Reactive oxygen species and SRO proteins as regulators of gene expression in Arabidopsis thaliana

Life on earth is largely dependent on plants, which provide us energy and oxygen. Plants are sessile organisms adapted to their respective growth environments. However, these environmental conditions are rarely constant and changes in growth conditions require modifications in plant metabolism. Abiotic environmental factors may suddenly become limited or excessive, or a pathogen attack may cause biotic stress. Reactive oxygen species (ROS) are by-products of normal aerobic metabolism and their production is enhanced by biotic and abiotic stresses. ROS serve as signaling molecules, which regulate expression of stress-responsive genes together with other signaling pathways in order to achieve appropriate responses to the suboptimal environment. Human activities also modify the environment for instance by increasing levels of air pollutant ozone (O3) which is a ROS causing foliar damage in sensitive species and cultivars. Therefore, understanding the mechanisms governing plant stress tolerance is of increasing importance. Importantly for this work, O3 is known to cause production of apoplastic ROS in plants similar to other stresses and was therefore used here as a tool to study stress signaling. The Arabidopsis thaliana mutant radical-induced cell death1 (rcd1) harbors several stress phenotypes related to ROS signaling. In order to identify novel genes and signaling pathways regulating plant stress responses, RCD1 and its homologs were studied further. RCD1 belongs to a plant-specific SIMILAR TO RCD-ONE (SRO) gene family present in all land plants analyzed. The molecular function of SROs remains unknown as they have no poly(ADP-ribose) polymerase (PARP) activity and lack catalytic amino acids in the conserved PARP catalytic domain. However, SROs are able to interact with transcription factors via their C-terminal RST domain which suggests that they may regulate gene expression. In addition to altered rosette morphology, rcd1 has changes in gene expression in normal growth conditions which may partially be attributed to RCD1 protein-protein interactions. However, sro1 and sro5 plants are similar to wild-type Col-0 regarding their growth habitus and gene expression. A. thaliana paralogs RCD1 and SRO1 share unequal genetic redundancy: the rcd1 sro1 double mutant is stunted and SRO1 can partially complement rcd1. Transcriptomic analysis of apoplastic ROS-induced signaling triggered by O3 treatment revealed altered expression of thousands of genes in a time-dependent manner. In rcd1, this response was exaggerated, which may explain the triggering of programmed cell death in O3-treated rcd1. Transcription factor WRKY70 was identified as a positive regulator of cell death, putatively acting through altered balance of salicylic acid and jasmonic acid signaling. A transient decrease in auxin signaling together with altered expression of auxin-responsive transcripts by apoplastic ROS was observed. Decreased auxin signaling did not affect the extent of cell death caused by acute O3, but caused more severe morphological changes in chronically O3-treated plants. Altogether, these results suggest that auxin-ROS interaction modulates plant development under stress.Elämä maapallolla on suurilta osin riippuvaista kasvien tuottamasta hapesta ja energiasta. Kasvit ovat elinolosuhteisiinsa sopeutuneita, kuitenkin nämä ympäristöolosuhteet pysyvät harvoin täysin samankaltaisina. Koska kasvit ovat juurtuneita kasvupaikkaansa, niiden ainoa keino sopeutua uusiin elinoloihin on muuttaa solujen aineenvaihduntaa. Reaktiiviset happilajit ovat normaalin aerobisen aineenvaihdunnan sivutuotteita, joita tuotetaan soluissa jatkuvasti. Lisäksi useat ympäristöperäiset stressit, kuten kasvipatogeenit, kuivuus ja kylmyys, lisäävät reaktiivisten happilajien aktiivista tuottoa kasvisoluissa. Näillä reaktiivisilla happilajeilla on tärkeä rooli solusignaloinnissa, joka johtaa muutoksiin geenien ilmenemisessä ja edelleen sopeutumisessa stressitekijöihin. Ilmansaaste otsonin jatkuvasti lisääntyvä pitoisuus alailmakehässä johtuu ihmistoiminnasta ja aiheuttaa paikoitellen merkittäviä kasvivaurioita ja satotappioita. Otsonin haitallisuus kasveille johtuu sen hajoamisesta lehtien soluväleissä reaktiivisiksi happilajeiksi, jotka aikaansaavat solujen stressivasteen sekä lisääntyneen reaktiivisten happilajien tuoton. Tämä ominaisuus mahdollistaa otsonin käytön kasvien stressisignaloinnin tutkimuksen työkaluna, jota hyödynnettiin myös tässä väitöskirjatyössä. Tutkimalla mallikasvi lituruohon (Arabidopsis thaliana) mutanttien stressivasteita voidaan tunnistaa uusia geenejä ja signalointireittejä, joiden avulla kasvi sopeutuu ympäristötekijöihin. Otsoniherkällä radical-induced cell death1 (rcd1) mutantilla on muutoksia sekä sen kehityksessä että stressikestävyydessä. Tässä työssä selvitettiin edelleen RCD1-proteiinin sekä muiden rakenteeltaan samankaltaisten SIMILAR TO RCD1-ONE (SRO)-proteiinien esiintymistä ja toimintaa. Yhdessä nämä proteiinit muodostavat kasvispesifisen SRO-geeniperheen, joka löytyi kaikista tässä työssä analysoiduista maakasveista. SRO-proteiinien biokemiallisen toiminnan ja sekvenssien analyysi osoitti, etteivät ne pysty toimimaan poly-(ADP-riboosi) polymeraaseina. SRO-proteiinit sitoutuvat muihin proteiineihin niin sanotun RST-domeenin avulla ja koska merkittävä osa SRO-proteiineihin sitoutuvista proteiineista oli geenien ilmenemistä sääteleviä transkriptiofaktoreita, näiden proteiini-proteiini vuorovaikutusten voidaan olettaa olevan todennäköisesti keskeisiä SRO-proteiinien toimintamekanismien kannalta. SRO-proteiinit eroavat kyvyssään säädellä geenien ilmenemistä, koska suurimmat muutokset transkriptimäärissä havaittiin rcd1 mutantissa ja sro1 sekä sro5 mutantit olivat hyvin villityypin kaltaisia. Kuitenkin mutanttianalyysi osoitti, että SRO1 proteiinin toiminta on osittain päällekkäistä RCD1:n kanssa. Laajamittaisessa aikasarjakokeessa otsonikäsittelyn aikaansaaman reaktiivisten happilajien tuoton havaittiin muuttavan tuhansien geenien ilmenemistä. Tämän vasteen ja signaloinnin suurempi voimakkuus rcd1-mutantissa voi selittää ohjelmoidun solukuoleman, joka havaitaan otsonikäsitellyissä kasveissa kuolleina lehtilaikkuina, synnyn. Transkriptiofaktori WRKY70:n puuttuminen alensi ohjelmoidun solukuoleman määrää rcd1-mutantissa, mikä osoittaa ohjelmoidun solukuoleman olevan yhteydessä geenien ilmenemiseen. Kasvihormoni auksiinin signaloinnin havaittiin alenevan reaktiivisten happilajien vaikutuksesta, mutta auksiinivasteella ei havaittu olevan suoraa vaikutusta nopeasti muodostuvien otsonivaurioiden syntyyn. Kuitenkin pitkäaikainen otsonikäsittely aiheutti eniten muutoksia lehtien ilmiasussa auksiinivasteiltaan puutteellisissa mutanteissa, joten auksiinin ja reaktiivisten happilajien vuorovaikutus säätelee kasvien kehitystä stressiolosuhteissa

The RST and PARP-like domain containing SRO protein family : Analysis of protein structure, function and conservation in land plants

Peer reviewe

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana : Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.Peer reviewe

Transcriptomics and Functional Genomics of ROS-Induced Cell Death Regulation by RADICAL-INDUCED CELL DEATH1

Peer reviewe

Parsimonious Model of Vascular Patterning Links Transverse Hormone Fluxes to Lateral Root Initiation : Auxin Leads the Way, while Cytokinin Levels Out

An auxin maximum is positioned along the xylem axis of the Arabidopsis root tip. The pattern depends on mutual feedback between auxin and cytokinins mediated by the PIN class of auxin efflux transporters and AHP6, an inhibitor of cytokinin signalling. This interaction has been proposed to regulate the size and the position of the hormones' respective signalling domains and specify distinct boundaries between them. To understand the dynamics of this regulatory network, we implemented a parsimonious computational model of auxin transport that considers hormonal regulation of the auxin transporters within a spatial context, explicitly taking into account cell shape and polarity and the presence of cell walls. Our analysis reveals that an informative spatial pattern in cytokinin levels generated by diffusion is a theoretically unlikely scenario. Furthermore, our model shows that such a pattern is not required for correct and robust auxin patterning. Instead, auxin-dependent modifications of cytokinin response, rather than variations in cytokinin levels, allow for the necessary feedbacks, which can amplify and stabilise the auxin maximum. Our simulations demonstrate the importance of hormonal regulation of auxin efflux for pattern robustness. While involvement of the PIN proteins in vascular patterning is well established, we predict and experimentally verify a role of AUX1 and LAX1/2 auxin influx transporters in this process. Furthermore, we show that polar localisation of PIN1 generates an auxin flux circuit that not only stabilises the accumulation of auxin within the xylem axis, but also provides a mechanism for auxin to accumulate specifically in the xylem-pole pericycle cells, an important early step in lateral root initiation. The model also revealed that pericycle cells on opposite xylem poles compete for auxin accumulation, consistent with the observation that lateral roots are not initiated opposite to each other.Peer reviewe

High levels of auxin signalling define the stem-cell organizer of the vascular cambium

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium(1). Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown(2). Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium(3). Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.Peer reviewe

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.</p

AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins

Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.Peer reviewe

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