Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.     

Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm.

A previous study [Rahman, Shewry & Miflin (1982) J. Exp. Bot. 33, 717-728] showed differential accumulation of the major storage proteins (called B and C hordeins) in developing endosperms of barley (Hordeum vulgare). To determine how this accumulation is regulated, we have studied mRNA fractions prepared from similar endosperms. Hordein-related mRNA species were detected some days before the deposition of hordeins in vivo. The translation products in vivo directed by polyribosomes, polysomal RNA and total cellular RNA showed similar changes in the proportions of the hordein products to those observed in the accumulations of the proteins in vivo. There was a relative increase in one of the subfamilies of B hordeins (called B1 hordein) and a decrease in the second subfamily of B hordeins (B3 hordein) and in C hordeins. The populations of RNA species related to these three groups of hordeins were studied by 'dot hybridization', with specific complementary-DNA probes for B1-, B3- and C-hordein-related sequences. This showed a 10-15-fold increase in sequences related to the B1 hordein during endosperm development, but only a 4-fold increase in sequences related to B3 and C hordeins. These results indicate that the rates of synthesis of hordeins are related to the abundance of their respective mRNA species. The different results observed for the two subfamilies of B hordeins are of interest, since they indicate differential expression of two subfamilies of genes present at a single multigenic locus.     

油菜裂外壁小孢子胚在分子水平上具有胚体/胚柄分化

早期合子胚取材困难, 难以开展相关研究。前人的工作表明, 油菜(Brassica napus)裂外壁小孢子胚胎发生系统能够较好地模拟合子胚的分化模式, 因而可替代早期合子胚胎作为研究材料。但目前尚缺乏该胚胎发生系统中胚胎具有胚体/胚柄分化的分子水平的证据。该文首次证明了油菜WOX家族基因能够用于标记胚体/胚柄的分化过程, 利用胚柄标记基因BnWOX8的表达模式, 从分子水平上证明了带胚柄的裂外壁小孢子胚的确存在胚体/胚柄的分化。研究结果为充分利用油菜裂外壁小孢子胚胎发生系统, 解决早期胚胎取材困难的问题奠定了坚实的基础。同时, 建立了活体激光切割分离特定细胞的技术, 结合用于少量细胞RNA提取的活体特异细胞RNA提取技术, 为鉴定少量特异分化细胞的基因表达模式提供了一个可行且明确的解决方案。     

Young microspore-derived maize embryos show two domains with defined features also present in zygotic embryogenesis

In this work we report the existence of two domains in early microspore maize proembryos displaying similar features of zygotic embryogenesis. The large or so-called endosperm-like domain exhibits specific features: coenocytic organisation, synchronous mitosis, vacuolated cytoplasm, starch granules, incomplete walls containing callose and differential tubuline organisation. The small or embryo-like domain displays small polygonal uninucleate cells with typical organisation of proliferating cells. The structural organisation and the subcellular localization of specific cytoplasmic and cell wall components (starch, tubuline and callose) in both proembryo domains have been determined by using specific cytochemical and immunocytochemical methods. Four morphological types of proembryos containing both domains have been characterised. Taking into account their relative size, the high asynchrony of the culture and the homologies between structural features of endosperm-like domain and zygotic endosperm development, they could represent different stages in microspore embryogenesis development. The ZmAE1 and ZmAE3 genes expressed in the Embryo Surrounding Region of the endosperm during zygotic embryogenesis (Magnard et al., 2000), were revealed to be expressed in early microspore proembryos by in situ hybridization at light and electron microscopy levels. This data supports the existence of an endosperm-like function during early microspore embryogenesis and provides new insights into the onset of microspore embryogenesis in maize, and its parallelism with zygotic embryogenesis.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

Spatio-temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis, seed development, and germination in sandalwood

Western-blot analysis and protein kinase assays identified two Ca(2+)-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages; the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes.     

Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.     

Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

Comparative ultrastructural analysis of the in vitro microspore embryoids and in vivo zygotic embryos of wheat as a basis for understanding of cytophysiological aspects of their development

Ultrastructures of in vitro microspore embryoids and in vivo zygotic embryos of spring wheat have been analyzed and compared. Along with the similarity of ultrastructural characteristics of embryoid and embryo cells at the corresponding developmental stages, some differences have been revealed. Unlike embryos, embryoid cells are characterized by lipid inclusions and numerous mitochondria with well-developed internal membranes. According to our hypothesis, lipids represent an alternative energy source required for active cell divisions in the forming embryoids. Unlike embryos, since the earliest developmental stages, embryoid cells accumulate a significant amount of starch and then utilize it during the organogenesis and germination. A conclusion has been made that embryoid cells create their own reserve of carbohydrates, which is then mobilized during their development. The concept of T.B. Batygina (1987, 1997, 2014) about the universal character of the plant morphogenesis in vivo, in situ, and in vitro has been confirmed. The prospects for the use of microspore embryoidogenesis in vitro as a model to study cytophysiological aspects of zygotic embryogenesis in vivo are discussed.     

Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare)

We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.     

Comparative Morphological Study of Zygotic and Microspore-derived Embryos ofBrassica napusL. as Revealed by Scanning Electron Microscopy

A comparative morphological study of microspore-derived (MD)and zygotic embryos ofBrassica napusL. was conducted, illustratingsubstantial similarities in external morphology of these embryosthroughout their development. Haploid embryos were producedfrom isolated microspores cultured on high molecular weightpolyethylene glycol (PEG), replacing sucrose as an osmoticum.Morphological changes during the time-course of microspore embryodevelopment induced on PEG (25%) and sucrose (13%) are describedin detail as revealed by scanning electron microscopy (SEM)and compared to the corresponding stages of zygotic embryosdevelopedin ovulo. At the heart, torpedo and early cotyledonarystages, microspore-derived (MD) embryos on PEG closely resembletheir zygotic counterparts. In contrast, the external morphologyof embryos induced on high sucrose medium differs from thatof PEG and zygotic embryos indicating that a high concentrationof sucrose in culture has a morphogenetic effect on MD embryodevelopment inB. napus. Fragments of the original pollen wallare regularly observed at the root pole region and at the tipsof suspensors in MD embryos throughout their development. Thissuggests that polarity in MD embryos might originate from structurallypolarized late uninuclear microspores and early bicellular pollen.Copyright1998 Annals of Botany Company Brassica napusL., scanning electron microscopy, microspore-derived embryo, zygotic embryo, morphology, microspore, suspensor, exine, sucrose, polyethylene glycol.     

Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15

The MADS domain protein AGL15 (AGAMOUS-Like 15) has been found to preferentially accumulate in angiosperm tissues derived from double fertilization (i.e. the embryo, suspensor, and endosperm) and in apomictic, somatic, and microspore embryos. Localization to the nuclei supports a role in gene regulation during this phase of the life cycle. To test whether AGL15 is involved in the promotion and maintenance of embryo identity, the embryogenic potential of transgenic plants that constitutively express AGL15 was assessed. Expression of AGL15 was found to enhance production of secondary embryos from cultured zygotic embryos, and constitutive expression led to long-term maintenance of development in this mode. Ectopic accumulation of AGL15 also promoted somatic embryo formation after germination from the shoot apical meristem of seedlings in culture. These results indicate that AGL15 is involved in support of development in an embryonic mode.     

Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize ( Zea mays L.)

Expression of knotted1 ( kn1) and ZmLEC1, a maize homologue of the Arabidopsis LEAFY COTYLEDON1 ( LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize ( Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5-10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize.     

Differential gene expression during seed germination in barley (Hordeum vulgare L.)

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process. Electronic Publication     

Comparative analysis of early embryonic sunflower cDNA libraries

To gain information concerning cell functions and activities during sunflower embryogenesis, an expressed sequence tag (EST) approach was used to analyse gene expression in the early stages of sunflower embryos development. Confocal microscopy observations of whole-mounted embryos allowed us to identify precisely the major steps of the zygotic embryonic development. A time-course analysis was then employed to collect the embryonic material. Three cDNA libraries were constructed from microdissected embryos, and three other cDNA libraries were created using a classical day after pollination schedule. A total of 7106 ESTs were produced and assembled. The total number of putative different genes represents about 43.1 (3064 tentative contigs and singlets) of the analysed sequences. The unigenes that showed similarity to proteins with known or predicted functions (50.3) were classified into 15 different functional categories. The functional profiles were found to be quite similar for all studied embryo stages but statistical analysis revealed that successive and coordinate sets of genes are expressed at each embryonic stage. The analysis allowed us to identify abundant and differentially expressed genes at the early stages of embryos development as well as some putatively interesting genes, showing strong similarities with genes playing key roles in plant and animal embryogenesis. The data presented in this study not only provide a first global overview of the genes expression profile during sunflower embryogenesis but also represent an original and valuable tool for developmental genomics studies on exalbuminous dicots.