Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

甘薯蔗糖转运蛋白的功能分析

甘薯(Ipomoea batatas)是重要的粮食和工业加工原料作物。蔗糖是植物体内碳水化合物长距离转运的主要形式,蔗糖转运蛋白(sucrose transporter,SUT)在植物的生长代谢中调控蔗糖的跨膜运输和分配,在韧皮部介导的源-库蔗糖运输和为库组织供应蔗糖的生理活动中起关键作用。本研究根据不同淀粉性状甘薯块根中差异表达的2个SUT基因转录本,进行cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)克隆,获得IbSUT62788和IbSUT81616的全长cDNA序列;通过系统发育分析明确其分类;通过在本氏烟草(Nicotiana benthamiana)中瞬时表达明确其亚细胞定位;通过酵母功能互补系统鉴定IbSUT62788和IbSUT81616是否具有吸收、转运蔗糖和己糖的能力。通过实时荧光定量PCR(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)分析IbSU62788和IbSUT81616在甘薯各器官中的表达特征;通过蘸花法得到外源表达IbSUT62788和IbSUT81616基因的拟南芥(Arabidopsis thaliana)植株,比较与野生型拟南芥的淀粉和糖含量的差异。结果表明,IbSUT62788和IbSUT81616分别编码505个和521个氨基酸的SUT蛋白,均属于SUT1亚家族。IbSUT62788和IbSUT81616均定位于细胞膜,在酵母系统中具有转运蔗糖、葡萄糖和果糖的能力。此外,IbSUT62788还具有转运甘露糖的能力。IbSUT62788在甘薯叶片、侧枝和茎中的表达量更高,IbSUT81616在侧枝、茎和块根中表达量更高。IbSUT62788和IbSUT81616在拟南芥中异源表达后,植株可以正常生长,但生物量增加。IbSUT62788的异源表达增加了拟南芥植株叶片可溶性糖含量、叶片大小和种子千粒重;IbSUT81616的异源表达增加了拟南芥植株叶片、根尖的淀粉积累量和种子千粒重,但减少了可溶性糖含量。本研究结果表明,IbSUT62788和IbSUT81616可能是调控甘薯蔗糖和糖含量性状的重要基因,在细胞膜上进行着蔗糖的跨膜运输、蔗糖进出库组织、韧皮部蔗糖的运输与卸载等生理功能,在拟南芥中异源表达造成的性状改变说明其在提高其他植物或作物产量中的应用潜力。本研究为揭示甘薯淀粉和糖代谢及重要品质性状形成机制提供了重要信息。     

干旱胁迫下紫花苜蓿幼苗非结构性碳水化合物代谢对NO的响应

以紫花苜蓿（Medicago sativa）为材料,采用盆栽试验方法,用聚乙二醇（PEG-6000）作为渗透介质模拟干旱胁迫,外源喷施NO供体硝普钠,NO清除剂（carboxy-PTIO,cPTIO）,对紫花苜蓿幼苗叶片、根系中非结构性碳水化合物含量及相关酶活性的变化进行研究,探讨NO对紫花苜蓿耐旱机制的作用。结果表明：外源NO促进了紫花苜蓿叶片中淀粉的分解、根系中淀粉的积累,提高叶片及根系中可溶性糖（蔗糖、果糖和葡萄糖）含量,降低了渗透势,促进细胞吸水,缓解干旱造成的损伤。此外,外源NO能提高干旱胁迫下紫花苜蓿叶片中蔗糖合成酶（SS）、酸性转化酶（AI）和中性转化酶（NI）活性,降低了蔗糖磷酸合成酶（SPS）的活性,提高根系中SS、SPS和转化酶活性,使蔗糖的合成与分解处于高水平的动态平衡,增强了紫花苜蓿的抗旱性。而NO清除剂cPTIO则会不同程度的抑制紫花苜蓿幼苗中非结构性碳水化合物（NSC）及其相关酶活性。因此,NO可以通过调控NSC的代谢响应干旱胁迫,缓解干旱胁迫造成的不利影响,在紫花苜蓿的抗旱中扮演着重要的角色。     

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.     

Spatial Patterns of Sucrose-Inducible and Polygalacturonic Acid-Inducible Expression of Genes That Encode Sporamin and r{beta}-Amylase in Sweet Potato

Two major proteins of tuberous roots of sweet potato, sporaminand rß-amylase, were detected in storage parenchymacells, which contain a large amount of starch. In both the leavesand petioles of sweet potato, the sucrose-induced accumulationof mRNAs for sporamin and rß-amylase, and of starchoccurred in a wide variety of cells, first in cells within andaround the vascular tissue and then in various cells distalto them, with the exception of epidermal cells. In the mesophyllcells of leaves treated with sucrose, the accumulation of largenumbers of well-developed starch granules occurred in the preexistingchloroplasts. These results, together with the previous observationthat the sucrose-induced accumulation of sporamin, of rß-amylaseand of starch occurs with similar dependency on the concentrationof sucrose, suggest that an excess supply of sugars to varioustypes of cell triggers a cellular transition that induces thesimultaneous accumulation of these reserve materials that arenormally present in tuberous roots. Accumulation of mRNAs forsporamin and rß-amylase, but not the accumulationof starch, in leaves and petioles can be also induced when leaf-petiolecuttings are supplied with low concentrations of polygalacturonicacid (PGA) at their cut edges. The spatial patterns of accumulationof mRNAs for sporamin and rß-amylase in leaves andpetioles after treatment with PGA were found to be similar tothose observed upon treatment with sucrose. These results suggestthat most of the cells in leaves and petioles have the capacityto respond to both a carbohydrate metabolic signal and a PGA-derivedsignal that is transmitted by diffusion from the vascular system. ⁴Present address: Department of Molecular Biology, NationalInsustitute of Agrobiological Resources, Tsukuba City, Ibaraki,305 Japan.     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.     

Magnesium deficiency in sugar beets alters sugar partitioning and phloem loading in young mature leaves

Magnesium deficiency has been reported to affect plant growth and biomass partitioning between root and shoot. The present work aims to identify how Mg deficiency alters carbon partitioning in sugar beet (Beta vulgaris L.) plants. Fresh biomass, Mg and sugar contents were followed in diverse organs over 20 days under Mg-sufficient and Mg-deficient conditions. At the end of the treatment, the aerial biomass, but not the root biomass, of Mg-deficient plants was lower compared to control plants. A clear inverse relationship between Mg and sugar contents in leaves was found. Mg deficiency promoted a marked increase in sucrose and starch accumulation in the uppermost expanded leaves, which also had the lowest content of Mg among all the leaves of the rosette. The oldest leaves maintained a higher Mg content. [¹⁴C]Sucrose labelling showed that sucrose export from the uppermost expanded leaves was inhibited. In contrast, sucrose export from the oldest leaves, which are close to, and export mainly to, the roots, was not restricted. In response to Mg deficiency, the BvSUT1 gene encoding a companion cell sucrose/H⁺ symporter was induced in the uppermost expanded leaves, but without further enhancement of sucrose loading into the phloem. The observed increase in BvSUT1 gene expression supports the idea that sucrose loading into the phloem is defective, resulting in its accumulation in the leaf.     

Soybean leaf responses to threecornered alfalfa hopper petiole girdling

Specific leaf weight, percent moisture, and free sugar, starch, and amino nitrogen content of soybean (Glycine max (L.) Merrill) leaves were measured at 1, 3, 7, and 10 day(s) after petiole girdling by threecornered alfalfa hopper,Spissistilus festinus (Say) (Homoptera: Membracidae) nymphs. Leaf starch was increased at 1, 3, and 7 day(s) after girdling and largely accounted for corresponding increases in specific leaf weight and decreases in percent moisture, free sugars, and amino nitrogen. Specific leaf weight was increased at 10 days after girdling despite no increase in starch. Amino nitrogen content was decreased 10 days after girdling. When leaf dry weights were corrected for starch, free sugar content was not affected by girdling, and amino nitrogen content was reduced only at 3 and 10 days. The amino nitrogen: free sugar ratio was reduced only at 10 days after girdling. Changes in leaf starch indicated a rapid but reversible effect of girdling on leaf carbohydrate metabolism.     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.     

Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum)

Here we show that fructose 2,6-bisphosphate cannot be reliably measured in mature leaves of tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.), or stinging nettle (Urtica dioica L.) using conventional extraction techniques, since the recoveries of fructose 2,6-bisphosphate added during extraction are poor. However, fructose 2,6-bisphosphate could be extracted by boiling leaves in ethanol and aqueous buffer. Evidence for the reliability of this technique is provided by high recovery measurements of fructose 2,6-bisphosphate added to the leaves before extraction. This extraction method was used to measure changes in the level of fructose 2,6-bisphosphate throughout the photoperiod in tobacco and potato leaves. These changes are compared with the rate of accumulation of sucrose and starch in the leaf samples. Variations in the levels of fructose 2,6-bisphosphate, and the relationship between this metabolite and sucrose and starch accumulation in these leaves during the photoperiod are similar to the pattern observed in leaves of other plant species.Abbreviations BSA bovine serum albumin - Fru-2,6-P₂ fructose 2,6-bisphosphate This research was supported by the Agricultural and Food Research Council (Grant no. PG43/531), and the Royal Society.     

Osmotic regulation of starch synthesis in potato tubers?

Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 mM but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300–400 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 mM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.     

Effect of aluminium on metabolism of starch and sugars in growing rice seedlings

The effect of increasing concentrations of Al₂(SO₄)₃ in situ on the content of starch, sugars and activity behaviour of enzymes related to their metabolism were studied in growing seedlings of two rice cvs. Malviya-36 and Pant-12 in sand cultures. Al₂(SO₄)₃ levels of 80 and 160 μM in the growth medium caused an increase in the contents of starch, total sugars as well as reducing sugars in roots as well as shoots of the rice seedlings during a 5–20 days growth period. The activities of the enzymes of starch hydrolysis α-amylase, β-amylase and starch phosphorylase declined in Al-exposed seedlings, whereas the activities of sucrose hydrolyzing enzymes sucrose synthase and acid invertase increased in the seedlings due to Al³⁺ treatment. The enzyme of sucrose synthesis, sucrose phosphate synthase showed decreased activity in Al³⁺ treated seedlings compared to controls. Results suggest that Al³⁺ toxicity in rice seedlings impairs the metabolism of starch and sugars and favours the accumulation of hexoses by enhancing the activities of sucrose hydrolyzing enzymes.     

Starch self-processing in transgenic sweet potato roots expressing a hyperthermophilic α-amylase

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered.