Plant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations... Show morePlant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations modulate plant metabolism. Factorial combinations of different plant species, including Arabidopsis thaliana (model plant), Brassica oleracea var. italica (crop) and Artemisia annua (medicinal plant), and phylogenetically distinct rhizobacterial species, including Pseudomonas fluorescens SS101 (Pf SS101), Microbacterium and three Paraburkholderia species, were used as study model systems in this thesis. Untargeted metabolomics was used to assess the impact of these rhizobacteria on the shoot chemistry of the host plant species. This study revealed that root treatment of different plant species with rhizobacteria altered 18-78% of the detected plant secondary metabolites in the shoot. We also assessed the impact of a known bacterial trait on plant phenotype and chemistry. cysH mutation of Pf SS101 affected the chain elongation of aliphatic glucosinolate biosynthesis in Arabidopsis whereas it led to an accumulation of indolic glucosinolates and flavonoids in Broccoli. To further discover the bacterial traits affected during the interaction with Broccoli roots, genome wide transcriptome analysis was carried out, resulting in upregulation of genes involved in flagellar assembly, chemotaxis, and motility together with nutrient uptake and (an)ion transporter in Paraburkholderia species. Show less
The plant hormone auxin plays a central role in the growth and development of plants. Auxin acts in a concentration dependent manner and polar cell-to-cell transport of this hormone determines its... Show moreThe plant hormone auxin plays a central role in the growth and development of plants. Auxin acts in a concentration dependent manner and polar cell-to-cell transport of this hormone determines its distribution in the tissues of plants. This polar auxin transport is mediated by several families of auxin transporters, including the PIN FORMED (PIN) auxin efflux carriers that determine the direction of transport by their polar localization at the plasma membrane. The plasma membrane abundance and polarity of PINs (and thereby of polar auxin transport) is regulated by their post-translational modification, of which phosphorylation is best studied. PIN proteins in Arabidopsis consist of two transmembrane domains separated by a ‘long’ (PIN1,2,3,4,6,7) or by a ‘short’ (PIN5,8) hydrophilic loop. Phosphorylation of ‘long’ PINs in their central hydrophilic loop by the AGC3 kinases PINOID, WAG1 and WAG2 triggers shootward (apical) or outer-lateral polarity. The AGC1- type D6 kinases also phosphorylate the PIN hydrophilic loop, however this was reported to result in auxin transport activation rather than subcellular polarity establishment. Here we investigate the conservation and phylogeny of AGC3 and D6 kinases and their phosphorylation sites in PINs from the earliest land plants to flowering plants. In early land plants, many of the same proteins and conserved motifs can be found, however it is in monocots and dicots that conservation of PIN phosphorylation by AGC3 and D6 kinases is strongest. The expansion and increased conservation of AGC3 and D6 kinases and PINs in later lineages such as monocot and dicot flowering plants, is in line with their important role in the formation of reproductive organs and in the tropic growth responses that allow plants to adapt to changes in their environment. Show less
In this thesis the evolutionary background, function and localization of the domesticated transposase DAYSLEEPER are described. We found that DAYSLEEPER-like genes can be found in angiosperms, but... Show moreIn this thesis the evolutionary background, function and localization of the domesticated transposase DAYSLEEPER are described. We found that DAYSLEEPER-like genes can be found in angiosperms, but not in lower plants. We also found that DAYSLEEPER interacts with several proteins and is probably involved in regulating protein degradation through involvement in ubiquitin-mediated protein degradation. Show less
Salicylic acid (SA) is involved in mediating defense against biotrophic pathogens. The current knowledge of the SA-mediated signaling pathway and its effect on the transcriptional regulation of... Show moreSalicylic acid (SA) is involved in mediating defense against biotrophic pathogens. The current knowledge of the SA-mediated signaling pathway and its effect on the transcriptional regulation of defense responses are reviewed in this thesis. PR-1 is a marker gene for systemic acquired resistance (SAR) and we have shown that the plant-specific transcription factor WRKY50 binds to the Arabidopsis PR-1 promoter at two positions close to binding sites of TGA proteins, bZIP class transcription factors previously shown to be important for PR-1 expression. We found that WRKY50 interacts with TGAs and together they synergistically activate PR-1 expression. In addition, a number of W-boxes in the PR-1 promoter have been shown to be also important for expression. We have shown that WRKY28 is able to bind to these W-boxes and to activate PR-1 gene expression in protoplasts. We also describe the effects of overexpression of the WRKY50 and WRKY28 proteins in transgenic plants. High constitutive expression of the WRKY50 gene resulted in accumulation of PR-1 mRNA when the plants were treated with SA. The overexpression of WRKY50 and WRKY28 had no clear effect on the plants__ resistance to the biotrophic pathogens, and neither was this the case with WRKY50 T-DNA knockout plants. To investigate the WRKYs__ influence on metabolite composition, a 1H NMR spectroscopy-based metabolomic approach was applied. The WRKY50 overexpression plants contained two- to three-fold more sinapic acid derivatives. This indicates a possible involvement of WRKY50 on metabolite production, in particular of hydroxycinnamates such as sinapic acid. As these compounds are components of lignin, this may point to a role of WRKY50 in stress-induced lignin modification. Show less
Jasmonates (JAs) are plant signaling molecules that play important roles in defense against insects and necrotrophic pathogens. The receptor-repressor-transcription factor module COI1-JAZ-MYC is a... Show moreJasmonates (JAs) are plant signaling molecules that play important roles in defense against insects and necrotrophic pathogens. The receptor-repressor-transcription factor module COI1-JAZ-MYC is a key component in JAs signaling in Arabidopsis thaliana. JAs stimulate their own biosynthesis by inducing the expression of genes encoding the biosynthetic enzymes. The research aim was to study the molecular mechanisms controlling this positive feedback loop. The results show that the AP2/ERF-domain transcription factor ORA47 is the key regulator. Overexpression of ORA47 resulted in increased expression of all JAs biosynthesis genes tested and in elevated levels of several JAs, including jasmonic acid (JA) and the main bioactive compound JA-Isoleucine. The expression of the ORA47 gene and the activity of the ORA47 protein are stimulated by JA. The JA-responsive expression of the ORA47 gene is controlled by the transcript ion factors MYC2, MYC3 and MYC4 which interact with a G-box in the ORA47 promoter. The expression of ORA47 target genes is negatively affected by members of the JAZ repressor family, suggesting that they regulate ORA47 activity. However ORA47 does not directly interact with JAZ repressors in yeast two-hybrid assays. Therefore it is hypothesized that ORA47 is regulated via interaction with an adaptor protein that recruits certain members of the JAZ family. Members of the BTB-TAZ (BT) protein family were found to interact with ORA47 and with certain JAZ proteins. However analysis of quadruple bt mutants failed to detect an effect on the expression of JAs biosynthesis genes, indicating that BT proteins are not the hypothetical adaptor proteins. Show less
Jasmonic acid (JA) is a plant hormone that plays an important role in defense against wounding, insects and microbial pathogens. In defense responses against microbial pathogens JA acts... Show moreJasmonic acid (JA) is a plant hormone that plays an important role in defense against wounding, insects and microbial pathogens. In defense responses against microbial pathogens JA acts synergistically with the stress hormone ethylene. ORA59, belonging to the plant-specific class of AP2-domain transcription factors, is the main regulator of JA/ethylene-responsive defense gene expression in the model plant species Arabidopsis thaliana. The aim of the research described in this thesis was to study how the activity of ORA59 is regulated by JA. Studies on the promoter of the defense gene PDF1.2 showed that ORA59 regulates gene expression by interaction with GCCGCC motifs. Studies of ORA59 at the protein level indicated that JA controls ORA59 activity by promoting the nuclear localization and stabilization of the protein. Based on the findings it was postulated that there is an F-box protein that targets ORA59 for degradation, and a repressor protein that sequesters ORA59 in the cytoplasm. A protein interacting with ORA59 was identified by yeast two-hybrid screening. Further studies showed that this protein acts as a repressor of ORA59 by retaining it in the cytoplasm, which leads to fine-tuning of basal resistance against pathogens. Show less
The study has shown that it is possible to introduce the heterologous CHS gene in Arabidopsis thaliana and common multicopies of transgenes containing plants were obtained. Analysis of the change... Show moreThe study has shown that it is possible to introduce the heterologous CHS gene in Arabidopsis thaliana and common multicopies of transgenes containing plants were obtained. Analysis of the change in metabolome of CHS transgenic plants, high expression transgenic lines can be identified by markers such as flavonoids and phenylpropanoids. It is also clear that UV-A/blue light stress does not further increase the levels of these marker compounds in CHS transgenic Arabidopsis plants, whereas in wild type plants such a treatment results in increased levels of these compounds, in fact similar to that in the transgenic plants. There are certain physiological limitations in the accumulation of certain products. This thesis starts with a review of the function of CHS in plants and especially in plant resistance (Chapter 2). Chapter 3 deals with the work on Agrobacterium-mediated transformation of heterologous chalcone synthase in Arabidopsis thaliana Col. 0. The effect of overexpression of CHS on the transcriptional level is discribed in this chapter. The activity of the CHS enzyme in the transgenic plants is reported in Chapter 4. In Chapter 5 metabolic profiling of Arabidopsis thaliana using nuclear magnetic resonance spectroscopy (NMR) is described. In this chapter the primary and secondary metabolites of Arabidopsis thaliana Col. 0 which can be detected by NMR are reported. Chapter 6 reports the metabolic profiling of CHS transgenic Arabidopsis. Metabolomic changes upon UV-A/blue light treatment of Arabidopsis thaliana were investigated (Chapter 7). Chapter 8 deals with the study of the effect of the non-pesticide chemical, Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) on the Arabidopsis metabolome. Finally, the general summary and discussion of thesis are given in Chapter 9. Show less
In view of their predominant sessile lifestyle, plants need to be able to adapt to changes in their environment. Environmental signals such as light and gravity modulate plant growth and... Show moreIn view of their predominant sessile lifestyle, plants need to be able to adapt to changes in their environment. Environmental signals such as light and gravity modulate plant growth and architecture by redirecting polar cell-to-cell transport of auxin, thus causing changes in the distribution of this plant hormone. The PIN auxin efflux carriers are key drivers of auxin transport that determine the direction of auxin flow through their asymmetric subcellular distribution. An important component in PIN polarity establishment is the plant protein kinase PINOID (PID). PID instructs apical (shoot meristem facing) PIN polarity by phosphorylating the central hydrophylic loop of PIN proteins (PINHL). In this thesis we investigated modulation of PID activity by the calcium binding proteins TCH3 and PBP1, and by the protein kinase PDK1. All three proteins were found to regulate both the enzymatic activity and the sub-cellular localisation of PID in response to calcium and phospholipids, respectively, and as such they are likely to be involved in translating environmental signals into PIN polarity changes. In addition, we show that PID and its close homologs act both redundantly and differentially in orienting plant development by instructing the subcellular distribution of PINs. Show less
Het onderzoek beschreven in het proefschrift van B.I. Lindhout heeft zich gericht op het maken van kunstmatige eiwitten die expressie van genen be_nvloeden, zogeheten zinkvinger bevattende artifici... Show moreHet onderzoek beschreven in het proefschrift van B.I. Lindhout heeft zich gericht op het maken van kunstmatige eiwitten die expressie van genen be_nvloeden, zogeheten zinkvinger bevattende artifici_le transcriptiefactoren. Met dit type eiwitten kan worden onderzocht hoe bepaalde processen in een organisme gereguleerd worden, maar ook om op een nieuwe manier eigenschappen van een organisme te veranderen. Een speciaal type DNA-bindende zinkvingers vormt een onderdeel van deze eiwitten. Het proefschrift beschrijft experimenten die zijn gericht op de verdere uitwerking van deze techniek, vooral voor het plantenbiologisch onderzoek. Er wordt aangetoond dat zinkvinger transcriptiefactoren inderdaad goed gebruikt kunnen worden om een specifiek gen te reguleren, maar ook hoe een grote verzameling daarvan gebruikt kan worden om te zoeken naar verborgen genetische eigenschappen. Deze speciale toepassing houdt in dat specifieke kenmerken die een plant in principe van zichzelf zou kunnen hebben, maar die ontbreken omdat het eigen genetische materiaal van de plant onvoldoende tot uitdrukking komt, plotseling zichtbaar kunnen worden. Zo is gebleken dat introductie van een bepaalde transcriptiefactor de plant in staat stelt zijn DNA veel nauwkeuriger te repareren. Eveneens is beschreven hoe lichtgevende zinkvingers gebruikt kunnen worden om __live__ naar het genetische materiaal van planten- en muizencellen te kunnen kijken. Show less
Lateral roots are an important means for the plant to increase its absorptive area and the volume of substrate exploited. Lateral roots originate in the pericycle, the outermost layer of the... Show moreLateral roots are an important means for the plant to increase its absorptive area and the volume of substrate exploited. Lateral roots originate in the pericycle, the outermost layer of the vascular cylinder, and by growing penetrate the overlaying cell layers before emergence. This process is mainly controlled by the plant hormone auxin. In this thesis we studied the putative role of three Arabidopsis (a model plant) genes: AIR1A, AIR1B and AIR3, which are specifically expressed in the outer cell layers at the sites of lateral root formation and emergence. AIR1A and AIR1B are putative plasma membrane associated proteins related structural proteins that are believed to link the plasma membrane to the cell wall. AIR3 encodes a putative serine protease belonging to the family of subtilisins. We provide genetic evidence of the link between lateral root formation and AIR1 gene expression. We also show that AIR3 is part of the nitrate-signalling network regulating lateral root growth. Show less
