Browsing by Subject "Signaling"

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Identification of regulatory factors determining nutrient acquisition in Arabidopsis
(2011) Giehl, Ricardo Fabiano Hettwer; von Wirén, Nicolaus
The acquisition and translocation of mineral nutrients involves the orchestrated action of a series of physiological and biochemical mechanisms, which are, in turn, regulated by nutrient availability and demand. Furthermore, root morphological changes play an outstanding role for nutrient acquisition, especially when the availability of a certain nutrient is low. Although for most nutrients the molecular mechanisms involved in their acquisition from soils have been described, much less is known about the regulatory pathways underlying the uptake and translocation of nutrients in plants. Thus, the main aim of the present study was to characterize root morphological responses to nutrient supply and to identify novel regulatory components. The first part of the present thesis describes the morphological response of Arabidopsis roots to the essential element iron (Fe), which has a particularly low solubility in soils. Relative to a homogenous supply of Fe, localized Fe supply to horizontally-separated agar plates doubled lateral root length without a particular effect on lateral root number. The internal tissue Fe rather than external Fe triggered the local elongation of lateral roots. In addition, the Fe-stimulated emergence of lateral root primordia and root cell elongation was accompanied by a higher activity of the auxin reporter DR5:GUS in lateral root apices. A crucial role of the auxin transporter AUX1 in Fe-triggered lateral root elongation was indicated by Fe-regulated AUX1 promoter activities in lateral root apices and by the failure of aux-1 mutants to elongate lateral roots into Fe-enriched agar patches. Furthermore, a screening was designed to identify novel regulatory components involved in the Fe-dependent stimulation of lateral roots. One member of the GATA family of transcription factors was found to play a role in the local, root-endogenous regulation of lateral root development in response to local supplies of Fe. It was concluded that a Fe sensing mechanism in roots regulates lateral root development by modulating auxin transport. The second part of the thesis describes the use of multi-elemental analyses to identify novel regulators of nutrient accumulation in Arabidopsis. Firstly, it is shown that the disruption of transcription factors expression can lead to significant alterations in the accumulation of one or more nutrients in shoots. In addition, this approach allowed the identification of a so-far uncharacterized transcription factor ? NGAL1 ? that regulates primary root elongation in response to phosphorus (P) supply. The loss of NGAL1 resulted in hypersensitive inhibition of primary root growth under low P and a P-independent increase in lateral root elongation. The results presented here indicate that NGAL1 participates in a signaling pathway that modulates meristematic activity by controlling the expression of important root patterning regulators according to the local availability of P.
Intracellular regulation of Wnt and FGF signal transduction by the late endosomal compartment in Xenopus laevis
(2022) Kreis, Jennifer; Feistel, Kerstin
The endosomal network depicts a vast playground of multiple processing capabilities in terms of signaling. Distinct compartments of the endosomal machinery exert specific functions and thus contribute in signal termination, transduction, attenuation or amplification. Initially, these functions were attributed to early endosomes but recent research likewise considers late endosomes to be just as relevant in mediating such processes. Functionality as well as the molecular identity of these intracellular membranous platforms are orchestrated by a large superfamily of small Ras like GTPases. The collected data of this study particularly highlight the involvement of late endosomes and its associated regulator Rab7 in the early development of the African clawed frog Xenopus laevis. In particular, the first two chapters address the Rab7-dependent specification of the mesodermal germ layer by regulating intracellular pathway activity of Wnt and FGF/MAPK signaling. After fertilization formation of the germ layers is one of the first processes to be initiated. An essential part of mesoderm development comprises subdivision into different mesodermal regions, thus clustering it into ventrolateral and dorsal mesoderm. This patterning is crucial to promote further differentiation into various tissues arising from the mesodermal germ layer. It turned out, Rab7 regulates ventrolateral fates in a Wnt-dependent manner. The small GTPase exerts its function upstream of the Wnt co-transcription factor Ctnnb1 to ensure its nuclear relocalization. In addition to that, Rab7-positive endosomes are likewise required to mediate intracellular FGF/MAPK signal transduction in order to specify dorsal mesoderm. Here, Rab7 regulates proper signaling at the level or downstream of Ras and upstream of Erk/Mapk1. The last chapter then elicits further regulative properties of the late endosomal platform, concerning Cd63 function. The tetraspanin Cd63, which constitutes a transmembrane protein, associates with late endolysosomal compartments and exhibits a similar expression pattern like the small GTPase Rab7 in Xenopus laevis. Contrary to Rab7, function of Cd63 seems to be dispensable whilst gastrulation. However, the presented studies in this chapter suggest a vital function of the tetraspanin Cd63 during axial elongation and correct eye development. Therefore, these investigations regarding Cd63 demonstrated an involvement of the regulative function of late endosomes as signaling platforms for embryonic development beyond mesoderm specification and gastrulation. Overall, the summarized data of this study provides further insights into the determining capacity of Rab7-positive endosomal platforms in intracellular signal transduction of different pathways during early embryonic development.
The formation of an apoplastic diffusion barrier in Arabidopsis seeds is regulated by peptide hormone signaling
(2022) Royek, Stefanie; Schaller, Andreas
Diffusion barrier formation is a critical factor in plant development. The most well described diffusion barriers in Arabidopsis are the Casparian strip and the cuticle. They function in the formation of organ boundaries, prevent water and molecule loss, and protect the plant against environmental stresses. The Casparian strip surrounds the root vascular tissue, whereas the cuticle covers aerial plant organs and is formed de novo during seed development. Embryonic cuticle formation is regulated by a peptide hormone signaling pathway, involving the leucine rich repeat receptor like kinases GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) and the subtilisin-like serine protease ABNORMAL LEAF SHAPE 1 (ALE1). Whereas the latter pathway components have been identified in 2001 and 2008, the peptide hormone mediating the signaling has remained elusive. One aim of this work was to identify the missing pathway element. It was hypothesized that the peptide hormone is released from a larger precursor by ALE1 protease activity to trigger cuticle formation via interaction with the GSO receptors. To uncover the unknown element, the signaling pathway for Casparian strip formation, prooved to be a useful lead. Remarkably, Casparian strip and embryonic cuticle formation employ the same receptor (GSO1), and for Casparian strip formation the GSO1 ligands are known to be members of the CASPARIAN STRIP INTEGRITY FACTOR (CIF) protein family (Doblas et al. 2017, Nakayama et al. 2017). Based on its similarity to the mature CIF peptides and on its phenotypic appearance, it was speculated that a seed expressed protein, called TWISTED SEED1 (TWS1), could serve as the sought ALE1 substrate. As it can be challenging to link proteases to their physiological substrates, this work describes methods how to identify protease specific cleavage sites. One of them was applied to test if TWS1 serves as ALE1 substrate. GFP-tagged TWS1 was transiently coexpressed with ALE1 in Nicotiana benthamiana via agroinfiltration. An ALE1-specific TWS1 cleavage product was detected in the protein extract of coinfiltrated leaves. It was identified by pull down via GFP immunoprecipitation, subsequent separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) analysis. Another method, described in this work, is the identification of protease cleavage sites by in-gel reductive dimethylation: cleavage product-containing gel bands are treated with formaldehyde and cyanoborohydride, prior to in-gel tryptic digest, to achieve a dimethylation of N-terminal free amino groups. The chemically modified N-termini can rapidly be identified and assigned to previous cleavage by the protease of interest. With the method described above, it was found that TWS1 is c-terminally cleaved by ALE1. The two amino acids directly flanking the cleavage site were found to be important for ALE1 cleavage site selection, as their substitution caused a loss of ALE1- dependent cleavage. Our cooperation partners demonstrated an interaction of mature TWS1 with the GSO receptors. The binding affinity of mature TWS1 was reduced by a 3 amino acid C-terminal extension, demonstrating the biological relevance of ALE1-mediated TWS1 processing. Like the CIFs, TWS1 contains a DY tyrosine sulfation motive at its N-terminal processing site. The role of tyrosine sulfation in precursor processing is largely unexplored and was addressed in this work by comparing in-vitro cleavage of different sulfated versus nonsulfated TWS1 precursors. SBT1.8 was found to cleave TWS1 at the N-terminal processing site, and cleavage site selection was influenced by the sulfation state of TWS1 P2´ tyrosine. A homology based 3D model of SBT1.8 was created, which suggested that SBT1.8 interacts with the negatively charged sulfate via a positively charged arginine residue (R302). The role of R302 in substrate binding and recognition was confirmed by in-vitro cleavage assays with mutated SBT1.8 versions, in which R302 was replaced. N-terminal TWS1 cleavage was no longer observed when R302 was substituted. Likewise, no N-terminal cleavage was observed for two other seed expressed Arabidopsis subtilases (SBT1.1 and SBT5.4) that feature an arginine at the corresponding position, indicating that the sole presence of R302 is not sufficient for N terminal cleavage site recognition.