Majer, Petra

English

Majer Petra

University of Szeged

Volume 55(1):197-212, 2011 Acta Biologica Szegediensis http: / /www.sci .u-szeged.hu/ABS Functional characterisation of AnHMGB-A and AnHMGB-B „high mobil ity g r o u p " prote ins of Aspergillus nidulans Zoltán Karácsony Depar tmen t of Microb io logy, University o f Szeged, Szeged, Hungary Aspergillus nidulans is an importan! modell-organism, many metabolic and regulation pathways and the genome sequence of this organism is known. The two proteins of our interest arc members of the ..high mobility group B" protein family that are present in both lower and higher eukaryotes.These proteins affect the expression of various genes on chromatin. Due to their DNA and protein binding ability they have an effect on the stability of chromatin-remodelling and transcription-initation complexes on chromatin. Our main goal was to explore the phsyological role of AnHMGB-A and AnHMGB-B proteins. We deleted the coding sequences of the two proteins from the genome and constructed single and double deleted mutants that were subjected to various experiments to compare their phenotype to the wild type (wt). The mycelia of AAnhmgB-A strain showed decrased growth rate, abnormal shape of cell wall and altered osmotic tolerance in comparison to the wt. Abnormal distribution of reactive oxygen species within the mycelia (central location for mutant and peripheric for the wt) was observed by nitro-blue tetrazolium staining that could result the decreased growth rate and compact growing of the mutant. We observed decreased trehalose level of mycelia by thin layer chromatography (TLC) and HPLC analysis that could be accounted for the aberrant cell wall formation and the elevated osmotic sensitivity. In case of AAnlwigB-B strain we observed abnormal morphology of the mycelia, drastic decrcasement of viability of the asexual spores (52% in wt and 0.35% in mutant at 37°C) and increased sensitivity of the mycelia to oxidative stress. The spores of wild-type and mutant strains were subjected to metabolite analysis by GC-MS analysis that revealed a singinficantly lower level of xylitol and trehalose content in the deleted spores, which could explain the decresed viability. To lind out the reason of the oxidative stress sensitivity of mutants, SOD (superoxide-dismutase) content of deleted and wild-type strains were compared, and significant differences in relative ratio of SOD isoe-nzymes were detected. The mycelia of double-deleted mutant showed nearly complete inhibition of growth in thermo-stress condition (at 42°C). To lind out the cause of the thermosensitivity. a comparative transcriptome sequencing was carried out by „Next Generation Sequencing" (NGS). The analysis process of transcriptom data are in progress. The preliminary results has already revealed that the mutant strain cannot maintain the expression activity of that of wt either at 37 or 42°C. It seems that the transcriptional discrepancies affect the whole chromatin in the mutant. For example, wt strain increases the transcription of 293 genes and decreases the transcription of 540 genes when temperature is shifted from 37 to 42°C. When these data are compared to that of the mutant we observed that out of the normally upregulated 293 genes in wt 52 genes were downregulated and 82 genes were overexpressed. Similarly, transcription activity changed in the downregulated popu-lation of 540 genes, where 94 genes were upregulated instead of downregulation and 123 genes were significantly downregulated than that was observed in the wt. As a preliminary result of the NGS analysis the metabolic pathways leading to secondary metabolite production was assumed to be disturbed in the mutant. To prove our finding experimentally, the total metabolite composition of wt and mutant strain was monitored by TLC and we found that certain intermediate metabolic compounds are extremely accumulated in the mutant strain. We expect that several further experiments will be carried out on the basis of the results of the transcriptome analysis. In the future, we would like to purify the two proteins to carry out ..pull down" assays and DNA-binding experiments. The identification of the interacting proteins and DNA sequences would give a deeper insight into the function of the proteins at the molecular level. Supervisor: Dr. Zsuzsanna Hamari E-mail: karzol@citromail.hu Changes in plant antioxidants and photosynthesis in response to abiotic stresses Petra Majer Inst i tu te o f Plant Biology, Biological Research Center, Hungar ian Academy o f Sciences, Szeged, Hungary As a consequence of their sedentary habit, plants are exposed to changing environmental conditions which may cause stress. A common effect of these abiotic stress factors is the rapid generation of reactive oxygen species (ROS) which are also found at very low concentrations in unstressed plants. Plants have evolved different enzymatic (SOD. POD, etc.) and non-enzymatic (ascorbic acid, phenols, carotenoids. etc.) antioxidants to reduce the amount of these potentially harmful agents. The elevated amount of ROS in plants is a signal of the antioxidant system being overwhelmed and is thus an important indicator of severe stress. Under these conditions, ROS are detectable directly, by a variety of modern biophysical methods. These, however, are not sensitive enough for detecting the ROS assumed to accompany moderate, acclimatory stress. Under these conditions, measuring changes in activity and quantity of the antioxidants is a widely used stress indicator in plant physiology. The aim of our work was to connect antioxidant 205 Volume 55(1):197-212, 2011 Acta Biologica Szegediens is h t t p : / /www.sc i . u - szeged .hu /ABS content , specific ROS scavenging capaci t ies and accl imation potential of leaves under various modera te stress condi t ions . Methods already exist for measur ing total antioxidant capacities and for detect ing enzymes specific to a certain ROS (like peroxidases o r superoxide dismutases) . however some of the ROS are not a imed by specific enzymes and are scavenged by the c o m m o n ef fec t of d i f ferent compounds . The re fo re we developed new methods for measur ing antioxidant capaci t ies of plant samples specif ic to the hydroxy! radical (Maje r et al. 2010b) and the singlet oxygen . (Hideg and Majer 2010) . By correlat ing the ROS specif ic and total ant ioxidant parameters w e showed the impor tance of s tudying the former besides measur ing the widely used latter parameters as well (Ma je r et al. 2010b) . In addi-t ion. w e developed a new method which serves us with an initial screening of photosynthesis in leaves based on digital images (Ma je r et al. 2010a) . Using the above techniques and chlorophyll-fluorescence-based assessment of leaf photochemistry, we studied contributions of preventive and antioxidant processes to high-light tolerance in linden tree (Tilia platyphyllos L.) leaves col lected f r o m sun-exposed and shaded parts of the same tree. Accord ing to our results, l inden sun leaves had 2- t imes s tronger singlet oxygen neutral izing capaci t ies than shade leaves and were able to avoid non-regulated loss of energy under high PAR (Hideg and Majer . 2010) . In sun linden leaves significantly h igher amoun t s of flavonoid glycosides were found and contr ibut ions of various phenol ics to specific ROS capaci t ies are currently investigated. In an other series of exper iments we studied the accl imation potential to UV-B radiation in younge r and older grapevine leaves (Vitis vinifera L. cv. Chardonnay) and found that younger leaves were able to mobi l ize screening p igments and ant ioxidants bet ter and therefore suffered less damage f rom UV-B as compared to o lder ones (Maje r and Hideg 2011) . Hideg É. Majer P (20I0) Factors contributing to the high light tolerance of leaves in vivo - Involvement of photo-protective energy dissipation and singlet oxygen scavenging. Acta Biol Hung 6l(Suppl.): 69-80. Majer P. Sass L . Horváth V G . Hideg É (20i0a) Leaf hue measurements offer a fast, high-throughput initial screening of photosynthesis in leaves. J Plant Physiol 167(I):74-76, Majer P. Stoyanova S, Hideg E (20l()b) Do leaf total antioxidant capacities ( T A C ) reflect specific antioxidant potentials? - A comparison of T A C and reactive oxygen scavenging in tobacco leaf extracts. Journal of Photochem Photobiol В I00( I ):38-43. Majer P. Hideg É ( 2 0 l I ) Are young ones more vigorous? A comparative study of photosynthesis and antioxidant responses of younger and older Vitis leaves under UV-B radiation in a green-house experiment. Abstracts of the 1st Annual Meeting of C O S T Action FA0906 UV4growth; Szeged. Hungary. Supervisors: Éva Hideg. Gábor V. Horváth E-mail: pmajer@brc.hu The role of oxidative stress in antibiotic resistance evolution Orsolya Katinka Mehi E v o l u t i o n a r y Systems B i o l o g y G r o u p , I n s t i t u t e o f B iochemis t ry , B io log i ca l Research Cen t re , H u n g a r i a n A c a d e m y o o f Sciences, Szeged, H u n g a r y T h e emergence of bacteria that are resistant to multiple antibiotics represents an increasingly signif icant threat to public health today. There are a number of mechan i sms whereby bacteria can develop antibiotic resistance including the physical exchange of genet ic material with another organism, the activation of latent mobile genetic e lements ( t ransposons or cryptic genes) and the mutagenesis of its own D N A . Chro-mosomal mutagenesis may arise directly f rom antibiotic-induced oxidative stress, or indirectly, as a consequence of the interaction be tween antibiotic and its target molecu le o r f rom the activity of bac ter ium's er ror-prone D N A polymerases dur ing the repair of D N A lesions. There are at least three major mutational mechanisms which facilitate gradual evolution of bacterial antibiotics resistance. These mecha-nisms include mutators (genotypes with increased, constitutive mutat ion rates). S O S response (a global response that min imizes the lethal and mutagenic consequences of the exposure of cell to DNA d a m a g i n g agents ) and the direct mutagenic effect of reactive oxygen species (ROS) . We investigated how far these three above ment ioned mechan i sms are linked to each other, and what their relative contr ibut ion to ant ibiot ics resistance evolution is. It is known f rom literature that bactericidal (but not bacteriostatic) antibiotics share a c o m m o n lethal pa thway that involves the genera-t ion/accumulat ion of ROS. In our study we focused on the role of ROS in antibiotic resistance evolution. The ef fec t of ROS has a doub le edged sword feature. On one hand ROS production contributes significantly to the killing ef fec t of bactericidal antibiotics. On o ther hand, by directly damaging DNA. ROS accumula t ion increases the muta t ion supply of the bacterial cell which p romotes the appearance of an-tibiotic resistant strains. We employed a series of 10-15 days- long laboratory evolut ionary exper iments with E. coli BW25II3. T h e used strains were initially sensitive to antibiotics and di f fer only in their respective consti tutive genomic mutat ion rates (wild type BW25II3 versus AmutS) and/or activities of the SOS response (wild type versus L e x A 3 expressing strain or AmutSLexA3) . % independent l ineages of each bacterial strain were al lowed to evolve in microti ter plates to successively higher antibiotic concentrat ions by t ransferr ing daily 1% of each cul ture. We employed three antibiotics: c iprof loxacin, ampicil l in. tobramycin, representing three major c lasses of bactericidal ant ibiot ics (quinolones . P- lactams and aminoglycosides respectively) known to stimulate the product ion of ROS. As a negat ive control , w e a lso tested the bacter io-static t r imethoprim, a folic acid biosynthesis inhibitor. In order to test the contribution of ROS format ion on evolvability we added thiourea to the medium. Thiourea is a hydroxyl radical scavenger which mitigates the damage caused by ROS format ion upon antibiotic t rea tment . 206 

Changes in plant antioxidants and photosynthesis in response to abiotic stresses

http://acta.bibl.u-szeged.hu/23207/1/biologica_055_numb_001_205-206.pdf

Changes in plant antioxidants and photosynthesis in response to abiotic stresses

Abstract

Similar works

Full text

Available Versions

University of Szeged