Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/−/−; +/+/−; +/+/+) or incapable (−/−/−) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.     

Plant nucleases. IV. Genetic control of ribonuclease activity in corn endosperm

Ribonuclease activity in the endosperms of 14 corn (Zea mays L.) inbreds ranged from 285 to 1305 units/g fresh weight 50 days after pollination. Activity is low in the inbred M14 and high in the inbred WF9. Hybrid endosperms contain intermediate levels of ribonuclease, and segregation occurs in the F₂ generation. The ribonuclease contents of the opaque-2 versions of nine inbred lines ranged from 1288 to 5110 units/g. The floury-2 mutation apparently does not affect ribonuclease content. Two or more genes may be involved in the control of ribonuclease content of developing endosperms.Cooperative investigations of the Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture, and the Illinois Agricultural Experiment Station, Urbana, Illinois.     

Maternal effects influencing DNA endoreduplication in developing endosperm of Zea mays.

A large proportion of the nuclei in developing endosperm of Zea mays L. undergoes endoreduplication. Nuclear preparations of the entire endosperm from maize kernels of inbred lines, their reciprocal hybrids, and in some cases, F2 and F3 endosperm tissue were evaluated using flow cytometry. Data relative to DNA endoreduplication patterns, percentage of nuclei undergoing endoreduplication, and mean DNA content per nucleus were obtained. The patterns of endoreduplication and extent of DNA amplification differ among some inbreds. In all experiments, the endoreduplication patterns show that the F1 endosperm is more similar to the maternal parent than to the paternal parent. F2 endosperms reveal little difference in endoreduplication patterns among individuals within an F2 family and no more variation than the F1 endosperms. In contrast, F3 endosperms showed greater variation among their endoreduplication patterns. These results indicate a maternal effect on endoreduplication; that is, the genotype of the maternal parent's nuclear genome exerts control over the endoreduplication activities of endosperm tissue.     

Nonadditive expression and parent-of-origin effects identified by microarray and allele-specific expression profiling of maize endosperm

Plant endosperm cells have a nuclear ratio of two maternal genomes to one paternal genome. This 2 to 1 dosage relationship provides a unique system for studying the additivity of gene expression levels in reciprocal hybrids. A combination of microarray profiling and allele-specific expression analysis was performed using RNA isolated from endosperm tissues of maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal hybrids at two developmental stages, 13 and 19 d after pollination. The majority of genes exhibited additive expression in reciprocal hybrids based on microarray analyses. However, a substantial number of genes exhibited nonadditive expression patterns, including maternal like, paternal like, high parent like, low parent like, and expression patterns outside the range of the parental inbreds. The frequency of hybrid expression patterns outside of the parental range in maize endosperm tissue is much higher than that observed for vegetative tissues. For a set of 90 genes, allele-specific expression assays were employed to monitor allelic bias and regulatory variation. Eight of these genes exhibited evidence for maternally or paternally biased expression at multiple stages of endosperm development and are potential examples of differential imprinting. Our data indicate that parental effects on gene expression are much stronger in endosperm than in vegetative tissues.     

Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.     

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.     

硬粒小麦与珍珠栗远缘杂交的受精作用和胚胎发育

对硬粒小麦（ＴｒｉｔｉｃｕｎｄｕｒｕｍＤｅｓｆ．）和珍珠栗（Ｐｅｎｎｉｓｅｔｕｍｇｌａｕｃｕｍｓｙｎ．Ｐ．ａｍｅｒｉｃａｎｕｍ）远缘杂交的受精作用和胚胎发育进行了研究。对授粉后的１９２个硬粒小麦子房进行制片观察,７．８１％发生双受精,具胚和胚乳,但胚乳发育往往落后于胚的发育;３．１３％只发生了单卵受精,只产生胚而无胚乳;１．０４％发生了单极核受精,只产生胚乳而无胚。总受精率为１１．９８％,成胚率为１０．９４％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子     

A debranching enzyme deficiency in endosperms of the sugary-1 mutants of maize

Many of the sugary-1 mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme [α-(1, 6)-glucosidase] activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing α-1,6 branch points into the linear α-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one of these peaks of activity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (0 to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant.     

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.     

薏苡胚乳发育及营养物质积累的研究

薏苡 ( Coix lacryma- jobi)授粉后 2 d,游离核胚乳已转变为细胞胚乳。授粉后 3d,中央细胞被胚乳细胞充满。起初 ,全部胚乳细胞均进行分裂 ,一定时期后 ,细胞分裂主要发生在胚乳周边区。授粉后 1 0 d,表皮停止平周分裂变为糊粉层 ,内方的数层形成层状细胞行平周分裂直到颖果接近成熟。胚乳内部生长则依赖于细胞体积扩大。胚乳基部 (颖果基部的胚乳 )形成了数层传递细胞。授粉后 9d,淀粉积累。授粉后 1 0 d,糊粉层及其内方数层细胞产生了脂体 ,后者的脂体以后又消失。授粉后 1 3、1 5 d,糊粉层细胞的液泡积累蛋白质。授粉后 2 0 d,液泡变为糊粉粒。授粉后 1 5 d淀粉胚乳细胞产生蛋白质体 ,营养物质积累持续到颖果成熟。还观察了胚和胚乳发育的对应关系。     

Expression of zein in long term endosperm cultures of maize

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by poly-acrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.     

Endosperm response to pollen irradiation in kiwifruit

 Cytological details of endosperm development after pollination with irradiated pollen were studied in Actinidia deliciosa (kiwifruit) cultivar Hayward. Pollinations were carried out involving five different sources of pollen (Matua, Tomuri, Burt, Berryman, and fruiting male) irradiated with gamma rays at doses of 700 and 900 Gy. Non-irradiated crosses were used as controls. Irradiated pollen induced development of approximately 25–30% of the ovules. Two types of ovules were observed: (1) with both embryo and endosperm and (2) with endosperm only. No mitotic abnormalities were found in control or irradiated endosperms. Mitotic divisions were regular and nuclei spherical and evenly spaced. However, the cells of irradiated endosperm usually contained low amounts of storage products. Ploidy level of the endosperms was evaluated by nuclear size (volume) with the use of image analyzis. Mean nuclear size in control and irradiated endosperms was 1598.3 and 750.9 μm³, respectively. It is concluded that endosperm produced after pollination with irradiated pollen is autonomous and represents the 2n level. Received: 14 October 1998 / Revision accepted: 10 March 1999     

Multivariate analyses of polypeptide synthesis in developing maize embryos

Summary Variation in polypeptide synthesis was examined in developing maize embryos of two inbred and two hybrid genotypes. Multivariate analyses were used to evaluate the variation among two-dimensional, electrophoretic separations of polypeptides. Several features of the data set were revealed. Similar developmental patterns were exhibited by all genotypes and no evidence was obtained for differential rates of development for inbreds and hybrids. The differential synthesis of two subsets of polypeptides during embryo development was observed. The multivariate methods employed in this study were a valuable aid in interpreting the results from a large and complex data set.     

Two Additional Phosphorylases in Developing Maize Seeds

Two additional phosphorylases (III and IV) have been detected in developing seeds of maize. Phosphorylase IV is found only in the embryo (with scutellum). It is also present in the embryo of the germinating seed where its activity is 90-fold greater than the activity in the developing embryo 22 days after pollination. Phosphorylase IV is eluted from a DEAE-cellulose column in the same fraction as phosphorylase I of the endosperm, and the 2 enzymes are similar in many respects. Phosphorylase IV is distinguished from phosphorylase I by electrophoretic mobility, by pH optimum, and because its properties are not affected by the shrunken-4 mutation.Phosphorylase III is found both in the endosperms and embryos of developing seeds. Activity for this enzyme is not detected in crude homogenates nor eluates from a DEAE-cellulose column apparently because it complexes with a non-dialyzable, heat-labile inhibitor. High activity is found after protamine sulfate fractionation. Phosphorylase III is bound to protamine sulfate and is then removed by washing with 0.3 m phosphate buffer. Phosphorylase III activity in the endosperm is not detectable 8 days after pollination but is present 12 days after pollination. Phosphorylase III differs from phosphorylases I, II, and IV in several respects-pH optimum, pH-independent ATP inhibition, time of appearance in the endosperm, and because purine and pyrimidine nucleotides are equally inhibitory. In common with phosphorylase II, phosphorylase III apparently does not require a primer to initiate the synthesis of an amylose-like polymer.     

STARCH GRANULE (AMYLOPLAST) DEVELOPMENT IN ENDOSPERM OF SEVERAL ZEA MAYS L. GENOTYPES AFFECTING KERNEL POLYSACCHARIDES

Endosperm cell and starch granule (amyloplast) development of six maize (Zea mays L.) genotypes, normal, amylose-extender (ae), sugary (su), waxy (wx), amylose-extender sugary (ae su), and amylose-extender waxy (ae wx), was compared. Endosperms of all genotypes were indistinguishable at 14 days after pollination. Cells were highly vacuolated and those in the central crown area of the kernel contained small starch granules in close association with the nucleus. Cellular and nuclear enlargement occurred during endosperm development in all genotypes, and major and minor gradients in physiological age of endosperm cells were observed in all kernels. Amyloplast development varied with genotype. Plastid development in normal and wx cells was characterized by an initial starch granule formation followed by granule enlargement to cell maturity. Endosperms homozygous for ae (ae, ae su, and ae wx) developed abnormal plastid-granules. Secondary granule formations preceded development of abnormality in ae and ae su, but not in ae wx endosperms. In contrast to ae and ae su starch granules, ae wx granules were highly birefringent indicating a high degree of crystallinity. In all three ae genotypes, abnormality increased as a function of kernel and physiological cell age. The su mutant had two distinct effects on amyloplast development. First, a mobilization of the initially formed starch, and second a synthesis and accumulation of phytoglycogen and the formation of large rounded plastids. In ae su plastid development, there was a mobilization of the starch initially formed (resulting in irregularly shaped, nonbirefringent granules) but only small amounts of phytoglycogen were produced.     

Correlation of cytokinin levels in the endosperms and roots with cell number and cell division activity during endosperm development in rice

Cell number and cell division activity in rice (Oryza sativa) endosperms are possibly regulated by cytokinin levels in the endosperm and its source in the roots. This study tried to find the possible correlations among them. Six rice genotypes were grown in nutrient solution. Two patterns of endosperm cell division, synchronous and asynchronous, were observed among the genotypes based on the cell division rate of superior and inferior spikelets. Contents of zeatin (Z) + zeatin riboside (ZR) were much higher than those of N6-isopentenyladenine (iP) and N6-isopentenyladenosine (iPR) in both endosperms and roots. Changes in Z + ZR levels in endosperms were significantly correlated with those in roots, and both were very significantly correlated with the cell division rate. Changes in iP + iPR contents in the roots were not significantly correlated with those in the endosperms and the cell division rate. When roots were treated with kinetin, endosperm cell number and grain weight were increased. Such enhancement was more significantly achieved by the root kinetin treatment than by spraying kinetin on leaves and panicles. The results suggest that the cell number and cell division activity in rice endosperms are regulated by cytokinin levels in the endosperm and that root-derived Z + ZR play a pivotal role.     

Activity and distribution of nitrogen-metabolism enzymes in the developing maize kernel

The activities of glutamine synthetase (EC 6.3.1.2), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and soluble protein content in the developing endosperm and embryo of normal (Oh-43) and mutant (Oh-430₂) maize were investigated. Maize inbred lines were grown under field conditions and all plants were self-pollinated. Ears for experiments were harvested over the period of 15 lo 45 days after pollination. After pollination kernel capacity for soluble protein synthesis is located mainly in the endosperm. This progressively decreases and about 40 days after pollination soluble protein synthesis is taken over by the embryo. Comparative data on the activity of the investigated enzymes in the embryo and endosperm indicate that the capacity for synthesis of glutamine and glutamate predominates in the embryo tissue, whereas transamination processes at the initial stages of the embryo development are less intensive than their counterparts in the endosperm. The roles of embryo and endosperm subsequently interchange. Biosynthetic processes of soluble precursors for protein synthesis in the embryo and endosperm of the developing kernel are mutually coordinated.