The role of cysteine-rich receptor-like protein kinases in ROS signaling in Arabidopsis thaliana

Responses to environmental changes are mediated via complex signaling networks in plants. Overlapping signaling pathways guarantee information flow from many simultaneous stress factors leading to both synergistic and antagonistic responses in order to maintain the most optimal conditions for growth under non- or suboptimal conditions. Adaptation to stressful environmental conditions is based on flexible interactions between hormone and reactive oxygen species (ROS) signaling. During the recent years researchers have started to understand the complexity of the crosstalk needed for stress tolerance. However, there are still many fundamental questions unanswered. For example, how are the intertwined signal transduction networks regulated, and how are ROS sensed and signaling specificity achieved? Receptor-like protein kinases (RLKs) are plasma membrane proteins which have a role in signal sensing. RLKs have been linked to many different physiological processes, such as plant development, pathogen defense and abiotic stress response. RLKs are involved in ROS signaling and it has been suggested that members of the cysteine-rich protein kinase (CRK) subfamily could be involved in direct ROS sensing due to the redox regulation possibilities in their extracellular protein domain. The large number of CRKs and their protein similarity suggests partly overlapping functions and possibilities for fine-tuning the stress responses. In this study, Arabidopsis CRKs, especially CRK6 and CRK7, have been characterized and their involvement in ROS signaling studied. Based on the presence of conserved kinase subdomains, this study suggests that Arabidopsis CRKs are active kinases and verifies kinase activity for CRK6 and CRK7 in vitro. This study shows that in addition to stress responses, as previously suggested, CRKs are involved also in many important developmental processes, such as germination and senescence. This novel finding broadens our understanding of the role of CRKs in plants. Despite the observed redundancy in crk phenotypes due to sequence similarity, some crk mutants, such as crk2 and crk5, showed clear individual phenotypes suggesting specific functions for these CRKs. crk6 and crk7 phenotypes were partly disguised by redundancy effect. Based on the obtained results and proposed redox regulation possibilities of CRK ectodomain, it can be proposed that CRKs are essential regulatory elements of cellular redox circuits that relay environmental information to the cell. Therefore the role of CRKs in cellular crosstalk is essential for maintaining the delicate balance between growth and defense. The loss of CRK function disrupts the information flow and leads to impaired stress tolerance. Thus, the obtained results suggest protective roles for CRKs. Furthermore, the large number of CRKs and their specific yet partly overlapping functions could bring operational reliability to the signal transduction and suggests specificity and fine tuning opportunities for signal transduction.Kasvien sopeutuminen alati muuttuviin ympäristöolosuhteisiin vaatii jatkuvia muutoksia kasvien metaboliassa ja kuluttaa energiaa, minkä takia puhutaan stressistä ja stressinsietokyvystä, kun viitataan siihen kuinka hyvin kasvit pystyvät sopeutumaan muutoksiin. Kasvisolujen pinnalla olevat reseptorit toimivat ikään kuin kasvien silminä ja korvina välittäen tietoa ympäröivästä maailmasta. Reseptorit aistivat myös kasvin sisäistä tilaa ja kasvin vaste ympäristöolosuhteiden muutoksiin riippuu aina myös kasvin fysiologisesta tilasta ja kehitysvaiheesta. Päällekkäiset viestinvälitysreitit takaavat tiedonkulun useasta samanaikaisesta eri lähteestä mahdollistaen vastakkaistenkin viestien kulun, mikä johtaa parhaaseen mahdolliseen kasvuun epäoptimaalisissakin olosuhteissa. Reaktiiviset happiyhdisteet (ROS) toimivat viestinvälitysmolekyyleinä ja muutokset ympäristöolosuhteissa, niin kuin kasvin kehityksessä, aistitaan ROS molekyylien aiheuttamien hapetus-pelkistystilojen muutoksina, jotka johtavat paikallisiin solunsisäisiin vasteisiin, mutta jotka voivat johtaa myös laajempiin koko kasvin käsittäviin vasteisiin. ROS molekyyleihin perustuva viestintäjärjestelmä on vuorovaikutuksessa hormonaalisen viestintäjärjestelmän kanssa ja yhdessä nämä viestintäjärjestelmät koordinoivat kasvien kasvua ja sopeutumista. Reseptorityyppiset proteiinikinaasit (RLK:t) ovat solukalvolla olevia proteiineja, jotka aistivat monenlaisia viestejä. RLK proteiinit ovat mukana monessa erilaisessa fysiologisessa tapahtumassa, kuten kasvien kehityksessä, patogeenipuolustuksessa ja (a)bioottisessa stressivasteessa. RLK proteiinien on todettu osallistuvan ROS-välitteiseen viestintään ja on ehdotettu, että RLK proteiiniperheeseen kuuluvat kysteiini-rikkaat proteiinikinaasit (CRK:t) voisivat aistia hapetus-pelkistystilojen muutoksia solunulkoisella osallaan. CRK proteiinien suuri lukumäärä ja proteiinien samankaltaisuus antaa viitteitä osittain päällekkäisistä tehtävistä ja stressivasteiden hienosäätömahdollisuuksista. Tässä tutkimuksessa tutkittiin lituruohon (Arabidopsis thaliana) CRK proteiinien, erityisesti CRK6 ja CRK7 proteiinien, ominaisuuksia ja niiden yhteyttä ROS-välitteiseen viestinvälitykseen. Tämä tutkimus osoitti, että lituruohon CRK6 ja CRK7 proteiinit ovat aktiivisia kinaaseja in vitro olosuhteissa ja ennustaa konservoituneiden kinaasialueiden perusteella kinaasiaktiivisuutta myös muille CRK proteiineille. Lisäksi tämä tutkimus osoitti että, stressiviestinvälityksen lisäksi CRK proteiineilla on tärkeä tehtävä myös useassa kasvinkehitykseen ja kasvuun liityvässä viestinvälitystehtävässä, kuten itämisessä, ikääntymisessä ja ilmarakojen säätelyssä. CRK proteiinien puutos johti usean CRK:n kohdalla stressinsietokyvyn laskuun. Tulosten perusteella voidaan päätellä, että CRK proteiinin puuttuminen katkaisee tärkeän viestinvälitysketjun ja johtaa puutteelliseen tiedonsaantiin ja täten virheelliseen vasteeseen. Tulosten mukaan CRK proteiinit suojaavat lituruohoa usealta ympäristön aiheuttamalta stressiltä mahdollistamalla stressiviestin kulkeutumisen solun sisälle ja edelleen tumaan, missä lopullinen päätös solun vasteesta tehdään

Transcriptional regulation of the CRK/DUF26 group of Receptor-like protein kinases by ozone and plant hormones in Arabidopsis

Peer reviewe

Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.Peer reviewe

Spreading the news: subcellular and organellar reactive oxygen species production and signalling

This review aims to depict the current knowledge on signalling events that are mediated by reactive oxygen species originating in the organelles.As plants are sessile organisms that have to attune their physiology and morphology continuously to varying environmental challenges in order to survive and reproduce, they have evolved complex and integrated environment-cell, cell-cell, and cell-organelle signalling circuits that regulate and trigger the required adjustments (such as alteration of gene expression). Although reactive oxygen species (ROS) are essential components of this network, their pathways are not yet completely unravelled. In addition to the intrinsic chemical properties that define the array of interaction partners, mobility, and stability, ROS signalling specificity is obtained via the spatiotemporal control of production and scavenging at different organellar and subcellular locations (e.g. chloroplasts, mitochondria, peroxisomes, and apoplast). Furthermore, these cellular compartments may crosstalk to relay and further fine-tune the ROS message. Hence, plant cells might locally and systemically react upon environmental or developmental challenges by generating spatiotemporally controlled dosages of certain ROS types, each with specific chemical properties and interaction targets, that are influenced by interorganellar communication and by the subcellular location and distribution of the involved organelles, to trigger the suitable acclimation responses in association with other well-established cellular signalling components (e.g. reactive nitrogen species, phytohormones, and calcium ions). Further characterization of this comprehensive ROS signalling matrix may result in the identification of new targets and key regulators of ROS signalling, which might be excellent candidates for engineering or breeding stress-tolerant plants

The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress

International audienc

Plant development is affected in several <i>crk</i> mutants.

<p><b>(A)</b> Representative pictures of 17-day old seedlings of Col-0 wild type and <i>crk2</i>. Complementation of <i>crk2</i> with 35S::<i>CRK2-CDS</i>:YFP rescued the growth defect of the mutant. Plants were grown under the following conditions: 250 μmol m^-2 s^-1 light intensity under 12 h-day length (day: 23°C, 70% relative humidity; night: 18°C, 90% relative humidity). Bar = 1 cm. Pictures are representative of three independent experiments. <b>(B)</b> A selection of <i>crk</i> mutant lines showing earlier senescence compared to Col-0 wild type. Results are means ± SE (<i>n</i> = 8). <b>(C)</b> Several <i>crk</i> mutants flowered earlier compared to wild type while <i>crk2</i> flowered later. Results are means ± SE (<i>n</i> = 8). <b>(D)</b> Time course analysis of endosperm rupture showed delayed germination in several <i>crk</i> mutants compared to wild type. Results represent means from three independent biological experiments (<i>n</i> = 30). Testa and endosperm rupture were assessed every 5 hours up to 51 hours of imbibition. A seed was considered as germinated when the radicle protruded through both envelopes. <b>(E)</b> Several <i>crk</i> mutants exhibit a lower pavement cell density (number of pavement cells / mm²) in cotyledons. Results are means ± SE (<i>n</i> = 15). <b>(F)</b> Three <i>crks</i> showed slightly longer roots compared to wild type (measured eight days after stratification). Results are means ± SE (<i>n</i> = 16). (<b>B-F)</b> Differences between mutants and Col-0 wild type were compared and analysed using one-way-ANOVA (<i>post hoc</i> Dunnett, asterisks indicate statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001) for <b>(B, C, E</b>) and linear model with single step p-value adjustment (<b>F</b>). All experiments were repeated three times with similar results.</p

Stomatal development and responses are impaired in specific <i>crks</i>.

<p><b>(A)</b> A subset of the <i>crk</i> mutants showed altered water loss (shown as decrease of fresh weight) compared to Col-0 wild type plants after detachment of shoots from roots as evaluated from rosette weight. Complementation of the <i>crk2</i><b>(B)</b>, <i>crk5</i><b>(C)</b> or <i>crk45</i><b>(D)</b> mutants restored a wild type-like water loss phenotype as interpreted from decrease of fresh weight of excised rosettes. Asterisks indicate differences between <i>crk</i> mutants or complementation lines and Col-0 with statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 according to one-way ANOVA with <i>post hoc</i> Tukey HSD. The experiment was repeated three times with similar results. <b>(E)</b> Stomatal apertures were measured 2 h after abscisic acid (ABA) treatment. Some <i>crk</i> mutants are impaired in stomatal closure 2 h after treatment with 10 μM ABA. Results are means of % stomatal aperture ratio (width/length) ± SE (average number of stomata measured = 250). Asterisks indicate statistical significance between control and ABA treatment at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (linear model, single-step p-value adjustment). Lowercase letters indicate statistical significance between wild type Col-0 and <i>crk</i> mutant at <i>P</i><0.05 (a), <i>P</i><0.01 (b) and <i>P</i><0.001 (c) according to one-way ANOVA with <i>post hoc</i> Dunnett’s test. <b>(F)</b> Stomatal density (number of stomata/mm²) is correlated with stomatal length (μm). Most of the <i>crks</i> exhibit a smaller stomata density which correlates with longer stomata (Pearson correlation -0.69, p-value = 0.04). Results are means (average number of stomata measured = 500). <b>(G-I)</b> Time courses of stomatal conductance (relative units) in response to a 3 min pulse of 500–600 ppb of O₃ (<b>G</b>), darkness (<b>H</b>) and elevation of CO₂ from 400 ppm to 800 ppm <b>(I)</b> in a subset of <i>crk</i> mutants and Col-0. Stimuli were applied at 0 time point, which is indicated by an arrow; pre-treatment stomatal conductance was used for normalization. Graph shows the mean of two experiments (<i>n</i> = 6). (<b>J</b>) Overexpression of CRK5 led to lower stomatal conductance compared to Col-0 wild type. (<b>K</b>) Complementation of the <i>crk5</i> mutant restored wild type-like phenotype in the response to a 3-min pulse of 500–600 ppb of O₃, darkness, and elevating CO₂ from 400 to 800 ppm. Asterisks indicate differences between <i>crk</i> mutants or complementation lines and Col-0 with statistical significance at *<i>P</i><0.05 according to one-way ANOVA with <i>post hoc</i> Tukey HSD. The experiment was repeated three times with similar results.</p

Models of CRK function and how they could provide specificity of stomatal aperture regulation.

<p><b>(A)</b> CRKs might act as pathway-specific or multi-pathway regulators of stomatal aperture in response to the PAMPs flg22 and chitin but also the stress hormone ABA and the abiotic stimuli O₃, darkness and CO₂. The figure has been created from data presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.g010" target="_blank">Fig 10</a>. <b>(B)</b> CRKs are involved in the response to pathogens downstream of extracellular ROS production. PAMPs are recognized by pattern recognition receptor complexes. Subsequently, intracellular signalling leads to activation of extracellular superoxide production by NADPH oxidases. ROS perception subsequently leads to intracellular signalling and ultimately stomatal closure. CRKs are implicated in linking extracellular ROS production to intracellular signalling and might regulate and/or interact directly with the recognition receptor complexes.</p