Engineering plants for elevated CO(2): a relationship between starch degradation and sugar sensing

In the future, plants will have additional CO(2) for photosynthesis. However, plants do not take maximal advantage of this additional CO(2) and it has been hypothesized that end product synthesis limitations and sugar sensing mechanisms are important in regulating plant responses to increasing CO(2). Attempts to increase end product synthesis capacity by engineering increased sucrose-phosphate synthase activity have been generally, but not universally, successful. It was found that plants benefited from a two- to three-fold increase in SPS activity but a 10-fold increase did not increase yield. Despite the success in increasing yield, increasing SPS did not increase photosynthesis. However, carbon export from chloroplasts was increased during the day and reduced at night (when starch provides carbon for sucrose synthesis. We develop here a hypothesis that starch degradation is closely sensed by hexokinase because a newly discovered pathway required for starch to sucrose conversion that involves maltose is one of few metabolic pathways that requires hexokinase activity.     

Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch

Starch is an important renewable raw material with an increasing number of applications. Several attempts have been made to obtain plants that produce modified versions of starch or higher starch yield. Most of the approaches designed to increase the levels of starch have focused on the increment of the amount of ADP-glucose or ATP available for starch biosynthesis. In this work, we show that the overexpression of starch synthase class IV (SSIV) increases the levels of starch accumulated in the leaves of Arabidopsis by 30%-40%. In addition, SSIV-overexpressing lines display a higher rate of growth. The increase in starch content as a consequence of enhanced SSIV expression is also observed in long-term storage starch organs such as potato tubers. Overexpression of SSIV in potato leads to increased tuber starch content on a dry weight basis and to increased yield of starch production in terms of tons of starch/hectare. These results identify SSIV as one of the regulatory steps involved in the control of the amount of starch accumulated in plastids.     

Diurnal changes in allocation of newly fixed carbon in exporting sugar beet leaves

Storage of newly fixed carbon as starch and sucrose follows a regular daily pattern in exporting sugar beet leaves under constant day length and level of illumination. Up to the final two hours of the light period, when starch storage declines, a nearly constant proportion of newly fixed carbon was allocated to carbohydrate storage, principally starch. Sucrose is stored only early in the light period, when there is little accumulation of starch. Pulse labeling with ¹⁴CO₂ revealed that considerable starch synthesis was taking place at this time. Starch made the previous day was not mobilized during this period but breakdown of newly synthesized starch may occur when carbon flow into sucrose synthesis increases early in the day. At the end of the day, starch storage declined from the constant level observed during most of the day, but no diversion of label into export of specific alternative compounds could be detected. Lowered storage of starch persisted when the 14-hour light period was lengthened. Changed allocation of recently fixed carbon to sucrose and starch at the beginning and end of the light period was not the result of outright inactivation of pathways but of regulation of carbon flow.     

玉米淀粉生物合成及其遗传操纵

淀粉是许多植物重要的储藏物质。淀粉突变体以及转基因植物中淀粉变异的特点使我们对淀粉生物合成的过程有了较深入的了解,许多研究的结果揭示了玉米淀粉的生物合成涉及4类酶--ADPG焦磷酸化酶、淀粉合成酶、淀粉分支酶和去分支酶。随着编码这些酶的基因的克隆,利用转基因技术对淀粉合成过程进行遗传操纵业已成为可能,并且在提高淀粉产量以及不同特性淀粉品质的种质资源创新等方面展示出巨大的潜力。 Abstract:Starch is the most important source of calories and a vital storage component in plants.The characterization and production of starch variants from mutation and with transgenic technology has improved our understanding of the synthesis of starch granule.In starch biosynthesis in plants,four enzymes,including ADP-glucose pyrophosphorylase,starch synthase,starch branching enzyme and starch debranching enzyme,are widely accepted from an enormous amount of research aimed primarily at enzyme characterization.As many genes encoding the enzymes and their multiple isoforms in starch biosynthesis pathway have been isolated,genetic manipulation of the starch biosynthesis pathway shows to be a practical way by which starch quantity is increased and starch with novel properties can be created.     

Starch synthesis and carbon partitioning in developing endosperm

The biosynthesis of starch is the major determinant of yield in cereal grains. In this short review, attention is focused on the synthesis of the soluble substrate for starch synthesis, ADPglucose (ADPG). Consideration is given to the pathway of ADPG production, its subcellular compartmentation, and the role of metabolite transporters in mediating its delivery to the site of starch synthesis. As ADPG is an activated sugar, the dependence of its production on respiration, changes which occur during development, and the constraints which ATP production may place on carbon partitioning into different end-products are discussed.     

Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves

Starch accumulation and sucrose synthesis and export were measured in leaves of sugar beet (Beta vulgaris L.) during a period of prolonged irradiance in which illumination was extended beyond the usual 14-hour day period. During much of the 14-hour day period, approximately 50% of the newly fixed carbon was distributed to sucrose, about 40% to starch, and less than 10% to hexose. Beginning about 2 hours before the end of the usual light period, the portion of newly fixed carbon allocated to sucrose gradually increased, and correspondingly less carbon went to starch. By the time the transition ended, about 4 hours into the extension of the light period, nearly 90% of newly fixed carbon was incorporated into sucrose and little or none into starch. Most of the additional sucrose was exported. Gradual cessation of starch accumulation was not the result of a futile cycle of simultaneous starch synthesis and degradation. Neither was it the result of a decrease in the extractable activity of adenosine diphosphoglucose pyrophosphorylase or phosphoglucose isomerase, enzymes important in starch synthesis. Nor was there a notable change in control metabolites considered to be important in regulating starch synthesis. Starch accumulation appeared to decrease markedly because of an endogenous circadian shift in carbon allocation, which occurred in preparation for the usual night period and which diverted carbon from the chloroplast to the cytosol and sucrose synthesis.     

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose‐accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter‐relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone‐like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.     

Starch turnover: pathways, regulation and role in growth

Many plants store part of their photosynthate as starch during the day and remobilise it to support metabolism and growth at night. Mutants unable to synthesize or degrade starch show strongly impaired growth except in long day conditions. In rapidly growing plants, starch turnover is regulated such that it is almost, but not completely, exhausted at dawn. There is increasing evidence that premature or incomplete exhaustion of starch turnover results in lower rates of plant growth. This review provides an update on the pathways for starch synthesis and degradation. We discuss recent advances in understanding how starch turnover and the use of carbon for growth is regulated during diurnal cycles, with special emphasis on the role of the biological clock. Much of the molecular and genetic research on starch turnover has been performed in the reference system Arabidopsis. This review considers to what extent information gained in this weed species maybe applicable to annual crops and perennial species.     

Influence of Carbon Dioxide Concentration on Growth, Carbohydrate Content, Translocation and Photosynthesis of White Clover

White clover ramets were grown at various carbon dioxide concentrations(200, 350 and 1000 µl 1^–1), defoliated and regrownat the same concentrations. Morphological characteristics, dryweights and non-structural carbohydrate contents of plant organs,diurnal variation of sugar and starch content of leaves, translocationof assimilates and photosynthesis were determined. Carbon dioxide concentration influenced the dry weights, butnot the number and size of the plant organs. However, defoliationof plants at low carbon dioxide concentration resulted in decreasedleaf size and stolon length. Carbon dioxide concentration influencedthe content and diurnal variation of starch and sugar in theleaves. Starch was accumulated at medium carbon dioxide concentrationand sugar at a higher concentration when the storage capacityfor starch seemed to be exceeded. Starch was preferentiallyaccumulated in the first and sugar in the second half of thelight period. Translocation was decreased during the periodsof accumulation. Sugar accumulation in the leaves seemed tobe a consequence of the imbalance between sink and source, whereasstarch accumulation seemed to follow an in-built diurnal pattern.Accumulation of both starch and sugar during the photoperiodwas followed by degradation and export during the dark period.Decreased dark export occurred at low carbon dioxide concentrationwhen neither starch nor sugar was accumulated during the photoperiod. Carbon dioxide, white clover, Trifolium repens L., growth, carbohydrates, starch, sugar, translocation, photosynthesis     

过表达NtAGPase小亚基基因提高烟叶淀粉含量和生物量

发展以非粮食作物为原料制备乙醇等生物燃料既可缓解全球能源危机,又能减低粮食作物用于生物燃料对粮食安全的威胁。烟草Nicotiana tabacum是一种生物量较高的经济作物,培育富含淀粉的新型烟草,可专用于燃料乙醇生产。文中克隆了烟草控制淀粉生物合成的ADP-葡萄糖焦磷酸化酶 (ADP-glucose pyrophosphorylase,NtAGPase) 小亚基基因NtSSU,并构建了NtSSU基因植物表达载体。通过农杆菌介导叶盘转化法在烟草中超表达NtSSU基因。转基因烟草植株表型鉴定显示,过表达NtSSU基因促进烟叶淀粉富集,烟叶淀粉含量从野生型17.5%升高到41.7%。转基因烟草的生长速率和生物量也显著增加。研究结果揭示,过表达NtSSU基因能有效调动光合产物碳通量更多地进入淀粉合成途径,提高生物质产量,且未对其他农艺性状产生负效应。因此,NtSSU基因可作为优异靶标基因应用于植物代谢工程以促进营养器官中淀粉的合成积累,从而开发专用于生产燃料乙醇的新种质。     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.     

Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii

Microalgal oils have attracted much interest as potential feedstocks for renewable fuels, yet our understanding of the regulatory mechanisms controlling oil biosynthesis and storage in microalgae is rather limited. Using Chlamydomonas reinhardtii as a model system, we show here that starch, rather than oil, is the dominant storage sink for reduced carbon under a wide variety of conditions. In short-term treatments, significant amounts of oil were found to be accumulated concomitantly with starch only under conditions of N starvation, as expected, or in cells cultured with high acetate in otherwise standard growth medium. Time-course analysis revealed that oil accumulation under N starvation lags behind that of starch and rapid oil synthesis occurs only when carbon supply exceeds the capacity of starch synthesis. In the starchless mutant BAFJ5, blocking starch synthesis results in significant increases in the extent and rate of oil accumulation. In the parental strain, but not the starchless mutant, oil accumulation under N starvation was strictly dependent on the available external acetate supply and the amount of oil increased steadily as the acetate concentration increased to the levels several-fold higher than that of the standard growth medium. Additionally, oil accumulation under N starvation is saturated at low light intensities and appears to be largely independent of de novo protein synthesis. Collectively, our results suggest that carbon availability is a key metabolic factor controlling oil biosynthesis and carbon partitioning between starch and oil in Chlamydomonas.     

Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape

The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.     

Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol

Many microalgae and plants have the ability to synthesize large amounts of triacylglycerol (TAG) that can be used to produce biofuels. Presently, TAG-based biofuel production is limited by the feedstock supply. Metabolic engineering of lipid synthesis pathways to overproduce TAGs in oleaginous microalgae and oil crop plants has achieved only modest success. We demonstrate that inactivation of ADP-glucose pyrophosphorylase in a Chlamydomonas starchless mutant led to a 10-fold increase in TAG, suggesting that shunting of photosynthetic carbon partitioning from starch to TAG synthesis may represent a more effective strategy than direct manipulation of the lipid synthesis pathway to overproduce TAG.     

岳桦幼苗光合特性和非结构性碳水化合物积累对干旱胁迫的响应

以长白山林线树种岳桦为对象,利用生长控制试验进行干旱处理,研究干旱对岳桦幼苗光合特性及非结构性碳水化合物(NSC)积累的影响.结果表明:干旱显著降低了岳桦幼苗的净光合速率和气孔导度,提高了其水分利用效率;干旱显著增加了岳桦幼苗叶、皮、干和根中的可溶性糖和总NSC的含量,但显著降低了淀粉含量;随着干旱的持续,叶片的气孔导...     

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L⁻¹ D⁻¹. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.     

The diurnal metabolism of leaf starch

Starch is a primary product of photosynthesis in leaves. In most plants, a large fraction of the carbon assimilated during the day is stored transiently in the chloroplast as starch for use during the subsequent night. Photosynthetic partitioning into starch is finely regulated, and the amount of carbohydrate stored is dependent on the environmental conditions, particularly day length. This regulation is applied at several levels to control the flux of carbon from the Calvin cycle into starch biosynthesis. Starch is composed primarily of branched glucans with an architecture that allows the formation of a semi-crystalline insoluble granule. Biosynthesis has been most intensively studied in non-photosynthetic starch-storing organs, such as developing seeds and tubers. Biosynthesis in leaves has received less attention, but recent reverse-genetic studies of Arabidopsis (thale cress) have produced data generally consistent with what is known for storage tissues. The pathway involves starch synthases, which elongate the glucan chains, and branching enzymes. Remarkably, enzymes that partially debranch glucans are also required for normal amylopectin synthesis. In the last decade, our understanding of starch breakdown in leaves has advanced considerably. Starch is hydrolysed to maltose and glucose at night via a pathway that requires recently discovered proteins in addition to well-known enzymes. These sugars are exported from the plastid to support sucrose synthesis, respiration and growth. In the present review we provide an overview of starch biosynthesis, starch structure and starch degradation in the leaves of plants. We focus on recent advances in each area and highlight outstanding questions.     

Water-deficit accumulates sugars by starch degradation--not by de novo synthesis--in white clover leaves (Trifolium repens)

Labeling 13CO2 in steady-state condition was used to estimate quantitative mobilization of recently fixed carbon or stored sugar during water-deficit in white clover (Trifolium repens L.). Water-deficient gradually decreased leaf-water parameters and total amount of recently fixed carbon. Amount of 13C incorporated into glucose, sucrose and soluble sugars fraction rapidly decreased after 3 days of water-deficit treatment. In contrast, the previously stored soluble sugars significantly increased after 5 days of water-deficit with a coincidence of significant decrease in starch concentration. A highly significant (P < or = 0.001) relationship between the decrease in leaf-water potential caused by water-deficit and the increase in ratio of soluble sugar/starch concentration was observed in water deficit-stressed plants. The data indicate that soluble carbohydrate accumulated by water-deficit treatment is mainly because of the hydrolysis of previously stored starch rather than to de novo synthesis.     

Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii

Starch and neutral lipids are two major carbon storage compounds in many microalgae and plants. Lipids are more energy rich and have often been used as food and fuel feedstocks. Genetic engineering of the lipid biosynthesis pathway to overproduce lipid has achieved only limited success. We hypothesize that through blocking the competing pathway to produce starch, overproduction of neutral lipid may be achieved. This hypothesis was tested using the green microalga Chlamydomonas reinhardtii and its low starch and starchless mutants. We discovered that a dramatic increase in neutral lipid content and the neutral lipid/total lipid ratio occurred among the mutants under high light and nitrogen starvation. BAFJ5, one of the mutants defective in the small subunit of ADP‐glucose pyrophosphorylase, accumulated neutral and total lipid of up to 32.6% and 46.4% of dry weight (DW) or 8‐ and 3.5‐fold higher, respectively, than the wild‐type. These results confirmed the feasibility of increasing lipid production through redirecting photosynthetically assimilated carbon away from starch synthesis to neutral lipid synthesis. However, some growth impairment was observed in the low starch and starchless mutants, possibly due to altered energy partitioning in PSII, with more excitation energy dissipated as heat and less to photochemical conversion. This study demonstrated that biomass and lipid production by the selected mutants can be improved by physiological manipulation. Biotechnol. Bioeng. 2010;107: 258–268. © 2010 Wiley Periodicals, Inc.