Spatiotemporal mechanisms of root branching

The fundamental tasks of the root system are, besides anchoring, mediating interactions between plant and soil and providing the plant with water and nutrients. The architecture of the root system is controlled by endogenous mechanisms that constantly integrate environmental signals, such as availability of nutrients and water. Extremely important for efficient soil exploitation and survival under less favorable conditions is the developmental flexibility of the root system that is largely determined by its postembryonic branching capacity. Modulation of initiation and outgrowth of lateral roots provides roots with an exceptional plasticity, allows optimal adjustment to underground heterogeneity, and enables effective soil exploitation and use of resources. Here we discuss recent advances in understanding the molecular mechanisms that shape the plant root system and integrate external cues to adapt to the changing environment

A Rho GTPazok foszforilációjának szerepe a kórokozókkal szembeni védekezésben és a növényi egyedfejlődésben = The role and significance of Rho GTPase phosphorylation during pathogenesis and normal plant growth

A Rop GTPázok a sejten belüli jelátviteli hálózatok molekuláris kapcsolói. Alapvető szerepük van számos sejtfolyamat szabályozásában, de a kapcsolódó jelátviteli mechanizmusok növényekben még alig ismertek. Kutatásaink során azonosítottunk lucernában olyan kináz fehérjéket (RRK), amelyeket a Rop GTPázok specifikusan aktiválnak. Pályázatunk ezek jellemzéséhez kapcsolódott. Kimutattuk, hogy a kináz nem foszforilálja a Rop GTPázt. Ennek ellenére megvizsgáltuk a Rop GTPáz potenciális foszforilációjának az esetleges szerepét a GTP-kötésre, GTPáz aktivitásra és fehérje-fehérje kölcsönhatásokra. Úgy találtuk, hogy az adott mutáció befolyásolja a fenti jellemzőket. Az RRK kinázokat indukálható promóterrel kifejező, illetve a kináz fehérjéket nem termelő transzgenikus növényeket állítottunk elő, melyek vizsgálata azonban még nem fejeződött be. A német partnertől segítséget kaptunk fehérjék tömegspektroszkópia vizsgálatában, a bimolekuláris fluoreszcencia komplementáció technika honosításában, a GTP-kötés fluoreszcencia energia transzferen alapuló vizsgálatában illetve kutatási anyagok cseréjén keresztül. Az együttműködés hozadéka egy közösen beadott európai pályázat, amelynek a második változata még elbírálás alatt áll. | Rop GTPases are molecular switches involved in many basic cellular processes in plants. Despite their central role in cellular signaling, our knowledge on their signaling partners is scarce. We identified for the first time plant kinases (RLCK VI_A) directly activated upon binding to Rop GTPases. Our research activity was linked to the characterization of this signaling interaction. It was investigated whether the kinases can phosphorylate the Rop GTPase. Although it could not be established, the potential phosphorylation of the GTPases on a specific residue was further investigated using a phosphomimic GTPase mutant. This mutation had a negative effect on GTP-binding and hydrolysis, and differentially affected protein-protein interactions. Furthermore, transgenic plants have been created that overexpress some of the RLCK VI_A kinases under the control of an inducible promoter, or, alternatively, do not expressthe kinase proteins as a result of RNA-interference. However, the characterization of these plants could not be accomplished yet. Collaboration with the German partner was successful. In addition to the exchange of research material, we had access to and learned technologies like MALDI-TOF analysis of protein phosphorylation, bimolecular fluorescence complementation, GTP-binding assay based on fluorescent energy transfer. The collaboration resulted in a common application for a European training grant, the second version of which is currently under evaluation

Rho-típusú GTPázokat szabályozó receptor-szerű citoplazmatikus kinázok: egy új jelátviteli kapcsolat növényekben = Receptor-like cytoplasmic kinases phosphorylating Rho GTPases: a new signal transduction link in plants

A Rop GTPázok a sejten belüli jelátviteli hálózatok molekuláris kapcsolói. Alapvető szerepük számos sejtfolyamat szabályozásában, de a kapcsolódó jelátviteli mechanizmusok növényekben még alig ismertek. Kutatásaink során elsőként azonosítottunk lucernában olyan kináz fehérjéket, amelyeket a Rop GTPázok specifikusan aktiválnak. Ezek a Rop-aktivált kinázok (RLCK VI_A) és a kinázok aktiválási mechanizmusa teljesen más, mint amit élesztő vagy állati sejtekben eddig leírtak. Vizsgáltuk a kölcsönhatás specificitását és meghatároztunk olyan fehérje régiókat amelyek a kölcsönhatásban aktiválásban részt vesznek. Megvizsgáltuk az Arabidopsis RLCK VI_A kinázok génexpressziós profilját, hogy betekintést kapjunk biológiai funkcióikba. Továbbá transzgenikus növényeket hoztunk létre, amelyekben ezeket a funkciókat potntosan meghatározhassuk. Elsődleges eredményeink alapján a kinázok között vannak olyanok, amelyek a sejtek polaritását szabályozzák, amiben a Rop GTPázok szerepe is ismert. Felismertük, hogy a Rop-aktivált kinázok kölcsönhatnak a Rop GTPázokat aktiváló RopGEF fehérje faktorokkal. Így egy potenciális molekuláris szabályozó kört sikerült feltárni. Megvizsgáltuk, hogy a Rop GTPázok maguk lehetnek-e foszforilációs szabályozás alanyai. Foszforilációt mimikáló mutációkkal igazoltuk ennek a lehetőségét és potenciális biológiai jelentőségét. Ugyanakkor igazoltuk, hogy nem az RLCK VI_A kinázok azok, amelyek a Rop GTPázokat foszforilálhatják. | Rop GTPases are molecular switches involved in many basic cellular processes in plants. Despite their central role in cellular signaling, our knowledge on their signaling partners is stills scarce. We identified for the first time plant kinases (RLCK VI_A) directly activated upon binding to Rop GTPases. These kinases are different from the Rho GTPase-activated kinases of other eukariotes and their activation mechanism seems to be also unique. We investigated the specificity of the GTPase-to-kinase interaction as well as determined some regions/motifs involved in the interaction and kinase activation. Furthermore, we determined the gene expression profile of all Arabidopsis RLCK VI_A kinases in order to have an insight into their biological function. For the same aim we produced several transgenic Arabidopsis lines. Preliminary investigations indicate that at least some of these kinases has a role in polar cell growth also regulated by Rop GTPases themselves. We realized that the Rop-activated kinases also interact with the Rop-activating RopGEF protein factors. In this way we could lay down the basis of a potential regulatory protein circuit. We also investigated the potential role for Rop GTPase activity regulation by phosphorylation. A phosphomimic Rop GTPase mutation was created and its negative effect on GTP hydrolisis as well as its differential effects on protein-protein interactions was established. It could have been revealed, however, that they are not phosphorylated by the RLCK VI_A kinases in vitro

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro

AbstractPlants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta

Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture

Nitrate commands genome-wide gene expression changes that impact metabolism, physiology, plant growth, and development. In an effort to identify new components involved in nitrate responses in plants, we analyze the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. 176 phosphoproteins show significant changes at 5 or 20 min after nitrate treatments. Proteins identified by 5 min include signaling components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with transporter activity or hormone metabolism functions, among others. The phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants was significantly altered as compared to wild-type plants, confirming its key role in nitrate signaling pathways that involves phosphorylation changes. Integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We validated a new phosphorylation site in PIN2 and provide evidence that it functions in primary and lateral root growth responses to nitrate

Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport

Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate‐dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments

Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage

Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine‐tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re‐organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin‐sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells