PECTATE LYASE LIKE12 patterns the guard cell wall to coordinate turgor pressure and wall mechanics for proper stomatal function in Arabidopsis.

Plant cell deformations are driven by cell pressurization and mechanical constraints imposed by the nanoscale architecture of the cell wall, but how these factors are controlled at the genetic and molecular levels to achieve different types of cell deformation is unclear. Here, we used stomatal guard cells to investigate the influences of wall mechanics and turgor pressure on cell deformation and demonstrate that the expression of the pectin-modifying gene PECTATE LYASE LIKE12 (PLL12) is required for normal stomatal dynamics in Arabidopsis thaliana. Using nanoindentation and finite element modeling to simultaneously measure wall modulus and turgor pressure, we found that both values undergo dynamic changes during induced stomatal opening and closure. PLL12 is required for guard cells to maintain normal wall modulus and turgor pressure during stomatal responses to light and to tune the levels of calcium crosslinked pectin in guard cell walls. Guard cell-specific knockdown of PLL12 caused defects in stomatal responses and reduced leaf growth, which were associated with lower cell proliferation but normal cell expansion. Together, these results force us to revise our view of how wall-modifying genes modulate wall mechanics and cell pressurization to accomplish the dynamic cellular deformations that underlie stomatal function and tissue growth in plants.

Genome-wide identification of the pectate lyase-like (PLL) gene family and functional analysis of two PLL genes in rice.

Pectate lyase catalyses the eliminative cleavage of de-esterified pectin, which is a major component of primary cell walls in many higher plants. Pectate lyase-like (PLL) genes have been identified in various plant species and are involved in a broad range of physiological processes associated with pectin degradation. Previous studies have functionally identified two PLL genes in rice (Oryza sativa. L). However, the knowledge concerning genome-wide analysis of this family remains limited, and functions of the other PLL genes have not been thoroughly elucidated to date. In this study, we identified 12 PLL genes based on a genome-wide investigation in rice. A complete overview of this gene family is presented, including chromosomal locations, exon-intron structure, cis-acting elements and conserved motifs. PLL protein sequences from multiple plant species were compared and divided into five groups based on phylogenetic analysis. Quantitative RT-PCR analysis revealed that only a portion of OsPLL genes (4 of 12) exhibits detectable expression levels. Notably, OsPLL1, OsPLL3, OsPLL4 and OsPLL12 exhibit strong and preferential expression in panicles suggesting that the potential roles of these genes are crucial during rice panicle development. Moreover, knockdown of OsPLL3 and OsPLL4 by artificial microRNA (amiRNA) disrupted normal pollen development and resulted in partial male sterility. These results could provide valuable information for characterising the functions and dissecting the molecular mechanisms of the OsPLL genes.

Characterization and fine mapping of the rice premature senescence mutant ospse1.

Premature senescence can limit crop productivity by limiting the growth phase. In the present study, a spontaneous premature senescence mutant was identified in rice (Oryza sativa L.). Genetic analysis revealed that the premature senescence phenotype was controlled by a recessive mutation, which we named Oryza sativa premature senescence1 (ospse1). The ospse1 mutants showed premature leaf senescence from the booting stage and exhibited more severe symptoms during reproductive and ripening stages. Key yield-related agronomic traits such as 1,000-grain weight and seed-setting rate, but not panicle grain number, were significantly reduced in ospse1 plants. Chlorophyll content, net photosynthetic rate, and transpiration rate of ospse1 flag leaves were similar to the wild-type plants in vegetative stages, but these parameters decreased steeply in the mutant after the heading stage. Consistent with this, the senescence-associated genes OsNYC1 and OsSgr were up-regulated in ospse1 mutant during premature leaf senescence. The ospse1 locus was mapped to a 38-kb region on chromosome 1 and sequence analysis of this region identified a single-nucleotide deletion in the 3' region of an open reading frame (ORF) encoding a putative pectate lyase, leading to a frame shift and a longer ORF. Our results suggested that the premature senescence of the ospse1 may be regulated by a novel mechanism mediated by pectate lyase.

Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination.

Alginates are polysaccharides composed of 1-4-linked beta-D-mannuronic acid and alpha-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a beta-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca(2+). All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis epidermis induces cell death and suggests a critical role for phospholipase A2 in this process.

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific beta-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.

Enzymatic deconstruction of backbone structures of the ramified regions in pectins.

The pectic enzymes are a diverse group of enzymes that collectively degrade pectin, a mixture of highly heterogeneous and branched polysaccharides rich in D: -galacturonic acids forming a major component of the primary cell wall of plants. This review covers key enzymes that function to deconstruct the "ramified region" of pectin. The enzymes include glycoside hydrolases and polysaccharide lyases that degrade complex pectic domains consisting of rhamnogalacturonans, xylogalacturonans, and other heterogeneous polymers. The chemical nature of the pectic substrates for the enzymes is presented. The biochemical properties of the enzymes, the mechanisms of enzyme actions, and related structures and functions, are described. Applications of these enzymes in fruit juice processing and in the production of bioactive compounds, as well as their technological relevance to the deconstruction of cell wall structures for biomass conversion are discussed.

A family 2 pectate lyase displays a rare fold and transition metal-assisted beta-elimination.

The family 2 pectate lyase from Yersinia enterocolitica (YePL2A), solved to 1.5A, reveals it to be the first prokaryotic protein reported to display the rare (alpha/alpha)(7) barrel fold. In addition to its apo form, we have also determined the structure of a metal-bound form of YePL2A (to 2.0A) and a trigalacturonic acid-bound substrate complex (to 2.1A) Although its fold is rare, the catalytic center of YePL2A can be superimposed with structurally unrelated families, underlining the conserved catalytic amino acid architecture of the beta-elimination mechanism. In addition to its overall structure, YePL2A also has two other unique features: 1) it utilizes a metal atom other than calcium for catalysis, and 2) its Brønstead base is in an alternate conformation and directly interacts with the uronate group of the substrate.

The role of pectate lyase and the jasmonic acid defense response in Pseudomonas viridiflava virulence.

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coil-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coil-1 relative to Col-0, indicating that P. viridiflava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.

Expression profiling of auxin-treated Arabidopsis roots: toward a molecular analysis of lateral root emergence.

Treating Arabidopsis roots with exogenous auxin results in dramatic changes in cellular processes including de novo induction of lateral roots which later emerge through the overlying cells. Microarray experiments reveal approximately 80 genes that are substantially up-regulated in the root over the first 12 h following auxin treatment. We hypothesize that the observed increase in expression of pectate lyase family genes leads to degradation of the pectin-rich middle lamellae, allowing cells in the parent root to separate cleanly. Differences in the degree of pectin methylation in lateral and parent roots may explain why lateral roots are not degraded themselves.

Contribution of hemagglutinin/protease and motility to the pathogenesis of El Tor biotype cholera.

Vibrio cholerae is a highly motile organism that secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HapA). HapA has been shown to have mucinase activity and contribute to the reactogenicity of live vaccine candidates, but its role in cholera pathogenesis is not yet clear. The contribution of motility to pathogenesis is not fully understood, since conflicting results have been obtained with different strains, mutants, and animal models. The objective of this work was to determine the contribution of HapA and motility to the pathogenesis of El Tor biotype cholera. To this end we constructed isogenic motility (motY) and mucinase (hapA) single and double mutants of an El Tor biotype V. cholerae strain. Mutants were characterized for the expression of major virulence factors in vitro and in vivo. The motility mutant showed a remarkable increase in cholera toxin (CT), toxin coregulated pilus major subunit (TcpA), and HapA production in vitro. Increased TcpA and CT production could be explained by increased transcription of tcpA, ctxA, and toxT. No effect was detected on the transcription of hapA in the motility mutant. The sodium ionophore monensin diminished production of HapA in the parent but not in the motility mutant. Phenamil, a specific inhibitor of the flagellar motor, diminished CT production in the wild-type and motY strains. The hapA mutant showed increased binding to mucin. In contrast, the motY mutation diminished adherence to biotic and abiotic surfaces including mucin. Lack of HapA did not affect colonization in the suckling mouse model. The motility and mucinase defects did not prevent induction of ctxA and tcpA in the mouse intestine as measured by recombinase-based in vivo expression technology. Analysis of mutants in the rabbit ileal loop model showed that both V. cholerae motility and HapA were necessary for full expression of enterotoxicity.

Chemotaxis between Vibrio cholerae O1 and a blue-green alga, Anabaena sp.

The chemotactic response of Vibrio cholerae O1 towards the mucilaginous sheath of Anabaena sp. was investigated by capillary tube method using a virulent strain of V. cholerae O1, El Tor, Ogawa (3083-T) and its isogenic mutant (HAP-1-T) that lacks the hap gene, which codes for mucinase (HA/protease). Homogenates of Anabaena sp. and purified mucin were used in this study as chemoattractants. Results showed 5.7% bacterial accumulation of wild-type V. cholerae O1 towards 4% homogenates of Anabaena sp. whereas, its mutant (hap-) showed 2.9% accumulation after 90 min. The higher percentage of attraction of wild-type V. cholerae O1 than the mutant (hap-) towards mucin and the homogenates of Anabaena sp. might be due to the activity of mucinase. These results indicate the role of mucinase in the chemotactic motility of V. cholerae O1 towards Anabaena sp.

Role of the Pseudomonas fluorescens alginate lyase (AlgL) in clearing the periplasm of alginates not exported to the extracellular environment.

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa secretes a capsule-like polysaccharide called alginate that is important for evasion of host defenses, especially during chronic pulmonary disease of patients with cystic fibrosis (CF). Most proteins for alginate biosynthesis are encoded by the 12-gene algD operon. Interestingly, this operon also encodes AlgL, a lyase that degrades alginate. Mutants lacking AlgG, AlgK, or AlgX, also encoded by the operon, synthesize alginate polymers that are digested by the coregulated protein AlgL. We examined the phenotype of an DeltaalgL mutation in the highly mucoid CF isolate FRD1. Generating a true DeltaalgL mutant was possible only when the algD operon was under the control of a LacI(q)-repressed trc promoter. Upon induction of alginate production with isopropyl-beta-D-thiogalactopyranoside, the DeltaalgL mutant cells were lysed within a few hours. Electron micrographs of the DeltaalgL mutant showed that alginate polymers accumulated in the periplasm, which ultimately burst the bacterial cell wall. The requirement of AlgL in an alginate-overproducing strain led to a new model for alginate secretion in which a multiprotein secretion complex (or scaffold, that includes AlgG, AlgK, AlgX, and AlgL) guides new polymers through the periplasm for secretion across the outer membrane. In this model, AlgL is bifunctional with a structural role in the scaffold and a role in degrading free alginate polymers in the periplasm.

Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4.

Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves alpha-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acids in the backbone of rhamnogalacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RG-lyase from Aspergillus aculeatus has been determined to 1.5 A resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows structural homology to non-catalytic domains from other carbohydrate active enzymes.

Mucinases and sialidases: their role in the pathogenesis of sexually transmitted infections in the female genital tract.

BACKGROUND: Mucinases and sialidases contribute to the process of invasion and colonisation in many conditions and infections of the female reproductive tract by degrading the protective cervical mucus. The role of hydrolytic enzymes in the pathogenesis of sexually transmitted diseases and their effect on cervical mucus are discussed in this review. METHODS: Articles were searched for using the keywords "sialidase," "mucinase," "protease," and "sexually transmitted infections." As well as review and other articles held by our group, searches were conducted using PubMed, Grateful Med, and the University of Bath search engine, BIDS. RESULTS: Numerous publications were found describing the production of hydrolytic enzymes in sexually transmitted diseases. Because the number of publications exceeded the restrictions imposed on the size of the review, the authors selected and discussed those which they considered of the most relevance to sexually transmitted infections.

Structure and function of pectic enzymes: virulence factors of plant pathogens.

The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.

Characterization of SotA and SotB, two Erwinia chrysanthemi proteins which modify isopropyl-beta-D-thiogalactopyranoside and lactose induction of the Escherichia coli lac promoter.

The expression, in Escherichia coli, of variants of the Erwinia chrysanthemi secretion genes outB and outS under the Ptac promoter is toxic to the cells. During attempts to clone E. chrysanthemi genes able to suppress this toxicity, I identified two genes, sotA and sotB, whose products are able to reduce the isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the E. coli lac promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of sotA and sotB is not regulated by their substrates, but sotA is activated by the cyclic AMP receptor protein (CRP), while sotB is repressed by CRP. Lactose is weakly toxic for E. chrysanthemi. This toxicity is increased in a sotB mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.

Control of virulence gene expression by plant calcium in the phytopathogen Erwinia carotovora.

Plant calcium can modulate a particular plant-pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora, the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora, the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant-pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.

Heparan sulfate isomers in cerebral arteries of Japanese women with aging and with atherosclerosis--heparitinase and high-performance liquid chromatography determinations.

Composition of heparan sulfate (HS) isomers from unaffected and atherosclerotic cerebral arteries (isolated by autopsy) of Japanese women, of various ages, was studied. HS isomers were separated as disaccharide units by high-performance liquid chromatography after degeneration with HS and heparin lyases. Heparitinase facilitated differentiation of eight unsaturated disaccharides (deltaDi-S(HS)) of vascular HS isomers. The HS isomers in the cerebral arteries consisted of approximately half the total glycosaminoglycans (GAGs). Both HS (and GAGs) tended to increase with the processes of aging but decreased with the advancing development of atherosclerosis. The HS isomers consisted of a higher proportion of non-sulfated disaccharide, about 2/3 of the total HS, followed by mono-sulfated and bis-sulfated saccharides; in addition, heparin existed, albeit in minute amounts. The proportion of deltaDi-S(HS) rich in sulfate compared with HS isomers tended to increase with aging but most decreased during formation of the atheroma. Putative functions of HS isomers in cerebral arteries are discussed, based on the characteristic distribution of HS components.