A mitokondriumok redox állapotának hatása az Arabidopsis stressztűrő képességére = Effect of mitochondrial redox state on Arabidopsis stress tolerance

Pályázatomban az Arabidopsis thaliana mitokondriális funkcióiban, elsősorban a redox állapotának változásában bekövetkező eltérések és a környezeti stresszválasz szabályozása közötti kapcsolatot tanulmányoztam. Munkám során a mitokondriális légzési folyamatok és a az oxidatív és ozmotikus stresszel szembeni ellenálló képesség közötti kapcsolatokat kutattam. A korábbi kutatásaink során azonosított ppr40-1 mutáns és vad típusú növények mitokondriumainak összehasonlításához először megvizsgáltam a Halliwell-Asada ciklus enzimeinek aktivitását. Az APX aktivitás és aszkorbát tartalom lecsökkent, míg a MDAR, DHAR és GR aktivitás és GSH tartalom magasabb szintű volt a ppr40-1 mutáns mitokondriumában a vad típushoz képest. Proteomikai módszerekkel jellemeztük a ppr40-1 mutánsban a légzési komplexek összetételének és működésének megváltozását stressz hatására. Új-generációs szekvenálást felhasználva tanulmányoztuk a ppr40-1 mutánsra jellemző génexpressziós változásokat, elsősorban a H2O2 jelátvitelt érintő szabályozó gének működését. Több mitokondriális mutáns és mitokondriális fehérjéket túltermelő transzgenikus vonal fiziológiai jellemzését végeztem el. A PPR40 túltermelő vonalak vizsgálatakor azt tapasztaltuk, hogy a nagy mennyiségben jelen levő PPR40 fehérje stressz körülmények között képes csökkenteni a reaktív oxigén gyökök képződését és az oxidatív károsodás mértékét a mitokondriális elektron transzport stabilizálása révén. | In the present proposal the interaction of mitochondrial redox state and responses to environmental stress was studied in Arabidopsis thaliana. During this research we studied the connections among mitochondrial respiration and regulatory circuits that control oxidative stress and drought tolerance. The activities of the Halliwell-Asada cycle enzymes were compared in mitochondria of the previously identified ppr40-1 mutant and wild-type plants. APX activity and ascorbic acid content decreased, while MDAR, DHAR and GR activities and GSH content were enhanced in the ppr40-1 mitochondria compared to wild type. Changes under stress conditions in the composition and functioning of the respiratory complexes in ppr40-1 mutant was characterized by proteomic methods. Next-generation sequencing was used to study the gene expression patterns in ppr40-1 mutant. Results suggested that changes primarily affected the functions of regulatory genes in H2O2 signaling. Several mitochondrial mutants and transgenic lines were analyzed by physiological characterization. Analysis of PPR40 overexpressing lines suggested that higher amounts of PPR40 protein can diminish the generation of reactive oxygen species by stabilizing the mitochondrial electron transport. PPR40 can therefore protect plants by reducing oxidative damage during stress

Az ozmotikus stresszválasz szabályozása magasabbrendű növényekben. = Regulation of osmotic stress responses in higher plants

Pályázatunkban egy új genetikai rendszert dolgoztunk ki, amely alkalmas a stressz jelátvitelben szerepet játszó növényi gének azonosítására. RT-PCR módszerrel jellemeztük több stressz indukált Arabidopsis gén aktivitását különböző stressz és homonkezelés után. A gének 5? promoter régióját megklónoztuk, és promoter nélküli luciferáz (LUC) illetve zöld fluorescens protein (GFP) riporter génekhez kapcsoltuk. A riporter gének aktivitását transzgenikus Arabidopsis növényekben tanulmányoztuk. Az új szabályozó faktorok azonosítása érdekében egy Arabidopsis cDNS könyvtárat hoztunk létre a pER8-GW expressziós vektorban, ami ösztradiol által indukálható expressziós kazettát hordoz. A cDNS könyvtár segítségével egy transzgenikus Arabidopsis növény populációt hoztunk létre. A transzgenikus növényeket só rezisztenciára, ABA érzékenységre illetve a már korábban beépített riporter gén aktivitásának megváltozására teszteltük. Több olyan Arabidopsis vonalat sikerült azonosítani, amelyekben az ösztradiol adása megnövekedett só vagy ABA toleranciával, illetve a riporter gén aktivitásával járt együtt. A C38-33 vonalban megemelkedett só toleranciát kaptunk a beépült cDNS transzkripciójának aktiválásával. A cDNS egy új, S1 domén-t tartalmazó fehérjét kódol. Az ADH-121 vonalban egy AP típusú transzkripció faktort azonosítottunk, ami képes volt az ADH-LUC riporter gén kontrukció aktiválására a külső környezeti tényezőktől függetlenül. | We have developed a genetic system to identify new regulatory factors, controlling stress responses in higher plants, namely in Arabidopsis. Using quantitative RT-PCR, we have characterized the expression of several stress-responsive genes in different conditions and hormonal treatments. The 5? promoter sequences of 5 stress-induced genes have been cloned and fused to promoterless reporter genes, such as the firefly luciferase (LUC) or the green fluorescence protein (GFP). Activity of the reporter gene constructs was characterized in transgenic Arabidopsis plants, using non-destructive assays. In order to identify new regulatory factors, a transformation-competent cDNA library was created in the plant expression vector pER8-GW, carrying an estradiol-responsive expression cassette. Large-scale Arabidopsis transformation generated a collection of transgenic plants, each carrying a cDNA clone. Transgenic plants were screened for salt tolerance, ABA insensitivity or activation of reporter gene constructs. Several salt tolerant or ABA insensitive lines were obtained and characterized. In some lines reporter genes were activated upon the induction of transgene expression, in the absence of stress. In the line C38-33 increased salt tolerance was obtained by the activation of a full length cDNA, coding for a previously unknown protein with S1 domain. In the line ADH-121, activation of an AP transcription factor lead to the increased expression of the ADH-LUC reporter construct

Modulating Visuomotor Sequence Learning by Repetitive Transcranial Magnetic Stimulation: What Do We Know So Far?

Predictive processes and numerous cognitive, motor, and social skills depend heavily on sequence learning. The visuomotor Serial Reaction Time Task (SRTT) can measure this fundamental cognitive process. To comprehend the neural underpinnings of the SRTT, non-invasive brain stimulation stands out as one of the most effective methodologies. Nevertheless, a systematic list of considerations for the design of such interventional studies is currently lacking. To address this gap, this review aimed to investigate whether repetitive transcranial magnetic stimulation (rTMS) is a viable method of modulating visuomotor sequence learning and to identify the factors that mediate its efficacy. We systematically analyzed the eligible records (n = 17) that attempted to modulate the performance of the SRTT with rTMS. The purpose of the analysis was to determine how the following factors affected SRTT performance: (1) stimulated brain regions, (2) rTMS protocols, (3) stimulated hemisphere, (4) timing of the stimulation, (5) SRTT sequence properties, and (6) other methodological features. The primary motor cortex (M1) and the dorsolateral prefrontal cortex (DLPFC) were found to be the most promising stimulation targets. Low-frequency protocols over M1 usually weaken performance, but the results are less consistent for the DLPFC. This review provides a comprehensive discussion about the behavioral effects of six factors that are crucial in designing future studies to modulate sequence learning with rTMS. Future studies may preferentially and synergistically combine functional neuroimaging with rTMS to adequately link the rTMS-induced network effects with behavioral findings, which are crucial to develop a unified cognitive model of visuomotor sequence learning

Light Control of Salt-Induced Proline Accumulation is Mediated by Elongated Hypocotyl 5 in Arabidopsis

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Delta 1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O-2(center dot-)) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O-2(center dot-) (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed

The Heat Shock Factor A4A confers salt tolerance and is regulated by oxidative stress and the Mitogen-Activated Protein kinases, MPK3 and MPK6

Heat-shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis. Estradiol-induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays HSFA4A shows homomeric interaction which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, Ser309 being the major phosphorylation site. Activation of the MPK3 and MPK6 MAPK pathway led to the transcriptional activation of the heat-shock protein gene HSP17.6A. In agreement that mutation of Ser309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HSP17.6A. These data suggest that HSFA4A is a substrate of the MPK3/6 signalling and it regulates stress responses in Arabidopsis

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses

Heat shock factors regulate responses to high temperatures, salinity, water deprivation or heavy metals. Their function in stress combinations is however not known. The Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature and combination of these conditions. Fast translocation of HSFA4A-YFP protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by MAP kinases MPK3/6 but also by MPK4 and Ser309 is the dominant MAPK phosphorylation site. In vivo data suggest that HSFA4A can be substrate of other kinases as well. Changing Ser309 to Asp or Ala has altered intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes

Small Paraquat Resistance Proteins Modulate Paraquat and ABA Responses and Confer Drought Tolerance to Overexpressing Arabidopsis Plants

Adaptation of higher plants to extreme environmental conditions is under complex regulation. Several small peptides have recently been described to modulate responses to stress conditions. The Small Paraquat resistance protein (SPQ) of Lepidium crassifolium has previously been identified due to its capacity to confer paraquat resistance to overexpressing transgenic Arabidopsis plants. Here, we show that overexpression of the closely related Arabidopsis SPQ can also enhance resistance to paraquat, while the Arabidopsis spq1 mutant is slightly hypersensitive to this herbicide. Besides being implicated in paraquat response, overexpression of SPQs enhanced sensitivity to abscisic acid (ABA), and the knockout spq1 mutant was less sensitive to ABA. Both Lepidium‐ and Arabidopsis‐derived SPQs could improve drought tolerance by reducing water loss, stabilizing photosynthetic electron transport and enhancing plant viability and survival in a water‐limited environment. Enhanced drought tolerance of SPQ‐overexpressing plants could be confirmed by characterizing various parameters of growth, morphology and photosynthesis using an automatic plant phenotyping platform with RGB and chlorophyll fluorescence imaging. Our results suggest that SPQs can be regulatory small proteins connecting ROS and ABA regulation and through that influence responses to certain stresses