Interaction between Hormone and Apoplastic ROS Signaling in Regulation of Defense Responses and Cell Death

Regulation of cellular homeostasis is crucial for proper development, survival, defense responses, programmed cell death and ultimately survival. Maintaining cellular homeostasis requires tight regulation of multiple highly interactive signaling pathways. The apoplast lies at the frontier between the cell and the environment, where the plant perceives environmental cues. Since the apoplast is also a site for cell-to-cell communication, it has an important role in mediating plant-environment interactions. Reactive oxygen species (ROS) are known as both toxic agents and indispensable signaling molecules in all aerobic organisms. A ROS burst in the apoplast is one of the first measurable events produced in response to different biotic and abiotic stresses, eventually leading to the initiation of signal transduction pathways and altered gene expression. Apoplastic ROS signaling is well known to dynamically coordinate multiple signaling pathways in the activation of defense responses in plants. Dissection of the signaling crosstalk within such a signaling network could therefore reveal the molecular mechanisms underlying defense responses. Treatments with ozone (O3) have been adopted as an efficient tool to study apoplastic ROS signaling. Plants exposed to O3 trigger a ROS burst in the apoplast and induce extensive changes in gene expression and alteration of defense hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene. Genetic variation in O3 sensitivity among Arabidopsis thaliana accessions or mutants highlights the complex genetic architecture of plant responses to ROS. To gain insight into the genetic basis of apoplastic ROS signaling, a recombinant inbred line (RIL) population from a reciprocal cross between two Arabidopsis accessions C24 (O3 tolerant) and Tenela (O3 sensitive) was used for quantitative trait loci (QTL) mapping. Through a combination of QTL mapping and transcriptomic analyses in the response to apoplastic-ROS treatment, three QTL regions containing several potential candidate genes were identified in this study. In addition, multiple mutants with varying O3-sensitivities were employed to dissect the signaling components involved in the early apoplastic ROS signaling and O3-triggered cell death. A combination of global and targeted gene expression profiling, genetic analysis, and cell death assays was performed to dissect the contribution of hormone signaling and various transcription factors to the regulation of apoplastic ROS-triggered gene expression and cell death. The contributions of SA, JA and ethylene were assessed through analysis of mutants deficient in these hormones, mutants with constitutively activated hormone signaling and the exogenous application of hormones. Plants with elevated SA levels were found to be associated with an attenuated O3 response, whereas simultaneous elimination of SA-dependent and SA- independent signaling components enhanced the response to apoplastic ROS treatment. JA could act as both a positive and negative modifier of apoplastic ROS signaling, which was enhanced when ethylene signaling was also impaired. However, transcriptome analysis of a triple mutant deficient in SA, JA and ethylene revealed that these hormones signaling only contributed part (about 30%) of early-apoplastic ROS-triggered changes in gene expression, suggesting multiple signaling pathways could be required to regulate the apoplastic ROS response via combinatorial or overlapping mechanisms.Solutason homeostaasin säätely on tärkeää yksilönkehityksen, puolustusvasteiden, ohjelmoidun solukuoleman ja lopulta selviytymisen kannalta. Homeostaasin säilyttäminen vaatii useiden keskenään vuorovaikutteisten viestinvälitysreittien tarkkaa säätelyä. Apoplasti sijaitsee solun ja sen ympäristön välisellä rajalla, josta kasvi aistii ympäristön muutoksia. Lisäksi apoplastin kautta kulkee osa solujen välisestä viestinnästä, joten sillä on tärkeitä tehtäviä kasvin ja sen ympäristön välisessä vuorovaikutuksessa. Reaktiiviset happilajit (reactive oxygen species, ROS) tunnetaan sekä myrkyllisinä yhdisteinä että korvaamattomina viestintämolekyyleinä kaikissa happea tarvitsevissa eliöissä. Yksi ensimmäisistä havaittavista vasteista erilaisiin bioottisiin ja abioottisiin stresseihin on apoplastissa tapahtuva reaktiivisten happilajien purkaus (ROS- purkaus), joka johtaa viestinvälitysreittien aktivaatioon ja muutoksiin geenien ilmentymisessä. Apoplastissa tapahtuva ROS-viestintä koordinoi dynaamisesti useita viestinvälitysketjuja kasvin puolustusvasteiden aktivoinnin aikana. Viestinvälitysketjujen ristivaikutusten analyysi saattaa siten paljastaa puolustusvasteen molekyylimekanismeja. Kasvien käsittelyä otsonilla (O3) käytetään tehokkaana työkaluna apoplastisen ROS-viestinnän tutkimuksessa. Kasvin otsonikäsittely laukaisee apoplastissa ROS-purkauksen ja aikaansaa suuria muutoksia geenien ilmentymisessä ja puolustukseen liittyvissä hormoneissa, kuten salisyylihapossa, jasmiinihapossa ja etyleenissä. Lituruohon (Arabidopsis thaliana) perinnöllinen muuntelu otsoniherkkyydessä korostaa kasvien ROS-vasteiden monimutkaista geneettistä rakennetta. Tässä työssä apoplastisen ROS-viestinnän geneettistä taustaa selvitettiin kvantitatiivisiin ominaisuuksiin vaikuttavien lokusten (QTL) kartoittamisella kahden Arabidopsis-ekotyypin, C24 (otsonikestävä) ja Tenela (otsoniherkkä), välisessä risteytysjälkeläistössä. Yhdistelemällä apoplastisen ROS-käsittelyn vaikutuksen QTL- kartoitusta ja transkriptomin tutkimusta löydettiin kolme QTL-aluetta, joissa on useita kandidaattigeenejä. Apoplastisen ROS-viestinnän ja otsonin käynnistämän solukuoleman parempaa ymmärtämistä varten tutkittiin lisäksi useita mutanttilinjoja, jotka eroavat toisistaan otsoniherkyydeltään. Laajaa ja kohdistettua geenien ilmentymisen profilointia, geneettistä analyysiä ja solukuolemakokeita käytettiin erittelemään hormoniviestinnän ja useiden transkriptiotekijöiden vaikutusta reaktiivisten happilajien käynnistämään apoplastiseen geenien ilmenemiseen ja solukuolemaan. Salisyylihapon, jasmiinihapon ja etyleenin tehtäviä tutkittiin analysoimalla mutanttilinjoja, joilta kyseisten hormonien viestintä oli estetty tai tehostettu, sekä lisäämällä hormoneja kasveille ulkoisesti. Kasvien, joilla oli korkeammat salisyylihappotasot, huomattiin reagoivan otsoniin vaimeammin, kun taas samanaikainen salisyylihaposta riippuvan ja riippumattoman viestinnän estäminen voimisti vastetta apoplastiseen ROS-käsittelyyn. Jasmiinihappo toimi sekä positiivisena että negatiivisena säätelijänä apoplastisessa ROS-viestinnässä, ja sen toiminta tehostui, kun myös etyleeniviestintä oli vaimennettu. Toisaalta kolmoismutantin, jolta oli vaimennettu kaikkien kolmen edellä mainitun hormonin viestintä, transkriptomitason analyysi paljasti, että näiden hormonien kautta kulkeva viestintä vaikuttaa vain osaan (noin 30%) varhaisista geenien ilmentymisen muutoksista vasteena apoplastiseen ROS-viestintään. Tästä voidaan päätellä, että useat viestinvälitysketjut säätelevät apoplastista ROS-vastetta päällekkäisin mekanismein

Salicylic acid signaling inhibits apoplastic reactive oxygen species signaling

Peer reviewe

Simultaneous Ozone and High Light Treatments Reveal an Important Role for the Chloroplast in Co-ordination of Defense Signaling

Plants live in a world of changing environments, where they are continuously challenged by alternating biotic and abiotic stresses. To transfer information from the environment to appropriate protective responses, plants use many different signaling molecules and pathways. Reactive oxygen species (ROS) are critical signaling molecules in the regulation of plant stress responses, both inside and between cells. In natural environments, plants can experience multiple stresses simultaneously. Laboratory studies on stress interaction and crosstalk at regulation of gene expression, imply that plant responses to multiple stresses are distinctly different from single treatments. We analyzed the expression of selected marker genes and reassessed publicly available datasets to find signaling pathways regulated by ozone, which produces apoplastic ROS, and high light treatment, which produces chloroplastic ROS. Genes related to cell death regulation were differentially regulated by ozone versus high light. In a combined ozone + high light treatment, the light treatment enhanced ozone-induced cell death in leaves. The distinct responses from ozone versus high light treatments show that plants can activate stress signaling pathways in a highly precise manner.Peer reviewe

A genetic framework for H2O2 induced cell death in Arabidopsis thaliana

Background: To survive in a changing environment plants constantly monitor their surroundings. In response to several stresses and during photorespiration plants use reactive oxygen species as signaling molecules. The Arabidopsis thaliana catalase2 (cat2) mutant lacks a peroxisomal catalase and under photorespiratory conditions accumulates H2O2, which leads to activation of cell death. Methods: A cat2 double mutant collection was generated through crossing and scored for cell death in different assays. Selected double mutants were further analyzed for photosynthetic performance and H2O2 accumulation. Results: We used a targeted mutant analysis with more than 50 cat2 double mutants to investigate the role of stress hormones and other defense regulators in H2O2 -mediated cell death. Several transcription factors (AS1, MYB30, MYC2, WRKY70), cell death regulators (RCD1, DND1) and hormone regulators (AXR1, ERA1, SID2, EDS1, SGT1b) were essential for execution of cell death in cat2. Genetic loci required for cell death in cat2 was compared with regulators of cell death in spontaneous lesion mimic mutants and led to the identification of a core set of plant cell death regulators. Analysis of gene expression data from cat2 and plants undergoing cell death revealed similar gene expression profiles, further supporting the existence of a common program for regulation of plant cell death. Conclusions: Our results provide a genetic framework for further study on the role of H2O2 in regulation of cell death. The hormones salicylic acid, jasmonic acid and auxin, as well as their interaction, are crucial determinants of cell death regulation.Peer reviewe

Ozone and nitrogen dioxide regulate similar gene expression responses in Arabidopsis but natural variation in the extent of cell death is likely controlled by different genetic loci

High doses of ozone (O-3) and nitrogen dioxide (NO2) cause damage and cell death in plants. These two gases are among the most harmful air pollutants for ecosystems and therefore it is important to understand how plant resistance or sensitivity to these gases work at the molecular level and its genetic control. We compared transcriptome data from O-3 and NO2 fumigations to other cell death related treatments, as well as individual marker gene transcript level in different Arabidopsis thaliana accessions. Our analysis revealed that O-3 and NO2 trigger very similar gene expression responses that include genes involved in pathogen resistance, cell death and ethylene signaling. However, we also identified exceptions, for example RBOHF encoding a reactive oxygen species producing RESPIRATORY BURST OXIDASE PROTEIN F. This gene had increased transcript levels by O-3 but decreased transcript levels by NO2, showing that plants can identify each of the gases separately and activate distinct signaling pathways. To understand the genetics, we conducted a genome wide association study (GWAS) on O-3 and NO2 tolerance of natural Arabidopsis accessions. Sensitivity to both gases seem to be controlled by several independent small effect loci and we did not find an overlap in the significantly associated regions. Further characterization of the GWAS candidate loci identified new regulators of O-3 and NO2 induced cell death including ABH1, a protein that functions in abscisic acid signaling, mRNA splicing and miRNA processing. The GWAS results will facilitate further characterization of the control of programmed cell death and differences between oxidative and nitrosative stress in plants.Peer reviewe

Simultaneous Ozone and High Light Treatments Reveal an Important Role for the Chloroplast in Co-ordination of Defense Signaling

Plants live in a world of changing environments, where they are continuously challenged by alternating biotic and abiotic stresses. To transfer information from the environment to appropriate protective responses, plants use many different signaling molecules and pathways. Reactive oxygen species (ROS) are critical signaling molecules in the regulation of plant stress responses, both inside and between cells. In natural environments, plants can experience multiple stresses simultaneously. Laboratory studies on stress interaction and crosstalk at regulation of gene expression, imply that plant responses to multiple stresses are distinctly different from single treatments. We analyzed the expression of selected marker genes and reassessed publicly available datasets to find signaling pathways regulated by ozone, which produces apoplastic ROS, and high light treatment, which produces chloroplastic ROS. Genes related to cell death regulation were differentially regulated by ozone versus high light. In a combined ozone + high light treatment, the light treatment enhanced ozone-induced cell death in leaves. The distinct responses from ozone versus high light treatments show that plants can activate stress signaling pathways in a highly precise manner

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.Peer reviewe

Author Correction: Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10−9 mutations per site per generation was used, whereas 9.5 × 10−9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice

Justice et Liberté

19 juin 19361936/06/19 (N25)