AGPase Small Subunit Research Articles

Preparation of Barley AGPS2b Antibody and Its Application in Hormone Regulation Research

ADP-glucose pyrophosphorylase (AGPase), which is a key enzyme in the starch biosynthesis pathway, plays a critical role in barley grain development. Despite its importance, the regulatory mechanisms governing AGPase expression, particularly the influence of plant hormones, remain poorly understood in barley. To address this, we identified and characterized the HvAGPS2b gene, which encodes the AGPase small subunit. The full-length HvAGPS2b gene was cloned from the barley database and expressed as a recombinant protein using the pET-30a system. Polyclonal antibodies were prepared against HvAGPS2b to facilitate detailed analysis. Our findings revealed that HvAGPS2b, as a small subunit of the rate-limiting enzyme AGPase, is integral to the later stages of grain development. Furthermore, RT-qPCR and Western blotting analyses showed that the phytohormones ABA, GA, ETH, and BR significantly upregulated the expression of AGPase small subunits. These results underscore the vital role of plant hormones in modulating AGPS2b expression, thereby influencing grain development. This study provides significant insights into the hormonal regulation of starch biosynthesis and establishes a foundation for further investigation into the functional dynamics of AGPase in barley.

Effect of Phosphorylation Sites Mutations on the Subcellular Localization and Activity of AGPase Bt2 Subunit: Implications for Improved Starch Biosynthesis in Maize

ADP-Glc pyrophosphorylase (AGPase) is a pivotal enzyme catalyzing the conversion of ATP and glucose-1-phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc), thereby serving as a rate-limiting factor in starch biosynthesis in crops. Although previous investigations have suggested phosphorylation-based regulation of AGPase in maize, the explicit modulation mechanisms have yet to be elucidated. This research evaluated the effect of point mutations at phosphorylation sites (identified using iTRAQTM AB SCIEX, Framingham, MA, USA) on the subcellular localization and activity of the AGPase small subunit Bt2, and its interaction with the large subunit Sh2, in maize. Despite the induction of point mutations, subcellular localization of the Bt2 subunit remained unaltered, primarily within the cytoplasm and nucleus. The interaction between Bt2 and Sh2 subunits continued, mainly in the chloroplast. Notably, an increase in AGPase activity was observed in the case of simulated phosphorylation point mutations, whereas dephosphorylation activity significantly diminished relative to the wild type. These findings demonstrate that point mutations do not affect the subcellular localization of the Bt2 subunit or its interaction with the Sh2 subunit, but substantially modulate AGPase activity. This study provides critical insights into the role of point mutations in enhancing AGPase activity, thus potentially accelerating the production of ADP-Glc, the primary substrate for starch synthesis, promising implications for improved starch biosynthesis in maize.

The 218th amino acid change of Ser to Ala in TaAGPS-7A increases enzyme activity and grain weight in bread wheat

ADP-glucose pyrophosphorylase (AGPase) influences cereal productivity. There are few reports on the function of cytosolic AGPase small subunit in bread wheat (TaAGPS). In the present study, TaAGPS was preferentially expressed in developing endosperm during grain-filling stages in bread wheat. TaAGPS allelic variations were characterized in 143 wheat accessions by PacBio RS II sequencing. Two haplotypes (TaAGPS-7A-TG and TaAGPS-7A-CT) of TaAGPS-7A were identified and corresponding functional markers were developed, whereas no variants of TaAGPS-7B and TaAGPS-7D were detected. TaAGPS-7A was associated with thousand-kernel weight (TKW) by haplotype–trait association analysis in two populations. Near-isogenic lines (NILs) with TaAGPS-7A-TG showed higher TKW and total kernel starch content than those with TaAGPS-7A-CT, owing to the higher AGPase activity of TaAGPS-7A-TG than TaAGPS-7A-CT both in vitro and in vivo. Overexpression of TaAGPS-7A-TG in bread wheat doubled the transcription levels of TaAGPS and increased AGPase activity by 55.7%, resulting in a 3.0-g higher TKW than in the wild type (WT). Knockdown of TaAGPS led to reduced expression of TaAGPS, AGPase activity, and TKW than in the WT. Thus, owing to the 218th amino acid change of Ser to Ala in TaAGPS-7A, the favorable haplotype TaAGPS-7A-TG showed higher AGPase activity, resulting in higher kernel starch content and grain weight. This finding could be applied to increasing starch content and grain weight in bread wheat.

Proteomic analysis reveals a role of ADP-glucose pyrophosphorylase in the asynchronous filling of rice superior and inferior spikelets

The poor grain filling of inferior spikelets (IS) situated on the lower secondary rachis branch leads to a remarkable decrease in rice yield and quality. The AGPase small subunit 2 (AGPS2) encodes a small subunit of adenosine diphosphate-glucose pyrophosphorylase (AGPase) enzyme, which plays an important role in sucrose-starch conversion and starch biosynthesis in the grain filling of rice. In the present study, qPCR analysis showed low expression abundance of AGPS2 in IS, compared to the superior spikelets (SS), which was consistent with the lower grain weight of IS. To evaluate the molecular mechanism of AGPS2, we first identified the AGPS2 interaction network through Co-immunoprecipitation (Co-IP). In total, 29 proteins of AGPS2 interaction network were characterized by LC–MS/MS. Bioinformatics analysis revealed that, the characterized proteins in the interaction network are likely to be related to starch synthesis, sugar conversion, energy pathway, and folding/modification, and most of them were involved in the grain filling of rice. The sequent Co-IP analysis showed that AGPS2 can bind to starch branching enzyme (SBE), pullulanase (PUL) and starch debranching enzyme (DBE) and assemble into starch synthesizing protein complex (SSPC). In addition, the 14-3-3 protein GF14e was also found to interact with AGPS2. Further analysis by qPCR showed that the expression of GF14e was much higher on IS than on SS. The qPCR results also showed that the expression of GF14e was relatively stable in SS, but changed significantly in IS under alternate wetting and moderate soil drying (WMD), which is consistent with the AGPS2 expression pattern. Our present work provides direct molecular evidence for the different expression patterns of AGPS2 in SS and IS, which could be greatly helpful for the molecular amelioration of the poor grain filling of IS in rice.

Cassava AGPase genes and their encoded proteins are different from those of other plants.

Cassava AGPase and AGPase genes have some unique characteristics. ADP-glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme for starch synthesis. In this study,cassava AGPase genes (MeAGP) were analyzed based on six cultivars and one wild species. A total of seven MeAGPs was identified, including four encoding AGPase large subunits (MeAGPLs 1, 2, 3 and 4) and three encoding AGPase small subunits (MeAGPSs 1, 2 and 3). The copy number of MeAGPs varied in cassava germplasm materials. There were 14 introns for MeAGPLs 1, 2 and 3, 13 introns for MeAGPL4, and 8 introns for other three MeAGPSs. Multiple conservative amino acid sequence motifs were found in the MeAGPs. There were differences in amino acids at binding sites of substrates and regulators among different MeAGP subunits and between MeAGPs and a potato AGPase small subunit (1YP2:B). MeAGPs were all located in chloroplasts. MeAGP expression was not only associated with gene copy number and types/combinations, regions and levels of the DNA methylation but was also affected by environmental factors with the involvement of various transcription factors in multiple regulation networks and in various cis-elements in the gene promoter regions. The MeAGP activity also changed with environmental conditions and had potential differences among the subunits. Taken together, MeAGPs differ in number from those of Arabidopsis, potato, maize, banana, sweet potato, and tomato.

Gene expression profiling of adp-glucose pyrophosphorylase (AGPase) in sink and source organs of some cassava varieties with different starch contents in Vietnam

Starch is the most widespread and abundant storage carbohydrate in plants. We depend upon starch for our nutrition, exploit its unique properties in industry, and use it as a feedstock for bio-ethanol production. Starch is stored in the form of osmotically inactive, water-insoluble granules in amyloplasts (storage starch) and chloroplasts (transitory starch). The biosynthesis of starch involves not only the production of the composite glucans but also their arrangement into an organized form within the starch granule. Understanding the specific functions played by individual isoforms of enzymes involved in starch biosynthesis pathways will provide important basis for regulation of starch production in plant. A transcript-level analysis of the genes which encode starch-synthesis enzymes is fundamental for assessment of enzyme function and the regulatory mechanism for starch biosynthesis in source and sink organs. In this work, the expression level of the genes encoding ADP-glucose pyrophosphorylase (AGPase) in two local varieties Do Dia Phuong (Do DF) and Trang Hoa Binh (Trang HB) as well as two imported varieties KM94 (Rayong1 X Rayong 90) and KM140 (KM98-1 x KM36) with different starch contents were evaluated by quantitative real-time PCR method. The result of transcript level analysis made the expression profiles of cassava AGPS and AGPL genes (encoding AGPase small and large subunits) during three development periods, 90, 180 and 270 DAP (day after planting). The transcriptional activities of these genes exhibited tissue-specific expression patterns. In particular, AGPS2 and AGPL1 transcripts were predominant in leaves, whereas expression of AGPS1, AGPL2, and AGPL3 appeared to be mostly confined to storage roots. Despite of having disparities between development stages, expression patterns of both AGPS2 and AGPL1 in leaves did not show significant differences amongst investigated cassava varieties. In contrast, transcriptional activities of AGPS1 and AGPL3 in tubers had patterns directly related to the starch contents of the cultivars. These results indicated that AGPS1 and AGPL3 genes likely play an important role in the starch biosynthesis pathway and have potential for regulation of starch production in cassava.

Enhancement of starch content by constitutive expression of GmTrxF in transgenic Arabidopsis

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant carbohydrate metabolism biosynthesis. In this study, a gene encoding the TrxF protein, named GmTrxF, was isolated from soybean. The open reading frame (ORF) contained 540 nucleotides encoding 179 amino acids. The coding region of GmTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis. The starch content in GmTrxF expressing plants was increased by 57–109% compared to that in wild-type (WT). Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmTrxF up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that GmTrxF may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of GmTrxF expression might be used for increasing starch accumulation of plants in the future.

A soybean plastidic ATP/ADP transporter gene, GmAATP, is involved in carbohydrate metabolism in transgenic Arabidopsis

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.

StTrxF, a potato plastidic thioredoxin F-type protein gene, is involved in starch accumulation in transgenic Arabidopsis thaliana

ABSTRACTThe plastidic thioredoxin F-type (TrxF) protein plays a key role in plant carbohydrate metabolism. In this work, a gene encoding the TrxF protein, named StTrxF, was successfully isolated from potato and transformed into Arabidopsis thaliana to obtain transgenic plants. Constitutive expression of StTrxF significantly increased the starch accumulation in the transgenic plants. Real-time quantitative polymerase chain reaction analysis showed that constitutive expression of StTrxF up-regulated the expression of the adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic A. thaliana plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild-type. These results suggest that StTrxF may improve the starch content of A. thaliana by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. The StTrxF gene may be applied for increasing starch accumulation of plants in the future.

Comparative Analysis of AGPase Genes and Encoded Proteins in Eight Monocots and Three Dicots with Emphasis on Wheat.

ADP-glucose pyrophosphorylase (AGPase) is a heterotetrameric enzyme with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. We are reporting here detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots. “True” orthologs of maize Sh2 (AGPase LS) and Bt2 (AGPase SS) were identified in seven other monocots and three dicots; structure of the enzyme at protein level was also studied. Novel findings of the current study include the following: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) “QTCL” motif present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while “LGGG” motif in LS was present in all species except T. urartu and chickpea; SS provides thermostability to AGPase, while LS is involved in regulation of AGPase activity; (iv) heterotetrameric structure of AGPase was predicted and analyzed in real time environment through molecular dynamics simulation for all the species; (v) several cis-acting regulatory elements were identified in the AGPase promoters with their possible role in regulating spatial and temporal expression (endosperm and leaf tissue) and also the expression, in response to abiotic stresses; and (vi) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The results of the present study have allowed better understanding of structure and evolution of the genes and the encoded proteins and provided clues for exploitation of variability in these genes for engineering thermostable AGPase.

Activation of ADP-Glucose Pyrophosphorylase Gene Promoters by a WRKY Transcription Factor, AtWRKY20, in Arabidopsis thaliana L. and Sweet Potato (Ipomoea batatas Lam.)

ADP–glucose pyrophosphorylase (AGPase) catalyzes the first limiting step in starch biosynthesis in plants. However, the direct transcriptional activator of the AGPase genes has not yet been determined. We have isolated a WRKY transcription factor cDNA, AtWRKY20, from Arabidopsis thaliana and purified the corresponding protein. Transient expression of AtWRKY20 by particle bombardment enhanced expression of the promoter of ApL3, encoding a sugar-inducible AGPase large subunit gene of A. thaliana, in leaves of A. thaliana. AtWRKY20 bound to the ApL3 promoter in vitro. The expression of AtWRKY20 was strongly induced by sucrose or, to a lesser extent, by mannitol, and the expression pattern of the ApL3 gene mimicked that of the AtWRKY20 gene. Transient expression experiments demonstrated that AtWRKY20 also activated the promoter of Koganesengan ibAGP1 encoding an AGPase small subunit gene of sweet potato var. Koganesengan. A 5’–end deletion analysis revealed a negative regulatory region from –1371 to –641 and a positive regulatory region from –640 to –180 in the Koganesengan ibAGP1 promoter. AtWRKY20 interacted directly with the region between positions –623 and –490 in the Koganesengan ibAGP1 promoter. These results suggest that AtWRKY20 functions directly as a transcriptional activator of the ApL3 promoter and regulates the expression of ApL3 induced by sucrose or osmoticum in A. thaliana. Moreover, AtWRKY20 can enhance the expression of the Koganesengan ibAGP1 promoter directly in sweet potato.

Contrasted patterns of selection since maize domestication on duplicated genes encoding a starch pathway enzyme

Maize domestication from teosinte (Zea mays ssp. parviglumis) was accompanied by an increase of kernel size in landraces. Subsequent breeding has led to a diversification of kernel size and starch content among major groups of inbred lines. We aim at investigating the effect of domestication on duplicated genes encoding a key enzyme of the starch pathway, the ADP-glucose pyrophosphorylase (AGPase). Three pairs of paralogs encode the AGPase small (SSU) and large (LSU) subunits mainly expressed in the endosperm, the embryo and the leaf. We first validated the putative sequence of LSU(leaf) through a comparative expression assay of the six genes. Second, we investigated the patterns of molecular evolution on a 2 kb coding region homologous among the six genes in three panels: teosintes, landraces, and inbred lines. We corrected for demographic effects by relying on empirical distributions built from 580 previously sequenced ESTs. We found contrasted patterns of selection among duplicates: three genes exhibit patterns of directional selection during domestication (SSU(end), LSU(emb)) or breeding (LSU(leaf)), two exhibit patterns consistent with diversifying (SSU(leaf)) and balancing selection (SSU(emb)) accompanying maize breeding. While patterns of linkage disequilibrium did not reveal sign of coevolution between genes expressed in the same organ, we detected an excess of non-synonymous substitutions in the small subunit functional domains highlighting their role in AGPase evolution. Our results offer a different picture on AGPase evolution than the one depicted at the Angiosperm level and reveal how genetic redundancy can provide flexibility in the response to selection.

Arabidopsis AMY1 expressions and early flowering mutant phenotype

The homozygous T-DNA mutant of the AMY1 gene in Arabidopsis was identified and importantly, shown to cause an early flowering phenotype. We found that the disruption of AMY1 enhanced expression of CO and FT. The expression analyses of genes related to starch metabolism revealed that expression of the AGPase small subunit APS1 in the wild type was higher than in the amy1 mutant. However, there were no significant differences in expression levels of the AGPase large subunit genes ApL1, AMY2, or AMY3 between wild type and the amy1 mutant. Expression profiling showed that AMY1 was highly expressed in leaves, stems, and flowers, and expressed less in leafstalks and roots. Furthermore, the level of AMY1 mRNA was highly elevated with age and in senescing leaves. RT-PCR analyses showed that the expression of AMY1 was induced by heat shock, GA, and ABA, while salt stress had no apparent effect on its expression.

Kinetic and regulatory properties of plant ADP-glucose pyrophosphorylase genetically modified by heterologous expression of potato upreg mutants in vitro and in vivo

In this study, the uses of the mutated genes, upreg1 and upreg2, encoding upregulated ADP-glucose pyrophosphorylase (AGPase) large subunits with increased enzymatic activity, to improve crop yield productivity was evaluated in vitro and in planta. For in vitro examination, wild type and upregs were co-expressed with three different AGPase small subunit genes from potato and perilla to produce nine AGPase isoforms. In kinetic experiments, 3-Phosphoglycerate increased the V max and decreased the K M for the recombinant AGPase. Regardless of the specific small subunit, Upreg-type AGPases had much larger increases in enzymatic activity with concomitant decreases in values as compared to the wild type enzyme. Transformation of lettuce with the upreg1 gene altered the regulatory properties of leaf AGPase. AGPases from transgenic lettuce showed greater 3-PGA activation and lower Pi inhibition than was observed for wild type AGPase. Fresh weights of the aerial parts of transgenic plants were larger than non-transgenic controls. Based on these results, upreg mutant genes could be used for the genetic improvement of plant AGPases other than potato and effectively increase crop yield productivity.

Isolation of a heat-stable maize endosperm ADP-glucose pyrophosphorylase variant

Heat stress reduces maize yield and several lines of evidence suggest that the heat lability of maize endosperm ADP-glucose pyrophosphorylase (AGPase) contributes to this yield loss. AGPase catalyzes a rate-limiting step in starch synthesis. Herein, we present a novel maize endosperm AGPase small subunit variant, termed BT2-TI that harbors a single amino acid change of residue 462 from threonine to isoleucine. The mutant was isolated by random mutagenesis and heterologous expression in a bacterial system. BT2-TI exhibits enhanced heat stability compared to wildtype maize endosperm AGPase. The TI mutation was placed into another heat-stable small subunit variant, MP. MP is composed of sequences from the maize endosperm and the potato tuber small subunit. The MP-TI small subunit variant exhibited greater heat stability than did MP. Characterization of heat stability as well as kinetic and allosteric properties suggests that MP-TI may lead to increased starch yield when expressed in monocot endosperms.

고구마에서 ADP-Glucose Pyrophosphorylase Small Subunit cDNA의 Antisense 발현에 의한 전분생합성 저해

고구마 유래 ibAGP1, ibAGP2 유전자가 antisense 방향으로 각각 도입된 벡터를 섞어서 particle bombardment 방법으로 고구마 배발생 캘러스에 형질전환을 수행하였다. 선발배지에서 형성된 배발생 캘러스로부터 체세포 배발생 과정을 거쳐 식물체로 재분화된 개체에서 PCR과 Southern 분석으로 고구마의 염색체 게놈에 ibAGP1, ibAGP2 유전자가 도입되었음을 확인하였고, western blot을 통해 형질전환체의 AGPase small subunit 효소 생산이 전반적으로 대조구에 비해 감소하였다. 또한, 형질전환체의 잎과 괴근 절편체의 전분염색 결과에서도 대조구에 비해 부분적으로 전분염색이 이루어짐을 알 수 있었다. 특히, ibAGP1, ibAGP2 유전자가 동시에 들어 있는 식물체 잎에서는 ibAGP1만 들어있는 것에 비해 전분합성이 더욱 감소됨을 확인하였다. 고구마의 경우 감자를 비롯한 다른 식물들과는 달리 외형적인 생장자체를 심하게 저해하였으며 괴근이 형성되지 않아 ibAGP1, 2의 발현 여부가 식물체의 생장에 절대적인 영향을 미침을 시사한다. Embryogenic calluses derived from shoot apical meristem explants of sweetpotato were subjected to particle bombardment to generate transgenic plants for antisense expression of cDNAs encoding two different AGPase small subunit (ibAGP1 and ibAGP2). Plants were generated via somatic embryogenesis. PCR and Southern analysis demonstrated that the incorporation of ibAGP1 and ibAGP2 into the genome in an antisense orientation. Immunoblot analysis confirmed reduced levels of AGPase small subunit in transgenic plant leaves. Plants with both ibAGP1 and ibAGP2 produced a lower level of the protein than plants with ibAGP1 alone. iodine test demonstrated that transgenic plant leaves and storage root accumulated reduced amounts of starch. Iodine staining of leaf tissues indicated that transgenic plants accumulated less amount of starch than control. In accordance with western blot analysis, plants with both ibAGP1 and ibAGP2 accumulated a lower amount of starch than plants with ibAGP1 alone. Both transgenic plants exhibited a severely retarded growth, resulting in bare survival. It is suggested that disrupted expression of the gene encoding AGPase small subunit is lethal to the growth of sweetpotato contrast to other species including potato.

The Two AGPase Subunits Evolve at Different Rates in Angiosperms, yet They Are Equally Sensitive to Activity-Altering Amino Acid Changes When Expressed in Bacteria

The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis.

A mutant of rice lacking the leaf large subunit of ADP-glucose pyrophosphorylase has drastically reduced leaf starch content but grows normally.

A mutant of rice was identified with a Tos17 insertion in OsAPL1, a gene encoding a large subunit (LSU) of ADP-glucose pyrophosphorylase (AGPase). The insertion prevents production of a normal transcript from OsAPL1. Characterisation of the mutant (apl1) showed that the LSU encoded by OsAPL1 is required for AGPase activity in rice leaf blades. In mutant leaf blades, the AGPase small subunit protein is not detectable and the AGPase activity and starch content are reduced to <1 and <5% of that in wild type blades, respectively. The mutation also leads to a reduction in starch content in the leaf sheaths but does not significantly affect AGPase activity or starch synthesis in other parts of the plant. The sucrose, glucose and fructose contents of the leaves are not affected by the mutation. Despite the near absence of starch in the leaf blades, apl1 mutant rice plants grow and develop normally under controlled environmental conditions and show no reduction in productivity.

Mechanism of Up-regulation of Starch Synthesis in Mature Leaves of Transgenic Apple Trees with Decreased Sorbitol Synthesis

The reaction catalyzed by ADP-glucose pyrophosphorylase (AGPase) to form ADP-glucose is a regulatory and rate-limiting step in starch synthesis in plants. In response to decreased sorbitol synthesis, starch synthesis was up-regulated in the transgenic apple plants. In this study, we examined both redox and metabolite regulation of AGPase to understand the mechanism responsible for the up-regulation of starch synthesis. No difference in the monomerization/dimerization of apple leaf AGPase small subunits was observed between the transgenic plants and the untransformed control. NADP-dependent malate dehydrogenase, indicative of chloroplastic redox status, did not show significant change in the transgenic plants either. Determination of key metabolites with nonaqueous fractionation indicated that concentrations of hexose phosphates (mainly glucose-6-phosphate and fructose-6-phosphate) were higher in both the cytosol and chloroplasts of the transgenic plants than in the control, whereas 3-phosphoglycerate (PGA) concentration in the chloroplast was not higher in the transgenic plants. We conclude that accumulation of hexose-phosphates results in a decrease in inorganic phosphate (Pi) concentration and an increase in PGA/Pi ratio in the chloroplast, leading to up-regulation of starch synthesis via activating AGPase.

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.