Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

A comprehensive expression analysis of the starch synthase gene family in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the roles of the isogenes encoding starch synthase (EC 2.4.1.21) in rice (Oryza sativa L.), a comprehensive expression analysis of the gene family was conducted. Extensive searches for starch synthase genes were done in the databases of both the whole genome and full-length cDNAs of rice, and ten genes were revealed to comprise the starch synthase gene family. Multi-sequence alignment analysis of the starch synthase proteins from rice and other plant species suggested that they were grouped into five classes, soluble starch synthase I (SSI), SSII, SSIII, SSIV and granule-bound starch synthase (GBSS). In rice, there was one gene for SSI, three for SSII and two each for SSIII, IV and GBSS. The expression pattern of the ten genes in the developing caryopsis was examined by semi-quantitative RT–PCR analysis. Based on the temporal expression patterns, the ten genes could be divided into three groups: (i) early expressers (SSII-2, III-1, GBSSII), which are expressed in the early stage of grain filling; (ii) late expressers (SSII-3, III-2, GBSSI), which are expressed in the mid to later stage of grain filling; and (iii) steady expressers (SSI, II-1, IV-1, IV-2), which are expressed relatively constantly during grain filling. Within a caryopsis, the three gene groups spatially share their expression, i.e. early expressers in the pericarp, the late expressers in the endosperm and the steady expressers in both tissues. In addition, this grouping was reflected in the expression pattern of various rice tissues: expression in non-endosperm, endosperm or all tissues examined. The implications in this spatio-temporal work sharing of starch synthesis isogenes are discussed.Abbreviations DAF Days after flowering - GBSS Granule-bound starch synthase - SS Soluble starch synthase     

Subtle structural differences crucial for function in similarly engineered ADP-glucose pyrophosphorylase larger subunit in rice and maize

ADP ?C glucose pyrophosphorylase (AGPase) is a key enzyme for starch synthesis in plants. Heterotetrameric plant AGPase is encoded by two genes, Shrunken-2 (Sh2) and Brittle-2 (Bt2). The Sh2 gene encodes regulatory larger subunit and the Bt2 gene encodes smaller subunit having catalytic properties. A specific mutation in Sh2 gene involving insertion of six nucleotides, without changing the reading frame, resulted in the insertion of two additional amino acid residues serine and tyrosine at specific position at carboxyl end and also in an increase in seed weight up to 11?C17%. No increase in seed weight with the same insertion in larger subunit of AGPase enzyme in rice was observed even though the rice and maize subunits have 93% of sequence similarity. In this study, the predicted 3D-structures of larger subunit of normal as well as mutated AGPase in maize and rice, were analyzed and superposed. The segment of six amino acid residues long secondary structure just before the site of insertion of additional amino acids (serine and tyrosine) got reduced in case of mutated maize but not in mutated rice. Therefore, the six residue sequence and corresponding subtle secondary structural difference might be the key factors for functional disparity of engineered larger subunits in the two crops.     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

Two Paralogous Genes Encoding Small Subunits of ADP-glucose Pyrophosphorylase in Maize, <Emphasis Type="Italic">Bt2</Emphasis> and <Emphasis Type="Italic">L2</Emphasis>, Replace the Single Alternatively Spliced Gene Found in Other Cereal Species

Two types of gene encoding small subunits (SSU) of ADP-glucose pyrophosphorylase, a starch-biosynthetic enzyme, have been found in cereals and other grasses. One of these genes encodes two SSU proteins. These are targeted to different subcellular compartments and expressed in different organs of the plant: the endosperm cytosol and the leaf plastids. The SSU gene encoding two proteins evolved from an ancestral gene encoding a single protein by the acquisition of an alternative first exon. Prior to the work reported here, this type of SSU gene had been found in all grasses examined except maize. In maize, two separate genes, Bt2 and L2, were known to have the same roles as the alternatively spliced gene found in other grasses. The evolutionary origin of these maize genes and their relationship to the SSU genes in other grasses were unclear. Here we show that Bt2 and L2 are paralogous genes that arose as a result of the tetraploidization of the maize genome. Both genes derive from an ancestral alternatively spliced SSU gene orthologous to that found in other grasses. Following duplication, the Bt2 and L2 genes diverged in function. Each took a different one of the two functions of the ancestral gene. Now Bt2 encodes the endosperm cytosolic SSU but does not contribute significantly to leaf AGPase activity. Similarly, L2 has lost the use of one of its two alternative first exons. It can no longer contribute to the endosperm cytosolic SSU but is probably responsible for the bulk of the leaf AGPase SSU. Reviewing Editor: Dr. Patrick Keeling Sequences have been deposited in GenBank under accession numbers AY727927, DQ118037, and DQ118038.     

Kinetic and regulatory properties of plant ADP-glucose pyrophosphorylase genetically modified by heterologous expression of potato<Emphasis Type="Italic"> upreg</Emphasis> 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.     

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.     

Over‐expression of mutated ZmDA1 or ZmDAR1 gene improves maize kernel yield by enhancing starch synthesis

Grain weight and grain number are important crop yield determinants. DA1 and DAR1 are the ubiquitin receptors that function as the negative regulators of cell proliferation during development in Arabidopsis. An arginine to lysine mutant at amino acid site 358 could lead to the da1‐1 phenotype, which results in an increased organ size and larger seeds. In this study, the mutated ZmDA1 (Zmda1) and mutated ZmDAR1 (Zmdar1) driven by the maize ubiquitin promoter were separately introduced into maize elite inbred line DH4866. The grain yield of the transgenic plants was 15% greater than that of the wild‐type in 3 years of field trials due to improvements in the grain number, weight and starch content. Interestingly, the over‐expression of Zmda1 and Zmdar1 promoted kernel development, resulting in a more developed basal endosperm transfer cell layer (BETL) than WT and enhanced expression of starch synthase genes. This study suggests that the over‐expression of the mutated ZmDA1 or ZmDAR1 genes improves the sugar imports into the sink organ and starch synthesis in maize kernels.     

The 14‐3‐3 protein GF14f negatively affects grain filling of inferior spikelets of rice (Oryza sativa L.)

In rice (Oryza sativa L.), later flowering inferior spikelets (IS), which are located on proximal secondary branches, fill slowly and produce smaller and lighter grains than earlier flowering superior spikelets (SS). Many genes have been reported to be involved in poor grain filling of IS, however the underlying molecular mechanisms remain unclear. The present study determined that GF14f, a member of the 14‐3‐3 protein family, showed temporal and spatial differences in expression patterns between SS and IS. Using GF14f–RNAi plants, we observed that a reduction in GF14f expression in the endosperm resulted in a significant increase in both grain length and weight, which in turn improved grain yield. Furthermore, pull‐down assays indicated that GF14f interacts with enzymes that are involved in sucrose breakdown, starch synthesis, tricarboxylic acid (TCA) cycle and glycolysis. At the same time, an increase in the activity of sucrose synthase (SuSase), adenosine diphosphate‐glucose pyrophosphorylase (AGPase), and starch synthase (StSase) was observed in the GF14f–RNAi grains. Comprehensive analysis of the proteome and metabolite profiling revealed that the abundance of proteins related to the TCA cycle, and glycolysis increased in the GF14f–RNAi grains together with several carbohydrate intermediates. These results suggested that GF14f negatively affected grain development and filling, and the observed higher abundance of the GF14f protein in IS compared with SS may be responsible for poor IS grain filling. The study provides insights into the molecular mechanisms underlying poor grain filling of IS and suggests that GF14f could serve as a potential tool for improving rice grain filling.     

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

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

Understanding the regulation of cereal grain filling: The way forward

During grain filling,starch and other nutrients accumulate in the endosperm;this directly determines grain yield and grain quality in crops such as rice(Oryza sativa),maize(Zea mays),and wheat(Triticum aestivum).Grain filling is a complex trait affected by both intrinsic and environmental factors,making it difficult to explore the underlying genetics,molecular regulation,and the application of these genes for breeding.With the development of powerful genetic and molecular techniques,much has bee...