Apoplastic ROS and transcriptional response in plant stress signaling

The air pollutant ozone (O3) enters plant leaves through stomata and activates apolastic reactive oxygen species (ROS) signaling. Depending on growth conditions and genotype, this results in large transcriptional reprogramming,closure of stomatal pores and activation of cell death programs. These responses are also regulated through plant stress hormones. This thesis sheds light on how stress hormone signaling is connected with apoplastic ROS signaling in the model plant Arabidopsis thaliana, and investigates regulatory mechanisms which generate specificity among sequence-specific transcription factors (TFs), the executers of apoplastic ROS -induced transcriptional reprogramming. The essential methods of the thesis include O3 exposures of Arabidopsis wild type and mutant plants followed by quantification of cell death and characterization of transcriptional responses supplemented with several protein-level analyses of selected WRKY family TFs. The O3-induced cell death was found to be inhibited by plant hormone salicylic acid, and genes RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and WRKY70 were found to be required for O3-induced cell death in jasmonic acid insensitive genetic background. Even though stress hormones were verified to play important roles in the regulation of cell death, the transcriptional response to apoplastic ROS in a hormone deficient/insensitive mutant was highly similar to wild type, suggesting that much of the signaling involved is independent of the studied hormones jasmonic acid, salicylic acid, and ethylene. The potential major executers of transcriptional response to apoplastic ROS, WRKY family TFs, were studied for their transcriptional regulation, DNA-binding preferences, protein-protein interactions, subcellular localization, and effects on transcriptome. The results showed that the DNA-binding preferences of WRKYs vary substantially between phylogenetic groups, implying that the specificity in signaling between different WRKYs can be partly achieved through DNA binding preferences. Transcriptomic analyses of mutants with altered expression levels of the strongly ROS-inducible WRKY75 implicate this TF as a positive regulator of well-known pathogen-responsive genes, such as PATHOGENESIS-RELATED GENE 1 (PR1) and PATHOGENESIS-RELATED GENE 2 (PR2), and as a negative regulator of several hormone signaling pathways and TFs.Ilmansaaste otsoni tunkeutuu kasvin lehteen ilmaraoista ja käynnistää siellä reaktiivisiin happilajeihin perustuvan viestintäketjun. Kasvuoloista ja perimästä riippuen tämä viestintä voi johtaa ilmarakojen sulkeutumiseen, ohjelmoidun solukuoleman käynnistymiseen ja suuriin muutoksiin geenien ilmentymisessä. Reaktiivisten happilajien lisäksi näitä vasteita säädellään kasvihormonien välityksellä. Tämä väitöstyö valaisee kasvin stressihormoniviestinnän ja reaktiivisiin happilajeihin perustuvan viestinnän yhteyksiä lituruohossa (Arabidopsis thaliana) ja tutkii, mitkä mekanismit luovat spesifisyyttä niiden geenien ilmentymisen säätelytekijöiden välille, jotka suurelta osin vastaavat reaktiivisten happilajien aiheuttamista muutoksista geenien ilmentymisessä. Väitöstyön keskeisiä menetelmiä ovat lituruohon sekä lituruohomutanttien otsonikäsittelyt ja niiden aiheuttaman ohjelmoidun solukuoleman mittaaminen. Lisäksi tutkittiin otsonikäsittelyn aiheuttamia muutoksia geenien ilmentymisessä sekä proteiinitason muutoksia WRKY-perheen säätelytekijöissä. Kasvihormoni salisyylihapon havaittiin estävän otsonin käynnistämää solukuolemaa lituruohossa. Lisäksi havaittiin, että geenit RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) ja WRKY70 vaaditaan otsonin käynnistämän ohjelmoidun solukuoleman toteuttamiseksi otsoniherkässä liturohomutantissa, joka on kykenemätön aistimaan kasvihormoni jasmiinihappoa. Vaikka stressihormonien keskeinen rooli solukuoleman säätelyssä vahvistettiinkin, muutokset geenien ilmentymisessä vasteena reaktiivisiin happilajeihin olivat yllättävän samanlaisia lituruohossa ja lituruohomutantissa, jolta puuttui kyky valmistaa/aistia keskeisiä stressihormoneja jasmiinihappoa, salisyylihappoa ja etyleeniä. Tulos antaa ymmärtää, että suuri osa geenien ilmentymisen muutoksiin johtavasta viestinnästä vasteena reaktiivisiin happilajeihin kulkee reittejä, jotka eivät vaadi kyseisiä hormoneja toimiakseen. WRKY-perheen säätelytekijät ovat keskeisessä osassa apoplastin reaktiivisten happilajien aiheuttamassa geenien ilmentymisen muutoksessa. Väitöstutkimuksessa WRKYjä tutkittiin geenisäätelyn, DNA-sitoutumistaipumusten, proteiini-proteiinivuorovaikutusten ja solunsisäisen jakautumisen suhteen. Tulos, jonka mukaan DNA-sitoutumistaipumukset erosivat merkittävästi fylogeneettisten alaryhmien välillä, viittaa siihen, että viestinvälityksen spesifisyys WRKY-perheen jäsenten välillä saavutetaan ainakin osittain DNA-sitoutumisen tasolla

Ozone responses in Arabidopsis : beyond stomatal conductance

Tropospheric ozone (O-3) is a major air pollutant that decreases yield of important crops worldwide. Despite long-lasting research of its negative effects on plants, there are many gaps in our knowledge on how plants respond to O-3. In this study, we used natural variation in the model plant Arabidopsis (Arabidopsis thaliana) to characterize molecular and physiological mechanisms underlying O-3 sensitivity. A key parameter in models for O-3 damage is stomatal uptake. Here we show that the extent of O-3 damage in the sensitive Arabidopsis accession Shahdara (Sha) does not correspond with O-3 uptake, pointing toward stomata-independent mechanisms for the development of O-3 damage. We compared tolerant (Col-0) versus sensitive accessions (Sha, Cvi-0) in assays related to photosynthesis, cell death, antioxidants, and transcriptional regulation. Acute O-3 exposure increased cell death, development of lesions in the leaves, and decreased photosynthesis in sensitive accessions. In both Sha and Cvi-0, O-3-induced lesions were associated with decreased maximal chlorophyll fluorescence and low quantum yield of electron transfer from Photosystem II to plastoquinone. However, O-3-induced repression of photosynthesis in these two O-3-sensitive accessions developed in different ways. We demonstrate that O-3 sensitivity in Arabidopsis is influenced by genetic diversity given that Sha and Cvi-0 developed accession-specific transcriptional responses to O-3. Our findings advance the understanding of plant responses to O-3 and set a framework for future studies to characterize molecular and physiological mechanisms allowing plants to respond to high O-3 levels in the atmosphere as a result of high air pollution and climate change.Peer reviewe

Cyclodextrin-complexed curcumin exhibits anti-inflammatory and antiproliferative activities superior to those of curcumin through higher cellular uptake (vol 80, vol 1021, 2010)

Retraction notice to “Cyclodextrin-complexed curcumin exhibits anti-inflammatory and antiproliferative activities superior to those of curcumin through higher cellular uptake” [Biochem. Pharmacol. 80 (2010) 1021–1032].Non peer reviewe

Natural Variation in Arabidopsis Cvi-0 Accession Reveals an Important Role of MPK12 in Guard Cell CO2 Signaling

Author Summary Human activities have increased the concentrations of CO2 and harmful air pollutants such as ozone in the troposphere. These changes can have detrimental consequences for agricultural productivity. Guard cells, which form stomatal pores on leaves, regulate plant gas exchange. To maintain photosynthesis, stomata open to allow CO2 uptake, but at the same time, open stomata lead to loss of water and allow the entrance of ozone. Elevated atmospheric CO2 levels reduce stomatal apertures, which can improve plant water balance but also increases leaf temperature. Using genetic approaches—in which we exploit natural variation and mutant analysis of thale cress (Arabidopsis thaliana)—we find that MITOGEN-ACTIVATED PROTEIN KINASE 12 (MPK12) and its inhibitory interaction with another kinase, HIGH LEAF TEMPERATURE 1 (HT1) (involved in guard cell CO2 signaling), play a key role in this regulatory process. We have therefore identified a mechanism in which guard cell CO2 signaling regulates how efficiently plants use water and cope with the air pollutant ozone.Peer reviewe

Cell wall integrity maintenance during plant development and interaction with the environment

Cell walls are highly dynamic structures that provide mechanical support for plant cells during growth, development and adaptation to a changing environment. Thus, it is important for plants to monitor the state of their cell walls and ensure their functional integrity at all times. This monitoring involves perception of physical forces at the cell wall–plasma membrane interphase. These forces are altered during cell division and morphogenesis, as well as in response to various abiotic and biotic stresses. Mechanisms responsible for the perception of physical stimuli involved in these processes have been difficult to separate from other regulatory mechanisms perceiving chemical signals such as hormones, peptides or cell wall fragments. However, recently developed technologies in combination with more established genetic and biochemical approaches are beginning to open up this exciting field of study. Here, we will review our current knowledge of plant cell wall integrity signalling using selected recent findings and highlight how the cell wall–plasma membrane interphase can act as a venue for sensing changes in the physical forces affecting plant development and stress responses. More importantly, we discuss how these signals may be integrated with chemical signals derived from established signalling cascades to control specific adaptive responses during exposure to biotic and abiotic stresses

Dissecting Contrasts in Cell Death, Hormone, and Defense Signaling in Response to Botrytis cinerea and Reactive Oxygen Species

Plants require interaction between signaling pathways to differentiate and integrate stress responses and deploy appropriate defenses. The hormones ethylene, salicylic acid (SA), and jasmonic acid (JA) are important regulators of plant defenses. Numerous interactions between these signaling pathways are the cornerstone of robust plant immunity. Additionally, during the early response to pathogens, reactive oxygen species (ROS) act as signaling molecules. Here, we examined the extent of signal interaction in the early stages of Botrytis cinerea infection. To enable a comparison between B. cinerea infection with ROS signaling, we subjected plants to ozone treatment, which stimulates an apoplastic ROS burst. We used a collection of single, double, and triple signaling mutants defective in hormone signaling and biosynthesis and subjected them to B. cinerea infection and ozone treatment at different timepoints. We examined lesion size, cell death, and gene expression (both quantitatively and spatially). The two treatments shared many similarities, especially in JA-insensitive mutants, which were sensitive to both treatments. Unexpectedly, a B. cinerea- susceptible JA-insensitive mutant (coil), became tolerant when both SA biosynthesis and signaling was impaired (coil npr1 sid2), demonstrating that JA responses may be under the control of SA. Extensive marker gene analysis indicated JA as the main regulator of both B. cinerea and ozone defenses. In addition, we identified the transcription factor SRI_ as a crucial regulator of PLANT DEFENSIN expression and cell-death regulation, which contributes to resistance to B. cinerea. Overall, our work further defines the context of ROS in plant defense signaling.Peer reviewe

Dissecting Contrasts in Cell Death, Hormone, and Defense Signaling in Response to Botrytis cinerea and Reactive Oxygen Species

Plants require interaction between signaling pathways to differentiate and integrate stress responses and deploy appropriate defenses. The hormones ethylene, salicylic acid (SA), and jasmonic acid (JA) are important regulators of plant defenses. Numerous interactions between these signaling pathways are the cornerstone of robust plant immunity. Additionally, during the early response to pathogens, reactive oxygen species (ROS) act as signaling molecules. Here, we examined the extent of signal interaction in the early stages of Botrytis cinerea infection. To enable a comparison between B. cinerea infection with ROS signaling, we subjected plants to ozone treatment, which stimulates an apoplastic ROS burst. We used a collection of single, double, and triple signaling mutants defective in hormone signaling and biosynthesis and subjected them to B. cinerea infection and ozone treatment at different timepoints. We examined lesion size, cell death, and gene expression (both quantitatively and spatially). The two treatments shared many similarities, especially in JA-insensitive mutants, which were sensitive to both treatments. Unexpectedly, a B. cinerea–susceptible JA-insensitive mutant (coi1), became tolerant when both SA biosynthesis and signaling was impaired (coi1 npr1 sid2), demonstrating that JA responses may be under the control of SA. Extensive marker gene analysis indicated JA as the main regulator of both B. cinerea and ozone defenses. In addition, we identified the transcription factor SR1 as a crucial regulator of PLANT DEFENSIN expression and cell-death regulation, which contributes to resistance to B. cinerea. Overall, our work further defines the context of ROS in plant defense signaling

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. Here we show that the cysteine-rich RLK CRK2 kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RBOHD in Arabidopsis. Functional CRK2 is required for the full elicitor-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment and substitution of S703 with alanine reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating MAMP-triggered ROS production.Peer reviewe

The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana

Cell walls surround all plant cells, and their composition and structure are modified in a tightly controlled, adaptive manner to meet sometimes opposing functional requirements during growth and development. The plant cell wall integrity (CWI) maintenance mechanism controls these functional modifications, as well as responses to cell wall damage (CWD). We investigated how the CWI system mediates responses to CWD in Arabidopsis thaliana. CWD induced by cell wall–degrading enzymes or an inhibitor of cellulose biosynthesis elicited similar, turgor-sensitive stress responses. Phenotypic clustering with 27 genotypes identified a core group of receptor-like kinases (RLKs) and ion channels required for the activation of CWD responses. A genetic analysis showed that the RLK FEI2 and the plasma membrane–localized mechanosensitive Ca2+ channel MCA1 functioned downstream of the RLK THE1 in CWD perception. In contrast, pattern-triggered immunity (PTI) signaling components, including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2, repressed responses to CWD. CWD induced the expression of PROPEP1 and PROPEP3, which encode the precursors of AtPep1 and AtPep3, and the release of PROPEP3 into the growth medium. Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration-dependent manner. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism. This suppression was alleviated when PTI signaling downstream of PEPR1 and PEPR2 was impaired. Defense responses controlled by the CWI maintenance mechanism might thus compensate to some extent for the loss of PTI signaling elements

Correction to: Functional characterization of genes mediating cell wall metabolism and responses to plant cell wall integrity impairment

Following publication of the original article [1], the author reported that the two curves in the sub-diagram WSR4 in Fig. 2a should be the other way round.</p