APX Genes Research Articles

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress‐related genes

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas (Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15-17 days after inoculation) and (iii) when all plants had developed disease symptoms (20-22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier (i.e. GR and SOD) or to higher levels (i.e. DAO) in the incompatible interaction. Expression of apx, cat, sod, lipoxygenase (lox) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier (lox) or higher (actin) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.

SOD와 APX를 동시에 엽록체에 발현시킨 형질전환 감자 (cv. Superior)의 산화스트레스 내성 증가

산화스트레스 유도성 SWPA2 프로모터 조절하에 항산화 효소 SOD와 APX 유전자를 동시에 엽록체에 발현시킨 형질전환 감자 (품종 수미)를 대상으로 methyl viologen (MV) 처리에 의해 유도되는 산화스트레스 내성을 잎절편체, 소식물체 및 식물체 수준에서 조사하였다. 잎 절편에 <TEX>$3{\mu}M$</TEX> MV를 처리하였을 때 SSA 식물체의 잎절편체는 비형질전환 (NT) 식물체에 비해 40% 정도 상해를 적게 받았다. 소식물체 수준에서 MV에 의한 산화스트레스 내성을 조사하기 위하여 SSA감자 shoot을 <TEX>$0.3{\mu}M$</TEX> MV 첨가 배지에 배양하였을 때 뿌리의 생장에서 내성이 나타났다. 또한 온실에서 4주 생장한 식물체에 <TEX>$350{\mu}M$</TEX> MV를 처리하였을 경우에도 SSA 식물체는 NT 식물체에 비해 약 75% 손상을 적게 입은 것으로 나타내었다. 추후 SSA 식물체를 이용하여 건조, 고온 등의 복합재해에 내성을 분석이 진행되어야 할 것이며 그 결과 복합스트레스 내성 감자 품종 (수미)을 개발할 수 있을 것으로 기대한다. Oxidative stress is a major damaging factor for plants exposed to environmental stresses. Previously, we have generated transgenic potato (cv. Superior) plants expressing both CuZnSOD and APX genes in chloroplast under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants) and selected the transgenic potato plant lines with tolerance against methyl viologen (MV)-mediated oxidative stress. When leaf discs of SSA plants were subjected to <TEX>$3{\mu}M$</TEX> methyl viologen (MV), they showed approximately 40% less damage than non-transgenic (NT) plants. SSA plantlets were treated with <TEX>$0.3{\mu}M$</TEX> MV stress, SSA plants also exhibited reduced damage in root growth. When 350 MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage, which was approximately 75% less damage than leaves of NT plants. These plants will be used for further analysis of tolerance to environmental stresses, such as high temperature and salt stress. These results suggest that transgenic potato (cv. Superior) plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions.

Ascorbate peroxidase gene family in tomato: its identification and characterization

The antioxidative response, where ascorbate peroxidase (APX) is a key enzyme, is an integral part of the plant tolerance response to environmental stresses. As a first step towards the study of the physiological role and the regulation of the members of the Apx gene family, the orthologs of the stress-sensitive cultivated tomato Solanum lycopersicum cv. M82 (Slm) and of the wild salt-tolerant species S. pennellii acc. Atico (Spa) were identified by utilizing the tomato EST database, and characterized. A redundant list of 16 virtual Apx transcripts and four singleton ESTs was shown to correspond to seven genuine Apx genes. The complete tomato Apx gene family is comprised of genes encoding three cytosolic, two peroxisomal, and two chloroplastic APXs. These genes attained differential regulatory patterns in various Slm organs. More detailed study of Apx1 and Apx2 genes, that are the products of a recent gene duplication event, shows that they have already attained differential regulation within and between Slm and Spa under control and stress conditions. It is also suggested that due to lineage-specific gene duplication and lose events, intricate phylogenetic relationships exist among the members of the Apx gene families.

Manganese Deficiency in Chlamydomonas Results in Loss of Photosystem II and MnSOD Function, Sensitivity to Peroxides, and Secondary Phosphorus and Iron Deficiency

For photoheterotrophic growth, a Chlamydomonas reinhardtii cell requires at least 1.7 x 10(7) manganese ions in the medium. At lower manganese ion concentrations (typically <0.5 microm), cells divide more slowly, accumulate less chlorophyll, and the culture reaches stationary phase at lower cell density. Below 0.1 microm supplemental manganese ion in the medium, the cells are photosynthetically defective. This is accompanied by decreased abundance of D1, which binds the Mn(4)Ca cluster, and release of the OEE proteins from the membrane. Assay of Mn superoxide dismutase (MnSOD) indicates loss of activity of two isozymes in proportion to the Mn deficiency. The expression of MSD3 through MSD5, encoding various isoforms of the MnSODs, is up-regulated severalfold in Mn-deficient cells, but neither expression nor activity of the plastid Fe-containing superoxide dismutase is changed, which contrasts with the dramatically increased MSD3 expression and plastid MnSOD activity in Fe-deficient cells. Mn-deficient cells are selectively sensitive to peroxide but not methyl viologen or Rose Bengal, and GPXs, APX, and MSRA2 genes (encoding glutathione peroxidase, ascorbate peroxidase, and methionine sulfoxide reductase 2) are slightly up-regulated. Elemental analysis indicates that the Mn, Fe, and P contents of cells in the Mn-deficient cultures were reduced in proportion to the deficiency. A natural resistance-associated macrophage protein homolog and one of five metal tolerance proteins were induced in Mn-deficient cells but not in Fe-deficient cells, suggesting that the corresponding gene products may be components of a Mn(2+)-selective assimilation pathway.

Distribution of RTX toxin genes in strains of [ Actinobacillus] rossii and [ Pasteurella] mairii

Strains of [ Actinobacillus] rossii, [ Pasteurella] mairii and [ Pasteurella] aerogenes can be isolated from abortion in swine. The RTX toxin Pax has previously been found only in those [ P.] aerogenes strains isolated from abortion. Nothing is known about RTX toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [ A.] rossii and seven [ P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [ P.] mairii but not in [ A.] rossii strains. No apx genes were found in [ P.] mairii but different gene combinations for apx were detected in [ A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [ A.] rossii strains. Phylogenetically [ A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [ P.] mairii strains clustered with [ P.] aerogenes but forming a separate branch. The fact that [ P.] aerogenes, [ P.] mairii and [ A.] rossii can phylogenetically clearly be identified and might contain distinct RTX toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.

Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in different subcellular compartments

Aerobic organisms evolved a complex antioxidant system, which protect the cells against oxidative damage caused by partially reduced oxygen intermediates, also known as reactive oxygen species. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H(2)O(2) to H(2)O, using ascorbate as the specific electron donor in this enzymatic reaction. Previously, eight APx genes were identified in the rice (Oryza sativa L.) genome through in silico analysis: two cytosolic isoforms, two putative peroxisomal isoforms, and four putative chloroplastic ones. Using gene-specific probes, we confirmed the presence of the eight APx genes in the rice genome by Southern blot hybridization. Transcript accumulation analysis showed specific expression patterns for each member of the APx family according to developmental stage and in response to salt stress, revealing the complexity of the antioxidant system in plants. Finally, the subcellular localization of rice APx isoforms was determined using GFP-fusion proteins in BY-2 tobacco cells. In agreement with the initial prediction, OSAPX3 was localized in the peroxisomes. On the other hand, the OSAPX6-GFP fusion protein was found in mitochondria of the BY-2 cells, in contrast to the chloroplastic location predicted by sequence analysis. Our findings reveal the functional diversity of the rice APx genes and suggest complementation and coordination of the antioxidant defenses in different cellular compartments during development and abiotic stress.

Serotype distribution of Actinobacillus pleuropneumoniae isolated from porcine pleuropneumonia in the Czech Republicduring period 2003-2004

A total of 245 isolates of Actinobacillus pleuropneumoniae included in this study were isolated from lung tissues of pigs which died of porcine pleuropneumonia. The isolates were obtained from 52 swine herds in the Czech Republic over the period 2003&amp;ndash;2004. All the serotypes were serotyped by the coagglutination test. All isolates with cross-reactivity between serotypes 9 and 11 or 1, 9, and 11 in the coagglutination test were further serotyped by the quantitative coagglutination test. The results of serotyping showed that serotype 9 was dominant (46.5%) followed by serotypes 2 (18.5%), and 11 (14.2%). Two-way cross-reaction between serotypes 9 and 11 was found for 7.5% of the isolates. The serotypes 4, 5, 7, and 12 were found infrequently (2.4%). The isolates that could not be serotyped (11%) were classified by PCR typing system based on the apx and omlA genes into the serotype groups 1, 9, 11 (7.5%); 2, 8 (2.3%), and 7, 13 (1.1%).

Expression of the Apx gene family during leaf senescence of Arabidopsis thaliana

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H2O2 scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.

Development and Use of a Multiplex Polymerase Chain Reaction Assay Based on Apx Toxin Genes for Genotyping of Actinobacillus Pleuropneumoniae Isolates

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.

Generation and scavenging of reactive oxygen species in chloroplasts: a submolecular approach

The specific traits of oxygen chemistry are examined as a basis of the step-wise electron acceptance by O 2 , giving rise to incompletely reduced highly reactive oxygen species (ROS) comprising both free radical (O 2 _ - ,OH_, HO 2 _) and non-radical (molecular) forms (H 2 O 2 ). The reversal of electron spin of oxygen, induced by energy transfer from triplet state chlorophyll, is considered as a generator of another non-radical ROS, singlet oxygen ( 1 O 2 ). The view that ROS are primarily agents of damage is questioned by data proving their beneficial role and particularly their signaling function. In this context the importance of the equilibrium between ROS production and scavenging, i.e. the regulation of ROS is emphasized. Particular attention is given to the chloroplast, a major source of ROS, which harbors ROS-producing centers (triplet chlorophyll, ETC in PSI and PSII) and a diversified ROS-scavenging network (antioxidants, SOD, APX-glutathione cycle, and a thioredoxin system). The electron carriers for ROS generation are specified. These include Fe-S clusters and ferredoxin in PSI, and Q A and Q B in PSII. The mechanisms and the molecular targets of damaging effect of ROS in chloroplasts are described. The photoprotective role of ROS formation is explained in terms of the use of oxygen as an electron acceptor, as an alternative to NADP, in high-light conditions producing excess electron flow, overreduction of the ETC and overproduction of NADPH. Adequate scavenging of ROS essentially contributes to photoprotection. Stress factors limiting CO 2 fixation and hence decreasing NADPH utilization and NADP regeneration exacerbate high-light stress; ROS formation and scavenging become unbalanced, and oxidative damage may occur. The water-water cycle in chloroplasts and photorespiration as alternative mechanisms of ROS scavenging, NADP regeneration, and dissipation of excess excitation energy are briefly examined. The defensive role of avoidance of ROS generation through alternative oxidases, reducing O 2 directly to water, is also described. The signaling function of ROS is illustrated by data proving the high-light induced systemic induction of APX genes and acclimation phenomena mediated by H 2 O 2 . The use of transgenic approaches for modeling ROS-tolerant plants is discussed.

Analysis of non-porcine isolates of Actinobacillussuis

Twenty-four Actinobacillus suis isolates obtained from several species of non-porcine mammals were compared to the representative porcine strains, ATCC 15557 (serotype O1) and H89-1173 (serotype O2), by O serotyping, DNA fingerprinting, PCR amplification of apxICA, apxIICA and apxIIICA toxin genes and by rrs (16S rRNA) gene sequencing. Only two strains, both equine, reacted with O1 antiserum while two others, one canine and the other feline, reacted with O2 antiserum. One equine strain reacted weakly with both antisera. No amplification of apx genes was found with the non-porcine O1 or the “not O1/O2” strains but amplification of the apxICA and apxIICA genes was observed with the two O2 strains. In addition, these two O2 strains had both BamHI and BglII fingerprints that were very similar to the porcine O2 reference strain, H89-1173 and rrs gene sequences that were identical to the A. suis reference strain ATCC 15557. Taken together, these data suggest that although many non-porcine A. suis isolates are not A. suis (sensu stricto), some isolates are genotypically as well as phenotypically similar to A. suis.

Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization.

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes — evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein ( omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 ( omlA l), Group II consisted of serotypes 2 and 8 ( omlA II), Group III included serotypes 3, 6 and 7 ( omlA III), Group IV ( omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 ( omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/ omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/ apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.

Serotype and apx genotype profiles of Actinobacillus pleuropneumoniae field isolates in Korea

A total of 100 field isolates of Actinobacillus pleuropneumoniae isolated from lung tissues of pigs with severe pleuropneumonia were serotyped by slide agglutination and precipitation tests. Polymerase chain reactions for...

Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains

A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes. The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.

RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains

Actinobacillus pleuropneumoniae serotype reference strains and 204 A. pleuropneumoniae field strains representing all 12 serotypes and both biovars 1 and 2, obtained from laboratories from various countries worldwide, were analyzed for the presence of the toxin genes apxIC, apxIA, apxIB, apxID, apxIIC, apxIIA, apxIIIC, apxIIIA, apxIIIB, and apxIIID by DNA-DNA hybridization with specific gene probes. Expression of the toxins ApxI, ApxII, and ApxIII was assessed by immunoblot analysis with monoclonal antibodies. The results show that the patterns of apx genes and those of the expressed Apx toxins in biovar 1 field strains are the same as those of the genes and toxins of corresponding serotype reference strain. We found only three strains which had certain apx genes missing compared with the genes in their serotype reference strains. Analysis of the expression of the three toxins showed that nearly all strains expressed their apx genes and produced the same Apx toxins as their serotype reference strain. We found only one strain that did not produce ApxI, although it contained the apxICABD genes, and one strain which did not express ApxII but which contained apxIICA. Several field strains which initially showed that their serotype did not correspond to the apx gene profile of the reference strain and which had an unexpected virulence for the given serotype revealed that their initial serotyping was erroneous. We show that the apx gene profiles are inherent to a given serotype. The method cannot differentiate between all 12 serotypes. However, it allowed us to distinguish five groups of toxin gene patterns which showed pathological, toxicological, and epidemiological significance. None of the biovar 2 strains contained apxIII genes. The apxI and apxII genes in the biovar 2 strains, however, were the same as those found in the serotype reference strains of biovar 1.

Effect of sucrose on expression of Apx genes upon heat stress

Aim. Sucrose plays an important role as a signaling molecule that causes changes in gene expression. However, the effect of sucrose on gene expression of antioxidant enzymes is poorly understood. The purpose of our study was to investigate the effect of sucrose on the expression of the multigenic family Apx in Arabidopsis thaliana under heat stress conditions. Methods. Leaves of A. thaliana were exposed to heat stress at 37ºC in the presence or in absent of sucrose. Subsequently, mRNA levels were determined in using quantitative RT-PCR. Results. It was found that absence of sucrose in the incubation buffer resulted in a decrease of mRNA for most Apx genes, both in heat stress-treated samples and after incubation at room temperature. The strongest effect was shown for Apx2: without sucrose the level of mRNA under heat stress was 30 times lower. Conclusions. Obtained data showed that sucrose can perform a signaling role in regulating the stress response upon heat stress, and, in particular, enhance the temperature-dependent gene expression of Apx1 and Apx2. Keywords: Arabidopsis thaliana, multigenic family Apx, heat stress, mRNA, qPCR.