| | Abstract | Results and discussion | Conclusions and future research | Acknowledgements | References
Abstract
Starch accumulated in the barley endosperm is composed of two types of granules: large A-type lenticular and small B-type oval shaped granules. The distribution of the A- and B-type starch granules in mature barley endosperm is controlled by several loci on the barley genome. To identify the factors controlling starch granule size, we have focused our studies on genes encoding starch biosynthetic enzymes that are associated with the granules. The ultimate goal is to obtain molecular markers that will aid development of malting barley lines with a lower content of B-type granules, as they yield less extract than large A-type granules and cause problems in the mashing process. One of the putative determinants for starch granule size is a 150 kD starch branching enzyme (SBEIc) that is preferentially incorporated into A-type starch granules. A 100 kb genomic region carrying the SBEIc gene in barley has been characterized and found to be genetically linked to genes encoding the 87 kD SBEI and a novel SBEI-like protein. Studies of wheat lines expressing anti-sense Sbe1c construct support a role for SBEIc or related protein in starch granule formation. In addition, we have characterized two genes encoding starch granule bound proteins encoded from a quantitative trait locus (QTL) on barley chromosome 2H. This locus is closely associated with starch granule size distribution in barley.
Results and Discussion
Starch granules and malting quality
The malting quality of barley grain is dependent on a complex set of traits that have been mapped to several regions on the seven barley chromosomes. Among the most important traits are starch degrading enzymes, such as alpha-amylases, beta-amylases, alpha-glucosidases and debranching enzymes, and their activities during malting. Other important factors affecting yield and quality of the malt extract are the properties of the starch granules.
The starch of mature barley endosperm is composed of two types of granules; large lenticular (A-type; >10 m) and small round (B-type; < µ10 m) granules. The large granules are initiated before 15 days-post-anthesis (dpa), whereas B granules are formed after 15 dpa. In mature grain, only 10-15% of the granules are of the A-type, but they account for most of the starch by weight (~90%). Large and small starch granules differ slightly in their amylose content, the overall lipid composition, and the amount of proteins attached to the surface (Morrison 1995; Meredith et al. 1981). During the mashing process in brewing, hydrolytic enzymes are more active on the large granules as compared to small granules (Borem et al. 1999), and therefore, A-type starch granules contribute more to malting quality than B-type granules. Small starch granules may also cause problems during malting as they adhere easily to proteins leading to clogging of filters. Thus, there has been a desire from the malting industries to produce malt from barley lines with a lower content of small starch granules (Pierce 1991).
In an effort to identify genomic regions determining starch granule size in barley, Borem et al. (1999) analyzed a series of doubled haploid (DH) barley lines derived from a cross between Morex (malting) and Steptoe (feed), two cultivars that differ in starch granule size distribution. Marker analysis of the DH lines led to the identification of one region on chromosome 2H that was associated with A-type granule volume and proportion of A-type and B-type granules. The mean maximum diameter of A-type granules correlated with a region on chromosome 5H and the shape of B granules was linked to a region on chromosome 4H. A similar analysis of doubled haploid wheat lines has also identified QTL’s affecting starch granule size (Igrejas et al., 2002). One of these OTL’s is located on chromosome 4DS and corresponds to 4H region identified in barley. The two additional wheat QTL’s were located to chromosomes 7AS and 1BL. The 7AS region carries the Wx-A1 gene encoding granule bound starch synthase 1 (GBSSI), but the region could not directly be linked to the GBSSI gene as wheat lines carrying null GBSSI alleles show normal granule size distribution. Recently, an analysis of the barley Riso 17 and Notch-2 mutants have indicated that isoamylase, which is also encoded by the 7H chromosome, has a role in starch granule formation (Burton et al. 2002). The mutants studied lack isoamylase activity in the endosperm and produce significant amounts of phytoglycogen instead of starch. The A-type granules are smaller than normal and pack together to form compound granules, whereas initiation of B-type granules appears to be impaired leading to uni-modal starch granule size distribution. Thus, isoamylase seems to have some role in starch granule initiation, probably acting in concert with other starch biosynthetic enzymes.
A large starch branching enzyme is preferentially associated with A-type starch granules
Production of the two size fractions of starch granules is only found in plants belonging to the Triticeae tribe, including wheat, barley rye and triticale. Our previous studies on wheat starch granules have revealed a difference in the abundance of SBEIc, an isoform of starch branching enzyme, in small and large granules (Båga et al. 2000; Peng et al. 2000). SBEIc is a large (~150 kD) protein that, in contrast to the 87 kD SBEI, is preferentially incorporated into the large starch granules. SBEIc is only found in wheat, barley and rye starch granules, and thus, is associated with plants showing bimodal starch granule size distribution in the endosperm. Our interest is to determine if SBEIc plays a role in determining the size of starch granules in this group of plants.
DNA sequence analysis of genomic region encoding barley SBEIc
Three bacterial artificial chromosomes (BAC) clones carrying Sbe1c sequences were analyzed by restriction analysis and found to be overlapping. The DNA sequence of the largest clone, carrying a 100-kb insert, has been obtained using a shot-gun cloning strategy combined with robotics sequencing technology. The Sbe1 region carries a complete Sbe1 gene encoding the 87 kD mature SBEI. In addition, two Sbe1-like genes were found (Sbe1-like gene 1 and 2 in Fig. 1). The first eight exons of Sbe1-like gene 2 were found to correspond in sequence to the first half of the barley Sbe1c transcript. The latter part of the Sbe1c mRNA corresponded to exons 3-12 of the Sbe1 gene. Therefore it is possible that Sbe1c is expressed in the barley endosperm as a 16.5 kb long primary transcript that is spliced as outlined in Fig. 1 to produce a 4.6 kb Sbe1c mRNA. Alternatively, the Sbe1c mRNA may be formed by a trans-splicing event involving the Sbe1-like gene 2 and the Sbe1 primary transcripts.
Figure 1. Schematic organization of Sbe1 loci in rice and barley genomes.
Expression of novel Sbe1-like gene in developing barley endosperm
Comparison of the gene organization in the barley Sbe1 region to that of rice revealed striking similarities between putative genes 1, 2 and 5 (Fig. 1) surrounding the Sbe1 locus. However, rice, which produces compound small granules, does not seem to carry a gene similar to the barley Sbe1-like gene 1. Expression analysis of the barley Sbe1-like gene 1 by RT-PCR revealed that the gene is active throughout endosperm development (Fig. 2) and putatively encodes an isoform of SBEI-like proteins not previously been reported. Further studies are needed to determine if this novel protein has a role in starch biosynthesis.
Figure 2. Expression analysis of Sbe1-like gene 1 during endosperm development in the barley cultivar Steptoe. Primers used in the analysis spans exons 6 and 7 and amplified product corresponds to expected 1.0 kb fragment.
Studies on wheat lines expressing anti-sense Sbe1c support a role for SBEIc in determination of starch granule size distribution
Wheat lines expressing Sbe1c in the anti-sense orientation have been produced to determine the role of SBEIc in starch biosynthesis. Initial studies of a dozen lines showed an increased percentage of large starch granules in the anti-sense SBEIc plants, which suggested that SBEIc or closely related protein is involved in determining starch granule size. The profiles of amylolytic activities produced in the endosperm of the anti-sense Sbe1c lines were found to differ from those of the parent line during initiation of small granules (18 DPA), but not during initiation of large granules (10 DPA). Several enzymes contributed to the total increase in amylolytic activities in the anti-sense lines and identification of these up-regulated activities is underway. To eliminate genetic variation within the seed population of the antisense-Sbe1c lines, we have used these to produce doubled haploid lines, which will be used in future studies of SBEIc expression.
Sbe1c maps to chromosome 5H in barley genome
Results from Southern blot analysis of barley DNA suggests that all SBEI genes are present at one locus in genome. Comparison of genes surrounding the Sbe1 loci in rice and barley (Fig. 1) supports that the barley Sbe1 locus is located at the end of the long arm of chromosome 7H. This location also corresponds to Sbe1 gene cluster on chromosome 7D in wheat (Rahman et al. 2000). The Sbe1 locus does not correspond to any QTL identified so far for starch granule size in barley or wheat, but this does not necessary rule out a role for this region determining starch granule size. For example, it is possible that parents of mapped populations do not differ at the Sbe1 locus or that Sbe1 regulatory factors are encoded elsewhere in the barley genome.
Biochemical and molecular characterization of two genes associated with QTL for starch granule distribution
Close examination of genes located at QTL for starch granule size distribution on the barley chromosome 2H revealed two genes (gene A and B) likely to have a role in starch biosynthesis. Published studies of protein A isoforms expressed in leaves have shown that the enzyme is involved in carbohydrate metabolism and exists in different glycosylated forms. Several cDNA clones encoding gene products A and B in barley endosperm have been isolated and characterized by DNA sequencing. Both gene A and B proteins have been expressed as His-tagged proteins in E. coli, purified and used for production of antibodies in rabbits. Immunoblot analyses using antisera raised against proteins A and B revealed high antibody titers against respective protein. However, no enzymatic activity could be detected from protein A when expressed in E. coli, which may be due to lack of protein glycosylation in the bacterial system.
Analysis of gene A and gene B activities in barley
Most of the enzymes involved in starch biosynthesis can be extracted from the starch granules or from the protein network attached to the granules. To determine the localization of gene product A and B during endosperm development, we have isolated and analyzed proteins attached to or trapped inside starch granules of mature barley kernels. This analysis showed that gene A and gene B products are both present on starch granules covered with protein network (crude starch granule preparation; lanes 4, Fig. 3). Both gene products can be released from the network/granules by SDS washes (lanes 1, Fig. 3). The close association of gene product A and B to the surface of starch granules further support that gene A and B have a role in starch granule biogenesis.
Figure 3. Immunoblot analysis of protein A and B present in crude starch granule preparations of barley cultivars Steptoe and Morex. Proteins released from crude granules by washes containing SDS or lacking SDS are shown in lanes 1 and 2, respectively. Proteins remained attached to starch granules after +SDS wash and -SDS wash are shown in lanes 3 and 4, respectively. The antibodies raised against protein A and B show some cross-reaction with major proteins found in starch granules.
Conclusions and Future Research
In conclusion, we have characterized the barley locus encoding SBE1c, which is preferentially accumulated in A-type starch granules. Preliminary studies on wheat lines expressing anti-sense Sbe1c contruct suggests that lower level of SBEIc or related protein reduces the amount of small granules formed in the endosperm. In addition, we have associated two other genes with a QTL for starch granule size in barley endosperm. These gene products are involved in starch biosynthesis and encode proteins bound to granules. We have raised antibodies against SBEIc/SBEI, protein A and B and they will be used to study the interaction between these proteins in mutant wheat and barley lines with altered starch granule size distribution. These data are expected to reveal the roles SBEIc, SBEI, small SBEI-like proteins, protein A and B play in determining starch granule size in barley. The molecular markers developed from these studies will be used to screen barley germplasm to identify genotypes with higher amount of A-type starch granules. Germplasms with desired starch granule characteristics will be transferred to barley breeders to introgress the genes responsible for increased A-type starch granules into adapted malting barley cultivars.
Acknowledgements
NRCC# 45264. Brewing and Malting Barley Research Institute, Winnipeg, MB and the Saskatchewan Agriculture Development Fund, Regina, SK are gratefully acknowledged for financial support of this research.
References
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Monica Båga1 and Ravindra N Chibbar2
1 Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8
2 Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, Saskatoon, SK S7N 0W9
Presented at the 3rd Canadian Barley Symposium, June 19-20, 2003 |
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