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