Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants

Main conclusion

PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.     

Phthalic Acid Esters in Soils from Vegetable Greenhouses in Shandong Peninsula,East China

Soils at depths of 0 cm to 10 cm, 10 cm to 20 cm, and 20 cm to 40 cm from 37 vegetable greenhouses in Shandong Peninsula, East China, were collected, and 16 phthalic acid esters (PAEs) were detected using gas chromatography-mass spectrometry (GC-MS). All 16 PAEs could be detected in soils from vegetable greenhouses. The total of 16 PAEs (Σ₁₆PAEs) ranged from 1.939 mg/kg to 35.442 mg/kg, with an average of 6.748 mg/kg. Among four areas, including Qingdao, Weihai, Weifang, and Yantai, the average and maximum concentrations of Σ₁₆PAEs in soils at depths of 0 cm to 10 cm appeared in Weifang, which has a long history of vegetable production and is famous for extensive greenhouse cultivation. Despite the different concentrations of Σ₁₆PAEs, the PAE compositions were comparable. Among the 16 PAEs, di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), di-n-butyl phthalate (DnBP), and diisobutyl phthalate (DiBP) were the most abundant. Compared with the results on agricultural soils in China, soils that are being used or were used for vegetable greenhouses had higher PAE concentrations. Among PAEs, dimethyl phthalate (DMP), diethyl phthalate (DEP) and DnBP exceeded soil allowable concentrations (in US) in more than 90% of the samples, and DnOP in more than 20%. Shandong Peninsula has the highest PAE contents, which suggests that this area is severely contaminated by PAEs.     

Contamination of Phthalate Esters (PAEs) in Typical Wastewater-Irrigated Agricultural Soils in Hebei,North China

The Wangyang River (WYR) basin is a typical wastewater irrigation area in Hebei Province, North China. This study investigated the concentration and distribution of six priority phthalate esters (PAEs) in the agricultural soils in this area. Thirty-nine soil samples (0–20 cm) were collected along the WYR to assess the PAE residues in soils. Results showed that PAEs are ubiquitous environmental contaminants in the topsoil obtained from the irrigation area. The concentrations of Σ₆PAEs range from 0.191 μg g⁻¹ dw to 0.457 μg g⁻¹ dw with an average value of 0.294 μg g⁻¹ dw. Di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP) are the dominant PAE species in the agricultural soils. Among the DEHP concentrations, the highest DEHP concentration was found at the sites close to the villages; this result suggested that dense anthropogenic activities and random garbage disposal in the rural area are possible sources of PAEs. The PAE concentrations were weakly and positively correlated with soil organic carbon and soil enzyme activities; thus, these factors can affect the distribution of PAEs. This study further showed that only dimethyl phthalate (DMP) concentrations exceeded the recommended allowable concentrations; no remediation measures are necessary to control the PAEs in the WYR area. However, the PAEs in the topsoil may pose a potential risk to the ecosystem and human health in this area. Therefore, the exacerbating PAE pollution should be addressed.     

Analysis of phosphate acquisition efficiency in different Arabidopsis accessions

The morphological and physiological characteristics of Arabidopsis accessions differing in their phosphate acquisition efficiencies (PAEs) when grown on a sparingly soluble phosphate source (hydroxylapatite) were analyzed. A set of 36 accessions was subjected to an initial PAE evaluation following cultivation on synthetic, agarose-solidified media containing potassium phosphate (soluble) or hydroxylapatite (sparingly soluble). From the five most divergent accessions identified in this way, C24, Co, and Cal exhibited high PAEs, whereas Col-0 and Te exhibited low PAEs. These five accessions were analyzed in detail. Significant differences were found in root morphology, phosphate uptake kinetics, organic acid release, rhizosphere acidification, and the ability of roots to penetrate substrates. Long root hairs at high densities, high uptake per unit root length, and high substrate penetration ability in the efficient accessions C24 and Co mediate their high PAEs. The third accession with high PAE, Cal, exhibits a high shoot-to-root ratio, long roots with long root hairs, and rhizosphere acidification. These results are consistent with previous observations and highlight the suitability of using Arabidopsis accessions to identify and isolate genes determining the PAE in plants.     

Postantibiotic effects of rifampin, amikacin, clarithromycin and ethambutol used alone or in various two-, three- and four-drug combinations against Mycobacterium avium

The postantibiotic effects (PAEs) of rifampin, amikacin, clarithromycin, and ethambutol were determined radiometrically against five AIDS-associated isolates of Mycobacterium avium. and were found to be 20.8+/-3.4. 18.4+/-2.5, 11.8+/-1.7. and 2.4+/-0.9 h, respectively. Various two-, three- or four-drug combinations were also screened: the PAEs for a two-drug combination were generally longer than individual drugs (mean PAE of 13.8+/-1.5 to 29.2+/-7.4 h instead of 2.4+/-0.9 to 18.4+/-2.5 h for single drugs). The addition of a third drug further increased the mean PAE to a range of 21.0+/-2.6 to 32.4+/-6.1 h. Both rifampin+clarithromycin and rifampin+amikacin were the most potent two-drug combinations resulting in longer PAEs than individual drugs, whereas rifampin+amikacin+clarithromycin was the most potent three-drug combination. Parallel viable count determinations showed a good correlation between the PAE results obtained by the radiometric method or by bacterial viability assessment. These results are useful in planning future clinical investigations to clarify the possible implication of PAE in drug schedule and dosage, a line of information that is urgently needed to guide the drug administration in M. avium-infected AIDS patients, who are presently over-burdened with the administration of too many drugs for HIV-treatment and opportunistic infections.     

Comparative genomics of grasses tolerant to aluminum

The family Poaceae includes over 10,000 species, among which are the most economically important cereals: maize, sorghum, rice, wheat, rye, barley, and oat. These cereals are very important components of human and animal food. Although divergence of the members of this family occurred about 40 million years ago, comparative genome analyses demonstrated that gene orders among species of this family remain largely conserved, which can be very useful for understanding their roles and evolution. Even with an intricate evolutionary history in which chromosome fragments, losses and duplications have to be considered at the ploidy level, grasses present a genetic model system for comparative genomics. The availability of mapped molecular markers, rice genome sequences and BAC and EST libraries from several grass species, such as rice, wheat, sorghum, and maize, facilitates biology and phylogeny studies of this group. The value of using information from different species in modern plant genetics is unquestionable, especially in the study of traits such as tolerance to aluminum in soils, which affects plant growth and development. Comparative genomic approaches to aluminum tolerance can identify genomic regions and genes responsible for aluminum tolerance in grasses.     

Modeling the mechanism of postantibiotic effect and determining implications for dosing regimens

A stochastic model is proposed to explain one possible underlying mechanism of the postantibiotic effect (PAE). This phenomenon, of continued inhibition of bacterial growth after removal of the antibiotic drug, is of high relevance in the context of optimizing dosing regimens. One clinical implication of long PAE lies in the possibility of increasing intervals between drug administrations. The model describes the dynamics of synthesis, saturation and removal of penicillin binding proteins (PBPs). High fractions of saturated PBPs are in the model associated with a lower growth capacity of bacteria. An analytical solution for the bivariate probability of saturated and unsaturated PBPs is used as a basis to explore optimal antibiotic dosing regimens. Our finding that longer PAEs do not necessarily promote for increased intervals between doses, might help for our understanding of data provided from earlier PAE studies and for the determination of the clinical relevance of PAE in future studies.      

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties ofSalmonella strains

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied forSalmonella typhimurium andS. enteritidis strains. PAE was induced by 2× and 4×MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenicS. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed withS. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4×MIC of amikacin withS. enteritidis (6.9 h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASME's were different while concentrations of 2×MIC+0.2×MIC and 0.3×MIC, and of 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin did not allow any regrowth ofS. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by bothSalmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenicS. typhimurium strain. We noted the influence of Congo red binding after applying 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin forS. typhinurium and 2×MIC+0.1×MIC forS. enteritidis.     

Removal of phthalate esters by alpha-cyclodextrin-linked chitosan bead

Removal of phthalate esters (PAEs) by alpha-cyclodextrin (CD)-linked chitosan bead in aqueous solution was studied. Results of kinetic experiments indicated that diheptyl phthalate (DHpP) was adsorbed most efficiently (3.21 mg/g) among the six PAEs. DHpP recovery was 94.6% from alpha-CD-linked chitosan bead by shaking both with a mixture of methanol and water (v/v = 8/2). The recovered alpha-CD chitosan bead was reusable as an adsorbent 20 times in the batch tests. The adsorbed PAE by alpha-CD-linked chitosan bead decreased as temperature increased. However, coexisting pH, NaCl, and Ca2+ did not affect adsorption efficiency. It was concluded that the application of low cost alpha-CD-linked chitosan bead could have the potential to effectively remove PAEs from different aquatic environments.     

A multi-type branching model with varying environment for bacterial dynamics with postantibiotic effect

A multi-type branching process with varying environment was used to construct a pharmacokinetic/pharmacodynamic (PK/PD) model that captures the postantibiotic effect (PAE) seen in bacterial populations after exposure of antibiotics. This phenomenon of continued inhibition of bacterial growth even after removal of the antibiotic from the growth medium is of high relevance in the context of optimizing dosing regimens. The clinical implication of long PAEs lies in the interesting possibility of increasing the intervals between drug administrations.The model structure is generalizable to most types of antibiotics and is useful both as a theoretical framework for understanding the time properties of PAE and to explore optimal antibiotic dosing regimens. Data from an in vitro study with Escherichia coli exposed to different dosing regimens of cefotaxime were used to evaluate the model.     

Polycyclic aromatic hydrocarbons and phthalic acid esters in the soil-radish (Raphanus sativus) system with sewage sludge and compost application

We studied the accumulation of polycyclic aromatic hydrocarbons (PAHs) and phthalic acid esters (PAEs) in a latosolic red soil and radish (Raphanus sativus) with application of sewage sludge at rates of 10, 20 and 40 g kg(-1) soil or compost at rate of 10 g kg(-1) soil. In radish the concentrations of individual PAHs and PAEs varied from non-detectable to 803 microg kg(-1) dry weight (d.w.) and from non-detectable to 2048 microg kg(-1) d.w., respectively. Compared to the control, higher application rates of sewage sludge resulted in pronounced increases in shoot, root and soil concentrations of PAHs and PAEs. PAE concentrations in radish grown in soil spiked with sludge compost were higher while the PAH concentrations were comparable to those receiving 10 g kg(-1) of sewage sludge. However, the root biomass of radish in soil amended with compost was significantly higher and the shoot-to-root ratio was significantly lower than in the other treatments. The bioconcentration factors (BCFs, the ratio of contaminant concentration in plant tissue to the soil concentration) of di-n-butyl phthalate and di(2-ethylhexyl) phthalate in both shoots and roots and of total PAH concentrations in roots were less than 1.0, but some BCFs for individual PAHs were high with a maximum value of 80.     

农业土壤中邻苯二甲酸酯污染研究进展

由邻苯二甲酸酯(PAEs)引起的环境污染和食品安全问题已经引起全球性关注.我国既是邻苯二甲酸酯生产大国,又是消费大国,其环境污染问题不容忽视.本文综述了美国国家环保署所列出的6种优先控制PAEs污染物在我国农田土壤中的污染现状,分析了其来源,重点阐述了不同类型农作物对PAEs化合物的吸收累积特征及PAEs类污染物的生物毒害效应.我国多数地区农业土壤中PAEs的含量显著高于美国和欧洲等国家.大气沉降、农用薄膜、施用污泥和污水灌溉是我国农业土壤中PAEs的主要来源.不同作物对PAEs的吸收、累积和分配特征具有显著的差异性.PAEs不但影响土壤质量、作物生长和生理生化性质,而且具有生物累积效应.最后指出了当前研究中的不足及对今后研究的展望,建议扩大PAEs污染调查范围,深入揭示PAEs对农作物的毒害机理,重点研发PAEs污染土壤的原位修复技术.     

Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction

Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.     

Differential herbivory on C3 versus C4 grasses by the grasshopper Ageneotettix deorum (Orthoptera: acrididae)

Summary The hypothesis that graminivorous grasshoppers select C₃ grasses over C₄ grasses was tested with experiments in the field. It was found that the generalist, graminivorous grasshopper Ageneotettix deorum typically chooses C₃ as opposed to C₄ grasses when both types are equally available. This preference is attributed to the differences in leaf anatomies of C₃ and C₄ plants since crude protein, water, lignin, fiber, and silicon content or the size of the individual leaves tested do not explain the observed feeding pattern. However, examination of the actual food plant selection of actual field inhabiting grasshoppers indicates that food plant prefernce may only be a minor component of food selection in natural settings.     

Food of three upland game birds in Selengei Area, Kajiado District, Kenya

Foods taken by yellow-necked spurfowl, crested francolin and helmeted guinea-fowl were investigated over a 22-month period. Bulblets of sedge (Cyperus sp.) were the major plant item in the diet of all three species. Other foods were seeds and fruits of forbs, florets and fruits of grasses, leaves of forbs and grasses and insects. Insect use increased following the rains, as did the use of florets of the grasses. Except for Cyperus sp. there was little overlap of food items taken by the crested francolin and the guinea-fowl, but both were in competition with the yellow-necked spurfowl for some species of plants. This was probably due to size of food items, that is, there was a direct relationship between size of bird and size of foods taken. Forag ing habits also influence food intake.     

Postantibiotic effects of gentamicin and netilmicin onSerratia marcescens: Effects on hydrophobicity and motility

The impact of postantibiotic effect (PAE) of aminoglycosides (gentamicin, netilmicin) on cell-surface hydrophobicity and motility of a clinical isolateSerratia marcescens was evaluated. For the induction of PAE 2× and 4×MIC concentrations of both antibiotics were used. Gentamicin and netilmicin induced a PAE of similar duration after 2×MIC concentration (2.7 and 2.8 h, respectively). Both aminoglycosides demonstrated concentration-dependent PAE. At a concentration of 4×MIC they produced PAEs of 5.9 and 8.2h, respectively. The evaluation of hydrophobic properties ofS. marcescens after affecting PAE showed that both aminoglycosides inhibited adherence to xylene. This inhibition was also concentration-dependent. More expressive, was netilmicin which inhibited the adhesion by 70.5% at 2×MIC and by 85.2% at 4×MIC. Netilmicin inhibited also the adhesion to nitrocellulose filter by 34.7% at 4×MIC. Exposure of the bacterial cells to suprainhibitory concentrations of both aminoglycosides resulted only in moderate inhibition of motility of strain tested compared to the unexposed cells.     

Use of beta-glucuronidase reporter gene for gene expression analysis in turfgrasses

The beta-glucuronidase (GUS) gene has been successfully used as a reporter gene in innumerable number of plant species. The functional GUS gene produces blue coloration in plants upon integration into the plant genome. Because of the ease it provides to analyze the gene expression (as no expensive equipment is needed), GUS gene is surely plant biotechnologist's first choice as a reporter gene. The turfgrass family contains the world's most economically important horticultural crops. There is a world-wide drive for genetic modification of grasses due to its huge economic importance. GUS gene can be transiently or stably expressed in grasses for the purpose of promoter analysis and to study tissue-specific and developmental gene expression. This paper summarizes the use of GUS gene for transient and stable expression studies in various turfgrass species.     

Comparison of the post-antibiotic effect (PAE) induced by ceftizoxime, ceftriaxone, cefoxitin, ampicillin-sulbactam, and ticarcillin-clavulanate against selected isolates of Bacteroides fragilis and B. thetaiotaomicron

The exposure of bacteria to various groups of antimicrobials at different concentrations can produce damage to the bacteria that persists even after removal of the antimicrobial agent. The post antibiotic effect (PAE) of beta-lactams on aerobic gram-negative bacilli is relatively short (<1 h), however, little information is available regarding anaerobic gram-negative bacilli. We studied the PAE of ceftizoxime, ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, and ceftriaxone against strains of Bacteroides fragilis and B. thetaiotaomicron at antimicrobial concentrations 4x, 8x, and 16x the MIC values using colony count determinations of treated and untreated cultures. Against B. fragilis H931, ceftizoxime-induced PAE values were 2 h, 3 h 24 min, and 11 h 36 min at 4xMIC, 8xMIC, and 16xMIC while for the B. thetaiotaomicron isolates PAEs ranged from 2 h 27 min to 6 h 12 min at the same concentrations. Cefoxitin PAE values were 3 h 6 min and 2 h 18 min for the clinical isolates at 16xMIC and 3 h 24 min and 1 h 12 min against the laboratory strains at 16xMIC respectively, and for ceftriaxone 1 h 12 min and 5 h 12 min, respectively, for the B. thetaiotaomicron D933 and B. fragilis H931 strains at 16xMIC. With ampicillin-sulbactam, the longest PAE values were observed at 16xMIC with all the test isolates of B. fragilis and B. thetaiotaomicron. PAE values induced by ticarcillin-clavulanate overall were the shortest for the two clinical isolates. These studies indicate substantial PAE values for beta-lactams against selected anaerobes which may be an important factor in the dosing regimen of these test agents.     

城市污泥与稻草堆肥中邻苯二甲酸酯（PAEs）的研究

将广州城市污泥与稻草进行翻堆、接菌-翻堆、连续通气和间歇通气4种方式的堆肥,应用GC／MS技术对堆肥中6种属于USEPA优控污染物的邻苯二甲酸醇化合物(PAEs)进行分析,探讨堆肥产物中PAEs的含量分布以及不同方式堆肥对PAEs的降解效果,结果表明,4种方式堆肥中PAEs总含量(∑PAEs)在9．815～17．832mg·kg^-1之间,依次为翻堆(17．832mg·kg^-1)>接菌-翻堆(13．927mg·kg^-1)>间隙通气(10．765mg·kg^-1)>连续通气(9．815mg·kg^-1),堆肥中PAEs以邻苯二甲酸正二辛酯(DhOP)为主,占∑PAEs的82．2％～89．696,不同方式堆肥中∑PAEs的降解率为连续通气(45．71％)>间隙通气(40．4696)>接菌-翻堆(22．97％)>翻堆(1．3796)(平均降解率为27．63％),其中邻苯二甲酸二乙醇(DEP)、邻苯二甲酸正二丁酯(DnBP)和邻苯二甲酸丁基苄基酯(BBP)的降解率分别为95．7696～98．6896、79．5696～99．46％和87．42％～98．42％;但邻苯二甲酸二甲酯(DMP)和邻苯二甲酸正二辛酯的含量反而增加,邻苯二甲酸(2-乙基己基)酯(DEHP)在所有堆肥中均未检出。