ADP-glucose Pyrophosphorylase Small Subunit Research Articles

Genome-wide association analysis and transgenic characterization for amylose content regulating gene in tuber of Dioscorea zingiberensis

BackgroundAmylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients’ diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation.ResultsTo uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value < 0.004) were clustered in a narrow window on the chromosome 6 (chr6). The peak SNP at the position 75,609,202 on chr6 could explain 63.15% of amylose variation in the population and fell into the first exon of the ADP-glucose pyrophosphorylase (AGPase) small subunit gene, causing a non-synonymous modification of the resulting protein sequence. Allele segregation analysis showed that accessions with the rare G allele had a higher amylose content than those harboring the common A allele. However, AGPase, a key enzyme precursor of amylose biosynthesis, was not expressed differentially between accessions with A and G alleles. Overexpression of the two variants of AGPase in Arabidopsis thaliana resulted in a significantly higher amylose content in lines transformed with the AGPase-G allele.ConclusionsOverall, this study showed that a major genetic variant in AGPase probably enhances the enzyme activity leading to high amylose content in D. zingiberensis tuber. The results provide valuable insights for the development of amylose-enriched genotypes.

Starch deficiency in tomato causes transcriptional reprogramming that modulates fruit development, metabolism and stress responses.

Tomato fruit (Solanum lycopersicum) store carbon as starch during early development and mobilize it at the onset of ripening. Starch accumulation has been suggested to buffer fluctuations in carbon supply to the fruit under abiotic stress, and contribute to sugar levels in ripe fruit. However, the role of starch accumulation and metabolism during fruit development is still unclear. Here we show that the tomato mutant adpressa (adp) harbors a mutation in a gene encoding the small subunit of ADP-glucose pyrophosphorylase (AGPase), which abolishes starch synthesis. The disruption of starch biosynthesis causes major transcriptional and metabolic remodeling in adp fruit but only minor effects on fruit size and ripening. Changes in gene expression and metabolite profiles indicate that the lack of carbon flow into starch increases levels of soluble sugars during fruit growth, triggers a readjustment of central carbohydrate and lipid metabolism, and activates growth and stress protection pathways. Accordingly, adp fruits are remarkably resistant to blossom-end rot, a common physiological disorder induced by environmental stress. Our results provide insights into the effects of perturbations of carbohydrate metabolism on tomato fruit development, with potential implications for the enhancement of protective mechanisms against abiotic stress in fleshy fruit.

Sweet potato ADP-glucose pyrophosphorylase small subunit affects vegetative growth, starch content and storage root yield

The development of storage roots is a key factor determining the yields of crop plants, including sweet potato. Here, using combined bioinformatic and genomic approaches, we identified a sweet potato yield-related gene, ADP-glucose pyrophosphorylase (AGP) small subunit (IbAPS). We found that IbAPS positively affects AGP activity, transitory starch biosynthesis, leaf development, chlorophyll metabolism, and photosynthesis, ultimately affecting the source strength. IbAPS overexpression in sweet potato led to increased vegetative biomass and storage root yield. RNAi of IbAPS resulted in reduced vegetative biomass, accompanied with a slender stature and stunted root development. In addition to the effects on root starch metabolism, we found that IbAPS affects other storage root development-associated events, including lignification, cell expansion, transcriptional regulation, and production of the storage protein sporamins. A combinatorial analysis based on transcriptomes, as well as morphological and physiological data, revealed that IbAPS affects several pathways that determine development of vegetative tissues and storage roots. Our work establishes an important role of IbAPS in concurrent control of carbohydrate metabolism, plant growth, and storage root yield. We showed that upregulation of IbAPS results in superior sweet potato with increased green biomass, starch content, and storage root yield. The findings expand our understanding of the functions of AGP enzymes and advances our ability to increase the yield of sweet potato and, perhaps, other crop plants.

Revisiting maize Brittle endosperm-2 reveals new insights in BETL development and starchy endosperm filling

Rerouting the starch biosynthesis pathway in maize can generate specialty types, like sweet corn and waxy corn, with a drastically increasing global demand. Hence, a fine-tuning of starch metabolism is relevant to create diverse maize cultivars for end-use applications. Here, we characterized a new maize brittle endosperm mutant, referred to as bt1774, which exhibited decreased starch content but a dramatic increase of soluble sugars at maturity. Both endosperm and embryo development was impaired in bt1774 relative to the wild-type (WT), with a prominently arrested basal endosperm transfer layer (BETL). Map-based cloning revealed that BRITTLE ENDOSPERM2 (Bt2), which encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase), is the causal gene for bt1774. A MuA2 element was found to be inserted into intron 2 of Bt2, leading to a severe decrease of its expression, in bt1774. This is in line with the irregular and loosely packed starch granules in the mutant. Transcriptome of endosperm at grain filling stage identified 1,013 differentially expressed genes in bt1774, which were notably enriched in the BETL compartment, including ZmMRP1, Miniature1, MEG1, and BETLs. Gene expression of the canonical starch biosynthesis pathway was marginally disturbed in bt1774. Combined with the residual 60 % of starch in this nearly null mutant of Bt2, this data strongly suggests that an AGPase-independent pathway compensates for starch synthesis in the endosperm. Consistent with the BETL defects, zein accumulation was impaired in bt1774. Co-expression network analysis revealed that Bt2 probably has a role in intracellular signal transduction, besides starch synthesis. Altogether, we propose that Bt2 is likely involved in carbohydrate flux and balance, thus regulating both the BETL development and the starchy endosperm filling.

Genetic analysis and molecular characterization of a novel maize &amp;lt;italic&amp;gt;Bt2 &amp;lt;/italic&amp;gt;gene mutant

<p id="C3">The research of the molecular mechanism underlying maize kernel development is particularly important for the genetic improvement of maize yield and quality traits. In this study, we characterized a new shrunken kernel mutant <italic>5601Q</italic>, which was generated by a random transposon insertion. Genetic analysis indicated that the kernel phenotype was stably controlled by a single recessive gene. F₂ segregating population was constructed by crossing <italic>5601Q</italic> into B73 inbred line, and the mutant gene was located in the genetic interval of 60.19-62.58 Mb on chromosome 4. Sequence annotation showed that the <italic>BRITTLE ENDOSPERM2 </italic>(<italic>Bt2</italic>) gene, previously reported to be involved in maize kernel development, was located in this region. Maize<italic> Bt2</italic> gene encoded the small subunit of ADP-glucose pyrophosphorylase (AGPase), the first rate-limiting enzyme in the starch biosynthetic pathway of higher plants. Compared with wild type, 100-grain weight and starch content of mutant <italic>5601Q</italic> decreased significantly, but the soluble sugar content increased dramatically 4.67 times. We confirmed that <italic>5601Q</italic> was a new allele mutant of <italic>Bt2</italic> by allelic test of <italic>Bt2</italic> mutant <italic>1774 </italic>and<italic> 5601Q</italic>. Sequencing analysis revealed that <italic>Mutator 19 </italic>transposon was inserted in the 2nd exon of <italic>Bt2</italic> gene. In summary, our results indicated that the shrunken kernel in <italic>5601Q </italic>was caused by the loss-of-function of <italic>Bt2 </italic>gene, which provided a new germplasm resource to elucidate the mechanism of maize <italic>Bt2 </italic>gene in endosperm storage substance accumulation.

Starch accumulation in bean fruit pericarp is mediated by the differentiation of chloroplasts into amyloplasts

The sucrose supply to bean fruits remains almost constant during seed development, and the early stages of this process are characterized by a significant amount of starch and soluble sugars (glucose, fructose and sucrose) accumulated in the pericarp. Bean fruits are photosynthetically active; however, our results indicated that starch synthesis in the pericarp was largely dependent on the photosynthetic activity of the leaves. The photosynthetic activity and the amount of the Rubisco large subunit were gradually reduced in the fruit pericarp, and a large increase in the amount of the ADP-glucose pyrophosphorylase small subunit (AGPase SS) was observed. These changes suggested differentiation of chloroplasts into amyloplasts. Pericarp chloroplasts imported glucose 1-P to support starch synthesis, and their differentiation into amyloplasts allowed the surplus sucrose to be used in the synthesis of starch, which was later degraded to meet the needs of fast-growing seeds. Starch stored in the bean fruit pericarp was not degraded in response to drought stress, but it was rapidly used under severe nutrient restriction. Together, this work indicated that starch accumulation in the pericarp of bean fruits is important to adjust the needs of developing seeds to the amount of sucrose that is provided to fruits.

Selection and validation of reference genes for quantitative real-time PCR in the green microalgae Tetraselmis chui.

Quantitative real-time reverse transcription PCR (RT-qPCR) is a highly sensitive technique that can be applied to analyze how genes are modulated by culture conditions, but identification of appropriate reference genes for normalization is a critical factor to be considered. For this reason, the expression stability of 18 candidate reference genes was evaluated for the green microalgae Tetraselmis chui using the widely employed algorithms geNorm, NormFinder, BestKeeper, the comparative ΔCT method, and RefFinder. Microalgae samples were collected from large scale outdoor photobioreactors during the growing phase (OUT_GP), and during the semi-continuous phase at different times of the day (OUT_DC). Samples from standard indoor cultures under highly controlled conditions (IND) were also collected to complement the other data. Different rankings for the candidate reference genes were obtained depending on the culture conditions and the algorithm employed. After comparison of the achieved ranks with the different methods, the references genes selected for samples from specific culture conditions were ALD and EFL in OUT_GP, RPL32 and UBCE in OUT_DC, and cdkA and UBCE in IND. Moreover, the genes EFL and cdkA or EFL and UBCE appeared as appropriate combinations for pools generated from all samples (ALL). Examination in the OUT_DC cultures of genes encoding the large and small subunits of ADP-glucose pyrophosphorylase (AGPL and AGPS, respectively) confirmed the reliability of the identified reference genes, RPL32 and UBCE. The present study represents a useful contribution for studies of gene expression in T. chui, and also represents the first step to set-up an RT-qPCR platform for quality control of T. chui biomass production in industrial facilities.

Two ADP-glucose pyrophosphorylase subunits, OsAGPL1 and OsAGPS1, modulate phosphorus homeostasis in rice.

Plant acclimatory responses to phosphate (Pi) starvation stress include the accumulation of carbohydrates, namely sugar and starch. However, whether altered endogenous carbohydrate profile could in turn affect plant Pi starvation responses remains widely unexplored. Here, two genes encoding the large and small subunits of an ADP-glucose pyrophosphorylase (AGP) in rice (Oryza sativa), AGP Large Subunit 1 (AGPL1) and AGP Small Subunit 1 (AGPS1), were functionally characterized with regard to maintenance of phosphorus (P) homeostasis and regulation of Pi starvation signaling. AGPL1 and AGPS1 were both positively responsive to nitrogen (N) or Pi deprivation, and expressed in almost all the tissues except in the meristem and mature zones of root. AGPL1 and AGPS1 physically interacted in chloroplast, and catalyzed the rate-limiting step of starch biosynthesis. Low-N- (LN) and low-Pi (LP)-triggered starch accumulation in leaves was impaired in agpl1, agps1 and apgl1 agps1 mutants compared with the wild-type plants. By contrast, mutation of AGPL1 and/or AGPS1 led to an increase in the content of the major sugar, sucrose, in leaf sheath and root under control and LN conditions. Moreover, the Pi accumulation was enhanced in the mutants under control and LN conditions, but not LP conditions. Notably, the LN-induced suppression of Pi accumulation was compromised attributed to the mutation of AGPL1 and/or AGPS1. Furthermore, the increased Pi accumulation was accompanied by the specific suppression of OsSPX2 and activation of several Pi transporter genes. These results indicate that a balanced level of carbohydrates is vital for maintaining plant P homeostasis.

Correlation between expression and activity of ADP glucose pyrophosphorylase and starch synthase and their role in starch accumulation during grain filling under drought stress in rice

ADP glucose pyrophosphorylase (AGPase, EC 2.7.7.27) and starch synthase (SS, EC 2.4.1.21) are key regulatory enzymes involved in the starch biosynthesis. Comprehensive analysis of transcription levels of ADP-glucose pyrophosphorylase and starch synthase genes was performed in leaves, roots, and developing grains of drought susceptible (IR64) and drought-tolerant (N22) cultivars under applied water deficit stress (WDS). AGPase and SS genes are differentially regulated in leaves, roots, and grains under the drought stress. The expression pattern of SS and AGPase genes was correlated with the activity of both AGPase, SS, and starch content of developing grains under the drought. Drought stress reduced transitory starch in leaves and enhanced storage starch in developing grains. An increase in the activity of AGPase in developing grains was due to induced expression of ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in N22 and both ADP glucose pyrophosphorylase small subunit 2 (AGPS2) & ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in IR64 and a positive correlation was established with starch content. Similarly, an increase in the SS activity in developing grains was due to induced expression of soluble starch synthase (SSIIB, SSIVA, and SSIVB) in N22 and SSIVB in IR64.

The NAC transcription factor NAC019-A1 is a negative regulator of starch synthesis in wheat developing endosperm.

Starch is a major component of wheat (Triticum aestivum L.) endosperm and is an important part of the human diet. The functions of many starch synthesis genes have been elucidated. However, little is known about their regulatory mechanisms in wheat. Here, we identified a novel NAC transcription factor, TaNAC019-A1 (TraesCS3A02G077900), that negatively regulates starch synthesis in wheat and rice (Oryza sativa L.) endosperms. TaNAC019-A1 was highly expressed in the endosperm of developing grains and encoded a nucleus-localized transcriptional repressor. Overexpression of TaNAC019-A1 in rice and wheat led to significantly reduced starch content, kernel weight, and kernel width. The TaNAC019-A1-overexpression wheat lines had smaller A-type starch granules and fewer B-type starch granules than wild-type. Moreover, TaNAC019-A1 could directly bind to the 'ACGCAG' motif in the promoter regions of ADP-glucose pyrophosphorylase small subunit 1 (TaAGPS1-A1, TraesCS7A02G287400) and TaAGPS1-B1 (TraesCS7B02G183300) and repress their expression, thereby inhibiting starch synthesis in wheat endosperm. One haplotype of TaNAC019-B1 (TaNAC019-B1-Hap2, TraesCS3B02G092800) was positively associated with thousand-kernel weight and underwent positive selection during the Chinese wheat breeding process. Our data demonstrate that TaNAC019-A1 is a negative regulator of starch synthesis in wheat endosperm and provide novel insight into wheat yield improvement.

A New SNP in Rice Gene Encoding Pyruvate Phosphate Dikinase (PPDK) Associated with Floury Endosperm.

Rice varieties with suitable flour-making qualities are required to promote the rice processed-food industry and to boost rice consumption. A rice mutation, Namil(SA)-flo1, produces grains with floury endosperm. Overall, grains with low grain hardness, low starch damage, and fine particle size are more suitable for use in flour processing grains with waxy, dull endosperm with normal grain hardness and a high amylose content. In this study, fine mapping found a C to T single nucleotide polymorphism (SNP) in exon 2 of the gene encoding cytosolic pyruvate phosphate dikinase (cyOsPPDK). The SNP resulted in a change of serine to phenylalanine acid at amino acid position 101. The gene was named FLOURY ENDOSPERM 4-5 (FLO4-5). Co-segregation analysis with the developed cleaved amplified polymorphic sequence (CAPS) markers revealed co-segregation between the floury phenotype and the flo4-5. This CAPS marker could be applied directly for marker-assisted selection. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the flo4-5 mutant than in the wild type during the grain filling stage. Plastid ADP-glucose pyrophosphorylase small subunit (AGPS2a and AGPS2b) and soluble starch synthase (SSIIb and SSIIc) also exhibited enhanced expression in the flo4-5 mutant.

On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

Cloning and expression analysis of cDNAs encoding ADP-glucose pyrophosphorylase large and small subunits from hulless barley (Hordeum vulgare L. var. nudum).

As an important plateau cereal crop, hulless barley is the principal food for the Tibetan people in China. ADP-glucose pyrophosphorylase (AGPase) is considered as the key enzyme for starch biosynthesis in plants. In this study, cDNAs encoding the small subunit (SSU I) and large subunit (LSU I) of AGPase were isolated from hulless barley. The results showed that SSU I and LSU I were 1438 and 1786 bp in length with a complete open reading frame (ORF) of 1419 and 1572 bp. The ORF-encoded polypeptides of 472 and 523 amino acids were having calculated molecular masses of 52.01 and 58.23 kDa, and the pI values were 5.59 and 6.30. In addition, phylogenetic analysis showed that SSU I and LSU I had the same phylogenetic trends with some species. Furthermore, expression levels in different growth periods and tissues of two hulless barley varieties were analyzed by quantitative reverse transcription-polymerase chain reaction. Gene expression levels of SSU I and LSU I were consistent with the total starch accumulation rate in endosperm. In conclusion, our data confirmed that SSU I and LSU I played an important role in hulless barley starch synthesis.

Cloning and characterization of Agp1, the gene encoding the small subunit of ADP-glucose pyrophosphorylase from wheat and its relatives

AbstractADP-glucose pyrophosphorylase (AGP) consists of two large (AGP-L) and two small (AGP-S) subunits; it regulates the limiting step in the biosynthesis of starch. Here, we isolated the full-length cDNA sequence of the AGP-S gene (designed as

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.

Sugar Potentiation of Fatty Acid and Triacylglycerol Accumulation.

Photosynthetically derived sugar provides carbon skeletons for lipid biosynthesis. We used mutants of Arabidopsis (Arabidopsis thaliana) and the expression of oleogenic factors to investigate relationships among sugar availability, lipid synthesis, and the accumulation of triacylglycerol (TAG) in leaf tissue. The adg1 mutation disables the small subunit of ADP-glucose pyrophosphorylase, the first step in starch synthesis, and the suc2 mutation disables a sucrose/proton symporter that facilitates sucrose loading from leaves into phloem. The adg1suc2 double mutant increases glucose plus sucrose content in leaves 80-fold relative to the wild type, total fatty acid (FA) content 1.8-fold to 8.3% dry weight, and TAG more than 10-fold to 1.2% dry weight. The WRINKLED1 transcription factor also accumulates to higher levels in these leaves, and the rate of FA synthesis increases by 58%. Adding tt4, which disables chalcone synthase, had little effect, but adding the tgd1 mutation, which disables an importer of lipids into plastids to create adg1suc2tt4tgd1, increased total leaf FA to 13.5% dry weight and TAG to 3.8% dry weight, demonstrating a synergistic effect upon combining these mutations. Combining adg1suc2 with the sdp1 mutation, deficient in the predominant TAG lipase, had little effect on total FA content but increased the TAG accumulation by 66% to 2% dry weight. Expression of the WRINKLED1 transcription factor, along with DIACYLGLYCEROL ACYLTRANSFERASE1 and the OLEOSIN1 oil body-associated protein, in the adg1suc2 mutant doubled leaf FA content and increased TAG content to 2.3% dry weight, a level 4.6-fold higher than that resulting from expression of the same factors in the wild type.

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.

Constitutive expression of StAATP, a potato plastidic ATP/ADP transporter gene, increases starch content in transgenic Arabidopsis

ABSTRACTThe plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in an increase in the ATP supply to facilitate the anabolic synthesis in heterotrophic plastids of dicotyledonous plants. In this study, a gene encoding the AATP protein, StAATP, was successfully isolated from potato and transformed into Arabidopsis. Constitutive expression of StAATP significantly increased the starch accumulation in the transgenic plants. Real-time quantitative polymerase chain reaction analysis showed that constitutive expression of StAATP up-regulated the expression of key genes involved in the starch biosynthesis pathway in the transgenic Arabidopsis plants: 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. Furthermore, the major enzymes involved in starch biosynthesis (AGPase, GBSS, SSS and SBE) exhibited higher catalytic activities in the transgenic plants compared to the wild type. These results indicate that StAATP may improve the 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 findings suggest that the StAATP gene may be applied for increasing starch accumulation of plants in the future.

GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development.

Rice grain filling determines grain weight, final yield and grain quality. Here, a rice defective grain filling mutant, gif2, was identified. Grains of gif2 showed a slower filling rate and a significant lower final grain weight and yield compared to wild-type. The starch content in gif2 was noticeably decreased and its physicochemical properties were also altered. Moreover, gif2 endosperm cells showed obvious defects in compound granule formation. Positional cloning identified GIF2 to encode an ADP-glucose pyrophosphorylase (AGP) large subunit, AGPL2; consequently, AGP enzyme activity in gif2 endosperms was remarkably decreased. GIF2 is mainly expressed in developing grains and the coded protein localizes in the cytosol. Yeast two hybrid assay showed that GIF2 interacted with AGP small subunits OsAGPS1, OsAGPS2a and OsAGPS2b. Transcript levels for granule-bound starch synthase, starch synthase, starch branching enzyme and starch debranching enzyme were distinctly elevated in gif2 grains. In addition, the level of nucleotide diversity of the GIF2 locus was extremely low in both cultivated and wild rice. All of these results suggest that GIF2 plays important roles in the regulation of grain filling and starch biosynthesis during caryopsis development, and that it has been preserved during selection throughout domestication of modern rice.

Constitutive expression of SlTrxF increases starch content in transgenic Arabidopsis

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (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 SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.