Subunit Of ADP-glucose Pyrophosphorylase Research Articles

Genomic organizations of two small subunit ADP-glucose pyrophosphorylase genes from sweetpotato

The genomic features of the small subunit ADP-glucose pyrophosphorylase (AGPase) isoforms are different in barley and maize. The two isoforms found in barley originated from one single gene through alternative splicing, while two independent genes encode the two isoforms in maize. To ascertain the genomic organizations of two small subunit AGPase isoforms in sweetpotato ( ibAGP1 and ibAGP2), we isolated genomic DNAs containing the entire coding regions of two genes. Complete genomic structures of ibAGP1 and ibAGP2 were ascertained by the sequencing of these genomic regions. The transcribed regions of ibAGP1 and ibAGP2, comprising nine exons and eight introns, were distributed over 3.9 and 4.0 kb, respectively. The eight introns differed in length, from 76 to 946 bp in ibAGP1, and from 76 to 811 bp in ibAGP2, while the locations of introns in ibAGP1 and ibAGP2 were identical. There was 46–58% sequence identity between the intron sequences of the two genes. Intron sequence analyses suggested that either duplication in each intron, or gene conversion between introns of two isoforms, might cause major intron size differences between the two genes. Altogether, these results indicate that two small subunit AGPase isoforms in sweetpotato are encoded by two independent genes, in a fashion similar to that of maize small subunit AGPase genes.

Characterization of a gene from chromosome 1B encoding the large subunit of ADPglucose pyrophosphorylase from wheat: evolutionary divergence and differential expression ofAgp2genes between leaves and developing endosperm

A full-length genomic clone containing the gene encoding the large subunit of the ADPglucose pyrophosphorylase (Agp2), was isolated from a genomic library prepared from etiolated shoots of hexaploid wheat (Triticum aestivum L., cv, Chinese Spring). The coding region of this gene is identical to one of the cDNA clones previously isolated from a developing wheat grain cDNA library and is therefore an actively transcribed gene. The sequence represented by the cDNA spans 4.8 kb of the genomic clone and contains 15 introns. 2852 bp of DNA flanking the transcription start site of the gene was cloned upstream of the GUS (beta-glucuronidase) reporter gene. This Agp2::GUS construct and promoter deletions were used to study the pattern of reporter gene expression in both transgenic tobacco and wheat plants. Histochemical analysis of GUS expression in transgenic tobacco demonstrated that the reporter gene was expressed in guard cells of leaves and throughout the seed. In transgenic wheat, reporter gene expression was confined to the endosperm and aleurone with no expression in leaves. The cloned Agp2 gene was located to chromosome 1B by gene-specific PCR with nullisomic-tetrasomic lines. Northern analysis demonstrated that the Agp2 genes are differentially expressed in leaves and developing endosperm; while all three classes of Agp2 genes are transcribed in developing wheat grain endosperm, only one is transcribed in leaves. The differences between the Agp2 genes are discussed in relation to the evolution of hexaploid wheat.

The small subunit ADP-glucose pyrophosphorylase ( ApS) promoter mediates okadaic acid-sensitive uidA expression in starch-synthesizing tissues and cells in Arabidopsis.

Transgenic plants of Arabidopsis thaliana Heynh., transformed with a bacterial beta-glucuronidase (GUS) gene under the control of the promoter of the small subunit (ApS) of ADP-glucose pyrophosphorylase (AGPase), exhibited GUS staining in leaves (including stomata), stems, roots and flowers. Cross-sections of stems revealed GUS staining in protoxylem parenchyma, primary phloem and cortex. In young roots, the staining was found in the root tips, including the root cap, and in vascular tissue, while the older root-hypocotyl axis showed prominent staining in the secondary phloem and paratracheary parenchyma of secondary xylem. The GUS staining co-localized with ApS protein, as found by tissue printing using antibodies against ApS. Starch was found only in cell and tissue types exhibiting GUS staining and ApS labelling, but not in all of them. For example, starch was lacking in the xylem parenchyma and secondary phloem of the root-hypocotyl axis. Sucrose potently activated ApS gene expression in leaves of wild-type (wt) plants, and in transgenic seedlings grown on sucrose medium where GUS activity was quantified with 4-methylumbelliferyl-beta-glucuronide as substrate. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, completely blocked expression of ApS in mature leaves of wt plants and prevented GUS staining in root tips and flowers of the transgenic plants, suggesting a similar signal transduction mechanism for ApS expression in various tissues. The data support the key role of AGPase in starch synthesis, but they also underlie the ubiquitous importance of the ApS gene for AGPase function in all organs/tissues of Arabidopsis.

Isolation and Characterization of Two cDNAs for Large and Small Subunits of ADP-glucose Pyrophosphorylase from Kidney Bean

Abstract: Two cDNA clones for the large and small subunits of ADP-glucose pyrophosphorylase (AGPase), designated pvagpLl and pvagpSl, respectively, were isolated from developing seeds of kidney bean (Phaseolus vulgaris L.). Each deduced amino acid sequence showed significant identity (65-86%) with those of legumi nous AGPase large or small subunits. Northern blot analysis revealed that both mRNA species accumulated abundantly at the early-to mid-stages of seed development, indicating that both genes are synchronically ex pressed during seed maturation. Both gene products were also detected in leaves, stems and roots of kidney bean. In leaves, the pvagpLl mRNA level was constant during the diurnal cycle, whereas the pvagpSl mRNA level decreased markedly during the dark period. These results indicate that the pvagpLl and pvagpSl genes are differentially regulated by environmental and spatial conditions.

Sucrose regulation of ADP-glucose pyrophosphorylase subunit genes transcript levels in leaves and fruits

ADP-glucose pyrophosphorylase (AGPase, EC2.7.7.27) is a key regulatory enzyme in starch biosynthesis. The enzyme is a heterotetramer with two S and two B subunits. In tomato, there are three multiple forms of the S subunit gene. Agp S1, S2 and B are highly expressed in fruit from 10 to 25 days after anthesis. Agp S3 is only weakly expressed in fruit. Sucrose significantly elevates expression of Agp S1, S2 and B in both leaves and fruits. Agp S1 exhibits the highest degree of regulation by sucrose. In fact, sucrose may be required for Agp S1 expression. For excised leaves incubated in water, no transcripts for Agp S1 could be detected in the absence of sucrose, whereas it took up to 16 h in water before transcripts were no longer detectable for Agp S2 and B. Neither Agp S3 nor the tubulin gene is affected by sucrose, demonstrating that this response is specifically regulated by a carbohydrate metabolic signal, and is not due to a general increase in metabolism caused by sucrose treatment. Truncated versions of the promoter for Agp S1 indicate that a specific region 1.3–3.0 kb upstream from the transcription site is responsible for sucrose sensitivity. This region of the S1 promoter contains several cis-acting elements present in the promoters of other genes that are also regulated by sucrose.

Induction of ApL3 expression by trehalose complements the starch-deficient Arabidopsis mutant adg2-1 lacking ApL1, the large subunit of ADP-glucose pyrophosphorylase.

The disaccharide trehalose has strong effects on plant metabolism and development. In Arabidopsis seedlings, growth on trehalose-containing medium leads to an inhibition of root elongation, an accumulation of starch in the shoots, an increased activity of ADP-Glc pyrophosphorylase (AGPase), and an induction of the expression of the AGPase gene, ApL3 (A. Wingler, T. Fritzius, A. Wiemken, T. Boller, R.A. Aeschbacher [2000] Plant Physiol 124: 105-114). We used Arabidopsis mutants deficient in starch synthesis to examine whether the primary effect of trehalose was to affect carbohydrate allocation by the induction of AGPase in the photosynthetic tissue. In a mutant lacking the large AGPase subunit, ApL1, (adg2-1 mutant) growth on trehalose restored AGPase activity and led to a strong accumulation of starch in the shoots. In contrast, starch synthesis could not be induced in a mutant lacking the small AGPase subunit, ApS, (adg1-1 mutant) or in a mutant lacking plastidic phosphoglucomutase (pgm1-1 mutant). These results indicate that ApL3 can substitute for ApL1 in the AGPase complex. In addition, root elongation in the mutants, especially in the adg1-1 mutant, was partially resistant to trehalose, suggesting that the induction of ApL3 expression and the resulting accumulation of starch in the shoots were partially responsible for the effects of trehalose on the growth of wild-type plants.

Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling.

Plants both produce and utilize carbohydrates and have developed mechanisms to regulate their sugar status and co-ordinate carbohydrate partitioning. High sugar levels result in a feedback inhibition of photosynthesis and an induction of storage processes. We used a genetic approach to isolate components of the signalling pathway regulating the induction of starch biosynthesis. The regulatory sequences of the sugar inducible ADP-glucose pyrophosphorylase subunit ApL3 were fused to a negative selection marker. Of the four impaired sucrose induction (isi) mutants described here, two (isi1 and isi2) were specific to this screen. The other two mutants (isi3 and isi4) showed additional phenotypes associated with sugar-sensing screens that select for seedling establishment on high-sugar media. The isi3 and isi4 mutants were found to be involved in the abscisic acid signalling pathway. isi3 is allelic to abscisic acid insensitive4 (abi4), a gene encoding an Apetala2-type transcription factor; isi4 was found to be allelic to glucose insensitive1 (gin1) previously reported to reveal cross-talk between ethylene and glucose signalling. Here we present an alternative interpretation of gin1 as an allele of the ABA-deficient mutant aba2. Expression analysis showed that ABA is unable to induce ApL3 gene expression by itself, but greatly enhances ApL3 induction by sugar. Our data suggest a major role for ABA in relation to sugar-signalling pathways, in that it enhances the ability of tissues to respond to subsequent sugar signals.

Phosphate status affects the gene expression, protein content and enzymatic activity of UDP-glucose pyrophosphorylase in wild-type and pho mutants of Arabidopsis.

The effects of inorganic phosphate (Pi) deficiency on the expression of the UDP-glucose pyrophosphorylase (UGPase) gene (Ugp), involved in sucrose synthesis/metabolism, and on carbohydrate status were investigated in different tissues of Arabidopsis thaliana (L.) Heynh. For leaves, a decrease in internal Pi status caused by growth of plants on a medium lacking Pi (-P conditions) led to an increase in the overall content of glucose and starch, but had little effect on sucrose content. The Pi deficiency also led to an increased carbohydrate content in stems/flowers, but not in roots. The expression of Ugp was upregulated in both leaves and roots, but not in stems/flowers. The effects of Pi status on Ugp expression were confirmed using leaves of both pho1-2 and pho2-1 mutants of Arabidopsis (Pi-deficient and Pi-accumulating, respectively) and by feeding the leaves with D-mannose, which acts as a sink for Pi. The Pi-status-dependent changes in Ugp expression followed the same patterns as those of ApS, a gene encoding the small subunit of ADP-glucose pyrophosphorylase, a key enzyme of starch synthesis. The changes in Ugp mRNA levels, depending on internal Pi status, were generally correlated with changes in UGPase protein content and enzymatic activity. This was demonstrated both for wild-type plants grown under Pi-deficiency and for Pi mutants. The data suggest that, under Pi-deficiency, UGPase represents a transcriptionally regulated step in sucrose synthesis/metabolism, involved in homeostatic mechanisms readjusting the nutritional status of a plant under Pi-stress conditions.

Isolation and characterisation of cDNAs encoding the large and small subunits of ADP-glucose pyrophosphorylase from cassava (Manihot esculenta Crantz)

Screening of a tuber specific cassava cDNA library resulted in the isolation of full length cDNA clones with homology to the genes encoding the small and large subunits of ADP glucose pyrophosphoryalse. Sequence analysis revealed that AGPase B the clone with homology to the small subunit shared 54% homology at amino acid level with the AGPase S clone that is more closely related to the large subunit. Segregation analysis of a cross between the cassava cultivars TMS 30572 and CM 2177-2 revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. AGPase B is a low copy gene of which one member is localised on the female derived linkage group P. The two genes are expressed in all cassava tissues but AGPase B exhibits a higher steady state mRNA level than AGPase S and is highly expressed in leaf and tuber tissue. The AGPase enzyme activity was much higher in young cassava leaves as compared to older leaves and tubers. Cassava AGPase was activated by 3-PGA and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA but was highly inhibited by Pi. Transformation of potato (Solanum tuberosum) plants with an antisense AGPase B construct resulted in 10 out of 134 antisense AGPase B plants having on average 3.5 times more tubers than the control non transgenic plants. Analysis of these transgenic plants revealed they had greatly reduced levels of AGPase B mRNA, 1.5 to 3 times less starch, and five times higher levels of soluble sugars, sucrose, glucose and fructose, to those found in control plants.

Partial male sterility induced in tobacco by overproduction of mRNA of sweet potato small subunit ADP-glucose pyrophosphorylase

Summary Previously we generated transgenic tobacco plants overexpressing sweet potato ADP-glucose pyrophosphorylase (AGPase) small subunit cDNA under control of CaMV 35S promoter. In order to understand the role of small subunit AGPase in pollen development, these transgenic tobacco plants were studied. Nine out of ten transgenic tobacco plants produced more than 50 percnt; aborted pollen grains. The two transgenic lines T4 and T27 produced more than 75 percnt; aborted pollen grains. Analysis of inheritance of the transgene inserts in T4 and T27 revealed that transgene co-segregated with the pollen abortion phenotype. Microscopic examination of aborted pollen grains showed that their cytoplasm did not contain starch granules, nuclei or any other organelles. Transcript levels of the transgene were very high in stamen tissues of transgenic plants. Transcript levels were correlated with the severity of the pollen abortion phenotype in transgenic plants, indicating that the pollen abortion phenotype probably resulted from overproduction of sweet potato small subunit AGPase mRNAs. Western blot analysis revealed that AGPase small subunit polypeptide levels were significantly reduced in stamen tissues of T4 and T27 compared with control plant. These results suggest that AGPase small subunit gene may play an important role in pollen development.

Quantitative trait loci affecting amylose, amylopectin and starch content in maize recombinant inbred lines

The loci explaining the variability of quantitative traits related to starch content and composition (amylose, amylopectin and water soluble fraction) were searched for in maize kernels. Multifactorial genetic methods were used to detect and locate QTLs (quantitative trait loci) on a genetic map consisting mainly of RFLP markers for genes with known function. The genetic material was recombinant inbred lines originating from parents differing in starch structure (dent vs. flint). Kernels were harvested from field grown plants for two successive years and under two pollination systems. Main effect and epistasis QTLs were detected using two methods, composite interval mapping (MQTL) and ANOVA. Despite large year-to-year differences, physiologically meaningful co-locations were observed between trait QTLs. Moreover, the number of expressed sequences on our map allowed the search for co-locations between QTLs and genes involved in carbohydrate metabolism. The main co-location was between an amylose QTL and Shrunken 2 (SH2) locus, on chromosome 3 (SH2 encoding for the large subunit of ADPglucose pyrophosphorylase). The importance of this locus as a candidate gene for a starch QTL is in agreement with previous studies based either on QTL co-locations or on revertant analysis. Other co-locations were observed between amylose and amylopectin QTLs and the two loci of IVR1 invertase genes on chromosomes 2 and 10. Further comparison with previously detected QTLs for carbohydrate metabolism in maize leaves showed consistent co-location in map regions devoid of candidate genes, such as near chromosome 1S telomere. The possible contribution of regulatory genes in this region is discussed.

Molecular cloning and spatial expression of an ApL1 cDNA for the large subunit of ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

A cDNA, ApL1a, corresponding to a homologue of the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated/characterised by screening a cDNA library prepared from leaves of Arabidopsis thaliana, followed by rapid amplification of cDNA 3'-ends (3'-RACE). Within the 1685 nucleotide-long sequence (excluding polyA tail), an open reading frame encodes a protein of 522 amino acids (aa), with a calculated molecular weight of 57.7 kDa. The derived aa sequence does not contain any discernible transit peptide cleavage site motif, similarly to two other recently sequenced full-length Arabidopsis homologues for AGPase, and shows ca. 58-78% identity to homologous proteins from other plants/tissues. The corresponding gene was found to be expressed in all tissues examined (rosette and stem leaves, stems, flowers and fruits). The ubiquitous expression of the gene is consistent with its critical role in starch synthesis in Arabidopsis.

Characterization of Transgenic Tobacco Plants Carrying Small Subunit ADPglucose Pyrophosphorylase cDNAs from Sweet Potato

Summary In order to test a possibility of forming an active ADPglucose pyrophosphorylase (AGPase) by homotetrameric composition of single subunit in plant, transgenic tobacco plants were generated with two previously isolated cDNAs ( sTL1 and sTL2 ) encoding the small subunit of sweet potato AGPase. Transformation was carried out with a mixture of the binary vectors, pMBP1- sTL1 and -sTL2 , under the control of CaMV 35S promoter. The PCR analysis of the randomly selected fifteen transgenic plants showed that ten transgenic lines were transformed by sTL1 and seven lines by sTL2 . Only two transgenic lines were transformed with sTL1 and sTL2 . The integrated sTL1 and/or sTL2 were actively transcribed. The steadystate transcript levels of the transgenic plants were higher than those of nontransgenic plants. The sTL1 -transformed lines showed higher transcript levels than sTL2 -transformed lines did although the transcript levels were variable among transgenic lines. Despite the differential expression of AGPase mRNA between nontransgenic and transgenic plants, there was no significant difference in the activities of AGPase, indicating no sweet potato AGPase activity in transgenic tobacco plants. The immunoblot analysis of the small subunit polypeptide showed that the protein levels remained unchanged in transgenic plants. The overexpressed transcripts of sweet potato AGPase small subunit in tobacco plants do not seem to produce an active form of AGPase.

Expression of a yeast-derived invertase in developing cotyledons of Vicia narbonensis alters the carbohydrate state and affects storage functions.

In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage-active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in Vicia narbonensis under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP-glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin-mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP-glucose pyrophosphorylase-mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C-sucrose in vitro, the invertase-expressing cotyledons partitioned less carbon into starch compared to the wild-type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose-mediated induction of storage-associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.

Pinpointing towards improved transformation and regeneration of cassava ( Manihot esculenta Crantz)

Friable embryogenic callus (FEC) of the cassava genotype 60444 was transformed by particle bombardment with DNA from the plasmid constructs pHB1 and pJIT100. Both plasmids contained the luciferase ( luc) marker gene under the control of the CaMV 35S promoter. In addition pJIT100 had the CaMV35S driven phosphinothricin acetyl transferase ( pat) gene, while pHB1 contained the cassava cDNA coding for the small subunit of ADP glucose pyrophosphorylase (AGPase B) in antisense orientation under the control of a double CaMV35S promoter. A total of 2 weeks after bombardment, luciferase (LUC) positive FEC units (spots) were isolated and subcultured separately for further proliferation. A total of 4 weeks later, those cultures having at least four positive LUC spots were subjected to three different selection regimes namely: stringent LUC selection, non stringent LUC selection and combined LUC/phosphinothricin (PPT) selection. A total of 16 weeks after bombardment, stringent LUC selection gave rise to cultures in which 92% of the FEC units were LUC positive. Within the same time period non stringently LUC selected cultures and LUC/PPT selection had only 1 and 41% of the units that were LUC positive, respectively. The number of LUC positive mature somatic embryos formed was similar to the percentage of LUC positive FEC units, within a culture, found with each selection method. Stringent LUC selection enabled transgenic plants to be produced in 28–36 weeks compared to 32–41 weeks for LUC/PPT selection and 53–78 weeks for non stringent LUC selection. This indicates that stringent selection is a more efficient method for obtaining transgenic cassava plants. Southern blot analysis of transgenic cassava plants revealed that they had between one to seven copies of the pHB1 and pJIT100 construct. The production of the first cassava plants carrying an agronomically important trait affecting starch biosynthesis is reported. Expression of the antisense AGPase B gene resulted in cassava plants within which the AGPase mRNA steady state levels was greatly decreased or even absent. The plants with no AGPase mRNA expression also had extremely low levels of starch, compared to control plants, as shown by iodine staining of in vitro thickened stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer of in vitro thickened stems.

The sugar‐mediated regulation of genes encoding the small subunit of Rubisco and the regulatory subunit of ADP glucose pyrophosphorylase is modified by phosphate and nitrogen

Tobacco seedlings were grown in nutrient agar at a range of ammonium nitrate concentrations either without added sucrose, or with 100 mol m–3 sucrose. In the absence of added sucrose, nitrogen‐limited plants had increased levels of glucose, fructose and sucrose, decreased chlorophyll, decreased protein, and decreased Rubisco activity, but the level of the transcript for the small subunit of Rubisco (RbcS) did not decrease compared with nitrogen‐sufficient plants. When sucrose was added to nitrogen‐sufficient seedlings, there was an increase of sucrose, glucose and fructose in the leaves, growth was increased, and the chlorophyll and protein content, Rubisco activity, and the RbcS transcript level did not change. When sucrose was added to nitrogen‐limited seedlings, there was a further increase of sucrose, glucose and fructose, growth was not increased, and there was a further decrease of chlorophyll, protein and Rubisco activity, and a marked decrease of the RbcS transcript level. To check that the decrease of the RbcS transcript level was not an indirect effect due to changes of nitrogen metabolites after adding sugars, glucose was added to Chenopodium cells in the presence and absence of glutamine or azaserine. Changes of glutamine that suffice to increase and decrease the level of the transcript for nitrate reductase (Nia) do not affect the RbcS transcript concentration, and glucose addition still led to a decrease of the RbcS transcript level when the internal glutamine concentration was high. Tobacco seedlings were also grown in nutrient agar at a range of phosphate concentrations either without added sucrose, or with 100 mol m–3 sucrose. Phosphate‐limited seedlings did not show a decrease of chlorophyll, protein, Rubisco activity, or the level of the RbcS transcript, compared with phosphate‐sufficient seedlings. The addition of sucrose to phosphate‐limited plants led to a similar increase of sugars to that seen after adding sucrose to nitrogen‐limited seedlings, but did not alter chlorophyll, protein, Rubisco activity, or the level of the RbcS transcript. The addition of sucrose to phosphate‐limited plants led to a slight increase of the level of the transcript for nitrate reductase (Nia), increased nitrate reductase activity, and a marked increase of the amino acid content. Phosphate limitation led to an increased level of the transcript for the regulatory subunit of ADP glucose pyrophosphorylase (AgpS2), and this response was strengthened when sucrose was added. The regulation of AgpS2 expression by phosphate and sucrose was further investigated by feeding sucrose and phosphate to detached source leaves via the transpiration stream. The level of the AgpS2 transcript decreased after feeding phosphate and increased after feeding sucrose, and the effect of sucrose was antagonised by phosphate. It is concluded that the response to sugar signalling is modulated by nitrogen and phosphate in a gene‐specific manner. The significance of these results for understanding the visual phenotype of nitrogen‐ and phosphate‐limited plants, and the response of photosynthesis and starch synthesis to the plant nutrient status is discussed.

Molecular cloning and characterization of small and large subunits of ADP-glucose pyrophosphorylase from oriental melon

We isolated three cDNAs encoding one small subunit and two large subunits of ADP-glucose pyrophosphorylase (AGP) from oriental melon.msf1 encoding the small subunit was 2,027 bp in length, andmlf1 andmlf2 encoding the large subunits were 2,009 and 2,227 bp, respectively. Each of the three clones had a long, uninterrupted open reading frame, deriving 57–58 kDa polypeptide. The strongest expression ofmsf1 occurred in leaves, and a relatively weak expression in roots and stems.mlf1 was highly expressed in stems and roots, whereas the expression was much weaker in leaves. The expression pattern ofmlf2 was similar to that ofmsf1, but the expression level ofmlf2 transcripts was much lower than that ofmsf1 transcripts. In fruits,msf1, mlf1, andmlf2 were expressed in all stages investigated with a similar expression profile. During fruit development, all three genes showed the highest expression at the early stage, and the transcripts decreased as the fruits matured.

Integration of carbohydrate and nitrogen metabolism during legume seed development: Implications for storage product synthesis

Summary This paper reviews recent data from our laboratory on aspects of carbon and nitrogen metabolism in developing legume embryos. The specific accumulation patterns of the major storage products starch and protein are different. Starch deposition is correlated with cell size and is arranged as a gradient increasing from the outer to the inner cotyledon side. Storage proteins start to accumulate in the outer epidermal and subepidermal cells and further accumulation does not follow the cell size gradient. Genes associated with storage activities and related to assimilate transport are expressed in a specific temporal pattern. In the embryo a H+-ATPase and a sucrose transporter are coordinately expressed together with sucrose synthase during early cotyledon stage. Expression of an amino acid transporter is highest at the mid to late cotyledon stage when legumin B transcripts start to accumulate. In order to identify branch points between C and N metabolism we changed the sugar and nitrogen state using in vitro culture of storage phase-cotyledons. Hexose sugars as well as nitrogen starvation induced transcripts of sucrose phosphate synthase and of the small subunit of ADP-glucose pyrophosphorylase, whereas a sucrose medium including nitrogen increased PEP-carboxylase and legumin B-mRNA. Hexose sugars as well as nitrogen starvation elevated sucrose and decreased amino acid levels in the cotyledons indicating that sugars and nitrogen are involved in the control of carbohydrate partitioning into starch and amino acids. In an in vivo approach, the sugar state of Vicia narbonensis embryos was changed by expressing apoplastically a legumin B4 promoter-yeast invertase construct. Embryos containing invertase have lower sucrose and higher hexose levels. Starch and protein content per seed are reduced. An increased fresh weight to dry weight ratio with severely wrinkled seeds indicates altered osmotic conditions due to a higher concentration of reducing sugars.

Differential processing of homologues of the small subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues.

ADP-glucose pyrophosphorylase (AGPase), a two-gene-encoded enzyme, is the key component of starch synthesis in all plants. In the present study, we have used an E. coli expression system for the (over)production of proteins derived from both full length and specifically truncated cDNAs encoding small subunits of AGPase from seed endosperm (AGPase-B1) and leaves (AGPase-B2) of barley (Hordeum vulgare). Based on immunoblot analyses, the molecular mass of the expressed AGPase-B1 (52 kD) was similar to that from endosperm extracts, whereas the expressed AGPase-B2 (56 kD) was larger than that in barley leaves (51 kD). Expression of truncated cDNAs for both the seed and leaf proteins has allowed for a direct verification of molecular masses that were earlier proposed for mature AGPases in barley tissues. The data suggest that seed AGPase-B1 does not undergo any post-translational proteolytic processing in barley, whereas the leaf homologue is processed to a smaller protein. Possible implications of these findings are discussed.

Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley ( Hordeum vulgare ) leaves

A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley ( Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54 815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley.