The integration of developmental signals during root procambial patterning in Arabidopsis thaliana

The vascular system of plants functions as a transportation route for water, nutrients and signaling molecules while also forming a support structure and generating most of the radial growth by increasing the number of cell files through periclinal cell divisions. These features have transformed life on Earth by enabling plants to colonize land and grow larger. In mature plants, the conductive tissues xylem and phloem are produced from stem cells in the vascular cambium, which develops from the procambium formed during early development. The vascular cylinder of the Arabidopsis root comprises a central xylem axis with a peripheral phloem pole on either side and procambial cells located between the xylem and phloem. Formation of the vascular pattern requires high auxin and cytokinin signaling domains in the xylem and phloem/procambium positions, respectively. However, the gene regulatory network acting downstream of these hormonal cues has remained unknown. I investigated procambium patterning in the Arabidopsis root. Our research group discovered that radial growth is activated in the peripheral phloem domain by six mobile DOF transcription factors that we named PHLOEM EARLY DOF (PEAR) proteins, consisting of PEAR1, PEAR2, and their four homologues. PEAR proteins form an inverse concentration gradient to the HD-ZIP III transcription factors, which inhibit periclinal cell divisions in the central domain partially by inhibiting the movement of PEAR proteins. HD-ZIP III expression is promoted by auxin in the xylem axis and inhibited by endodermis-derived mobile microRNA165/166 in the periphery. The PEAR and HD-ZIP III genes form a feedback loop in which the PEAR proteins promote HD-ZIP III transcription while the HD-ZIP IIIs inhibit PEAR transcription and protein movement. The PEAR-HD-ZIP III regulatory module decodes hormonal and microRNA signals to result in the formation of a highly active peripheral zone and a more quiescent central zone during procambium development. We also determined that a member of the DOF family, DOF2.1, acts downstream of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY-dependent cytokinin biosynthesis to regulate periclinal cell divisions in the outer procambial cells in contact with the xylem axis. Together, PEAR and DOF2.1 proteins control all of the periclinal divisions in the procambium through their activity in partially distinct domains. We also identified SUPPRESSOR OF MAX2 1-LIKE 3 (SMXL3), a member of SMXL subclade 2 which is expressed in the early phloem and procambium cells, as a putative direct target of PEAR2 that is sufficient to promote periclinal divisions. Characterization of SMXL subclade 2 identified SMXL3, 4 and 5 as essential regulators of phloem formation that act very early in development and thus are required for all aspects of phloem development. Phloem specification requires periclinal divisions in the procambium. SMXL3, 4 and 5 act in both the periclinal divisions and phloem specification in a partially redundant manner. Furthermore, analysis of regulators downstream of the PEARs revealed that they not only promote cell proliferation but also specify the identity of the surrounding cells non-cell autonomously, including procambial and phloem pole pericycle identity. Our work highlights the importance of cell-to-cell communication in plant development. The interaction of mobile hormones, transcription factors and microRNAs originating from different tissues is required to coordinate developmental processes in the vascular cylinder. We have assembled the most complete understanding to date of the regulatory network coordinating procambial development and have identified the protophloem sieve elements as the organizers of radial growth during the early stages of vascular development in the Arabidopsis root. These findings can potentially be used to increase yields in forestry and agriculture.Kasvien johtosolukko kuljettaa vettä, ravinteita ja viestimolekyylejä, sekä toimii tukirakenteena, joka tuottaa suurimman osan kasvin paksuuskasvusta solun pitkittäisen akselin suuntaistesti tapahtuvien ns. periklinaalisten solunjakautumisten avulla. Näiden ominaisuuksien ansiosta kasvit pystyivät siirtymään maalle ja kasvamaan kooltaan suuremmiksi, minkä seurauksena elämä maanpäällä muuttui täysin. Täysikasvuisilla kasveilla johtosolukon puu- ja nilasolut kehittyvät kantasoluja sisältävästä jällestä, jota edeltää varhaisen kasvuvaiheen esijälsi. Arabidopsis thalianan eli lituruohon keskuslieriössä puu- eli ksyleemisolut muodostavat akselin keskelle, nilasolut ovat molempien puolien reunoilla ja esijällen solut sijaitsevat näiden välissä. Korkea auksiinipitoisuus ksyleemissä ja korkea sytokiniinipitoisuus nilassa ja esijällessä tarvitaan johtosolukon normaaliin kehitykseen. Näiden hormonaalisten viestien ohjaama geeninsäätelyverkosto on ollut kuitenkin tuntematon. Tutkin esijällen muodostumista lituruohon juuressa. Tutkimusryhmämme havaitsi paksuuskasvun aktivoituvan keskuslieriön reunoilla olevissa varhaisen vaiheen nilan siiviläsoluissa ja tietyissä niitä ympäröivissä soluissa. Määritimme että näiden solujen jakautumisesta on vastuussa kuuden liikkuvan DOF-transkriptiotekijän perhe, jolle me annoimme nimeksi PHLOEM EARLY DOF (PEAR). Nämä koostuvat PEAR1 ja PEAR2 proteiineista ja niiden neljästä homologista. PEAR proteiinit muodostavat vastakkaisen pitoisuusgradientin ennestään tunnettujen HZ-ZIP III transkriptiotekijöiden kanssa. HD-ZIP III proteiinit estävät periklinaalisia solunjakautumisia keskuslieriön keskiosassa osittain estämällä PEAR proteiinien liikkumista. Auksiini edistää HD-ZIP III:n ilmenemistä keskuslieriön keskellä sijaitsevissa ksyleemisoluissa, kun taas keskuslieriön ulkopuolelta liikkuva mikroRNA165/166 heikentää HD-ZIP III:n ilmenemistä sen reunoilla. PEAR ja HD-ZIP III muodostavat takaisinkytkentämekanismin: PEAR lisää HD-ZIP III:n ilmenemistä, kun taas HD-ZIP III estää PEAR geenien ilmenemistä ja PEAR proteiinin liikkumista. PEAR-HDZIP III säätelymoduuli tulkitsee hormonaalisia ja mikroRNA signaaleja minkä seurauksena esijälteen muodostuvat aktiivisesti jakautuva reuna-alue ja hiljainen keskialue. Osoitimme myös, että TARGET OF MONOPTEROS 5/LONESOME HIGHWAY transkriptiotekijäparin aktivoima sytokiniinin biosynteesi aktivoi DOF perheeseen kuuluvan DOF2.1 geenin ilmenemistä. DOF2.1 säätelee periklinaalisia solujakautumisia ksyleemin viereisissä reuna- alueen esijälsisoluissa. Osittain eri alueilla toimivat PEAR ja DOF2.1 proteiinit säätelevät kaikkia esijällen periklinaalisia solunjakautumisia. Tutkimuksessamme havaitsimme, että PEAR2 aktivoi SUPPRESSOR OF MAX2 1-LIKE (SMXL) proteiinien toiseen alaluokkaan kuuluvan SMXL3 geenin ilmenemistä. SMXL3 toimii varhaisen vaiheen nilan ja esijällen soluissa ja on riittävä aktivoimaan periklinaalisia solunjakautumisia. SMXL proteiinien toisen alaluokan jäsenet SMXL3, 4 ja 5 säätelevät lisäksi nilan kehitystä. Ne toimivat jo kehityksen varhaisessa vaiheessa ja täten ovat tarpeellisia kaikkiin nilan kehitysvaiheisiin. Esijällen periklinaaliset solunjakautumiset ovat tärkeä osa nilan kehitystä. Tutkimuksemme osoittaa, että SMXL3, 4 ja 5 toimivat sekä solunjakautumisten että erilaistumisen säätelyssä osittain päällekkäisesti. PEAR proteiinien säätelykohteiden analyysi myös paljasti, että PEAR proteiinit toimivat sekä solunjakautumisten säätelyssä että ympäröivien solujen, kuten esijällen ja nilan viereisten perisyklisolujen, identiteetin määrittämisessä liikkumalla solusta toiseen. Tuloksemme korostavat solujenvälisen viestinnän tärkeyttä: eri solukoista peräisin olevien liikkuvien kasvihormonien, säätelytekijöiden ja mikroRNA-molekyylien vuorovaikutus tarvitaan ohjaamaan kehitystä. Työmme luo tähän mennessä kattavimman ymmärtämyksen säätelyverkostoista, jotka ohjaavat esijällen kehitystä ja osoittaa varhaisen vaiheen nilan siiviläsolujen järjestävän paksuuskasvun aktivoinnin lituruohon juuren johtosolukon varhaisessa kehityksessä. Näitä löydöksiä voidaan mahdollisesti hyödyntää maa- ja metsätaloudessa lisäämään tuotantoa

Plasmodesmata-mediated intercellular signaling during plant growth and development

Peer reviewe

Strigolactone- and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation

Plant stem cell niches, the meristems, require long-distance transport of energy metabolites and signaling molecules along the phloem tissue. However, currently it is unclear how specification of phloem cells is controlled. Here we show that the genes SUPPRESSOR OF MAX2 1-LIKE3 (SMXL3), SMXL4, and SMXL5 act as cell-autonomous key regulators of phloem formation in Arabidopsis thaliana. The three genes form an uncharacterized subclade of the SMXL gene family that mediates hormonal strigolactone and karrikin signaling. Strigolactones are endogenous signaling molecules regulating shoot and root branching [1] whereas exogenous karrikin molecules induce germination after wildfires [2]. Both activities depend on the F-box protein and SCF (Skp, Cullin, F-box) complex component MORE AXILLARY GROWTH2 (MAX2) [3-5]. Strigolactone and karrikin perception leads to MAX2-dependent degradation of distinct SMXL protein family members, which is key for mediating hormonal effects [6-12]. However, the nature of events immediately downstream of SMXL protein degradation and whether all SMXL proteins mediate strigolactone or karrikin signaling is unknown. In this study we demonstrate that, within the SMXL gene family, specifically SMXL3/4/5 deficiency results in strong defects in phloem formation, alteredsugar accumulation, and seedling lethality. By comparing protein stabilities, we show that SMXL3/4/5 proteins function differently to canonical strigolactone and karrikin signaling mediators, although being functionally interchangeable with those under low strigolactone/karrikin signaling conditions. Our observations reveal a fundamental mechanism of phloem formation and indicate that diversity of SMXL protein functions is essential for a steady fuelling of plant meristems.Peer reviewe

DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW

To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3-7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.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

Cell-to-cell communication via plasmodesmata in vascular plants

In plant development, cell-to-cell signaling is mediated by mobile signals, including transcription factors and small RNA molecules. This communication is essential for growth and patterning. Short-range movement of signals occurs in the extracellular space via the apoplastic pathway or directly from cell-to-cell via the symplastic pathway. Symplastic transport is mediated by plant specific structures called plasmodesmata, which are plasma membrane-lined pores that traverse the cell walls of adjacent cells thus connecting their cytoplasms. However, a thorough understanding of molecules moving via plasmodesmata and regulatory networks relying on symplastic signaling is lacking. Traffic via plasmodesmata is highly regulated, and callose turnover is known to be one mechanism. In Arabidopsis, plasmodesmata apertures can be regulated in a spatially and temporally specific manner with the icals3m, an inducible vector system expressing the mutated CalS3 gene encoding a plasmodesmata localized callose synthase that increases callose deposition at plasmodesmata. We discuss strategies to use the icals3m system for global analyses on symplastic signaling in plants

Specification and regulation of vascular tissue identity in the Arabidopsis embryo

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.Peer reviewe

DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW

Smet et al. capture the transcriptional responses upon simultaneous TMO5/LHW induction and identify DOF2.1 as part of the cytokinin-dependent downstream responses. Furthermore, they show that DOF2.1 and its closest homologs control periclinal and radial procambium divisions in distinct zones of this tissue.</p

Multisite gateway-compatible cell type-specific gene-inducible system for plants

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.</p