The interaction of auxin and cytokinin signalling regulates primary root procambial patterning, xylem cell fate and differentiation in Arabidopsis thaliana

The interaction of auxin and cytokinin signalling regulates primary root procambial patterning, xylem cell fate and differentiation in Arabidopsis thaliana. Plants contribute to the Earth s atmosphere by binding carbon dioxide and releasing oxygen. Trees produce biomass, which is a renewable source of energy. The Arabidopsis root vasculature is a good model system for studying biomass formation, as it contains the same cell types that are also found in trees: xylem, phloem and intervening pluripotent procambial cells. In Arabidopsis thaliana roots, these cells arise from stem cells within the root meristem. The wild type root radial pattern is bisymmetric, and the regulation of xylem formation is controlled by phytohormones, especially auxin and cytokinin. Our findings show that the vascular pattern is set by a symmetry-breaking event during embryogenesis and is initiated by auxin accumulation and signalling at the cotyledon initials. As the embryo grows, the high auxin signalling promotes the expression of AHP6. Upregulation of AHP6 in specific cells leads to inhibition of cytokinin signalling and might be a key factor in symmetry breakage. Mutants with altered cotyledon numbers or altered cotyledon anatomy fail to establish the bisymmetric pattern and often show altered root symmetry. In growing roots, the bisymmetric pattern is actively reinforced by polar auxin transport and long distance cytokinin transport/translocation from the apical parts of the plant. Cytokinin movement via the phloem and unloading at the root apical meristem promotes cytokinin signalling in the procambial cells in the proximal meristem. Both cytokinin and auxin are required during root procambial patterning, and the interaction of these two phytohormones is mutually inhibitory. According to our model (described in the first part of this thesis), auxin signalling is critical for protoxylem identity formation. In turn, the results from the procambial re-patterning experiments (second part of this thesis) show that cytokinin is the key hormone in promoting cell proliferation in the proximal meristem. Epistasis experiments illustrate that a fine balance between these two hormones affects the fate of all vascular cells. We are beginning to understand the complexity and interdependencies of signalling pathway interactions during proximal meristem vascular patterning, yet the temporal aspect is still largely unexplored. In the last part of this thesis, I discuss the role ROS signalling might have in stele patterning and temporal regulation of programmed cell death. While our published GRI-MC9-PRK5 module might not be directly linked to primary root proximal meristem procambial patterning, one cannot exclude the possibility that it might be required in the final stages of protoxylem differentiation or that a similar signalling mechanism could regulate initial stele patterning and meristem growth dynamics. This thesis describes the auxin-cytokinin interaction in vascular initial patterning and the mechanism by which the hormonal signalling domains are maintained in the proximal meristem. The unpublished data demonstrate how procambial cells can be manipulated to generate new tissues by affecting the homeostasis of auxin and cytokinin signalling. The last part of the thesis describes a cell death signalling module and speculates that it (or similar module) might be involved with primary root meristem maturation.Auksiini-sytokiniini-signaloinnin vuorovaikutus Arabidopsis thalianan primääri-juuren johtojänteen solutyyppien identiteetin muodostumisen ja puusolukon erilaistumisen säätelyssä. Kasvit vaikuttavat maapallon ilmakehän koostumukseen sitomalla itseensä hiilidioksidia sekä vapauttamalla happea. Puut tuottavat biomassaa, joka on uusiutuva energianlähde. Puusolukon muodostumista säätelevät kasvihormonit, erityisesti auksiini ja sytokiniini. Arabidopsis thalianan eli lituruohon juuren johtojänne on hyvä malli puunmuodostuksen tutkimiselle, sillä se sisältää samat solutyypit kuin suuremmat puuvartiset kasvit. Nämä keskeiset solutyyppit ovat ksyleemi (puu), nila ja jälsi, joka sijaitsee ksyleemi- ja nilasolujen välissä. Nämä solutyypit kehittyvät lituruohon juurissa kärkikasvupisteiden kantasoluista. Villityypin lituruohon johtojänne on rakenteeltaan bisymmetrinen. Johtojänteen rakenne muodostuu varhain alkiokehityksen aikana, ja juurten symmetria määräytyy verson sirkkalehtien perusteella. Auksiini akkumuloituu sirkkalehtien aiheisiin. Kun alkio kasvaa suuremmaksi, korkea auksiini-pitoisuus edistää AHP6-geenin ekspressiota sirkkalehdissä ja alkion juuren johtojänteessä. Tämä soluspesifinen AHP6 ilmentyminen johtaa sytokiniinisignaloinnin inhibitioon, mikä on kriittistä bisymmetrian muodostumiselle. Mutanteilla joiden sirkkalehtien lukumäärä tai muoto poikkeaa normaalista, on havaittu ongelmia sekä bisymmetrisen rakenteen muodostumisessa alkionkehityksen aikana, että juuren normaalin rakenteen ylläpidossa itämisen jälkeen. Kasvavien juurten rakennetta pidetään aktiivisesti yllä auksiinin ja sytokiniinin kuljetuksella versoista juuriin. Sytokiniinin liikkuminen nilan mahlavirtauksen mukana juurten kärkiin edistää sytokiniinisignalointia kärkikasvupisteen kantasoluissa ja niiden tytärsoluissa. Sekä sytokiniinia että auksiinia tarvitaan johtosolukon erilaistumiseen ja nämä hormonit vaikuttavat toisiinsa inhiboivasti. Esittämämme mallin mukaan (kuvattu ensimmäisessä osiossa) auksiinisignalointi on kriittistä protoksyleemin identiteetin muodostumiselle. Sytokiniini on puolestaan tärkeää juuren kärkikasvupisteen solujen jakautumisen ja erilaistumattomien jälsisolujen identiteetille, kuten tulokset johtojänteen uudelleen-järjestäytymiskokeista osoittavat (väitöskirjan toisessa osuudessa). Näiden kahden hormonin välinen epistasia säätelee kaikkien johtojänteen solujen kehitystä. Tämän väitöskirjan viimeinen osuus keskittyy happiradikaali-signaloinnin ja kontrolloidun solukuoleman rooliin juuren meristeemin kehityksessä. Vaikka julkaisemamme GRI-MC9-PRK5-moduuli ei vaikuta liittyvän kärkikasvupisteen kantasolujen identiteetin ja johtojänteen rakenteen säätelyyn, on mahdollista että sitä tarvitaan protoksyleemin erilaistumisessa myöhemmissä vaiheissa. Väitöskirjan viimeisessä osuudessa spekuloidaan sillä, mikä rooli solukuolemaan liittyvällä signaloinnilla on juuren kärkikasvupisteen kypsymisen säätelyssä. Tämä väitöskirjatyö havainnollistaa auksiini-sytokiniini-vuorovaikutuksen roolia johtojänteen kantasolujen identiteetin muodostumisessa ja mekanismin, jolla hormonisignalointidomeenit vuorovaikuttavat toisiinsa. Tulokset osoittavat, että juuren rakennetta voidaan muuttaa keinotekoisesti manipuloimalla auksiini-sytokiniini hormonisignalointia. Ymmärryksemme eri hormonisignalointireittien monimutkaisuudesta ja niiden välisistä vuorovaikutuksista juuren johtojänteen eri solutyyppien identiteettien muodostumisessa on lisääntynyt merkittävästi viime vuosien aikana, mutta juuren kärkikasvupisteen eri solujen kypsymisen ajallinen säätely kaipaa lisää tutkimusta

Coded Acoustic Microscopy to Study Wood Mechanics and Development

We have developed a coded excitation scanning acoustic microscope (CESAM) that operates in range of 0.1 to 1 GHz. We used a focusing transducer with 375 MHz central frequency to image two different tree species (birch and hybrid aspen) at different stem height to study their micromechanical difference. The method was able to capture the fresh wood anatomy with cellular resolution. A full stem section scan revealed the heterogeneity of micromechanical properties throughout tissues, and highlighted the higher stiffness of the phloem fibers compared to other vascular cells. This demonstrates the applicability of the method for plant developmental biology.Peer reviewe

The reduction of selenium(IV) by boreal Pseudomonas sp. strain T5-6-I – Effects on selenium(IV) uptake in Brassica oleracea

Peer reviewe

A core mechanism for specifying root vascular patterning can replicate the anatomical variation seen in diverse plant species

Pattern formation is typically controlled through the interaction between molecular signals within a given tissue. During early embryonic development, roots of the model plant Arabidopsis thatiana have a radially symmetric pattern, but a heterogeneous input of the hormone auxin from the two cotyledons forces the vascular cylinder to develop a diarch pattern with two xylem poles. Molecular analyses and mathematical approaches have uncovered the regulatory circuit that propagates this initial auxin signal into a stable cellular pattern. The diarch pattern seen in Arabidopsis is relatively uncommon among flowering plants, with most species having between three and eight xylem poles. Here, we have used multiscale mathematical modelling to demonstrate that this regulatory module does not require a heterogeneous auxin input to specify the vascular pattern. Instead, the pattern can emerge dynamically, with its final form dependent upon spatial constraints and growth. The predictions of our simulations compare to experimental observations of xylem pole number across a range of species, as well as in transgenic systems in Arabidopsis in which we manipulate the size of the vascular cylinder. By considering the spatial constraints, our model is able to explain much of the diversity seen in different flowering plant species.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

Mobile PEAR transcription factors integrate positional cues to prime cambial growth.

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4-the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.Gatsby Foundation [GAT3395/PR3)] University of Helsinki [award 799992091] ERC Grant SYMDEV [No. 323052] NSF-BBSRC MCSB 1517058 etc

The spatial scale of the cytokinin gradients.

<p>The spatial steady state profile of cytokinin in a source-diffusion-decay model, for different degradation rates. The lower panel is zoomed in (50x) to the dotted area within the upper panel; the root sections are drawn to scale for comparison with the gradient. Dotted lines mark the characteristic length of each gradient. The blue line shows the cytokinin distribution with degradation rate and diffusion coefficient similar to auxin; the red and green lines represent gradients that could be informative at the transverse scale of the root meristematic zone.</p

Auxin-driven cytokinin biosynthesis parameters.

<p>Auxin-driven cytokinin biosynthesis parameters.</p

Active auxin import is required for correct protoxylem formation.

<p>(A) Auxin concentration was plotted for the cells of the xylem axis, marked with a white line. (B) Plot of the auxin concentration along the xylem axis in simulations of wild type (black), <i>pin7</i> (green), and the importer mutant (blue). px = protoxylem; mx = metaxylem. (C, D) Frequency of protoxylem defects in auxin importer mutants (C) without and (D) with a 48 h treatment of 10<i>n</i>m 6-benzylaminopurine. ‘Defective’ protoxylem has breaks in one or both strands; ‘normal’ protoxylem is continuous. Asterisks indicate a significant difference from wild type (Col-0). (E) Chloral hydrate cleared <i>aux1</i> roots with long gaps in one (top) or both (bottom) protoxylem strands. Yellow arrows indicate protoxylem; white arrows indicate missing protoxylem. (F–H) Expression of <i>DR5::GFP</i> in the (F, G) <i>aux1lax1lax2</i> mutant and (H) wild type. Scale bar: 10<i>μ</i>m.</p

Auxin leaks out of the stele via the apoplast.

<p>(A) A diagram showing the orientations used in depicting the fluxes. Angular position is measured counter-clockwise from the line labelled 0°. Negative radial fluxes are inwards; positive are outwards. Negative angular fluxes are clockwise; positive are counter-clockwise. (B) The auxin flux integrated across the cells in each tissue layer. (C, D) Heatmap of auxin levels in the stele of (C) static and (D) dynamic simulations of <i>wol</i>. (E, F) Zoomed-in quadrant of the heatmap of the (E) static and (F) dynamic simulations of wol to facilitate comparison of apoplastic auxin levels.</p