Cloning of β-1,3-glucanases expressed during Cichorium somatic embryogenesis

Three different -1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid `474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular -1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56–63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1. Southern blot analysis revealed the presence of 3–4 genes coding for -1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid `474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid `474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa -1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3 and 5 RACE-PCR, and its sequence revealed that it encodes a -1,3-glucanase that is equally homologous to both class III and class IV plant -1,3-glucanases.     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

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     

Morphogenic events during indirect somatic embryogenesis in coffee “Catimor”

Summary The main goal of this research was to identify and describe the morphological and histological events during coffee somatic embryogenesis. Leaf sections of coffee Catimor (Coffea arabica CV. Red Caturra X hybrid of Timor) were cultivated in vitro on solid medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine. After 4 months, the calli produced were transferred to a medium containing naphthalene acetic acid. During the process of somatic embryogenesis, calli were sampled for histological observation. After four days of culture, the expiant produced a callus in the cut edges, where cell division occurred in the spongy parenchyma and in the perivascular parenchyma. After two months of culture, the first sign of organization within the growing callus was evidenced by the formation of densely stained cell groups appearing physically isolated, surrounded by thick cell walls. Two months later, proembryogenic clumps were formed by groups of dividing cells, unconnected to the callus. These cells were small, relatively isodiametric, with a dense cytoplasm, large nucleus, prominent nucleoli and thick cell walls. Afterwards, embryogenic calli formed somatic embryos going through the typical stages of development: globular, heart, and torpedo shapes. Histological observations revealed that the somatic embryos originated from a single cell, with dense cytoplasm, prominent nucleus and with signs of isolation evidenced by the presence of a thick cell wall.Abbreviations BAP 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthalene acetic acid - SEM scanning electron microscopy     

Distinct ethylene- and tissue-specific regulation of β-1,3-glucanases and chitinases during pea seed germination

The expression of β-1,3-glucanase (βGlu) and chitinase (Chn) was investigated in the testa, cotyledons, and embryonic axis of germinating Pisum sativum L. cv. `Espresso generoso' seeds. High concentrations of βGlu and Chn activity were found in the embryonic axis. Treatment with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that an early, 4-fold induction of βGlu activity in the embryonic axis during the first 20 h after the start of imbibition is ethylene-independent. This initial increase was followed by a later 4-fold ethylene-dependent induction in the embryonic axis starting at 50 h, which is after the onset of ethylene evolution and after completion of radicle emergence. The βGlu activity in cotyledons increased gradually throughout germination and was ethylene-independent. In contrast, the ethylene-independent Chn activity increased slightly after the onset of radical emergence in the embryonic axis and remained at a constant low level in cotyledons. Immunoinactivation assays and immunoblot analyses suggest that early βGlu activity in the embryonic axis is due to a 54-kDa antigen, whereas late induction is due to a 34.5-kDa antigen, which is likely to be the ethylene-inducible class I βGlu G2 described for immature pea pods. Increases in Chn in the embryonic axis were correlated with a 26-kDa antigen, whereas amounts of the additional 32- and 20-kDa antigens remained roughly constant. Thus, ethylene-dependent and ethylene-independent pathways regulate βGlu and Chn during pea seed germination. The pattern of regulation differs from that of leaves and immature pods, and from that described for germinating tobacco seeds. The functional significance of this regulation and its underlying mechanisms are discussed. Received: 12 January 1999 / Accepted: 22 March 1999     

Genes encoding acidic and basic class III β-1,3-glucanases are expressed in tomato plants upon viroid infection

-1,3-glucanases are hydrolytic enzymes considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants. We have isolated and characterized two complementary DNA clones, corresponding to new -1,3-glucanases from tomato plants (Lycopersicon esculentum) which are expressed upon challenge with citrus exocortis viroid. Amino acid sequence comparison revealed that they are most similar to -1,3-glucanases from tobacco, particularly to PR-Q, the unique component of the class III -1,3-glucanase. The deduced amino acid sequences of the two tomato -1,3-glucanases indicate that, although being highly similar in amino acid sequence, they have different isoelectric points: pI 10.5 for the basic isoform (Tom PR-Q b) and pI 5.2 for the acidic one (Tom PR-Q a). The expression of these two -1,3-glucanase messenger RNAs (mRNAs) in response to viroid infection and ethephon treatments was examined. mRNAs for these two isoforms are coordinately expressed and induced similarly to mRNAs for other PR proteins, indicating that they are part of a general and coordinate mechanism of response of tomato plants susceptible to viroid infection.     

Nutrition supply affects the activity of pathogenesis-related β-1,3-glucanases and chitinases in wheat

Nitrogen (N) is an essential mineral for plants and both its deficiency and excess causes serious problems in agriculture. As stress-inducible defense is costly, N conditions likely affect the trade-off between the growth and defense. Previous studies identified a few defense-related enzymes dependent on N nutrition. Chitinases (EC 3.2.1.14) and glucanases (EC 3.2.1.39) are typical plant defense enzymes belonging to the group of pathogenesis-related (PR) proteins with multiple functions in plants. Since a comprehensive study on the impact of N nutrition on their activity is missing, we studied their profiles and activities at isoforms level in wheat plants grown hydroponically at N doses corresponding to limited (0, 0.75 and 5.25 mM N), optimal N (7.5 mM N) as well as excess (15, 30 and 35 mM N) N supply in the form of nitrate. Our results show that several isoforms of both enzymes in wheat leaves and/or shoots clearly depended on N supply, while their activities rather depended on organ type. Furthermore, glucanases and chitinases appeared to be regulated in an opposite way. The activities of particular chitinases and glucanases correlated with a proline content that has multiple functions in plants. Proline typically accumulated with increasing the N supply when certain excessive N doses induced the gene for proline synthase (P5CS) in shoots and that for ornithine aminotransferase (OAT) in roots. This work points to a N-dependent activity of several defense-related compounds suggesting the possibly of altered plant defense potential under various N regimes.     

The extracellular system of β-1,3-glucanases ofAlternaria tenuissima andAspergillus vesicolor

During cultivation in a minimal medium with glucoseAlternaria tenuissima andAspergillus vesicolor produce constitutively α- and β-glucanases. Fractions of β-1,3-glucanases exhibiting affinity for laminarin were separated by means of gel filtration chromatography. Two neutral β-1,3-glucanases with affinity for yeast glucan were isolated by affinity chromatography and further characterized.     

Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host β-1,3-glucanases

Plant viruses spread cell-to-cell by exploiting and modifying plasmodesmata, coaxial membranous channels that cross cell walls and interlink the cytoplasm, endoplasmic reticulum and plasma-membranes of contiguous cells. To facilitate viral spread, viruses encode for one or more movement proteins that interact with ER and ER derived membranes, bind vRNA and target to Pd. Mounting evidence suggests that RNA viruses that do not spread as virions employ the same basic mechanism to facilitate cell-to-cell spread. In light of the research reviewed here, we propose a general functional model for the cell-to-cell spread of these viruses. This model posits that MPs have multiple functions: one function involves directing virus induced β-1,3-glucanases which accumulate in ER derived vesicles to the cell wall to hydrolyze Pd associated callose in order to gate open the Pd; independently, the MPs form ER-associated protein rafts which transport bound vRNA by diffusion along ER to adjacent cells via the ER component of the plasmodesmata. The driving force for spread is the diffusion gradient between infected and non-infected adjacent cells.     

Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review

Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus) is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-β-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-β-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The βglII and βglII _A genes from strain DSM 10297 (also known as O. xanthineolytica LL G109) encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the β-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73–14) encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that βglII and βglII _A have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both βglII and the 54.5 kDa β-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to β-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, βglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans β-1,3-glucanases has been compiled and compared.     

Characterization of the type of action of β-1,3-glucanases from marine invertebrates

• 1.1. Ten species of marine invertebrates possessing the highest laminarinase activity have been investigated.
• 2.2. The following approaches were used in determining the type of action of partially purified β-1,3-glucanases: effect on modified substrates, comparative study of enzymolysis products by the Nelson's and glucose oxidase methods and stereochemistry of the enzymolysis products.
• 3.3. Experimental results indicate that the examined marine invertebrates contain β-1,3-glucanases with endo-type activity; exo-enzymes are apparently absent.

     

Catalytic efficiency diversification of duplicate β-1,3-1,4-glucanases from Neocallimastix patriciarum J11

Four types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designated bglA13, bglA16, bglA51, and bglM2, were found in the cDNA library of Neocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias to Streptococcus equinus. The presence of expansion and several predicted secondary structures in the 3' untranslated regions (3'UTRs) of bglA16 and bglM2 suggest that these two genes were duplicated recently, whereas bglA13 and bglA16, which contain very short 3'UTRs, were replicated earlier. These findings indicate that the β-glucanase genes from N. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes of Streptococcus equinus, Ruminococcus albus 7, and N. patriciarum J11 were cloned and expressed by Escherichia coli. The recombinant β-glucanases cloned from S. equinus, R. albus 7, and N. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higher k(cat) values than others. These results support the notion that duplicated β-glucanase genes, namely, bglA16 and bglM2, increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms in N. patriciarum J11.     

Cell–cell communication in Arabidopsis early embryogenesis

The basic body plan of the adult plant is established during embryogenesis, resulting in the juvenile form of the seedling. Arabidopsis embryogenesis is distinguished by a highly regular pattern of cell divisions. Some of these divisions are asymmetric, generating daughter cells with different fates. However, their subsequent differentiation might still depend on cell–cell communication to be fully accomplished or maintained. In some cases, cell fate specification solely depends on cell–cell communication that in general plays an important role in the generation of positional information within the embryo. Although auxin-dependent signalling has received much attention, other ways of cell–cell communication have also been demonstrated or suggested. This review focuses on aspects of pattern formation and cell–cell communication during Arabidopsis embryogenesis up to the mid-globular stage of development.     

Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi

β-1,3-Glucanases are abundant in plants and have been characterized from a wide range of species. They play key roles in cell division, trafficking of materials through plasmodesmata, in withstanding abiotic stresses and are involved in flower formation through to seed maturation. They also defend plants against fungal pathogens either alone or in association with chitinases and other antifungal proteins. They are grouped in the PR-2 family of pathogenesis-related (PR) proteins. Use of β-1,3-glucanase genes as transgenes in combination with other antifungal genes is a plausible strategy to develop durable resistance in crop plants against fungal pathogens. These genes, sourced from alfalfa, barley, soybean, tobacco, and wheat have been co-expressed along with other antifungal proteins, such as chitinases, peroxidases, thaumatin-like proteins and α-1-purothionin, in various crop plants with promising results that are discussed in this review.     

Plant hydrolytic enzymes (chitinases and β-1,3-glucanases) in root reactions to pathogenic and symbiotic microorganisms

Within the last decade, a great deal of attention has been devoted to the role of chitinases and -1,3-glucanases in plant/microbe interactions. While there is strong evidence that these hydrolases are antifungal proteins, there are also recent indications of roles in both plant morphogenesis and plant/microbe signal perception. This paper reviews recent findings pertinent to root/microbe interactions, and discusses the nature and significance of specific hydrolase isoforms in symbioses with arbuscular mycorrhizal (AM) fungi.     

Pathogenesis-related functions of plant β-1,3-glucanases investigated by antisense transformation — a review

Plant β-1,3-glucanases (βGlu) have been implicated in several physiological and developmental processes, e.g., cell division, microsporogenesis, pollen germination, fertilization and seed germination. These enzymes, particularly the antifungal class-I vacuolar isoforms, are also believed to be part of the defences of plants against fungal infection. The function of βGlu in tobacco and Nicotiana sylvestris has been investigated by antisense transformation. Transformation with GLA, the gene encoding the A isoform of tobacco class-I βGlu, in reverse orientation regulated by the strong cauliflower mosaic virus 35S RNA promoter effectively and specifically blocked the induction of class-I βGlu. This induction was in response to ethylene treatment and following infection with the pathogenic fungus, Cercospora nicotianae, tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV). Nevertheless, the plants compensated for this deficiency by producing a functionally equivalent (i.e., ‘ersatz’) enzyme or enzymes. The fact that compensation occurred specifically in response to infection suggests that βGlu activity has an important role in pathogenesis. Antisense transformation substantially reduced lesion size and number in virus-infected local-lesion hosts. These results suggest novel antisense-based strategies for protecting plants against virus infection. They also raise the intriguing possibility that viruses use a defence mechanism of the host, production of antifungal βGlu, to promote their own replication and spread.     

Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung

Background  

The aim of this study was to analyze the cell-specific expression of E- and N-cadherin and β-catenin in developing human lung tissues from 12 to 40 weeks of gestation.