Novel Regulators of Vascular Development in Arabidopsis thaliana

Plant vascular tissues are supporting and conductive tissues composed of two major components, xylem and phloem. These tissues transport water, food, hormones and minerals within the plant. In my thesis work, I used the Arabidopsis root as a model system to study vascular tissue formation. The first part of my thesis work is focused on the formation of xylem, the water transporting tissue. In the Arabidopsis root, the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. It has been previously reported that high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. In this work, we provide evidence that auxin biosynthesis promotes HD-ZIP III expression and metaxylem formation. We observed that plants with mutations in auxin biosynthesis genes, such as trp2-12, wei8 tar2, or the quintuple yucca mutant, as well as plants treated with a pharmacological inhibitor of auxin biosynthesis, show reduced expression of the HD-ZIP III genes accompanied by specific defects in metaxylem formation. We were able to induce a partial rescue of the metaxylem defects by introducing an endogenous auxin supply. In addition, some of the patterning defects can be suppressed by synthetically elevating HD-ZIP III expression in the stele of the Arabidopsis root. The second part of my thesis work is focused on phloem tissue formation. Phloem is the tissue responsible for long-distance molecular transport and signaling. The conductive components of the phloem, the sieve elements, rely on specific junctions between the conducting cells in the form of highly perforated sieve areas. We identified mutations in the CHER1 (CHOLINE TRANSPORTER LIKE 1) locus of Arabidopsis which result in altered phloem conductivity, reduced sieve pore density, and defects in sieve pore formation. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We provide data showing that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and is associated with the late stage of phragmoplast formation during cytokinesis. Interestingly, CHER1 has a sustained, polar localization in forming sieve plates, which is consistent with its function in the elaboration of the sieve areas.Kasvien johtojänteet koostuvat pääosin kahdesta solutyypistä, puu- ja nilasoluista, jotka tukevat kasvia ja ovat erilaistuneet veden, ravintoaineiden, kasvihormonien sekä mineraalien kuljettamiseen kasvin eri osien välillä. Väitöstyössäni tutkin johtosolukon muodostumista käyttäen mallina lituruohon juurta. Väitöstyöni ensimmäinen osio pureutuu puusolukon eli ksyleemin, vettä kuljettavan putkiston muodostumiseen. Lituruohon pääjuuressa kahdenlaiset puusolut muodostavat keskuslieriöön poikittaisen akselin jonka kummassakin päädyssä on yksi ns. protoksyleemi solu ja niiden välissä metaksyleemi soluja. Aiempien tutkimusten perusteella on tiedetty että HD-ZIP III transkriptiofaktorin korkea ilmenemistaso edistää metaksyleemin erilaistumista kun taas alhainen taso on kytköksissä protoksyleemiin. Väitöstyöni puitteissa selvitimme että kasvihormoni auksiinin tuotanto edistää HD-ZIP III ilmenemistä ja siten myös metaksyleemin muodostumista. Poikkeavan alhainen auksiinin määrä kasveissa joiden auksiinin tuotantoa oli heikennetty joko geneettisin tai kemiallisin menetelmin alensi HD-ZIP III ilmenemistä, mikä puolestaan johti poikkeavuuksiin metaksyleemin erilaistumisessa. Näitä poikkeavuuksia voitiin osittain korjata nostamalla joko auksiinin määrää tai HD-ZIP III ilmenemistasoa juuren keskuslieriössä. Väitöstyöni toisessa osassa tutkin nilan muodostumista. Nila on erikoistunut useiden molekyylien pitkänmatkan kuljetukseen, mikä perustuu siiviläputkien huokosellisten poikkiseinien eli siivilälevyjen ominaisuuteen yhdistää peräkkäiset siiviläputkisolut toisiinsa pitkäksi solujonoksi. Havaitsimme että lituruoholla siivilälevyjen huokoisuus ja siten siiviläputkien kuljetuskyky poikkeaa normaalista jos CHER1 (CHOLINE TRANSPORTER LIKE 1) geenin toiminta on estynyt. CHER1 proteiini paikallistuu kehittyviin siiviläputkiin polaarisesti ennakoiden siivilälevyn sijaintia, ja on siis siivilälevyjen kehittymisen kannalta tärkeä jo varhaisessa vaiheessa. Lisäksi osoitimme että CHER1 osallistuu koliinin kuljetukseen, paikallistuu solutasolla Golgi verkostoon, ja liittyy tiettyjen rakenteiden muodostumiseen solunjakautumisen yhteydessä

Plant Vascular Biology 2013: vascular trafficking.

About 200 researchers from around the world attended the Third International Conference on Plant Vascular Biology (PVB 2013) held in July 2013 at the Rantapuisto Conference Center, in Helsinki, Finland (http://www.pvb2013.org). The plant vascular system, which connects every organ in the mature plant, continues to attract the interest of researchers representing a wide range of disciplines, including development, physiology, systems biology, and computational biology. At the meeting, participants discussed the latest research advances in vascular development, long- and short-distance vascular transport and long-distance signalling in plant defence, in addition to providing a context for how these studies intersect with each other. The meeting provided an opportunity for researchers working across a broad range of fields to share ideas and to discuss future directions in the expanding field of vascular biology. In this report, the latest advances in understanding the mechanism of vascular trafficking presented at the meeting have been summarized

Tryptophan-dependent auxin biosynthesis is required for HD-ZIP III-mediated xylem patterning.

The development and growth of higher plants is highly dependent on the conduction of water and minerals throughout the plant by xylem vessels. In Arabidopsis roots the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. During vascular development, high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. Protoxylem specification is determined by both mobile, ground tissue-emanating miRNA165/6 species, which downregulate, and auxin concentrated by polar transport, which promotes HD-ZIP III expression. However, the factors promoting high HD-ZIP III expression for metaxylem identity have remained elusive. We show here that auxin biosynthesis promotes HD-ZIP III expression and metaxylem specification. Several auxin biosynthesis genes are expressed in the outer layers surrounding the vascular tissue in Arabidopsis root and downregulation of HD-ZIP III expression accompanied by specific defects in metaxylem development is seen in auxin biosynthesis mutants, such as trp2-12, wei8 tar2 or a quintuple yucca mutant, and in plants treated with L-kynurenine, a pharmacological inhibitor of auxin biosynthesis. Some of the patterning defects can be suppressed by synthetically elevated HD-ZIP III expression. Taken together, our results indicate that polar auxin transport, which was earlier shown to be required for protoxylem formation, is not sufficient to establish a proper xylem axis but that root-based auxin biosynthesis is additionally required.This work was funded by the Academy of Finland, Tekes, the University of Helsinki (Y.H.); Helsinki Graduate Program in Biotechnology and Molecular Biology (R.U.); the European Molecular Biology Organisation [ALTF 450-2007 to J.D.]; and the Japan Society for the Promotion of Science Research Fellowships for Young Scientists (to S.M.)

Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells.

In vascular plants, the root endodermis surrounds the central vasculature as a protective sheath that is analogous to the polarized epithelium in animals, and contains ring-shaped Casparian strips that restrict diffusion. After an initial lag phase, individual endodermal cells suberize in an apparently random fashion to produce 'patchy' suberization that eventually generates a zone of continuous suberin deposition. Casparian strips and suberin lamellae affect paracellular and transcellular transport, respectively. Most angiosperms maintain some isolated cells in an unsuberized state as so-called 'passage cells', which have previously been suggested to enable uptake across an otherwise-impermeable endodermal barrier. Here we demonstrate that these passage cells are late emanations of a meristematic patterning process that reads out the underlying non-radial symmetry of the vasculature. This process is mediated by the non-cell-autonomous repression of cytokinin signalling in the root meristem, and leads to distinct phloem- and xylem-pole-associated endodermal cells. The latter cells can resist abscisic acid-dependent suberization to produce passage cells. Our data further demonstrate that, during meristematic patterning, xylem-pole-associated endodermal cells can dynamically alter passage-cell numbers in response to nutrient status, and that passage cells express transporters and locally affect the expression of transporters in adjacent cortical cells

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization

Publisher Copyright: Copyright © 2021 The Authors, some rights reserved;In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.Peer reviewe

Plant development: how long Is a root?

The plant hormone cytokinin controls root growth by balancing the division and differentiation of stem cells. But what controls accumulation of cytokinin? A new study has identified a regulatory loop between a transcription factor, PHABULOSA, and cytokinin biosynthesis that creates robust domains of cytokinin activity

Plant vascular biology 2013: vascular trafficking

About 200 researchers from around the world attended the Third International Conference on Plant Vascular Biology (PVB 2013) held in July 2013 at the Rantapuisto Conference Center, in Helsinki, Finland (http://www.pvb2013.org). The plant vascular system, which connects every organ in the mature plant, continues to attract the interest of researchers representing a wide range of disciplines, including development, physiology, systems biology, and computational biology. At the meeting, participants discussed the latest research advances in vascular development, long- and short-distance vascular transport and long-distance signalling in plant defence, in addition to providing a context for how these studies intersect with each other. The meeting provided an opportunity for researchers working across a broad range of fields to share ideas and to discuss future directions in the expanding field of vascular biology. In this report, the latest advances in understanding the mechanism of vascular trafficking presented at the meeting have been summarized

An inducible genome editing system for plants

Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type-specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a new tool with which target genes can efficiently and conditionally be knocked out by genome editing at any developmental stage. Target genes can also be knocked out in a cell-type-specific manner. Our tool is easy to construct and will be particularly useful for studying genes having null alleles that are non-viable or show pleiotropic developmental defects.Peer reviewe

CRISPR/Cas-mediated in planta gene targeting: current advances and challenges

Gene targeting can be used to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant gene targeting. Several studies have recently demonstrated improvements in gene targeting efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified gene-targeting-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated gene targeting in plants and discuss potential efficiency improvements. Increasing the efficiency of gene targeting technology will help pave the way for increased crop yields and food safety in environmentally friendly agriculture.This work was financially supported by Ayudas Ramón y Cajal (RYC2021-033414-l) to RU. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 945043 to NPA. This work was also supported by grant CEX2019-000902-S funded by MCIN/AEI/10.13039/501100011033, and by the CERCA Programme/Generalitat de Catalunya to NB and NPA.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000902-S)Peer reviewe

The suberinteresting role of GELP proteins

Trabajo presentado a la XVI Reunión de Biología Molecular de Plantas-Meeting of Plant Molecular Biology (RBMP) celebrada en Sevilla entre el 14 y el 16 de diciembre de 2022.Plants roots take up essential nutrients and block out unwanted compounds from the soil by using a selective barrier in the roots known as the endodermis. Endodermis contains ring-shaped and lignin-based Casparian strip network that acts as a difusion barrier (Barbosa et al., 2019). Later in development, endodermal cells suberize to produce ‘patchy’ suberization that eventually leads to a zone of continuous suberin deposition (Serra and Geldner 2022). The two impermeable polymers, lignin and suberin, afect paracellular and transcellular transport, respectively. Suberin is a lipophilic polyester composed of fatty acids, glycerol and some aromatics. It’s deposited as a hydrophobic layer between the primary cell wall and plasma membrane. Despite suberin being a major plant polymer, fundamental aspects of its biosynthesis and plasticity have remained unclear. Plants shape their root system via lateral root formation, an auxin-induced process requiring local degradation and re-sealing of endodermal suberin. We demonstrated that diferentiated endodermis has a specifc, auxin-mediated transcriptional response, dominated by cell wall remodelling genes. We identifed two sets of auxin-regulated GELP proteins (GDSL-lipases). Using an optimized CRISPR-Cas9 gene editing toolset (Ursache et al., 2021), we discovered that one set of GELPs is required for suberin polymerization, while the other can drive suberin degradation (Ursache et al., 2021). These enzymes constitute novel core players of suberisation, driving root suberin plasticity during plant growth and in response to the environmental stress.EMBO long term fellowship (EMBO ALTF 1046-2015), University of Lausanne (UNIL) and Centre for Research in Agricultural Genomics (CRAG)