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

Abstract 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.

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

Functional Plant Biology ◽

10.1071/fp06257 ◽

2007 ◽

Vol 34 (6) ◽

pp. 480 ◽

Cited By ~ 37

Author(s):

Sandrine Rösti ◽

Brendan Fahy ◽

Kay Denyer

Keyword(s):

Environmental Conditions ◽

No Reduction ◽

Starch Content ◽

Large Subunit ◽

Starch Synthesis ◽

Small Subunit ◽

Wild Type ◽

Gene Encoding ◽

Subunit Protein ◽

Adp Glucose Pyrophosphorylase

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.

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Molecular Cloning and Spatial Expression of an ApL1 cDNA for the Large Subunit of ADP-Glucose Pyrophosphorylase from Arabidopsis thaliana

Zeitschrift für Naturforschung C ◽

10.1515/znc-1999-5-610 ◽

1999 ◽

Vol 54 (5-6) ◽

pp. 353-358 ◽

Cited By ~ 3

Author(s):

Leszek A. Kleszkowski ◽

Lubomir N. Sokolov ◽

Cheng Luo ◽

Per Villand

Keyword(s):

Arabidopsis Thaliana ◽

Large Subunit ◽

Critical Role ◽

Starch Synthesis ◽

Transit Peptide ◽

Homologous Proteins ◽

Peptide Cleavage ◽

Reading Frame ◽

Calculated Molecular Weight ◽

Adp Glucose Pyrophosphorylase

Abstract A cDNA, A p L 1a , corresponding to a homologue of the large subunit of ADP-glucose pyrophosphorylase (AG Pase), has been isolated/characterised by screening a cDNA library prepared from leaves of Arabidopsis thaliana, followed by rapid amplification of cDNA 3′-ends (3′-RACE). Within the 1685 nucleotide-long sequence (excluding polyA tail), an open reading frame encodes a protein of 522 amino acids (aa), with a calculated molecular weight of 57.7 kDa. The derived aa sequence does not contain any discernible transit peptide cleavage site motif, similarly to two other recently sequenced full-length Arabidopsis homo-logues for AGPase, and shows ca. 58–78 % identity to homologous proteins from other plants/tissues. The corresponding gene was found (rosette and stem leaves, stems, flowers and fruits), consistent with its critical role in starch synthesis in

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Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives

Genome ◽

10.1139/gen-2016-0007 ◽

2016 ◽

Vol 59 (7) ◽

pp. 501-507 ◽

Cited By ~ 6

Author(s):

Xiao-Wei Zhang ◽

Si-Yu Li ◽

Ling-Ling Zhang ◽

Qiang Yang ◽

Qian-Tao Jiang ◽

...

Keyword(s):

Large Subunit ◽

Full Length ◽

Starch Accumulation ◽

Coding Region ◽

D Genome ◽

Reading Frame ◽

B Genome ◽

Genome Donor ◽

Adp Glucose Pyrophosphorylase ◽

Donor Species

ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.

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Suppression of starch synthesis in rice stems splays tiller angle due to gravitropic insensitivity but does not affect yield

Functional Plant Biology ◽

10.1071/fp14159 ◽

2015 ◽

Vol 42 (1) ◽

pp. 31 ◽

Cited By ~ 12

Author(s):

Masaki Okamura ◽

Tatsuro Hirose ◽

Yoichi Hashida ◽

Ryu Ohsugi ◽

Naohiro Aoki

Keyword(s):

Double Mutant ◽

Oryza Sativa L ◽

Starch Content ◽

Large Subunit ◽

Starch Synthesis ◽

Starch Accumulation ◽

Gravitropic Response ◽

Rice Mutant ◽

Matter Production ◽

Tiller Angle

In rice (Oryza sativa L.), tiller angle – defined as the angle between the main culm and its side tillers – is one of the important factors involved in light use efficiency. To clarify the relationship between tiller angle, gravitropism and stem-starch accumulation, we investigated the shoot gravitropic response of a low stem-starch rice mutant which lacks a large subunit of ADP-glucose pyrophosphorylase (AGP), called OsAGPL1 and exhibits relatively spread tiller angle. The insensitive gravitropic response exhibited by the mutant led us to the conclusion that insensitivity of gravitropism caused by stem-starch reduction splayed the tiller angle. Furthermore, since another AGP gene called OsAGPL3 was expressed at considerable levels in graviresponding sites, we generated a double mutant lacking both OsAGPL1 and OsAGPL3. The double mutant exhibited still lower stem-starch content, less sensitive gravitropic response and greater tiller angle spread than the single mutants. This indicated that the expansion of the tiller angle caused by the reduction in starch level was intense according to the extent of the reduction. We found there were no significant differences between the double mutant and wild-type plants in terms of dry matter production. These results provided new insight into the importance of stem-starch accumulation and ideal plant architecture.

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A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare)

Biochemical Journal ◽

10.1042/bj3130149 ◽

1996 ◽

Vol 313 (1) ◽

pp. 149-154 ◽

Cited By ~ 68

Author(s):

Tine THORBJØRNSEN ◽

Per VILLAND ◽

Leszek A. KLECZKOWSKI ◽

Odd-Arne OLSEN

Keyword(s):

Hordeum Vulgare ◽

Genomic Library ◽

Single Gene ◽

Starch Synthesis ◽

Transit Peptide ◽

Small Subunit ◽

Peptide Sequence ◽

Subunit Gene ◽

Starchy Endosperm ◽

Adp Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepsF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5ʹ ends. In blps14, the unique 5ʹ end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5ʹ end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5ʹ ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5ʹ-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing.

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Characterization of the AGPase large subunit isoforms from tomato indicates that the recombinant L3 subunit is active as a monomer

Biochemical Journal ◽

10.1042/bj20091777 ◽

2010 ◽

Vol 428 (2) ◽

pp. 201-212 ◽

Cited By ~ 11

Author(s):

Marina Petreikov ◽

Miriam Eisenstein ◽

Yelena Yeselson ◽

Jack Preiss ◽

Arthur A. Schaffer

Keyword(s):

Single Gene ◽

Large Subunit ◽

Starch Synthesis ◽

Active Role ◽

Size Exclusion ◽

Starch Accumulation ◽

Gene Encoding ◽

Interaction Modelling ◽

L Proteins ◽

Time Specific

The enzyme AGPase [ADP-Glc (glucose) pyrophosphorylase] catalyses a rate-limiting step in starch synthesis in tomato (Solanum lycopersicon) fruit, which undergoes a transient period of starch accumulation. It has been a generally accepted paradigm in starch metabolism that the enzyme naturally functions primarily as a heterotetramer comprised of two large subunits (L) and two small subunits (S). The tomato genome harbours a single gene encoding S and three genes for L proteins, which are expressed in both a tissue- and time-specific manner. In the present study the allosteric contributions of the different L subunits were compared by expressing each one in Escherichia coli, in conjunction with S and individually, and characterizing the resulting enzyme activity. Our results indicate different kinetic characteristics of the tomato L1/S and L3/S heterotetramers. Surprisingly, the recombinant L3 protein was also active when expressed alone and size-exclusion and immunoblotting showed that it functioned as a monomer. Subunit interaction modelling pointed to two amino acids potentially affecting subunit interactions. However, directed mutations did not have an impact on subunit tetramerization. These results indicate a hitherto unknown active role for the L subunit in the synthesis of ADP-Glc.

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22KD Zein Content Coordinates Transcriptional Activity during Starch Synthesis in Maize Endosperm

Agronomy ◽

10.3390/agronomy10050624 ◽

2020 ◽

Vol 10 (5) ◽

pp. 624

Author(s):

Ada Menie Nelly Sandrine ◽

Hailiang Zhao ◽

Yao Qin ◽

Qin Sun ◽

Dianming Gong ◽

...

Keyword(s):

Sweet Corn ◽

Large Subunit ◽

Starch Synthesis ◽

Glycogen Metabolism ◽

Starch Accumulation ◽

Transferase Activity ◽

Maize Endosperm ◽

F2 Population ◽

Expression Of Genes ◽

Corn Inbred

Starch, the main form of stored energy in plants, plays an important role in maize (Zea mays L.) kernel development. The Shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme ADP-glucose pyrophosphorylase (AGPase). The sh2 mutant exhibits impaired AGPase activity, resulting in the partial or complete loss of starch synthesis. Here, we investigated the transcriptional regulatory framework of sh2 through transcriptome and co-expression network analysis using an F2 population derived from the maize reference line B73 and sweet corn inbred line HZ508. We identified 5175 differentially expressed genes (DEGs), including 2878 upregulated and 2297 downregulated genes in sh2 mutant lines. DEGs are associated with various biological processes including nutrient reservoir activity, transferase activity, catalytic activity, water deprivation and glycogen metabolism. At the genetic level, 2465 DEGs, including 357 transcription factors, were involved in transcription. In addition, the maize floury and opaque mutant genes fl1, ndk2, o7 and o2, which regulate the biosynthesis of 22KD zein, were co-expressed with the differential expressed transcription factor genes, thus suggesting that zein content might be a key regulator coordinating the expression of genes determining starch accumulation in maize endosperm.

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Increasing Starch Accumulation via Genetic Modification of the ADP-glucose Pyrophosphorylase

10.32747/2009.7591740.bard ◽

2009 ◽

Author(s):

Arthur Schaffer ◽

Jack Preiss ◽

Marina Petreikov ◽

Ilan Levin

Keyword(s):

Starch Content ◽

Large Subunit ◽

Tomato Fruit ◽

Sugar Content ◽

Soluble Sugar ◽

Starch Synthesis ◽

Regulatory Subunit ◽

Starch Accumulation ◽

Research Project ◽

Immature Fruit

The overall objective of the research project was to utilize biochemical insights together with both classical and molecular genetic strategies to improve tomato starch accumulation. The proposal was based on the observation that the transient starch accumulation in the immature fruit serves as a reservoir for carbohydrate and soluble sugar content in the mature fruit, thereby impacting on fruit quality. The general objectives were to optimize AGPase function and activity in developing fruit in order to increase its transient starch levels. The specific research objectives were to: a) perform directed molecular evolution of the limiting enzyme of starch synthesis, AGPase, focussing on the interaction of its regulatory and catalytic subunits; b) determine the mode of action of the recently identified allelic variant for the regulatory subunit in tomato fruit that leads to increased AGPase activity and hence starch content. During the course of the research project major advances were made in understanding the interaction of the small and large subunits of AGPase, in particular the regulatory roles of the different large subunits, in determining starch synthesis. The research was performed using various experimental systems, including bacteria and Arabidopsis, potato and tomato, allowing for broad and meaningful conclusions to be drawn. A novel discovery was that one of the large subunits of tomato AGPase is functional as a monomer. A dozen publications describing the research were published in leading biochemical and horticultural journals. The research results clearly indicated that increasing AGPase activity temporally in the developing fruit increase the starch reservoir and, subsequently, the fruit sugar content. This was shown by a comparison of the carbohydrate balance in near-isogenic tomato lines differing in a gene encoding for the fruit-specific large subunit (LS1). The research also revealed that the increase in AGPase activity is due to a temporal extension of LS1 gene expression in the developing fruit which in turn stabilizes the limiting heterotetrameric enzyme, leading to sustained starch synthesis. This genetic variation can successfully be utilized in the breeding of high quality tomatoes.

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

Journal of the American Society for Horticultural Science ◽

10.21273/jashs03944-16 ◽

2017 ◽

Vol 142 (2) ◽

pp. 119-125 ◽

Cited By ~ 1

Author(s):

Shanshan Seng ◽

Jian Wu ◽

Jiahui Liang ◽

Fengqin Zhang ◽

Qiuyan Yang ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Abscisic Acid ◽

Starch Content ◽

Large Subunit ◽

Starch Synthesis ◽

Transcript Level ◽

Starch Accumulation ◽

Sucrose Content ◽

Gladiolus Hybridus ◽

Rate Limiting

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.

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