agpase in starch biosynthesis
TRANSCRIPT
IDENTIFICATION OF THE ADP
GLUCOSE PYROPHOSPHORYLASE
ISOFORMS ESSENTIAL FOR STARCH
SYNTHESIS IN THE LEAF AND SEED
ENDOSPERM OF RICE
Presented by
Sangeeta Sarma
PhD 2ndYear 1st Semester
Dept Of Agril.Biotechnology
Assam Agricultural University
Jorhat
INTRODUCTION TO ADP
GLUCOSE
PYROPHOSPHORYLASE
Key enzyme in starch
biosynthesis
Fig- Structure of a LS and b SS of rice AGPase
Exist as a heterotetramer (α2β2) containing two large (LSU) and two small
(SSU) subunits with slightly different molecular masses
SSU- catalytic activity; LSU- regulatory properties
Undergo post translational redox modification; activated in light
Inactivated at night by formation of interchain disulphide bond between
the SSUs
Gene duplication events followed by divergence
Mutation in specific SSU or LSU genes leads to a severe
depression of starch synthesis
AGP activity is normally located in the plastids except for
cereal endosperm where it is cytoplasmic and plastidic
In rice endosperm, ADP-Glc is largely produced in the
cytosol then transported across the plastid envelope
RICE AGP
GENE
OsAGPS1 OsAGPS2 OsAGPL1 OsAGPL2
OsAGPL3 OsAGPL4
OsAGPS2a OsAGPS2b
Classification of OsAGP isoforms
APPROACHESSub cellular localization of OsAGP proteins using
OsAGP::GFP fusion constructs, and examination of the
transformed plants tissues by laser-scanning confocal
microscopy
Isolation and analysis of T-DNA insertion mutants, including
genetic complementation of the mutants
Western blotting analysis of the osagp mutants and wild type
plants
Determination of starch content in the leaves and
endosperm
Measurement of AGP activity in the leaves and developing
endosperm
RESULTS
Subcellular localization of the OsAGP isoforms
Fig- The subcellular localization of OsAGP::GFP fusion proteins in rice plants and
maize protoplasts
osagpl2 and osagps2 mutants
T-DNA insertion mutants previously isolated were used to
determine the role of the genes during starch synthesis in
developing seeds endosperm
Fig- (A) Location of the T-DNA insertion in the OsAGPS2 genomic region of
the osagps2-1 mutant
(B) Position of insertions in the OsAGPL2 genomic region
Recovery of the normal endosperm in the mutants complemented with
wild type OsAGPS2, therefore normal function of OsAGPS2 is essential
for starch synthesis in rice endosperm
Starch composition in the seed endosperms
(iodine staining)
Seed endosperm
phenotype
Iodine stained
phenotype
Scanning electron micrographs of endosperm
starch granulesWild type osagps2-1
osagpl2-1 osagpl2-2
Fig- Scanning electron micrographs of endosperm starch
granules
Small and round
starch granules in
the mutant
endosperm, due to
reduced starch
synthesis
AGP activity and Starch contents in rice
plants
Fig- AGP activity and starch contents of rice leaves and endosperms
Western blotting analysis of OsAGP
isoforms
Western blot analysis
showed that developing rice
endosperm contains two
AGP SS isoforms,
OsAGPS1 and OsAGPS2b
that differ slightly in
molecular size (respectively
49 and 53 kDa proteins) and
an LS isoform OsAGPL2 (a
57 kDa species)
Western blotting analysis of wild type,
osagps2, and osagpl2 mutants
A model for spatiotemporal complex formation of
OsAGP isoforms in the leaf and endosperm of rice
Fig- A model of spatiotemporal
complex formation between
OsAGP SSU and LSU isoforms
in leaf and in developing
endosperm of rice
DISCUSSION
Two SSU isoforms OsAGPS1 and OsAGPS2b, and an LSU isoform
OsAGPL2, function in starch synthesis in rice seed endosperm
OsAGPL1 and OsAGPS1 in the amyloplasts of developing rice endosperm,
OsAGPL2 and OsAGPS2b in the cytosol of the endosperm
Tissue-preferential transcriptional control mechanisms and spatial
subcellular compartmentation of these OsAGP proteins are important for
appropriate starch synthesis in the leaf and endosperm of rice plants
In the developing endosperm, at an early stage, the amyloplast-targeted
OsAGPS1/OsAGPL1 heterotetramer has the main functional role and the
cytosolic OsAGPS2b/OsAGPL2 complex plays a relatively minor role due
to its low levels
As the endosperm matures, the cytosolic OsAGPS2b/OsAGPL2 complex
confers the dominant enzyme activity in starch synthesis