Oil Body Biogenesis during Brassica napus Embryogenesis

Although the oil body is known to be an important membrane enclosed compartment for oil storage in seeds, we have little understanding about its biogenesis during embryogenesis. In the present study we investigated the oil body emergence and variations in Brassica napus cv. Topas. The results demonstrate that the oil bodies could be detected already at the heart stage, at the same time as the embryos began to turn green, and the starch grains accumulated in the chloroplast stroma. In comparison, we have studied the development of oil bodies between Arabidopsis thaliana wild type （Col） and the low-seed-oil mutant wrinkled1-3. We observed that the oil body development in the embryos of Col is similar to that of B. napus cv. Topas, and that the size of the oil bodies was obviously smaller in the embryos of wrinkled1-3. Our results suggest that the oil body biogenesis might be coupled with the embryo chloroplast.     

Oil Body Biogenesis during Brassica napus Embryogenesis

Although the oil body is known to be an important membrane enclosed compartment for oil storage in seeds, we have little understanding about its biogenesis during embryogenesis. In the present study we investigated the oil body emergence and variations in Brassica napus cv. Topas. The results demonstrate that the oil bodies could be detected already at the heart stage, at the same time as the embryos began to turn green, and the starch grains accumulated in the chloroplast stroma. In comparison, we have studied the development of oil bodies between Arabidopsis thaliana wild type (Col) and the low-seed-oil mutant wrinkled1–3 . We observed that the oil body development in the embryos of Col is similar to that of B. napus cv. Topas, and that the size of the oil bodies was obviously smaller in the embryos of wrinkled1–3 . Our results suggest that the oil body biogenesis might be coupled with the embryo chloroplast.     

Changes in Nuclear DNA Content, Cell and Nuclear Size, and Frequency of Cell Division in the Cotyledons of Brassica napus L. during Embryogenesis

Cellular behaviour was examined during embryogenesis in Brassicanapus to test whether or not polyploidy occurs in the cotyledonsduring the phase of oil deposition. Nuclear DNA content, nuclearand cell size, and the mitotic index were measured in the cotyledonson various days post anthesis (dpa). In squashed monolayersfrom 15 dpa cotyledons, a polyploid (>5C) population wasdetected together with a substantial number of cells in G2 (4C).Nuclear volume was measured on sectioned tissues and, at 15dpa, the range of values from the cotyledons (40–500 *m³)contrasted with that in the vestigial suspensor and endosperm(50–> 600 µm³). At 15 dpa the nuclear volumedata suggest that whilst cells in the cotyledons were in Gland G2 many endosperm and suspensor cells were polyploid. Thus,polyploidy observed in the squashed monolayers was probablydue to contaminating endosperm/suspensor cells. At 25 and 35dpa, polyploidy was not detected; all cells were in Gl (2C)and cell area increased. The mitotic index peaked at 20 dpabefore declining and given the narrower distribution of nuclearvolumes at 25 and 35 dpa (50–300 µm³), these dataare consistent with cell arrest in Gl. Thus, polyploidy wasnot detected in the cotyledons of B. napus which differs fromwhat is known about cellular development in legume cotyledons. Key words: Brassica napus L., DNA, nuclear volume     

甘蓝型油菜游离小孢子培养的胚胎发生

以生长在非控温控光下的４个冬性甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）品种为供体,进行游离小孢子培养。研究发现,多数品种在开花３－７天取材最为适宜。在甘蓝型油菜小孢子培养中只有单核晚期的小孢子才可能发育成胚状体,而花药培养时处于单核早期的小孢子易于发育成胚状体。在适当花期选取发育比较一致的单核晚期小孢子培养,经数小时后,部分小孢子便开始膨大,这是小孢子发育成胚的最早标志,膨大的小孢子中,有部分形成多细胞球并进一步发育成胚。用春性甘蓝型油菜为材料进行蔗糖浓度的实验结果表明：培养３天后,在１６％蔗糖培养基中存活的小孢子最多,达１６．１３％;培养３０天后,胚状体诱导频率则以１３％蔗糖浓度为最高,每花蕾可达１４４个胚状体。如果在１６％蔗糖培养基中培养３天后,添加等体积的１３％蔗糖培养基,能够大大提高胚状体的诱导频率,为仅用１３％蔗糖培养基培养的３．７倍。这一实验体系正在用于抗菌核病的诱变与筛选,并作为外源基因导入的实验体系。     

Spatio-Temporal Plasticity in Chromatin Organization in Mouse Cell Differentiation and during Drosophila Embryogenesis

Cellular differentiation and developmental programs require changing patterns of gene expression. Recent experiments have revealed that chromatin organization is highly dynamic within living cells, suggesting possible mechanisms to alter gene expression programs, yet the physical basis of this organization is unclear. In this article, we contrast the differences in the dynamic organization of nuclear architecture between undifferentiated mouse embryonic stem cells and terminally differentiated primary mouse embryonic fibroblasts. Live-cell confocal tracking of nuclear lamina evidences highly flexible nuclear architecture within embryonic stem cells as compared to primary mouse embryonic fibroblasts. These cells also exhibit significant changes in histone and heterochromatin binding proteins correlated with their distinct epigenetic signatures as quantified by immunofluorescence analysis. Further, we follow histone dynamics during the development of the Drosophila melanogaster embryo, which gives an insight into spatio-temporal evolution of chromatin plasticity in an organismal context. Core histone dynamics visualized by fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence anisotropy within the developing embryo, revealed an intriguing transition from plastic to frozen chromatin assembly synchronous with cellular differentiation. In the embryo, core histone proteins are highly mobile before cellularization, actively exchanging with the pool in the yolk. This hyperdynamic mobility decreases as cellularization and differentiation programs set in. These findings reveal a direct correlation between the dynamic transitions in chromatin assembly with the onset of cellular differentiation and developmental programs.     

Auxin Polar Transport Is Essential for the Establishment of Bilateral Symmetry during Early Plant Embryogenesis

We used an in vitro culture system to investigate the effects of three auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, trans-cinnamic acid, and 2,3,5-triiodobenzoic acid) on the development of early globular to heart-shaped stage embryos of Indian mustard (Brassica juncea) plants. Although the effective concentrations vary with the different inhibitors, all of them induced the formation of fused cotyledons in globular ([less than or equal to]60 [mu]m) but not heart-shaped embryos. Inhibitor-treated Brassica embryos phenocopy embryos of the Arabidopsis pin-formed mutant pin1-1, which has reduced auxin polar transport activity in inflorescence axes, as well as embryos of the Arabidopsis emb30 (gnom) mutant. These results indicate that the polar transport of auxin in early globular embryos is essential for the establishment of bilateral symmetry during plant embryogenesis. Based on these observations, we propose two possible models for the action of auxin during cotyledon formation.     

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichter's (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichter's medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

The Mechanisms and Frequencies of Cell Nuclei Division in the Mouse Trophoblast and Decidua during Postimplantation Embryogenesis

Our finding of amitotic division of trophoblast cell nuclei in blastocysts of American mink Mustela vison, which has an obligatory period of delay in implantation (obligatory embryonic diapause) in its ontogenesis, led us to study the mechanisms and frequencies of division of trophoblast and decidua cell nuclei during the postimplantation embryogenesis of mouse Mus musculus, which does not exhibit an obligatory diapause nor amitosis in blastocysts. It has been established that the main mechanism underlying the cell nuclei division in both tissues (trophoblast and decidua) forming the placenta is amitosis. These data suggest that the occurrence of an obligate diapause in ontogenesis of certain animal species is related not only to the delay in implantation, but also to the alteration in the chronology of all processes of embryogenesis.     

A Plausible Microtubule-Based Mechanism for Cell Division Orientation in Plant Embryogenesis

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秋水仙碱对甘蓝型油菜离体小孢子胚胎发生的影响

研究了秋水仙碱不同浓度和处理时间对甘蓝型油菜23个基因型离体小孢子胚胎发生的影响.3个基因型的小孢子被10、50和100mg/L秋水仙碱处理24h或48h,胚产量是2.55～14.75胚/蕾,10～50mg/L处理72h则是0.94～2.43胚/蕾.这表明处理72h对小孢子胚发生有抑制作用.用200、400、500和800mg/L处理2个基因型小孢子16～48h,胚产量为0.6～1.33胚/蕾,未处理对照是6.25和9.36胚/蕾.可见200～800mg/L浓度对胚再生有不同程度的阻碍效应.结果还证明,小孢子对秋水仙碱的反应与其基因型有关.当用10、20、50和100mg/L处理48h时,22B5-6和903-3小孢子的胚产量为37.09～69.47胚/蕾,而F1-29、W592和SF10-12是0.28～1.45胚/蕾,相互之间差异很大.秋水仙碱处理小孢子的目的是使其再生植株的染色体高频率加倍,因此应根据胚产量和染色体加倍率来确定秋水仙碱浓度和处理时间.本试验中,采用10～50mg/L处理48h或者用100mg/L处理24h,约80%基因型的小孢子胚产量在5胚/蕾以上,约70%基因型的再生植株加倍率达60%以上,可有效地用于油菜遗传和育种研究等领域.     

Enhancement of Naphthaleneacetic Acid-Induced Rhizogenesis in T(L)-DNA-Transformed Brassica napus without Significant Modification of Auxin Levels and Auxin Sensitivity

Determination of the abscisic acid and indoleacetic acid (IAA) contents of floral stem segments of nontransformed and pRi A₄ T_L-DNA-transformed rape (Brassica napus L. var oleifera, cv Brutor) using a high performance liquid chromatography-enzyme-linked immunosorbent assay procedure and mass spectrometry controls showed that IAA levels were not modified. The regeneration abilities of the in vitro cultured explants were compared on media supplemented with several plant growth regulator combinations. No regeneration occurred on hormone-free media, and shoot production was similar in both genotypes when supplemented with benzyladenine. In the presence of naphthaleneacetic acid (NAA), transformed explants were characterized by faster root regeneration and reduced shoot organogenesis. The optimum for root formation was the same in nontransformed and transformed plants, but the sensitivity threshold was slightly lower in the latter. The NAA inductive period was shorter (14 versus 22 h) with transformed tissue. Root neoformation occurred about 72 h earlier on transformed explants. Our results suggest mainly that there is an acceleration of the auxinic signal transduction and/or that the events preliminary to the formation of roots occur faster in the transformed tissues than in the normal ones.     

A Division in PIN-Mediated Auxin Patterning during Organ Initiation in Grasses

The hormone auxin plays a crucial role in plant morphogenesis. In the shoot apical meristem, the PIN-FORMED1 (PIN1) efflux carrier concentrates auxin into local maxima in the epidermis, which position incipient leaf or floral primordia. From these maxima, PIN1 transports auxin into internal tissues along emergent paths that pattern leaf and stem vasculature. In Arabidopsis thaliana, these functions are attributed to a single PIN1 protein. Using phylogenetic and gene synteny analysis we identified an angiosperm PIN clade sister to PIN1, here termed Sister-of-PIN1 (SoPIN1), which is present in all sampled angiosperms except for Brassicaceae, including Arabidopsis. Additionally, we identified a conserved duplication of PIN1 in the grasses: PIN1a and PIN1b. In Brachypodium distachyon, SoPIN1 is highly expressed in the epidermis and is consistently polarized toward regions of high expression of the DR5 auxin-signaling reporter, which suggests that SoPIN1 functions in the localization of new primordia. In contrast, PIN1a and PIN1b are highly expressed in internal tissues, suggesting a role in vascular patterning. PIN1b is expressed in broad regions spanning the space between new primordia and previously formed vasculature, suggesting a role in connecting new organs to auxin sinks in the older tissues. Within these regions, PIN1a forms narrow canals that likely pattern future veins. Using a computer model, we reproduced the observed spatio-temporal expression and localization patterns of these proteins by assuming that SoPIN1 is polarized up the auxin gradient, and PIN1a and PIN1b are polarized to different degrees with the auxin flux. Our results suggest that examination and modeling of PIN dynamics in plants outside of Brassicaceae will offer insights into auxin-driven patterning obscured by the loss of the SoPIN1 clade in Brassicaceae.     

Drought-Induced Changes in Protein Patterns of Brassica napus var. oleifera Roots

Drought-induced changes in two-dimensional silver stained protein patterns of Brassica napus L. var. oleifera M. root system were detected both at quantitative and qualitative levels. Particularly, 13 new polypeptides of low molecular weight were evidenced in the drought-stressed tap root, 12 of which were also present in the short tuberized roots, a specific drought-induced root type. The reversibility of these modifications, observed after 3 days rehydration, suggests that they might be involved in drought tolerance.     

植物生长素与体细胞胚发生

简要介绍了植物生长素对体细胞胚发育的作用及其调控机制的研究进展。     

Acyl-CoA elongase expression during seed development in Brassica napus

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.