Records 1 - 20 / 299
Functional analyses of plant-specific histone deacetylases : Their role in root development, stress responses and symbiotic interactions
Li, Huchen - \ 2017
Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): O. Kulikova. - Wageningen : Wageningen University - ISBN 9789463436816 - 188
plants - histones - enzymes - roots - development - symbiosis - gene expression - molecular biology - root nodules - mycorrhizas - planten - histonen - enzymen - wortels - ontwikkeling - symbiose - genexpressie - moleculaire biologie - wortelknolletjes - mycorrhizae
Plants have a sessile lifestyle. To ensure survival, they develop a potential to respond to environmental cues to set up an adaptive growth and development. This adaptation involves transcriptional reprogramming of the genome through chromatin-based mechanisms relying on the dynamic interplay of transcription factors (TFs), post-translational modification of histones, the deposition of histone variants, DNA methylation, and nucleosome remodeling. This thesis is focused on a role of one group of histone post-translational modifiers, plant-specific histone deacetylases (HDTs), in plant development under control condition and variable stresses/symbiotic interactions.
It is well known that HDTs are involved in plant responses to environmental stresses. However, whether they play a role in regulating plant growth and development is elusive. In this thesis it is shown that Arabidopsis thaliana AtHDT1/2 regulate the cell fate switch from division to expansion in the Arabidopsis root. Knock-down of AtHDT1/2 (hdt1,2i) causes that this switch occurs earlier and results in less cells in the root meristem. This process slows down root growth. One target of AtHDT1/2, AtGA2ox2, is identified here. Its overexpression displays the same root phenotype as hdt1/2i , and its knock-out partially rescues hdt1,2i root meristem phenotype. AtGA2ox2 inactivates gibberellin (GA4) whose application increases root meristem cell number in WT, but not in hdt1,2i. Based on these data, we conclude that AtHDT1/2 repress the transcription of AtGA2ox2, and likely fine-tunes GA homeostasis to regulate the switch from cell division to expansion in root tips.
HDTs respond to salt stress in Arabidopsis seedlings. Halotropism is a novel reported tropism allowing roots to avoid a saline environment. Whether the AtHDT1/2-AtGA2ox2 module is operational in halotropism is studied here. We show that hdt1,2i mutants respond more severe in halotropism. AtHDT1/2, as well as AtGA2ox2 display asymmetric localization patterns in halotropism with AtHDT1/2 reduced and AtGA2ox2 induced at high salt side of root tips. Our data indicate that their asymmetric patterns likely results in less GA at high salt side of root tips and this is required for halotropism establishment. In line with this, both constitutive expression of AtHDT2 and exogenous GA application reduce halotropic response. A reduction of GA in root tips causes an earlier switch from cell division to expansion. We discuss that this earlier switch enables roots rapidly to bend away from saline environment.
It has been shown that HDTs play a role under biotic stress in rice and tobacco leaves. We demonstrate that they are also involved in response to biotic stress in Arabidopsis leaves. Arabidopsis hdt2 mutants are more susceptible to virulent Pseudomonas syringae pv. tomato PstDC3000, whereas AtHDT2 overexpression mutants are more resistant. In addition, we detected a translocation of AtHDT2 from nucleolus to nucleoplasm after the perception of flagellin22 in Arabidopsis leaf cells. This translocation is not observed under abiotic stress. A mechanism controlling this translocation is identified. AtMPK3 is activated under biotic stress, it interacts with and phosphorylates AtHDT2. This leads to the accumulation of AtHDT2 in nucleoplasm where it contributes to the repression of defense genes.
During the interaction with symbiotic microorganisms, plants could develop a symbiotic organ/structure. For example, legumes of which Medicago truncatula is a model, can form root nodules or arbuscules by interacting with rhizobia or arbuscular mycorrhiza.
We show that nodule-specific knock-down of MtHDT1/2/3 (MtHDTs RNAi) blocks nodule primordia development and affects the function of nodule meristem. This is consistent with their roles in controlling cell division during root development and suggests that the function of nodule and root meristems is closely related. However, MtHDT2 gains a new sub-nuclear localization pattern in nodule meristem by using a not yet known mechanism, different from that in root meristem. This suggests that these two meristems have different transcriptional landscapes. In the nodule infection zone MtHDTs are also expressed and in MtHDTs RNAi the intracellular release of rhizobia is markedly reduced. Expression of MtHMGR1 and its paralogs, encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductases are down-regulated in MtHDTs RNAi. It has been shown MtHMGR1 interacts with MtDMI2, a component of Nod factor signalling pathway, to control rhizobial infection. Knock-down of MtHMGR1/MtDMI2, as well as inhibiting MtHMGRs enzymatic activity blocks nodule primordia development and rhizobial infection in nodule primordia/mature nodules. This phenotype partially resembles MtHDTs RNAi phenotype. We discuss that MtHDTs regulate expression of MtHMGRs and in this way affect Nod factor signalling and control nodule development.
Similar to nodule symbiosis, during arbuscular mycorrhizal symbiosis cells in the cortex are also intracellularly infected. We show that MtHDT2 is also induced in these arbuscule containing cells. Knock-down of MtHDT2 (MtHDT2i) significantly reduces the intracellular infection of the hyphae on the mycorrhized root segments, indicating that MtHDT2 control mycorrhizal intracellular infection. We discuss whether MtHDTs can regulate mycorrhizal/rhizobial infection in a similar way.
The data obtained in this thesis and the published information related to these subjects are discussed at the end. HDTs are key players in plant responses to environmental cues, whereas they respond to abiotic factors and biotic factors differently. They are also key regulators of plant growth and development that is clearly demonstrated in this thesis on examples of root and nodule development. I also propose a role of AtHDT1/2 in response to salt signal to fine-tune the switch from cell division to expansion in root tips during halotropism.
Quickscan zodekwaliteit dijkgrasland Afsluitdijk op basis van visuele beoordeling van doorworteling, vegetatietype en bedekking; situatie 2016
Huiskes, H.P.J. ; Vries, Daisy de - \ 2016
Wageningen UR Alterra - 42
graslanden - dijken - graslanden, conditie - wortels - vegetatie - graslandbeheer - afsluitdijk - nederland - grasslands - dykes - grassland condition - roots - vegetation - grassland management - afsluitdijk - netherlands
In februari 2016 is door Alterra, onderdeel van Wageningen Universiteit en Researchcentrum, een quickscan uitgevoerd om de kwaliteit van de zode van de Afsluitdijk te bepalen. Gegevens over de doorworteling van de zode, het graslandtype en de vegetatiebedekking vormen de basis om tot een kwaliteitsoordeel van de zode te komen. Daarnaast is een korte vergelijking gemaakt tussen de huidige uitkomst en de situatie van 2010. Afsluitend wordt een beheeradvies gegeven.
Roots in the tundra : relations between climate warming and root biomass and implications for vegetation change and carbon dynamics
Wang, Peng - \ 2016
Wageningen University. Promotor(en): Frank Berendse, co-promotor(en): Monique Heijmans; Liesje Mommer. - Wageningen : Wageningen University - ISBN 9789462578609 - 168
roots - biomass - climatic change - vegetation - carbon - global warming - tundra - ecosystems - decomposition - siberia - wortels - biomassa - klimaatverandering - vegetatie - koolstof - opwarming van de aarde - toendra - ecosystemen - decompositie - siberië
Global climate has been warming up for the last decades and it will continue in this century. The Arctic is the part of the globe that warms fastest and is more sensitive to climate warming. Aboveground productivity of Arctic tundra has been shown to increase in response to warmer climates. However, belowground responses of tundra vegetation are still unclear. As the major part of plant biomass in tundra lies belowground, it is pivotal to investigate changes in the belowground parts of tundra vegetation for our understanding of climate warming effects on tundra ecosystems.
To get a general idea of how belowground plant biomass may change in a warmer climate, we synthesized published data on the belowground biomass of tundra vegetation across a broad gradient of mean annual air temperature from −20 to 0 °C. We found that aboveground biomass of tundra biomass indeed increases with mean annual temperature as well as summer air temperature, while belowground biomass did not show a significant relationship with temperature. The increases in the aboveground biomass were significantly larger than belowground biomass, resulting in reduced below/above ratios at higher temperatures. The shifted biomass allocation with temperature can influence the carbon dynamics of tundra ecosystems. Future tundra studies need to focus more on the species or functional type composition of belowground biomass and species or functional type specific belowground responses to climate warming.
To determine the seasonal changes and vertical distribution of root biomass of different plant functional types, we sampled roots at a Siberian tundra site in the early and late growing season, from vegetation types dominated by graminoids and shrubs respectively. We distinguished the roots of graminoids and shrubs, and found that shrub roots grew earlier and shallower than graminoid roots, which enables shrubs to gain advantage over graminoids at the early growing season when nutrient pulses occur during snowmelt and soil thaw. The deeper roots of graminoids can help them to be more competitive if climate warming induces more nutrient release in the deeper soil.
In a soil thawing and fertilization experiment, we further investigated the effects of increased thawing depth and nutrient supply in the upper soil, which can be the consequences of climate warming, on root biomass and its vertical distribution. In this study we distinguished between the roots of grasses, sedges, deciduous shrubs and evergreen shrubs. The study was done in a moist tussock tundra site with similar abundance of the different plant functional types. We found that only sedges benefited from the increased thawing depth, probably through their deepest root distribution among the four functional types, while the shrubs, which were shallower-rooted, benefited from the increased nutrient availability in the upper soil. The deep-rooted grasses had the highest plasticity in vertical root distribution, which enabled them also to benefit greatly from the fertilization. Our results show that tundra plants with different rooting strategies can show different responses to climate warming dependent on the relative warming impacts on the nutrient supply in shallow and deeper soil layers. This insight can help to predict future tundra vegetation dynamics.
The carbon balance of tundra ecosystems also depends on the decomposition of plant litter, particularly the root litter, which may account for a larger part of annual litter input than leaf litter in tundra ecosystems. Vegetation shifts also change litter quality which ultimately influences carbon dynamics. To investigate the differences in the decomposition of leaves and roots of graminoids and shrubs, we performed a litter transplant experiment. We found that although the decomposability of leaf litter did not differ between the graminoid and shrub, root decomposability might be lower for the shrub. However, this cannot be extrapolated to the overall decomposition in different vegetation types, as these different plant communities differ in rooting depths. We also found evidence of home-field advantage in the decomposition in Arctic tundra, and we show that the early stage of litter decomposition at our research site could be driven by the phosphorus concentration of the litter. To get a full understanding of the carbon balance of tundra ecosystems, much more efforts are needed to quantify litter input and decomposition.
In this thesis we show that belowground parts, which account for a major part of plant biomass in tundra, can show a different response to climate warming from aboveground parts. Belowground responses to climate warming can have crucial impacts on the competitive balance between tundra plants, and consequently result in vegetation shifts in tundra. Such shifts in species composition can have large effects on carbon dynamics through altered input and decomposability of plant litter, particularly root litter.
White root tips supply plants with oxygen, water and nutrients : healthy roots are fundamental for a healthy plant
Heuvelink, E. ; Kierkels, T. - \ 2016
In Greenhouses : the international magazine for greenhouse growers 5 (2016)3. - ISSN 2215-0633 - p. 44 - 45.
tuinbouw - glastuinbouw - worteloppervlak - wortelharen - wortels - wateropname (planten) - voedselopname - opname (uptake) - calcium - tomaten - pythium - plantenontwikkeling - horticulture - greenhouse horticulture - rhizoplane - root hairs - roots - water uptake - food intake - uptake - calcium - tomatoes - pythium - plant development
The main, most important function of roots belonging to horticultural crops is the uptake of water and nutrients. Healthy roots are essential for a healthy plant. After all, if the uptake of water and nutrients is not functioning properly, then other aspects also leave a lot to be desired
Stem cell organization in Arabidopsis : from embryos to roots
Wendrich, J.R. - \ 2016
Wageningen University. Promotor(en): D. Weijers, co-promotor(en): B.P.M. de Rybel. - Wageningen : Wageningen University - ISBN 9789462577350 - 192
arabidopsis - stem cells - roots - plant embryos - morphogenesis - biological development - cellular biology - plant cell biology - arabidopsis - stamcellen - wortels - plantenembryo's - morfogenese - biologische ontwikkeling - celbiologie - plantencelbiologie
Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or meristems stay active throughout plant-life. Specification of stem cells occurs very early during development of the emrbyo and they are maintained during later stages. The Arabidopsis embryo is a highly predictable and relatively simple model to study several developmental processes. Chapter 1 discusses the Arabidopsis embryo as a model for development and morphogenesis and describes the currently known factors involved in these processes.
Molecular cloning is a vital technique of today’s plant biological research. The ability to quickly produce reliable constructs for follow-up analyses can greatly accelerate biological research. In Chapter 2, we describe the optimization of a highly efficient Ligation Independent Cloning method. This method makes use of sticky overhangs that enable in vivo ligation of cloning products. We present a step-by-step protocol that enables generating plant transformation-ready constructs in a semi-high-throughput manner, within two to three days. This method can for example facilitate follow-up analysis of genome-wide approaches.
Proteins regularly function as part of larger protein-complexes and their interaction partners can often be indicative of functionality. Unbiased, in vivo analysis of protein complexes can therefore be very informative for the functional characterization of a protein of interest. In Chapter 3, we describe an optimized method for immunoprecipitation followed by tandem mass-spectrometry. By performing mass-spectrometry measurements on at least three biological replicates, relative abundance of proteins in GFP-tagged sample compared to background controls can be statistically evaluated to identify high-confidence interactors. In this step-by-step protocol we detail the entire procedure from plant material to data analysis and visualization.
The establishment of distinct cellular identities is of critical importance for multicellular organisms. The first step that leads to cell identity is the activation of a unique set of transcripts and this often exploited in order to infer cell identity. In Chapter 4, we have generated 12 gene expression marker lines and describe their expression domain in the Arabidopsis embryo. We divided them into four different categories based on their expression domain: (I) ground tissue; (II) root stem cell; (III) shoot apical meristem; and (IV) post-embryonic. In addition, we used two stem cell markers to show their use as marker lines in genetic studies.
A central player in development of the Arabidopsis root meristem is the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP). Several downstream targets of this transcription factor have been characterized, but the main focus has been on targets that were themselves transcription factors. An open question remains, therefore, how MP can orchestrate cellular responses during development. Chapter 5 describes the in-depth functional and biochemical characterization of a group of IQ-domain proteins. We show that their expression is regulated by the hormone auxin and that they bind microtubules and Calmodulins, in vivo. In addition, we show that the subcellular localization of IQD18 is cell cycle dependent. Loss- and gain-of-function analysis resulted in differential auxin- and calcium-signaling output, suggesting these proteins may form a bridge between these two major signaling pathways. Furthermore, this indicates a mode for how MP may be affecting cellular responses, during root development.
In Chapter 6, we take a step back and re-evaluate the currently prevailing model for stem cell organization in the Arabidopsis (embryonic) root. Using different gene expression markers, we were able to generate non-cell type specific and cell type specific transcriptomic datasets from systematically obtained ontogenetic cell populations in the root meristem. Follow-up analyses give support for an extended model for stem cell organization in the root.
Finally, in Chapter 7, we discuss the novel findings of this thesis and suggestions are made for future research directions.
Arriving at the right time : a temporal perspective on above-belowground herbivore interactions
Wang, Minggang - \ 2016
Wageningen University. Promotor(en): Wim van der Putten, co-promotor(en): T.M. Bezemer; A. Biere. - Wageningen : Wageningen University - ISBN 9789462578142 - 174
herbivores - aboveground belowground interactions - herbivory - defence mechanisms - roots - leaves - mycorrhizas - population dynamics - soil biology - herbivoren - boven- en ondergrondse interacties - herbivorie - verdedigingsmechanismen - wortels - bladeren - mycorrhizae - populatiedynamica - bodembiologie
Prof. Liesje Mommer over groei en overlevingskansen bij planten
Mommer, L. - \ 2016
biodiversiteit - plantenecologie - landbouwkundig onderzoek - plantenontwikkeling - openbare redes - microbiële interacties - grondvegetatie - wortels - planteninteractie - biodiversity - plant ecology - agricultural research - plant development - public speeches - microbial interactions - ground vegetation - roots - plant interaction
Planten jutten elkaar op. Verschillende gewassen die samen een vegetatie vormen, groeien beter dan wanneer er maar één soort groeit. Maar hoe werkt dat? Welke processen onder de grond zorgen ervoor dat deze gewassen samen beter groeien? Prof. Liesje Mommer licht een tip van de sluier op tijdens haar inaugurele rede als persoonlijk hoogleraar bij de leerstoelgroep Plantenecologie en natuurbeheer aan Wageningen
Moleculaire inzichten vergroten sturingsmogelijkheden wortelgroei : Auxine speelt een sleutelrol
Scheres, B.J.G. ; Heuvelink, E. - \ 2015
Onder Glas 12 (2015)12. - p. 16 - 17.
botany - agricultural research - arabidopsis - auxins - proteins - roots - shoots - growth regulators - growth promoters - plantkunde - landbouwkundig onderzoek - arabidopsis - auxinen - eiwitten - wortels - scheuten - groeiregulatoren - groeibevorderaars
Het wortelstelsel van gewassen is vaak ‘de verborgen helft’ genoemd. Lange tijd bestond er een enorm gebrek aan kennis over wortelgroei. Dat maakt het moeilijk er op te veredelen of om er in de teelt anders mee om te gaan. De afgelopen jaren is het inzicht belangrijk gegroeid, met name op genetisch en moleculair niveau.
Goud, J.C. - \ 2015
Gewasbescherming 46 (2015)5. - ISSN 0166-6495 - p. 145 - 146.
gewasbescherming - plantenziektebestrijding - kassen - kasproeven - landbouwkundig onderzoek - dijken - wortels - klimaatverandering - abiotische beschadigingen - solanum dulcamara - maïs - capsicum - slakkenbestrijding - plant protection - plant disease control - greenhouses - greenhouse experiments - agricultural research - dykes - roots - climatic change - abiotic injuries - solanum dulcamara - maize - capsicum - mollusc control
Onlangs is bij de Radboud Universiteit Nijmegen een nieuw kassencomplex met faciliteiten in gebruik genomen. Er worden voedselkeuzeproeven gedaan met slakken, rupsen en coloradokevers, er wordt gewerkt aan planten die tolerant zijn tegen abiotische stress, zoals hitte, droogte en overstroming, er worden invasieve plantensoorten bestudeerd, zoals de waterhyacint, en er blijkt al een halve eeuw een grote genenbank van nachtschade-soorten (Solanaceae) te zijn. Genoeg interessants om eens een kijkje te gaan nemen bij de informatiedag. Bezoekers werden niet teleurgesteld. Maar het woord ‘plantenziektekunde’ is niet gevallen.
Linkages between plant traits and soil ecology in the rhizosphere and through litter decomposition
Brolsma, K.M. - \ 2014
Wageningen University. Promotor(en): Ellis Hoffland, co-promotor(en): Ron de Goede. - Wageningen : Wageningen University - ISBN 9789462571068 - 112
bodemecologie - rizosfeer - ligstro - decompositie - wortels - bodembiologie - nematoda - solanum tuberosum - globodera pallida - biofumigatie - genotypen - soil ecology - rhizosphere - litter - decomposition - roots - soil biology - nematoda - solanum tuberosum - globodera pallida - biofumigation - genotypes
Physiology and genetics of root growth, resource capture and resource use efficiency in lettuce (Lactuca sativa L.)
Kerbiriou, P.J. - \ 2014
Wageningen University. Promotor(en): Edith Lammerts van Bueren; Paul Struik, co-promotor(en): Tjeerd-Jan Stomph. - Wageningen : Wageningen University - ISBN 9789462570863 - 179
lactuca sativa - slasoorten - cultivars - groei - wortels - scheuten - plantenontwikkeling - nutriëntengebruiksefficiëntie - voedingsfysiologie - watergebruiksrendement - genetische variatie - droogteresistentie - tolerantie van variëteiten - genotype-milieu interactie - biologische plantenveredeling - lactuca sativa - lettuces - cultivars - growth - roots - shoots - plant development - nutrient use efficiency - nutrition physiology - water use efficiency - genetic variation - drought resistance - varietal tolerance - genotype environment interaction - organic plant breeding
Mycorrhiza: duurzaam bodembeheer bij peer (met poster)
Heijne, B. ; Maas, M.P. van der; Anbergen, R.H.N. - \ 2013
fruitteelt - pyrus - plantenontwikkeling - mycorrhizae - wortels - bodemschimmels - biologische bodemactiviteit - plantenvoeding - fruit growing - pyrus - plant development - mycorrhizas - roots - soil fungi - biological activity in soil - plant nutrition
Fruitkennisdag Wageningen 22 november 2013 voor 350 mensen
Visualizing brassinosteroid receptor hetero-oligomers in Arabidopsis roots
Bücherl, C.A. - \ 2013
Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Janwillem Borst. - S.l. : s.n. - ISBN 9789461736543 - 195
brassinosteroïden - biochemische receptoren - arabidopsis - wortels - beeldanalyse - signaalpeptide - signaaltransductie - fluorescentiemicroscopie - brassinosteroids - biochemical receptors - arabidopsis - roots - image analysis - signal peptide - signal transduction - fluorescence microscopy
Living matter is continuously challenged by the dynamics of its environment and intrinsic fluctuations. In the course of evolution, cells have developed mechanisms to detect and adapt to environmental and endogenous cues by the use of a wide array of receptors (Afzal et al., 2008). These receptors perceive specific signals, which, in turn, initiate a sequence of molecular events within the cells that convert signal perception into an adequate physiological response. Collectively, these processes of signal perception, signal transmission and cell adaptation represent so-called signal transduction pathways.
For the perception of signals such as hormones or pathogens cells are equipped with receptors that are often located at the cell surface. In plants, many of these receptors belong to the class of leucine-rich repeat receptor-like kinases (LRR-RLKs) (Shiu and Bleecker, 2001). They comprise an extracellular LRR domain for ligand binding, a transmembrane domain, which anchors them within the plasma membrane (PM) of their host cells, and an intracellular kinase domain for transducing the event of ligand binding into the cell interior. One of the best-described plant LRR-RLKs is the Brassinosteroid insensitive 1 (BRI1) receptor. Since the discovery in 1997 (Li and Chory, 1997) its mode of action has been studied extensively and has resulted in the elucidation of a complete set of molecular components constituting the brassinoteroid (BR) signal transduction pathway (Clouse, 2011).
BRs, the ligands of BRI1, are a group of polyhydroxy lactones that are structurally similar to animal steroid hormones (Grove et al., 1979). This class of phytohormones regulates several aspects of plant growth and development (Kutschera and Wang, 2012). During the last decade it has been shown that BRI1 indeed perceives BRs at the PM (Kinoshita et al., 2005), however, initiation of BR signal transduction requires interaction of BRI1 with other, non-ligand binding receptors (Nam and Li, 2002; Wang et al., 2008; Gou et al., 2012). These coreceptors belong to the family of Somatic embryogenesis receptor-like kinases (SERKs) and have a related structural architecture to BRI1, but with a smaller extracellular domain. Three members of this protein family are involved in BR signaling: SERK1, SERK3 (also known as BAK1 for BRI1-associated kinase 1), and SERK4 (also known as BKK1 for BAK1-like kinase 1). Besides their role as coreceptors of BRI1, the SERKs have also been implicated in various other signaling processes like somatic embryogenesis, male fertility, cell death regulation and plant immunity (Chinchilla et al., 2009).
In the first Chapter of this thesis, the BR signaling pathway was introduced in further detail and it was highlighted how genetic and biochemical approaches attributed to the identification of cellular components that link signal perception of BRs at the PM to BR dependent transcriptional regulation in the nucleus. Based on these findings a model for BRI1-mediated signal transduction was established, which often serves as a paradigm for plant PM receptor signaling. Even though the molecular determinants of BR signaling have been revealed, full mechanistic detail is still missing. The aim of this thesis was to describe BRI1-mediated signal transduction and the respective role of SERK3, the main coreceptor of BR signaling (Albrecht et al., 2008), at (sub)cellular level in Arabidopsis roots. For this purpose different fluorescence imaging techniques were applied, which allowed investigating the spatiotemporal localization and interaction dynamics of BRI1 and SERK3 in their natural environment.
One of the main microscopic methods applied throughout this thesis was fluorescence lifetime imaging microscopy (FLIM). Most imaging approaches, like confocal microscopy, only rely on fluorescence intensities as read-outs. However, the fluorescence lifetime τ is an additional parameter of fluorescence microscopy. This parameter is sensitive to the local environment of fluorescent probes and therefore can be exploited to illuminate cellular processes in live cells and tissues. In Chapter 2, the theoretical background of FLIM was introduced and it was illustrated how this technique can be used to reveal protein-protein interactions in Arabidopsis mesophyll protoplasts based on Förster resonance energy transfer (FRET). Next to a protocol for protoplast isolation and transient transfection, we provided a tutorial for analyzing time-resolved fluorescence intensity images using the software package SPCImage (Becker & Hickl). By determining the fluorescence lifetimes of a FRET donor fluorophore in the absence and the presence of a FRET acceptor chromophore physical interaction between the fluorescently tagged proteins of interest can be deduced. If the two proteins of interest, and thus the conjugated fluorophores, reside in close proximity FRET can occur and will result in a decrease of donor fluorescence lifetime. Besides the applicability to live cells and organisms, another important advantage of FRET-FLIM is the possibility to spatially resolve protein interactions within the two-dimensional imaging plane.
In Chapter 3, this technique was applied to live Arabidopsis roots. In our attempt to visualize the molecular events upon initiation of BR signaling, we performed FRET-FLIM on a double transgenic plant line expressing BRI1-GFP (Friedrichsen et al., 2000) and SERK3-mCherry. In accord with the current model of BR signal transduction (Jaillais et al., 2011a), a time-dependent and ligand-induced hetero-oligomerization between BRI1 and SERK3 was observed, similar to previous reports using coimmunoprecipitation (Wang et al., 2005; 2008; Albrecht et al., 2012). In addition, the spatially resolved FLIM images enabled us to localize these BRI1-SERK3 receptor complexes to restricted areas within the PM of live epidermal root cells, a cell file known to exhibit active BR signaling (Hacham et al., 2011). Application of brefeldin A (BFA), a fungal toxin reported to inhibit recycling (Nebenführ et al., 2002), allowed the visualization of intracellular receptor oligomers, which were most likely endocytosed from the PM. In contrast to the established BRI1 signaling model, FRET-FLIM revealed that a substantial amount of the BRI1-SERK3 hetero-oligomers was preformed. Constitutive receptor oligomerization is a well-established concept in animal signaling research (Gadella and Jovin, 1995; Martin-Fernandez et al., 2002; Issafras et al., 2002; Van Craenenbroeck et al., 2011), however in the plant field only a single study reported similar findings (Shimizu et al., 2010).
Besides the physical interaction between BRI1 and SERK3, also their localization and colocalization patterns were investigated (Chapter 3). As expected, most of the fluorescently tagged receptors localized to the PM. The intracellular fraction of BRI1-GFP mainly resided in punctate endosomal structures as documented previously (Geldner et al., 2007; Viotti et al., 2010; Irani et al., 2012). Similar endomembrane compartments were also observed for SERK3-mCherry, though to a lesser extent. In contrast to BRI1, for SERK3 an additional intracellular compartment was elucidated, the tonoplast. A further difference in the localization patterns of BRI1 and SERK3 was revealed when BFA was applied. Whereas BRI1-GFP strongly accumulated in BFA bodies, SERK3-GFP was only marginally affected, which hints at a differential endocytic pathway for both receptors. Although BRI1 and SERK3 showed distinct localization patterns, the two fluorescently tagged proteins also overlapped to some degree. Comparative colocalization analysis revealed that both the PM and the intracellular overlap between both LRR-RLKs is responsive to the BR signaling status. Application of brassinolide (BL), an endogenous BRI1 ligand, as well as BFA, which was demonstrated to elevate BR signaling activity (Geldner et al., 2007; Irani et al., 2012), resulted in an increased number of colocalizing BRI1 and SERK3 proteins. Thus FRET-FLIM and confocal imaging based colocalization analysis indicated that activation of the BR signaling system is accompanied by spatially distinct association of the two signal transduction inducing receptors BRI1 and SERK3.
As just illustrated, fluorescence microscopy is a valuable tool for investigating signal transduction processes in the natural environment of the executing molecular components. Unfortunately, a major drawback of the various techniques is that often only qualitative read-outs are obtained. Therefore we examined (Chapter 4) two different quantitative colocalization approaches in their ability to discriminate varying colocalizing proteinpopulations. First, the cytosolic colocalization of BRI1-GFP with the endosomal markerproteins ARA6 and ARA7 was investigated. Both tested and freely available ImageJ plugins Coloc2 and PSC Colocalization (French et al., 2008) revealed that BRI1-GFP preferentially localized to ARA7-mRFP labeled endosomal compartments. This finding was confirmed by manual counting of the respective endosomal structures and verified the reliability of the two quantitative colocalization methods. A biological explanation of the obtained result is given by the identity of the labeled endomembrane compartments. ARA7 localizes to both early endosomes (EEs), which enable recycling to the PM, and late endosomes (LEs; also known as multivesicular bodies [MVBs]), which are determined for vacuolar fusion. In contrast, ARA6 labels mainly LEs/MVBs. Thus both markers overlap to a certain degree during the maturation of LE but still have distinct localization patterns (Ueda et al., 2004; Ebine et al., 2011). Since BRI1 undergoes constitutive recycling (Geldner et al., 2007), our finding of preferential colocalization between BRI1 and ARA7 is plausible. In addition, similar observations were recently also reported for Flagellin sensing 2 (FLS2), an LRR-RLK involved in plant immunity, which also exhibits constitutive recycling (Beck et al., 2012).
After establishing the applicability of both colocalization approaches, we also intended to confirm our previous observation of increased BRI1 and SERK3 colocalization in response to BFA obtained with the Coloc2 plugin (Chapter 3). The application of PSC Colocalization indeed confirmed our initial colocalization results. The elevated colocalization of BRI1 and SERK3 upon drug treatment mostly like reflects the PM-stabilizing effect of BFA (Irani et al., 2012), which may also account for SERK3, since both Manders’ colocalization coefficients were increased. Nonetheless, a difficulty of quantitative colocalization analysis is the interpretation of colocalization coefficients obtained for individual images. However, they enable to assess image data sets, recorded under the same imaging conditions, in a comparative manner and that way allows drawing quantitative conclusions (Dunn et al., 2011). Colocalization analysis is not the only approach that suffers from qualitative read-outs and interpretations. The same accounts for FRET-FLIM studies. In particular the observation of preformed BRI1-SERK3 hetero-oligomers triggered our interest in developing a quantitative FLIM analysis procedure, which would be able to resolve ligand-independent and ligand-induced receptor complex populations. The details of our approach, which is based on time-correlated single photon count (TCSPC) measurements, were described in Chapter 4. Using this novel FLIM analysis procedure enabled us to estimate the different populations of BRI1 and SERK3 complexes. Upon BL stimulation around 10% of PM-located BRI1-GFP receptors were in complex with SERK3-mCherry. This finding is in line with recently reported data based on an in silico modeling approach (van Esse et al., 2012) and semi-quantitative coimmunoprecipitation (Albrecht et al., 2012), which suggested that active BR signal transduction involves between 1-10% of BRI1 receptors. Unfortunately, there are no quantitative data available for constitutive BRI1-SERK3 hetero-oligomers, even though their existence was proposed (Wang et al., 2005). Based on our imaging approach and analysis procedure we estimate that approximately 70% of PM BRI1-SERK3 heterooligomers are preformed. Finding such a considerable amount of preformed BRI1-SERK3 receptor complexes in the PM of root epidermal cells was intriguing since it contradicts the current view on BR signaling, which assumes a strictly ligand-dependent association of the two LRR-RLKs (Jaillais et al., 2011a). This posed the question when or where these preformed complexes are established. To address this point we investigated in Chapter 5 which cellular compartments harbor individual BRI1 and SERK3 receptors, and in which organelles these two receptors colocalize. Comparative colocalization analysis in live Arabidopsis roots revealed that both LRR-RLKs follow the traditional secretory and retrograde transport routes. These observations confirmed and extended previous findings for BRI1 using live cell (Friedrichsen et al., 2000; Geldner et al., 2007; Viotti et al., 2010; Irani et al., 2012) and electron microscopy (Viotti et al., 2010). For SERK3, to date only localization to EEs was suggested (Russinova et al., 2004).
Using the transient expression system of Arabidopsis protoplasts we could moreover show that both receptors also colocalize in the various endomembrane compartments of anterograde and retrograde trafficking. However, using electron microscopy a striking difference between their localization in retrograde endosomal compartments was elucidated. Whereas BRI1 was previously shown to reside at the membranes of the enclosed vesicles (Viotti et al., 2010), SERK3 was visualized at the limiting membrane of prevacuolar compartments (PVCs). This finding also explains, why SERK3, but not BRI1, was observed at the tonoplast (Chapter 3). Fusion of MVBs with the vacuole results in the release of BRI1 along with the inner MVB vesicles into the vacuolar lumen. PVC-localized SERK3 instead is incorporated into the tonoplast after membrane fusion. Collectively, the colocalization analysis of BRI1 and SERK3 with respect to endomembrane compartments revealed that subpopulations of both LRR-RLKs probably follow the same route to the PM, but that after endocytosis from the PM, possibly during the maturation of TGN/EEs to LEs/MVBs, a separation occurs. Still, these findings do not answer where or when BRI1-SERK3 hetero-oligomers are established. For that reason we applied FRET-FLIM on the subcellular compartment, in which BRI1 and SERK3 colocalized for the first time, the endoplasmic reticulum (ER). Similar to our observations at the PM of root epidermal cells (Chapter 3), most of the ER membrane did not show BRI1-SERK3 receptor complexes. Still, in restricted ER membrane regions strongly reduced donor fluorescence lifetimes were observed, indicating that BRI1-SERK3 hetero-oligomers are established already in the ER before entering the anterograde trafficking pathway. Finally, using a heat-shock inducible plant system we could confirm the establishment of BRI1-SERK3 hetero-oligomers shortly after biogenesis on their way to the PM. Thus, the observed preformed receptor complexes in the PM of root epidermal cells (Chapter 3) mostly likely originated from the ER and were inserted via targeted transport into the PM, the site where they fulfill their function as BR signaling units.
Plant reguleert zelf bodemleven rond de wortels
Heuvelink, E. ; Kierkels, T. - \ 2013
Onder Glas 10 (2013)5. - p. 14 - 15.
glastuinbouw - cultuurmethoden - bodembeheer - wortels - potplanten - cultuur zonder grond - ph - plant-water relaties - plantenvoeding - greenhouse horticulture - cultural methods - soil management - roots - pot plants - soilless culture - ph - plant water relations - plant nutrition
De plant beïnvloedt in vergaande mate zijn eigen wortelmilieu; of hij nu op substraat groeit, in een pot of in de volle grond. De wortels scheiden tal van stoffen uit, die de pH veranderen, maar ook de samenstelling van het bodemleven, zodat de plant er beter baat bij heeft.
Rhizoctonia solani in Delphinium
Bulle, A.A.E. ; Lans, A.M. van der; Breeuwsma, S.J. - \ 2013
Lisse : Praktijkonderzoek Plant en Omgeving BBF - 23
delphinium - schimmelziekten - bodemschimmels - rhizoctonia - aantasting - wortels - chemische bestrijding - pesticiden - proeven - zomerbloemen - delphinium - fungal diseases - soil fungi - rhizoctonia - infestation - roots - chemical control - pesticides - trials - summer flowers
Rhizoctonia solani (dradenschimmel) is een algemeen in de grond voorkomende bodemschimmel zowel in de open teelten als onder glas. Delphinium is één van de vele waardplanten van Rhizoctonia solani. Een aantasting van Rhizoctonia solani begint meestal aan de plantvoet op de grens van lucht en grond. Bij een ernstige aantasting vallen planten volledig weg. Nadat in Alchemilla groeiremming (zonder ziekteverschijnselen) was gezien als gevolg van Rhizoctonia, kwam de vraag of groeiremming in Delphinium, die tot nu toe in verband werd gebracht met herinplantziekte, ook een gevolg kon zijn van Rhizoctonia solani. In een kasteelt en in een buitenteelt van Delphinium zijn monsters genomen van gewas en wortels. Een slechte groei is op beide locaties gezien, maar in de monsters die op deze plaatsen zijn genomen is geen enkele keer Rhizoctonia aangetroffen. Uit analyses van de wortels en de grond bleek dat vooral op de plekken waar slechte groei werd gezien, veel wortellesie-aaltjes (Pratylenchus penetrans) voorkwamen. Ook de grondanalyses gaven een sterke aanwijzing dat wortellesie-aaltjes een rol spelen bij de slechte groei van Delphinium op deze bedrijven. Bekend is dat Delphinium waardplant is voor Pratylenchus penetrans en dat schade in de vorm van groeiremming optreedt. Naast Pratylenchus penetrans kwamen aaltjes uit de groep van Paratylenchus (speld- of naaldaaltjes) voor. Bekend is dat Paratylenchus-aaltjes groeiproblemen kunnen geven in enkele andere zomerbloemen en in schermbloemigen zoals peen, selderij en venkel. De waardplantstatus van Delphinium voor Paratylenchus-soorten is niet bekend. Nader onderzoek naar de relatie tussen Paratylenchus en slechte groei in Delphinium wordt aanbevolen.
Modeling the effects of saline groundwater and irrigation water on root zone salinity and sodicity dynamics in agro-ecosystems
Shah, S.H.H. - \ 2013
Wageningen University. Promotor(en): Sjoerd van der Zee, co-promotor(en): R.W. Vervoort. - S.l. : s.n. - ISBN 9789461735256 - 201
grondwater - zout water - modelleren - irrigatiewater - wortels - zoutgehalte - agro-ecosystemen - bodemfysica - bodemzoutgehalte - groundwater - saline water - modeling - irrigation water - roots - salinity - agroecosystems - soil physics - soil salinity
Recent trends and future projections suggest that the need to produce more food and fibre for the world’ s expanding population will lead to an increase in the use of marginal-quality water and land resources (Bouwer, 2000; Gupta and Abrol, 2000; Wild, 2003). This is particularly relevant to less-developed, arid and semi-arid countries, in which problems of soil and water quality degradation are common (Qadir and Oster, 2004). The aim, therefore, should be to increase yield per unit of land rather than the area cultivated. More efforts are needed to improve productivity as more lands are becoming degraded. It is estimated that about 15% of the total land area of the world has been degraded by soil erosion and physical and chemical degradation, including soil salinization (Wild, 2003).
The main sources of soil salinity and sodicity development are groundwater and irrigation water. In discharge areas of the landscape, water exits from groundwater to the soil surface bringing the salts dissolved in it. The driving force for upward movement of water and salts is evaporation from the soil plus plant transpiration. Salt accumulation is high when the water table depth is less than a threshold. However, this threshold depth may vary depending on soil hydraulic properties and climatic conditions. Groundwater associated salinity and sodicity affects around 350 X 104km2in the world (Szabolcs, 1989).
In this thesis, the focus is to quantify and understand the salinity and sodicity dynamics, and the feedback on dynamics in groundwater dependent agro-ecosystems. First we have considered the impact of salt coming from groundwater on capillary fluxes and on the root zone water and salt dynamics. Groundwater can be a source of both water and salts in semi-arid areas, and therefore capillary pressure induced upward water flow may cause root zone salinization. To identify which conditions result in hazardous salt concentrations in the root zone, we combined the mass balance equations for salt and water, further assuming a Poisson-distributed daily rainfall and brackish groundwater quality. For the water fluxes (leaching, capillary upflow, and evapotranspiration), we account for osmotic effects of the dissolved salt mass using Van‘t Hoff’s law. Root zone salinity depends on salt transport via capillary flux and on evapotranspiration, which concentrates salt in the root zone. Both a wet climate and shallow groundwater lead to wetter root zone conditions, which in combination with periodic rainfall enhances salt removal by leaching. For wet climates, root zone salinity (concentrations) increases as groundwater is more shallow (larger groundwater influence). For dry climates, salinity increases as groundwater is deeper due to a drier root zone and less leaching. For intermediate climates, opposing effects can push the salt balance in either way. Root zone salinity increases almost linearly with groundwater salinity. With a simple analytical approximation, maximum concentrations can be related with the mean capillary flow rate, leaching rate, water saturation and groundwater salinity, for different soils, climates and groundwater depths.
A Soil sodicity (quantified by ESP) model based on the soil salinity model (as discussed above) has been developed. For sodicity calculations, we have used the Gapon equation favored in salinity research. The simulation results show that soil salinity and sodicity development in groundwater driven agro-ecosystems play a major role in soil structure degradation. To identify which conditions can make soil sodic, we have modeled the coupled water, salt, and cation balances. The root zone salinity Cand sodicity ESPgradually change to their long term average values. These long term average values are independent of the cation exchange capacity CEC. The rate of change depends inversely on the size of the root zone reservoir, i.e., on root zone thickness for C, and additionally on CEC, for ESP.Soil type can have a large effect on both the rate of approach of the long term steady state salinity and sodicity, and on the long term levels, as it affects the incoming and out-going water and chemical fluxes. Considering two possible sources of salts, i.e., groundwater and irrigation water (here represented by rainfall), the long term salt concentration Cof the root zone corresponds well with a flux weighted average of infiltrating and upflowing salt mass divided by the average water drainage. In full analogy, the long term ESPcan be approximated very well for different groundwater depths and climates. A more refined analytical approximation, based on the analytical solution of the water balance of Vervoort and Van der Zee(2008), leads to a quite good approximation of long term salinity and sodicity, for different soils, groundwater depths, and climates.
Modeling is an efficient tool to investigate water and solute movement in groundwater driven agro-ecosystems. However, in most available models (SWAP,MODFLOW/MT3D) continuing degradation of soil hydraulic properties as a result of rising Na+concentrations is ignored. Disregarding the soil hydraulic degradation due to sodicity level in some cases makes modeling water and solute movement within the soil profile questionable. We have translated the effects of soil salinity and sodicity into reduction in saturated hydraulic conductivity to quantify the feedback effects of reduction in saturated hydraulic conductivity on root zone fluxes, salinity, and sodicity under different groundwater depths and climates of Oenpelli and Tennant Creek Airport located in the North Territory of Australia. The reduction in saturated hydraulic conductivity due to salinity and sodicity (Ks(C,ESP))has been calculated by using the procedure developed by McNeal(1968). The significant feedback effects of Ks(C,ESP) on salt concentration and soil ESPdepend on many important parameters like groundwater depth, leaf area index, weather seasonality and non-seasonality, and soil type. Out of these important parameters, weather seasonality is the main driver that can develop significant feedback effects of Ks(C,ESP) onsalt concentration and soil ESP. Furthermore, Ks(C,ESP) although decreasing the capillary flux, leaching flux, and evapotranspiration, it increases the magnitude of runoff. Also when Ks(C,ESP)affects both capillary and leaching flux under seasonal rainfall, the feedback effects are significant compared to the partial feedback (Ks(C,ESP)affects only leaching flux, but not capillary flux).
In the second theme of this thesis, we have focused on optimizing irrigation water between two farms under water scarcity and salinity regimes. In arid and semi-arid regions, irrigation water is scarce and often saline. To reduce negative effects on crop yields, the irrigated amounts must include water for leaching and therefore exceed evapotranspiration. The leachate (drainage) water returns to water sources such as rivers or groundwater aquifers and increases their level of salinity and the leaching requirement for irrigation water of any sequential user. We develop a sequential (upstream-downstream) model of irrigation that predicts crop yields and water consumption and tracks the water flow and level of salinity along a river dependent on irrigation management decisions. The model incorporates an agro-physical model of plant response to environmental conditions including feedbacks. For a system with limited water resources, the model examines the impacts of water scarcity, salinity and inefficient application on yields for specific crop, soil, and climate conditions. As a general pattern we find that, as salinity level and technical inefficiency increase, the system benefits when upstream farms use less water than is available to them, to provide downstream farms with more and better quality water. We compute the marginal value of water, i.e. the price water that would command on a market, for different levels of water scarcity, salinity and levels of water loss.
In summary this thesis aims to understand theoretically how soil salinity and sodicity develop under different climates, groundwater depths, soil types, root zone thicknesses, and different groundwater salinities. The developed salinity sodicity model can be applied in potential salt affected areas to predict the long term salinity, sodicity trends. Furthermore, quantification of feedback effects of reduction in saturated hydraulic conductivity (Ks(C,ESP)) on root zone fluxes, salinity, and sodicity guide us towards better management of soil, vegetation, and irrigation/groundwater.
Effecten van Disappyr op bruinverkleuring en beworteling van stek van sierheesters
Derkx, M.P.M. - \ 2012
houtachtige planten - struiken - stekken - vermeerderingsmateriaal - fenolen - bruinkleuring - wortels - ziektebestrijding - woody plants - shrubs - cuttings - propagation materials - phenols - browning - roots - disease control
Bij het snijden van stekmateriaal wordt plantenweefsel beschadigd. Hierdoor komen chemische verbindingen vrij, zoals fenolen. Deze fenolen kunnen onder invloed van enzymen en zuurstof omgezet worden in schadelijke verbindingen en brui nige pigmenten. In stek van pruimenonderstammen was eerder gevonden dat fenolen een negatief effect hebben op de beworteling. De schadelijke verbindingen kunnen weggevangen worden door Disappyr, met daarin het actieve bestanddeel polyvinylpolypyrrolidone ( PVPP). In stek van vijf soorten sierheesters zijn effecten van Disappyr op bruinverkleuring en beworteling onderzocht.
Onderzoek naar de relaties tusen snoeimethode, klimaat en wortelrot bij Snijanthurium
Garcia Victoria, N. - \ 2012
Bleiswijk : Wageningen UR Glastuinbouw (Rapporten GTB 1211) - 74
anthurium - wortelrot - afsterving - wortels - snoeien - teelt onder bescherming - nederland - anthurium - root rots - dieback - roots - pruning - protected cultivation - netherlands
Niet door pathogeen veroorzaakte wortelafsterving leidt bij Anthurium tot plantvergeling, plantstrekking, en het soms massaal wegvallen van planten in de praktijk. Het Productschap Tuinbouw heeft een onderzoek gefinancieerd dat tussen April 2010 en April 2011 bij Wageningen UR Glastuinbouw in Bleiswijk is uitgevoerd om inzicht te krijgen in de oorzaken van de wortelafsterving. Onderzocht is of de methode van bladsnoei een rol speelt in het optreden van wortelrot bij de soorten Tropical en Midori. Met de drie toegepaste methodes “Jong Blad Breken Continu”, “Jong Blad Breken met vervanging van 1 blad” en “Blad halveren” is wortelrot opgetreden in beide cultivars, waarbij Midori veel gevoeliger bleek dan Tropical. De mate waarin wortelrot zich voordeed was, vooral na de winter, het ergste in de behandeling “continu Jong Blad Breken”. Bij deze behandeling waren de planten ook het slechtst in bladkwaliteit, hadden het minste blad en produceerden ze iets minder bloemen, van slechtere kwaliteit en houdbaarheid. De oorzaak lijkt eerder een verstoring van de assimilaten- dan van de hormonalehuishouding van de plant. Vervolg onderzoek moet uitwijzen of andere methodes voor bladsnoei en teeltsystemen met meer ruimte voor de wortels ook tot wortelrot leiden
Effecten van Disappyr op bruinverkleuring en beworteling van stek van sierheesters
Derkx, M.P.M. - \ 2012
Lisse : Praktijkonderzoek Plant & Omgeving, Bollen en Bomen - 17
houtachtige planten - struiken - stekken - vermeerderingsmateriaal - fenolen - bruinkleuring - wortels - ziektebestrijding - woody plants - shrubs - cuttings - propagation materials - phenols - browning - roots - disease control
Bij het snijden van stekmateriaal wordt plantenweefsel beschadigd. Hierdoor komen chemische verbindingen vrij, zoals fenolen. Deze fenolen kunnen onder invloed van enzymen en zuurstof omgezet worden in schadelijke verbindingen en bruinige pigmenten. In stek van pruimenonderstammen was eerder gevonden dat fenolen een negatief effect hebben op de beworteling. De schadelijke verbindingen kunnen weggevangen worden door D isappyr,met daarin het actieve bestanddeel polyvinylpolypyrrolidone (PVPP). In stek van vijf soorten sierheesters zijn effecten van Disappyr op bruinverkleuring en beworteling onderzocht
Epigenetic control of root and nodule development : the role of plant-specific histone deacetylases and LHP1 in root cell reprogramming
Schilderink, S. - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Olga Kulikova; Joan Wellink. - S.l. : s.n. - ISBN 9789461734259 - 131
cellen - epigenetica - wortels - knobbeltjes - plantenontwikkeling - apicale meristemen - medicago - arabidopsis - wortelknolletjes - histonen - cells - epigenetics - roots - nodules - plant development - apical meristems - medicago - arabidopsis - root nodules - histones
In plants, unlike in animals, most organs develop post embryonically. These organs originate from clusters of undifferentiated dividing cells that form so-called meristems. Differentiated cells can be re-activated to enter the cell cycle and to ultimately give rise to new meristems. These differentiated cells reprogram to become pluripotent cells that are able to give rise to all cell types of the new organ. Examples are the formation of lateral roots from pericycle cells in Arabidopsis and the formation of root nodules from cortical cells in Medicago. In the latter organ rhizobia bacteria in symbiosis with Medicago fix atmospheric nitrogen. The reprogramming of differentiated cells is associated with large changes in gene expression. In this thesis the role of two chromatin modifiers in these reprogramming events is studied; the plant-specific histone deacetylases (HDTs) and LIKE HETEROCHROMATIN PROTEIN I (LHP1).
In Chapter 2 we show that in Arabidopsis during lateral root formation all four members of the AtHDT family are strongly induced in lateral root founder cells already before the first divisions occur. Furthermore, AtHDT1 and AtHDT2 were shown to be necessary for maintenance of the root stem cell niche and meristem.
Nodules are formed from fully differentiated root cortical cells. The reprogramming of cortical cells during the formation of nodules is unique to legumes, as cortical cells in other plants are not able to change their cell fate. In Medicago we characterized 3 HDTs and showed that these MtHDTs are involved in cell fate changes and are essential for the formation of nodules (Chapter 3). These MtHDTs are involved in early steps of reprogramming of the cortical cells in nodule initiation, since RNAi knock-down expression of all 3 MtHDTs simultaneously, strongly reduced nodule formation. Microscopic analysis of the small nodules that were still formed revealed these had a normal wt-like proximal part and a disturbed distal part.
To explain this hybrid phenotype we created a fate map of nodule development by analyzing early stages of nodule formation (Chapter 4). This analysis revealed that the proximal part of nodules contain about 8 layers of fully differentiated cells that directly originate from the nodule primordium, which is derived from the two innermost cortical layers. The nodule meristem originates from the middle cortical layer and further growth of the nodule depends of differentiation and infection of cells originating from this meristem. At the disturbed distal part of the RNAi hybrid nodules, meristem-derived cells do not properly differentiate and are not infected with rhizobia indicating a disturbed meristem function.
The second chromatin modifier we studied is LHP1, which is known to be involved in several developmental processes in plants, possibly as part of the plant analog of Polycomb Repressive Complex 1 (PRC1). We characterized the Medicago LHP1 homolog (Chapter 5) and showed that LHP1 forms euchromatic complexes that most likely regulate gene expression. MtLHP1 might be involved in proper differentiation of meristem-derived cells in the nodule since knock-down of MtLHP1 resulted in a similar phenotype as seen in MtHDT knock-down nodules.
Our findings provide new insights on the role of the chromatin modifiers HDTs and LHP1 in reprogramming events in Arabidopsis and Medicago organ development.