Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Temporal dynamics of tuber formation and related processes in a crossing population of potato (Solanum tuberosum)

The chronological relationships between stolon formation, stolon tip swelling, tuber initiation, flowering, senescence, growth and resorption of tubers were studied under field conditions in a diploid population of potato with 238 genotypes, the parental clones and seven tetraploid cultivars. Timing of tuber initiation was not closely related to the timing of stolon formation, flowering and duration of the plant cycle. Tuber initiation very often preceded stolon branching. The number and size distribution of tubers were largely influenced by the degree of stolon branching, the length of the stolon swelling period and tuber resorption. The peak production of stolons and swollen stolon tips largely took place within the flowering period, although in most genotypes, some stolon tip swelling took place until the end of the plant cycle. More information on the general temporal relationships between events related to tuber formation and plant development will contribute to a better understanding of the physiological and genetic basis of the processes leading to the production of harvestable tubers.     

Effect of Carbohydrate Demand on the Remobilization of Starch in Stolons and Roots of White Clover (Trifolium repens L.) after Defoliation

White clover plants were grown from stolon tips in growth cabinetsand then defoliated. Thereafter, changes in the contents ofnon-structural carbohydrates such as starch, sucrose, glucose,fructose, maltose, and pinitol in stolons and roots were monitored.Initial contents of carbohydrate reserves, photosynthetic supplyof new carbohydrates and carbohydrate demand after defoliationwere varied by growing the plants at various CO₂ partial pressures,by varying the extent of defoliation and by removing eitherroots or stolon tips at the time of defoliation. Remobilization of carbohydrate reserves in stolons increasedproportionally to their initial contents and was greater whenplants had been severely defoliated, suggesting that carbohydrateswere remobilized according to availability and demand. Starchwas the predominant reserve carbohydrate. Starch degradationwas associated with decreased contents of sucrose, glucose andfructose in young stolon parts and roots but not in old stolonparts suggesting that starch degradation was not strictly controlledby the contents of these sugars. A decrease in the demand forcarbohydrates by removal of roots did not decrease starch degradationbut increased the contents of sucrose, glucose, and fructose.Removal of stolon tips decreased starch degradation and contentsof sucrose, glucose, and fructose. The results suggest thatstarch degradation was controlled by a factor other than sucrose,glucose, and fructose which was exported from stolon tips, e.g.gibberellin. Key words: White clover, storage carbohydrates, remobilization, regrowth     

Tuber morphology and starch accumulation are independent phenomena: Evidence from ipt-transgenic potato lines

Tuber formation and carbohydrate metabolism in potatoes were studied using transgenic potato plants carrying the Agrobacterium tumefaciens ipt gene, involved in cytokinin biosynthesis. Three independent transformants, viz. clones 1, 11 and 13, whose cytokinin and auxin content had previously been shown to be different from each other and from the wild-type, were analysed in vitro. Clones 11 and 13 showed a higher ability to form stolons and tubers, as evident from: (1) stolon development in whole plants grown under non-inductive conditions, (2) total number and weight of tubers formed by cuttings of this clone in darkness, (3) tubers appeared earlier than tubers of wild-type plants and at a lower sucrose concentration in the medium. Clone 1 did not form stolons or tubers under any conditions tested, but rather formed short shoots. A series of metabolic changes, known to be characteristic for tubers, were analysed in leaves, stems and developing buds. It was found that the short type of shoots, formed by clone 1, had metabolic characteristics very similar to tubers formed in wild-type or clones 11 and 13, including glucose, fructose, sucrose, and starch levels, and activities of invertase, sucrose synthase and fructokinase. It is concluded that the regulation of the stolon swelling and of carbohydrate metabolism, normally occurring simultaneously, can be uncoupled, and are thus, at least partly independent phenomena. The present data obtained with a high-cytokinin line indicate that cytokinins (probably in concert with auxins) might be mainly involved in the regulation of tuber morphology.     

A crosstalk of auxin and GA during tuber development

Several hormones have been studied for their effect on tuber initiation and development. Until recently, the hormone with the most prominent role in tuber initiation was attributed to GA. Genes involved in GA degradation do exhibit an upregulated profile during early stages of tuber development, leading to a rapid decrease of active GA content, thereby facilitating stolon-tip swelling. While GA is known to be involved in shoot and stolon elongation, the development of the new tuberorgan requires changes in meristem identity and the reorientation ofthe plane of cell division. In other developmental processes, such as embryo patterning, flower development and lateral root initiation auxin plays a key role. Recent evidence on the involvement of auxin in tuber formation was providedby the measurement of auxin content in swelling stolons. Auxin content in the stolon tips increased several fold prior to tuber swelling. In vitro tuberisation experiments with auxin applications support the role of auxin during tuber initiation. Taken together, it is becoming clear that the initiation and induction of tubers in potato is a developmental process that appears to be regulated by a crosstalk between GA and auxin.     

StGA2ox1 is induced prior to stolon swelling and controls GA levels during potato tuber development

The formation and growth of a potato ( Solanum tuberosum ) tuber is a complex process regulated by different environmental signals and plant hormones. In particular, the action of gibberellins (GAs) has been implicated in different aspects of potato tuber formation. Here we report on the isolation and functional analysis of a potato GA 2-oxidase gene ( StGA2ox1 ) and its role in tuber formation. StGA2ox1 is upregulated during the early stages of potato tuber development prior to visible swelling and is predominantly expressed in the subapical region of the stolon and growing tuber. 35S-over-expression transformants exhibit a dwarf phenotype, reduced stolon growth and earlier in vitro tuberization. Transgenic plants with reduced expression levels of StGA2ox1 showed normal plant growth, an altered stolon swelling phenotype and delayed in vitro tuberization. Tubers of the StGA2ox1 suppression clones contain increased levels of GA₂₀, indicating altered GA metabolism. We propose a role for StGA2ox1 in early tuber initiation by modifying GA levels in the subapical stolon region at the onset of tuberization, thereby facilitating normal tuber development and growth.     

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.     

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.     

Cytokinin Activity in Stolons and Tubers of Solanum tuberosum during the Period of Tuberization

In water-culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), changes in cytokinin activity in the stolon tips and newly formed tubers during tuberization were studied. Tuberization was induced by withdrawing nitrogen from the nutrient solution. — The cytokinin activity was low in the stolon tips prior to tuberization, but increased considerably in both stolon tips and young tubers during tuberization. At the same time qualitative changes in the cytokinin spectrum occurred. These qualitative changes are reversible if ‘regrowth’ of young tubers is brought about by a sudden high supply of nitrogen. — Despite the close correlation between tuberization and cytokinin activity, it is assumed that cytokinins are not directly responsible for the onset of tuberization, although they play an important role in tuber growth.     

Dynamic changes in carbohydrate metabolism and endogenous hormones during <Emphasis Type="Italic">Tulipa edulis</Emphasis> stolon development into a new bulb

The stolon is the main asexual reproductive organ of Tulipa edulis (Miq.) Baker. It has a special morphology and can develop into a new bulb for propagation. In the current greenhouse experiment, the dynamic changes in carbohydrates and related enzymes, protein and endogenous hormones during T. edulis stolon development were investigated. The results showed that soluble sugar levels were basically declining, whereas starch and protein content rose continuously during stolon development. The adenosine diphosphoglucose pyrophosphorylase (AGPase) activity peaked in the initial swelling stage and stayed a relative high level in the middle swelling stage; sucrose synthase (SS), soluble starch synthase (SSS) and granule-bound starch synthase (GBSS) activities followed the same law that showed rising trends during stolon development. SS activity was significantly inversely related to sucrose content but had significantly positive relations with starch content, SSS and GBSS activities. Gibberellin (GA), indole-3-acetic acid (IAA) and zeatin riboside (ZR) peaked in the initial swelling stage and maintained high levels in the middle swelling stage; they then decreased significantly in the later swelling stage. A substantial increase was observed in abscisic acid (ABA) content until the middle swelling stage, followed by a significant reduction in the later swelling stage. The ratios of ABA to IAA, GA and ZR reached their lowest levels in the initial swelling stage. In conclusion, T. edulis stolon development is a process of new bulb morphogenesis along with the starch accumulation catalyzed by AGPase, SSS and GBSS, using the product of sucrose cleavage caused by SS. Initial low ABA content and low ratios of ABA to IAA, GA and ZR, together with the GA, IAA and ZR of high-content, soluble sugars worked more efficiently to induce new bulb formation.     

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.     

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.     

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.     

马铃薯块茎发育机理及其基因表达

马铃薯(Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成。对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日照、较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化。然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后。已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关。综述了这一领域现有的研究进展。     

马铃薯块茎发育机理及其基因表达

马铃薯（Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成,对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日 照,较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化,然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后,已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关,综述了这一领域现有的研究进展。     

Enhancing vacuolar sucrose cleavage within the developing potato tuber has only minor effects on metabolism

Modification of tuber carbohydrate metabolism by the tuber-specific expression of a yeast invertase targeted to the cytosol or apoplast has previously been demonstrated to have diverse effects on tuber growth and metabolism. In the current study, we generated plants exhibiting tuber-specific expression of the same enzyme targeted to the vacuole. Enzymatic analysis of the carbohydrate levels of the tuber revealed dramatic decreases in sucrose content coupled with large increases in the levels of glucose and hexose phosphates, but unaltered starch content in the transformants. Analysis of the key enzyme of glycolysis suggests that this pathway is down-regulated in the transformants. Despite these changes in metabolite pools and enzyme activity, few consistent changes could be observed in the estimated metabolic fluxes following incubation of isolated tuber discs in labelled glucose. The analysis of the relative levels of a wide range of metabolites using a gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling method revealed large changes in the levels of fructose and decreases in a range of other sugars, but very few changes in the contents of organic and amino acids. This metabolic profile is remarkably consistent with that obtained following expression of the invertase in the apoplastic compartment, providing circumstantial evidence for the endocytotic trafficking of sugars within potato tuber parenchyma. Finally, the results of this study are compared with those from other plant species and the relative roles of the vacuolar isoform of the enzyme are contrasted.