Identification of a transitional cell state in the developmental pathway to carrot somatic embryogenesis

We have located a novel carbohydrate epitope in the cell walls of certain single cells in embryogenic, but not in non-embryogenic, suspension cultures of carrot. Expression of this epitope, recognized by the mAb JIM8, is regulated during initiation, proliferation, and prolonged growth of suspension cultures such that changes in the abundance of JIM8-reactive cells always precede equivalent changes in embryogenic potential. Therefore, a direct correlation exists between the presence of the JIM8-reactive cell wall epitope and somatic embryo formation. The JIM8-reactive cell wall epitope is expressed in the cell walls of three types of single cells and one type of cell cluster. One of the single cell types seems able to follow one of two phytohormone-controlled developmental pathways, either a cell elongation pathway that eventually leads to cell death, or a cell division pathway that gives rise to proembryogenic masses. We demonstrate that all JIM8-reactive cell types in embryogenic carrot suspension cultures are developmentally related, and that the switch by one of them to somatic embryogenesis is accompanied by the immediate dissipation of the JIM8-reactive cell wall epitope. The cell wall carbohydrate epitope recognized by JIM8 therefore represents a cell wall marker for a very early transitional cell state in the developmental pathway to carrot somatic embryogenesis.     

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.     

Characterization of a dehydrin-like phosphoprotein (ECPP-44) relating to somatic embryogenesis in carrot

Somatic embryogenesis in carrot can be induced by the treatment of shoot apices with various kinds of stress chemicals. Using this system, we previously identified a phosphoprotein (ECPP-44) that appears to be involved in the induction of somatic embryogenesis. We have also isolated and characterized a cDNA encoding ECPP-44. In this study, to further characterize ECPP-44, we performed Western blot and immuno-precipitation analyses. Western blot analysis revealed that ECPP-44 was present in embryogenic cells, stress- and non-stress-treated tissues, and somatic embryos but was absent in non-embryogenic cells. Furthermore, ECPP-44 was found in some parts of the carrot plant, such as tap roots, leaves, and flowers (18–26 days after fertilization) but not in mature dry seeds. Interestingly, we could detect phosphorylated ECPP-44 in embryogenic cells and somatic embryos but not in non-embryogenic cells, tap roots, and non-stress-treated shoot apices by immunoprecipitation analysis, even though the protein existed. Our results suggest that ECPP-44 may perform some role in the induction or maintenance of embryogenic competence.     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L.

Daucus carota L. cell lines secrete a characteristic set of arabinogalactan proteins (AGPs) into the medium. The composition of this set of AGPs changes with the age of the culture, as can be determined by crossed electrophoresis with the specific AGP-binding agent, β-glucosyl Yariv reagent. Addition of AGPs isolated from the medium of a non-embryogenic cell line to an expiant culture initiated the development of the culture to a non-embryogenic cell line. Without addition of AGPs or with addition of carrot-seed AGPs an embryogenic cell line was established. Three-month-old embryogenic cell lines usually contain less than 30% of dense, highly cytoplasmic cells, i.e. the embryogenic cells, but when carrot-seed AGPs were added this percentage increased to 80%. Addition of carrot-seed AGPs to a two-year-old, non-embryogenic cell line resulted in the re-induction of embryogenic potential. These results show that specific AGPs are essential in somatic embryogenesis and are able to direct development of cells.     

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot

Somatic embryogenesis in carrot ( Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.     

Embryo-specific Proteins in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> Analyzed with 2-D DIGE

Somatic embryogenesis can be used to produce artificial seeds of Cyclamen persicum, one of the most important ornamental plants for the European market, both as a potted plant in northern Europe and a bedding plant in the cool winters in southern Europe. The aim of this study was to obtain new insights into the molecular biology of somatic embryogenesis, which in turn can be useful for the improvement of tissue culture methodology. Total proteins were characterized from two isogenic cell lines of Cyclamen persicum, one that was embryogenic and one that never has shown any embryogenic capacity. The extracted proteins were separated by two-dimensional differential gel electrophoresis (2-D DIGE) and selected proteins were treated using the ETTAN Dalt Spot Handling Workstation. Protein identification was performed using MALDI-TOF-MS. More than 1200 Cyclamen proteins were detected; 943 proteins were common to both lines. The different protein patterns of the embryogenic and non-embryogenic cell lines were obvious: One hundred eight proteins were more abundant in the embryogenic cells, and 97 proteins in the non-embryogenic cells. Among the differentially expressed proteins, 128 were identified. MALDI-TOF-MS analysis enabled 27 spots to be proposed as candidates for embryo-specific proteins, as they were unique to the embryogenic cell line. The proteins identified are involved in a variety of cellular processes, including cell proliferation, protein processing, signal transduction, stress response, metabolism, and energy state, but the majority are involved in protein processing and metabolism. The main functions of the putative embryo-specific proteins have been discussed in proportion to their role in the somatic embryogenesis process. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Lyngved and J. Renaut contributed equally to this work.     

Initial identification of a phosphoprotein that appears to be involved in the induction of somatic embryogenesis in carrot

We examined changes in the absence versus presence patterns of phosphoproteins with respect to the acquisition of embryogenic competence during somatic embryogenensis in carrot (Daucus carota L.). To characterize a possible correlation between the induction of embryogenic competence and protein phosphorylation, we examined the patterns of protein phosphorylation in embryogenic cells (EC) and non-embryogenic cells (NC) that had lost the ability to form somatic embryos. Two-dimensional polyacrylamide gel electrophoresis and subsequent autoradiography revealed the presence of 31 phosphoproteins in EC but not in NC. Furthermore, when we examined the induction of somatic embryogenesis by certain stress compounds in the absence of phytohormones, we identified one specific phosphoprotein (ECPP-44). ECPP-44 was found to be induced in all treatments that resulted in embryogenic competence. The partial amino acid and nucleotide sequence of ECPP-44 shows partial homology to two dehydrins (ERD10 and ERD14) from Arabidopsis. Received: 1 October 1999 · Accepted: 3 November 1999     

Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems

Abstract

Whatever the in vitro regeneration pathway, it would be of interest to be able to distinguish regeneration-competent from non-regenerating cells and tissues, and as early in culture as possible, as this would allow a dramatic improvement of biotechnology breeding, particularly for the so-called recalcitrant species. With this aim, we examined a range of genotypes of pea (Pisum sativum) and grass pea (Lathyrus sativus), and of the two model species Medicago truncatula, another legume, and Arabidopsis thaliana. This was done by comparing cell suspension cultures of different ages (young [<6 transfers-old]vs. mature [>6 transfers-old]), densities (dense [>10⁷ cells/ml] or sparse [<10⁶ cells/ml]) and regeneration abilities (non-embryogenic vs. embryogenic), in order to identify early indicators of competence for somatic embryogenesis. All such cell suspensions were subcultured every 14 days and several parameters were assessed every 3–4 days during each 14-day cycle. These included the time course pH and osmolarity of the culture medium, the internal osmolarity of cells, the cell surface and the cell wall thickness (by examining cellulose accumulation in Calcofluor White-stained cells under UV light). As cells underwent embryogenesis they enlarged. Cellulose accumulated in the walls of non-embryogenic cells, but walls became thinner with the onset of embryogenesis, and diminished further as embryos matured. Although medium osmolarity decreased at the onset of embryogenesis, this was never observed for non-embryogenic cell suspensions. Conversely, there was a concomitant increase in intracellular osmolarity for embryogenic cells. Medium pH (analysed with the model species only) was not significantly correlated with regeneration competence of cells. For all genotypes and species, the kinetics of cell wall thickness and cell surface, and that of medium and cell osmolarity were reliable early indicators of the competence of cells to undergo somatic embryogenesis. The implication of these results for biotechnological breeding of grain legumes and for plant regeneration competence in general are discussed.     

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.     

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot ( Daucus carota L.), an embryogenic, a non-embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non-embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota , 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

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.     

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L^?1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L^?1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.     

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture. Received: 13 March 1998 / Revision received: 6 June 1998 / Accepted: 1 July 1998     

Developmental localization and methylesterification of pectin epitopes during somatic embryogenesis of banana (Musa spp. AAA)

Background

The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development.

Methodology/Principal Findings

Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment.

Conclusions/Significance

These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana.     

Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot

Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall.     

尾巨桉胚状体诱导及愈伤组织差异研究

以尾巨桉优良无性系无菌苗茎段为外植体,通过对多种不同浓度生长调节剂组合的优化,进行胚状体诱导研究;并对胚性与非胚性愈伤组织进行形态解剖学观察、相关生理指标检测以及相关基因荧光定量PCR分析,以揭示尾巨桉胚性愈伤组织非胚性化发生的机理,为建立尾巨桉体细胞胚胎再生体系提供参考。结果表明:(1)胚性愈伤组织在MS+0.1mg/L NAA+0.01mg/L TDZ培养基中诱导得到胚状体,外植体经过0.5mol/L蔗糖处理12h有助于胚性愈伤组织产生胚状体,胚状体最高发生率为16.7%。(2)尾巨桉胚性与非胚性愈伤组织石蜡切片观察发现,两者的细胞形态特征存在明显的差异,胚性愈伤组织细胞体积小,排列紧密,表现出典型的胚性细胞特征,而非胚性细胞比较大,排列疏松,细胞呈不规则形状。(3)生理生化指标检测结果表明,非胚性愈伤组织中蛋白质含量、SOD、PPO及CAT活性均显著低于胚性愈伤组织,非胚性愈伤组织中木质素、可溶性糖含量以及PAL和POD活性要高于胚性愈伤组织,二者的反肉桂酸4-单加氧酶基因、淀粉磷酸化酶基因、谷胱甘肽硫转移酶基因、葡萄糖-1-磷酸腺苷酸转移酶基因、葡萄糖六磷酸异构酶基因、分支酸合酶基因以及苯丙氨酸解氨酶基因表达差异也达到显著水平。     

Peroxidase activity in non-embryogenic and embryogenic calli and in developing somatic embryos of white fir (Abies concolor Gord. et Glend)

ABSTRACT

Peroxidase activity was monitored during somatic embryogenesis of white fir (Abies concolor Gord. et Glend) starting from a non-embryogenic callus. Results revealed profound differences between non-embryogenic and embryogenic calli with an elevated level of enzyme activity in non-embryogenic ones. Precotyledonary, early cotyledonary and late cotyledonary stages of somatic embryogenesis were characterized by a substantially reduced peroxidase activity compared to callus tissues and regenerated plantlets. Changes in peroxidase activity are as a rule paralleled by variation in isoenzyme composition. The utility of the enzyme in the induction stage of somatic embryogenesis in white fir is proposed.