Three differentially expressed basic peroxidases from wound-lignifying Asparagus officinalis

The activity of ionically bound peroxidases from an asparagus spear increased from 5-24 h post-harvest. Isoelectric focusing showed that the post-harvest increase of the total peroxidase activity was due to the increase of several distinct isoperoxidases. Concomitantly, a decrease in the activity of two anionic peroxidases was observed. Peroxidases with pI 5.9, 6.4 and 9.2 were detected only at 24 h post-harvest, whereas four peroxidases, with pI 8.7, 8.1, 7.4, and 6.7, detected throughout the time-course, increased in their activity. Histochemical staining demonstrated that lignin and peroxidase activity were located in the vascular bundles throughout the period of measurement. Lignin was detected in the cell walls of the protoxylem in the vascular bundles of the asparagus stem. A cDNA library of mRNA isolated from asparagus spears 24 h post-harvest was screened for peroxidases using homologous and heterologous probes. Three clones were isolated and the corresponding mature asparagus peroxidases displayed 70%, 76% and 81% amino acid sequence identity to each other. These new asparagus peroxidases are typical class III plant peroxidases in terms of conserved regions with a calculated pI >9.2, which is consistent with most basic peroxidases. One of the genes was shown to be a constitutively expressed single-copy gene, whereas the others showed an increased expression at post-harvest. The highest similarity in the amino acid sequence (71-77%) was found in peroxidases from roots of winter grown turnip TP7, to Arabidopsis AtP49, to an EST sequence from cotton fibres and to TMV-infected tobacco.     

Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana

Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E.C. 1.11.1.7). These enzymes have been implicated in a wide array of physiological processes such as H(2)O(2) detoxification, auxin catabolism and lignin biosynthesis and stress response (wounding, pathogen attack, etc.). During the last 10 years, molecular cloning has allowed the isolation and characterization of several genes encoding peroxidases in plants. The achievement of the large scale Arabidopsis genome sequencing, combined with the DNA complementary to RNA (cDNA) expressed sequence tags projects, provided the opportunity to draw up the first comprehensive list of peroxidases in a plant. By screening the available databases, we have identified 73 peroxidase genes throughout the Arabidopsis genome. The evolution of the peroxidase multigene family has been investigated by analyzing the gene structure (intron/exon) in correlation with the phylogenetic relationships between the isoperoxidases. An evolutionary pattern of extensive gene duplications can be inferred and is discussed. Using a cDNA array procedure, the expression pattern of 23 peroxidases was established in the different organs of the plant. All the tested peroxidases were expressed at various levels in roots, while several were also detected in stems, leaves and flowers. The specific functions of these genes remain to be determined.     

Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain

A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.     

Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots

PCR with oligonucleotide primers that corresponded to two highly homologous regions, in terms of amino acid sequence, of plant peroxidases was used to amplify a specific DNA fragment from a mixture of rice (Oryza sativa L.) cDNAs. We then screened a cDNA library prepared from mRNAs of rice shoots utilizing the product of PCR as probe. Two cDNA clones, prxRPA and prxRPN, were isolated. They encode distinct isozymes of peroxidase. Sequence analysis indicated that the clones encode mature proteins of approximately 32 kDa, both of which possess a putative signal peptide. Comparison of the amino acid sequences of the two rice peroxidases showed that they are about 70% similar to each other but are only 40% to 50% similar to other plant peroxidases. RNA blot hybridization revealed that mRNAs that corresponded to prxRPA and prxRPN cDNAs accumulate at high levels in roots but only at low levels in stems and leaves. In various tissues of rice plants, levels of both mRNAs were stimulated by wounding and by ethephon. These results indicate that at least two isozymes of peroxidase are expressed not only in shoots but also in roots of rice plants, and that the expression of these genes is influenced by ethylene which is the simplest plant hormone.     

Root growth and lignification of two wheat species differing in their sensitivity to NaCl, in response to salt stress

Application of a 100-mM NaCl salt stress to wheat seedlings of a salt-tolerant (Triticum durum var. Ben Béchir) and a salt-sensitive (Triticum aestivum var. Tanit) species decreases the fresh and dry weights of roots especially in the salt-sensitive species, and slightly increases the ratio of dry to fresh weight, especially in the salt-resistant species. All peroxidase activities are increased by salt stress, the water-soluble peroxidase activity being increased much more in the salt-sensitive than in the salt-tolerant species, while the opposite result is observed with the cell-wall peroxidase activity. Some water-soluble peroxidases have been hypothesised to have auxin oxidase activity (which might explain the effect observed on the root biomass), while the cell-wall peroxidases would be involved in lignification. Histochemical observation confirms a more intense lignification in the root cells of the salt-tolerant species compared to the sensitive species, under the effect of NaCl.     

Identification of a novel peptide motif that mediates cross-linking of proteins to cell walls

A cDNA clone representing a member of a novel class of cell wall proteins was isolated from tobacco plants. We have designated this protein NtTLRP for tyrosine- and lysine-rich protein. It is structurally related to the previously identified TLRP from tomato plants, sharing a high amino-acid sequence similarity at the C-terminal region. This region contains what appears to be a novel peptide motif which we call CD for cysteine-rich domain, and which is common to several other cell-wall proteins. By using a functional test in transgenic plants, we demonstrate that the presence of the CD domain is per se sufficient to cross-link previously soluble proteins to the cell wall. We present evidence that NtTLRP is cross-linked and specifically localizes to the cell wall of lignified cells. The highly localized deposition of NtTLRP in these cells indicates that this class of cell-wall proteins may have a specialized function in the formation of xylem tissue.     

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1–5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.     

Phylogenetic diversity of non-nodulating Rhizobium associated with pine ectomycorrhizae

Most Rhizobium species described are symbionts that form nodules on legume roots; however, non-nodulating strains of Rhizobium are also widespread in nature. Unfortunately, knowledge of non-nodulating Rhizobium is quite limited compared with nodulating Rhizobium . Here, we studied the phylogenetic diversity of Rhizobium species that inhabit Japanese red pine roots ( Pinus densiflora ). Because fine roots of pine trees are usually colonized by ectomycorrhizal fungi in nature, we mainly used ectomycorrhizal root tips for bacterial isolation. Out of 1195 bacteria isolated from 75 independent root samples from the field and greenhouse experiments, 102 isolates were confirmed to be Rhizobium following partial 16S rRNA gene analysis. Rhizobium species were occasionally dominant in culturable bacterial communities, whereas no Rhizobium species were isolated from the soil itself. Molecular phylogenetic analyses using 16S rRNA, atpD , and recA gene sequences revealed that isolated Rhizobium strains were phylogenetically diverse and that several were distantly related to known Rhizobium species. Considering that a single species of pine is associated with unique and phylogenetically diverse Rhizobium populations, we should pay more attention to non-nodulating strains to better understand the diversity, ecology, and evolution of the genus Rhizobium and plant– Rhizobium associations.     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

cDNA cloning,characterization and expression of an endosperm-specific barley peroxidase

A barley peroxidase (BP 1) of pI ca. 8.5 and M _r 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.     

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

 A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein. Received: 15 March 1999 / Accepted: 12 July 1999     

cDNA cloning of bradykinin-potentiating peptides-C-type natriuretic peptide precursor, and characterization of the novel peptide Leu3-blomhotin from the venom of Agkistrodon blomhoffi.

A cDNA clone, 1.8 kb long, was isolated from a venom gland cDNA library of Agkistrodon blomhoffi that encodes a large plurifunctional precursor composed of 263 amino-acid residues. Nucleotide sequence analysis of this clone revealed that sequences which code for blomhotin and a novel peptide Leu3-blomhotin are located in the N-terminal region of the precursor polypeptide, followed by four tandemly aligned sequences which code for three types of bradykinin-potentiating peptide. In the C-terminal region, the sequence for the C-type natriuretic peptide was located along with a preceding processing signal. The deduced amino-acid sequences for the four bradykinin-potentiating peptides coincided exactly with previously known sequences for potentiator B, potentiator C and potentiator E. The actual Leu3-blomhotin peptide was subsequently isolated from the venom of A. blomhoffi and characterized. Leu3-blomhotin possesses contractile activity in isolated rat stomach fundus smooth muscle in the same manner as blomhotin. Furthermore, it was shown that blomhotin and Leu3-blomhotin retained activity to inhibit the angiotensin-converting enzyme.