴C]sugar introduced, and was loaded and distributed throughoutthe tubers by the internal phloem system. After 20 h the proportionsof labelled sugars bore no relationship to those of the unlabelledendogenous sugars. However, with time the percentage of ¹⁴Cin sucrose fell while that in glucose increased and by 65 dthe proportions of the labelled sugars more closely resembledthe endogenous pools. Fructose represented a consistently lowproportion of both the labelled and unlabelled sugars. By 21d a considerable proportion of the soluble ¹⁴C had been convertedto starch (approx. 25% of the total tuber 14C), this value remainingrelatively constant for the remainder of the storage period.Sprouts which formed on the tubers contained up to 6% of thetotal tuber ¹⁴C but less than 0.2% of the tuber dry matter.It is suggested that the bulk of the translocated [¹⁴C]sucroseentered the symplast and exchanged slowly with the bulk of thesugars in the storage cell vacuoles. [¹⁴C]sugars, phloem loading, starch, potato tuber, Solunum tuberosum, cold storage     

Effect of sink isolation on sugar uptake and starch synthesis by potato-tuber storage parenchyma

Import into potato (Solarium tuberosum L. cv. Record) tubers was terminated by removing the sink at its connection with the stolon. The ability of discs of storage tissue from the excised tubers to take up exogenous sugars and convert them to starch was compared with that of discs from untreated tubers from the same plant population. In rapidly-growing control tubers, glucose and fructose were taken up to a greater extent than sucrose, 77% of the glucose being converted to starch within 3 h (compared with 64% and 27% for fructose and sucrose, respectively). These values fell as the tubers aged but the ranking (glucose > fructose > sucrose) was maintained, emphasising a severe rate-limiting step following the import of sucrose into the growing tuber. Sink isolation had little effect on the ability of the storage cells to take up exogenous sucrose across the plasmalemma for up to 7 d after sink isolation. However, the ability of the same cells to convert the sucrose to starch was severely inhibited within 24 h, as was the sensitivity of starch synthesis to turgor. In the case of glucose, sink isolation inhibited both the uptake and the conversion to starch, the latter being inhibited to a greater degree. A detailed metabolic study of tubers 7 d after excision showed that, with sucrose as substrate, 94% of the radioactivity in the soluble sugar pool was recovered in sucrose following sink isolation (92% in control tubers). However, with glucose as substrate, 80% of the radioactivity was recovered as sucrose following tuber excision (28% in control tubers), providing evidence that sucrose synthesis acts as a major alternative carbon sink when starch synthesis is inhibited. In the same tubers, sucrose-synthase activity decreased by 70% following sink isolation, compared with a 45% reduction in ADP-glucose pyrophosphorylase. Activities of UDP-glucose pyrophosphorylase, starch phosphorylase, starch synthase nd both PPi- and ATP-dependent phosphofructokinases remained unchanged. Acid-invertase activity increased fivefold.     

Assimilate Conversion in Potato Tubers in Relation to Starch Deposition and Cell Growth

The activity of enzymes involved in the conversion of sucrose to starch together with the distribution of ¹⁴C-labelled photosynthate and ⁴C-sucrose was studied in potato tubers showing a range of growth rates and growth patterns. Within a particular tuber the uptake of ¹⁴C from labelled photosynthate and the conversion to ethanol-insoluble ¹⁴C was greatest in the apical tissue where both the rate of production of new storage cells and starch synthesis were likely to be greatest. Uptake and conversion of ¹⁴C was lowest in the older tissue of the tuber base. Pre-treatment of tubers with gibberellic acid reduced the total input of ¹⁴C from labelled photosynthate, reversed the gradient in ¹⁴C uptake between apical and basal tuber tissue, increased the amount of ¹⁴C per g fresh weight in the basal tissue and decreased the conversion of labelled sugars to starch. For tubers with different growth rates both the total uptake of ¹⁴C from labelled photosynthate and the ratio ethanol-insoluble ¹⁴C/ethanol-soluble ¹⁴C appeared to be correlated with growth rate. In contrast when tubers were fed directly with ¹⁴C-sucrose via the tuber surface, total uptake was independent of growth rate but the correlation between growth rate and the ratio ethanol-insoluble ¹⁴C/ethanol-soluble ¹⁴C persisted. Within a particular tuber there was a decreasing gradient in sucrose synthetase activity between youngest tissue of the tuber apex and the older tissue at the tuber base but there was no clear correlation between mean enzyme activity and tuber growth rate. ADPG-pyrophosphorylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase showed some correlation with tuber growth rate. Starch synthase, starch phosphorylase and UDPG-pyro-phosphorylase activities per g fresh weight of tuber tissue appeared to be relatively constant. The results suggest that the transport of sugar from the phloem sieve tubes to the tuber storage parenchyma cells, in particular the phloem unloading step, and the conversion of sugar into starch are subject to separate regulation in the potato tuber.     

Characterization of gene expression during potato tuber development in individuals and populations using the luciferase reporter system

Analysis of gene expression and enzyme activity in pooled tuber samples has previously indicated different developmental events occurring in a fixed sequential order during tuber development, starting with the up-regulation of starch synthesis then induction of protein storage followed by cell division and cell enlargement. In this report we analysed in vivo promoter activity of genes related to cell division and storage of reserves during tuber development in individual in vitro tubers, using the non invasive firefly luciferase reporter system. The average activity of the storage related promoters (AGPaseS and Pat21) was up-regulated prior to visible swelling, while the average activity of both cell cycle genes (cycB1;1 and CDC2a) showed an up-regulation after the onset of swelling. However, this novel system allowed expression analysis in individual tubers, which showed a variable up-regulation of both storage genes in relation to the moment of swelling, from 4 days before to 10 days after the onset of swelling. We conclude that during the first stages of tuber development, the moment of storage gene induction is independent from swelling. These results indicate that the developmental program of potato tubers does not consist of a fixed sequential order of events, but consists of independent developmental programs (storage and swelling), together resulting in the formation of a potato tuber. It is concluded that analysis of developmental programs by studying individuals may result in new insights, possibly obscured when using pooled samples.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.