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postgraduate thesis: Functional study of two ferredoxin homologs with extended C-termini in Arabidopsis thaliana

TitleFunctional study of two ferredoxin homologs with extended C-termini in Arabidopsis thaliana
Authors
Issue Date2016
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Guan, X. [官小茜]. (2016). Functional study of two ferredoxin homologs with extended C-termini in Arabidopsis thaliana. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractFerredoxins, the small ancient [2Fe-2S] cluster-containing proteins at the stroma of thylakoid membrane, transfer electrons from PSI to multiple downstream redox-driven enzymes. They support the assimilation of carbon, nitrogen, and sulfur; the synthesis of amino acids and fatty acids; and the modulation of the ATP/NADPH ratio by switching between photosynthetic cyclic and linear electron routes. In the genome of Arabidopsis thaliana, four functionally diverse ferredoxins (Fds) have been classified as leaf-type (AtFd1, At1g10960; AtFd2, At1g60950) and root-type (AtFd3, At2g27510; AtFd4, At5g10000). Recently, two novel ferredoxin homologs with extended C-termini have been found and termed ferredoxin C 1 and 2 (FdC1, At4g14890; FdC2, At1g32550). FdC1 was presumed to be a substitute electron acceptor of PSI in extremely ferredoxin-deficient conditions (Voss et al. 2011), and FdC2 and its homologs in rice, were essential for the assimilation of copper and the synthesis of chloroplast grana stacks and chlorophyll (Goss 2014, Li et al. 2015, Zhao et al. 2015). However, the functional differences between FdCs and well-identified leaf-type ferredoxins in electron transfer remain obscure. Here, to explore the functions of these two novel Fd homologs with C-termini, a study adopting biochemical, histological, biomolecular, and genetic methodologies was performed. The phylogenic analysis has distinguished the divergent evolutionary distances between two FdC isoforms. FdC2 appeared in cyanobacteria while FdC1 appeared later in green algae. They were identified as chloroplast proteins by the eYFP approach in tobacco leaves. The spatial-temporal expression patterns of both FdCs in Arabidopsis were investigated by in silico expression profiling, immunoblotting, and promoter-GUS analysis. Both FdCs were detected in chlorenchyma, flower, and siliques throughout the life cycles. Additionally, FdC1 was detected in roots and anthers. The consistent results of yeast two-hybrid and bimolecular fluorescence complementation showed that two FdC isoforms can differentially interact with the PSI “stromal bridge” subunits (PsaC, D, and E), which was confirmed by their ability to receive electrons from PSI to support the photoreduction of cytochrome c. FdCs also shared some but not all of the electron receptors of both leaf-type Fds, including ferredoxin-thioredoxin reductase A/B, sulfite reductase, and nitrite reductase but not leaf-type and root-type FNRs, which was in agreement with the in vitro enzymatic assays on nitrite reductase, sulfite reductase, and the photoreduction of NADP^+. FdC1 exhibited a wider range of downstream electron acceptors than FdC2. This could be explained by the conserved negatively-charged residues and specific hydrophobic residues in the proposed three-dimensional complexes containing FdCs and their interacting partners based on the well-resolved crystal structures. Though no morphological changes were observed, the overexpression or suppression of FdC has significantly affected the chlorophyll fluorescence parameters in transgenic plants. To summarize, the heterogeneity of PsaD and PsaE which evolved in higher plants might function as branching points of electron flows to different acceptors (Fd, FdC1, and FdC2) with varied binding affinities, to regulate the routes of photosynthetic electron transfer to downstream biological processes in plastids, such as the assimilation of sulfate and nitrate, and the regulation of light-dependent enzymes in the Calvin cycle.
DegreeDoctor of Philosophy
SubjectArabidopsis thaliana - Genetics
Dept/ProgramBiological Sciences
Persistent Identifierhttp://hdl.handle.net/10722/250349
HKU Library Item IDb5838460

 

DC FieldValueLanguage
dc.contributor.authorGuan, Xiaoqian-
dc.contributor.author官小茜-
dc.date.accessioned2017-12-30T15:01:03Z-
dc.date.available2017-12-30T15:01:03Z-
dc.date.issued2016-
dc.identifier.citationGuan, X. [官小茜]. (2016). Functional study of two ferredoxin homologs with extended C-termini in Arabidopsis thaliana. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/250349-
dc.description.abstractFerredoxins, the small ancient [2Fe-2S] cluster-containing proteins at the stroma of thylakoid membrane, transfer electrons from PSI to multiple downstream redox-driven enzymes. They support the assimilation of carbon, nitrogen, and sulfur; the synthesis of amino acids and fatty acids; and the modulation of the ATP/NADPH ratio by switching between photosynthetic cyclic and linear electron routes. In the genome of Arabidopsis thaliana, four functionally diverse ferredoxins (Fds) have been classified as leaf-type (AtFd1, At1g10960; AtFd2, At1g60950) and root-type (AtFd3, At2g27510; AtFd4, At5g10000). Recently, two novel ferredoxin homologs with extended C-termini have been found and termed ferredoxin C 1 and 2 (FdC1, At4g14890; FdC2, At1g32550). FdC1 was presumed to be a substitute electron acceptor of PSI in extremely ferredoxin-deficient conditions (Voss et al. 2011), and FdC2 and its homologs in rice, were essential for the assimilation of copper and the synthesis of chloroplast grana stacks and chlorophyll (Goss 2014, Li et al. 2015, Zhao et al. 2015). However, the functional differences between FdCs and well-identified leaf-type ferredoxins in electron transfer remain obscure. Here, to explore the functions of these two novel Fd homologs with C-termini, a study adopting biochemical, histological, biomolecular, and genetic methodologies was performed. The phylogenic analysis has distinguished the divergent evolutionary distances between two FdC isoforms. FdC2 appeared in cyanobacteria while FdC1 appeared later in green algae. They were identified as chloroplast proteins by the eYFP approach in tobacco leaves. The spatial-temporal expression patterns of both FdCs in Arabidopsis were investigated by in silico expression profiling, immunoblotting, and promoter-GUS analysis. Both FdCs were detected in chlorenchyma, flower, and siliques throughout the life cycles. Additionally, FdC1 was detected in roots and anthers. The consistent results of yeast two-hybrid and bimolecular fluorescence complementation showed that two FdC isoforms can differentially interact with the PSI “stromal bridge” subunits (PsaC, D, and E), which was confirmed by their ability to receive electrons from PSI to support the photoreduction of cytochrome c. FdCs also shared some but not all of the electron receptors of both leaf-type Fds, including ferredoxin-thioredoxin reductase A/B, sulfite reductase, and nitrite reductase but not leaf-type and root-type FNRs, which was in agreement with the in vitro enzymatic assays on nitrite reductase, sulfite reductase, and the photoreduction of NADP^+. FdC1 exhibited a wider range of downstream electron acceptors than FdC2. This could be explained by the conserved negatively-charged residues and specific hydrophobic residues in the proposed three-dimensional complexes containing FdCs and their interacting partners based on the well-resolved crystal structures. Though no morphological changes were observed, the overexpression or suppression of FdC has significantly affected the chlorophyll fluorescence parameters in transgenic plants. To summarize, the heterogeneity of PsaD and PsaE which evolved in higher plants might function as branching points of electron flows to different acceptors (Fd, FdC1, and FdC2) with varied binding affinities, to regulate the routes of photosynthetic electron transfer to downstream biological processes in plastids, such as the assimilation of sulfate and nitrate, and the regulation of light-dependent enzymes in the Calvin cycle.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.subject.lcshArabidopsis thaliana - Genetics-
dc.titleFunctional study of two ferredoxin homologs with extended C-termini in Arabidopsis thaliana-
dc.typePG_Thesis-
dc.identifier.hkulb5838460-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiological Sciences-
dc.description.naturepublished_or_final_version-

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