Genetic control of wheat starch biosynthesis

S. Rahman*, Z. Li, A. Regina, B. Kosar-Hashemi, S. McMaugh., C. Konik-Rose and M. Morell

CSIRO Plant Industry, GPO Box 1600, ACT 2601, Australia

* Corresponding author: Sedequr Rahman
CSIRO Plant Industry, GPO Box 1600, ACT 2601, Australia
E-mail address: Sadequr.Rahman@csiro.au

Key words: starch biosynthesis, starch synthase, starch branching enzyme, isoamylase, pullulanase

Introduction

Starch makes up over 70% of the wheat grain. Starch is composed of polymers of glucose where the predominant link is alpha 1,4. Such chains are linear but side branches can be introduced by joining chains through alpha 1,6 links. Starch consists of two main types of polymers- amylose and amylopectin. Amylose is essentially linear and has few branches; the molecular mass is typically about 10⁴-10⁵. Amylopectin is the more highly branched form of the polymer and it has a molecular mass of about 10⁵.10⁶. In amylopectin these branches are clustered and this gives rise to a crystalline polymer that, along with amylose, is deposited in granules. The size and shape of starch granules differ from species to species (Singh et al. 2003).

Starch can be divided into two types, based on the mode of synthesis and utilization. Transitory starch is synthesized in leaves during the day and broken down at night. Reserve starch is laid down in storage organs for utilization later by the plant; grain starch is of this type. Starch biosynthesis in the grain endosperm takes place in the amyloplast-a double membraned organelle related to the chloroplast. The amyloplast, however, is not capable of photosynthesis and does not contain chlorophyll. In wheat and rice, multiple starch granules can be formed per amyloplast (Bechtel and Wilson 2003; Kawagoe et al. 2005). Studies to identify all the polypeptides present in wheat amyloplasts are well underway (Andon et al. 2002).

In wheat endosperm, the ratio of amylose to amylopectin is typically 1:3, although wheat starches that are composed only of amylopectin are known. Wheat granules are found in two classes: A granules which are lenticular and have a modal diameter of 2Oum and B granules which are spherical and have a modal diameter of about 3um. Typically wheat has low peak and final viscosities compared to potato starch and a gelatinsation temperature of about 65°C. Cereal starches have low levels of phosphorylation compared to tuber starches and this has been related to their lower viscosity (Singh et al. 2003)

Alteration of the starch quality can add value to the wheat crop. Parameters that are important in the physiological utility of starch include the Glycemic Index, which is a measure of how easily the starch is digested and the proportion of resistant starch, the starch that is not digested until it reaches the large intestine. Consumer preference of foods can be strongly influenced by the properties of the starch (Suwansri and Meullenet 2004). The polyploidy of wheat allows the production of mutants for starch biosynthetic genes in some or all of the genomes and this allows control of the severity of the effect. In this paper the current knowledge of the genetic basis of wheat starch biosynthesis will be briefly surveyed and potential for alteration of wheat starch functionality through genetics will be discussed.

Genes involved in starch biosynthesis in the wheat grain

Three principal approaches have been. taken in the study of proteins involved in starch biosynthesis in wheat. Firstly, enzymes known to be involved in starch biosynthesis have been purified on the basis of their activity (Morell et al. 1997). Secondly, proteins found associated with the starch granule have been analysed and identified (Denyer et al. 1995; Rahman et al. 1995; Li et al. 1999a, b). Thirdly, heterologous probes for known starch biosynthetic enzymes have been used to isolate the cognate wheat sequences (Regina et al. 2005). These approaches have led to the following picture of starch biosynthesis in wheat shown in Fig 1. The genes involved will now be described in more detail (see also Table 1).

The priming reaction

ADP-glucose pyrophosphorylase uses ATP and glucose-1-phosphate to produce ADP-glucose. This is the first committed step in starch biosynthesis. In the leaves of plants studied so far, ADP-glucose pyrophosphorylase is found in the amyloplast. However, in cereal grains, the evidence is that the enzyme is found in both the cytosol and the amyloplast, with the main activity in the cytoplasm (Burton et al. 2002; Thorbjornsen 1996). This indicates that most of the ADP-glucose is imported into the cell, probably through the ADP-glucose transporter.

ADP-glucose pyrophosphorylase is a tetrameric enzyme, being composed of two large and two small subunits. The large and small subunits are similar in terms of molecular mass, being composed of 522 and 473 amino acids respectively (Ainsworth et al. 1993b, 1995). The identity between the subunits is approximately 60% at the nucleotide level; The genes for the large and small subunits from the endosperm are located on chromosomes of the homoeologous groups 1 and 7 respectively. The plastidic and cytoplasmic isoforms for the large subunit appear to be produced by alternate processing from the same gene; it is not known if the situation is similar for the small. The sequence identity between homeologous isoforms encoded by the A, B and D genomes is of the order of 95%. In rice there are 15 exons in the large subunit and 9 in the small. Multiple putative gene sequences have been identified in rice from scrutiny of the rice genome (http://www.tigr.org). It remains to be seen if they are all expressed and if so, in what tissues.

In leaves ADP-glucose pyrophosphorylase is the rate limiting step in starch biosynthesis and the starch content has been increased in potato tubers through the alteration of the properties of this enzyme (Stark et al. 1992). However, it is not clear if this enzyme is the rate limiting enzyme in starch biosynthesis in cereals, although in wheat increased grain size has been associated with altered ADP-glucose pyrophosphorylase (Smidansky et al. 2002).

Loss of the activity of this enzyme in the endosperm does not appear to alter starch quality, leading instead to a loss of the total starch synthesized. In maize such mutants are known as the shrunken-1 and brittle-2 mutants, due to lack of the large and small subunits respectively. As the cytoplasmic and plastidic isoforms appear to be generated by the processing of a single gene, the mutants of the enzyme are presumably lacking both isoforms. It may be of interest to produce and investigate the effect of knocking out either the plastidic or cytoplasmic isoforms; there could conceivably be effects on the quality of the starch. However, this has not yet been investigated. Mutants in this enzyme have not been described in wheat.

Starch synthases

Starch synthase activity is required to add residues to the non-reducing end of a pre-existing glucan chain. A number of starch synthases are known in wheat and described below.

GBSS: Granule bound starch synthase (GBSS) is responsible for the elongation of glucose chains by adding on a glucose unit at the non-reducing end. It is also known as the waxy protein. GBSS is a single chain polypeptide of about 60 kDa. Grain and leaf specific isoforms show approximately 60% nucleotide identity and are known as GBSS I and II respectively (Ainsworth et al. 1993 a; Vrinten and Nakamura 2000).

Loss of GBSS I in the endosperm leads to the well-known waxy phenotype, where amylose is missing from the starch (e.g. Nakamura et al. 1995). In addition branching of the amylopectin fraction is slightly affected through the reduction of the fraction of short chains (DP6-16) (Fujita et al. 2001) consonant with results observed in Chlamydomonas (Delrue et al. 1992).

Genome specific isoforms for GBSS I have been isolated and these show 95% nucleotide identity. The gene consists of 11 exons but is relatively small, spanning approximately 3 kb from the first to the last exon (Yan et al. 2000). The genes for GBSS are located on the tip of the short arm of chromosomes 7A and 7D and on the long arm of chromosome 4A; this position arises because of the ancient translocation of the distal end of the short arm of 7B. The genes for GBSS stand apart from other starch biosynthetic genes in wheat because of their relatively small size; this is due to the much smaller average intron length.

The GBSS polypeptide is the most prominent when proteins attached to the starch granule from wheat are examined by SDS-PAGE. The genome specific isoforms can be separated by electrophoresis under modified conditions (Nakamura et al. 1995; Zhao et al. 1998) and this led to the realization that Australian wheats that were particularly suited to the preparation of the Japanese Udon noodles were missing the isoform encoded by chromosome 4A. Analysis of chromosome engineered lines led to the deduction that in Chinese Spring wheat loss of the 7A, 4A and 7D isoforms led to reductions of 2%, 3% and 2% of amylose respectively (Miura and Sugawara 1996); however, it is possible that there are allele-specific differences between cultivars. It would be of interest to investigate the suitability of nulli-7A and nulli-7D isoforms for the production of Udon noodles.

SS I: Starch synthase I is found in wheat as a 70 kDa polypeptide, both associated with the starch granule in the mature wheat grain and in the stroma. The gene has been characterized from Aegilops tauschii, the D genome donor to wheat, and is located on the short arm of group 7 chromosomes, proximal to the GBSS genes. The gene covers 10kb and consists of 15 exons. The sequence identity between the homoeologous isoforms is of the order of 95% (Li et al. 1999a). The effect of the lack of this enzyme on starch structure in wheat is not known although work in vitro using maize SS I suggests that SS I elongates small chains less than 10 DP (Commuri and Keeling 2001). Only one gene for SS I has been reported in rice.

SS II: In the mature wheat grain SS II is found as a set of polypeptides of 100/105 kDa associated with the starch granule (Li et al. 1999b). The polypeptides run anomalously on SDS-PAGE electrophoresis as the deduced mass is of the order of 87 kDa. The polypeptides are predominantly in the soluble phase during early grain development (Li et al. 1999b). There are two closely related polypeptides, SS IIa and SS llb. SS lIb is not expressed in the endosperm.

The gene for SS IIa is located on the short arm of group 7 chromosomes, proximal to the SS I gene. The gene consists of 8 exons and extends over 9 kb (Li et al. 2003).

Yamamori et al. (2000) have produced mutant lines of wheat that entirely lack SS II This was achieved by separating genome-specific isoforms by SDS-PAGE under modified conditions and then identifying single nulls in each of the isoforms. These lines were then crossed and combined to produce the triple null line, missing all three isoforms.

The starch from lines of wheat missing SS II is highly modified. The apparent amylose of these lines is elevated to 35% and the gelatinization temperature is reduced by 10°C. The starches also display reduced swelling. There is an alteration in the branching of the amylopectin and there is a reduction in the proportion of branches of size 5-9. Examination of the starch granule by microscopy reveals a distorted morphology and it has been observed that there is also a reduction of starch granule associated proteins (Yamamori et al. 2000). It maybe debated therefore that the effects observed are not a direct effect of the lack of SS II but due to associated effects caused by the absence of other starch biosynthetic proteins from the granule. An analogous mutation in barley (Morell et al.2003) produces a more extreme phenotype and produces increased resistant starch which in turn is associated with improved bowel health.

SS III: In wheat genes for SS III are located on chromosome arm 1AS in wheat, unlike other genes for starch synthases which are located on the group 7 chromosomes. The gene consists of 16 exons with a deduced molecular mass of 184 kDa-a polypeptide of this molecular mass can be detected using specific antibodies on the soluble fraction in endosperm (Li et al. 2000). Like the other starch biosynthetic genes in wheat, the enzyme exists in genome specific isoforms but the identity between isoforms has not been investigated. In maize the loss of SS III leads to starch with a reduction of the average chain length of the amylopectin fraction and an increased viscosity (Wang et al. 1993) but the effect on wheat starch is not known.

Rice SS III exists in two isoforms, one of which is preferentially expressed in leaves and the other in the endosperm (Hirose and Terao 2004; Dian et al. 2005); only the gene expressed in the endosperm has been reported in wheat (Li et al. 2000). The effect of single and double nulls on starch properties are also not known in wheat. The gene is expressed early in endosperm development.

SS IV: SS IV has been described from rice but not so far from wheat. In rice it exists in two isoforms and these differ in the degree of expression in the endosperm (Hirose and Terao 2004; Dian et al. 2005). So far from wheat only one sequence has been submitted to the database (accession AY044844. 1 ) and this is expressed predominantly in the leaf. The deduced molecular mass for this enzyme is 103 kDa. No mutant phenotype has been associated in cereals with the loss of this enzyme and it is possible that the change in phenotype is slight.

Branching enzymes

Branching activity is required during starch biosynthesis to add alpha 1,4 linked glucose chains to the 6 position of a glucose molecule that is already linked alpha 1,4 in another chain. Branching activity can be separated on ion-exchange column into three activities- branching activity I and branching activities IIa and llb. IIa and IIb elute close to each other. The enzymes associated with these branching activities have been purified and the genes for these have been characterized in wheat as in maize, barley and rice.

SBE I: Starch branching enzyme I is an enzyme searching for a role in the grain. Loss of this enzyme in rice leads to minor alterations in branch chain length distribution in rice endosperm but no increase in amylose (Satoh et al. 2003). In wheat, mutants have been produced that essentially lack SBE I but no significant effects on grain starch properties were observed (Regina et al. 2004). A second related form of the enzyme but of much greater molecular mass has been reported from Canadian cultivars (Peng et al. 2000).

The gene for SBE I is located on the long arm of the group 7 chromosomes (Rahman et al. 1997, 1999). The locus is complex as it consists of four SBE I like genes or gene fragments located over a distance of 53 kb (Suzuki et al. 2003).

One of the genes, wSBE I-D2, appears to be a transcribed pseudogene. A transcript comprising of the pseudogene fused to the SBE I gene has also been isolated that corresponds to a large SBE I variant reported so far only from wheat; this would consist of 22 exons (Baga et al. 2000). The protein product of this variant SBE I has been reported to be associated with large starch granules (Peng et al. 2000) but loss of the SBE I gene (Regina et al. 2004) which would also lead to the loss of this transcript was not associated with any alteration in starch granule size distribution. The function of this enzyme is still unresolved.

The identity between genome specific isoforms of SBE I is of the order of 95%.

SBE II: All the cereals appear to have two isoforms of starch branching enzyme II (SBE II)- IIa and lIb . In maize, rice and barley endosperm, SBE lIb appears to be the predominant isoform. In maize the relative amounts of SBE llb versus SBE IIa is estimated to be about 50: 1 (Gao et al. 1997). However, in contrast to this SBE IIa represents the predominant starch branching II activity in the wheat endosperm. This is because of the large excess of SBE IIa over SBE llb in the amyloplast stroma. In the polypeptides bound to the granule however, SBE IIb is more abundant than SBE lla by a ratio of about 3:2 (Regina et al. 2005). The reason for this difference is not yet clear.

Both SBE IIa and SBE IIb in wheat have identical molecular masses and cannot be separated easily by denaturing electrophoresis. However, the two isoforms can be separated by ion-exchange chromatography. The two enzymes have 85% amino acid identity over the central third of the sequence and about 70% overall; most of the variation is at the 5' end (Rahman et al. 2001; Regina et al. 2005).

The SBE IIa gene consists of 22 exons spread over about 17 kb. The entire gene sequence for SBE lIb has not yet been obtained but it also consists of 22 exons spread out over at least 17 kb. In wheat both SBE IIa and SBE IIb are located on the long arm of the group 2 chromosomes; this co-location is another point of difference with maize and barley. The genes for rice are located on chromosomes 4 and 2 for IIa and IIb isoforms respectively (www.tigr.org)

Debranching enzymes

A major difference between starch and glycogen (which are both polymers of glucose linked alpha 1,4) is that the branches in starch occur in clusters which are regularly spaced. There is strong evidence from mutants in maize and rice that debranching enzymes are involved in the generation of this regular spacing (James et al. 1995; Kubo et al. 1999).

Two main types of debranching activity have been described in cereals. These are the isoamylase type and the pullulanase type. They differ in their activity towards branched substrates-isoamylase is active against amylopectin but weakly active against pullulan which is a more open structure. Pullulanase is active against pullulan but less so against amylopectin.

Isoamylase : There are three isoamylases so far reported in rice but only one has been characterized in wheat (Rahman et al. 2003). This corresponds to ISA-1, the isoform that is missing in sugary-1 mutants of maize and rice. The gene consists of 18 exons and encodes a putative protein of 89 kDa. The gene for ISA-1 from wheat is on the group 7 chromosome short arm, proximal to the SS II gene. The identity between genome specific isoforms is about 95%.

The loss of ISA-1 enzyme in maize and rice is associated with the production of phytoglycogen, a highly branched glycogen like polymer (James et al. 1995; Kubo et al. 1999). There is also an increase in the amount of free glucose that is found, hence the description of the mutants as sugary. A corresponding mutation has not yet been described in wheat and it is not clear what the effect of partial mutants would be. Introduction of wheat ISA-1 equivalent into rice mutants missing isoamylase restored the ability to produce starch but structure of the starch was subtly different (Kubo et al. 2005). It is not clear if this is due to differences in the amount or nature of the isoamylase activity between rice and wheat.

Two other isoforms of isoamylase, ISA-2 and ISA-3 have been described in rice (Kubo et al. 2005). It is not clear what the effects of loss of ISA-2 and ISA-3 would be in wheat and mutant phenotypes have not been reported from wheat.

Pullulanase: Pullulanase is an enzyme that is also involved in debranching starch and glycogen. The molecular mass is about 100 kDa in rice; the enzyme has not been characterized from wheat. In rice, loss of ISA-1 appears to bring about a concomitant reduction of the amount of this enzyme, suggesting the involvement in a complex. In rice the gene of this enzyme consists of 28 exons and is found on chromosome 4 (Nakamura et al. 1996; Francisco et al. 1998).

Reports in maize suggest that loss of this enzyme alone does not lead to any substantial changes in starch quality, although it appears to act in concert with isoamylase in controlling the accumulation of phytoglycogen (Dinges et al. 2003).

Other enzymes

The use of model systems such as Chlamydomonas and Arabidopsis has led to the demonstration of the involvement of other enzymes in starch biosynthesis. These include the D-enzyme, phosphorylase and glucan-water dikinases. However, the role of these enzymes in starch biosynthesis is still being resolved and no mutants in these enzymes have been reported from cereals.

Conclusions

Wheat has an extremely large genome and most genes are found on all three genomes. The development of full mutants in wheat is traditionally a difficult exercise, involving the combination of mutants in each of the genomes. Mutants in starch biosynthetic enzymes have been obtained in this way in a limited number of cases (Waxy, SS-II, SBE I). The development of techniques such as TILLING has now allowed the relatively facile production of wheat lines missing the functional product from all three genomes and the utility of the method has been demonstrated by the production of waxy wheats (Slade et al. 2005). The ability to transform wheat plants has also improved significantly in the last few years leading to the improvement of ability to produce mutants through this method (Jones 2005). The use of RNAi techniques is especially promising in wheat because multiple copies of nearly identical genes can be knocked out simultaneously. Thus the production of mutants in wheat will in the future be far less problematic than before and we can expect to see wheats with novel starch properties in the near future.

One advantage of wheat because of the presence of three genomes is that partial mutants can be developed, often with advantages in properties not seen in the full mutant. A clear case of this is demonstrated by the use of partial GBSS mutants for Udon noodle biosynthesis; wheat lines missing one isoform of GBSS, encoded at the 4A locus, display the swelling characteristics of starch that are most suited to this product. This ability to obtain partial mutants represents an advantage that is not easily obtained in other cereals.

The starch biosynthetic pathway in cereals involves many enzymes. There have been reports that these enzymes exist in supramolecular complexes. Such complexes have been reported for starch biosynthetic activity in wheat (Tetlow et al. 2004) and suggested for debranching activity in rice (Fujita et al. 1999). Regulation of such complexes may occur through phosphorylation (Sehnke et al. 2001; Tetlow et al. 2004) and this is an exciting area of research.

Wheat starch is the predominant component in the grain. With increasing information about the genes involved in starch biosynthesis in the grain and improvements in screening and gene regulation technologies there is also increasing possibility of altering wheat starch to increase its value. Such increase in value may become from enhanced health benefits through lowering the Glycemic index of the starch or increasing the proportion of resistant starch. Alterations in starch structure could also lead to greater efficiency of traditional processes that involve starch or lead to the opening of new opportunities for its use. Starch is edible, abundant and relatively inexpensive. It is likely that the future will lead to greater use of this versatile biopolymer.

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