Auxin coordinates cell division and cell fate specification during lateral root initiation

Met behulp van hun wortels zijn planten in staat zich te verankeren in de bodem, waar ze ook dienen voor de opname van water en nutriënten en verankering in de bodem. Het volume bodem dat door de wortels efficiënt gebruikt wordt, hangt in sterke mate af van de complexiteit van het wortelsysteem. De complexiteit van wortelarchitectuur kan verhoogd worden door de vorming van vertakkingen, zijwortels. Naast het fundamentele biologisch belang van zijwortelvorming voor planten, kan dit ontwikkelingsproces ook dienen als een uitstekend model om cruciale vragen te beantwoorden zoals, welke positionele signalen liggen aan de basis van patroonvorming, groei-as-bepaling en de herspecificatie van celidentiteit en –polariteit. Daarom is het belangrijk om de moleculaire mechanismen van zijwortelvorming te bestuderen. Endogene en omgevingsfactoren controleren samen de vorming van een nieuwe zijwortel. In Arabidopsis thaliana, ontstaan zijwortels in een specifiek weefsel binnenin de wortel, de pericyclus. De eerste gebeurtenissen die geobserveerd kunnen worden tijdens zijwortelvorming zijn de gecoördineerde asymmetrische celdelingen in twee aangrenzende xyleempool-geassocieerde pericycluscellen (Beeckman et al., 2001). De signaal molecule in planten, auxine, is één van de belangrijkste regulatoren van zijwortelontwikkeling en is betrokken in alle stappen van dit ontwikkelingsproces (Blakely et al., 1988). Desalniettemin is er weinig geweten over de moleculaire mechanismen waarmee auxine zijwortelvorming beïnvloedt (Hoofdstuk 1 & 2). Mijn doctoraatsonderzoek was gericht op de identificatie en karakterisering van nieuwe moleculaire regulatoren van zijwortelontwikkeling. Eén van de grootste uitdagingen was de analyse van transcriptionele veranderingen tijdens de vroege stappen van auxine-geïnduceerde zijwortelvorming. Om dit gericht te kunnen doen hebben we een systeem ontwikkeld om dit proces te manipuleren. Met behulp van het zijwortelinduceerbaar systeem (ZWIS) zijn we in staat zijwortelinitiatie gesynchroniseerd te induceren over gans de lengte van de wortel (Himanen et al., 2002). Via transcriptionele analyse op ongeveer 4600 genen konden we aantonen dat binnen het ZWIS, signaaltransductie en celcyclusactivatie van elkaar konden gescheiden worden in de tijd (Himanen et al., 2004). Vervolgens gebruikten we het ZWIS in een bijna genoom-wijde analyse van transcriptionele veranderingen die optreden in wortels van wild type en een auxine-ongevoelige mutant, solitary-root-1 (slr-1), die geen zijwortels kan maken (Hoofdstuk 3; Vanneste et al., 2005). Onze data toonde aan dat auxine-afhankelijke celcyclusactivatie sterk verstoord was in slr-1. Om te testen of celdelingsstimulatie in de pericyclus voldoende zou zijn om het zijworteldefect in slr-1 te complementeren, hebben we de G1-S regulatoren, CYCD3;1 en E2Fa/DPa, tot overexpressie gebracht in deze mutant. In plaats van het induceren van zijwortels, kon enkel proliferatieve celdeling waargenomen worden in de pericyclus. Dit suggereerde dat celdeling niet voldoende is voor auxine-geïnduceerde zijwortelvorming (Vanneste et al., 2005). Om de rol van celcyclusregulatoren in zijwortelontwikkeling verder te onderzoeken analyseerden we de functie van de CYCA2 genfamilie via mutanten. In specifieke drievoudige mutanten vonden we een sterke reductie in zijworteldensiteit. Daarenboven vonden we dat zijwortels van de mutanten opgebouwd waren uit minder cellen. Desondanks het cellulaire defect, waren alle celtypes van een functioneel zijwortelmeristeem aanwezig. Dit suggereert dat celidentiteit bepaald wordt, onafhankelijk van celdelingsvoortgang. In de epidermis van CYCA2 mutanten vonden we unicellulaire structuren die sluitcelkarakteristieken vertoonden. Dit is in overeenstemming met de zijworteldefecten in de mutanten aangezien ook hier celidentiteitsbepaling en celdelingsvoortgang onafhankelijk gebeurden (Hoofdstuk 6). In de literatuur werden mutanten en behandelingen beschreven waarin celidentiteitsbepaling in ontwikkelende zijwortels verstoord was (Benková et al., 2003; Geldner et al., 2004). Beide rapporten toonden aan dat transport- afhankelijke lokale auxine-accumulatie essentieel is voor celidenteiteitsbepaling tijdens zijwortelvorming. Regulatoren van polair auxine-transport van de PIN-familie hebben specifieke en overlappende expressie patronen tijdens zijwortelvorming (Benková et al., 2003). Onze bijna-genoom-wijde analyse van transcriptionele veranderingen tijdens zijwortelinitiatie leverde ook in belangrijke mate bij tot nieuwe inzichten in dit mechanisme. We vonden dat PIN-genen bij de vroegst auxine-geïnduceerde genen horen. Daarenboven vonden we dat hun transcriptionele opregulatie afhankelijk was van de klassieke auxinesignaalcascade. In deze signaaltransductieweg beïnvloedt auxine transcriptie door de destabilisering van labiele repressoreiwitten (Hoofdstuk 4; Vieten et al., 2005). Deze bevinding bleek uiteindelijk een essentiële component te zijn voor computermodellen van auxinetransport-afhankelijke ontwikkelingsprocessen (Jonsson et al., 2006). Daarenboven vonden we dat ook het AUX1 homoloog LAX3 auxine-induceerbaar was. Onlangs werd door Swarup et al. (2008) aangetoond dat LAX3 betrokken is in zijworteluitgroei. LAX3 is essentieel voor de opname van auxine in de endodermis- en cortexcellen in de nabijheid van zijwortelprimordia, waar het vervolgens transcriptie van celwandmodificerende enzymen stimuleert, zodat de zijwortel kan uitgroeien. Bovenop de celcyclusregulatoren en auxinetransport-componenten hebben we verschillende transcriptiefactoren geïdentificeerd die potentieel een belangrijke rol spelen in auxinesignalisatie. Zo vonden we dat de stomatale differentiatie transcriptiefactor, FOUR LIPS/MYB124 (Lai et al., 2005), naast de eigenschap van auxine-induceerbaar te zijn, eveneens tot expressie komt in alle stadia van zijwortelvorming. De mutant vertoonde zijworteldefecten die deden denken aan defecten in polair auxinetransport. We konden inderdaad aantonen dat FLP gerecruteerd wordt naar het chromatine in de PIN3-promotor, waar het PIN3-transcriptie stimuleert. Deze bevinding verhoogt ons inzicht in de mechanismen van auxine-gestuurde zijwortelvorming (Hoofdstuk 5). Tot besluit kunnen we stellen dat onze gedetaileerde analyse van vroege auxine-geïnduceerde transcripten in wortels geleid heeft tot verscheidene belangrijke nieuwe inzichten in zijwortelinitiatie. Het zal dus interessant zijn om in de verkregen dataset te zoeken naar belangrijke regulatoren van zijwortelontwikkeling. Beeckman, T., Burssens, S. and Inzé, D. (2001). The peri-cell-cycle in Arabidopsis. J. Exp. Bot. 52: 403-11. Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G. and Friml, J. (2003). Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: 591-602. Blakely, L. M., Blakely, R. M., Colowit, P. M. and Elliott, D. S. (1988). Experimental studies on lateral root formation in radish seedling roots II. Analysis of the dose-response to exogenous auxin. Plant Physiol. 87: 414-419. Geldner, N., Richter, S., Vieten, A., Marquardt, S., Torres-Ruiz, R. A., Mayer, U. and Jürgens, G. (2004). Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis. Development 131: 389-400. Himanen, K., Boucheron, E., Vanneste, S., de Almeida Engler, J., Inzé, D. and Beeckman, T. (2002). Auxin-mediated cell cycle activation during early lateral root initiation. Plant Cell 14: 2339-51. Himanen, K., Vuylsteke, M., Vanneste, S., Vercruysse, S., Boucheron, E., Alard, P., Chriqui, D., Van Montagu, M., Inzé, D. and Beeckman, T. (2004). Transcript profiling of early lateral root initiation. Proc. Natl. Acad. Sci. USA 101: 5146-51. Jonsson, H., Heisler, M. G., Shapiro, B. E., Meyerowitz, E. M. and Mjolsness, E. (2006). An auxin-driven polarized transport model for phyllotaxis. Proc. Natl. Acad. Sci. USA 103: 1633-8. Lai, L. B., Nadeau, J. A., Lucas, J., Lee, E. K., Nakagawa, T., Zhao, L., Geisler, M. and Sack, F. D. (2005). The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage. Plant Cell 17: 2754-67. Swarup, K., Benková, E., Swarup, R., Casimiro, I., Péret, B., Yang, Y., Parry, G., Nielsen, E., De Smet, I., Vanneste, S., Levesque, M.P., Carrier, D., James, N., Calvo, V. Ljung, K., Kramer, E.M., Roberts, R., Graham, N., Marillonnet, S., Patel, K., Jones, J.D.G., Taylor, C.G., Schachtman, D.P., May, S.T., Sandberg, G., Benfey, P.N., Friml, J., Kerr, I.D., Beeckman, T., Laplaze, L. & Bennett, M.J. (2008) The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. (11 Jul 2008), doi: 10.1038/ncb1754 Vanneste, S., De Rybel, B., Beemster, G. T., Ljung, K., De Smet, I., Van Isterdael, G., Naudts, M., Iida, R., Gruissem, W., Tasaka, M., Inzé, D., Fukaki, H. and Beeckman, T. (2005). Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana. Plant Cell 17: 3035-50

Pericyclic versus endodermal lateral roots : which came first?

Digging into the limited literature on lateral root (LR) formation in early vascular plants, we came to the novel conclusion that the pericycle, rather than the endodermis as commonly assumed, represents the ancestral tissue that was evolutionarily recruited to form LRs

Review : membrane tethers control plasmodesmal function and formation

Cell-to-cell communication is crucial in coordinating diverse biological processes in multicellular organisms. In plants, communication between adjacent cells occurs via nanotubular passages called plasmodesmata (PD). The PD passage is composed of an appressed endoplasmic reticulum (ER) internally, and plasma membrane (PM) externally, that traverses the cell wall, and associates with the actin-cytoskeleton. The coordination of the ER, PM and cytoskeleton plays a potential role in maintaining the architecture and conductivity of PD. Many data suggest that PD-associated proteins can serve as tethers that connect these structures in a functional PD, to regulate cell-to-cell communication. In this review, we summarize the organization and regulation of PD activity via tethering proteins, and discuss the importance of PD-mediated cell-to-cell communication in plant development and defense against environmental stress

Cellular mechanisms for cargo delivery and polarity maintenance at different polar domains in plant cells

The asymmetric localization of proteins in the plasma membrane domains of eukaryotic cells is a fundamental manifestation of cell polarity that is central to multicellular organization and developmental patterning. In plants, the mechanisms underlying the polar localization of cargo proteins are still largely unknown and appear to be fundamentally distinct from those operating in mammals. Here, we present a systematic, quantitative comparative analysis of the polar delivery and subcellular localization of proteins that characterize distinct polar plasma membrane domains in plant cells. The combination of microscopic analyses and computational modeling revealed a mechanistic framework common to diverse polar cargos and underlying the establishment and maintenance of apical, basal, and lateral polar domains in plant cells. This mechanism depends on the polar secretion, constitutive endocytic recycling, and restricted lateral diffusion of cargos within the plasma membrane. Moreover, our observations suggest that polar cargo distribution involves the individual protein potential to form clusters within the plasma membrane and interact with the extracellular matrix. Our observations provide insights into the shared cellular mechanisms of polar cargo delivery and polarity maintenance in plant cells

Pharmacological strategies for manipulating plant Ca2+ signalling

Calcium is one of the most pleiotropic second messengers in all living organisms. However, signalling specificity is encoded via spatio-temporally regulated signatures that act with surgical precision to elicit highly specific cellular responses. How this is brought about remains a big challenge in the plant field, in part due to a lack of specific tools to manipulate/interrogate the plant Ca2+ toolkit. In many cases, researchers resort to tools that were optimized in animal cells. However, the obviously large evolutionary distance between plants and animals implies that there is a good chance observed effects may not be specific to the intended plant target. Here, we provide an overview of pharmacological strategies that are commonly used to activate or inhibit plant Ca2+ signalling. We focus on highlighting modes of action where possible, and warn for potential pitfalls. Together, this review aims at guiding plant researchers through the Ca2+ pharmacology swamp

The ins and outs of Ca2+ in plant endomembrane trafficking

Trafficking of proteins and lipids within the plant endomembrane system is essential to support cellular functions and is subject to rigorous regulation. Despite this seemingly strict regulation, endomembrane trafficking needs to be dynamically adjusted to ever-changing internal and environmental stimuli, while maintaining cellular integrity. Although often overlooked, the versatile second messenger Ca2+ is intimately connected to several endomembrane-associated processes. Here, we discuss the impact of electrostatic interactions between Ca2+ and anionic phospholipids on endomembrane trafficking, and illustrate the direct role of Ca2+ sensing proteins in regulating endomembrane trafficking and membrane integrity preservation. Moreover, we discuss how Ca2+ can control protein sorting within the plant endomembrane system. We thus highlight Ca2+ signaling as a versatile mechanism by which numerous signals are integrated into plant endomembrane trafficking dynamics

A conserved but plant-specific CDK-mediated regulation of DNA replication protein A2 in the precise control of stomatal terminal division

The R2R3-MYB transcription factor FOUR LIPS (FLP) controls the stomatal terminal division through transcriptional repression of the cell cycle genes CYCLIN-DEPENDENT KINASE (CDK) B1s (CDKB1s), CDKA; 1, and CYCLIN A2s (CYCA2s). We mutagenized the weak mutant allele flp-1 seeds with ethylmethane sulfonate and screened out a flp-1 suppressor 1 (fsp1) that suppressed the flp-1 stomatal cluster phenotype. FSP1 encodes RPA2a subunit of Replication Protein A (RPA) complexes that play important roles in DNA replication, recombination, and repair. Here, we show that FSP1/RPA2a functions together with CDKB1s and CYCA2s in restricting stomatal precursor proliferation, ensuring the stomatal terminal division and maintaining a normal guard-cell size and DNA content. Furthermore, we provide direct evidence for the existence of an evolutionarily conserved, but plant-specific, CDK-mediated RPA regulatory pathway. Serine-11 and Serine-21 at the N terminus of RPA2a are CDK phosphorylation target residues. The expression of the phosphorylation-mimic variant RPA2a(S11,21/D) partially complemented the defective cell division and DNA damage hypersensitivity in cdkb1;1 1;2 mutants. Thus, our study provides a mechanistic understanding of the CDK-mediated phosphorylation of RPA in the precise control of cell cycle and DNA repair in plants

Enquiry into the topology of plasma membrane-localized PIN auxin transport components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five alpha-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants

The Arabidopsis Synaptotagmin1 is enriched in endoplasmic reticulum-plasma membrane contact sites and confers cellular resistance to mechanical stresses

Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca2+ influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses

Plasma membrane lipid remodeling during cold acclimation is mediated by the ER-PM contact sites-localized synaptotagmins 1 and 3

Cold acclimation is the capacity of certain plants to increase their freezing tolerance in response to a period of low non-freezing temperatures. Cold acclimation involves a series of biochemical and physiological adaptations, including a deep transcriptional reprogramming and drastic changes in the lipid composition of cellular membranes in order to prevent the freeze-induced damage (1). While a profound knowledge has been acquired on the regulation of gene expression triggered by cold-acclimation, very little is known about the mechanisms governing the cold-induced changes in membranes’ lipid composition. In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1 (SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for cold- acclimated freezing tolerance and for the lipid remodelling of the plasma membrane during cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1 expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata, and old primary root. TIRF microscopy analyses show that during cold acclimation there is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM. High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3 are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation, and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1 and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold acclimation, which in turn leads to freezing tolerance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech