Separation and Some Properties of Large and Small Amyloplasts throughout Development in Barley Endosperm

A method is described whereby amyloplasts from immature barley (Hordeum distichum L.) endosperm could be separated into two populations of large and small amyloplasts at all stages of development. The small amyloplasts had more amylopectin than the large at early stages, but by 60 days after anthesis, the large had the greater proportion of amylopectin. Starch synthetase activity was associated with both types of amyloplast. The nucleotide specificity of the starch synthetase associated with each population varied independently throughout development. At 25 days after anthesis, the large amyloplasts were more susceptible than the small to α-amylolysis; however, at 38 and 60 days, the small amyloplasts became more susceptible.     

Starch synthetase: Comparison of UDPG and ADPG as glucosyl donors in immature barley endosperm

Summary The activity of starch synthetase in developing barley endosperm was measured in amyloplasts and in the soluble endosperm fraction by incorporation of radioactively labelled glucose into starch. Both uridine diphosphate glucose (UDPG) and adenosine diphophate glucose (ADPG) were used as glucosyl donors. Enzyme activity was initially located in the soluble fraction, but increasing activity could be detected in the amyloplast fraction during endosperm maturation.     

Factors affecting dry weight accumulation in developing barley endosperm

Some factors that may be concerned in determining final grain weight in barley ( Hordeum vulgare L. var. distichum ) have been investigated. Variation in endosperm fresh and dry weight, volume and starch content have been recorded at different stages of grain development between anthesis and harvest-ripeness for two barleys, cvs Kym and Golden Promise, differing in final grain weight. Results were recorded under both field and glasshouse conditions. The results suggest that the higher final dry weight of Kym, in comparison with Golden Promise, is a function of both rate and duration of grain filling. Only at later stages of endosperm development did the differences in volume become significant and the Kym endosperms continued to increased rapidly in volume for two to three days after endosperm volume had reached a maximum in Golden Promise. The rates of starch accumulation in both cultivars were very similar but starch deposition continued in Kym endosperms for four to five days after deposition in Golden Promise endosperms had slowed down.     

Effects of the barley mutants Risø 1508 and 527 high lysine genes on the cellular development of the endosperm

The dry grain weight of the Risø barley ( Hordeum vulgare L. var. disticum ) high lysine mutants 1508 and 527 at maturity was 32 and 37% lower, respectively, than the grains of the cultivar Bomi. Dry grain weight of the double mutant 527/1508 was reduced by 57%. Total number of endosperm nuclei from cv. Bomi, mutants 1508, 527 and 527/1508 at 24 days after anthesis was 173 000,156 000,121 000 and 111 000, respectively. Transverse mid-grain sections from mutants 1508 and 527 contained fewer aleurone cells and approximately the same number of starchy endosperm cells as cv. Bomi. The cellular organization of the endosperm of the double mutant deviated substantially from the normal. Cell volume in the central starchy endosperm of cv. Bomi, mutants 1508 and 527 at 33 days after anthesis averaged 390 000, 270 000 and 180 000 um5, respectively. Cell volume in the double mutant was smaller than in 527, but could not be accurately estimated due to the irregular shape of the cells. The mean section area of individual large starch granules in the central endosperm of mutants 1508, 527 and 527/1508 at 33 days after anthesis was 30, 48 and 72% smaller, respectively, than those of cv. Bomi. The average aleurone cell volume in cv. Bomi at 33 days after anthesis was 6 200 μm³.     

Starch-branching enzymes preferentially associated with A-type starch granules in wheat endosperm

Two starch granule-bound proteins (SGP), SGP-140 and SGP-145, were preferentially associated with A-type starch granules (>10 microm) in developing and mature wheat (Triticum aestivum) kernels. Immunoblotting and N-terminal sequencing suggested that the two proteins were different variants of SBEIc, a 152-kD isoform of wheat starch-branching enzyme. Both SGP-140 and SGP-145 were localized to the endosperm starch granules but were not found in the endosperm soluble fraction or pericarp starch granules younger than 15 d post anthesis (DPA). Small-size starch granules (<10 microm) initiated before 15 DPA incorporated SGP-140 and SGP-145 throughout endosperm development and grew into full-size A-type starch granules (>10 microm). In contrast, small-size starch granules harvested after 15 DPA contained only low amounts of SGP-140 and SGP-145 and developed mainly into B-type starch granules (<10 microm). Polypeptides of similar mass and immunologically related to SGP-140 and/or SGP-145 were also preferentially incorporated into A-type starch granules of barley (Hordeum vulgare), rye (Secale cereale), and triticale (x Triticosecale Wittmack) endosperm, which like wheat endosperm have a bimodal starch granule size distribution.     

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.     

Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue

Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6-bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.

Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient.

     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

Endosperm cell number in cultivars of barley differing in grain weight

The number of endosperm cells in caryopses 30 ‘days’ after anthesis was determined and a positive correlation was found between endosperm cell number and 1000 grain weight, and between endosperm cell number and dry grain volume in a number of cultivars of field-grown barley. The genetic factors influencing grain weight are discussed in relation to these results and to observations made on transverse sections of immature caryopses.     

大麦胚和胚乳发育的相关性及贮藏营养物质的积累

大麦(Hordeum vulgare L.)开花后1d,见合子及退化助细胞,游离核胚乳尚未形成;开花后2～3d,胚为5及10个细胞,胚乳为游离核期;开花后4及5、6d,胚为梨形及长梨形,胚乳达细胞化期;开花后8d,胚为胚芽鞘期,糊粉层原始细胞产生;开花后10d,胚具1叶,糊粉层1～2层;开花后13d胚为2叶胚,亚糊粉层发生;开花后17d,3叶胚形成,糊粉层多为3层并停止分裂,菱柱形及不规则胚乳细胞分化;开花后21～29d,胚为4叶胚,胚乳进一步分化;开花后33d,胚为5叶成熟胚,胚乳亦成熟。淀粉、蛋白质在胚中积累始于开花后13d。在盾片中由基向顶发生,在胚芽鞘及叶原基中,首先在顶端出现。成熟盾片顶端的淀粉消失。开花后6d,胚乳开始积累淀粉;开花后10d,糊粉层及胚乳细胞积累蛋白质。开花17d后胚乳的蛋白质体多聚集,29d后蛋白质体显著减少。开花后17d,在盾片及糊粉层细胞中检测到油脂。果长或果长与稃片长之比和盾片长可作为不同发育期胚和胚乳的形态指标。     

Development of Embryo and Endosperm and Nutrient Accumulation in Vigna sesquipedalis (L.) Fruwirth

The structure of embryo sac, fertilization and development of embryo and endosperm in Vigina sesquipedalis (L.) Fruwirth were investigated. Pollization occures 7–10h before anthesis, and fertilization is completed 10 h after anthesis. After fertilization, wall ingrowths are formed at the micropylar and chalazal ends of the embryo sac. Embryo development conforms to the Onagrad type, and passes through 2 or more celled proembryo, long stick-shaped, globular, heart shaped, torpedo, young embryo, growing and enlarging embryo and mature embryo. Wall ingrowths are formed on the walls of basal cells and outer walls of the cells at basal region of suspenser. The suspensor remains as the seed reaches maturity. The starch grains accumulate in the cells of cotyledons by 9–16 days after anthesis, and proteins accumulate by 12–18 days after. The endosperm development follows the nuclear type. The endosperm ceils form at the micropylar end, and remain free nuclear phase at chalazal end. The outer cells are transfer cells. Those cells at the micropylar end form folded cells with wall ingrowths. At heartembryo stage, the endosperm begins to degenerate and disintegrates before the embryo matures.     

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     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale (x Triticosecale Wittmack)

Triticale (x Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase, soluble starch synthases, granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development. There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale. Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development. Dead cells within the endosperm were detected at 6 d post anthesis (DPA), and evidence of DNA fragmentation was first observed at 21 DPA. The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development. Cell death occurred stochastically throughout the whole endosperm, meanwhile, the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling. These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase

In this work we test the hypothesis that yield of rice ( Oryza sativa L.) can be enhanced by increasing endosperm activity of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch biosynthesis. The potential for increases in yield exist because rice initiates more seeds than are taken to maturity and possesses excess photosynthetic capacity that could be utilized if there were more demand for assimilate. Following an approach already shown to be successful in wheat, 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 AGP large 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, potentially leading to higher AGP activity in vivo. The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. Increases in AGP activity in transgenic seeds compared with controls were maximal between 10-15 days after anthesis. Starch content of individual seeds at harvest was not increased, but seed weight per plant and total plant biomass were each increased by more than 20%. Increased endosperm AGP activity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set. Results demonstrate that deregulation of endosperm AGP increases overall plant sink strength, leading to larger, more productive plants in a manner similar to that in wheat having similar genetic modification.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale(× Triticosecale Wittmack)

Triticale(× Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase,soluble starch synthases,granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development.There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale.Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development.Dead cells within the endosperm were detected at 6 d post anthesis(DPA),and evidence of DNA fragmentation was first observed at 21 DPA.The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development.Cell death occurred stochastically throughout the whole endosperm,meanwhile,the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling.These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

The role of alpha-glucosidase in germinating barley grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.     

Starch synthetases from Vitis vinifera and zea mays

ADPglucose: α-1,4-glucan α-4-glucosyltransferases (starch synthetases) from leaves of Vitis vinifera and leaves and kernels of Zea mays were chromatographed on DEAE-cellulose columns. One form of the enzyme was present in grape leaves having activity both in the presence and absence of primer. Two forms were present in both leaves and kernels of maize. The second peak of activity in maize leaves and the first peak in maize kernels synthesized a polyglucan in the absence of primer. A peak of branching enzyme (Q-enzyme) occurred between the two starch synthetase peaks with both tissues. When fractions containing starch synthetase and branching enzyme were added to the first leaf starch synthetase peak, up to 100-fold activation of the unprimed reaction occurred. Branching enzyme did not stimulate the unprimed activity of the first kernel peak and no branching enzyme could be detected in this peak. The unprimed product was a branched polyglucan with mainly α-1,4-links.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Soluble and particulate lysophospholipase in the aleurone and endosperm of germinating barley

Lysophospholipase was measured in extracts of germinating barley by determining the amount of free [¹⁴C]palmitate released from [1-¹⁴C] 1-palmitoyl-lysophosphatidylcholine (LPC). Soluble and particulate lysophospholipase activity was measured at 1-day intervals in extracts from the aleurone and endosperm of barley seeds germinated for 8 days. The soluble and particulate activities of the aleurone increase approximately in parallel with one another and after 8 days of germination have 20–30 times more activity than at day 1. The activity profiles and the distribution of the activity between the soluble and particulate forms of lysophospholipase in the endosperm are markedly different. With the exception of the first 2 days when the aleurone activity is low, the endosperm activity is less than that associated with the aleurone. The soluble activity increases during the first 3 days and is more active than that of the aleurone. Thereafter it diminishes and remains low. The particulate enzyme, however, increases dramatically between days 4 and 5 and remains moderately high. The fourth and fifth day represent that stage of germination when starch-bound LPC is released in concert with the increase in amylase activity. It is proposed that it is this particulate form of the endosperm activity which may be responsible for maintaining the level of free LPC low in the endosperm of the germinating seed.