Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

Alterations in cytosolic glucose-phosphate metabolism affect structural features and biochemical properties of starch-related heteroglycans

The cytosolic pools of glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate are essential intermediates in several biosynthetic paths, including the formation of sucrose and cell wall constituents, and they are also linked to the cytosolic starch-related heteroglycans. In this work, structural features and biochemical properties of starch-related heteroglycans were analyzed as affected by the cytosolic glucose monophosphate metabolism using both source and sink organs from wild-type and various transgenic potato (Solanum tuberosum) plants. In leaves, increased levels of the cytosolic phosphoglucomutase (cPGM) did affect the cytosolic heteroglycans, as both the glucosyl content and the size distribution were diminished. By contrast, underexpression of cPGM resulted in an unchanged size distribution and an unaltered or even increased glucosyl content of the heteroglycans. Heteroglycans prepared from potato tubers were found to be similar to those from leaves but were not significantly affected by the level of cPGM activity. However, external glucose or Glc-1-P exerted entirely different effects on the cytosolic heteroglycans when added to tuber discs. Glucose was directed mainly toward starch and cell wall material, but incorporation into the constituents of the cytosolic heteroglycans was very low and roughly reflected the relative monomeric abundance. By contrast, Glc-1-P was selectively taken up by the tuber discs and resulted in a fast increase in the glucosyl content of the heteroglycans that quantitatively reflected the level of the cytosolic phosphorylase activity. Based on (14)C labeling experiments, we propose that in the cytosol, glucose and Glc-1-P are metabolized by largely separated paths.     

A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)

The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step ¹⁴C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- α -arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.     

Double Knockout Mutants of Arabidopsis Grown under Normal Conditions Reveal that the Plastidial Phosphorylase Isozyme Participates in Transitory Starch Metabolism

In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1 × phs1a and mex1 × phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1 × phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.During the last two decades, biochemical analyses of starch metabolism in higher plants have been favored by the availability of large sets of insertion mutants deficient in a single starch-related gene product. Based on phenotypical characterization of these mutants followed by the identification of the respective locus in the genome, novel starch-related proteins were discovered that reside inside the plastid, in the cytosol, in the nucleus, and in the plastidial envelope membranes. Taken together, these results have largely altered the current view on starch metabolism (Zeeman et al., 2010; Fettke et al., 2012a; Smith, 2012).Despite this progress, phenotypical analyses of starch-related mutants are complex and, under certain circumstances, yield misleading conclusions. Loss of function of metabolic steps may cause the entire starch synthesizing or degrading process to become nonfunctional. In this case, mutants are expected to have starch levels that are significantly altered. If, however, single knockout mutants are capable of partially or fully compensating the loss of function by other routes, the resulting phenotypes are less obvious and more difficult to predict. Carbon fluxes through existing paths may be enhanced, or novel metabolic routes may be established that compensate the lost function. As an example, leaves of Arabidopsis (Arabidopsis thaliana) mutants constitutively lacking the plastidial hexose-phosphate isomerase strongly express a distinct plastidial Glc-6-P/orthophosphate antiporter isoform that in wild-type plants is found only in heterotrophic tissues (Kunz et al., 2010). In mesophyll cells of the mutant, the reductive pentose phosphate cycle cannot drive assimilatory starch biosynthesis, as chloroplasts are unable to convert Fru-6-P to Glc-6-P. However, their capacity of transporting Glc-6-P between the cytosolic and the chloroplastic compartment is strongly increased. Furthermore, nonfunctionality of some starch-related proteins can lead to enlarged or diminished metabolite pools that via sensing processes, lead to cellular alterations distant from central carbon metabolism. This complexity is evidenced by several starch-related Arabidopsis mutants that possess a largely altered plastidial ultrastructure and exhibit premature degradation of the entire chloroplast (Stettler et al., 2009; Cho et al., 2011).Furthermore, several starch-related enzymes are capable of forming homomeric or heteromeric complexes that are functionally relevant but, to some extent, variable (Delatte et al., 2005; Utsumi and Nakamura, 2006; Kubo et al., 2010; Emes and Tetlow, 2012; Nakamura et al., 2012; Streb et al., 2012).In starch or glycogen storing prokaryotic and eukaryotic cells, α-glucan phosphorylase (EC 2.4.1.1) is common. Initially, this enzyme was considered to be the main starch synthesizing activity (Hanes, 1940). Later, both starch and glycogen synthases have been discovered that utilize either ADPglucose or UDPglucose (or both; Deschamps et al., 2006) as hexosyl donor. Ample evidence has been presented that these enzymes are essential biosynthetic enzymes (Ballicora et al., 2003; Zeeman et al., 2010; Roach et al., 2012; Palm et al., 2013). Furthermore, it is widely accepted that in glycogen-storing cells, phosphorylase is indispensible for the degradation of the storage polysaccharide (Hwang et al., 1989; Alonso-Casajús et al., 2006; Wilson et al., 2010; Roach et al., 2012; Gazzerro et al., 2013).In plant cells, the metabolic function of phosphorylase is more complex and far from being clear. In lower and higher plants, two distinct phosphorylase types exist as plastid- and cytosol-specific isozymes and are designated as Pho1 (or, in Arabidopsis, PHS1) and Pho2 (PHS2), respectively. Based on the large differences in the affinities for glycogen, the plastidial and the cytosolic phosphorylases are also named as low-affinity (L-type) and high-affinity (H-type) isozymes, respectively. As starch is restricted to the plastids, only the Pho1 (PHS1) type appears to possess direct access to native starch and/or plastidial starch-derived α-glucans.Conflicting phenotypical features have been reported for several mutants possessing altered levels of the plastidial phosphorylase isozyme(s). In the starch-related mutant4 of the unicellular green alga Chlamydomonas reinhardtii, the lack of one plastidial Pho1 isozyme (designated as PhoB) was associated with a lower cellular starch content, abnormally shaped granules, a modified amylopectin structure, and an elevated amylose-to-amylopectin ratio when the cells were kept under nitrogen limitation (Dauvillée et al., 2006). These phenotypical features suggest an involvement of the plastidial phosphorylase PhoB in the biosynthesis of a storage polysaccharide resembling the reserve starch of higher plants. Similarly, a rapid incorporation of ¹⁴C into starch was observed when tuber discs from various transgenic potato lines were incubated with [U-¹⁴C]Glc-1-P. The rate of starch labeling was found to reflect the activity of the plastidial phosphorylase isozyme Pho1 (Fettke et al., 2010, 2012b). By contrast, transgenic potato (Solanum tuberosum) lines have been generated that due to expression of an antisense construct, possess a largely diminished total Pho1 activity in leaves. Leaf starch content is essentially unchanged compared with that of the wild-type plants, suggesting that under normal growth conditions, the plastidial phosphorylase is not necessarily involved in starch metabolism or, alternatively, can easily be replaced by other enzymes (Sonnewald et al., 1995). Likewise, the phenotype (including leaf starch content) of an Arabidopsis mutant lacking functional PHS1 has been reported not to differ from the wild type when the plants were grown under normal conditions. However, under water stress conditions, significantly more local leaf lesions have been reported to occur (Zeeman et al., 2004).When leaf discs from bean (Phaseolus vulgaris) or Arabidopsis plants were exposed to conditions favoring photorespiration (i.e. an atmosphere consisting of 30% [v/v] O₂ and 70% [v/v] N₂ but lacking CO₂), transitory starch was degraded in the light at a high rate and the plastidial Glc-6-P pool increased. In Arabidopsis mutants deficient in PHS1, the Glc monophosphate pool did not respond to photorespiratory conditions (Weise et al., 2006). These data lead to the conclusion that in illuminated leaves with very high rates of photorespiration, PHS1 is involved in the conversion of starch to Glc monophosphates but does not to participate in the nocturnal starch degradation.When studying several starch-related Arabidopsis mutants, we noticed that two single knockout mutations that both affect the maltose metabolism but differ in the subcellular location of the target protein possess a significantly increased PHS1 activity (Malinova et al., 2011a, 2011b). One mutant constitutively lacks the functional cytosolic transglucosidase (also designated as disproportionating enzyme2; DPE2) and, therefore, the cytosolic route of starch-derived maltose metabolism is impaired (Chia et al., 2004; Lu and Sharkey, 2004). The other mutant does not express the plastidial maltose transporter MEX1, resulting in a massively enlarged maltose pool (Niittylä et al., 2004). Thus, in the two mutants, the metabolism of starch-derived maltose is blocked at different subcellular sites, i.e. the cytosol and the chloroplast. The enhanced PHS1 activity as observed for the two mutants is difficult to explain unless a more general function of the phosphorylase isozyme in starch metabolism is assumed.For a detailed functional analysis of PHS1-related processes, we generated two types of constitutive PHS1-deficient double knockout mutants (DPE2 plus PHS1 or MEX1 plus PHS1) and studied their phenotypes in more detail under various experimental conditions. Shoot growth and leaf chlorophyll content are reduced when the plants are grown under a light-dark regime, but under continuous illumination, both effects are far less pronounced. Based on these data, we propose that the plastidial phosphorylase participates in both the turnover of transitory starch and in the maintenance of intact chloroplasts.     

Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana

Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.     

Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells

In plants several ‘starch-related’ enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.     

Vanadium distribution in roots and leaves of Phaseolus vulgaris: morphological and ultrastructural effects

In different plant species, vanadium has been considered either as beneficial or as a toxic element, or even as secondary metabolism elicitor, but the mechanisms involved are still not completely understood. In this study, the responses of Phaseolus vulgaris L. cv. Contender roots and leaves to different vanadyl sulfate concentrations were studied. The plants grown hydroponically with V had thicker roots, a less developed main root, and a smaller number of secondary roots than the control plants. The V content in roots and leaves was correlated with V supply concentration but the V content in leaf was always much lower than in the root, which leads us to conclusion that V accumulates in the roots and only small quantities are transferred to the leaves. However, thylakoid disorganisation was observed in the chloroplasts of plants grown with vanadyl sulphate.     

Acclimation of potato plants to polyethylene glycol-induced water deficit I. Photosynthesis and metabolism

The acclimation of photosynthesis and metabolism in response to water deficit is characterized using hydroponically grown potato plants (Solanum tuberosum cv. Désirée). Plants were subjected to a reduced water potential of the nutrient solution by adding 10% (w/v) PEG 6000. PEG-treated plants were retarded in growth. Leaves which had been fully developed before the PEG treatment and leaves grown during the PEG treatment showed different phenotypes and biochemical and physiological properties. Photosynthesis of all leaves decreased during the whole treatment. However, the decrease of photosynthesis in the two types of leaves had different causes indicated by differences in their metabolism. Leaves which were fully developed at the beginning of the PEG treatment began to wilt starting from the leaf rim. The apoplastic ABA content increased, coinciding with a decreased stomatal conductance. Increased energy charge of the cells indicated impaired chloroplastic metabolism, accompanied by a decrease of amounts of chloroplastic enzymes. The apoplastic and the symplastic ABA content were increased during water deficit and because ABA was concentrated in the cytosolic compartment it is suggested that ABA is involved in decreasing photosynthetic enzyme contents in old leaves. Young leaves, grown after the imposition of water deficit, were smaller than control leaves and had a curly surface. In young leaves apoplastic and cytosolic ABA contents were identical with control values. Carboxylation efficiency of photosynthesis was decreased, but the water use efficiency remained unchanged. Metabolic data of the photosynthetic pathways indicate a down-regulation of chloroplastic metabolism. It is concluded that in young leaves photosynthesis was non-stomatally limited. This limitation was not caused by ABA.     

Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme

During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased.     

Soilborne wheat mosaic virus movement protein and RNA and wheat spindle streak mosaic virus coat protein accumulate inside resting spores of their vector, Polymyxa graminis

To study virus-vector interactions between Soilborne wheat mosaic virus (SBWMV) or Wheat spindle streak mosaic virus (WSSMV) and Polymyxa graminis Ledingham, P. graminis was propagated in plants grown hydroponically. P. graminis accumulated to high levels in several barley cultivars tested. Multiple developmental stages of P. graminis could be identified in infected barley roots. Accumulation of SBWMV and WSSMV inside P. graminis sporosori in the roots of soil-grown winter wheat and hydroponically grown barley was compared to determine if data obtained from plants naturally infected plants and plants infected by manual inoculation were similar. WSSMV coat protein (CP), SBWMV RNAs, SBWMV movement protein but not SBWMV CP were detected in both soil-grown winter wheat and hydroponically grown barley roots. These data are the first direct evidence that SBWMV and WSSMV are internalized by P. graminis.     

Reduction of the Cytosolic Phosphoglucomutase in Arabidopsis Reveals Impact on Plant Growth,Seed and Root Development,and Carbohydrate Partitioning

Phosphoglucomutase (PGM) catalyses the interconversion of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and exists as plastidial (pPGM) and cytosolic (cPGM) isoforms. The plastidial isoform is essential for transitory starch synthesis in chloroplasts of leaves, whereas the cytosolic counterpart is essential for glucose phosphate partitioning and, therefore, for syntheses of sucrose and cell wall components. In Arabidopsis two cytosolic isoforms (PGM2 and PGM3) exist. Both PGM2 and PGM3 are redundant in function as single mutants reveal only small or no alterations compared to wild type with respect to plant primary metabolism. So far, there are no reports of Arabidopsis plants lacking the entire cPGM or total PGM activity, respectively. Therefore, amiRNA transgenic plants were generated and used for analyses of various parameters such as growth, development, and starch metabolism. The lack of the entire cPGM activity resulted in a strongly reduced growth revealed by decreased rosette fresh weight, shorter roots, and reduced seed production compared to wild type. By contrast content of starch, sucrose, maltose and cell wall components were significantly increased. The lack of both cPGM and pPGM activities in Arabidopsis resulted in dwarf growth, prematurely die off, and inability to develop a functional inflorescence. The combined results are discussed in comparison to potato, the only described mutant with lack of total PGM activity.     

Carbohydrate partitioning and metabolism during acclimatization of micropropagated Calathea

Regulation of carbohydrate metabolism and compartmentation were studied during the acclimatization of tissue cultured Calathea plantlets. At transplantation plants were characterised by a heterotrophic metabolism with roots and stems as the main storage organs for carbohydrates. As acclimatization proceeded, a switch to autotrophic growth was observed: leaves became source organs, which was among others reflected by significant increases of invertase, sucrose synthase and sucrose-P synthase activities. Mobilization of reserves in roots and stems was also observed during the same period. Sucrose and starch accumulation in leaves was positively correlated with increasing light intensity.     

Deficiency in a cytosolic ribose-5-phosphate isomerase causes chloroplast dysfunction, late flowering and premature cell death in Arabidopsis

The oxidative pentose phosphate pathway (oxPPP) is part of central metabolism, consisting of two distinct phases: the oxidative phase and the non-oxidative phase. The non-oxidative phase of the oxPPP generates carbon skeletons for the synthesis of nucleotides, aromatic amino acids, phenylpropanoids and their derivatives, which are essential for plant growth and development. However, it is not well understood how the non-oxidative phase of the oxPPP contributes to plant growth and development. Here, we report the characterization of Arabidopsis T-DNA knockout mutants of the RPI2 gene (At2g01290), which encodes a cytosolic ribose-5-phosphate isomerase (RPI) that catalyzes the reversible interconversion of ribulose-5-phosphate and ribose-5-phosphate in the non-oxidative phase of the oxPPP. Although recombinant Arabidopsis RPI2 protein exhibits marked RPI enzymatic activity, knockout of the RPI2 gene does not significantly change the total RPI activity in the mutant plants. Interestingly, knockout of RPI2 interferes with chloroplast structure and decreases chloroplast photosynthetic capacity. The rpi2 mutants accumulate less starch in the leaves and flower significantly later than wild-type when grown under short-day conditions. Furthermore, the rpi2 mutants display premature cell death in the leaves when grown at an above-normal temperature (26°C). These results demonstrate that a deficiency in the non-oxidative phase of the cytosolic oxPPP has pleiotropic effects on plant growth and development and causes premature cell death.     

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.     

The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants

Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C–N status in roots and developing flowers.     

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.     

Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The phosphoglucan, water dikinase

The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the beta-P of ATP to the phosphoglucan, whereas the gamma-P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD.