Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris.

The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-beta-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)). Here, we report the functional analysis of Delta(1-90)Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Delta(1-90)Cel16 in a pure form. The molecular mass of Delta(1-90)Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Delta(1-90)Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta(1-90)Cel16 had a pH optimum of 6.0. The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.

Two Arabidopsis thaliana genes, KOR2 and KOR3, which encode membrane-anchored endo-1,4-beta-D-glucanases, are differentially expressed in developing leaf trichomes and their support cells.

The Arabidopsis KOR gene encodes a membrane-anchored endo-1,4-beta-D-glucanase involved in cell wall assembly. To obtain a more detailed knowledge of the small gene family encoding membrane-anchored endo-1,4-beta-D-glucanases in Arabidopsis thaliana, we have characterized two additional membrane-anchored endo-1,4-beta-D-glucanase genes. Sequence comparison indicates that KOR2 is distantly related to KOR and other plant membrane-anchored endo-1,4-beta-D-glucanases. The expression of KOR2 and KOR3 was followed by the beta-glucuronidase (gusA) reporter-gene method. While the KOR gene is most often expressed throughout the plant, KOR2::gusA and KOR3::gusA are active only in restricted cell types. We demonstrate that KOR2::gusA is expressed very early in the development of root hairs within the root differentiation zone (specialization zone) but not in the root-hair-bearing epidermal cells at the root/shoot junction (transition zone). Furthermore, KOR2::gusA is expressed in the proximal parts of leaves and floral organs (rosette and cauline leaves, sepals, petals and stamens), and in trichomes, as they develop at the tip of young leaves and later in more basal regions of the leaf blade. The KOR3::gusA construct is expressed in the trichome support cells that form a ring at the base of each trichome and in the bundle sheath cells which surround the vascular bundle within the leaf mesophyll tissue. Reverse transcription-polymerase chain reaction of Arabidopsis RNA confirmed the expression of KOR2::gusA and KOR3::gusA. In conclusion, although KOR2 and KOR3 have more restricted expression patterns than the previously characterized KOR gene, they are expressed in cell types at time points where cell wall assembly is likely to occur and, interestingly, differentially expressed in leaf trichomes and their support cells.

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.

The cleavable N-terminal domain of plant endopolygalacturonases from clade B may be involved in a regulated secretion mechanism.

Polygalacturonases represent the most abundant carbohydrate hydrolase family in the Arabidopsis thaliana genome, and they are thought to be involved in nearly all of the developmental processes requiring cell wall modifications during the life cycle of the plant. By phylogenetic analysis, plant polygalacturonases fall into at least three groups, one of which is distinguished from the others by the presence of an additional N-terminal domain. We have used RDPG1, the polygalacturonase involved in pod dehiscence in oilseed rape (Brassica napus), as a model to investigate the function of this domain. We have confirmed that this domain is absent in the mature protein by determination of the N-terminal sequence of mature RDPG1 purified from oilseed rape pod. We have furthermore investigated the accumulation and subcellular localization of the precursor containing the N-terminal domain and of the mature protein throughout the development and maturation of the pod. Using recombinant expression in Pichia pastoris, we have produced the RDPG1 precursor, and we present evidence that the N-terminal domain of plant polygalacturonases is not involved in folding or inactivation of the precursor but may play a role in the intracellular transport of this protein family via a novel regulated secretion pathway.

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

Expression of a membrane-anchored endo-1,4-beta-glucanase from Brassica napus, orthologous to KOR from Arabidopsis thaliana, is inversely correlated to elongation in light-grown plants.

A PCR fragment derived from a membrane-anchored endo-1,4-beta-glucanase cDNA was amplified using degenerated oligonucleotides and mRNA from oilseed rape (Brassica napus L.) siliques. Sequence analysis of the corresponding gene, Cel16, showed that the predicted Cel16 protein has high identity with the Arabidopsis KOR protein (94%). High-stringency genomic Southern analysis further revealed that Cel16 and KOR are most likely orthologous genes performing a similar function in both species. Northern blot and GUS analysis of transgenic Arabidopsis containing a fusion between a 2.0 kb Cel16 promoter fragment and the GUS reporter gene showed that Cel16 was expressed at a low level in the primary raceme, the young lateral stems, the elongation zone of the primary root and the older root base. By contrast, a high level of Cel16 mRNA accumulation was found in the young root and in the main stem carrying flowers and young siliques. Cel16 transcripts were localized to the apical meristem, cambium, primary xylem and cortex of oilseed rape stem tissue by in situ RT-PCR. A similar pattern of activity was found in the GUS analysis of transgenic Arabidopsis. Cel16 mRNA accumulation in the main stem was lower in the zone of most rapid cell elongation than in the subjacent, fully elongated internodes. Similarly, Cel16 transcripts accumulated to a higher level in leaves as they reached full size than during early leaf expansion. Analysis of the expression pattern in elongating, light-grown seedlings showed that Cel16 mRNA accumulated at a lower level in the elongating upper third than elsewhere in the hypocotyl. This is contrary to etiolated hypocotyls, where we found a higher expression level in the rapidly elongating upper part. This difference in expression most probably reflects a difference in cell wall assembly between light- and dark-grown seedlings.

Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase.

Potato tuber pectin is rich in galactan (oligomer of beta-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.

In vitro biosynthesis of 1,4-beta-galactan attached to rhamnogalacturonan I.

The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[14C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [14C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-beta-galactanase released 65-70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [14C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-beta-galactanase. Thus, the majority of the 14C-labelled product was 1,4-beta-galactan. Compounds released by the endo-1,4-beta-galactanase treatment were mainly [14C]galactose and [14C]galactobiose, indicating that the synthesized 1,4-beta-galactan was longer than a trimer. In vitro synthesis of 1,4-beta-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn2+. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [14C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-beta-galactanase, radioactivity was not only found as [14C]galactose or [14C]galactobiose but also as larger fragments. The larger fragments were likely the [14C]galactose or [14C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with beta-galactosidase together with endo-1,4-beta-galactanase digested all radioactivity to the fraction eluting as [14C]galactose. The data indicate that the majority of the [14C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-beta-galactan sidechains in the endogenous pectic rhamnogalacturonan I.

Isolation and characterisation of a pod dehiscence zone-specific polygalacturonase from Brassica napus.

Seven distinct partial cDNAs, similar in sequence to previously described polygalacturonases (PGs), were amplified from cDNA derived from rape pod wall, dehiscence zone and leaves by the polymerase chain reaction. Northern analysis showed that one clone, PG35-8, was expressed at low levels in the dehiscence zone during the first five weeks after anthesis but was very abundantly expressed at week 6. In contrast, no PG35-8-related RNA was detected in the pod wall. Our data suggest that there are temporal and spatial correlations between the breakdown of the middle lamella, of the dehiscence zone cells and the pattern of synthesis of PG35-8 transcripts which may indicate a role for this particular PG in rape pod dehiscence. PG35-8 was used to isolate five cDNA clones from a rape dehiscence zone cDNA library. Restriction enzyme analysis and partial sequencing revealed that they were derived from four highly homologous transcripts which are probably allelic forms of a single gene. One full-length clone, RDPG1, was completely sequenced. The predicted protein of RDPG1 showed its highest identity with PG from apple fruit with an identity of 52%.

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.) : I. Spatial distribution of endogenous cytokinins in relation to leaf growth.

Immunoaffinity purification of zeatin, dihydrozeatin and isopentenyl-type cytokinins from expanding leaves of sweet pepper was accomplished using a single immobilized monoclonal antibody. Isopentenyl adenosine, zeatin, zeatin riboside and the N9-glucosides of zeatin and isopentenyl adenine were found to be the dominating endogenous cytokinins while the dihydrozeatin cytokinins were either absent or constituted a very minor group of cytokinin metabolites in pepper. Leaves were selected for analysis at an age where a range of developmental stages exist within a single leaf. The spatial distribution of endogenous cytokinins in rapidly expanding leaves at this stage was markedly different from the almost uniform distribution in expanded leaves. The distribution of zeatin and zeatin riboside in rapidly expanding leaves was found to be correlated with the rate of leaf expansion which is high (∼40%/24 h) in the basal leaf tissue and low (∼10%/24 h) near the leaf tip. Applied growtn factors supported a rate of expansion of excised discs comparable to the growth rates observed in situ, but did not affect the ability of the tissue to retain assimilated amino acids. The results are discussed in relation to sink-strength stimultation as a potential mode of cytokinin action in leaf development.

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.) : II. Sink metabolism in relation to cytokinin-promoted leaf expansion.

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.

Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas.

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1'-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1'-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.

Plant polypeptides reversibly glycosylated by UDP-glucose. Possible components of Golgi beta-glucan synthase in pea cells.

In pea membranes, UDP[14C]Glc glycosylates a approximately 40-kDa polypeptide doublet. This label rapidly disappears if excess unlabeled UDP-Glc, or UDP, is added, indicating that the glycosylation is reversible, and suggesting that the glycosylated polypeptides might be intermediates in a glycosyl transfer reaction. Glycosylation of the doublet requires a divalent cation, the effective ions being the same (except for Zn2+) as those that activate Golgi-localized beta-glucan synthase (GS-I) activity. Treatments that inhibit GS-I also inhibit doublet glycosylation. The doublet is associated with Golgi (and to a minor extent with plasma) membranes and occurs also in the soluble fraction. The Golgi-bound doublet may be a component of the GS-I system. Immunological, inactivation, and fractionation evidence indicates that at least one other polypeptide is required in GS-I activity.

Effect of detergents on the H(+)-ATPase activity of inside-out and right-side-out plant plasma membrane vesicles.

In search for a detergent to be used to assess the sidedness of plant plasma membrane vesicles by enzyme latency we tested the effect of 42 detergents on the ATPase activity of right-side-out and inside-out plasma membrane vesicles from sugar beet leaves. Most of the detergents seemed to inactivate the ATPase in addition to disrupting the permeability barrier to ATP. There were two main exceptions, namely long chain polyoxyethylene acyl ethers, such as detergents of the Brij series and Lubrol WX, and long chain lysophospholipids. These two types of detergents permeabilized the membranes at low concentrations and did not inhibit the ATPase at higher concentrations. Unmasking of latent active sites seemed to explain the activation of the plasma membrane H(+)-ATPase produced by long chain polyoxyethylene acyl ethers. These detergents should therefore be ideal for determination of vesicle orientation based on ATPase latency. By contrast, long chain lysophospholipids were found to be highly specific activators of the enzyme. In addition, long chain fatty acids were found to strongly inhibit ATP-dependent proton accumulation in the vesicles without inhibiting ATP hydrolysis. This uncoupling effect of the fatty acids could be abolished by the addition of fatty acid-free bovine serum albumin (BSA). Similarly, the proton transport capacity of ageing vesicles could be restored by addition of BSA. The latter findings may explain why isolated plasma membranes so often exhibit increased permeability to protons on ageing.

Preparation and Properties of Antibodies against Indoleacetic Acid (IAA)-C5-BSA, a Novel Ring-Coupled IAA Antigen, as Compared to Two Other Types of IAA-Specific Antibodies.

Two types of indoleacetic acid (IAA) antigens have been described in the literature which show marked differences with respect to antibody specificity. In this communication an alternative antigen design is described. In the so-called IAA-C5-BSA conjugate, both the acetic acid group and the pyrrole moiety are presented free, as the covalent linkage between IAA and the carrier molecule is introduced in the benzene moiety. Antibodies were elicited in rabbits against this novel antigen. Using cross-reactivity data and the strategy of successive approximation for the measurements of auxin levels in the internodes of light-grown broad bean (Vicia faba L. cv Superfine), the three types of antibodies are compared.