Sexual and apomictic development in Hieracium

 Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis. Received: 11 February 1998 / Revision accepted: 23 July 1998     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999     

Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy

Apomixis is facultative in characterized members of the genus Hieracium. The three components that comprise the apomictic mechanism include apospory followed by autonomous embryo and endosperm formation. The time of aposporous embryo sac initiation and mode of embryo sac formation are different in Hieracium piloselloides (D3) and Hieracium aurantiacum (A3.4). Genetic studies have shown that a single dominant locus encodes all three components of apomixis in both species (Bicknell et al. 2000). We histologically examined a range of related, genetically characterized apomictic Hieracium plants derived from D3 and A3.4 to assess conservation of the apomictic mechanism in different genetic backgrounds. The plants varied in ploidy, and also in the amount of DNA introduced from sexual Hieracium pilosella (P4). An apomictic hybrid from a cross between the two apomicts was also examined. The developmental processes observed in the parental apomicts were not conserved in the examined plants and alterations occurred in the components of apomixis. One plant also exhibited adventitious embryony. The results show that other genetic factors can modify apomixis with respect to time of initiation, spatial location, and mode of developmental progression. Both the apomictic locus and the modifiers are essential for efficient penetrance of the trait in Hieracium. Some of the findings in Hieracium correspond with observations in Ranunculus and this is discussed in terms of models for apomictic development and the control of apomixis in crops. Received: 21 June 1999 / Revision accepted: 17 November 1999     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

Sense transformation reveals a novel role for class I beta-1, 3-glucanase in tobacco seed germination

'Coat-enhanced' seed dormancy of many dicotyledonous species, including tobacco, is released during after-ripening. Rupture of the endosperm, which is the limiting step in tobacco seed germination, is preceded by induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm where the radicle will penetrate. Treating after-ripened tobacco seeds with abscisic acid (ABA) delays endosperm rupture and inhibits betaGLU I induction. Sense transformation with a chimeric ABA-inducible betaGLU I transgene resulted in over-expression of betaGLU I in seeds and promoted endosperm rupture of mature seeds and of ABA-treated after-ripened seeds. Taken together, these results provide direct evidence that betaGLU I contributes to endosperm rupture. Over-expression of betaGLU I during germination also replaced the effects of after-ripening on endosperm rupture. This suggests that regulation of betaGLU I by ABA signalling pathways might have a key role in after-ripening.     

Cloning and expression of a new Tibetan hulless barley (Hordeum vulgare) beta-1,3-glucanase gene

A new full-length -1,3-glucanase cDNA was obtained by RT-PCR and RACE techniques from Tibet hulless barley and its complete gene sequence obtained by DNA Walking. Sequence alignment with the BLAST program showed that cDNA has high similarity with barley -1,3-glucanase II. The gene was functionally expressed in E. coli and the recombinant protein catalysed the hydrolysis of -1,3-glucan with an action pattern characteristic of a -1,3-glucan endohydrolase (EC 3.2.1.39). Southern blot analysis indicated that the gene is a member of a small gene family. RT-PCR and northern blot analysis indicated it is constitutively expressed in barley shoots.     

Local expression of enzymatically active class I β-1, 3-glucanase enhances symptoms of TMV infection in tobacco

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.     

利用乳酸乳球菌AcmA表面展示β-1,3-1,4-葡聚糖酶

采用PCR扩增乳酸乳球(Lactococcus lactis)MBl91菌株的全长肽聚糖水解酶基因acmA,通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp,再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137,然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137.经SDS-PAGE检测.重组菌MB137可表达预期的分子量约74 kD的蛋白质.Western blotting、细胞分级分离组分的荧光活性测定和特异GFP 二抗标记的流式细胞仪检测证实GFP被成功锚定在重组茵细胞表面,被锚定蛋白约占总表达融合蛋白的35%.进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的β-1,3-1,4葡聚糖酶基因gls,来取代pMB137中的gfp,得到携带融合基因acmA-gls的重组质粒pMB138,经导入到乳酸乳球茵AS1.2829后得到重组菌MB138,其全细胞β-1,3-1,4-葡聚糖水解酶的活性约为12 U/mL茵液,明显高于对照茵株.     

Induction of chitinase and β-1,3-glucanase in Parthenocissus quinquefolia cells cultured in vitro

Chitin, chitosan and peptidoglycan induced chitinase (EC 3. 2. 1. 14) activity in Parthenocissus quinquefolia cells cultured in vitro, while cellulose did not. The real inducers seemed to be oligomers released from the large size polymers by hydrolytic enzymes secreted into the medium during the cell growth and division. This effect was mimicked by the addition to the medium of a partially purified Parthenocissus chitinase/lysozyme (EC 3. 2. 1. 17), which was also able to hydrolyse chitosan. Oligomers of chitin and of chitosan induced the activity to the same level and with the same time course, while peptidoglycan oligomers induced less activity. Oligomers also induced β-1,3-glucanase (EC 3. 2. 1. 6) activities. The changes with time of both activities and the relative effects of the three kinds of polymers suggested that the induction of both enzymes involves a common element early in the signal pathway.     

Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins

Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.     

Expression of soybean b-1,3-endoglucanase cDNA and effect on disease tolerance in kiwifruit plants

Kiwifruit was transformed with a soybean β-1,3-endoglucanase (EC 3.2.1.39) cDNA under the control of the cauliflower mosaic virus (CaMV) 35S RNA promoter. The introduced gene was expressed in young leaves of the transformants. Assays of protein extracts from young leaves showed an increase in enzyme activity in many transformants, the transformant with the highest level of enzyme activity having an about sixfold increase over the control plants. When leaves from control and three transformants were inoculated with Botrytis cinerea, which causes gray mold disease, the disease lesion areas for two transformants were smaller than on control plants. Received: 5 March 1998 / Revision received: 19 October 1998 / Accepted: 27 October 1998     

Inheritance of parental genomes in progenies of Poa pratensis L. from sexual and apomictic genotypes as assessed by RAPD markers and flow cytometry

 Moving gene(s) responsible for the apomictic trait into crop plants that naturally reproduce through a sexual process would open up new areas in plant breeding and agricultural systems. Kentucky bluegrass (Poa pratensis L.) is one of the most important forage and turf grasses in temperate climates. It reproduces through facultative aposporous parthenogenesis, but the reproductive behaviour ranges naturally from nearly obligate apomixis to complete sexuality. In addition to apomictic reproduction, sexual hybridization may take place. Selfing may also occur, and occasionally reduced egg cells may develop through parthenogenesis generating (poly)haploids. The inheritance of parental genomes was assessed in Kentucky bluegrass progenies by employing RAPD markers in combination with flow cytometry (FCM). Nine progenies from different crosses carried out between completely sexual and highly apomictic genotypes were evaluated in order to probe the reproductive behaviour of the mother plants and to distinguish the different classes of aberrant plants. Not only were maternals and balanced B_II hybrids recorded, but so were (poly)triploid B_III hybrids, selfs, and (poly)haploids. The application of these techniques demonstrated that FCM analysis accurately distinguishes the n, 2n, and 3n ploidy levels of progenies, and that RAPD markers unequivocally recognize progenies of apomictic and hybrid origin. The occurrence of aneusomaty was documented in one of the selected sexual genotypes, whose crossed progeny plants manifested two distinct classes of ploidy. The nomenclature B_I was adopted to refer to hybrids with a hypodiploid nuclear condition. On the whole, the FCM analysis confirmed most of the RAPD data. The combined evaluation of DNA markers and DNA contents proved to be an efficient screening tool for scoring maternal plants, assessing the genetic origin of aberrant plants, and quantifying the inheritance of parental genomes in Kentucky bluegrass. Hybrid populations from sexual×apomictic matings that segregate for the mode of reproduction represent a valuable basis for attempting to identify molecular markers linked to the apomixis gene(s). Received: 11 November 1996/Accepted: 22 November 1996     

Anti-apoptotic Activity of Laminarin Polysaccharides and their Enzymatically Hydrolyzed Oligosaccharides from Laminaria japonica

Laminarin polysaccharides (LP1) were prepared from Laminaria japonica, a marine brown alga with potential biological activities, by hot water extraction, ultrafiltration and gel chromatography; the molecular weights of the LP1s were between 5 and 10 kDa. Laminarin oligosaccharides (LO) derived by hydrolyzing LP1 with an endo-β-(1→3)-glucanase from Bacillus circulans were mainly di- and penta-oligosaccharides. Treatment of mouse thymocytes with LO or LP1 (1–4 mg ml⁻¹) suppressed apoptotic death around 3- or 2-fold and extended cell survival in culture at a rate of about 30 or 20%. A mouse cDNA microarray showing the genes coding for immune response proteins were induced and apoptotic cell death proteins were reduced significantly by LO provided preliminary information regarding the immunomodulatory mechanism of LO. These results suggest that laminarin oligosaccharides and polysaccharides can be utilized to develop new immunopotentiating substances and functional alternative medicines. Revisions requested 26 October 2005; Revisions received 19 December 2005     

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

A monoclonal antibody recognizes a 65 kDa higher plant membrane polypeptide which undergoes cation-dependent association with callose synthase in vitro and co-localizes with sites of high callose deposition in vivo

Summary A monoclonal antibody (MAb) capable of immobilizing detergent-solubilized UDP-glucose: (13)--glucan (callose) synthase activity from higher plants has been selected and characterized. On Western blots this MAb recognizes a polypeptide of about 65 kDa found in membranes isolated from a variety of plant sources. The polypeptide recognized by this MAb does not appear to bind the substrate UDP-glucose, and evidence is presented which indicates that this polypeptide associates with the enzyme complex in a cation-dependent manner under conditions where the callose synthase assumes a larger size. Indirect immunofluorescence localization with this MAb was positive with sieve plates of cucumber (Cucumis sativus) seedlings, and with plasmodesmata of onion (Allium cepa) epidermal cells, both being sites of localized, stress-induced callose deposition.Abbreviations BSA bovine serum albumine - DMSO dimethylsulf-oxide - DTT dithiothreitol - FITC fluorescein isothiocyanate - HB Hepes buffer - HBS Hepes buffer plus 0.15 M NaCl - IgG immuno-globulin G - MAb monoclonal antibody - MSB microtubule stabilizing buffer - NP 40 Nonidet P 40 - PBS phosphate buffered saline - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - UDP uridine diphosphate - UV ultraviolet