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Polycomb protein ZmEMF1a restricts endosperm proliferation and directs differentiation via stage-specific H2Aub1 and H3K27me3 landscapes in maize.

Polycomb group (PcG) proteins serve as pivotal epigenetic repressors that govern the transcriptional programs underlying cell growth and differentiation. However, their functional roles in maize endosperm remain largely unexplored. Here, we characterize the recessive maize small-kernel mutant sks1, which exhibits persistent endosperm cell hyperproliferation and compromised cell expansion during grain filling. Map-based cloning reveals that SKS1 encodes ZmEMF1a, a PcG protein that physically interacts with subunits of both PRC1 and PRC2. Integrated ChIP-seq and RNA-seq analyses were performed to investigate its epigenetic regulatory functions. ZmEMF1a orchestrates a stage-specific epigenetic regulatory program: it predominantly mediates H3K27me3 deposition at 6 d after pollination (DAP), while coordinately regulating the deposition of both H3K27me3 and H2Aub1 at 10 DAP. Loss of ZmEMF1a leads to ectopic hyperproliferation of differentiated endosperm tissues, specifically the basal endosperm transfer layer (BETL) and aleurone (AL), as well as elevated vitamin B content in the endosperm. Collectively, these findings establish ZmEMF1a as an epigenetic regulator that balances endosperm proliferation, cell fate specification, and nutrient accumulation through stage-specific histone modifications, thereby offering promising targets for enhancing maize yield and nutritional quality.

H2Aub1

[Patterns in the cellular organization of ovule growth in the pine, Pinus silvestris L. I. Growth processes in endosperm].

The dynamics of growth processes was studied in the endosperm of Pinus silvestris during the year of fertilization. The growth of endosperm is described by an diauxic curve where the period of delay corresponds, by time, to fertilization. The growth of tissue proceeds, mainly, at the expense of cell proliferation. The mitotic activity is preserved in the endosperm till the beginning of formation of reserve substances in the cells; cell division and elongation occur simultaneously. The unfavourable environmental conditions influence the mitotic activity of cells only. The size of fully grown cells is relatively constant and somewhat decreases later, during maturation. One can distinguish rather early those regions in the endosperm where the cell size is 2-3 times less than in the rest tissue; the size of cells and nuclei in these regions suffers only insignificant changes during the growth. The large nuclei are observed in the beginning of the cell endosperm formation and, then, their size decreases 1.5-2 times; the second increase in nuclear size in the endosperm is observed during the period prior to the formation of reserve substances.

Animals

Effects of floury-2 locus on zein accumulation and RNA metabolism during maize endosperm development.

Zein accumulation patterns during mutant and normal maize endosperm development were determined. Accompanying an increase in the number of floury-2 alleles present in the endosperm was a well-defined stepwise depression in zein accumulation. Analysis of the zein accumulated in endosperms containing zero, one, two, and three doses of the floury-2 allele by sodium dodecylsulfate--polyacrylamide gel electrophoresis revealed a proportionate reduction in the two major zein components, Z1 and Z2. In contrast, the relative proportions of the minor zein bands were altered. Membrane-bound polysomes isolated from kernels of floury-2 and normal maize were predominantly large size classes. The presence of increasing numbers of the floury-2 allele in the endosperm decreased recovery of membrane-bound polysomal material in a stepwise fashion. However, major alterations in polysome size-class distributions were not observed. The reduction in membrane-bound polysome material correlated linearly with reductions in in vitro zein synthesis and in vivo zein accumulation.

Electrophoresis, Polyacrylamide Gel

Actin in spindles of Haemanthus katherinae endosperm. II. Distribution of actin in chromosomal spindle fibres, determined by analysis of serial sections.

We have studied the arrangements of actin-containing filaments in 13 bundles of kinetochore microtubules in glycerinated, heavy meromyosin-treated Haemanthus endosperm cells: 7 bundles were in a cell at anaphase, and 6 were in a cell at metaphase. Actin-containing filaments were present in each of the 13 bundles of kinetochore microtubules: they were in amongst the microtubules in the bundle and seemed to be associated with the microtubules. Actin-containing filaments in each bundle seemed to terminate at the kinetochores. Actin-containing filaments associated with the kinetochore microtubules were of consistent polarity (the arrowheads pointed towards the kinetochores) whereas those associated with other microtubles and those not associated with microtubules did not have consistent polarity (some pointed towards the spindle pole, others pointed away from it). Roughly, there were as many individual stretches of actin-containing filaments identified per bundle of kinetochore microtubules as there were microtubules which terminated at the kinetochore. These data suggest that actin-containing filaments in spindles have a functional role. We used 2 glycerination procedures in our studies (one for each cell), and neither seemed to disrupt the basic microtubule arrangements: the arrangements of spindle microtubules seen after glycerination of Haemanthus endosperm were identical to those described previously by others in non-glycerinated glutaraldehyde-fixed Haemanthus endosperm. Thus we argue that spindle structure is not disrupted by the procedures, and therefore that the arrangements of actin-containing filaments are not artifacts of the glycerination procedures. The only difference between microtubules in glycerinated cells and microtubules in untreated cells is that there seem to be fewer in the glycerinated cells. The possible role of actin-containing filaments in the spindle is discussed.

Actins

Hypostase, embryonic sac and endosperm in Anethum graveolens L., and in various families.

The presence of pyriform hypostase tissue was investigated before double fertilization till the fusion of free endosperm nuclei in the embryonic sac of A. graveolens. Hypostase a glandular tissue, can secrete its cell contents into the embryonic sac during the development of free endosperm nuclei. Of 78 randomly selected families, 19 display different numbers of species with hypostase. In 15 of these families the hypostase is present together with the Polygonum type of embryonic sac and nuclear type of endosperm.

Cell Differentiation

Adaptation of the tRNA population of maize endosperm for zein synthesis.

Maize endosperm, 30 days after pollination is actively synthesizing zein, a storage protein containing high amounts of glutamine. leucine and alanine. Endosperm tRNAs have a higher accepting activity than embryo tRNAs for these three amino acids, but not for some other (control) amino acids. This increase in accepting activity is accompanied by a change in the distribution of the isoaccepting tRNA species corresponding to these three amino acids, but not of the isoacceptors corresponding to some other (control) amino acids. These results are in favor of the theory of functional adaptation of tRNA population.

Alanine

Cloning of double stranded DNAs derived from polysomal mRNA of maize endosperm: isolation and characterisation of zein clones.

Starting from polysomal RNA of developing maize endosperm and applying the cloning procedure of integrating dsDNA into the Pst I site of plasmid pBR 322, clones containing sequences complementary to endosperm mRNAs were obtained. 25 per cent of these clones were identified as containing zein specific DNA sequences which hybridized either with the zein mRNA coding for the 22 000 Mr protein or with the zein mRNA coding for the 19 000 Mr protein. The zein-specific DNA inserts of the recombinant plasmids were further characterized by restriction enzyme analysis.

DNA, Recombinant

Electrophoretic fractionation and translation in vitro of poly(rA)-containing RNA from maize endosperm. Evidence of two mRNAs coding for zein protein.

In the search for individual mRNAs coding for particular zein proteins, polysomal RNA was isolated from the endosperm of 22-days post-pollination maize kernels. This RNA was enriched for poly(rA)-containing RNAs and then submitted to preparative polyacrylamide gel electrophoresis under denaturing conditions. After electrophoresis, RNA fractions were eluted from the polyacrylamide gel and analyzed for zein mRNA activity by translation in vitro in the wheat germ system. The wheat germ system had previously been optimized for accurate translation of zein mRNAs. By a gel-electrophoretic analysis of immunoprecipitated products from the translation reactions in vitro, it could conclusively be shown that the endosperm of developing maize kernels contains two separable mRNAs for zein, one coding for the 22000-Mr protein and one coding for the 19000-Mr protein.

Electrophoresis, Polyacrylamide Gel

Actin in spindles of Haemanthus katherinae endosperm. I. General results using various glycerination methods.

We have studied actin-containing filaments in spindles in Haemanthus endosperm cells glycerinated by various methods; the actin-containing filaments were identified by their reaction with rabbit skeletal muscle heavy meromyosin (HMM) to form 'decorated' filaments. Actin-containing filaments in the spindle were seen in amongst microtubules in bundles (both non-kinetochore microtubule bundles and kinetochore microtuble bundles) and were also seen not associated with microtubules. There were very few extra-spindle actin-containing filaments in these cells. Actin-containing filaments seemed to interact with microtubules, because the filaments remained close to and parallel to microtubules even when the microtubules were sharply curved. Because of the close association between microtubules and actin-containing filaments we could not identify all the actin-containing filaments present in microtubule bundles: microtubules obscured actin-containing filaments. We studied Haemanthus endosperm cells as they were glycerinated. For some of these observations we used phase-contrast microscopy. Glycerination caused the cells to shrink, initially, and this was followed by rapid expansion, but the cells did not expand to as large a volume as before glycerination. Spindle structure was maintained despite these changes in cell size. Evidences for this are that relative chromosome positions were maintained during glycerination, that spindle birefringence was maintained during glycerination, and that individual chromosomal spindle fibres remained birefringent during glycerination. Electron-microscopic observations supported this in that kinetochore microtubule bundles and non-kinetochore microtubule bundle were maintained during glycerination, as was the helical arrangement of spindle ribosomes into polyribosomes. One-step glycerination procedures were used (cells were treated with mixtures containing 25% glycerol, Triton-X-100 and HMM), and such procedures might be of general use. Living cells were embedded in fibrin clots in making light-microscopic observations; this procedure, too, might be of general use.

Actins

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 chromosomes. With a slight modification the technique has been applied successfully to root tips and other tissues.

Chromosomes

Comparative susceptibility to amylases of starch granules of several single endosperm mutants representative of floury-opaque, starch-deficient, and modified starch types and their double-mutant combinations with opaque-2 in four inbred lines of maize.

Starch granules were prepared from kernels of eight single endosperm mutants, brittle-1, (bt1), brittle-2 (bt2), floury-1, floury-2, soft starch, opaque-1 (o1), shrunken-2 (sh2), and sugary-2 (su2), and their double-mutant combinations with opaque-2 (o2) of four inbred lines of maize (Zea mays L.), B37, C103, Oh43 and W64A. We compared the susceptibility of various starch granules to Rhizopus glucoamylase and pancreatin. Starch granules of the su2 and su2o2 mutants were digested by amylases much faster than those of the normal counterparts. Starch granules of the bt1, bt2, o1 and sh2 mutants tended to be digested by amylases faster than those of normal maize. Starch granules of double-mutant combinations with the o2 gene were, in general, digested to an extent very comparable to their respective non-opaque single mutant counterparts in each of their four inbred backgrounds. We followed the relative digestion of starch granules by using scanning electron microscopy. Starch granules of endosperm mutants susceptible to amylases showed numerous pin holes on the surface layer and the pores penetrated into the inner layers of the granules during the attack by amylases. In some of the granules the inner portion, which appeared terraced or step-shaped, could be seen. This may be indicative of layered internal structures of the granules.

Amylases

Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles.

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.

Adenosine Triphosphatases

Reaction of the anastral mitotic apparatus of endosperm cells of the plant Leucojum aestivum with antibodies to tubulin from porcine brain as revealed by immunofluorescence microscopy.

Structures binding an antibody against tubulin from porcine brain were localized in the giant anastral mitotic apparatus of endosperm cells of the monocotyledonous plant, Leucojum aestivum, by indirect immunofluorescence microscopy. Both continuous and chromosomal spindle fibers were strongly stained. Postive fluorescence was also noted in polar cap regions and, in prometaphase stages, to some extent at the fragmented nuclear envelope. Intermingling and branching of subfiber elements was frequently noted.

Animals

Diffusion of water in the endosperm tissue of wheat grains as studied by pulsed field gradient nuclear magnetic resonance.

Pulsed field gradient nuclear magnetic resonance has been used to measure water self-diffusion coefficients in the endosperm tissue of wheat grains as a function of the tissue water content. A model that confines the water molecules to a randomly oriented array of capillaries with both transverse dimension less than 100 nm has been used to fit the data and give a unique diffusion coefficient at each water content. The diffusion rates vary from 1.8 x 10(-10) m2s-1 at the lowest to 1.2 x 10(-9) m2s-1 at the highest moisture content. This variation can be explained in terms of an increase in water film thickness from approximately 0.5 to approximately 2.5 nm over the moisture range investigated (200-360 mg g-1).

Diffusion

Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor-bean endosperm.

Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.

Ricinus communis

Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings.

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 X 10(-5) M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 X 10(-4) M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 X 10(-4) M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a 'neutral' fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.

Adenosine Monophosphate