Starch Engineering

CEREAL STARCH ENGINEERING : 

CEREAL STARCH ENGINEERING Ritu Batra 2010BS44D

Starch: a principal component of grain : 

Starch Cereal Energy Endosperm Starch: a principal component of grain Understanding the way in which starch is made in grass endosperm is of fundamental biological interest and such knowledge may also benefit agriculture by providing the means to manipulate starch .

Contd… : 

Starch comprises -Amylose and Amylopectin . Based on life duration – Transitory and Reserve. Amylose - linear polymer having 1,4 linked a-glucan chain. 11-37% (depending upon species and site of storage). Amylopectin- A highly branched glucan having a-1,6 glucosidic linkage that connect linear chain. has a high degree of structural organization and is composed of repeated crystalline and amorphous structure. Contd…

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Fig- The connections of glucosyl units by a-1,4 and a-1,6 glycoside linkages.

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Fig- Cluster model of amylopectin structure.

Models for amylopectin : 

Models for amylopectin Multiple cluster model:- A chains- which lack other chains, are linked to other chains at their reducing ends B chains -carry one or more chains belonging to a cluster. B1 chains are present within single clusters B2 and B3 chains are long chains interconnecting a number of clusters. Polymodal chain-length distributions model with periodic waves of varying degrees of polymerization (DP). 6-12 (A) , 13-24 (B1) 25-36 (B2) , more than 37 (B3 or more).

Starch Biosynthesis. : 

Starch Biosynthesis.

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Pathways for synthesis of starch from sucrose

ADP glucose pyrophosphorylase : 

catalyzes the first key regulatory step. exists as a heterotetramer- two large and two small subunits. SSUs - enzymatic complex catalytic activity, whereas LSUs- modulate the enzymatic regulatory properties. Positive and Negative regulator- 3-PGA and Pi respectively. ADP glucose pyrophosphorylase

Alternate pathway and its functional significance. : 

In most species, ADPglucose, is made exclusively in the plastids by AGPase. However, in the endosperm of grasses, including the economically important cereals, it is also made in the cytosol via a cytosolic form of AGPase. Cytosolic ADPglucose is imported into plastids for starch synthesis via an ADPglucose/ADP antiporter in the plastid envelope. The genes encoding the two subunits of cytosolic AGPase and the ADPglucose transporter are unique to grasses. Thus, grass endosperm cells have two separate ways of providing ADPglucose for starch synthesis. Alternate pathway and its functional significance.

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The grass-endosperm specific pathway via cytosolic AGPase and the ADPglucose transporter will be referred to as the ‘alternate pathway’. functional significance- partitioning large amounts of carbon into starch when sucrose is plentiful.

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Fig:- Hexose transport from the cytosol into non-cereal or cereal plastids.

2. Starch synthase : 

2. Starch synthase utilizes ADPG to elongate linear chains by the formation of a-1,4 linkages by catalyzing the transfer of the glucosyl unit of ADP-Glc to the nonreducing end of a glucan chain. The various isoforms are- GBSS, SSI, SSII, SSIII and SSIV. Granule-bound starch synthase – Elongation of amylose chain that carry no branches. GBSSI encoded by the Waxy (Wx) locus in cereals, functions specifically to elongate amylose. wx mutants typically lack amylose and have starches comprised solely of amylopectin.

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GBSSII- expressed in tissue that accumulate starch transiently. These are also likely involved in amylopectin synthesis, particularly in the formation of the extralong unit chain (ELC) fraction. Soluble starch synthase 1 Accounting for about 70% of the total SS activity. It preferentially synthesizes short chains (DP 6-15). It prefers the shortest amylopectin chains as substrates.

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Short A and B1chains are extended by SSI up to a critical length. SSI is then tightly bound to longer amylopectin chains, whereupon it becomes entrapped within longer glucans as a relatively inactive protein in the starch granules. Thus, further glucan chain extension for amylopectin synthesis is most likely catalyzed by other SS enzymes. Soluble starch synthase 2 Synthesis of long cluster chains by elongating short A and B1 chains.

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Starch synthase III likely produces the relatively long chains of amylopectin B2, B3, and B4. A loss of SSIIIa function was found to enhance the endogenous SSI activity, thereby facilitating the synthesis of DP 9-15 and DP 22-29 from DP 6-8 and DP 16-20, respectively. Starch synthase IV Little is known about the contributions of SSIV isoforms to glucan chain lengths in cereals. No SSIV null mutant has yet been characterized in cereal plants, and only Arabidopsis ssIV mutants have so far been identified.

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The ssIV Arabidopsis mutants also show a striking reduction in the number of starch granules within their plastids but an increase in starch granule size, indicating that SSIV may be involved in the priming of starch granule formation and the regulation of starch granule numbers. Thus, SSIV may be required for establishing an initial structure which nucleates the crystallization and biosynthesis of a new starch granule.

3. Branching enzyme : 

BEs generate a-1,6 linkages by cleaving internal a-1,4 bonds and transferring the released reducing ends to C6 hydroxyls. BEI - produces longer chains with DP-16 branching lesser branched polyglucan. BEII- generates shorter chains with DP-12 branching highly branched polyglucans. 3. Branching enzyme

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BEI and BEIIa are expressed in the endosperm and several other cereal tissues whereas BEIIb is only expressed in the endosperm and reproductive tissues. BEIIb mutants of maize and rice were historically designated ‘amylose-extender’ (ae) because they have an apparent increase the relative proportion of amylose to amylopectin in the endosperm.

4. Debranching enzyme : 

Isoamylase (ISA) and Pullulanase (PUL)- directly hydrolyze the a-1,6 glucosic linkages of polyglucans. ` Both differ in substrate specificity. ISA- phytoglycogen and amylopectin PUL-pullulan and amylopectin. At least three ISA genes are present in plants, whilst only a single PUL gene has been identified. ISA plays a crucial role in proper amylopectin biosynthesis. ISA is assumed to function- in the editing of excessively branched chains or in removing improper branches of amylopectin formed by branching enzymes in order to maintain the cluster structure of amylopectin during 4. Debranching enzyme

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starch biosynthesis. As a result of this, amylopectin is competent to crystallize within the starch granule matrix. The physiological function of PUL is less well established. Although PUL is believed to function in starch degradation within the germinating grain. It partially compensates for the defect in ISA. suggesting that it may function during starch biosynthesis in cereal endosperm.

5. Plastidial starch phosphorylase : 

Pho1 and Pho2 - catalyze the transfer of glucosyl units from Glc-1-P to the non-reducing end of a-1,4-linked glucan chains. Pho1 and Pho2 localize to the plastid and cytosol, respectively. Pho1 is believed to act at the surface of the granule, where it functions to phosphorolytically modify the structure of starch. 5. Plastidial starch phosphorylase

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Summary of the starch biosynthesis processes in rice endosperm the glucan initiation process

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the starch amplification process.

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Schematic representation of amylopectin cluster synthesis in the rice endosperm.

Starch Engineering : 

Starch Engineering Enhanced starch biosynthesis greatly influences the grain yield of cereals that have starch as their principal storage reserve. The properties of starch granules in cereals also influence the eating and cooking qualities of these grains. The production of industrial polymers such as paints, varnishes, paper coverings, super absorbers and adhesives . Thus, a better understanding of the processes underlying starch biosynthesis is essential for improving grain yield and quality in cereals.

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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.

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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.

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Fig. Comparison of phenotypes of mature T3 seeds

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Experiments were designed to increase demand for assimilate by increasing the capacity for starch synthesis in endosperm. This was accomplished by transforming rice with a modified maize AGPlarge subunit sequence (Sh2r6hs) under control of an endosperm-specific promoter. This altered subunit confers upon AGP decreased sensitivity to allosteric inhibition by inorganic phosphate (Pi) and enhanced heat Stability.

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The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. The r6 mutation is a 6-bp insertion resulting in the addition of tyrosine and serine between amino acid positions 495 and 496 of the AGP large subunit. This renders the AGP less sensitive to Pi inhibition. The second change, hs, is a point mutation causing substitution at amino acid position 333 of tyrosine for histidine. This change results in more stable large subunit–small subunit interactions in the AGP heterotetramer. Increased endosperm AGPactivity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set.

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Fig- Northern blot analysis of 11 transgenic rice lines and control. Fig. 2 Southern blot analysis of five transgenic rice lines and untransformed control.

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Fig - Northern blot developmental stage analysis of mRNA from rice seeds. Fig - Immunoblot developmental stage analysis of SH2R6HS and BT2 proteins from rice seeds.

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AGP activity is from pooled groups of approximately 15 developing T2 generation transgenic line RS10 and untransformed M202 (UT) seeds

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Null mutants of OsSS111a/Flo5 were generated using T-DNA insertion.

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Fig. 2 Morphology of the seed endosperm in the flo5 mutants and their respective wild type controls

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Fig - Scanning electron micrographs of cut endosperms and endosperm starch granules of flo5-1 and flo5-2 and their respective wild type controls

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Fig- X-ray diffraction patterns of endosperm starch granules

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Fig- Chain-length distribution of amylopectin isolated from the endosperm of flo5 mutants and wild type controls.

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Journal of Experimental Botany Advance Access published July 25, 2011

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Most high amylose mutants in rice have been obtained by chemical mutagenesis or by exposure to sublethal doses of radiation (Yano et al., 1985; Kim et al., 2005; Shu et al., 2006). Mutation of SBEIIb in rice (Yano et al., 1985; Asaoka et al., 1986) and in maize (Moore and Creech, 1972; Boyer et al., 1976; Garwood et al., 1976) was accomplished by directly mutating the genome, hence the expression of active SBEIIb in the entire plant is affected. Possible effects on the whole plant from such genomic alterations can be avoided by RNA silencing using seed-specific promoters (Wang et al., 1998; Kawakatsu et al., 2008).

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RNA interference using hairpin RNA (hp-RNA) has been successfully demonstrated in wheat (Regina et al., 2006; Sestili et al., 2010) and in barley (Regina et al., 2010) endosperm. Another method that has been recently developed is RNA silencing by artificial microRNA (amiRNA) (Ossowski et al., 2008). Although this has not yet been successfully demonstrated in cereal endosperm, one study was able to silence the expression of three rice genes systemically in japonica (Nipponbare) and indica (IR64) backgrounds using an amiRNA driven by a ubiquitin promoter (Warthmann et al., 2008).

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Journal of Experimental Botany Advance Access published August 3, 2011

Food Security News : 

South Australia researchers working on designer wheat to reduce colon cancer. Food Security News

Future prospects : 

Future prospects Isozymes characterization. For example, the functional roles of SSIV isoforms. Coordinated actions of starch synthesis-related enzymes and analyses of the functional relationships between components of the starch biosynthetic system in the cereal endosperm. Little is known about starch biosynthesis enzymes controlled by post-translational modifications.