Seed Development in a Shrunken Endosperm Barley Mutant

Seg8 is one of eight barley (Hordeum vulgare L.) mutants whoseendosperm development is affected by the maternal plant genotype.This study was initiated to determine the nature and onset ofabnormal development to provide a basis for further studiesaimed at understanding the mechanism of genetic control. Seeddevelopment and synthesis and accumulation of reserve substanceswere compared between seg8 and its normal counterpart, cv. Klages.Light microscopic examination showed that the mutant phenotypeappeared as early as 4 d after anthesis (DAA), and seg8 graindry weight was significantly lower than cv. Klages by 8 DAA.Grain cell number was significantly lower in seg8 by 8 DAA,indicating an early termination of cell division. The mutanthad a lower starch concentration and higher sucrose concentration,also evident at 8 DAA. Rates of [¹⁴C]sucrose incorporation intostarch in excised half seeds were similar in both genotypesat 2 and 4 DAA, but at 8 and 12 DAA seg8 had a lower rate. Totalprotein concentration was not significantly different betweenthe two genotypes throughout endosperm development. These resultsindicate that the mutation affects cell division and starchaccumulation prior to 8 DAA. It is not known if the reductionin starch biosynthesis and accumulation results from a reducedcapacity for starch or a defect in starch biosynthesis. Hordeum vulgare L., barley, shrunken endosperm mutant, endosperm development, starch, protein, endosperm cell number     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms

The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.We are very grateful to Dr. Roger Ellis (SCRI, Dundee, Scotland) for the gift of barley seeds, and to Drs Roger Ellis, Alan Schulman and Cathie Martin for helpful advice and comments during the course of this work.     

Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules

Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent K_m (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10⁻⁵ M, which is compared to 3 × 10⁻³ M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent K_m estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction.     

ANATOMY OF IMMATURE GRAINS OF EIGHT MATERNAL EFFECT SHRUNKEN ENDOSPERM BARLEY MUTANTS

The anatomical basis for the shrunken endosperm phenotype of eight recessive maternal effect mutants (seg1-seg8) of barley (Hordeum vulgare L.) is described. Plants which are homozygous recessive for these nuclear genes produce only shrunken seeds regardless of pollen source. Light microscopy revealed that four of the mutants, seg1, seg3, seg6, and seg7, exhibited premature termination of grain filling because of the necrosis and crushing of the chalaza and nucellar projection of the pericarp early in the grain-filling period, resulting in thin, wrinkled seed. Endosperm growth of these mutants before the occurrence of chalazal necrosis seemed normal. The other four mutants exhibited characteristic abnormalities in the endosperm growth pattern but normal development of matemal-origin tissues. Endosperm size was severely reduced in seg2, in which two flat columns of tissue were present with no central endosperm. Seg4 and seg5 developed distorted, disorganized endosperms of variable size. Endosperms of seg8 developed as two well-filled lobes with no central endosperm, resulting in a distinct dorsal crease. We suggest that the mutants are useful as probes to study maternal effects on endosperm development.     

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants

Normal (nonglutinous) rice plants (Oryza sativa andO. glaberrima) contain more than 18% amylose in endosperm starch, whilewaxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even inwaxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with aMr of 60 kd, whereaswaxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is thewaxy protein encoded by theWx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands withMr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum againstWx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous toWx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from theWx gene expressed in the endosperm.     

C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley

Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [¹⁴C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-¹⁴C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains.     

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley ( Hordeum vulgare L. cv. Bomi-like), shx , which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.     

Waxy genes from spelt wheat: new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species

Background and Aims

Waxy proteins are responsible for amylose synthesis in wheat seeds, being encoded by three waxy genes (Wx-A1, Wx-B1 and Wx-D1) in hexaploid wheat. In addition to their role in starch quality, waxy loci have been used to study the phylogeny of wheat. The origin of European spelt (Triticum aestivum ssp. spelta) is not clear. This study compared waxy gene sequences of a Spanish spelt collection with their homologous genes in emmer (T. turgidum ssp. dicoccum), durum (T. turgidum ssp. durum) and common wheat (T. aestivum ssp. aestivum), together with other Asian and European spelt that could be used to determine the origin of European spelt.

Methods

waxy genes were amplified and sequenced. Geneious Pro software, DNAsp and MEGA5 were used for sequence, nucleotide diversity and phylogenetic analysis, respectively.

Key Results

Three, four and three new alleles were described for the Wx-A1, Wx-B1 and Wx-D1 loci, respectively. Spelt accessions were classified into two groups based on the variation in Wx-B1, which suggests that there were two different origins for the emmer wheat that has been found to be part of the spelt genetic make-up. One of these groups was only detected in Iberian material. No differences were found between the rest of the European spelt and the Asiatic spelt, which suggested that the Iberian material had a different origin from the other spelt sources.

Conclusions

The results suggested that the waxy gene variability present in wheat is undervalued. The evaluation of this variability has permitted the detection of ten new waxy alleles that could affect starch quality and thus could be used in modern wheat breeding. In addition, two different classes of Wx-B1 were detected that could be used for evaluating the phylogenetic relationships and the origins of different types of wheat.     

A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin.     

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity

Two mutant lines of barley, Risø 17 and Notch‐2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen‐accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A‐type or B‐type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild‐type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild‐type endosperm plays a role in determining the number, and hence the form, of starch granules.     

Development of tannin vacuoles in chalaza and seed coat of barley in relation to early chalazal necrosis in the seg1 mutant

Structural development of grain tissues of maternal origin in normal and seg1 barley (Hordeum vulgare L. cv. Betzes) was examined using light and electron microscopy. Chalaza and seedcoat cells of normal grains developed prominent tannin vacuoles which persisted throughout the grain-filling period. Tannins were present in the same tissues of seg1, but no large central vacuoles developed. Instead, the chalaza and nucellar projection degenerated and were crushed, presumably terminating sugar flow and causing formation of shrunken grains (35–55% normal dry weight). Tannins were localized using various histochemical stains. Extracts of chalaza and adjacent tissues contained proanthocyanidins which yielded delphinidin and cyanidin upon hydrolysis in boiling HCl. We suggest that the basis of the seg1 phenotype may be abnormal compartmentation of tannins causing precipitation of cytoplasmic proteins and early death of chalazal cells.Abbreviations FAA Formalin-acetic acid-ethanol - PAS periodic acid Schiffs reagent     

A 56-kDa protein is a novel granule-bound starch synthase existing in the pericarps, aleurone layers, and embryos of immature seed in diploid wheat (Triticum monococcum L.)

A novel 56-kDa granule-bound starch synthase (GBSS; NDPglucose-starch glucosyltransferase, EC 2.4.1.21) responsible for amylose synthesis was found in the pericarps, aleurone layers and embryos of immature diploid wheat (Triticum monococcum L.). The GBSS and other proteins bound to starch granules of various tissues of immature normal and waxy diploid wheat seeds were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their activities were examined. In the waxy mutant, the waxy protein (59.5 kDa, GBSSI) was absent, but amylose and GBSS activity were evident in all tissues except the endosperm. Of the proteins bound to starch granules, only the 56-kDa protein was associated with the presence of amylose and GBSS activities in the pericarps, aleurone layers and embryos. Mutations at the waxy locus did not affect the 56-kDa protein in these tissues. Changes in the amount of 56-kDa protein during the course of seed development, and the distribution of the 56-kDa protein in each tissue of immature seeds were quite different from those of the waxy protein. On the other hand, the N-terminal amino acid sequence of the 56-kDa protein had a 40–50% similarity to GBSSI of some other plant species and was antigenically related to the waxy protein. These results strongly suggest that the 56-kDa protein in diploid wheat is a GBSSI class enzyme and, hence, an isoform of the waxy protein. The waxy protein and 56-kDa protein, however, are expressed in different seed tissues and at different stages of seed development. Received: 15 May 1998 / Accepted: 18 June 1998     

Differences of Starch Granule Distribution in Grains from Different Spikelet Positions in Winter Wheat

Wheat starch development is a complex process and is markedly difference by changes in spikelet spatial position. The present study deals with endosperm starch granule distribution and spatial position during filling development. The study was conducted with pure starch isolated from wheat (Triticum aestivum L.), Jimai20 and Shannong1391, at 7–35 days after anthesis (DAA). The results showed that grain number, spikelet weight and grain weight per spikelet in different spatial position showed parabolic changes. Upper spikelets had highest starch and amylose content followed by basal spikelets, then middle spikelets. The paper also suggested the volume percents of B-type and A-type granule in grain of middle spikelets were remarkably higher and lower than those of basal and upper spikelets, respectively. However, no significant difference occurred in the number percents of the two type granule. The ratio of amylase to amylopectin was positively correlated with the volume proportion of 22.8–42.8 µm, but was negatively related to the volume proportion of <9.9 µm. The results indicated that the formation and distribution of starch granules were affected significantly by spikelet position, and grains at upper and basal spikelet had the potential of increasing grain weight through increasing the volume of B-type granules.     

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.     

Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase

The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between “waxy” proteins and starch-granule-bound starch synthases generally. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grant-in-aid to the John Innes Institute.     

The function of the Waxy locus in starch synthesis in maize endosperm

The soluble adenosine diphosphate glucose-starch glucosyltransferase of maize (Zea mays L.) endosperm uses adenosine diphosphate glucose as a sole substrate, but the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase utilizes both adenosine diphosphate glucose and uridine diphosphate glucose. The soluble glucosyltransferase can be bound to added amylose or to maize starch granules that contain amylose. However, binding of the soluble enzyme to the starch granules does not change its substrate specificity to that of the natural starch granule-bound glucosyltransferase. Furthermore, the soluble glucosyltransferase bound to starch granules can be removed by repeated washing without a change in specificity. The bound glucosyltransferase can be released by mechanical disruption of starch granules, and the released enzyme behaves in a manner similar to that of the bound enzyme in several respects. These observations suggest that the soluble and bound glucosyltransferases are different enzymes. The starch granule-bound glucosyltransferase activity is linearly proportional to the number of Wx alleles present in the endosperm. This is compatible with the hypothesis that the Wx allele is a structural gene coding for the bound glucosyltransferase, which is important for the normal synthesis of amylose.Journal Paper No. 4818 of the Purdue University Agricultural Experiment Station.