Molecular and functional evolution of class I chitinases for plant carnivory in the caryophyllales.

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.

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title: Transgenic Plants with Enhanced Resistance to the Fungal Pathogen Rhizoctonia solani
abstract: The production of enzymes capable of degrading the cell walls of invading phytopathogenic fungi is an important component of the defense response of plants. The timing of this natural host defense mechanism was modified to produce fungal-resistant plants. Transgenic tobacco seedlings constitutively expressing a bean chitinase gene under control of the cauliflower mosaic virus 35S promoter showed an increased ability to survive in soil infested with the fungal pathogen Rhizoctonia solani and delayed development of disease symptoms.

title: Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana.
abstract: Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ-specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue.

title: Sequence and Structural Analysis of the Chitinase Insertion Domain Reveals Two Conserved Motifs Involved in Chitin-Binding
abstract: Chitinases are prevalent in life and are found in species including archaea, bacteria, fungi, plants, and animals. They break down chitin, which is the second most abundant carbohydrate in nature after cellulose. Hence, they are important for maintaining a balance between carbon and nitrogen trapped as insoluble chitin in biomass. Chitinases are classified into two families, 18 and 19 glycoside hydrolases. In addition to a catalytic domain, which is a triosephosphate isomerase barrel, many family 18 chitinases contain another module, i.e., chitinase insertion domain. While numerous studies focus on the biological role of the catalytic domain in chitinase activity, the function of the chitinase insertion domain is not completely understood. Bioinformatics offers an important avenue in which to facilitate understanding the role of residues within the chitinase insertion domain in chitinase function.  Twenty-seven chitinase insertion domain sequences, which include four experimentally determined structures and span five kingdoms, were aligned and analyzed using a modified sequence entropy parameter. Thirty-two positions with conserved residues were identified. The role of these conserved residues was explored by conducting a structural analysis of a number of holo-enzymes. Hydrogen bonding and van der Waals calculations revealed a distinct subset of four conserved residues constituting two sequence motifs that interact with oligosaccharides. The other conserved residues may be key to the structure, folding, and stability of this domain.  Sequence and structural studies of the chitinase insertion domains conducted within the framework of evolution identified four conserved residues which clearly interact with the substrates. Furthermore, evolutionary studies propose a link between the appearance of the chitinase insertion domain and the function of family 18 chitinases in the subfamily A.

title: Biochemical and molecular characterization of three barley seed proteins with antifungal properties.
abstract: We have purified three proteins from barley (Hordeum vulgare L.) seeds which synergistically inhibit the growth of fungi measured in a microtiter well assay. The proteins are a 26-kDa chitinase, a 30-kDa ribosome-inactivating protein, and a 32-kDa (1-3)-beta-glucanase. Full-length cDNAs encoding them were isolated and sequenced to determine the complete primary structures of the proteins. Northern hybridizations with the cDNAs as probes showed that the corresponding mRNAs accumulate differentially during seed development and germination. Chitinase mRNA accumulates to high levels in aleurone cells during late seed development and early germination, while high levels of mRNA encoding the ribosome-inactivating protein accumulate only in the starchy endosperm during late seed development. The glucanase mRNA accumulates to low levels during seed development and to higher levels in aleurone and seedling tissues during germination. Southern hybridizations showed that the three proteins are encoded by small families of three to eight genes. Their biological roles and potential use in genetic engineering studies are discussed.

title: Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco.
abstract: cDNAs encoding three proteins from barley (Hordeum vulgare), a class-II chitinase (CHI), a class-II beta-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed antifungal proteins in vivo.

title: Isolation and characterization of chitinase genes from pitchers of the carnivorous plant Nepenthes khasiana.
abstract: The genus Nepenthes represents carnivorous plants with pitcher traps capable of efficient prey capture and digestion. The possible involvement of plant chitinases in this process was studied in Nepenthes khasiana. Two different types of endochitinases were identified in the liquid of closed traps exhibiting substrate specificity for either long chitin polymers or N-acetylglucosamine (GlcNAc) oligomers. Injection of chitin into such closed sterile pitchers induced the appearance of additional endochitinase isoenzymes, with substrate specificity only for long chitin polymers. No significant exochitinase (N-acetyl-beta-glucosaminidase) or chitobiosidase activity could be detected in the non-induced or induced trap liquid. Four genes representing two subgroups of basic chitinases, denoted as Nkchit1b and Nkchit2b, were isolated from the secretory region of N. khasiana pitchers. The main differences between the two subgroups are the presence of a proline-rich hinge region only in NkCHIT1b and a C-terminal putative vacuole targeting extension only in NkCHIT2b, indicating different compartmentalization of the two enzymes. Reverse transcription-polymerase chain reaction (RT-PCR) evaluation of mRNA levels showed that the Nkchit2b genes are constitutively expressed in the secretory cells while transcription of Nkchit1b genes is induced by chitin injection. These results show for the first time the involvement of genes encoding chitinases in prey-trap interaction and their differential expression and activity during prey trapping.

title: Structural and Evolutionary Relationships Among Chitinases of Flowering Plants

abstract: The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains—namely, a chitin-binding domain and a carboxy-terminal extension—flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host–pathogen interactions are discussed.

title: Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain
abstract: The endochitinases (E.C. 3.2.1.14, chitinase) are a structurally diverse group of enzymes believed to be important in the biochemical defense of plants against potential pathogens. The gene for a chitinase of Nicotiana tabacum L. cv. Havana 425 has been cloned and sequenced. The major transcription start is 11 bp upstream of the ATG codon and 28 bp downstream of the TATA box. The gene contains two introns and encodes a basic chitinase of 329 amino acids with a 23 amino acid N-terminal signal peptide followed by a 43 amino acid, cysteine-rich domain, which is linked by a hinge region to the main structure of the enzyme. This gene appears to be expressed because the exons are identical to the coding sequence of a cDNA which was isolated. Comparison of chitinase amino acid sequences from different plants indicates there are at least three classes of these enzymes: class I, basic chitinases with an N-terminal cysteine-rich domain and a highly conserved main structure; class II, chitinases similar to the main structure of class I chitinases but lacking the cysteine-rich domain; and, class III, chitinases with conserved sequences different from those of the class I and II enzymes. The sequences encoding the cysteine-rich domain in class I chitinases are flanked by 9–10 bp imperfect direct repeats suggesting that these domains arose from a common ancestral gene and were introduced into genes for class I enzymes by transposition events.

title: Structural features of plant chitinases and chitin‐binding proteins
abstract: Structural features of plant chitinases and chitin‐binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin‐binding hevein domain is a predominant one. X‐ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three‐dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b‐type (classes I, II and IV) and h‐type (classes III and V) chitinases, respectively.

title: Structural classification of plant chitinases: two subclasses in class I and class II chitinases.
abstract: For the classification of plant chitinases, phylogenetic relationships were analyzed besides the established classification of domain structure and C-terminal extension sequences. Two genetically different subclasses (high and low molecular weight) were found for the main structure of class I and class II chitinases in their phylogenetic trees. The genetic distance of these subclasses showed that the high molecular weight subclass may be an ancestral molecule.

title: Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chitinases.
abstract: Four endochitinases (poly[1,4-(N-acetyl-beta-D-glucosaminide)] glycanohydrolase, EC 3.2.1.14) have been purified from leaves of Nicotiana tabacum cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Two of them are acidic proteins of molecular weights 27,500 and 28,500 and have been identified as 2 of the 10 pathogenesis-related proteins that are known to accumulate in tobacco in response to stress or pathogen attack. These two pathogenesis-related proteins, named "P" and "Q" when their biological function was unknown, account for one-third of tobacco mosaic virus-induced chitinase activity of tobacco leaves. They are serologically closely related to the two other chitinases, which can be considered as new basic pathogenesis-related proteins. These two basic chitinases exhibit higher molecular weights (32,000 and 34,000) and higher specific enzyme activity than the two acidic isoforms.

title: The N-Terminal Cysteine-Rich Domain of Tobacco Class I Chitinase Is Essential for Chitin Binding but Not for Catalytic or Antifungal Activity
abstract: The vacuolar chitinases of class I possess an N-terminal cysteine-rich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urtica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana tabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified to homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the N-terminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitin-binding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for its substrate. The pH and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low pH values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD is not necessary for catalytic or antifungal activity of chitinase.

title: Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants
abstract: The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and β-1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.

title: A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.
abstract: Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.

title: Purification and characterization of abundant secreted protein in suspension-cultured pumpkin cells : abundant secreted protein may be a chitinase.
abstract: The abundant secreted protein with molecular weight of 32,000 was purified from the culture medium of suspension-cultured pumpkin (Cucurbita sp.) cells. Two steps, ammonium sulfate fractionation and Sepharose 6B column chromatography, were sufficient for purification to homogeneity. Antibodies against the pure protein were used to show that a protein of the same size is made by callus cells. There is considerable homology between the amino-terminal amino acid sequence of this secreted protein and chitinase isolated from tobacco (Nicotiana tabacum L.) or bean (Phaseolus vulgaris L.).

title: Subcellular localization of acidic and basic PR proteins in tobacco mosaic virus-infected tobacco
abstract: SummaryInfection of Samsum NN tobacco with tobacco mosaic virus (TMV) results in the induction of the synthesis of acidic and basic isoforms of many pathogenesis-related (PR) proteins. By immunogold-electronmicroscopy we have shown that PR proteins accumulate mainly in cells around the necrotic spots of TMV-induced lesions. The acidic chitinases, β-(1,3)-glucanases and thaumatin-like proteins were found to accumulate in extracellular “pocket-like” vesicles while the basic chitinases were found in electron dense inclusion bodies in the vacuoles. These structures were not detectable in PR-containing leaves devoid of virus nor in healthy plants.

title: Characterization of a wheat class Ib chitinase gene differentially induced in isogenic lines by infection with Puccinia graminis
abstract: Plant chitinases have been recognized to be involved in defense against pathogens. A class Ib genomic chitinase gene was isolated from a wheat (Triticum aestivum L.) genomic library using a wheat chitinase sequence amplified from DNA by PCR. The nucleotide sequence analysis showed that the wheat chitinase gene (Wchl) contains a 960-bp long coding region with no intron. The derived polypeptide includes a signal peptide, a cysteine-rich domain and a catalytic domain without a carboxy terminal extension. Primer extension experiments identified a single transcription start site 33 bp upstream of the translation initiation site. Southern blot analysis indicated that Wchl is one member of a small gene family in wheat. Northern blot hybridization and histological investigation of wheat leaves infected by Puccinia graminis f. sp. tritici revealed a close correlation between the accumulation of the chitinase transcripts and the inhibition of fungal growth when two isogenic wheat lines (Prelude Sr6 and Prelude sr6) differing at the Sr6 locus for race-specific incompatibility with the fungus were studied. The massive accumulation of the chitinase mRNA occurred only in the incompatible Sr6/P6 interaction, in parallel with a severe inhibition of mycelial growth in the leaves. In contrast, in the compatible sr6/P6 interaction the fungus grew rapidly throughout the leaf tissue and the chitinase transcripts were not detected. The chitinase mRNA was detectable in non-infected stems but not in non-infected leaves or roots.

title: Molecular Analysis of Two cDNA Clones Encoding Acidic Class I Chitinase in Maize
abstract: The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus grown on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions.

title: A class I chitinase from soybean seed coat.
abstract: Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.

title: A hevein-like protein and a class I chitinase with antifungal activity from leaves of the paper mulberry.
abstract: Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low-molecular-weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activity against Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit. The purification protocol employed (NH(4))(2)SO(4) precipitation, ion-exchange chromatography and hydrophobic-interaction chromatography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has no peptides similar to PMAPII. N-terminal amino acid sequencing revealed that PMAPI is a hevein-like protein, and PMAPII is a class I chitinase. They both had a half-maximal inhibitory concentration (IC50) of 0.1 µg/µL against T. viride. This is the first report of high-molecular-weight extracts with antifungal activity from paper mulberry.

title: Rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution.
abstract: Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions.

title: Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifoliaL.) show induction of chitinase activity upon mimicking the presence of prey
abstract: Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of non-treated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.

title: A Sticky Situation: Assessing Adaptations for Plant Carnivory in the Caryophyllales by Means of Stochastic Character Mapping
abstract: Phylogenetic relationships among carnivorous plants of the angiosperm order Caryophyllales have been explored, although a robust phylogeny encompassing all carnivorous genera is absent. We sample nuclear ribosomal spacer (internal transcribed spacer) and chloroplast intergenic spacer (PY-IGS), along with previously sequenced DNA from members of the noncore Caryophyllales, for use in Bayesian statistics and maximum likelihood–based searches of phylogeny. Taxonomic relationships across genera are refined, and three strongly supported clades are identified: monophyletic Droseraceae, Nepenthaceae, and a third clade containing Ancistrocladaceae, Dioncophyllaceae, and Drosophyllaceae. In combination with phylogenetic reconstruction, stochastic character mapping is used to assess evolutionary changes in the morphology of glands that are found on the lamina and involved in the digestion of prey. The presence of sessile glands is identified as the likely ancestral character state, and stalked and pitted glands are suggested to have been acquired independently by ingroup and outgroup taxa. Additionally, in some genera we found a lack of association between gland vasculature and plant carnivory, demonstrating that the internal architecture of glands is not indicative of whether the plant is a functional carnivore. Finally, we discuss how adaptive changes resulting in the evolution of the carnivorous gland may have occurred either by emargination of the leaf blade or homologous transformation of pinnae.

title: A rapid DNA isolation procedure for small quantities of fresh leaf tissue
abstract: A stopped flow kinetic analysis has been performed with a homogeneous protein fraction of plant glutamate dehydrogenase. The enzyme exerts strong negative cooperativity with ammonium as variable substrate. The limiting initial rate constants for low substrate concentrations, as calculated from the kinetic data, indicate that the catalytic efficiency of the enzyme increases at low ammonium concentrations. From this it becomes evident that the reductive amination reaction is highly adaptive to the ammonium environment.

title: A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.
abstract: The polymerase chain reaction (PCR) has revolutionised the rapid analysis of mammalian genomic DNA (1). However, PCR is less useful in the analysis of plant DNA due to the difficulties in extracting nucleic acids from limited amounts of plant tissue. We have developed a method for the rapid extraction of small amounts of plant genomic DNA suitable for PCR analysis. The method is applicable to a variety of plant species and has the added advantage of not requiring any phenol or chloroform extraction. Thus it is possible to complete an extraction within 15 minutes without handling any hazardous organic solvents. Samples for PCR analysis (usually leaf tissue) are collected using the lid of a sterile Eppendorf tube to pinch out a disc of material into the tube. This ensures uniform sample size and also reduces the possibilities of contamination arising from handling the tissue. DNA is extracted as follows: The tissue is macerated (using disposable grinders from Bel-art Products: Scienceware, Pequannock, NJ, 07440 USA. catalog no 992) in the original Eppendorf tube at room temperature, without buffer, for 15 seconds. 400 yl of extraction buffer (200 mM Tris HC1 pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) is added and the sample vortexed for 5 seconds. This mixture can then be left at room temperature until all the samples have been extracted (> 1 hour). The extracts are centrifuged at 13,000 rpm for 1 minute and 300 y\ of the supernatant transferred to a fresh Eppendorf tube. This supernatant is mixed with 300 /il isopropanol and left at room temperature for 2 minutes. Following centrifugation at 13,000 rpm for 5 minutes, the pellet is vacuum dried and dissolved in 100 /xl 1XTE. This DNA is stable at 4°C for greater than one year. 2.5 yX of this sample is sufficient for a standard 50 y\ PCR (. When older tissue is used this may be increased to 25 /xl without any deleterious effect on the PCR. Using this protocol we have found it possible to process hundreds of individual samples in a single working day.

title: How to usefully compare homologous plant genes and chromosomes as DNA sequences.
abstract: There are four sequenced and publicly available plant genomes to date. With many more slated for completion, one challenge will be to use comparative genomic methods to detect novel evolutionary patterns in plant genomes. This research requires sequence alignment algorithms to detect regions of similarity within and among genomes. However, different alignment algorithms are optimized for identifying different types of homologous sequences. This review focuses on plant genome evolution and provides a tutorial for using several sequence alignment algorithms and visualization tools to detect useful patterns of conservation: conserved non-coding sequences, false positive noise, subfunctionalization, synteny, annotation errors, inversions and local duplications. Our tutorial encourages the reader to experiment online with the reviewed tools as a companion to the text.

title: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
abstract: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.

title: Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds.
abstract: Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.

title: ProtTest: selection of best-fit models of protein evolution
abstract: Using an appropriate model of amino acid replacement is very important for the study of protein evolution and phylogenetic inference. We have built a tool for the selection of the best-fit model of evolution, among a set of candidate models, for a given protein sequence alignment.


AVAILABILITY
ProtTest is available under the GNU license from http://darwin.uvigo.es

title: HyPhy: hypothesis testing using phylogenies
abstract: The HyPhypackage is designed to provide a flexible and unified platform for carrying out likelihood-based analyses on multiple alignments of molecular sequence data, with the emphasis on studies of rates and patterns of sequence evolution.


AVAILABILITY
http://www.hyphy.org


CONTACT
muse@stat.ncsu.edu



HyPhydocumentation and tutorials are available at http://www.hyphy.org.

title: The SWISS-MODEL Repository and associated resources
abstract: SWISS-MODEL Repository (http://swissmodel.expasy.org/repository/) is a database of 3D protein structure models generated by the SWISS-MODEL homology-modelling pipeline. The aim of the SWISS-MODEL Repository is to provide access to an up-to-date collection of annotated 3D protein models generated by automated homology modelling for all sequences in Swiss-Prot and for relevant models organisms. Regular updates ensure that target coverage is complete, that models are built using the most recent sequence and template structure databases, and that improvements in the underlying modelling pipeline are fully utilised. As of September 2008, the database contains 3.4 million entries for 2.7 million different protein sequences from the UniProt database. SWISS-MODEL Repository allows the users to assess the quality of the models in the database, search for alternative template structures, and to build models interactively via SWISS-MODEL Workspace (http://swissmodel.expasy.org/workspace/). Annotation of models with functional information and cross-linking with other databases such as the Protein Model Portal (http://www.proteinmodelportal.org) of the PSI Structural Genomics Knowledge Base facilitates the navigation between protein sequence and structure resources.

title: The Protein Data Bank: a computer-based archival file for macromolecular structures.
abstract: The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.

title: X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases.
abstract: The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

title: The role of enzyme distortion in the single displacement mechanism of family 19 chitinases.
abstract: By using molecular dynamics simulations, we have examined the binding of a hexaNAG substrate and two potential hydrolysis intermediates (an oxazoline ion and an oxocarbenium ion) to a family 19 barley chitinase. We find the hexaNAG substrate binds with all sugars in a chair conformation, unlike the family 18 chitinase which causes substrate distortion. Glu 67 is in a position to protonate the anomeric oxygen linking sugar residues D and E whereas Asn 199 serves to hydrogen bond with the C2' N-acetyl group of sugar D, thus preventing the formation of an oxazoline ion intermediate. In addition, Glu 89 is part of a flexible loop region allowing a conformational change to occur within the active site to bring the oxocarbenium ion intermediate and Glu 89 closer by 4-5 A. A hydrolysis product with inversion of the anomeric configuration occurs because of nucleophilic attack by a water molecule that is coordinated by Glu 89 and Ser 120. Issues important for the design of inhibitors specific to family 19 chitinases over family 18 chitinases also are discussed.

title: Chitinolytic enzymes: catalysis, substrate binding, and their application.
abstract: After the epoch-making report on X-ray crystal structure of a lysozyme-N-acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 +) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate-assisted mechanism. Hen egg white lysozyme has the binding cleft represented by (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)(-1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application.

title: Purification and Characterization of an Antifungal Chitinase from Arabidopsis thaliana.
abstract: Plants exhibit an altered pattern of protein synthesis in response to pathogen invasion and abiotic stress. One of these ;pathogenesis-related' proteins has been identified as chitinase, which is capable of inhibiting fungal growth in vitro. This observation has led to the suggestion that the in vivo role of chitinases is to protect plants against fungal invasion. Here, we report the purification and characterization of a basic chitinase from Arabidopsis thaliana (L.) Heynh. Columbia wild type. The purified enzyme has a molecular mass of approximately 32 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and an apparent pl of approximately 8.7 as determined by isoelectric focusing. The purified protein is an effective inhibitor of the growth of Trichoderma reesei in vitro but does not affect the growth of several other fungi. Amino acid composition analysis of the intact protein as well as amino acid composition analysis and automatic Edman degradation of isolated tryptic fragments of the enzyme indicate that it may be identical to the product of a chitinase gene isolated from an Arabidopsis genomic library (Samac DA, Hironaka CM, Yallaly PE, Shah DM [1990] Plant Physiol 93: 907-914).

title: Identification of an essential tyrosine residue in the catalytic site of a chitinase isolated from Zea mays that is selectively modified during inactivation with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.
abstract: Chitinase isolated from Zea mays seeds is inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the absence of exogenous nucleophiles. Oligomers of N-acetylglucosamine,N,N',N",N"'-tetra-N-acetylchitotetraose (GlcNAc4), and to a lesser extent, N,N',N"-tri-N-acetylchitotriose (GlcNAc3) and N,N'-di-N-acetylchitobiose (GlcNAc2) provide partial protection against inactivation by the reagent. An examination of the concentration dependence of the protection afforded by GlcNAc4 revealed direct competition between the substrate analog and the reagent for the same binding sites on the enzyme. Isolation and Edman degradation of a "new" tryptic fragment, observed after inactivation of chitinase with EDC, revealed the sequence G-P-L-Q-I-S-W-N-*-N-Y-G-P-A-G-R, where the asterisk represents a cycle in which no amino acid was detected, presumably as a consequence of derivatization with EDC. In basic chitinases from dicotyledonous plants such as Arabidopsis thaliana, Phaseolis vulgaris (bean), Nicotiana tabacum (tobacco), and Solanum tuberosum (potato), as well as in the chitinase isolated from the monocotyledonous plant Hordeum vulgare (barley), this position is invariably occupied by a tyrosine. However, in the Oryza sativa (rice) basic chitinase, this position is occupied by a phenylalanine. The following additional evidence supports identification of this residue as tyrosine in Z. mays chitinase. (a) Inactivation of chitinase with EDC is reversible by treatment with hydroxylamine. (b) Liquid secondary ion mass spectrometric analysis of the isolated derivatized peptide revealed the presence of a molecular ion with a mass to charge ratio consistent with the peptide containing a derivatized tyrosine residue. These results provide evidence for an essential tyrosine residue at or near the catalytic site of chitinase that is selectively modified during inactivation with EDC.

title: Examination of the role of tyrosine-174 in the catalytic mechanism of the Arabidopsis thaliana chitinase: comparison of variant chitinases generated by site-directed mutagenesis and expressed in insect cells using baculovirus vectors.
abstract: Using the catalytic mechanism of lysozyme as a paradigm for the mechanism of other enzymes that catalyze the hydrolysis of beta-1,4-glycosidic linkages, including chitinase, we have examined the effect of chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) on the reaction catalyzed by Zea mays chitinase. Inactivation with EDC did not result in derivatization of essential carboxylic acid residues, but resulted in the selective modification of a single essential tyrosine residue (Verburg, J. G., Smith, C. E., Lisek, C. A., and Huynh, Q. K., 1991, ). Here, we examine the role of the homologous tyrosine residue in the catalytic mechanism of the Arabidopsis thaliana chitinase. Tyrosine-174 of the Arabidopsis chitinase was replaced, with phenylalanine, alanine, histidine, and methionine by site-directed mutagenesis, and the variant chitinases were expressed in insect cells using baculovirus transfer vectors. A comparison of the reaction catalyzed by each of the variant enzymes indicates that substitution of another amino acid for Tyr-174 alters, but does not eliminate, enzymatic activity. Estimates of the specific activities of the variant chitinases reveal that substitution of His for Tyr-174 has a minimal effect on catalysis, the specific activities of the Phe and Met variants are approximately equivalent to each other, but are 60% the specific activity of wild-type Arabidopsis chitinase, and the specific activity of the Ala variant is only 40% that of wild-type. The observation that the Arabidopsis chitinase is tolerant to mutagenesis at this position suggests that Tyr-174 does not participate directly in catalysis.

title: Populus chitinase genes: structure, organization, and similarity of translated sequences to herbaceous plant chitinases
abstract: Poplar trees have at least two different chitinase genes, win6 and win8, which are systemically wound-inducible and belong to multigene families [Proc Natl Acad Sci USA 86: 7895–7899]. On one genomic clone that we have partially sequenced, there are three win6 genes which are transcriptionally oriented in the same direction. Between two of the win6 genes is a gene that we have designated chitinase X (chiX), which appears to be a pseudogene belonging to a multigene family distinct from win6 and win8. The win6 and chiX genes we have sequenced contain two AT-rich introns that correspond in location to those in a basic chitinase gene from tobacco. The predicted Win6 proteins have a putative signal peptide, a cysteine-rich ‘hevein’ domain, a hinge region, and a catalytic domain as described in Shinshi et al.

title: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana.
abstract: Chitinases (EC3.2.1.14), found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed using basic local alignment search tool (BLAST) and simple modular architecture research tool (SMART), respectively. On the basis of the annotations of rice (Oryza sativa L.) and Arabidopsis genomic sequences and using the bio-software SignalP3.0, TMHMM2.0, TargetP1.1, and big-Pi Predictor, 25 out of 37 and 16 out of 24 open reading frames (ORFs) with chitinase activity from rice and Arabidopsis, respectively, were predicted to have signal peptides (SPs), which have an average of 24.8 amino acids at the N-terminal region. Some of the chitinases were secreted extracellularly, whereas some were located in the vacuole. The phylogenic relationship was analyzed with 61 ORFs and 25 known chitinases and they were classified into 6 clusters using Clustal X and MEGA3.1. This classification is not completely consistent when compared with the traditional system that classifies the chitinases into 7 classes. The frequency of distribution of amino acid residues was distinct in different clusters. The contents of alanine, glycine, serine, and leucine were very high in each cluster, whereas the contents of methionine, histidine, tryptophan, and cysteine were lower than 20%. Each cluster had distinct amino acid characteristics. Alanine, valine, leucine, cysteine, serine, and lysine were rich in Clusters I to VI, respectively.

title: Genome-wide identification, expression and chromosomal location of the genes encoding chitinolytic enzymes in Zea mays
abstract: Chitinolytic enzymes are important pathogenesis and stress related proteins. We identified 27 putative genes encoding endochitinases in the maize genome via in silico techniques and four exochitinases. Only seven of the endochitinases and segments of the exochitinases were heretofore known. The endochitinases included members of family 19 chitinases (classes I–IV of PR3, II of PR4) and members of family 18 chitinases (class III of PR8). Some similar enzymes were detected on adjacent regions of the same chromosome, and seem to result from duplication events. Most of the genes expressed were identified from EST libraries from plants exposed to biotic or abiotic stresses but also from libraries from tissues not exposed to stresses. We isolated proteins from seedlings of maize in the presence or absence of the symbiotic root colonizing fungus Trichoderma harzianum strain T22, and analyzed the activity of chitinolytic enzymes using an in-gel activity assay. The activity bands were identified by LC/MS/MS using the database from our in silico study. The identities of the enzymes changed depending on whether or not T22 was present. One activity band of about 95 kDa appeared to be a heterodimer between an exochitinase and any of several different endochitinases. The identity of the endochitinase component appeared to be dependent upon treatment.

title: Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases
abstract: The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.

title: Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes
abstract: Carnivory in plants is an adaptation strategy to nutrient-poor environments and soils. Carnivorous plants obtain some additional mineral nutrients by trapping and digesting prey; the genus Nepenthes is helped by its specialized pitcher traps. To make the nutrients available, the caught prey needs to be digested, a process that requires the concerted activity of several hydrolytic enzymes. To identify and investigate the various enzymes involved in this process, fluid from Nepenthes traps has been analysed in detail. In this study, a novel type of Nepenthes endochitinase was identified in the digestion fluid of closed pitchers. The encoding endochitinase genes have been cloned from eight different Nepenthes species. Among these, the deduced amino acid sequence similarity was at least 94.9%. The corresponding cDNA from N. rafflesiana was heterologously expressed, and the purified protein, NrChit1, was biochemically characterized. The enzyme, classified as a class III acid endochitinase belonging to family 18 of the glycoside hydrolases, is secreted into the pitcher fluid very probably due to the presence of an N-terminal signal peptide. Transcriptome analyses using real-time PCR indicated that the presence of prey in the pitcher up-regulates the endochitinase gene not only in the glands, which are responsible for enzyme secretion, but at an even higher level, in the glands’ surrounding tissue. These results suggest that in the pitchers’ tissues, the endochitinase as well as other proteins from the pitcher fluid might fulfil a different, primary function as pathogenesis-related proteins.

title: Preservation of duplicate genes by complementary, degenerative mutations.
abstract: The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. 1997 (p. 362)

title: Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.)
abstract: The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated.

title: Purifying selection and birth-and-death evolution in the ubiquitin gene family.
abstract: Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (p(S)) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown that p(S) is generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level of p(S) values, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-and-death evolution at the DNA level.

title: Phylogenetic Comparison of F-Box (FBX) Gene Superfamily within the Plant Kingdom Reveals Divergent Evolutionary Histories Indicative of Genomic Drift
abstract: The emergence of multigene families has been hypothesized as a major contributor to the evolution of complex traits and speciation. To help understand how such multigene families arose and diverged during plant evolution, we examined the phylogenetic relationships of F-Box (FBX) genes, one of the largest and most polymorphic superfamilies known in the plant kingdom. FBX proteins comprise the target recognition subunit of SCF-type ubiquitin-protein ligases, where they individually recruit specific substrates for ubiquitylation. Through the extensive analysis of 10,811 FBX loci from 18 plant species, ranging from the alga Chlamydomonas reinhardtii to numerous monocots and eudicots, we discovered strikingly diverse evolutionary histories. The number of FBX loci varies widely and appears independent of the growth habit and life cycle of land plants, with a little as 198 predicted for Carica papaya to as many as 1350 predicted for Arabidopsis lyrata. This number differs substantially even among closely related species, with evidence for extensive gains/losses. Despite this extraordinary inter-species variation, one subset of FBX genes was conserved among most species examined. Together with evidence of strong purifying selection and expression, the ligases synthesized from these conserved loci likely direct essential ubiquitylation events. Another subset was much more lineage specific, showed more relaxed purifying selection, and was enriched in loci with little or no evidence of expression, suggesting that they either control more limited, species-specific processes or arose from genomic drift and thus may provide reservoirs for evolutionary innovation. Numerous FBX loci were also predicted to be pseudogenes with their numbers tightly correlated with the total number of FBX genes in each species. Taken together, it appears that the FBX superfamily has independently undergone substantial birth/death in many plant lineages, with its size and rapid evolution potentially reflecting a central role for ubiquitylation in driving plant fitness.

title: Eukaryotic transposable elements and genome evolution.
abstract: The changes in DNA sequence that have taken place during the evolution of eukaryotic genomes cannot be accounted for simply by base substitutions; some more complex mutations must have taken place as well. Transposable elements can affect gene structure and expression in several ways that suggest that they may have contributed to these evolutionary events.

title: Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.).
abstract: To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0. 25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.

title: Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1
abstract: We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar Pieris rapae feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using α-mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by α-mannosidase. BjCHI1’s ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0 of wild-type specific activity. H211N and R361A resulted in considerable (>91) activity loss, implying these charged residues are also important in catalysis. E234A showed 36 retention of activity and substitution Y269D, 50. The least affected mutants were E349A and D360A, with 73 and 68 retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.

title: Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide Nod signals
abstract: The ability of 16 chitinases from seven different plant species to hydrolyze a collection of several structurally related lipochitooligosaccharides (LCOs) ofRhizobiumwas analyzed. It was found that the enzymes differed to a large extent in their activity on different LCOs. Differences were attributed to (i) the relative activity on different LCOs as substrate (e.g. sulfated versus non-sulfated LCOs); (ii) the relative cleavage site preference on a given LCO molecule (hydrolysis of either the second, third or fourth glycosidic bond from the non-reducing end of the molecule); and (iii) the stereochemistry of the reaction (retention or inversion of the anomeric configuration). A graphic representation of the different substrate specificities resulted in a ‘fingerprint’ that is characteristic for a given enzyme or a family of related enzymes. By comparing the LCO-fingerprint of unknown enzymes with those obtained for already characterized proteins, it is possible to identify new glycosyl hydrolases. The high diversity of substrate specificity found among plant chitinases may reflect variations in the natural substrates of the enzymes, such as substitutions on the chitin moiety of fungal cell walls or, in plants, the presence of putative endogenous substrates related to LCOs.

title: Mutation-selection models of codon substitution and their use to estimate selective strengths on codon usage.
abstract: Current models of codon substitution are formulated at the levels of nucleotide substitution and do not explicitly consider the separate effects of mutation and selection. They are thus incapable of inferring whether mutation or selection is responsible for evolution at silent sites. Here we implement a few population genetics models of codon substitution that explicitly consider mutation bias and natural selection at the DNA level. Selection on codon usage is modeled by introducing codon-fitness parameters, which together with mutation-bias parameters, predict optimal codon frequencies for the gene. The selective pressure may be for translational efficiency and accuracy or for fine-tuning translational kinetics to produce correct protein folding. We apply the models to compare mitochondrial and nuclear genes from several mammalian species. Model assumptions concerning codon usage are found to affect the estimation of sequence distances (such as the synonymous rate d(S), the nonsynonymous rate d(N), and the rate at the 4-fold degenerate sites d(4)), as found in previous studies, but the new models produced very similar estimates to some old ones. We also develop a likelihood ratio test to examine the null hypothesis that codon usage is due to mutation bias alone, not influenced by natural selection. Application of the test to the mammalian data led to rejection of the null hypothesis in most genes, suggesting that natural selection may be a driving force in the evolution of synonymous codon usage in mammals. Estimates of selection coefficients nevertheless suggest that selection on codon usage is weak and most mutations are nearly neutral. The sensitivity of the analysis on the assumed mutation model is discussed.