AGPase Genes Research Articles

ADP-glucose pyrophosphorylase gene family in soybean and implications in drought stress tolerance.

ADP-glucose pyrophosphorylase (AGPase) is the key rate-limiting enzyme in starch biosynthesis pathway, and has been identified as a potential target for manipulation strategies aimed at improving crop yield and quality. To identify the AGPase gene family members in soybean, and explore the potential implications of GmAGPS2 in drought stress tolerance. Thegenome-wide identification and sequence analysis of soybean AGPase gene family was carried out by bioinformatics methods. The GmAGP gene expression was analyzed using transcriptome data and quantitative real-time PCR (qRT-PCR). Furthermore, transgenic yeast strains overexpressing GmAGPS2 were generated, and their growth was observed under drought stress. In this study, we searched for AGPase genes (GmAGP) in the soybean genome and identified a total of 14 GmAGP genes. The GmAGP proteins had a unique conserved NTP_transferase domain and were mainly located in the chloroplast and cytosol. Evolutionarily, theGmAGP proteins can be clustered into two distinct subgroups; within the same subgroup, they displayed a similar distribution pattern of conserved motifs. The GmAGP genes exhibited an uneven distribution on 10 chromosomes, and segmental duplication contributed to AGPase gene family expansion in soybean. The GmAGP genes presented different tissue expression pattern, in which GmAGPL6, GmAGPL9, and GmAGPL10 mainly exhibited tissue-specific expression pattern. The promoter of GmAGP genes had multiple cis-acting elements related to phytohormones and stress responses, and 8 GmAGP genes contained drought-responsive cis-acting elements. qRT‒PCR analysis demonstrated a significant upregulation expression of GmAGPL6, GmAGPL10, and GmAGPS2 in response to drought stress. Further functional analysis indicated that GmAGPS2 gene could improve yeast growth under drought stress conditions and enhance the drought tolerance of yeast. These results will contribute to further elucidation of the function of GmAGP genes, and offer important candidate genes for the genetic improvement of starch and yield-related traits and the breeding of high drought stress tolerance varieties in soybean.

ADP-glucose pyrophosphorylase genes are differentially regulated in sugar-dependent or -independent manners in tomato (Solanum lycopersicum L.) fruit.

In early developing tomato (Solanum lycopersicum L.) fruit, starch accumulates at high levels and is used by various primary metabolites in ripening fruits. ADP-glucose pyrophosphorylase is responsible for the first key step of starch biosynthesis. Although it has been reported that AgpL1 and AgpS1 isoforms are mainly expressed in early developing fruit, their regulatory mechanism has not been elucidated. The present study investigated the transcriptional response of AgpL1 and AgpS1 to various metabolizable sugars, nonmetabolizable sugar analogues, hexokinase inhibitors and proline by an experimental system using half-cut fruits. AgpL1 was upregulated in response to sucrose and constituted hexoses such glucose, whereas the AgpS1 gene almost did not exhibit a prominent sugar response. Further analyses revealed that other disaccharides such maltose and trehalose did not show a remarkable effect on both AgpL1 and AgpS1 expressions. These results indicate that there are two distinct regulatory mechanisms, namely, sugar metabolism-dependent and -independent, for the regulation of AGPase gene expression. Interestingly, the ADP treatment, a hexokinase inhibitors, cancelled the sugar response of AgpL1, indicating that hexokinase-mediated sugar signaling should be involved in the sugar response of AgpL1. These results suggest that sugar-dependent (AgpL1) and sugar-independent (AgpS1) pathways coordinatively regulate starch biosynthesis in immature tomato fruit.

Exploiting AGPase genes and encoded proteins to prioritize development of optimum engineered strains in microalgae towards sustainable biofuel production.

Although ADP glucose pyrophosphorylase (AGPase), with two large subunits (ls) and two small subunits (ss), is a promising knockout target for increasing the neutral lipid content, the details regarding the sequence-structure features and their distribution within metabolic system in microalgae is rather limited. Against this backdrop, a comprehensive genome-wide comparative analysis on 14 sequenced microalgal genomes was performed. For the first time the heterotetrameric structure of the enzyme and the interaction of the catalytic unit with the substrate was also studied. Novel findings of the present study includes:(i) at the DNA level, the genes controlling the ss are more conserved than those controlling the ls; the variation in both the gene groups is mainly due to exon number, exon length and exon phase distribution; (ii) at protein level, the ss genes are more conserved relative to those for ls;(III) three putative key consensus sequences 'LGGGAGTRLYPLTKNRAKPAV', 'WFQGTADAV' and 'ASMGIYVFRKD' were ubiquitously conserved in all the AGPases; (iv) molecular dynamics investigations revealed thatthe modeled AGPase heterotetrameric structure, from oleaginous algae Chlamydomonas reinharditii, was completely stable in real time environment; (v) The binding interfaces of catalytic unit, ssAGPase, from C. reinharditii with α-D-glucose 1-phosphate(αGP) was also analyzed. The results of the present study have provided system-based insights into the structure-function of the genes and encoded proteins, which provided clues for exploitation of variability in these genes that, could be further utilized to design site-specific mutagenic experiments for engineering of microalgal strains towards sustainable development of biofuel.

Biogas effluent treatment by Landoltia punctata for starch biomass production

Due to the high potential of using Landoltia punctata in biogas effluent treatment and starch accumulation, three integrated processes were proposed: (1) bioethanol production from duckweed, (2) biogas production from vinasse, and (3) biogas effluent treatment with sequentially increasing starch-rich duckweed biomass. This recent work focuses only on the third process, which involves the treatment of wastewater from biogas effluent and the production of L. punctata with high starch content. L. punctata showed a significantly higher TKN removal and biomass production than Lemna aequinoctialis. After nine days of wastewater treatment and five days of starch accumulation, L. punctata grew by 4.3 g/m2/day, with 44.4% starch content. NH3, NH4+, and PO43− in the biogas effluent were rapidly utilized in the first three days for L. punctata’s growth. The upregulation of AGPase genes in the starch biosynthesis pathway was observed when the environmental conditions changed in both nutrient starvation and population reduction. Production of starch-rich L. punctata using biogas effluent is a sustainable feedstock for the bioethanol process. Moreover, L. punctata-based stabilization pond is a promising technique for biogas effluent treatment.

Difference and molecular mechanism of soluble sugar metabolism and quality of different rice panicle in <italic>japonica</italic> rice

<p id="C3">This study investigated the mechanism of starch and rice quality differences on different parts of spikelet in <italic>japonica</italic> rice. According to the internodes of the primary stem on main panicle, rice grains on the panicles were divided into top, middle, and the bottom spikelets. The dynamic changes of soluble carbohydrate and starch, starch biosynthesis related enzyme activity, gene expression on different parts of panicles at different filling stage and the starch content, chalkiness degree after harvest of six varieties were measured. The results showed that the amylose content of the five varieties was in an order as top &gt; middle &gt; bottom except Xiushui 134. The chalkiness degree of Jia 58 and Zhongjia 8 was higher than the other varieties, and the chalkiness degree of rice grains on the middle part of panicles was lower than that on the bottom part, and the rice grains on the top part had the highest chalkiness degree. Rice grains of high chalkiness degree had significant difference in starch granules size and shape. We found that the shape of starch granules was semi-ellipsoid or other spherical curved in rice grains with high chalkiness degree and mostly regular polyhedra in rice grains with low chalkiness degree. The contents of sucrose, glucose, and fructose in grains of different varieties and parts were different during grain filling period, but the dynamic changes of sucrose content were first decreased and then slightly increased, while glucose and fructose were basically increased first and then decreased. The contents of invertase and AGPase were significantly different among rice grains on top, middle, and bottom parts of panicles. However, the relative expression levels of AGPase were in an order as top &gt; middle &gt; bottom during the whole filling stage. The relative expression levels of invertase were in an order as top &lt; middle &lt; bottom during the early filling stage, and the rule was completely the opposite at late filling stage. The expression levels of <italic>OsCIN2 </italic>and<italic> OsCIN5</italic> encoding sucrose invertase genes were in an order as middle &gt; bottom &gt; top, and the expression levels of <italic>OsAGPL1</italic>,<italic> OsAGPL2</italic>, and <italic>OsAGPS1a</italic> encoding AGPase genes were in an order as top &gt; middle ≈ bottom. It concluded that the amylose content and chalkiness degree of grains in different panicles had significant difference and also affected by varieties. The content of soluble carbohydrate, starch biosynthesis related enzyme activity, and the gene expression of encoding related enzymes had an effect on starch biosynthesis. The genes of <italic>OsCIN2, OsCIN5, OsAGPL1</italic>,<italic> OsAGPL2</italic>, and<italic> OsAGPS1a</italic> played an important role in the difference of starch content and chalkiness degree in different panicles.

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.

Comprehensive analysis of AGPase genes uncovers their potential roles in starch biosynthesis in lotus seed

BackgroundStarch in the lotus seed contains a high proportion of amylose, which endows lotus seed a promising property in the development of hypoglycemic and low-glycemic index functional food. Currently, improving starch content is one of the major goals for seed-lotus breeding. ADP-glucose pyrophosphorylase (AGPase) plays an essential role in regulating starch biosynthesis in plants, but little is known about its characterization in lotus.ResultsWe describe the nutritional compositions of lotus seed among 30 varieties with starch as a major component. Comparative transcriptome analysis showed that AGPase genes were differentially expressed in two varieties (CA and JX) with significant different starch content. Seven putative AGPase genes were identified in the lotus genome (Nelumbo nucifera Gaertn.), which could be grouped into two subfamilies. Selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of AGPase genes. Expression analysis revealed that lotus AGPase genes have varying expression patterns, with NnAGPL2a and NnAGPS1a as the most predominantly expressed, especially in seed and rhizome. NnAGPL2a and NnAGPS1a were co-expressed with a number of starch and sucrose metabolism pathway related genes, and their expressions were accompanied by increased AGPase activity and starch content in lotus seed.ConclusionsSeven AGPase genes were characterized in lotus, with NnAGPL2a and NnAGPS1a, as the key genes involved in starch biosynthesis in lotus seed. These results considerably extend our understanding on lotus AGPase genes and provide theoretical basis for breeding new lotus varieties with high-starch content.

Changes in Starch Synthesis and Metabolism Within Developing Bulbs of Lycoris radiata During the Vegetative Growth Stage

Lycoris is a genus of plants with flowering bulbs that have high ornamental, medicinal, and ecological value. The slow growth of Lycoris bulbs under natural conditions is a constraint to the Lycoris industry. Thus, accelerating the development of Lycoris bulbs is extremely important. Starch provides the energy and material basis for bulbs to enlarge. Previous studies have shown reduced starch metabolism capacity in Lycoris bulbs when dormant, but changes within bulbs during the vegetative growth stage remain poorly understood. In our present study, four groups of developing Lycoris radiata bulbs during the vegetative growth phase and with the same genetic background were created and used to investigate changes in carbohydrate content, starch synthesis, metabolic enzyme activity, and the gene expression patterns of several genes encoding these enzymes. Increases in enzyme activity during starch synthesis, especially for AGPase, were observed and correlated with the expression levels of the AGPase genes. Starch levels initially increased and then declined. This may be due to the increasing activity of amylase, which resulted in a rapid increase in sugar concentration during bulb development. We also detected changes in starch synthesis and metabolism from different layers of the bulbs during development, with increases in starch synthesis and starch degradation activity being more significant in the middle scales. The sugar within the middle scales may be transported to the inner scales, which increases the inner scale sugar content, promotes the expression levels of CycD genes, and accelerates cell division during bulb development.

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.

MePHD1 as a PHD-Finger Protein Negatively Regulates ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava.

ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in the starch synthesis pathway. Its enzyme activity can determine the efficiency of starch biosynthesis. Cassava (Manihot esculenta Crantz) is the main staple crop worldwide and has a high starch content in its storage root. However, the inner regulatory mechanism of AGPase gene family is unclear. MePHD1; a plant homeodomain transcription factor; was isolated through a yeast one-hybrid screening using the promoter of ADP-glucose pyrophosphorylase small subunit1a (MeAGPS1a) as bait, and cassava storage root cDNA library as prey. This factor could bind to the MeAGPS1a promoter in vitro and in vivo, and its predicted binding region ranged from −400 bp to −201 bp, at the translation initiation site. The transcript level of MePHD1 could be induced by five plant hormones, and a temperature of 42 °C. This was down-regulated during the maturation process of the storage root. MePHD1 protein could repress the promoter activity of MeAGPS1a gene by a dual-luciferase assay; which indicated that MePHD1 is a negative regulator for the transcript level of MeAGPS1a gene.

TaGW2-6A allelic variation contributes to grain size possibly by regulating the expression of cytokinins and starch-related genes in wheat.

Functional allelic variants of TaGW2 - 6A produce large grains, possibly via changes in endosperm cells and dry matter by regulating the expression of cytokinins and starch-related genes via the ubiquitin-proteasome system. In wheat, TaGW2-6A coding region allelic variants are closely related to the grain width and weight, but how this region affects grain development has not been fully elucidated; thus, we explored its influence on grain development based mainly on histological and grain filling analyses. We found that the insertion type (NIL31) TaGW2-6A allelic variants exhibited increases in cell numbers and cell size, thereby resulting in a larger (wider) grain size with an accelerated grain milk filling rate, and increases in grain width and weight. We also found that cytokinin (CK) synthesis genes and key starch biosynthesis enzyme AGPase genes were significantly upregulated in the TaGW2-6A allelic variants, while CK degradation genes and starch biosynthesis-negative regulators were downregulated in the TaGW2-6A allelic variants, which was consistent with the changes in cells and grain filling. Thus, we speculate that TaGW2-6A allelic variants are linked with CK signaling, but they also influence the accumulation of starch by regulating the expression of related genes via the ubiquitin-proteasome system to control the grain size and grain weight.

Silencing GhAGPL1 Reduces the Quality and Quantity of Corms and Cormels in Gladiolus

Starch accumulation is important during com development. ADP-glucose pyrophosphorylase (AGPase) is the rate-limiting enzyme in starch synthesis. AGPL is the large subunit of AGPase. Here, we isolated and characterized the large subunit of AGPase gene GhAGPL1 in gladiolus (Gladiolus hybridus). GhAGPL1 was highly expressed in sink organs (cormels and corms). The expression of GhAGPL1 was induced by glucose, sucrose, and mannitol, and it was repressed by abscisic acid (ABA). Overexpression of GhAGPL1 in the arabidopsis (Arabidopsis thaliana) apl1 mutant resulted in complementation of AGPase activity and thus starch synthesis. Silencing GhAGPL1 in gladiolus decreased the transcript level of GhAGPL1 and GhSus, and resulted in the reduction of AGPase activity and starch content in gladiolus corm and cormel. Meanwhile, sucrose content was higher in GhAGPL1-silenced corm. Surprisingly, silencing GhAGPL1 in gladiolus produced smaller corms and fewer number of cormels. Overall, GhAGPL1 contributed to the quality and quantity of gladiolus corms and cormels.

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.

Activity and expression of ADP-glucose pyrophosphorylase during rhizome formation in lotus (Nelumbo nucifera Gaertn.).

BackgroundLotus root is a traditional and popular aquatic vegetable in China. Starch is an important component of the rhizome and directly affects the quality of processed products. ADP -glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme associated with starch biosynthesis in plants. Therefore, in the present study, AGPase activity and NnAGP expression during rhizome development of lotus were analyzed.ResultsAmong 15 cultivars analyzed, the contents of amylose and total starch in the rhizome were highest in ‘Mei Ren Hong’. ‘Su Zhou’ and ‘Zhen Zhu’ showed the lowest amylose, amylopectin and total starch contents. In the rhizome, activity of AGPase was highest at the middle swelling stage of development, and higher activity was observed in the ‘Hou ba’ leaf and terminational leaf at the same stage. Three AGPase genes, comprising two large subunit genes (NnAGPL1 and NnAGPL2) and one small subunit gene (NnAGPS), were isolated and identified. The deduced amino acid sequences showed 40.5 % similarity among the three genes. Full-length genomic DNA sequences of NnAGPL1, NnAGPL2, and NnAGPS were 4841, 11,346 and 4169 bp, respectively. Analysis of the temporal and spatial expression patterns revealed that the transcription levels of NnAGPL1 and NnAGPS were higher in the rhizome, followed by the ‘Hou ba’ leaf, whereas NnAGPL2 was significantly detected in the ‘Hou ba’ leaf and terminational leaf. The initial swelling stage of rhizome development was accompanied by the highest accumulation of mRNAs of NnAGPL1, whereas expression of NnAGPL2 was not detected during rhizome development. The transcript level of NnAGPS was highest at the initial swelling stage compared with the other rhizome developmental stages. Transcription of NnAGPL1, NnAGPL2, and NnAGPS was induced within 24 h after treatment with exogenous sucrose. The mRNA level of NnAGPL1 and NnAGPS was increased by exogenous ABA, whereas transcription of NnAGPL2 was not affected by ABA.ConclusionsThe three AGPase genes display marked differences in spatial and temporal expression patterns. Regulation of AGPase in relation to starch synthesis in lotus is indicated to be complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-016-0140-z) contains supplementary material, which is available to authorized users.

ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus

Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed that starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus.

Isolation and characterization of cDNAs and genomic DNAs encoding ADP-glucose pyrophosphorylase large and small subunits from sweet potato.

Sweet potato [Ipomoea batatas (L.) Lam.], the world's seventh most important food crop, is also a major industrial raw material for starch and ethanol production. In the plant starch biosynthesis pathway, ADP-glucose pyrophosphorylase (AGPase) catalyzes the first, rate-limiting step and plays a pivotal role in regulating this process. In spite of the importance of sweet potato as a starch source, only a few studies have focused on the molecular aspects of starch biosynthesis in sweet potato and almost no intensive research has been carried out on the AGPase gene family in this species. In this study, cDNAs encoding two small subunits (SSs) and four large subunits (LSs) of AGPase isoforms were cloned from sweet potato and the genomic organizations of the corresponding AGPase genes were elucidated. Expression pattern analysis revealed that the two SSs were constitutively expressed, whereas the four LSs displayed differential expression patterns in various tissues and at different developmental stages. Co-expression of SSs with different LSs in Escherichia coli yielded eight heterotetramers showing different catalytic activities. Interactions between different SSs and LSs were confirmed by a yeast two-hybrid experiment. Our findings provide comprehensive information about AGPase gene sequences, structures, expression profiles, and subunit interactions in sweet potato. The results can serve as a foundation for elucidation of molecular mechanisms of starch synthesis in tuberous roots, and should contribute to future regulation of starch biosynthesis to improve sweet potato starch yield.

Characterization of genes encoding Starch Branching Enzyme I from Triticum monococcum and its diploid wheat relatives

Abstract The Starch Branching Enzyme I (SBEI) gene plays an important role in amylopectin synthesis. Here, we isolated and characterized the full-length cDNA and DNA sequences of SBEI gene from diploid Triticeae species, Triticum monococcum, T. urartu, Aegilopsspeltoides, and Ae. tauschii. Then we predicted its protein structure, analyzed its evolutionary relationship with other species, and explored its expression patterns using real-time quantitative PCR. The SBEI cDNA includes a 2,490-bp open reading frame (ORF) encoding 829 amino acids. The genomic DNA of SBEI is 5,526-bp in length, containes fourteen exons and thirteen introns, and shares a similar structure with its homologous genes from other cereal plants. Sequence similarity ranging from 70.50% to 98.02% in exons and from 15.50% to 83.63% in introns was detected. Results of phylogenetic tree based on the deduced amino acid sequences from T. monococcum and other plants indicated that T. monococcum SBEI is more closely related to T. boeoticum and T. urartu. Expression analysis revealed that T. monococcum SBEI and AGPase genes were highly expressed in the seeds at middle developmental stage. This is the first report on characterization of the SBEI gene in T. monococcum. These results could be used to explore the roles of this enzyme in amylopectin synthesis.

Cloning and characterization of the genes encoding the small and large subunit of the ADP-glucose pyrophosphorylase in lotus (Nelumbo nucifera Gaertn)

In higher plants, ADP-glucose pyrophosphorylase (EC 2.7.7.27, AGPase), a heterotetrameric enzyme comprised of two small and two large subunits, carries out the first committed step of starch synthesis. To characterize the AGPase genes in lotus, we cloned a small subunit and a large subunit of AGPase using the rapid amplification of cDNA ends (RACE) technique, designated NnAGPS1 and NnAGPL1 genes, respectively. The full-length cDNA of NnAGPS1 was 2,168 bp which codes for a protein of 524 amino acids and of NnAGPL1 was 2,304 bp encoding a protein of 528 amino acids. Complete genomic structures of NnAGPS1 (comprising 9 exons and 8 introns) and NnAGPL1 (comprising 14 exons and 13 introns) were distributed over 3.8 and 4.8 kb, separately. Phylogenetic tree divided the AGPase subunits into two classes: small subunit (SSU) and large subunit (LSU). Sequence alignment of these two genes with other plant species displayed a vast dissimilarity in the N-terminal regions. Results of quantitative real-time RT-PCR (qRT-PCR) showed that both genes were expressed in all tissues, including leaf, leafstalk, rhizome and root. The highest expression of NnAGPS1 was observed in leaves and NnAGPL1 seemed to be preferentially expressed in rhizomes. Besides, transcripts of NnAGPS1 decreased significantly while NnAGPL1 kept a steady level during the early to middle rhizome-swelling stages. Our study will provide important molecular information for the genetic modification and regulation of starch content in lotus.

Raman spectroscopy provides a rapid, non-invasive method for quantitation of starch in live, unicellular microalgae.

Conventional methods for quantitation of starch content in cells generally involve starch extraction steps and are usually labor intensive, thus a rapid and non-invasive method will be valuable. Using the starch-producing unicellular microalga Chlamydomonas reinhardtii as a model, we employed a customized Raman spectrometer to capture the Raman spectra of individual single cells under distinct culture conditions and along various growth stages. The results revealed a nearly linear correlation (R(2) = 0.9893) between the signal intensity at 478 cm(-1) and the starch content of the cells. We validated the specific correlation by showing that the starch-associated Raman peaks were eliminated in a mutant strain where the AGPase (ADP-glucose pyrophosphorylase) gene was disrupted and consequentially the biosynthesis of starch blocked. Furthermore, the method was validated in an industrial algal strain of Chlorella pyrenoidosa. This is the first demonstration of starch quantitation in individual live cells. Compared to existing cellular starch quantitation methods, this single-cell Raman spectra-based approach is rapid, label-free, non-invasive, culture-independent, low-cost, and potentially able to simultaneously track multiple metabolites in individual live cells, therefore should enable many new applications.

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.