AtDeg2 – a chloroplast protein with dual protease/chaperone activity

Chloroplast protease AtDeg2 (an ATP-independent serine endopeptidase) is cytosolically synthesized as a precursor, which is imported into the chloroplast stroma and deprived of its transit peptide. Then the mature protein undergoes routing to its functional location at the stromal side of thylakoid membrane. In its linear structure AtDeg2 molecule contains the protease domain with catalytic triad (HDS) and two PDZ domains (PDZ1 and PDZ2). In vivo AtDeg2 most probably exists as a supposedly inactive haxamer, which may change its oligomeric stage to form active 12-mer, or 24-mer. AtDeg2 has recently been demonstrated to exhibit dual protease/chaperone function. This review is focused on the current awareness with regard to AtDeg2 structure and functional significance

Mitogen-Activated Protein Kinase Cascades in Plant Hormone Signaling

Mitogen-activated protein kinase (MAPK) modules play key roles in the transduction of environmental and developmental signals through phosphorylation of downstream signaling targets, including other kinases, enzymes, cytoskeletal proteins or transcription factors, in all eukaryotic cells. A typical MAPK cascade consists of at least three sequentially acting serine/threonine kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and finally, the MAP kinase (MAPK) itself, with each phosphorylating, and hence activating, the next kinase in the cascade. Recent advances in our understanding of hormone signaling pathways have led to the discovery of new regulatory systems. In particular, this research has revealed the emerging role of crosstalk between the protein components of various signaling pathways and the involvement of this crosstalk in multiple cellular processes. Here we provide an overview of current models and mechanisms of hormone signaling with a special emphasis on the role of MAPKs in cell signaling networks.One-sentence summary: In this review we highlight the mechanisms of crosstalk between MAPK cascades and plant hormone signaling pathways and summarize recent findings on MAPK regulation and function in various cellular processes

Chloroplast protease/chaperone AtDeg2 influences cotyledons opening and reproductive development in Arabidopsis

AtDeg2 is a chloroplast protein with dual protease/chaperone activity. Since data on how the individual activities of AtDeg2 affect growth and development of Arabidopsis thaliana plants is missing, two transgenic lines were prepared that express mutated AtDeg2 versions that have either only protease or chaperone activity and a comprehensive ontogenesis stage-based study was performed comprising wild type (WT) plants and insertional mutants that do not express AtDeg2, as well as the two transgenic lines. The repression of both AtDeg2 activities in deg2-3 mutants altered just a few phenotypic traits including the time when cotyledons were fully opened, the time when 10% flowers were open as well as the number of inflorescence branches and seed length in plants which have completed their generative development. It was demonstrated that complete opening of cotyledons as well as the number of inflorescence branches and seed length in plants which have completed their generative development required involvement of both AtDeg2 activities, whereas the time when 10% of flowers were open was controlled by AtDeg2 protease activity. These results show for the first time that the chaperone activity of AtDeg2 is needed for some elements of generative development of A. thaliana plants to proceed normally. So far, the chaperone activity of AtDeg2 was confirmed based on in vitro assays only

Insights into the expression of DNA (de)methylation genes responsive to nitric oxide signaling in potato resistance to late blight disease

Our previous study concerning the pathogen-induced biphasic pattern of nitric oxide (NO) burst revealed that the decline phase and a low level of NO, due to S-nitrosoglutathione reductase (GSNOR) activity, might be decisive in the upregulation of stress-sensitive genes via histone H3/H4 methylation in potato leaves inoculated with avr P. infestans. The present study refers to the NO-related impact on genes regulating DNA (de)methylation, being in dialog with histone methylation. The excessive amounts of NO after the pathogen or GSNO treatment forced the transient upregulation of histone SUVH4 methylation and DNA hypermethylation. Then the diminished NO bioavailability reduced the SUVH4-mediated suppressive H3K9me2 mark on the R3a gene promoter and enhanced its transcription. However, we found that the R3a gene is likely to be controlled by the RdDM methylation pathway. The data revealed the time-dependent downregulation of the DCL3, AGO4, and miR482e genes, exerting upregulation of the targeted R3a gene correlated with ROS1 overexpression. Based on these results, we postulate that the biphasic waves of NO burst in response to the pathogen appear crucial in establishing potato resistance to late blight through the RdDM pathway controlling R gene expression

Chaperone activity of chloroplast protease AtDeg2

Wydział BiologiiNa podstawie analiz in vitro potwierdzono pojedyncze doniesienie, pochodzące z innego laboratorium, że chloroplastowe białko AtDeg2 oprócz aktywności proteazy dysponuje także aktywnością białka opiekuńczego typu holdazy. Dowiedziono, ponadto, że AtDeg2 wykazuje in vitro także aktywność opiekuńczą dezagregazy. Za aktywność opiekuńczą holdazy i dezagregazy AtDeg2 in vitro w głównej mierze odpowiedzialne są domeny PDZ1 i PDZ2 oraz częściowo domena proteazowa. Wykazano, że in vivo, w warunkach ekspozycji roślin na światło o wysokim natężeniu AtDeg2 dysponuje aktywnością holdazy, dla której substratem jest podjednostka γ1 chloroplastowej syntazy ATP. Aktywność ta wyraża się zahamowaniem homoagregacji zmienionych oksydacyjnie cząsteczek podjednostki γ1 syntazy ATP poprzez niedopuszczenie do powstania mostków dwusiarczkowych między tymi cząsteczkami. W odniesieniu do cech fenotypowych dowiedziono, że w warunkach bezstresowych AtDeg2 przez swoją aktywność opiekuńczą bierze udział w regulacji szerokości nasion, a przez swoją aktywność opiekuńczą i proteolityczną jednocześnie uczestniczy w regulacji momentu rozprostowania się liścieni, liczby węzłów na pędzie kwiatostanowym i jego rozgałęzieniach oraz długości nasion.The results of our in vitro studies confirm a single report (coming from another lab) showing that a chloroplast protein AtDeg2, besides being a protease displayed a chaperone activity as well (holdase type). In addition, it was shown by us that AtDeg2 exhibited an efficient disaggregase chaperone activity in vitro. It was demonstrated that PDZ1 and PDZ2 domains were required for both types of AtDeg2 chaperone activity in vitro and that protease domain contributed to these activities. Moreover it was found that AtDeg2 had holdase chaperone activity in vivo, involving the inhibition of the formation of homoaggregates of γ1-subunit of ATP synthase (mediated by disulphide bridges) in chloroplasts of plants grown under high irradiance conditions. Based on comparative phenotypic screens of four genotypes of A. thaliana grown in comfortable conditions we found that AtDeg2 was involved in regulation of normal seed width through its chaperone activity as well as in regulation of the time it takes for cotyledons to fully open, for accomplishment of normal inflorescence branching and for normal seed length through both protease and chaperone activities

Chaperone activity of chloroplast protease AtDeg2

Wydział BiologiiNa podstawie analiz in vitro potwierdzono pojedyncze doniesienie, pochodzące z innego laboratorium, że chloroplastowe białko AtDeg2 oprócz aktywności proteazy dysponuje także aktywnością białka opiekuńczego typu holdazy. Dowiedziono, ponadto, że AtDeg2 wykazuje in vitro także aktywność opiekuńczą dezagregazy. Za aktywność opiekuńczą holdazy i dezagregazy AtDeg2 in vitro w głównej mierze odpowiedzialne są domeny PDZ1 i PDZ2 oraz częściowo domena proteazowa. Wykazano, że in vivo, w warunkach ekspozycji roślin na światło o wysokim natężeniu AtDeg2 dysponuje aktywnością holdazy, dla której substratem jest podjednostka γ1 chloroplastowej syntazy ATP. Aktywność ta wyraża się zahamowaniem homoagregacji zmienionych oksydacyjnie cząsteczek podjednostki γ1 syntazy ATP poprzez niedopuszczenie do powstania mostków dwusiarczkowych między tymi cząsteczkami. W odniesieniu do cech fenotypowych dowiedziono, że w warunkach bezstresowych AtDeg2 przez swoją aktywność opiekuńczą bierze udział w regulacji szerokości nasion, a przez swoją aktywność opiekuńczą i proteolityczną jednocześnie uczestniczy w regulacji momentu rozprostowania się liścieni, liczby węzłów na pędzie kwiatostanowym i jego rozgałęzieniach oraz długości nasion.The results of our in vitro studies confirm a single report (coming from another lab) showing that a chloroplast protein AtDeg2, besides being a protease displayed a chaperone activity as well (holdase type). In addition, it was shown by us that AtDeg2 exhibited an efficient disaggregase chaperone activity in vitro. It was demonstrated that PDZ1 and PDZ2 domains were required for both types of AtDeg2 chaperone activity in vitro and that protease domain contributed to these activities. Moreover it was found that AtDeg2 had holdase chaperone activity in vivo, involving the inhibition of the formation of homoaggregates of γ1-subunit of ATP synthase (mediated by disulphide bridges) in chloroplasts of plants grown under high irradiance conditions. Based on comparative phenotypic screens of four genotypes of A. thaliana grown in comfortable conditions we found that AtDeg2 was involved in regulation of normal seed width through its chaperone activity as well as in regulation of the time it takes for cotyledons to fully open, for accomplishment of normal inflorescence branching and for normal seed length through both protease and chaperone activities

The contribution of individual domains of chloroplast protein AtDeg2 to its chaperone and proteolytic activities

The thylakoid protease AtDeg2 is a non-ATP hydrolyzing chloroplast protease/chaperone peripherally connected with stromal side of thylakoid membrane. Its linear structure consists of protease domain and two PDZ domains. To unveil the significance of individual domains, chaperone and proteolytic activities of AtDeg2, its mutated recombinant versions have been developed and their ability to suppress protein aggregation and resolubilization of protein aggregates as well as the ability to degrade substrate protein was examined in vitro. Our work reveals for the first time that AtDeg2 is able not only to suppress aggregation of denatured proteins, but to resolubilize existing protein aggregates as well. We show that PDZ2 domain contributes significantly to both chaperone and protease activities of AtDeg2, whereas PDZ1 is required for chaperone but superfluous for proteolytic activity. Protease domain – but not S-268 in its catalytic site – contributes to chaperone activities of AtDeg2. These results show an entirely new function of AtDeg2 chaperone/protease (i.e., disaggregation of protein aggregates) and allow to identify structural motifs required for “old” and new functions of AtDeg2

Nitric Oxide Implication in Potato Immunity to Phytophthora infestans via Modifications of Histone H3/H4 Methylation Patterns on Defense Genes

Nitric oxide (NO) is an essential redox-signaling molecule operating in many physiological and pathophysiological processes. However, evidence on putative NO engagement in plant immunity by affecting defense gene expressions, including histone modifications, is poorly recognized. Exploring the effect of biphasic NO generation regulated by S-nitrosoglutathione reductase (GNSOR) activity after avr Phytophthora infestans inoculation, we showed that the phase of NO decline at 6 h post-inoculation (hpi) was correlated with the rise of defense gene expressions enriched in the TrxG-mediated H3K4me3 active mark in their promoter regions. Here, we report that arginine methyltransferase PRMT5 catalyzing histone H4R3 symmetric dimethylation (H4R3sme2) is necessary to ensure potato resistance to avr P. infestans. Both the pathogen and S-nitrosoglutathione (GSNO) altered the methylation status of H4R3sme2 by transient reduction in the repressive mark in the promoter of defense genes, R3a and HSR203J (a resistance marker), thereby elevating their transcription. In turn, the PRMT5-selective inhibitor repressed R3a expression and attenuated the hypersensitive response to the pathogen. In conclusion, we postulate that lowering the NO level (at 6 hpi) might be decisive for facilitating the pathogen-induced upregulation of stress genes via histone lysine methylation and PRMT5 controlling potato immunity to late blight

Nitric Oxide Implication in Potato Immunity to <i>Phytophthora infestans</i> via Modifications of Histone H3/H4 Methylation Patterns on Defense Genes

Nitric oxide (NO) is an essential redox-signaling molecule operating in many physiological and pathophysiological processes. However, evidence on putative NO engagement in plant immunity by affecting defense gene expressions, including histone modifications, is poorly recognized. Exploring the effect of biphasic NO generation regulated by S-nitrosoglutathione reductase (GNSOR) activity after avr Phytophthora infestans inoculation, we showed that the phase of NO decline at 6 h post-inoculation (hpi) was correlated with the rise of defense gene expressions enriched in the TrxG-mediated H3K4me3 active mark in their promoter regions. Here, we report that arginine methyltransferase PRMT5 catalyzing histone H4R3 symmetric dimethylation (H4R3sme2) is necessary to ensure potato resistance to avr P. infestans. Both the pathogen and S-nitrosoglutathione (GSNO) altered the methylation status of H4R3sme2 by transient reduction in the repressive mark in the promoter of defense genes, R3a and HSR203J (a resistance marker), thereby elevating their transcription. In turn, the PRMT5-selective inhibitor repressed R3a expression and attenuated the hypersensitive response to the pathogen. In conclusion, we postulate that lowering the NO level (at 6 hpi) might be decisive for facilitating the pathogen-induced upregulation of stress genes via histone lysine methylation and PRMT5 controlling potato immunity to late blight

DataSheet_1_Insights into the expression of DNA (de)methylation genes responsive to nitric oxide signaling in potato resistance to late blight disease.docx

Our previous study concerning the pathogen-induced biphasic pattern of nitric oxide (NO) burst revealed that the decline phase and a low level of NO, due to S-nitrosoglutathione reductase (GSNOR) activity, might be decisive in the upregulation of stress-sensitive genes via histone H3/H4 methylation in potato leaves inoculated with avr P. infestans. The present study refers to the NO-related impact on genes regulating DNA (de)methylation, being in dialog with histone methylation. The excessive amounts of NO after the pathogen or GSNO treatment forced the transient upregulation of histone SUVH4 methylation and DNA hypermethylation. Then the diminished NO bioavailability reduced the SUVH4-mediated suppressive H3K9me2 mark on the R3a gene promoter and enhanced its transcription. However, we found that the R3a gene is likely to be controlled by the RdDM methylation pathway. The data revealed the time-dependent downregulation of the DCL3, AGO4, and miR482e genes, exerting upregulation of the targeted R3a gene correlated with ROS1 overexpression. Based on these results, we postulate that the biphasic waves of NO burst in response to the pathogen appear crucial in establishing potato resistance to late blight through the RdDM pathway controlling R gene expression.</p