Fine structural observations of embryo development inPelargonium XHortorum bailey: with normal and mutant plastids

Summary The ultrastructural changes in the cotyledon, radicle and suspensor haustorium ofPelargonium, containing either normal or mutant plastids, are investigated from the heart stage of embryogenesis to the mature seed. The fine structure of parenchymatous cells from the cotyledon and radicle is essentially similar whereas that of the suspensor haustorium is very different.The cotyledon and radicle develop into one massive storage tissue possessing numerous lipid and several protein bodies per cell, and well developed starch grains. The suspensor haustorium has no storage function, rather it acts as a transitory tissue which dies off as the seed matures. The extensive chloroplast development suggests that, in addition to its traditional role, the suspensor haustorium also acts as a photosynthetic booster for the developing embryo.The development of surviving mutant embryos is similar to normal ones except that in cotyledon and radicle cells plastids develop only to vesicles, which associate into loose prolamellar bodies and sometimes small fenestrated thylakoids, and in the suspensor haustorium cells, only to small compact grana.     

Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.     

Gravitropism in a starchless mutant of Arabidopsis

The starch-statolith theory of gravity reception has been tested with a mutant of Arabidopsis thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (WT) seedlings, starch-filled plastids in the starch sheath of the hypocotyl and in three of the five columellar layers of the root cap were piled on the cell floors, and sedimented to the ceilings when the plants were inverted. However, starchless plastids of the mutant were not significantly sedimented in these cells in either upright or inverted seedlings. Gravitropism of light-grown seedling roots was vigorous: e.g., 10^o curvature developed in mutants rotated on a clinostat following a 5 min induction at 1 · g, compared with 14^o in the WT. Curvatures induced during intervals from 2.5 to 30 min were 70% as great in the mutant as the WT. Thus under these conditions the presence of starch and the sedimentation of plastids are unnecessary for reception of gravity by Arabidopsis roots. Gravitropism by hypocotyls of light-grown seedlings was less vigorous than that by roots, but the mutant hypocotyls exhibited an average of 70–80% as much curvature as the WT. Roots and hypocotyls of etiolated seedlings and flower stalks of mature plants were also gravitropic, although in these cases the mutant was generally less closely comparable to the WT. Thus, starch is also unnecessary for gravity reception in these tissues.Abbreviations PAR photosynthetically active radiation - PAS periodic acid-Schiff's reagent - PGM phosphoglucomutase - WT wild-type     

A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death

Seeds of the longcell mutant in maize (Zea mays L) have a defective-kernel phenotype: the embryo aborts at the early coleoptilar stage and the endosperm is reduced in size. Mutant embryos have severe alterations in morphogenesis. They have a suspensor-, an embryo axis- and a scutellum-like structure, but the shoot apical meristem (SAM) is not formed. Scanning electron microscopy showed that most of the cells in longcell embryos are tubular and abnormally enlarged. The level of expression of several genes involved in basic metabolism is not severely affected during early and mid embryogenesis, but storage molecule accumulation is reduced. Genes which in normal conditions are only expressed after germination, are expressed during kernel development in the longcell seeds. Mutant embryos undergo cell death in late embryogenesis. Nuclei in dying embryos are TUNEL positive, and different genes coding for hydrolytic enzymes are up-regulated. The expression of genes related to oxidative stress is also altered in longcell embryos. These results lead us to suggest that the longcell mutant may be cytokinesis-defective.     

Novel patterns of ectopic cell plate growth and lipid body distribution in the Arabidopsis gemini pollen1 mutant

The nature of aberrant gametophytic cell divisions and altered pollen cell fate in the gemini pollen1 (gem1) mutant was investigated through ultrastructural analysis. The earliest noticeable defect in gem1 was the appearance of extended membrane profiles at the early bicellular stage. These were replaced by ectopic internal walls, which divided the cytoplasm into twin or multiple cell compartments. Complete or partial internal walls were callosic with highly complex profiles, indicating failed guidance or deregulated cell plate growth. Extended membrane profiles and delayed callose synthesis at division sites further suggested a novel pattern of cell plate assembly in gem1. Multiple cell compartments in gem1 adopted vegetative cell fate with regard to lipid body distribution. In the wild type, lipid bodies appear specifically in the vegetative cell, whereas in gem1, lipid bodies accumulated in all cytoplasmic compartments. Our results support the hypothesis that altered pollen cell fate in gem1 results from abnormal inheritance of cell fate determinants as a result of disturbed cytokinesis.     

A mutation of the CRUMPLED LEAF gene that encodes a protein localized in the outer envelope membrane of plastids affects the pattern of cell division, cell differentiation, and plastid division in Arabidopsis

We identified a novel mutation of a nuclear-encoded gene, designated as CRUMPLED LEAF (CRL), of Arabidopsis thaliana that affects the morphogenesis of all plant organs and division of plastids. Histological analysis revealed that planes of cell division were distorted in shoot apical meristems (SAMs), root tips, and embryos in plants that possess the crl mutation. Furthermore, we observed that differentiation patterns of cortex and endodermis cells in inflorescence stems and root endodermis cells were disturbed in the crl mutant. These results suggest that morphological abnormalities observed in the crl mutant were because of aberrant cell division and differentiation. In addition, cells of the crl mutant contained a reduced number of enlarged plastids, indicating that the division of plastids was inhibited in the crl. The CRL gene encodes a novel protein with a molecular mass of 30 kDa that is localized in the plastid envelope. The CRL protein is conserved in various plant species, including a fern, and in cyanobacteria, but not in other organisms. These data suggest that the CRL protein is required for plastid division, and it also plays an important role in cell differentiation and the regulation of the cell division plane in plants. A possible function of the CRL protein is discussed.     

Turnover of cell-wall polysaccharides during somatic embryogenesis and development of celery (Apium graveolens L.)

Non-embryogenic cells (NEC) and embryogenc cells (EC) were separated from cell clusters derived from the hypocotyl segments of celery seedlings, which had been suspension-cultured in MS medium supplemented with 10⁵ M 2,4-D. The EC formed globular embryos in medium without 2,4-D. The globular embryo developed through heart-shaped, torpedo to cotyledonary embryos within 10 days. The EC and developing embryos were fractionated into symplastic [MeOH, hot water (HW), starch (S)] and apoplastic [pectin, hemicellulose, TFA (trifluoroacetic acid)-soluble and cellulose] fractions. The EC contained lower levels of sugar in the MeOH fraction and higher levels of starch than NEC. In the apoplastic fractions, there were no differences of total sugar amounts between NEC and EC. Cellulose contents were about 10% of the wall polysaccharides. During somatic embryogenesis, total sugar contents of the MeOH and HW fractions increased till the heart-shaped embryo stage, and then decreased during the torpedo and cotyledonary embryo stages. The sugar contents of the starch, pectin, TFA-soluble, and cellulose fractions did not change during the stages mentioned above. However, the hemicellulose substances remarkably increased during embryogenesis, and then decreased as the development proceeded. The neutral sugar components of the hemicellulosic fractions were analyzed. Arabinose increased markedly in EC to the globular embryo stage, but decreased as the development proceeded. Galactose increased only at the torpedo and cotyledonary embryo stages. Xylose was present at lower levels in all stages of embryogenesis than in the differentiated hypocotyl cell walls. These results suggest that there was a high turnover of arabinogalactan polysaccharides during embryogenesis, and that xylan accumulated in the cell walls of differentiated cells     

MIRO1 influences the morphology and intracellular distribution of mitochondria during embryonic cell division in Arabidopsis

Regulating the morphology and intracellular distribution of mitochondria is essential for embryo development in animals. However, the importance of such regulation is not clearly defined in plants. The evolutionarily conserved Miro proteins are known to be involved in the regulation of mitochondrial morphology and motility. We previously demonstrated that MIRO1, an Arabidopsis thaliana orthologue of the Miro protein, is required for embryogenesis. An insertional mutation in the MIRO1 gene causes arrest of embryonic cell division, leading to abortion of the embryo at an early stage. Here we investigated the role of MIRO1 in the regulation of mitochondrial behaviour in egg cells and early-stage embryos using GFP-labeled mitochondria. Two-photon laser scanning microscopy revealed that, in miro1 mutant egg cells, mitochondria are abnormally enlarged, although egg cell formation is nearly unaffected. After fertilization and subsequent zygotic cell division, the homozygous miro1 mutant two-celled embryo contained a significantly reduced number of mitochondria in its apical cell compared with the wild type, suggesting that the miro1 mutation inhibits proper intracellular distribution of mitochondria, leading to an arrest of embryonic cell division. Our findings suggest that proper mitochondrial morphology and intracellular distribution are maintained by MIRO1 and are vital for embryonic cell division.     

Trehalose-6-phosphate synthase/phosphatase regulates cell shape and plant architecture in Arabidopsis

The vacuole occupies most of the volume of plant cells; thus, the tonoplast marker delta-tonoplast intrinsic protein-green fluorescent protein delineates cell shape, for example, in epidermis. This permits rapid identification of mutants. Using this strategy, we identified the cell shape phenotype-1 (csp-1) mutant in Arabidopsis thaliana. Beyond an absence of lobes in pavement cells, phenotypes included reduced trichome branching, altered leaf serration and stem branching, and increased stomatal density. This result from a point mutation in AtTPS6 encoding a conserved amino-terminal domain, thought to catalyze trehalose-6-phosphate synthesis and a carboxy-terminal phosphatase domain, is catalyzing a two-step conversion to trehalose. Expression of AtTPS6 in the Saccharomyces cerevisiae mutants tps1 (encoding a synthase domain) and tps2 (encoding synthase and phosphatase domains) indicates that AtTPS6 is an active trehalose synthase. AtTPS6 fully complemented defects in csp-1. Mutations in class I genes (AtTPS1-AtTPS4) indicate a role in regulating starch storage, resistance to drought, and inflorescence architecture. Class II genes (AtTPS5-AtTPS11) encode multifunctional enzymes having synthase and phosphatase activity. We show that class II AtTPS6 regulates plant architecture, shape of epidermal pavement cells, and branching of trichomes. Thus, beyond a role in development, we demonstrate that the class II gene AtTPS6 is important for controlling cellular morphogenesis.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Structural changes during the first divisions of embryos resulting from anther and free microspore culture inBrassica napus

Summary Ultrastructural and cytochemical features of embryo development during anther and free microspore culture inBrassica napus have been followed from the late uninucleate microspore stage through the first embryonic division. On transfer to culture, the microspore cytoplasm possesses a large vacuole, often containing electron opaque aggregates, and a peripheral nucleus. Mitochondria, endoplasmic reticulum and starch-free plastids are distributed throughout the cytoplasm. The conditions of culture induce a number of major changes in the cytoplasmic organisation of the microspore. First, the central vacuole becomes fragmented allowing the nucleus to assume a central position within the cell. Secondly, starch synthesis commences in the plastids which, in turn, are seen to occupy a domain investing the nucleus. Thirdly, the cell develops a thick fibrillar wall, situated immediately adjacent to the intine of the immature pollen wall. Finally, the microspores develop large cytoplasmic aggregates of globular material. The nature of this substance remains unknown, but it remains present until the young embryos have reached the 30 cell stage. The first division of cultured microspores destined to become embryos is generally symmetrical, in contrast to the asymmetric division seen in normal development in vivo. Consideration is given to the differences observed between embryos developing from anthers and free microspores in culture.     

Ultrastructural Changes in Cotyledons of Pineapple Guava (Myrtaceae) During Somatic Embryogenesis

Ultrastructural studies (SEM and TEM) were performed on cotyledonsof pineapple guava ( Feijoa sellowiana Berg, Myrtaceae) inducedto form embryos on medium containing 1.0 mg l^-1(4.5µM2,4-D) and 0.3M sucrose. At the time of culture, the cells werefilled with protein and lipid bodies. Microbodies and poorlydifferentiated organelles could also be seen. In contrast togerminating cotyledons, where lipid and protein reserves werequickly metabolized, cells of the embryogenically induced cotyledonsshowed evidence of reserve consumption only after 5 d of culture.Subepidermal cells of the upper cotyledonary surface underwentseveral divisions giving rise to a meristematic layer of severalcells thickness from which somatic embryos developed. Embryoscould also be formed directly by successive divisions of epidermalcells. Cells involved in somatic embryo formation containeda large nucleus with a conspicuous nucleolus and dense cytoplasmwhere numerous ribosomes, mitochondria, plastids with starchand short profiles of rough endoplasmic reticulum were present.Plasmodesmata were present both in cell walls of the meristematiccells and in few celled embryos whereas in degenerating embryosor in more advanced stages of somatic embryo development noplasmodesmata could be found. Although oil bodies were not observedin the meristematic cells they were identified in very youngembryos, being the first reserve compounds to appear. Cellsnot involved in somatic embryo differentiation were characterizedby the presence of several microbodies containing a crystalloidinclusion and elongated mitochondria. Feijoa sellowiana ; pineapple guava; somatic embryogenesis; ultrastructural studies     

New Embryo Lethals in Arabidopsis thaliana: Basic Genetic and Morphological Study

Six different mutations with defects in immature seed development have been identified during screening of a T-DNA collection of Arabidopsis thaliana. The mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development, and therefore they belong to the embryo-lethal class of mutants. The genetic and morphological studies of T-DNA mutations affecting embryo development are presented. The youngest embryos with a defect were observed at the globular stage in the VIII-64 mutation. Externally located cells, precursor of the protoderm, were characterised by abnormal cell division. VIII-41 mutation with a defect at the late globular stage was arrested at the globular-heart stage transition. VIII-111 mutation showed defect at heart stage of embryogenesis with atypical development of cotyledon primordia. The defect was associated with abnormal pattern of cell division constituting the precursor of the shoot apical meristem. In VIII-82 mutation defect in torpedo stage with asymmetric cotyledons was observed. Cotyledon stage of embryos and chlorophyll defect were observed in VIII-75 mutant. Abnormal suspensor consisting of two columns of cells was observed in 280-4-4 mutation. Newly identified embryo-lethals can serve as starting material for more detailed genetic and molecular studies.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.