Phosphorylation of ADP-Glucose Pyrophosphorylase During Wheat Seeds Development

wheat endosperm

Next, the syringe tip was placed against the upper side of the leaf and pressed down slowly on the plunger, while the upper side of the leaf was supported with a finger. The infiltrations were repeated in the two different midrib regions of three leaves per plant. The infiltrated regions were marked in black for further identification. Primers were designed based on the LpAGP sequences without the regions encoding the plastid transit peptides . The purified PCR products of LpAGPSs and pGBKT7 yeast expression vectors were double-digested with Nco I/BamH I and separated by agarose gel electrophoresis.

Analysis of the expression of the genes by qPCR and semi RT-PCR shows that the three genes display significant differences in spatial and temporal expression patterns. We conclude that regulation of AGPase in the context of starch synthesis is extremely complex. The present findings when integrated with bioengineering approaches will assist in improving the quality of lotus root products. Generally, mRNAs that prematurely terminate translation are regulated by nonsense code to maintain a low abundance.

Data-mining, isolation and characterization of rice AGPase genes

In all cases, the variation in was directly related to the number of kernels that developed and supply of concurrent photosynthate. Collectively, these results suggest that kernel number may be limited by carbohydrate supply, particularly during drought stress at anthesis. According to the invention, enhancing sink strength of the immature ear and grain would make these limited assimilate supplies more accessible, maintain ear and seed growth, and as a consequence buffer this important vulnerable period of yield formation. Differential distribution of photoassimilates within the plant is termed partitioning. Partitioning of assimilated carbon amongst sink organs is a critical factor that controls rate and pattern of plant growth.

Alternatively, substrates may be imported into the plastid to support starch biosynthesis. Limited import of Glc1P into the chloroplast (Fettke et al., 2011) could explain the small amounts of starch observed in pgm1 mutants, but not adg1 mutants. It has also been suggested that ADPGlc could be synthesised by sucrose synthases in the cytosol and imported in the chloroplasts (Muñoz et al., 2005). SuSy is able to produce ADPGlc and fructose from sucrose and ADP in vitro , although its normal function is considered to be the production of UDPGlc and fructose from sucrose and UDP.

Responses of field-grown irrigated rice cultivars to varying levels of floodwater salinity in a semi-arid environment

In particular, the methods of the invention described herein may be applicable to any crop species including but not limited to barley, sorghum, wheat, maize, soybean, and rice. Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. (CRC Press, Inc., Boca Raton, 1993) pages 67–88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available.


Phosphate is concomitantly released by SEX4 and LSF2 to allow complete degradation. Image adapted from Zeeman et al. with permission from Annual Reviews. Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity. Lin, T. P., Caspar, T., Somerville, C. R. & Preiss, J. A starch deficient mutant of Arabidopsis thaliana with low ADPglucose pyrophosphorylase activity lacks one of the two subunits of the enzyme. GTTCTTGAAGCACCAGACAA. The CRISPR/Cas9 and gRNA sequences were cloned into the pHEE401E vector as previouslly described62,63.

Enhanced ADP-glucose pyrophosphorylase activity in wheat endosperm increases seed yield

ISA2 carries substitutions in amino acid residues important for catalysis, probably rendering it non-catalytic. ISA1 is therefore likely to be the catalytic subunit, with ISA2 being required for the enzyme’s stability, and possibly for its specificity and/or regulatory properties . Similarly, in potato, only a heteromultimeric complex of ISA1 and ISA2 was found , although potato ISA1 was active on its own when recombinantly expressed as an S-tagged protein in E.

  • However, like seeds turions provide energy storage for starting the next growing season.
  • Furthermore, the amount of starch synthesised differs depending on the environmental conditions, as shown by growing Arabidopsis in different light/dark regimes (Gibon et al., 2004a; Lu et al., 2005; Smith and Stitt, 2007; Stitt et al.,2007).
  • It is still unclear whether growth rings reflect periodic growth of the granule or simply a structural feature.
  • There are other intriguing proteins candidates which may be involved in protein complex formation.

These Regulation Of The Amount Of Starch In Plant Tissues By Adp Glucose Pyrophosphorylase carry mutations in one of four genes encoding plastidial enzymes linking the Calvin cycle and starch biosynthesis (see Table 1 and Section 4.2, below). The screen for starch-excess mutants differs only in that leaf starch content is visualized at the end of the dark phase, when the wild-type has metabolized almost all of its starch . Mutants which still contain starch at the end of the night are assumed to have either decreased starch breakdown or increased starch synthesis rates.

Starch-Branching Enzyme IIa Is Required for Proper Diurnal Cycling of Starch in Leaves of Maize

For transmission electron microscopy , 90 nm-thin sections were cut on a Leica EM UC6 ultramicrotome, stained with a saturated solution of uranyl acetate and lead citrate and scoped at 80 kV with a Philips CM 12 transmission electron microscope. Kinetic parameters obtained for Pi with the potato tuber ADP-Glc PPase and its mutants. Α-D-[U-14C]Glc-1P was purchased from GE Healthcare . Substrates, effectors and enzymes used for kinetic determinations were purchased from Sigma . Phusion DNA polymerase and restriction enzymes were purchased from New England Biolabs . StrataClone Blunt PCR cloning kit was purchased from Agilent Technologies .

  • The basis for these claims was in turn questioned by studies showing that UDP-glucose is more stable at moderately alkaline pH than claimed by Baroja- Fernández et al.25,52.
  • This latter characterization could be followed by the production of phosphomimetic forms of the TaeADP-Glc PPase, which would allow analyzing how phosphorylation modifies its kinetic, regulatory, and stability properties.
  • In some cases, the impact on starch may be an indirect one whereas in others, the proteins may be part of a hitherto undiscovered aspect of the starch-biosynthetic process.
  • In order to maintain the uniformity of small cell cluster size, suspension-cultured cells were passed through mesh and were subcultured every 7 d.
  • Because starch biosynthesis is an important feature for the developmental switch from fronds to turions, it also provides us with the first entry point to dissect the developmental regulation of turion formation.

Figure 1A shows the homologous residues positioned in the homotetrameric enzyme from Anabaena sp. These residues are located in loops adjacent to the ATP binding site . The Anabaena and potato tuber enzymes were selected to explore whether those residues play similar roles when the allosteric activator is the three carbon metabolite 3-PGA rather than the hexose-bisP. As a first approach, we constructed and characterized mutants of the Anabaena enzyme , specifically the Q58A and W96A mutant proteins. Interestingly, activation by 3-PGA of AnaQ58A and AnaW96A was significantly lower than that observed for the wild type enzyme .

Primers were designed based on the LpAGP sequences without the regions encoding the plastid transit peptide . About 5 mL YEB culture medium supplemented with appropriate selection antibiotics (rifampicin + kanamycin) was inoculated with Agrobacterium strain GV3101 that had previously been transformed with fusion protein binary vector. The culture was left to grow overnight at 28°C and 200 rpm. About six weeks old and healthy Nicotiana tabacum plants were prepared for BiFC analysis. First, the regions of the leaves were prepared for infiltration by puncturing them with a thin needle.

  • Furthermore, GWD1 activity was shown to stimulate the degradation of intact starch granules by recombinant glucan hydrolases (Edner et al., 2007).
  • According to the crystal structure of barley SSI, it was proposed that a disulfide bridge between cysteines 126 and 506 can be formed, blocking the active site .
  • The relatively easy harvesting process compared to algae is to skim of the floating fronds by net or collect them at the outlet of water by a grid .

The relative contribution of each SS class varies in different tissues and between species, which is believed to account at least partially for the structural variation between starches from different sources. In the maize endosperm, SSI and SSIII constitute the major apparent soluble SS activities , whereas in maize leaves no SSI transcript was detected . In contrast, SSII and SSIII are the major apparent soluble SS in the pea seed and potato tuber [130–132], while transcripts of potato SSI were almost exclusively detected in leaves . In Arabidopsis leaves, SSI is the major soluble SS, as judged by remaining SS activity of single ss mutants, followed by SSIII and SSII . Although expressed to a reasonable level, SSIV appears to contribute only little to total SS activity .