AGPase Genes Research Articles

Characterization of ADP-Glucose Pyrophosphorylase Encoding Genes in Source and Sink Organs of Maize

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis. Six cDNA sequences encoding three large subunits and three small subunits of maize AGPase from database were mined and subsequently named: agpl1, agpl2, agpl3, agps1, agps2, and agps3. To elucidate the roles of these isogenes, a comprehensive expression analysis of the gene family was conducted by quantitative real-time RT-PCR. Based on the expression patterns, the six genes can be divided into three groups: (1) steady expressers (agpl1, agps1, and agpl2), which were expressed relatively constantly both in leaf and grain; (2) tissue and development-specific expressers (agpl3 and agps2), which were expressed only in grain at middle and late development phases; (3) tissue-specific expressers (agps3), whose transcripts kept constant during grain filling and were observed only in grain. In order to clarify the effects of sugar and plant hormone on maize AGPase genes expression, a serial of treatments were used. The results showed that AGPase genes significantly differed in response to sugar and hormone inductions. Enormous transcript changes of these genes could be observed in glucose and sucrose treatments. Interestingly, synergistic effect of ABA and sucrose on these genes was observed.

Regulation of Expression of D3-type Cyclins and ADP-Glucose Pyrophosphorylase Genes by Sugar, Cytokinin and ABA in Sweet Potato (Ipomoea batatas Lam.)

The productivity of sweet potato is governed by both the rate of cell division and sink activity of its tuberous root. The aim of this study was to reveal the mechanisms that regulate cell division activity and sink activity during tuberous root formation. As an indicator of the cell division activity, we used the transcript level of two D3-type cyclins, which regulate cell cycle progression through the formation of the regulatory subunit of the cyclin-dependent kinase complex. As an indicator of photosynthetic product sink activity, we used the gene expression of ADP-glucose pyrophosphorylase (AGPase), one of the key enzymes of starch synthesis. During tuberous root formation, the expression of D3 cyclin genes increased to the maximal levels and then decreased. In contrast, the expression of the AGPase gene increased continuously. Sucrose enhanced the expression of D3 cyclin and AGPase genes, but a high concentration of sucrose repressed the expression of a D3 cyclin gene. In the presence of sucrose, cytokinin increased the expression of D3 cyclins, but abscisic acid (ABA) did not. However, cytokinin and ABA repressed the induction of AGPase gene expression by sucrose. These results suggested that sugars, cytokinin and ABA regulate the cell division activity and the sink activity in sweet potato.

Molecular cloning and analysis of the Thermus caldophilus ADP-glucose pyrophosphorylase

Previously, we have purified a highly thermostable ADP-glucose pyrophosphorylase (EC 2.7.7.27; AGPase) from Thermus caldophilus GK-24. In the present paper, we further report the molecular cloning and characterization of the thermostable bacterial AGPase. Using a 0.6-kb DNA probe obtained by polymerase chain reaction (PCR) with a primer set deduced from the N-terminal and internal amino acid sequence, the AGPase gene was cloned. The cloned AGPase gene had a 1245-bp open reading frame that encodes a protein of 414 amino acids. Then, the full-length AGPase gene was further cloned into the pHCE19T(II) vector and was expressed in Escherichia coli DH5α. Western analysis of the recombinant enzyme showed the same immunity as the wild-type enzyme with a molecular mass of ca. 46 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinetic parameters of the recombinant AGPase were basically similar to the wild type. The N-terminal region of T. caldophilus AGPase showed a partial similarity to that of E. coli, and R336 and P295 were identical to those of E. coli AGPase. In addition, sequence comparison revealed that R177 and P235 of T. caldophilus AGPase were aligned with K195 and K247 of E. coli, and K376 with R386, while K195 residue of E. coli was reported to be located in the glucose-1-phosphate (Glc-1-P) substrate binding region.

Different gene expression patterns of sucrose‐starch metabolism during pollen maturation in cytoplasmic male‐sterile and male‐fertile lines of rice

Starch biosynthesis, a very critical event during pollen development, is not well understood in terms of gene regulation and intracellular controls. Using the rice HL‐type cytoplasmic male sterile (HL‐CMS) line with starch deficiency in pollen and two related male‐fertile lines (Oryza sativa L.), we have studied two specific developmental stages: ‘early’, that is, before or during pollen mitosis I, without starch accumulation, and ‘late’, the actively starch‐filling postpollen mitosis I phase. Real‐time reverse transcription‐PCR analysis showed temporal changes in the expression patterns of six metabolic genes involved in the sucrose–starch pathway, i.e. sucrose synthase (EC 2.4.1.13), Rsus3; acid invertase (EC 3.2.1.26), OSINV2; phosphoglucomutase (EC 5.4.2.2), OsPGM; uridine diphosphate‐glucose pyrophosphorylase (EC 2.7.7.9), OsUGP; a subunit of adenosine diphosphate‐glucose pyrophosphorylase (AGPase) (EC 2.2.7.2), OsAGPL3; soluble starch synthase (EC 2.4.1.21), OsSSI and three transporter genes [sucrose transporter, OsSUT3; hexose transporter, OSMST7 and plasma membrane H+‐adenosine triphosphatase (ATPase) (EC 3.6.1.35), OSA2]. Regarding metabolic enzyme activities, lower activity of sucrose synthase was detected at ‘late’ compared with ‘early’ stage in the three rice lines; invertase and other downstream enzymes had higher activities at ‘late’ stage in male‐fertile but not male‐sterile lines. Meanwhile, significantly reduced sugars and ATP levels were detected at ‘early’ and ‘late’ stages in male‐sterile compared with male‐fertile tissues. Furthermore, transgenic data showed that AGPase gene can stimulate starch accumulation in pollen of male‐sterile host lines, showing the importance of AGPase in the metabolic pathway. Taken together, these data suggest that the combined effects of the reduced energy supply and sugar levels in starch biosynthesis may lead to the observed temporal changes in gene expression, and eventually to pollen sterility.

Modification of Sugar Composition in Strawberry Fruit by Antisense Suppression of an ADP-glucose Pyrophosphorylase

To modulate the soluble sugar content of strawberry fruits, we generated transgenic plants that incorporated an antisense cDNA of ADP-glucose pyrophosphorylase (AGPase) small subunit (FagpS) under the control of the strawberry fruit-dominant ascorbate peroxidase (APX) promoter (cv. Anther). Several independent transgenic lines were obtained and grown in the greenhouse for analysis of agronomic traits. Most transgenic fruit did not show significant differences in weight and hardness compared to control fruit. However, the starch content in fruit was decreased to 27–47% and the total soluble sugar content was increased to 16–37% in transgenic plants (analyzed by the HPLC of sugar composition at four different stages of fruit development). The sugar contents of fruits in transgenic lines were particularly higher than control fruits at the red stage. The results were consistent with northern analysis, which showed that the levels of AGPase mRNA drastically were reduced in the red stage of fruits in all the transgenic plants. In other tissues of transgenic plants, the FagpS mRNA expression level was similar to control plants. Our studies indicate that fruit-specific down-regulation of the AGPase gene might be an effective strategy for increasing sugar and decreasing starch content in strawberry.

Two sweetpotato ADP-glucose pyrophosphorylase isoforms are regulated antagonistically in response to sucrose content in storage roots

The transcriptional regulation of ADP-glucose pyrophosphorylase (AGPase) genes in detached leaves in response to exogenous sucrose has been investigated earlier; however the effects of endogenous sucrose on AGPase gene transcription in leaves or starch-accumulating tissues have not yet been determined. We therefore have investigated the relationship between endogenous sucrose content in the storage tissues of sweetpotato ( Ipomoea batatas cv. Yulmi) and the rate of transcription of the two sweetpotato AGPase isoforms, ibAGP1 and ibAGP2, by means of transient expression analysis of their promoters. Sequence analysis of the two promoters identified putative sucrose-responsive elements on the ibAGP1 promoter and, conversely, putative sucrose-starvation elements on the ibAGP2 promoter. Transient expression analyses on transverse storage root sections revealed that the ibAGP1 and ibAGP2 promoters directed strong expression in the sweetpotato storage roots (diameter: 1.5 cm). Sucrose contents of the sweetpotato storage roots were positively correlated with growth of the storage root. In the storage roots, ibAGP1 promoter activity became stronger with increasing endogenous sucrose levels, while ibAGP2 promoter activity became markedly weaker. Consequently, ibAGP2 was expressed primarily during the early stages of storage root development, whereas ibAGP1 was abundantly expressed in the later stages, during which a profound level of starch accumulation occurs. The antagonistic regulation of the two promoters in response to endogenous sucrose levels was also confirmed in carrot ( Daucus carota L. cv. Hapa-ochon) taproots.

Gene expression of the biosynthetic enzymes and biosynthesis of starch during Rice Grain development

Amylose and amylopectin are determinants of the physicochemical properties for starch and grain quality in rice. Their biosynthesis is catalyzed by the interplay of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), a starch branching enzyme (SBE), and a starch debranching enzyme (SDE). In this study, the genes for these enzymes were highly expressed 7 to 28 days after flowering during grain development, and their expression closely matched increases in both starch content and grain weight Among all the tested cultivars, amylose contents in the rice grains remained essentially constant throughout their development The AGPase gene was highly expressed in the high-yield cultivars of both glutinous and non-glutinous rice. The SSS gene was actively expressed when mature GBSS mRNA decreased. Genes responsible for amylopectin biosynthesis were simultaneously expressed in the late stage of grain development. We have now demonstrated that the expression patterns of starch biosynthetic genes differ between glutinous and non-glutinous rice, and between Tongil (a Japonica/ Indica hybrid) and Japonica types.

Gene Expression of ADP-glucose Pyrophosphorylase and Starch Contents in Rice Cultured Cells are Cooperatively Regulated by Sucrose and ABA

Six cDNA clones encoding two small subunits and four large subunits of ADP-glucose pyrophosphorylase (AGPase) were mined from the database of rice full-length cDNAs, cloned and subsequently named: OsAPS1, OsAPS2, OsAPL1, OsAPL2, OsAPL3 and OsAPL4. Expression patterns of the six genes were examined by Northern blot analysis with gene-specific probes. OsAPL3 was predominantly expressed in the middle phases of seed development, and OsAPS1, OsAPL1 and OsAPL2 were expressed later in seed development. OsAPS2 and OsAPL4 were constitutively expressed and these isoforms were coordinated with starch accumulation in the developing rice seed. In order to clarify the effect of sugars and plant hormones on AGPase gene expression more precisely, a rice cell culture system was used. OsAPL3 transcript significantly accumulated in response to increased levels of sucrose and abscisic acid (ABA) concentration in the medium; however, the transcripts of other AGPase genes did not show significant accumulation. Under identical conditions, starch contents in the cultured cells also increased. Interestingly, ABA alone did not affect the gene expression of OsAPL3 and starch content. Collectively, these results indicated that the expression level of OsAPL3 and starch content in the cultured cells were cooperatively controlled by alterations in the concentration of both sucrose and ABA.

Mutan produced in potato amyloplasts adheres to starch granules

Production of water-insoluble mutan polymers in Kardal potato tubers was investigated after expression of a full-length (GtfI) and a truncated mutansucrase gene referred to as GtfICAT (GtfI without glucan-binding domain) from Streptococcus downei. Subsequent effects on starch biosynthesis at the molecular and biochemical levels were studied. Expression of the GtfICAT gene resulted in the adhesion of mutan material on starch granules, which stained red with erythrosine, and which was hydrolysed by exo-mutanase. In addition, GtfICAT-expressing plants exhibited a severely altered tuber phenotype and starch granule morphology in comparison to those expressing the full-length GtfI gene. In spite of that, no structural changes at the starch level were observed. Expression levels of the sucrose-regulated, AGPase and GBSSI genes were down-regulated in only the GTFICAT transformants, showing that GtfICAT expression interfered with the starch biosynthetic pathway. In accordance with the down-regulated AGPase gene, a lower starch content was observed in the GTFICAT transformants. Finally, the rheological properties of the GTFICAT starches were modified; they showed a higher retrogradation during cooling of the starch paste.

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.

Starch synthesis in potato tubers transformed with the wheat genes for ADPglucose pyrophosphorylase.

The aim of this work was to study the role of ADPglucose pyrophosphorylase (AGPase) in starch biosynthesis of non-photosynthetic organs. Agrobacterium tumefaciens was used to transform potato plants (Solanum tuberosum L. cv. Desireé) with the wheat AGPase genes (AGP-S and AGP-L, coding for the small and large subunits, respectively). Neither of these genes contains a recognisable plastid targeting sequence. Southern analysis and analysis of starch content identified four lines that contained both wheat sequences. Immunoblotting indicated that, in the tubers, three lines expressed the wheat small subunit (AGP-S), but AGP-L cross-reacting protein was not apparent. The fourth transgenic line had reduced AGPase activity. AGPase activity in the AGP-transgenic tubers ranged from 15 to 165% of that found in β-glucuronidase (GUS) control lines.

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.

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggest that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.