An exo-β-d-glucanase derived from Zea coleoptile walls with a capacity to elicit cell elongation

Cell wall proteins were extracted from maize coleoptiles, Zea mays L. B37 x MO 17, with high concentrations of LiCl. Ion-exchange, chromatofocusing and gel-filtration chromatography were employed extensively to purify exo-β-glucanase activity from the extract. The purified enzyme functioned as an exo-(1→3)-β-glucanase (E.C. 3.2.1.58) and as a glucosidase (E.C. 3.2.1.21) capable of extensive hydrolysis of the native Zea wall (1→3), (1→4)-β- d -glucan, yielding glucose as the final product. The exoglucanase also enhances elongation of maize coleoptile sections in both the presence and absence of exogenous IAA.     

Coleoptile growth-inducing capacities of exo-β-(1→3)-glucanases from fungi

An exo-β-(1β3)-glucanase derived from Selerotinia libertiana induced growth of Avena sativa coleoptiles and degraded hemicelluloses and β-(1→4):(1→3) mixed linked glucan. However, neither endo-β-(1→4)- nor endo-β-(1→3)-glucanase activity could be detected in the enzyme preparation. Nojirimycin inhibited the glucan degradation caused by the enzyme but glucono-1,5-lactone did not. Another exo-β-(1→3)-glucanase derived from Basidiomycete QM 806 did not induce coleoptile growth and did not degrade the glucan. Growth-inducing properties of exo-β-(1→3)-glucanases are discussed.     

Expression of genes for cell-wall proteins in dividing and wounded tissues ofZea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.     

Purification and properties of apoplastic amylase from oat (Avena sativa) seedlings

The protein fraction extracted with a high ionic strength buffer from the cell wall preparation of oat ( Avena sativa L.) coleoptiles and first leaves contained starch-degrading (amylase) activity. The activity of apoplastic amylase in the coleoptiles and first leaves continued to increase in parallel with organ growth. One of the apoplastic amylases recovered from shoot cell wall preparations was purified by sequential ion exchange and gel filtration chromatography, and the catalytic properties of the enzyme were analysed. The purified enzyme gave a single 25 kDa protein band on SDS-PAGE. The enzyme exhibited maximum activity at pH 5.0 against maltooligosaccharides. The purified enzyme hydrolysed soluble starch and maltooligosaccharides larger than tetraose at maltose unit, but did not hydrolyse β -limit dextrin or p -nitrophenyl- α - d -glucopyranoside. These results as well as the findings that the molecular size and the catalytic properties of the purified enzyme are different from those of known amylases obtained from Gramineae caryopses suggest that this enzyme is a novel type of β -amylase present in cell walls of vigorously elongating Gramineae shoot organs.     

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)-β-D-glucans

Polyclonal antibodies, raised against ((1→3), (1→4)-β-D-glucans from oat ( Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed-linked β-D-glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β-D-glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β-D-glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β-D-glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)-β-D-glucans of cell walls.     

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice ( Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10–50°C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30–40°C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40°C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30–40°C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40°C were substantially higher than those grown at 10, 20 and 50°C. Furthermore, the activities of (1→3),(1→4)- β -glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1→3),(1→4)- β -glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30–40°C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of β -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of β -glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.     

Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase.

BACKGROUND: Cell walls of the starchy endosperm and young vegetative tissues of barley (Hordeum vulgare) contain high levels of (1-->3,1-->4)-beta-D-glucans. The (1-->3,1-->4)-beta-D-glucans are hydrolysed during wall degradation in germinated grain and during wall loosening in elongating coleoptiles. These key processes of plant development are mediated by several polysaccharide endohydrolases and exohydrolases. RESULTS:. The three-dimensional structure of barley beta-D-glucan exohydrolase isoenzyme ExoI has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosylated at three sites. The first 357 residues constitute an (alpha/beta)8 TIM-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three alpha helices on either side of the sheet. A glucose moiety is observed in a pocket at the interface of the two domains, where Asp285 and Glu491 are believed to be involved in catalysis. CONCLUSIONS: The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this active-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on the second domain that may bind carbohydrate, as suggested by previously published kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3, 1-->4)-beta-D-glucans.     

The place of brassinolide in the sequential response to plant growth regulators in elongating tissue

In the sequential response to plant growth regulators in young elongating tissue from peas and wheat the peak of sensitivity to 24-epi-brussinolide (1 μM) occurs after those of gibberellin and cytokinin and begins before that of auxin in isolated wheat ( Triticum vulgare L. ev. Egret) coleoptiles aged from 21-96 h. In dwarf pea ( Pisum sativum L. cv. Greenfeast) segments, the peak of sensitivity also lies between those of gibberellin and auxin, and it also occurs before sensitivity to auxin in sections from first leaves of wheat. All the leaf sections and all but the most mature coleoptiles and pea segments were sensitive to fusicocein (1 μM).     

THE FIBRILLAR POLYSACCHARIDES AND THEIR LINKAGE TO ALGAENAN IN THE TRILAMINAR LAYER OF THE CELL WALL OF COELASTRUM SPHAERICUM (CHLOROPHYCEAE)

Methylation and spectroscopical analyses of DMSO-LiCl-solubilized fractions of the fibrillar cell wall of Coelastrum sphaericum Näg. established the presence of cellulose and β-1,4-linked mannan. A small proportion (2–7%) of (1→2) linkages in β-mannan that introduced an interruption in the regular ribbon chain conformation was interpreted as a component that modulated the mechanical strength of the cell wall. The trilaminar layer fractions consisted mostly of algaenan and cellulose. Evidence for ether linkages between glucose C-6 in the β-1,4-glucan and algaenan was obtained.     

Effect of anti-wall protein antibodies on auxin-induced elongation, cell wall loosening, and β-D-glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin-induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize ( Zea mays L. cv. B73 x Mo17) coleoptiles by SP-Sephadex and Bio-Gel P-150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin-induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth-inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin-induced elongation of segments also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP-Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin-induced decrease in (1→3), (1→4)-β-D-glucans in the cell walls. Thus, the degradation of β-D-glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin-induced degradation of (1→3), (1→4)-β-D-glucanses, β-D-glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.     

Cell wall autolytic activities and distribution of cell wall glucanases in Zea mays L. seedlings

Exo- and endo-glucanases mediate specific degradation of cell wall (1,3)(1,4)-beta-D-glucans and these enzymes have been related to auxin-mediated growth and development of cereal coleoptiles. However, their distribution and functions have not been well established in other tissues. In this study the glucanase activities and cell wall autolytic activities of different maize organs were determined. Autolysis assays serve to evaluate the hydrolysis of cell wall polymers in situ by measuring the sugars released from the insoluble cell wall matrix resulting from the action of bound enzymes. Autolytic activities were observed in the cell walls of elongating young leaves, mesocotyl and roots of maize. Wall proteins extracted from all of these structures are enriched in several types of glucanases and other wall polysaccharide hydrolases. These enzymes therefore appear to have a widespread and fundamental role in wall metabolism in growing tissues.     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.     

<Emphasis Type="Italic">QUASIMODO1</Emphasis> is expressed in vascular tissue of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> inflorescence stems,and affects homogalacturonan and xylan biosynthesis

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities.     

Characterisation of maize peroxidases having differential patterns of mRNA accumulation in relation to lignifying tissues

Among other enzymes, peroxidases have been proposed to participate in the latest steps of lignin biosynthesis. In order to identify new proteins involved in such mechanism of lignification in maize, we have isolated three cDNAs coding for three different peroxidases, named ZmPox1, ZmPox2, and ZmPox3, respectively. Computational analyses of these three proteins correlate with features typically attributed to heme-containing plant peroxidases of approximately 300 amino acid residues. Although with different expression levels, ZmPox2 and ZmPox3 mRNAs are accumulated in the elongating region of young roots but not in the root tips. In addition, the ZmPox2 mRNA levels are up-regulated by wounding and ethylene treatments. However, ZmPox1 is also expressed in the root tip meristems, where lignification does not occur. Finally, in situ hybridisations indicate that ZmPox2 mRNA localises in vascular tissues and epidermis. Although ZmPox1 mRNA localises in the same regions as ZmPox2 mRNA in root tips, its mRNA is only detected in the epidermis but not in the vascular tissues of young roots, suggesting that the function of ZmPox1 is not correlated to lignification. In addition, although ZmPox3 mRNA is also detected in the regions where lignification occurs, the involvement of this peroxidase in such a mechanism remains to be further investigated due to its very low expression level. Therefore, based on its amino acid sequence and mRNA accumulation and localisation patterns, the involvement of ZmPox2 in the latest steps of lignification is discussed.     

Differential expression of two functional serine/threonine protein kinases from soyabean that have an unusual acidic domain at the carboxy terminus

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::β-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses. Received: 6 January 1997 / Accepted: 19 March 1997