Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

Nonsymbiotic hemoglobins in rice are synthesized during germination and in differentiating cell types

Summary Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots; however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. We report the immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decreased during leaf maturation. Roots and mesocotyls contained detectable, but low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are synthesized de novo during seed germination and are expressed in the absence of any signal originating from the embryo. Immunolocalization of ns-Hbs by confocal microscopy indicated the presence of ns-Hbs primarily in differentiated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the first report of the specific cellular localization of these proteins during seedling development.Abbreviations Hbs hemoglobins - Ns nonsymbiotic     

Expression of the QrCPE gene is associated with the induction and development of oak somatic embryos

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine-rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northern blot and in situ hybridization were used to determine the specific localisation of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype-dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localised expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved in the fate of root cap cells.     

9-kDa acidic and basic nsLTP-like proteins are secreted in the culture-medium conditioned by somatic embryogenesis in Cichorium

Direct somatic embryogenesis was induced in leaf fragments of the Cichorium ‘474’ genotype. Addition of glycerol to the induction medium allowed a relative synchronization of the first division of the embryogenic cells that only occurs after transfer at day 5 to a medium without glycerol. The abundant presence of 9-kDa extracellular proteins in the culture-medium conditioned by somatic embryogenesis is reported here. Such proteins were also secreted when embryogenesis was initiated in root but were never detected when a non-embryogenic genotype was used as control under the same conditions of culture. Among these proteins, one basic and one acidic isoform were separated through cation-exchange chromatography. Both proteins were recognized by an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). In addition, the partial N-terminal amino acid sequence of each isoform showed similarities with nsLTPs of different plant species. The presence of the acidic nsLTP-like protein was concomitant with the obtention of embryogenic cells during the induction step. The basic form was shown to have only accumulated during the expression step when first divisions of embryogenic cells have occurred. These results allowed us to report, for the first time, the secretion of a 9-kDa acidic nsLTP-like protein in the culture-medium conditioned by plant embryogenic cells.     

Effects of glycerol on somatic embryogenesis in Cichorium leaves

 Direct somatic embryogenesis was induced in leaf cells of a Cichorium hybrid (Cichorium intybus L var. sativum×Cichorium endivia L. var. latifolia) through a two-step procedure. Leaf tissue explants were cultured for 5 days in M17 liquid medium supplemented with 30 mM sucrose and 330 mM glycerol (M17S30Gly330 medium). Synchronised divisions of embryogenic cells occurred after transfer for 7 days onto glycerol free-medium (M17S30). By doubling the sucrose concentration (60 mM) in the presence of glycerol (M17S60Gly330) during the induction step, embryogenesis increased and the length of the induction step was reduced from 5 to 4 days. Compared to sucrose, glycerol as carbon source during the induction and the expression steps had an inhibitory effect on the embryogenic response. During culture, glycerol was not detected in M17S60 medium and was at a low level in leaf fragments incubated in this medium. Initially supplied as an osmoticum, glycerol disappeared from M17S60Gly330 medium during the 4-day induction period and penetrated into the tissues where most of was metabolised. Furthermore, glycerol modified it carbohydrate metabolism, particularly during the induction period of embryogenesis. Sucrose hydrolysis was affected in the medium and sucrose and hexose contents in tissues were higher than in glycerol-free medium. The effects of glycerol as osmoticum and as a molecule itself are discussed. Received: 30 January 1998 / Accepted after revision 25 February 1999     

Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. : Effects in Vivo and in Vitro of Glyphosate, Fluridone, and Paclobutrazol

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA₃; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Accumulation pattern of dehydrins during sugarcane (var. SP80.3280) somatic embryogenesis

The objective of the present study was to determine dehydrin protein levels in sugarcane var. SP80-3280 during somatic embryogenesis. Dehydrins from embryogenic and non-embryogenic cell cultures were analyzed using western blot and in situ immunolocalization microscopy. Both techniques employ antibodies raised against a highly conserved lysine-rich 15-amino acid sequence termed the K-domain, which is extensively used to recognize proteins immunologically related to the dehydrin family. In embryogenic cultures, western blot analysis of the heat-stable protein fraction revealed eleven major bands ranging from 52 to 17?kDa. They were already visible on the first days, gradually increasing until reaching peak values around day 14, when organogenesis begins, to later decrease in concurrence with the appearance of green plantlets (around day 28). These fluctuations indicate that this pattern of accumulation is under developmental control. Dehydrins were mainly immunolocalized in the nuclei. A phosphatase treatment of protein extracts caused a mobility shift of the 52, 49, and 43?kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation. In sugarcane embryogenic cultures, presence of dehydrins is a novel finding. Dehydrins were absent in non-embryogenic cultures. The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis. Key message The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis.     

Temporal Regulation of Somatic Embryogenesis by Adjusting Cellular Polyamine Content in Eggplant

Four critical stages of embryogenesis, including callus induction, cellular acquisition of morphogenetic competence, expression of embryogenic program, and development and maturation of somatic embryos during somatic embryogenesis from leaf discs of eggplant (Solanum melongena L.), were identified by scanning electron microscopy. Temporal changes in arginine decarboxylase (ADC) activity and polyamines (PAs) during critical stages of embryogenesis revealed that high levels of PAs (especially putrescine [PUT]), due to higher ADC activity in discs from the apical region (with high embryogenic capacity) than from the basal region of the leaf (with poor embryogenic capacity), were correlated with differential embryogenesis response. Kinetic studies of the up- and down-regulation of embryogenesis revealed that PUT and difluoromethylarginine pretreatments were most effective before the onset of embryogenesis. Basal discs pretreated with PUT for 4 to 7 d showed improved embryogenesis that was comparable to apical discs. PA content at various critical steps in embryogenesis from basal discs were found to be comparable to that of apical discs following adjustments of cellular PA content by PUT. In contrast, pretreatment of apical discs with difluoromethylarginine for 3 d significantly reduced ADC activity, cellular PA content, and embryogenesis to levels that were comparable to basal discs. Discs from the basal region of leaves treated with PUT for 3 d during the identified stages of embryogenesis improved their embryogenic potential.     

Quaternary structure of rice nonsymbiotic hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.     

Co-Localization of β-1,3-Glucanases and Callose During Somatic Embryogenesis in Cichorium

During direct somatic embryogenesis in leaves of Cichorium hybrid clone ‘474’, 38 kDa β-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of β-1,3-glucan linkages and so represented a possible substrate for β-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa β-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and β-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that β-1,3-glucanases could be involved in the degradation of this callose.Key Words: β-1,3-glucanases, callose, Cichorium, immunolocalizations, somatic embryogenesis     

The Glycine-Glomus-Rhizobium Symbiosis : VII. Photosynthetic Nutrient-Use Efficiency in Nodulated, Mycorrhizal Soybeans

Four consecutive trifoliate leaves of 56-day-old symbiotic or nonsymbiotic soybean plants were evaluated individually for CO₂ exchange rates (CER), leaf area and dry weight, and leaf N, P, and starch concentrations. Plants had been inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae and Rhizobium japonicum, with either of the endophytes alone, or with neither at time of planting. Plants lacking one or both endophytes received N and/or P fertilizers to produce plants of equal total leaf dry weight in all four treatments. Photosynthetic P-use efficiency (CER per unit leaf P) was higher in the leaves of VAM plants than in P-fertilized plants regardless of the N source (N₂ fixation or combined N). Photosynthetic N-use efficiency was also higher in VAM than in non-VAM plants, but it was affected by the N source, with higher CER in the nodulated plants. The greatest differences in CER, starch accumulation and leaf area were found between the nonsymbiotic plants and those with both endophytes. Statistical evaluations of leaf parameters for treatment or nutrient concentration (N and P) effects between the tri-partite and the nonsymbiotic treatments showed significant changes in concentration of P, but not N, with decreasing leaf age. Both endophytes apparently enhance CO₂ fixation at N and/or P concentrations lower than those of the nonsymbiotic plants. The effects of the endophytes on CO₂ fixation were additive.     

Crystal structures of Parasponia and Trema hemoglobins: differential heme coordination is linked to quaternary structure

Hemoglobins from the plants Parasponia andersonii (ParaHb) and Trema tomentosa (TremaHb) are 93% identical in primary structure but differ in oxygen binding constants in accordance with their distinct physiological functions. Additionally, these proteins are dimeric, and ParaHb exhibits the unusual property of having different heme redox potentials for each subunit. To investigate how these hemoglobins could differ in function despite their shared sequence identity and to determine the cause of subunit heterogeneity in ParaHb, we have measured their crystal structures in the ferric oxidation state. Furthermore, we have made a monomeric ParaHb mutant protein (I43N) and measured its ferrous/ferric heme redox potential to test the hypothesized link between quaternary structure and heme heterogeneity in wild-type ParaHb. Our results demonstrate that TremaHb is a symmetric dimeric hemoglobin similar to other class 1 nonsymbiotic plant hemoglobins but that ParaHb has structurally distinct heme coordination in each of its two subunits that is absent in the monomeric I43N mutant protein. A mechanism for achieving structural heterogeneity in ParaHb in which the Ile(101(F4)) side chain contacts the proximal His(105(F8)) in one subunit but not the other is proposed. These results are discussed in the context of the evolution of plant oxygen transport hemoglobins, and other potential functions of plant hemoglobins.     

Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader)

 Indirect somatic embryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somatic embryogenesis. Another cycle of somatic embryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were cultured on media containing different auxin/cytokinin ratios. The auxin/cytokinin ratio had an influence on the intensity of embryo formation, germination and the capability to regenerate plants. Somatic embryogenesis was generally more intensive on the medium with lower concentrations of 6-benzylamino-purine. Further, the percentage of regenerated plants was higher when embryos were induced on high-cytokinin, low-auxin medium. Secondary somatic embryogenesis was induced on embryos by culture in liquid hormone-free medium. Similar to direct embryogenesis the efficiency of secondary embryogenesis depended on the medium used for the induction of the primary embryos. In contrast to the mostly low frequencies of conversion of secondary embryos into plants that has been observed in other species, the percentage of regenerated plants from secondary embryos of H. maximiliani was quite high, although slightly lower than that obtained in primary embryos. Received: 28 March 2000 / Revision received: 1 September 2000 / Accepted: 2 October 2000     

Induction of somatic embryogenesis from different explants of shoot cultures derived from young <Emphasis Type="Italic">Quercus alba</Emphasis> trees

The induction of somatic embryogenesis from shoot apices and leaf explants of shoot cultures derived from 6- to 7-year-old white oak (Quercus alba L.) trees is reported in this study. Embryogenic response was obtained in two out of the three genotypes evaluated with embryo induction frequencies up to 50.7% for WOQ-1 and 3.4% for WOQ-5 genotypes. The embryogenic explants formed translucent nodular structures and cotyledonary-stage somatic embryos, which developed from callus tissue, indicating an indirect embryogenesis process. An efficient procedure was developed for WOQ-1 material on the basis of the most appropriate leaf developmental stage. Growing leaves excised from two nodes below the shoot apex showed the highest embryogenic induction index. These leaves contain cells in an undifferentiated state, as shown by the presence of precursor cells of stomata, absence of intercellular spaces and low starch content in the mesophyll cells. Nodular structures and/or somatic embryos began to arise 7–8 weeks after culture initiation, although most emerged after 9–12 weeks in culture. The sequence of application of media for somatic embryo induction was optimized with a two-step procedure consisting of culturing the explants in medium supplemented with 21.48 μM NAA and 2.22 μM BA for 8 weeks and transfer of explants into plant growth regulator-free medium for another 12 weeks. Clonal embryogenic lines were established and maintained by secondary embryogenesis. Embryo germination (30%) and plantlet conversion (16.6%) were achieved after cold storage for 2 months.