An analysis of seed development in Pisum sativum XIX. Effect of mutant alleles at the r and rb loci on starch grain size and on the content and composition of starch in developing pea seeds

The effects of mutant alleles at the r and rb loci on starchgrain size and the levels of starch and amylose in developingpea (Pisum sativum L.) seeds have been examined. Four lines,near-isogenic except for genes at these loci, have been usedto show that both mutations reduce levels of starch throughoutembryo development and reduce levels still further when combinedin the ‘double mutant’. The reduction in starchcontent was due, at least in part, to a reduction in starchgranule size. Although the proportion of starch in mature embryoswas similar in the rrRbRb and RRrbrb lines, the starch contentdiffered between these two lines during development, as a percentageof embryo dry weight. This difference was due to a reductionin the absolute growth of the embryo caused by the rb mutation.Lines homozygous for the mutant r allele with either wild-type(RbRb) or mutant (rbrb) alleles at the rb locus contained increasedproportions of amylose in their starch throughout development,due to a reduced production of amylopectin. The presence ofthe rb mutation, however, also reduced the amount of amylosein relation to the reduction in total starch levels. Mutantalleles at both loci also reduced starch levels in the testaduring development, the reduction due to rb being more extreme.Reciprocal crosses showed a maternal effect of the rb mutationon final seed size and on the absolute amount of starch in theembryo. Key words: Pisum sativum L., seed, starch, development, mutant     

Transgenic Arabidopsis plants expressing Escherichia coli pyrophosphatase display both altered carbon partitioning in their source leaves and reduced photosynthetic activity

The effects of the cytosolic expression of Escherichia coli pyrophosphatase (ppa) were investigated in the rosette leaves of transgenic Arabidopsis plants. During the daytime, glucose and fructose were found to accumulate at levels that were approximately two- to threefold higher in these plants than in the wild type. Interestingly, however, neither sucrose nor starch levels showed any distinctive build up in transgenic plants except under continuous white light growth conditions, during which they accumulated at high levels. Additionally, the leaves of transgenic Arabidopsis plants contain two- to threefold higher levels of inorganic phosphate (Pi) and two- to sixfold higher levels of uridine diphosphate-glucose than wild type plants during the diurnal cycle. In contrast, triose phosphate contents in the leaves of E. coli ppa transformants were either similar or slightly decreased when compared with wild type leaves. Furthermore, the photosynthetic activity of these transgenic plants was found to be reduced by 20–40% compared to normal levels. These results indicate that induction of ppa activity in the cytosol affects carbon partitioning between source and sink organs and also that the concomitant increase in Pi caused the accumulation of carbon metabolites and reduced photosynthetic activity.     

An Analysis of Seed Development in Pisum sativum XVII. The Effect of the rb Locus Alone and in Combination with r on the Growth and Development of the Seed

Near-isogenic lines have been produced in pea to examine theeffects of genes at the r and rb loci on the growth and developmentof the seed. The rb alleles were found to have an effect onthe growth of the testa as well as on that of the embryo incontrast to those at the r locus which only affect the latter.The relationship between the fresh and dry weights of the embryosof rb isoline and the double mutant differed from that of theround-seeded wild-type line. Furthermore, for this relationship,the double mutant line very closely resembled the rr isoline.Copyright1994, 1999 Academic Press Embryo, growth, pea, Pisum sativum L., rugosus loci, seed development, testa, wrinkled-seeded     

The role of the hexose transporter in the chloroplasts of Arabidopsis thaliana L.

The metabolism of wild-type Arabidopsis thaliana L. and its mutant TC265 were compared in order to reveal the role of the chloroplast glucose transporter. Plants were grown in a 12-h photoperiod. From 20 to 40 days after germination, starch per gram fresh weight of shoot in the mutant was four times that in the wild type. The extent of this difference did not alter during this period. Stereological analysis showed that the chloroplasts in the mutant were larger than those in the wild type; the thylakoids appeared to be distorted by the high starch content. [U-¹⁴C]Glucose and [U-¹⁴C]glycerol were supplied, separately, to excised leaves in the dark. [U-¹⁴C]Glucose was a good precursor of sucrose in the wild type and mutant; [U-¹⁴C]glycerol was a poor precursor of sucrose in both. The distribution of ¹⁴C in the wild type was used to calculate that the net flux was from hexose monophosphates to triose phosphates, not vice versa. During the first 4 h of the night the sugar content (75% sucrose, 20% glucose) of the leaves of the mutant dropped sharply, and at all times during the night it was less than that of the wild-type leaves. This drop in sugar coincided with a decrease in the rate of respiration. The growth rate of the mutant was less than that of the wild type. Addition of sucrose restored the rate of respiration at night and increased the rate of growth. It is argued that a major function of the glucose transporter in Arabidopsis chloroplasts is export of the products of starch breakdown that are destined for sucrose synthesis at night.We thank Professor C.R. Somerville for his generous gift of seed of the Arabidopsis mutant TC265. We are also grateful to Mr B. Chapman for assistance with the preparation of the sections for electron microscopy. R.N.T. thanks the Science and Engineering Research Council for a studentship.     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose     

Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1

We investigated the individual effect of null mutations of each of the four sucrose‐phosphate synthase (SPS) genes in Arabidopsis (SPSA1, SPSA2, SPSB and SPSC) on photosynthesis and carbon partitioning. Null mutants spsa1 and spsc led to decreases in maximum SPS activity in leaves by 80 and 13%, respectively, whereas null mutants spsa2 and spsb had no significant effect. Consistently, isoform‐specific antibodies detected only the SPSA1 and SPSC proteins in leaf extracts. Leaf photosynthesis at ambient [CO₂] was not different among the genotypes but was 20% lower in spsa1 mutants when measured under saturating [CO₂] levels. Carbon partitioning at ambient [CO₂] was altered only in the spsa1 null mutant. Cold treatment of plants (4 °C for 96 h) increased leaf soluble sugars and starch and increased the leaf content of SPSA1 and SPSC proteins twofold to threefold, and of the four null mutants, only spsa1 reduced leaf non‐structural carbohydrate accumulation in response to cold treatment. It is concluded that SPSA1 plays a major role in photosynthetic sucrose synthesis in Arabidopsis leaves, and decreases in leaf SPS activity lead to increased starch synthesis and starch turnover and decreased Ribulose 1,5‐bisphosphate regeneration‐limited photosynthesis but not ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco)‐limited photosynthesis, indicating a limitation of triose‐phosphate utilization (TPU).     

Carbon-partitioning inArabidopsis is regulated by the fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase enzyme

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P₂ levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P₂ were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P₂ contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.     

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi‐deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi‐sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi‐deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.     

Evidence that the rb Locus Alters the Starch Content of Developing Pea Embryos through an Effect on ADP Glucose Pyrophosphorylase

The aim of this work was to discover whether the rb locus of peas (Pisum sativum L.) affects seed starch content through action on an enzyme of starch synthesis in the developing embryo. The phenotypic effects of this locus are like those of the better characterised, unlinked r locus, which affects seed starch content through action on starch-branching enzyme. Embryos recessive at one or both of these loci (RRrbrb, rrRbRb, rrrbrb) have lower starch contents from an early stage of development than embryos dominant at these loci (RRRbRb). Maximum catalytic activities of enzymes of the pathway from sucrose to starch (sucrose synthase EC 2.4.1.13, UDP glucose pyrophosphorylase EC 2.7.7.9, ADP glucose pyrophosphorylase EC 2.7.7.27, ADP glucose-starch synthase EC 2.4.1.21, starch-branching enzyme EC 2.4.1.18) were compared in developing embryos of three lines of rbrb peas and four lines of RbRb peas. The only consistent difference between the two sorts of embryo was in the activity of ADP glucose pyrophosphorylase, which was at least tenfold lower in rbrb than in RbRb embryos. The activity in rbrb embryos was in most cases less than the estimated rate of starch synthesis of RRRbRb embryos. We conclude that the effect of the rb locus on the starch content of pea seeds is mediated through an alteration in the activity of ADP glucose pyrophosphorylase in the developing embryo.     

Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli

Summary The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding acpacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K _D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the K _D value was found to be 9–31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K _m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. V _max of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.Abbreviations AP alkaline phosphatase - Pi inorganic orthophosphate - Km kanamycin     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

The rb Mutation of Peas Causes Structural and Regulatory Changes in ADP Glucose Pyrophosphorylase from Developing Embryos

A mutation at the rb locus of pea (Pisum sativum L.) alters the shape, reduces the starch content, and increases the lipid and sucrose contents of the seed. These effects are probably all consequences of a reduction of up to 40-fold in the maximum catalytic activity of ADP glucose pyrophosphorylase in the developing embryo of the mutant relative to the wild type. We have investigated how the mutation brings about this reduction in activity. The purified enzyme from mutant embryos has a specific activity about 10-fold lower than that from wild-type embryos, and it is much more sensitive to the effectors inorganic phosphate and 3-phosphoglycerate than the wild-type enzyme. Both wild-type and mutant enzymes consist of polypeptides of around 50 kilodaltons. One of the polypeptides of the purified wild-type enzyme is missing from the mutant enzyme. We deduce that in the wild-type embryo this protein may interact with other subunits to confer a high specific activity and a low susceptibility to effectors on the enzyme.     

Comparisons of Phosphate, Carbohydrate, and Kjeldahl Nitrogen Content of Normal and Xantha Mutant Leaves of Helianthus annuus

Tissues of the xantha mutant of Helianthus annuus have an abnormally high phosphorus content compared to normally pigmented tissues of comparable age and position on the plant. A major portion of the observed differences can be accounted for in the acid-insoluble “7-minute” phosphate fraction, a fraction thought to be composed primarily of polyphosphates. Although this suggests that the xantha mutant may possess a mechanism for immobilizing excess phosphates as insoluble polymers, all qualitative tests for polyphosphates in extracts of mutant leaves have been negative. Carbohydrate levels in the mutant are lower than in wild type leaves due to the absence of photosynthesis in the mutant and its inability to form starch. Differences between mutant and wild type in Kjeldahl nitrogen appear to be a consequence of aging effects resulting from propagation by grafting onto physiologically older stocks.     

Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase

The aim of this work was to identify which aspects of photosynthetic metabolism respond most sensitively to leaf water deficit. Spinach (Spinacia oleracea L.) leaf discs were floated on sorbitol concentrations of increasing molarity and changes of the protoplast volume were estimated using [¹⁴C]sorbitol and ³H₂O penetration. Detached leaves were also wilted until 10% of their fresh weight was lost. Photosynthesis was studied at very high external CO₂ concentrations, to eliminate the effect of closing stomata. There was no large inhibition of CO₂ fixation after wilting leaves, or until the external water deficit was greater than-1.2 MPa. However, partitioning changed markedly at these moderate water deficits: more sucrose and less starch was made. When an inhibition of CO₂-saturated photosynthesis did appear at a water deficit of-2.0 MPa and above, measurements of chlorophyll-fluorescence quenching and metabolite levels showed the thylakoid reactions were not especially susceptible to short-term water stress. The inhibition was accompanied by a small increase of the triose phosphate: ribulose-1,5-bisphosphate ratio, showing regeneration of ribulose-1,5-bisphosphate was affected. However, there was also a general increase of the estimated concentrations of most metabolites, indicating that there is no specific site for the inhibition of photosynthesis. Increasing water deficit led to a large increase of fructose-2,6-bisphosphate. This is explained in terms of a simultaneous increase of fructose-6-phosphate and inorganic phosphate as the cell shrinks. The high fructose-2,6-bisphosphate led to the accumulation of triose phosphates, and the potential significance of this for protection against photoinhibition is discussed. There was an increase in the extractable activity of sucrose-phosphate synthase. This was only detected when the enzyme was assayed in conditions which distinguish between different kinetic forms which have previously been identified in spinach leaves. It is proposed that activation of sucrose-phosphate synthase is one of the first sites at which spinach leaves respond to a rising water deficit. This could be of importance for osmoregulation.Abbreviations Chl chlorophyll - Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA glycerate-3-phosphate - Pi inorgamic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - triose-P sum of glyceraldehyde-3-phosphate and dehydroxyacetone phosphate - UDPGlc uridine diphosphoglucose     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO₂ assimilation measured in individual leaves was substantially diminished only under conditions of high CO₂ and low O₂. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod. Received: 4 January 2000 / Accepted: 21 March 2000