Sadequr Rahman

Characterisation of the wheat Mr 15000 “grain-softness protein” and analysis of the relationship between its accumulation in the whole seed and grain softness

The Mr 15000 protein associated with water-washed wheat starch granules from soft wheats was shown to be heterogeneous: it could be divided into a fraction containing one or moreα-amylase inhibitor subunits and a fraction largely composed of a previously uncharacterised polypeptide(s) referred to as the “grainsoftness protein” (GSP). The major N-terminal sequence and sequences of peptides derived from protease digests of GSP are reported. An antiserum specific for GSP was used to show that GSP accumulated in both hard and soft wheat grains, but the GSP in soft grains associated more strongly with starch granules than the GSP in hard grains. A positive correlation between grain softness and accumulation of GSP in the seed was demonstrated for a range of cultivars. This differs from the qualitative relationship, based on the isolated starch fraction, between GSP and grain softness that has already been reported. Analysis of wholemeal extracts with the antiserum demonstrated that the accumulation of GSP in the seed was dependent on the short arm of chromosome 5D, which also encodes theHa locus. In addition, examination of near-isogenic lines differing in hardness indicated that the gene(s) controlling GSP was (were) linked with theHa locus. The findings indicate that GSP may be the product of theHa locus and thus be the major factor that determines the milling characteristics of bread wheats.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

Effects of starch synthase IIa gene dosage on grain, protein and starch in endosperm of wheat

Starch synthases (SS) are responsible for elongating the α-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose–lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.

Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct role for isoamylase1 in amylopectin biosynthesis.

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.

The structural organisation of the gene encoding class II starch synthase of wheat and barley and the evolution of the genes encoding starch synthases in plants

Abstract. Wheat and barley contain at least four classes of starch synthases in the endosperm, granule bound starch synthase I (GBSSI) and starch synthases I, II and III (SSI, SSII, SSIII). In this work, SSII in barley is shown to be associated with the starch granule by using antibodies. A cDNA from barley encoding SSII and the genes for SSII from barley and Aegilops tauschii (A. tauschii, the D genome donor to wheat) are characterised. Fluorescent in situ hybridisation (FISH) and PCR were used to localise the wheat SSII gene to the short arm of chromosome 7, showing synteny with the location of the rice SSII gene to the short arm of chromosome 6. Comparison of the genes encoding SSII of A. tauschii, barley and Arabidopsis showed a conserved exon-intron structure although the size of the introns varied considerably. Extending such comparison between the genes encoding starch synthases (GBSSI, SSI, SSII and SSIII) from A. tauschii and Arabidopsis showed that the exon-intron structures are essentially conserved. Separate and distinct genes for the individual starch synthases therefore existed before the separation of monocotyledons and dicotyledons.

Three sucrose transporter genes are expressed in the developing grain of hexaploid wheat

A family of three cDNAs, designated TaSUT1A, 1B and 1D, encoding sucrose transporter (SUT) proteins was isolated from a hexaploid wheat (Triticumaestivum) endosperm library. The cDNA sequences are 96% identical but are distinguishable from one another by virtue of a size polymorphism in the 3′-untranslated region (UTR). The predicted amino acid sequences are 98% identical and are highly similar to the sucrose transporters from rice, maize and barley. A gene for TaSUT1 was isolated from genomic libraries of Aegilopstauschii (the donor of the D genome of wheat) and the coding sequence found to be identical to that of TaSUT1D cDNA. There is only one copy of each TaSUT1 gene in hexaploid wheat and it is located on chromosome 4. Genomic Southern analysis and PCR analysis across the 3′ polymorphic region of hexaploid, tetraploid and progenitor diploid wheat DNAs established that the TaSUT1A gene was present in the putative A-genome progenitor, T.xa0monococcum, and that the TaSUT1B gene was present in the putative B-genome progenitor, T.xa0searsii. All three TaSUT1 genes are expressed at high levels in filling grain, showing a good correlation with the developmental time course of growth. This reinforces the view that in cereals a major role of SUT1 is in the post-phloem sugar transport pathway associated with seed filling.

Characterization of low-molecular-weight glutenin genes in Aegilops tauschii

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3′ and 5′ flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2xa0kDa and 39.6xa0kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundredxa0kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.

Regulation of gene expression in plants

This chapter discusses regulation of gene expression in plants, with a special focus on the processes which determine expression of particular genes in plants. Plant cells contain three distinct genomes; those of the nucleus; the plastid (that is, the chloroplast in green tissue); and the mitochondrion. The nucleus contains almost all the functional genes of the plant, and its genome is organized on the general model for eukaryotes. However, the plastid and mitochondrion contain genomes which have many features in common with prokaryotes, and are thus considered separately. A gene contains a transcribed sequence of deoxyribonucleic acid (DNA), which is bounded by bases at which transcription is initiated and terminated. Transcription of nuclear genes is carried out by one of three different types of RNA polymerase, according to the type of gene product. The ribosomal RNA genes are transcribed by RNA polymerase I. This chapter begins with a discussion on nuclear genes and organellar genes. Signaling mechanisms in gene regulation are then explained. The chapter concludes with a discussion on gene regulation in plant development and presenting the basic concepts of transgenic plants.

Early expression of grain hardness in the developing wheat endosperm

Abstract. Seeds from near-isogenic hard and soft wheat lines were harvested at regular intervals from 5xa0days post-anthesis to maturity and examined for hardness using the single kernel characterisation system (SKCS). SKCS analysis revealed that hard and soft lines could be distinguished from 15xa0days post-anthesis (dpa). This trend continued until maturity where the difference between the hard and soft lines was most marked. SKCS could not be applied to the small 5- and 10-dpa wheat kernels. Fresh developing endosperm material was examined using light microscopy and no visible differences between the cultivars were detected. When air-dried material was examined using scanning electron microscopy (SEM) differences between soft and hard lines were visible from as early as 5xa0dpa. Accumulation of puroindoline a and puroindoline b was investigated in developing seeds using both Western blotting and ELISA. Low levels of puroindoline a could be detected in the soft cultivar from 10xa0dpa, reaching a maximum at 32xa0dpa. In the hard cultivar, puroindoline a levels were negligible throughout grain development. Puroindoline b accumulates in both the soft and hard cultivars from 15xa0dpa, but overall contents were higher in the soft cultivar. These findings indicate that endosperm hardness is expressed very early in developing grain when few starch granules and storage proteins were deposited in the endosperm cells. Further, the near-isogenic soft and hard Heron lines could be differentiated by SEM at a stage in development when the accumulation of puroindolines could not be detected by the methods used in this study.

A microarray analysis of wheat grain hardness

Grain hardness is an important quality characteristic of wheat grain, and considerable research effort has focused on characterising the genetic and biochemical basis underlying the hardness phenotype. Previous research has shown that the predominant difference between hard and soft seeds is linked to the puroindoline (PIN) proteins. In this study the near-isogenic lines of Heron and Falcon, which differ only in the grain hardness character, were compared using a cDNA microarray consisting of approximately 5,000 unique cDNA clones that were isolated from wheat and barley endosperm tissue. Our analysis showed that major differences in gene expression were evident for puroindoline-a (Pina), with a minor but not consistent change in the expression of puroindoline-b (Pinb). These observations were confirmed using a 16,000 unique cDNA microarray in a comparison of hard wheats with either the Pinaxa0null or Pinb mutation.