A Squash Technique for Studying the Cytology of Maize Endosperm and Other Tissues

A new cytological procedure specifically suited to maize endosperms is presented. It uses 8-hydroxyquinoline with sucrose and aeration to pretreat the tissues. Glusulase is used to spread the cells. the procedure makes it possible to squash endosperms into a single cell layer and to photograph as many as 70 chromosomes in the same focal plane. It also allows identification of translocation chromsomes. with a slight modification the technique has been applied successfully to root tips and other tissues.     

Tissue-specific elimination of alternative whole parental genomes in one barley hybrid

Chromosome elimination was studied in squash preparations of seeds of two different Hordeum crosses between diploid parents whose karyotypes allowed identification with unusual ease for Hordeum of the parental origins of the chromosomes being eliminated in each mitosis in embryos and endosperms. In both crosses, the mean chromosome number in hybrid tissues fell during several mitoses until nuclei became haploid in embryos and diploid in endosperms. Elimination was always uniparental, i.e. all chromosomes eliminated from a given tissue in a given cross were from the same parent. In H. marinum x H. vulgare cv. Tuleen 346, elimination involved the Tuleen 346 genome in the endosperm, but the H. marinum genome in the embryo. This is a good example of alternative elimination, i.e. uniparental elimination involving different parental genomes in different tissue of the same cross. In Tuleen 346 x H. bulbosum, the H. bulbosum genome was eliminated from both embryos and endosperms. — In H. marinum x Tuleen 346 endosperms, eliminated Tuleen 346 chromosomes were individually identifiable and tended to be eliminated in non-random order: the nucleolar chromosomes, T3-7 and T6-2 first, followed by chromosomes T5-1, T7-3, T2-6 and 4, with chromosome T1-5 last. — The nucleolar constrictions were expressed in eliminated satellite chromosomes from Tuleen 346, but not in those from H. marinum or H. bulbosum. Eliminated chromosomes differed from retained ones in having smaller centromeres and tending before, during and after elimination to occupy more peripheral regions of mitoses. Elimination may result primarily from specific suppression of genes involved in centromere function, perhaps by DNA methylation.     

Structure and dynamics of natural populations of polyploid trilliums

To confirm predominant self-pollination in natural populations ofTrillium apetalon (2n=4x=20, SSUU), chromosome constitutions of endosperms (2n=30, SSSUUU) fertilized in nature were analyzed for the patterns of H-segments of cold-treated chromosomes. The endosperms were of two kinds: homo-constitutions in which three chromosomes in each of the ten sets of homologues were of the same patterns of H-segments and hetero-constitutions in which one chromosome in a set of three homologous chromosomes was different from the other two. Out of 30 endosperms, 27 (90%) were of homo-constitutions and only three (10%) were of hetero-constitutions. Since the population consisted of various homokaryotypes in which each of the ten pairs of homologous chromosomes was homozygous for the patterns of H-segments, the endosperms of homo-constitutions were the result of self-pollination, and those of hetero-constitutions were from cross-pollination. Due to pollination tests conducted in two natural populations, many good seeds were found in the fruits from both open-pollinated flowers and those covered with cotton-bags. In striking contrast, no good seeds were found in the fruits from flowers castrated and pollinated in the open-air. An additional evidence of predominant self-pollination and occasional cross-pollination was given by analyzing spatial distribution of plants of various karyotypes in a population.     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

Banded polytene chromosomes in developing endosperm of pearl millet

The occurrence of polyteny in the endosperm of field-grown plants as well as cultured endosperms of variety Vg272 of pearl millet (Pennisetum glaucum (L.) R.Br.) is recorded. There is a pronounced banded structure of these chromosomes similar to the ones observed in Dipteran salivary glands. Polyteny under physiologically controlled conditions also seems feasible in pearl millet.     

Ploidy barrier to endosperm development in maize

Maize kernels inheriting the indeterminate gametophyte mutant (ig) on the female side had endosperms that ranged in ploidy level from diploid (2x) to nonaploid (9x). In crosses with diploid males, only kernels of the triploid endosperm class developed normally. Kernels of the tetraploid endosperm class were half-sized but with well-developed embryos that regularly germinated. Kernels of endosperm composition other than triploid or tetraploid were abortive.-Endosperm ploidy level resulting from mating ig/ig x tetraploid Ig similarly was variable. Most endosperms started to degenerate soon after pollination and remained in an arrested state. Hexaploid endosperm was exceptional; it developed normally during the sequence of stages studied and accounted for plump kernels on mature ears. Since such kernels have diploid maternal tissues (pericarp) but triploid embryos, the present finding favors the view that endosperm failure or success in such circumstances is governed by conditions within the endosperm itself.-Whereas tetraploid endosperm consisting of three maternal genomes and one paternal genome is slightly reduced in size but supports viable seed development, that endosperm having two maternal and two paternal chromosome sets was highly defective and conditioned abortion. Thus, development of maize endosperm evidently is affected by the parental source of its sets of chromosomes.     

优质蛋白玉米02基因控制赖氨酸超量积累的分析

以３８个ＱＰＭ（或０２）和对照普通玉米为实验材料,进行０２基因控制赖氨酸超量积累的生化和遗传分析。主要实验结果如下：（１）ＱＰＭ玉米０２基因为隐性的单基因遗传,它控制着胚乳、雄穗和幼苗期叶片中赖氨酸的超量积累,一些修饰因子和遗传背景对胚乳物理性状产生影响;（２）ＱＰＭ玉米、普通玉米的胚较之胚乳,或者ＱＰＭ玉米胚乳较之普通玉米胚乳都含有较多的天门冬氮酸、甘氨酸、赖氨酸和精氨酸,含有较少的脯氨酸、谷氨酸、亮氨酸和苯丙氨酸;（３）两种玉米之间,在胚乳蛋白质含量及胚乳可溶性蛋白、醇溶蛋白、谷蛋白的赖氨酸含量方面没有什么不同;（４）已经育成一批ＱＰＭ或０２玉米自交系,并配制出几个强优势杂交组合。     

Aleurone cell identity is suppressed following connation in maize kernels

Expression of the cytokinin-synthesizing isopentenyl transferase enzyme under the control of the Arabidopsis (Arabidopsis thaliana) SAG12 senescence-inducible promoter reverses the normal abortion of the lower floret from a maize (Zea mays) spikelet. Following pollination, the upper and lower floret pistils fuse, producing a connated kernel with two genetically distinct embryos and the endosperms fused along their abgerminal face. Therefore, ectopic synthesis of cytokinin was used to position two independent endosperms within a connated kernel to determine how the fused endosperm would affect the development of the two aleurone layers along the fusion plane. Examination of the connated kernel revealed that aleurone cells were present for only a short distance along the fusion plane whereas starchy endosperm cells were present along most of the remainder of the fusion plane, suggesting that aleurone development is suppressed when positioned between independent starchy endosperms. Sporadic aleurone cells along the fusion plane were observed and may have arisen from late or imperfect fusion of the endosperms of the connated kernel, supporting the observation that a peripheral position at the surface of the endosperm and not proximity to maternal tissues such as the testa and pericarp are important for aleurone development. Aleurone mosaicism was observed in the crown region of nonconnated SAG12-isopentenyl transferase kernels, suggesting that cytokinin can also affect aleurone development.     

Location of chromosomal control of GA-induced enzyme release by distal half-grains of wheat

The three enzymes, -amylase, peroxidase, and acid phosphatase, are among those released by the aleurone of bread wheat, Triticum aestivum, as a direct response to gibberellins (GA) from the embryo during germination. Aneuploid genotypes are used to investigate the chromosomal location, nature, and extent of genetic control of the release of these enzymes in endosperms after induction by exogenous GA. Ditelosomics demonstrate the effect of removal of known chromosome arms, and tetrasomics show the effect of duplication of chromosome pairs. Quantitative analysis demonstrates complex control systems over and above those previously found using zymogram techniques. For -amylase, chromosome arms with net promoter effects and chromosomes with net inhibitor effects were found. These effects were not chromosome-dosage responsive, unlike the promoter-inhibitor system found for acid phosphatase control. Peroxidase levels in the endosperms were generally high in both types of aneuploids. With one exception, all chromosomes showing involvement as ditelosomics showed a similar effect as tetrasomics, indicating that in the euploid a balanced state restricting, rather than promoting, peroxidase levels existed.     

Chromosomal assignment of genes controlling salt-soluble proteins (albumins and globulins) in wheat and related species

Summary Salt-soluble proteins from the endosperms of wheat, barley, and rye have been separated by nonequilibrium electrofocusing x electrophoresis. Genes encoding 14 of the 25 components observed in wheat have been unambiguously assigned to 10 different chromosomes (1B, 3B, 3D, 4A, 4D, 5B, 6B, 6D, 7B, 7D) by analysis of the compensated nulli-tetrasomic series. Five more wheat proteins seem to be controlled by group 2 chromosomes. Analysis of wheat-barley and wheat-rye addition lines has led to the location of genes for 6 out of 20 barley proteins in 4 different chromosomes (1H, 3H, 4H, 6H; 1H is homoeologous to group 7 chromosomes of wheat) and of genes for 5 out of 20 rye proteins in two different chromosomes (2R, 4R). The relationship between the proteins reported here and previously characterized ones is discussed.     

Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm.

Grains of nine opaque (o) and floury (fl) mutants of maize (Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2) were examined for the weight proportions of their component tissues and the content of eight nitrogen fractions in their endosperms. A linear regression was found connecting the amounts (mg per endosperm) of zeins and true proteins (crude proteins minus non-protein nitrogen) for the non-opaque2 mutants. The data points connecting zeins to true proteins present in the mature endosperms of six wild-type (+) inbred lines and their o2 versions were located outside (+) or within (o2) the 95% confidence range of the regression line. The data obtained from the developing and mature endosperms of the W22o7 inbred line (Di Fonzo et al., Plant Sci. Lett., 1979, 77) and the floury portion of mature endosperms of three other wild-type inbred lines fell practically on the regression line. The effects of genotype and environmental factors upon the relative accumulation rate of zeins were assessed from the present results and the data taken from the literature concerning the quantitative interdependence between zeins and true proteins in immature and mature endosperms.     

Polyamine metabolism in hants : Arginine and omithine decarboxylase activity in ripeningTrlticum durum seeds

The pattern of the activity of arginine decarboxylase (ADC) and omithine decarboxylase (ODC) involved in polyamine synthesis in ripening wheat seeds was examined. The aim was to study the polyamines and the activity of the two enzymes in correlation with the growth processes occurring in the developing wheat seeds. The results obtained showed a very different pattern of polyamine content in the two organs of caryopsis, and that the two enzymes in the embryos have a higher activity than in the endosperms. Moreover, while in the embryos the ADC exhibits higher activity than the ODC, in the endosperms the activity of ODC is about similar to that of ADC. This pattern is discussed in relation to the different histological characteristics of embryo and endosperm tissues during seed development.     

Abscisic Acid Is an Endogenous Inhibitor in the Regulation of Mannanase Production by Isolated Lettuce (Lactuca sativa cv Grand Rapids) Endosperms

The production of mannanase, a cell-wall-degrading carbohydrase, can be manipulated in isolated lettuce (Lactuca sativa cv Grand Rapids) endosperms by changes in the volume of buffer in which they are incubated. The enzyme is produced when endosperms are incubated in a large volume, but not when incubated in a small volume, which is suggestive that an endogenous, diffusible inhibitor of mannanase production is being lost from the endosperms in a large volume (JD Bewley, P Halmer 1980/1981 Israel J Bot 29: 118-132). We have investigated the possibility that the phytohormone abscisic acid (ABA) is involved in this regulation of mannanase production in isolated lettuce endosperms. We find several correlations between the presence of the endogenous inhibitor and of ABA, i.e. (a) a `leachate' prepared from isolated lettuce endosperms induces synthesis of ABA-specific proteins in barley aleurone layers, indicating that incubation of endosperms in a large volume results in the diffusion of ABA therefrom into the surrounding medium; (b) fractionation of the components of a leachate by either polyvinylpyrrolidone-chromatography of C₁₈ reversed-phase high performance liquid chromatography fails to separate the endogenous inhibitor from authentic ABA; and (c) changes in the incubation volume of endosperms result in changes in the amount of extractable ABA in the endosperms, as detected by ELISA. These results are consistent with a role for endogenous ABA in the regulation of mannanase production in isolated lettuce endosperms.     

水稻 ADP-葡萄糖焦磷酸化酶小亚基 I 基因的启动子分析

水稻（Oryce sativa L．）基因组中的ADP-葡萄糖焦磷酸化酶小亚基（ADP-Glucose pyrophosphorylase small subunit,OsAgpS）由两个基因编码,即OsAgpSl和OsAgpS2。其中OsAgpSl基因产生两个转录本OsAgpSla和OsAgpSlb,区别在第一个外显子的位置不同。通过RT—PCR方法分析了两个转录本在水稻组织和胚乳不同发育时期的表达特性;同时通过报告基因GUS检测了两个转录本上游转录调节区DNA片段的转录启动特性。结果表明,两个启动子与其下游转录本的表达模式完全一致,即OsAgpSla转录本和OsAgpSla上游启动子控制的GUS基因主要在胚乳中高水平表达,在叶片中有很低水平的表达;而OsAgpSlb转录本和OsAgpSlb上游启动子控制的GUS基因主要在叶片和胚乳发育早期低水平表达。这说明OsAgpSl基因产生的两个转录本是由不同的启动子控制转录的,OsAgpSla上游启动子可以作为胚乳表达用启动子。     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Protein Bodies from Developing Seeds of Barley, Maize, Wheat and Peas: The Effects of Protease Treatment

When isolated protein bodies of barley were treated with proteinase-kthey were almost completely digested. Similar results were obtainedwhen fresh homogenates, prepared by rapid chopping of barleyor wheat endosperms into isolation medium, were incubated withthe protease. In contrast, the protein bodies of developingmaize endosperms, or pea cotyledons were resistant to proteaseattack. The results are interpreted as indicating that isolatedprotein bodies of developing wheat and barley endosperms arenot surrounded by a complete membrane whereas those of the peacotyledons and maize endosperms are.     

Isolation and purification of functional total RNA from blue-grained wheat endosperm tissues containing high levels of starches and flavonoids

Isolation of intact, functional total RNA from blue-grained wheat endosperms containing high levels of starches, polysaccharides, and flavonoids was extremely difficult using traditional methods. We describe here a modified SDS/phenol method that can be used to isolate total RNA from blue-grained wheat endosperms. This method solved the problems of RNA degradation, contamination, and low yield due to binding and/or co-precipitation with starches, polysaccharides, and flavonoids. The isolated total RNA was of high quality and undegraded as determined by spectrophotometric readings and denaturing agarose gel analysis. Quality of the total RNA isolated using our protocol was further assessed by RT-PCR and northern blot hybridization. Using this efficient procedure, 350–400 μg of total RNA was routinely obtained from 1 g of blue-grained wheat seeds.     

RNases and nucleases in embryos and endosperms from naturally aged wheat seeds stored in different conditions

Temperature and moisture content are particularly important factors influencing the longevity of seeds, and therefore the ageing of seeds is closely tied to storage conditions. The ageing process is characterised by many physiological and biochemical changes: membranes tend to leak, enzymes lose catalytic activity, and chromosomes accumulate mutations. Since viability loss is also associated with the breakdown of nucleic acids, the aim of the study was to determine whether the damage induced by ageing could be associated with changes in the activity of RNases and nucleases in embryos and endosperms of differently stored wheat seeds. In order to better characterise seed conditions, the damage to membranes during seed ageing was evaluated by measuring the conductivity of the soaking solution during imbibition, and by using the Evans Blue colorant; lipid peroxidation was also recorded. RNases and nucleases were studied by SDS-PAGE and activity staining. Ageing of seeds stored in a dry state involved a progressive loss of membrane integrity, which increased with the degree of ageing, while lipid peroxidation remained unchanged. Changes in nucleolytic enzyme activity were recorded in embryos: a decrease in RNases and an increase in nucleases. In the endosperm compartment there were no significant differences in ribonuclease and nuclease patterns during seed ageing. Moreover, neutral RNases were absent in endosperms of dry seeds and were activated following imbibition. Present studies reveal that embryos and endosperms have different enzymatic patterns, thus highlighting that the two seed compartments age independently. A different nucleolytic pattern was present in seeds of comparable viability and membrane damage, which were stored differently, and nuclease metabolism was subject to regulation according to both ageing and the length of the storage period.