Ed Etxeberria

Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124–13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1–4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.

Enhancing Sucrose Synthase Activity in Transgenic Potato ( Solanum tuberosum L.) Tubers Results in Increased Levels of Starch, ADPglucose and UDPglucose and Total Yield

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields

Structurally composed of the glucose homopolymers amylose and amylopectin, starch is the main storage carbohydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells. Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both a cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including production of adhesives, biodegradable materials, and first-generation bioethanol. This review provides an update on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophic organs, and provides emerging information about the networks regulating them and their interactions with the environment. Special emphasis is given to recent findings showing that volatile compounds emitted by microorganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starch metabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species. Finally we present some potential biotechnological strategies for enhancing starch content.

Vacuolar protein sorting mechanisms in plants

Plant vacuoles are unique, multifunctional organelles among eukaryotes. Considerable new insights in plant vacuolar protein sorting have been obtained recently. The basic machinery of protein export from the endoplasmic reticulum to the Golgi and the classical route to the lytic vacuole and the protein storage vacuole shows many similarities to vacuolar/lysosomal sorting in other eukaryotes. However, as a result of its unique functions in plant defence and as a storage compartment, some plant‐specific entities and sorting determinants appear to exist. The alternative post‐Golgi route, as found in animals and yeast, probably exists in plants as well. Likely, adaptor protein complex 3 fulfils a central role in this route. A Golgi‐independent route involving plant‐specific endoplasmic reticulum bodies appears to provide sedentary organisms such as plants with extra flexibility to cope with changing environmental conditions.

Microbial Volatile Emissions Promote Accumulation of Exceptionally High Levels of Starch in Leaves in Mono-and Dicotyledonous Plants

Microbes emit volatile compounds that affect plant growth and development. However, little or nothing is known about how microbial emissions may affect primary carbohydrate metabolism in plants. In this work we explored the effect on leaf starch metabolism of volatiles released from different microbial species ranging from Gram-negative and Gram-positive bacteria to fungi. Surprisingly, we found that all microbial species tested (including plant pathogens and species not normally interacting with plants) emitted volatiles that strongly promoted starch accumulation in leaves of both mono- and dicotyledonous plants. Starch content in leaves of plants treated for 2 d with microbial volatiles was comparable with or even higher than that of reserve organs such as potato tubers. Transcriptome and enzyme activity analyses of potato leaves exposed to volatiles emitted by Alternaria alternata revealed that starch overaccumulation was accompanied by up-regulation of sucrose synthase, invertase inhibitors, starch synthase class III and IV, starch branching enzyme and glucose-6-phosphate transporter. This phenomenon, designated as MIVOISAP (microbial volatiles-induced starch accumulation process), was also accompanied by down-regulation of acid invertase, plastidial thioredoxins, starch breakdown enzymes, proteins involved in internal amino acid provision and less well defined mechanisms involving a bacterial- type stringent response. Treatment of potato leaves with fungal volatiles also resulted in enhanced levels of sucrose, ADPglucose, UDPglucose and 3-phosphoglycerate. MIVOISAP is independent of the presence of sucrose in the culture medium and is strongly repressed by cysteine supplementation. The discovery that microbial volatiles trigger starch accumulation enhancement in leaves constitutes an unreported mechanism for the elicidation of plant carbohydrate metabolism by microbes.

Fluid Phase Endocytic Uptake of Artificial Nano-Spheres and Fluorescent Quantum Dots by Sycamore Cultured Cells

Fluid phase endocytic uptake of external solutes in plant cells was further substantiated using artificial polystyrene nano-spheres (40 nm) and CdSe/ZnS quantum dots (20 nm). Both types of artificial nano particles were taken up by sycamore cultured cells. However, whereas polystyrene nano-spheres were delivered to the central vacuole, CdSe/ZnS nano-dots were sequestered into cytoplasmic vesicular structures. Using dextran Texas Red (m.w. 3,000; d-TR) as additional marker, confocal micrographs confirmed the distinct topographic distribution of CdSe/ZnS quantum dots within the cell. Initially, d-TR and CdSe/ZnS quantum dots co-localized within cytoplasmic vesicles. After 18 h incubation, d-TR was distinctly localized in the vacuole whereas CdSe/ZnS quantum dots remained sequestered in cytoplasmic membranous compartments. The data provide a first evidence for the rapid distribution of solutes taken up by endocytosis to distinct intracellular compartments.

New enzymes, new pathways and an alternative view on starch biosynthesis in both photosynthetic and heterotrophic tissues of plants

Since the initial discovery showing that ADPglucose (ADPG) serves as the universal glucosyl donor in the reaction catalyzed by starch synthase, the mechanism of starch biosynthesis in both leaves and heterotrophic organs has generally been considered to be an unidirectional process wherein ADPG pyrophosphorylase (AGPase) exclusively catalyzes the synthesis of ADPG and acts as the major limiting step of the gluconeogenic process. There is however mounting evidence that ADPG linked to starch biosynthesis is produced de novo in the cytosol by means of sucrose synthase (SuSy). In this review we show and discuss the numerous pitfalls of the ‘classic’ view of starch biosynthesis. In addition, we describe many overlooked aspects of both ADPG and starch metabolism. With the overall data we propose an ‘alternative’ model of starch biosynthesis, applicable to both photosynthetic and heterotrophic tissues, according to which both sucrose and starch biosynthetic processes are tightly interconnected by means of an ADPG synthesizing SuSy activity. According to this new view, starch metabolism embodies catabolic and anabolic reactions taking place simultaneously in which AGPase plays a vital role in the scavenging of starch breakdown products.

In and out of the plant storage vacuole

The plant storage vacuole is involved in a wide variety of metabolic functions a great many of which necessitate the transport of substances across the tonoplast. Some solutes, depending on the origin, have to cross the plasma membrane as well. The cell is equipped with a complex web of transport systems, cellular routes, and unique intracellular environments that support their transport and accumulation against a concentration gradient. These are capable of processing a diverse nature of substances of distinct sizes, chemical properties, and origins. In this review we describe the various mechanism involved in solute transport into the vacuole of storage cells with special emphasis placed on solutes arriving through the apoplast. Transport of solutes from the cytosol to the vacuole is carried out by tonoplast-bound ABC transporters, solute/H(+) antiporters, and ion channels whereas transport from the apoplast requires additional plasma membrane-bound solute/H(+) symporters and fluid-phase endocytosis. In addition, and based on new evidence accumulated within the last decade, we re-evaluate the current notion of extracellular solute uptake as partially based on facilitated diffusion, and offer an alternative interpretation that involves membrane bound transporters and fluid-phase endocytosis. Finally, we make several assertions in regards to solute export from the vacuole as predicted by the limited available data suggesting that both membrane-bound carriers and vesicle mediated exocytosis are involved during solute mobilization.

Mannitol-enhanced, fluid-phase endocytosis in storage parenchyma cells of celery (Apium graveolens; Apiaceae) petioles

We recently demonstrated the occurrence of a sucrose-enhanced, fluid-phase endocytic (FPE) mechanism of nutrient uptake in heterotrophic cells. In the present work, the possible enhancement/induction of FPE by photoassimilates other than sucrose was investigated by measuring the incorporation of the fluorescent endocytosis marker d-TR (dextran-Texas red, 3000 mw) into celery (Apium graveolens) petiole storage parenchyma (CSP), a tissue that transports and accumulates mannitol. Mannitol uptake in these cells is biphasic, with a hyperbolic phase at concentrations below 20 mM and a linear phase above 20 mM external solute concentration. In the absence of mannitol, or in its presence at concentrations within the hyperbolic phase, CSP cells accumulated low levels of d-TR. Conversely, d-TR accumulation by CSP cells was greatly enhanced in the presence of mannitol at concentrations within the linear phase. At high external mannitol concentration, d-TR accumulation was prevented by the endocytic inhibitors LY294002 and latrunculin B. In addition, d-TR uptake was temperature dependent under high mannitol concentration. Microscopic observations revealed that d-TR accumulated in the vacuole. These data support the occurrence of an FPE mechanism in CSP cells that participates in trapping and transport of photoassimilates to the vacuole. The FPE mechanism is enhanced by high mannitol concentrations.

Fluid-phase endocytosis in Citrus juice cells is independent from vacuolar pH and inhibited by chlorpromazine, an inhibitor of PI-3 kinases and clathrin-mediated endocytosis

Summary Compared to their more acidic relatives, sweet lime (Citrus limettioides Tan.) is distinguished by having a high vacuolar pH and a low sugar content. To determine whether variations in vacuolar pH are linked to different sugar uptake systems, resulting in contrasting sugar levels, we investigated developmental changes in V- (tonoplast-bound) and P-(plasmalemma-bound) ATPase activities, and the capacity of energised plasmalemma and tonoplast vesicles to take-up sucrose. Both plasmalemma- and tonoplast-bound ATPase activities, increased during fruit development. Artificially energised plasmalemma vesicles accumulated sucrose against a concentration gradient, whereas tonoplast vesicles did not show a similar uptake capacity, even in the presence of ATP. When juice cells were incubated with sucrose for 18 h with the endocytic marker, dextran-Texas Red (d-TR), an intense red fluorescence was observed in the vacuole. Sucrose-inducible uptake of d-TR was suppressed by the clathrin- and PI-3 kinase-mediated endocytosis inhibitor, chlorpromazine, but not by the caveolin-mediated endocytosis inhibitor, filipin. However, 3-dimensional Z-stack confocal images demonstrated the formation and presence of endocytic vesicles 0.5 – 2 µm in diameter, which were considerably larger than vesicles formed by clathrin-assisted scaffolding. The data indicated that: (i) most sucrose uptake into the vacuole is not mediated by a tonoplast-bound carrier; (ii) uptake of external solutes into the vacuole of C. limettioides juice cells is independent of vacuolar pH; and (iii) sucrose uptake occurs largely by a non-selective endocytic mechanism composed of vesicles, consistent with pinocytosis.