Mechanisms of stomatal development: an evolutionary view

Plant development has a significant postembryonic phase that is guided heavily by interactions between the plant and the outside environment. This interplay is particularly evident in the development, pattern and function of stomata, epidermal pores on the aerial surfaces of land plants. Stomata have been found in fossils dating from more than 400 million years ago. Strikingly, the morphology of the individual stomatal complex is largely unchanged, but the sizes, numbers and arrangements of stomata and their surrounding cells have diversified tremendously. In many plants, stomata arise from specialized and transient stem-cell like compartments on the leaf. Studies in the flowering plant Arabidopsis thaliana have established a basic molecular framework for the acquisition of cell fate and generation of cell polarity in these compartments, as well as describing some of the key signals and receptors required to produce stomata in organized patterns and in environmentally optimized numbers. Here we present parallel analyses of stomatal developmental pathways at morphological and molecular levels and describe the innovations made by particular clades of plants

Symplastically transmitted signals regulate pattern formation during root development in Arabidopsis thaliana

The exchange of positional information largely determines the fate of plant cells during organ development. Plasmodesmata (PD) are membrane-lined channels that connect the plant cells and allow symplastic cell-to-cell movement of developmental regulators and other molecules. Currently, little is known about the role of symplastic signaling in plant morphogenesis. In this study, the Arabidopsis root was used to investigate molecular control of the pattern formation and to elucidate how symplastic signaling regulates root development. We identified four novel mutants, phb-7d, cals3-1d, -2d, and -3d (collectively referred as cals3-d) that display defective protoxylem development. Characterization of these mutants revealed that the protoxylem defects are a result of an altered regulatory pathway involving the transcription factor SHORT-ROOT (SHR). It has been shown that SHR moves from the stele into the endodermis and the quiescent centre (QC) where it regulates cell division of the endodermis/cortex initials and maintains the stem cell identity. We identified phb-7d as a novel gain-of-function allele of PHABULOSA (PHB), which is normally post-transcriptionally regulated by microRNA165/6 (miR165/6). phb-7d carries a point mutation which renders it insensitive to regulation through miR165/6 and results in a loss of protoxylem identity. We found that SHR is required in the endodermis to turn on the expression of MIR165/6, which in turn is essential for the formation of the proper concentration gradient of PHB in the stele. We demonstrated that bidirectional non-cell-autonomous actions of SHR and miR165/6 are essential for the regulation of PHB, and therefore, for the regulation of protoxylem development. The movement of SHR from the stele is impeded in cals3-d. We defined that the cals3-d mutations are in the CALLOSE SYNTHASE 3 (CALS3)/GLUCAN SYNTHASE-LIKE 12 (GSL12) gene, whose product participates in the biosynthesis of callose, β-1,3-glucan. The cals3-1d, -2d and -3d lead to amino acid changes in the predicted cytosolic chains of CALS3 resulting in an increased callose biosynthesis. In order to characterize the role of the cals3-d in cell-to-cell communication, we engineered a spatiotemporally controlled miss-expression system for cals3-d. Using this system, we demonstrated that the aperture of a PD is controlled by the callose biosynthesis at the surrounding cell wall domain. Moreover, we identified symplastically transmitted cell signaling events. Our study reveals that SHR and miR165 traffic via the PD. In addition, we show that the phloem-borne symplastically transported signals are essential for the root development. In conclusion, this work demonstrates the fundamental importance of symplastic connectivity for the regulation of root morphogenesis.Jokaisessa monisoluisessa eliössä soluista toiseen kulkee sekä tietoa, että solujen rakennusmateriaalia. Viestintä on kasvisoluille erityisen tärkeää, sillä ne erilaistuvat sijaintinsa perusteella; solun ympärillä olevat solut välittävät solulle tietoa siitä, millaiseksi sen tulee kehittyä. Nämä viesti voivat kulkea lyhyitä tai pitkiä matkoja. Viestit voivat kulkea solusta toiseen soluseinissä olevien aukkojen, plasmodesmien, kautta. Johtosolukot ovat pitkänmatkan kuljetukseen erikoistuneita solukoita, joiden avulla jopa useiden metrien päässä toisistaan sijaitsevat kasvisolut ovat yhteyksissä toisiinsa. Tutkin väitöskirjassani solujen välistä viestintää lituruohossa. Löysimme sarjan mutanttikasveja, joiden johtosolukoiden kehitys on puutteellinen; tietyt johtosolukon solut kehittyvät väärään kohtaan ja toiset solutyypit jäävät kehittymättä kokonaan. Ensimmäinen mutantti kantaa perimässään mutaatiota, joka estää sen koodaaman PHB mRNA:n rajoitusmekanismin toiminnan. Tämän seurauksena PHB mRNA leviää liian laajalle alueelle juuressa, mikä aiheuttaa johtosolukon solujen virheellisen erilaistumisen. Tämä osoittaa, että PHB mRNA:n oikea sijainti on tärkeää johtosolukon normaalille kehitykselle. Selvitimme, että sen sijainti määräytyy johtosolukon ja sitä ympäröivän perussolukon välisen kaksisuuntaisen viestinnän ohjaamana. Havaitsimme, että loput mutanteista kantavat perimässään muutoksia CALS3 geenissä, jonka koodaama proteiini tuottaa kalloosia, suurta sokeripolymeeria. Havaitsimme, että sekä proteiini, että sen tuottama kalloosi sijaitsevat plasmodesmeissa. Mutaatioiden seurauksena kalloosin tuotanto lisääntyy ja solujen välinen viestintä vähenee. Kehitimme muuttuneen proteiinin avulla kasvilinjoja, joissa kalloosia voidaan tuottaa tietyssä solukossa tiettynä ajankohtana altistamalla kasvit kemikaalille. Näissä kasvilinjoissa solujen välistä viestintää voidaan siis säädellä kohdennetusti. Tämän työkalun avulla tunnistimme, että kaksisuuntainen viestintä, joka säätelee juuren johtosolukon kehitystä, kulkee plasmodesmien läpi. Lisäksi tunnistimme muita solujen välisiä viestejä, jotka kulkevat plasmodesmien läpi sekä toisaalta viestejä, jotka kulkevat toisia viestireittejä pitkin. Tiettyjen solukoiden välisen viestinnän estäminen aiheutti kasvin ilmiasun muuttumisen. Tämä osoittaa, että näiden solukoiden välillä kulkee kehitystä sääteleviä viestejä, jotka ovat toistaiseksi tuntemattomia. Kehitetyllä työkalulla on mahdollista kartoittaa mitkä kaikista solujen välisistä viesteistä kulkevat plasmodesmeja pitkin. Tällä työkalulla on myös mahdollista muokata kasvien kasvua kohdistetusti, esimerkiksi muuttaa kasvin rakennetta, kukkimisen ajankohtaa tai taudinkestävyyttä

Modulation of Asymmetric Division Diversity through Cytokinin and SPEECHLESS Regulatory Interactions in the Arabidopsis Stomatal Lineage

Coordinated growth of organs requires communication among cells within and between tissues. In plants, leaf growth is largely dictated by the epidermis; here, asymmetric and self-renewing divisions of the stomatal lineage create two essential cell types—pavement cells and guard cells—in proportions reflecting inputs from local, systemic, and environmental cues. The transcription factor SPEECHLESS (SPCH) is the prime regulator of divisions, but whether and how it is influenced by external cues to provide flexible development is enigmatic. Here, we show that the phytohormone cytokinin (CK) can act as an endogenous signal to affect the extent and types of stomatal lineage divisions and forms a regulatory circuit with SPCH. Local domains of low CK signaling are created by SPCH-dependent cell-type-specific activity of two repressive type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR16 and ARR17, and two secreted peptides, CLE9 and CLE10, which, together with SPCH, can customize epidermal cell-type composition

Modulation of Asymmetric Division Diversity through Cytokinin and SPEECHLESS Regulatory Interactions in the Arabidopsis Stomatal Lineage

Coordinated growth of organs requires communication among cells within and between tissues. In plants, leaf growth is largely dictated by the epidermis; here, asymmetric and self-renewing divisions of the stomatal lineage create two essential cell types—pavement cells and guard cells—in proportions reflecting inputs from local, systemic, and environmental cues. The transcription factor SPEECHLESS (SPCH) is the prime regulator of divisions, but whether and how it is influenced by external cues to provide flexible development is enigmatic. Here, we show that the phytohormone cytokinin (CK) can act as an endogenous signal to affect the extent and types of stomatal lineage divisions and forms a regulatory circuit with SPCH. Local domains of low CK signaling are created by SPCH-dependent cell-type-specific activity of two repressive type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR16 and ARR17, and two secreted peptides, CLE9 and CLE10, which, together with SPCH, can customize epidermal cell-type composition

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.)

Varhaiskasvatuksen opettajien kokemuksia pedagogisesta dokumentoinnista yhteistyössä huoltajien kanssa

Tämän opinnäytetyön tarkoituksena on tarkastella varhaiskasvatuksen opettajien kokemuksia pedagogisesta dokumentoinnista: Millaisia pedagogisen dokumentoinnin tapoja he kertovat käyttävänsä suunnitelluissa tapaamisissa tai muussa sovitussa toiminnassa tapahtuvassa yhteistyössä huoltajien kanssa, sekä mikä näitä valintoja heidän kertomansa mukaan ohjaa. Opinnäytetyössä tarkastellaan myös, millaiset pedagogisen dokumentoinnin tavat toimivat varhaiskasvatuksen opettajien kertoman mukaan parhaiten, kun tavoitteena on huoltajien osallisuuden lisääminen. Tavoitteena tällä opinnäytetyöllä on tuottaa ajankohtaista tietoa pedagogisesta dokumentoinnista pääkaupunkiseudulla. Opinnäytetyö on toteutettu osana Metropolia Ammattikorkeakoulun hanketta Pedagoginen dokumentointi osana oman työn arviointia ja osallisuuden tukemista. Opinnäytetyön tutkimuksellinen osio toteutettiin laadullisena tutkimuksena, jonka aineisto kerättiin haastattelemalla varhaiskasvatuksen opettajia kuudessa varhaiskasvatusyksikössä pääkaupunkiseudulla. Haastateltujen varhaiskasvatuksen opettajien käsitys pedagogisesta dokumentoinnista ja sen tarjoamista mahdollisuuksista huoltajien kanssa oli monipuolinen. Monet vastaajista kokivat valokuvien ja videoiden tuottamisen yhtenä mielekkäimpänä dokumentointitapana, jota käytettiin useimmin myös huoltajien osallisuutta lisäämään pyrkivässä toiminnassa osana viestintää ja muuta vuorovaikutusta. Myös varhaiskasvatussuunnitelmakeskustelut sekä vanhempainillat nähtiin luontevina tilaisuuksina pedagogiselle dokumentoinnille, ja huoltajia kannustettiin osallistumaan. Varhaiskasvatussuunnitelman perusteiden määritelmään pedagogisesta dokumentoinnista oli perehdytty tarkasti, ja varhaiskasvatuksen opettajat olivat osallistuneet myös pedagogista dokumentointia koskevaan kehittämistyöhön esimerkiksi Pedagoginen dokumentointi -hankkeen seminaariin osallistuen tai aiheeseen omassa varhaiskasvatusyksikössä paneutuen. Haasteena koettiin henkilöstön määrään ja kiireeseen liittyvä resurssien vähyys sekä toisinaan myös asianmukaisten tai toimivien dokumentointivälineiden puuttuminen. Myös pedagogisen dokumentoinnin käsite herätti yhä osalle vastaajista kysymyksiä tai erimielisyyksiä. Haastatteluaineistomme perusteella varhaiskasvatuksen opettajien valmiuksia pedagogisen dokumentoinnin prosessiin yhteistyössä huoltajien kanssa tulisi vielä vahvistaa lisäämällä esimerkiksi tietoa huoltajien osallistumisen mahdollisuuksista ja tätä tukevista erilaisista pedagogisen dokumentoinnin tavoista.This Bachelor’s thesis studies, how the early childhood education teachers in the capital region are experiencing the pedagogical documentation. The thesis concentrates on the planned meetings or other agreed cooperation between the early childhood education teachers and the guardians. The thesis studies what kind of pedagogical documentation methods the early childhood education teachers are using according to them, what in their opinion motivates the selection of the methods, and which methods they see as the best methods when the aim is to increase the level of the participation of the guardians. The aim of this Bachelor’s thesis was to produce current information on pedagogical documentation in the capital region and it was done in cooperation with the Pedagogical Documentation project run by Metropolia University of Applied Sciences. The thesis was conducted a qualitative research. The data was collected by interviewing early childhood education teachers in six early childhood education centers in the capital region. The data indicated that early childhood education teachers had a diverse understanding of the pedagogical documentation and of the possibilities it offers for the cooperation with the guardians. Many of the early childhood education teachers felt that photos and videos were among the most pleasant ways to make documentation. Photos and videos were also one the most used documentation methods in communication and other interaction when the aim was to increase the participation of the guardians. The early childhood education discussions and the parents' evenings were seen as a natural situation for pedagogical documentation, and the guardians were encouraged to participate widely. The early childhood education teachers were familiar with the description of the pedagogical documentation presented in the National Core Curriculum for Early Childhood Education and Care, and they had also participated in development work as part of the Pedagogical Documentation project or in their own workplace. Limited resources and rush were seen as a challenge, and also the technical equipment were in some cases lacking or poor. The concept of pedagogical documentation still raised some questions or disagreements among the staff. On the basis of the interviews, the data indicate that early childhood education teachers would benefit from streghtening their skills in performing pedagogical documentation in cooperation with the guardians by providing them information of the possibilities and the different methods available for increasing the level of participation of the guardians

Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate

A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner

Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate

A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner

Vers une danse « citoyenne » ?

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes