The effect of the high lysine genes of the barley mutants Risø 1508 and 527 on embryo development

Dry weight of 34-day-old embryos from barley ( Hordeum vulgare L.) cv. Bomi and high lysine mutants 527, 1508 and the double mutant 527/1508 were 1.00, 1.13, 1.56 and 2.22 mg, respectively. Embryos of the four lines were compared by light and electron microscopy and two dimensional gel electrophoresis. Morphological differences were restricted to the scutellar tissue, which had an irregular form in 1508 and the double mutant. Average scutellar cell volumes in 34-day-old embryos from cv. Bomi, mutants 527, 1508, and 527/1508 were 4 200, 8 300, 4 900 and 23 400 μm³. respectively. The starch content of the scutellar parenchyma cells was slightly higher in mutant 527 than in cv. Bomi, and considerably higher in mutant 1508 and the double mutant. The two dimensional gel electrophoretic examination showed that seven of the embryo protein spots were of a significantly different size in the mutants relative to those from cv. Bomi. Among these seven proteins, one was apparent only in the double mutant, four were more abundant in the mutants than in cv. Bomi and two were less abundant. The isoelectric points and the molecular weights of these proteins do not correspond exactly to those of proteins previously described in barley seeds.     

Starch and prolamin level in single and double high-lysine barley mutants

At maturity the high-lysine barley (Hordeum vulgare L.) Ris0 mutants 1508, 527 and 29 kernels contained about 20% less starch and twice as much free sugars as the parent varieties Bomi and Carlsberg II. An enhanched effect on starch reduction and free sugar accumulation was observed during kernel development when the single mutants 527 and 29 are combined with the mutant 1508. At maturity, kernels of the double mutants 527/1508 and 29/1508 contained, respectively, 68 and 43% less starch than Bomi. The double mutant 29/1508 kernel had a slightly lower prolamin content than mutant 1508 which is the most prolamin-deficient single mutant. In the double mutant 527/1508, however, an almost complete suppression of prolamin synthesis was observed during kernel development. The percentage of lysine in the seed proteins of the double mutants was about the same as in the most extreme single mutant 1508. Based on the additive effect of the individual high-lysine genes in the double mutants, it is concluded that the influences of these genes on prolamin and starch synthesis are independent.     

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.     

Isolation of protoplasts from developing barley endosperm: a tool for transient expression studies

We have developed a method for the routine isolation of protoplasts from developing starchy endosperm of barley (Hordeum vulgare L.). Preplasmolysis of the intact endosperms, a low concentration of hydrolytic enzymes and gravity sedimentation before any centrifugation step, were crucial factors for a good preparation. Best yields were obtained early after pollination (8–13 days) or with mutants with low starch content. Transient expression of a reporter gene under the control of the 35S promoter, after polyethyleneglycol transfection of endosperm protoplasts, was of the same order as that found in coleoptile derived protoplasts. No significant difference in expression was found for a given tissue between cv. Bomi and its mutant Risø 1508.Abbreviations 2, 4D 2, 4 dichlorophenoxyacetic acid - dap days after pollination - MS Murashige and Skoog medium - pp protoplasts - PEG polyethylenglycol - GUS ß-glucuronidase - MUG 4-methylumbelliferyl-ß-D-glucuronide - X-gluc 5-bromo-4 chloro-3 indolyl glucuronide     

Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotypes of barley

Changes in gibberellin-like activity and content of indole type auxins were investigated during grain development of the two high-lysine barley (Hordeum vulgare L.) genotypes Sv 73608 and Risø 1508, and their corresponding normal cultivars Mona and Bomi. A peak in gibberellin-like activity was found in developing grains of the normal cultivars about 18 days after anthesis, whereas the grains of the high-lysine genotypes showed a two to five times higher maximum about 3–4 days later. The auxin content of the cultivar Bomi showed a maximum between the 22nd and the 29th day after anthesis, whereas, throughout their development the grains of the mutant Risø 1508 exhibited only about ¹/10 of the maximum level of auxin found in the grains of Bomi. The normal cultivar Mona also displayed higher contents of auxin than the high-lysine genotypes Sv 73608, particularly at the later stages of grain growth, but the differences in concentration were considerable smaller than for the pair ‘Bomi’—‘Risø 1508’. It is suggested that auxins play an important role in the development of barley grains.     

The maize disorganized aleurone layer 1 and 2 ( dil1, dil2) mutants lack control of the mitotic division plane in the aleurone layer of developing endosperm

The maize (Zea mays L.) endosperm consists of an epidermal like layer of isodiametric aleurone cells surrounding a central body of starchy endosperm cells. In disorgal1 (dil1) and disorgal2 (dil2) mutants the control of the mitotic division plane is relaxed or missing, resulting in mature grains with disorganized aleurone layers. In addition to orientation of the division plane, both the shape and size of the aleurone cells are affected, and often more than one layer of aleurone cells is present. Homozygous dil1 and dil2 grains are shrunken due to reduced accumulation of starchy endosperm and premature developmental arrest of the embryo, and mature mutant grains germinate at a very low rate and fail to develop into plants. However, homozygous mutant plants can be obtained through embryo rescue, revealing that both mutants have an irregular leaf epidermis as well as roots with a strongly reduced number of root hairs and aberrant root hair morphology. Our results suggest the presence of common regulatory mechanisms for the control of cell division orientation in the aleurone and plant epidermis.Abbreviations DAP days after pollination - dek defective kernel mutant - dil disorganized aleurone layer mutant - GUS -glucuronidase - LM light microscopy - PPB pre-prophase band - SEM scanning electron microscopy - TUSC Trait Utility System for Corn     

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。     

Positional cues specify and maintain aleurone cell fate in maize endosperm development

A genetic analysis of maize aleurone development was conducted. Cell lineage was examined by simultaneously marking cells with C1 for anthocyanin pigmentation in the aleurone and wx1 for amylose synthesis in the starchy endosperm. The aleurone and starchy endosperm share a common lineage throughout development indicating that positional cues specify aleurone fate. Mutants in dek1 block aleurone formation at an early stage and cause peripheral endosperm cells to develop as starchy endosperm. Revertant sectors of a transposon-induced dek1 allele showed that peripheral endosperm cells remain competent to differentiate as aleurone cells until late in development. Ds-induced chromosome breakage was used to generate Dek1 loss-of-function sectors. Events occurring until late development caused aleurone cells to switch fate to starchy endosperm indicating that cell fate is not fixed. Thus, positional cues are required to specify and maintain aleurone fate and Dek1 function is required to respond to these cues. An analysis of additional mutants that disrupt aleurone differentiation suggests a hierarchy of gene functions to specify aleurone cell fate and then control aleurone differentiation. These mutants disrupt aleurone differentiation in reproducible patterns suggesting a relationship to endosperm pattern formation.     

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

Rates of Cell Division in Developing Barley Endosperms

Counts of nuclei in enzyme digested endosperms of barley cultivarBomi show that the final number of cells, 170000, is reachedbetween 18 and 21d after anthesis. Based on the number of cellprofiles in transverse mid-grain sections, starchy endospermcells divide up to day 14. Thereafter, cell proliferation isrestricted to the aleurone layer. Hordeum vulgare, starchy endosperm, aleurone, mitotic activity, light microscopy     

Rice caryopsis development Ⅱ: Dynamic changes in the endosperm

The rice endosperm plays crucial roles in nourishing the embryo during embryogenesis and seed germination. Although previous studies have provided the general information about rice endosperm, a systematic investigation throughout the entire endosperm developmental process is still lacking. In this study, we examined in detail rice endosperm development on a daily basis throughout the 30‐day period of post‐fertilization development. We observed that coenocytic nuclear division occurred in the first 2 days after pollination (DAP), cellularization occurred between 3 and 5 DAP, differentiation of the aleurone and starchy endosperm occurred between 6 and 9 DAP, and accumulation of storage products occurred concurrently with the aleurone/starchy endosperm differentiation from 6 DAP onwards and was accomplished by 21 DAP. Changes in cytoplasmic membrane permeability, possibly caused by programmed cell death, were observed in the central region of the starchy endosperm at 8 DAP, and expanded to the whole starchy endosperm at 21 DAP when the aleurone is the only living component in the endosperm. Further, we observed that a distinct multi‐layered dorsal aleurone formed near the dorsal vascular bundle, while the single‐ or occasionally two‐cell layered aleurone was located in the lateral and ventral positions of endosperm. Our results provide in detail the dynamic changes in mitotic divisions, cellularization, cell differentiation, storage product accumulation, and programmed cell death that occur during rice endosperm development.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

Two-dimensional Separation of the Prolamins of Normal and High Lysine Barley (Hordeum vulgare L.)

The polypeptide components of the reduced prolamin fraction(hordein) of barley seed proteins have been separated, beforeand after alkylation, by polyacrylamide gel electrophoresisusing buffers containing urea and/or sodium dodecylsulphate(SDS). Alkylation of the protein with 4-vinylpyridine or acrylonitrileresults in a considerable sharpening of the protein bands andsome minor changes in the band pattern. The procedure has beenused to compare the hordeins of the normal commercial varieties,Julia and Bomi, to those of a high lysine mutant of Bomi (Rise,1508). Whereas the alkylated hordein fractions of Bomi and Julia containSDS bands of apparent molecular weights 13 000, 16 000, 20 000,30 000, 43 000, 51 000, 67 000, and 86 000, the mutant hordeinfractions contain predominantly the low molecular weight (13000, 16 000, and 20 000) and mol. wt. 51 000 bands. Further resolution of the fractions was obtained by two-dimensionalelectrophoresis using 6 M urea in glycine/acetate buffer atpH 4?6 as the first dimension and SDS in tris/borate bufferat pH 8?9 as the second. Separation of the Rise 1508 hordeinin this system demonstrated that the mol. wt. 51 000 band containsseveral closely similar components.     

Localization of carboxypeptidase I in germinating barley grain

Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain.     

Growth inhibitors in oat grains. I. Leakage of inhibiting factors from oat grains during imbibition and the effect of these on germination and growth

Spikes of barley ( Hordeum vulgare L.) cultivar Bomi and high-lysine mutants Riso 1508 and Riso 56 were cultured on liquid media at varying N and sucrose levels. Bomi accumulated N in response to increasing N levels in the medium and a higher level was reached than in spikes of intact plants. The distribution of N in salt-soluble, hordein, and non-protein N fractions appeared to be normal. Endosperm dry weight and starch were lower than in intact plants and declined at higher N levels. A linear relationship was observed between starch content and the concentration of sucrose in the endosperm water. Uptake of culture medium by the spikes was affected by both N and sucrose concentration. The mutants had lower dry weights and starch contents, and higher sucrose contents than Bomi. At high N levels, the mutants accumulated less hordein, and more non-protein N than Bomi.     

Endosperm differentiation in barley wild-type and sex mutants

The cereal endosperm is a storage organ consisting of the central starchy endosperm surrounded by the aleurone layer. In barley, endosperm development is subdivisible into four main stages, i.e. the syncytial (I), the cellularization (II), the differentiation (III) and the maturation stage (IV). During stage I, a multinucleate syncytium is formed, which in stage II develops into the undifferentiated cellular endosperm. During stage III the cells of the endosperm differentiate into two types of aleurone cells (peripheral and modified) and three different starchy endosperm cell types (irregular, prismatic and subaleurone). To elucidate the ontogenetic relationship between the endosperm tissues, the phenotypes of sex (shrunken endosperm mutants expressing xenia) mutant endosperms were studied. These mutants can be classified into two groups, i.e. those in which development is arrested at one of the four wild-type stages described above, and those with abnormal development with new organizational patterns in the endosperm or with novel cell types. Based on these studies, it is suggested that the two endosperm halves represent cell lines derived from the two daughter nuclei of the primary endosperm nucleus, and that the prismatic starchy endosperm cells arise from a peripheral endosperm meristematic activity during stage III. Finally, a model for the main molecular events underlying the morphogenetic processes is discussed.