Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

Water deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post-mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post-fertilization period. Greenhouse-grown maize was subjected to water-limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical-region kernels, but had little effect on middle-region endosperms. Rewatcring did not allow cell division to recover in apical-region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical-region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A-content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA-content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.     

Endoreduplication of nuclear DNA in the developing maize endosperm

Multiparametric flow cytometry was used to analyze the development of the endosperm in Zea mays L. during the period from 8 to 20 days after pollination (dap). Nuclear size, DNA content per nucleus, and frequencies of nuclei with varying properties were measured in preparations that included all of the endosperm nuclei of single kernels of the inbred strain Al88. Characteristics of nuclear populations from different kernels on the same ear showed minimal variation. The dynamic changes of non-mitotic cells involved in endosperm development consisted of alternating periods of DNA replication with non-replication. Seven rounds of DNA replication had occurred in some nuclei in the later developmental stages with the rate averaging approximately one round per 24-hour period. Analysis of the DNA levels in the nuclei showed an exact doubling pattern indicating an endoreduplication process, that is, replication of the entire genome during each round. The loosely organized polytenization of the chromatin occurred to varying extents among the nuclei within an endosperm. A weak positive correlation existed between DNA content and size of nuclei suggesting that DNA increases and nuclear growth may not be highly coordinated in this tissue. Increased proportions of the larger nuclei occurred in the later stages of endosperm development. Considering the entire endosperm, the average DNA content per nucleus at the 15-dap peak level was approximately 12.8 C constituting a 2.7-fold overall increase from 8 dap.     

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     

The female gametophyte and the endosperm control cell proliferation and differentiation of the seed coat in Arabidopsis

Double fertilization of the female gametophyte produces the endosperm and the embryo enclosed in the maternal seed coat. Proper seed communication necessitates exchanges of signals between the zygotic and maternal components of the seed. However, the nature of these interactions remains largely unknown. We show that double fertilization of the Arabidopsis thaliana female gametophyte rapidly triggers sustained cell proliferation in the seed coat. Cell proliferation and differentiation of the seed coat occur in autonomous seeds produced in the absence of fertilization of the multicopy suppressor of ira1 (msi1) mutant. As msi1 autonomous seeds mostly contain autonomous endosperm, our results indicate that the developing endosperm is sufficient to enhance cell proliferation and differentiation in the seed coat. We analyze the effect of autonomous proliferation in the retinoblastoma-related1 (rbr1) female gametophyte on seed coat development. In contrast with msi1, supernumerary nuclei in rbr1 female gametophytes originate mainly from the endosperm precursor lineage but do not express an endosperm fate marker. In addition, defects of the rbr1 female gametophyte also reduce cell proliferation in the ovule integuments before fertilization and prevent further differentiation of the seed coat. Our data suggest that coordinated development of the seed components relies on interactions before fertilization between the female gametophyte and the surrounding maternal ovule integuments and after fertilization between the endosperm and the seed coat.     

The dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila

We describe a recessive, maternal-effect lethal mutation of Drosophila, gnu. gnu uncouples nuclear division from many cytoplasmic events of mitosis in the Drosophila embryo. Embryos from homozygous females are defective in nuclear division, but not in DNA replication, and therefore develop a small number of giant nuclei. Centrosomes divide independently of nuclear division and migrate to the surface of the syncytial blastoderm. There they nucleate microtubules into asters, which appear normal at first but become very large. Only later, when the giant nuclei begin to break down, are spindles sometimes formed. The cortical actin of these embryos develops into a characteristic network.     

The Aspergillus nidulans bncA1 mutation causes defects in the cell division cycle, nuclear movement and developmental morphogenesis

Wild-type Aspergillus nidulans conidia are uninucleate. The mutation bncA1 (binucleated conidia) was first described as a single mutation located on chromosome IV that caused formation of approximately 25% binucleate and 1% trinucleate conidia. In this study, we show that bncA1 conidia exit G1 arrest earlier than the wild type. Germlings have hyphal elements with abnormal morphology, elevated numbers of randomly distributed nuclei and an irregular septation pattern. Older hyphal elements undergo mitotic catastrophe, suggesting the nuclear division cycle of internal (nonterminal) elements is not arrested. The bncA1 mutation also causes aberrant morphogenesis of the asexual reproductive structure, the conidiophore. Metulae and phialides are elongated and have incorrect numbers of nuclei. Phialides also have internal septation that appears to delineate hyphal-like elements. Heterokaryon analysis using strains with contrasting auxotrophic markers showed that the bncA1 mutation resulted in a higher frequency of diploid and multinucleated prototrophic conidia than control heterokaryons. These results suggest that in bncA1 strains multiple nuclei can move from the conidiophore vesicle to the metulae and/or from the phialide to the conidium. The bncA1 mutant also showed hypersensitivity to the anti-microtubule drugs thiabendazole and nocodazole, which is consistent with the defects in cell cycle regulation and nuclear movement. We propose that bncA has an important role in correctly regulating both the cell division cycle and nuclear movement.     

Biosynthesis of lysine and other amino acids in the developing maize endosperm

Tracer studies with aspartic acid-[4-¹⁴C], alanine-[1-¹⁴C] acetate-[2-¹⁴C] and diaminopimelic acid-[1,(7)-¹⁴C] injected into the developing endosperm of maize revealed that the biosynthesis of lysine and other amino acids occurs in this organ. The data suggest that lysine is synthesized via the diaminopimelic acid pathway.     

细胞分裂过程中的组蛋白H3磷酸化

组蛋白是染色质中主要的蛋白质组分,经过复杂的翻译后修饰,主要包括乙酰化、甲基化、磷酸化、泛素化和ADP-核糖基化后,会改变染色质的结构及功能特性。组蛋白H3的磷酸化是高度保守的,发生在有丝分裂和减数分裂中特定的时期和染色体部位。在真核生物的不同物种中,组蛋白的磷酸化在染色体上的分布和起始时期是不同的,但常在中期磷酸化水平达到最高。在有丝分裂或减数分裂将结束的时候,H3普遍发生去磷酸化现象。不同组蛋白的共价修饰有不同的表观遗传学效应。     

The low phytic acid1-241 (lpa1-241) maize mutation alters the accumulation of anthocyanin pigment in the kernel

The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355–368, 2000; Pilu et al. in Theor Appl Genet 107:980–987, 2003a; Shi et al. Nat Biotechnol 25:930–937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP₆) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo (about 3.8-fold) or in the aleurone layer (about 0.3-fold) in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and expression analysis showed a reduction of ZmMRP3 anthocyanins’ transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalisation in the kernel.     

The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line

To understand the molecular basis of sensory organ development and disease, we have cloned and characterized the zebrafish mutation dog-eared (dog) that is defective in formation of the inner ear and lateral line sensory systems. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders.     

Glutamine synthetase activity of the endosperm, embryo and pedicel-placento-chalazal regions of developing maize (Zea mays) kernels

Glutamine synthetase (GS, EC 6.3.1.2) activity in homogenates of the maize ( Zea mays L. hybrid A619 X W64A) kernel pedicel-placento-chalazal (PPCh), endosperm regions was characterized in order to optimize assay (hydroxylamine-dependent γ-glutamyl hydroxymate formation) conditions for quantitating maize kernel GS in crude extracts. The GS activities of all three tissue extracts exhibited optima at pH 7.0 with ATP:Mg²⁺ of 1:1.6. Assays of kernel tissue GS activity required relatively high concentrations of substrates to achieve saturation compared to GS from other plant tissue sources, a point which has not been considered in previous reports of maize kernel GS activity. When measured under optimal assay conditions. PPCh-GS increased to a peak of 51 nmol γ-glutamyl hydroxymate kernel⁻¹ min⁻¹ at 25 days after pollination and then declined throughout the remainder of kernel development. Embryo GS activity increased steadily throughout development to a maximum of 24 nmol γ-glutamyl hydroxymate embryo⁻¹ min⁻¹ by 50 days after pollination. In contrast, endosperm GS activity, which was 25 nmol γ-glutamyl hydroxymate endosperm⁻¹ min⁻¹ at 25 days after pollination, exhibited no discernable pattern of change during kernel development. These findings are discussed with respect to the possible roles PPCh, endosperm and embryo GS play in kernel development.     

The Escherichia coli cell division mutation ftsM1 is in serU.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.