4 research outputs found
Comparative analysis of thylakoid protein complexes in state transition mutants nsi and stn7: focus on PSI and LHCII
The photosynthetic machinery of plants can acclimate to changes in light
conditions by balancing light-harvesting between the two photosystems
(PS). This acclimation response is induced by the change in the redox
state of the plastoquinone pool, which triggers state transitions
through activation of the STN7 kinase and subsequent phosphorylation of
light-harvesting complex II (LHCII) proteins. Phosphorylation of LHCII
results in its association with PSI (state 2), whereas dephosphorylation
restores energy allocation to PSII (state 1). In addition to state
transition regulation by phosphorylation, we have recently discovered
that plants lacking the chloroplast acetyltransferase NSI are also
locked in state 1, even though they possess normal LHCII
phosphorylation. This defect may result from decreased lysine
acetylation of several chloroplast proteins. Here, we compared the
composition of wild type (wt), stn7 and nsi thylakoid
protein complexes involved in state transitions separated by Blue Native
gel electrophoresis. Protein complex composition and relative protein
abundances were determined by LCâMS/MS analyses using iBAQ
quantification. We show that despite obvious mechanistic differences
leading to defects in state transitions, no major differences were
detected in the composition of PSI and LHCII between the mutants.
Moreover, both stn7 and nsi plants show retarded growth
and decreased PSII capacity under fluctuating light as compared to wt,
while the induction of non-photochemical quenching under fluctuating
light was much lower in both nsi mutants than in stn7.</p
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THE ROLE OF KEA1 AND KEA2 TRANSPORTERS IN PLASTID ION HOMEOSTASIS AND GENE EXPRESSION
The maintenance of ion gradients across the chloroplast envelope plays a key role in the bioenergetics of photosynthesis by moderating proton motive force and membrane dynamics. However, this research supports the novel finding that plastid ion transporters are additionally involved in maintaining plastid gene expression (PGE), likely by impacting the stromal buffer conditions required for function of plastid RNA binding proteins. We discovered this by investigating mutant Arabidopsis thaliana lines with loss-of-function of two inner envelope membrane potassium-proton (K+/H+) antiporters KEA1 and KEA2. Simultaneous loss of both transporters results in a unique âvirescentâ phenotype in which young leaves have disproportionately lower photosynthetic efficiency, chlorophyll production, and underdeveloped chloroplasts compared to older leaves. The goal of this research was to determine how the loss of KEA1/2 transporters results in this peculiar chloroplast developmental phenotype. Preliminary experiments using Total-reflection X-ray Fluorescence (TXRF) revealed that loss of KEA1/2 perturbs overall plastid ion homeostasis. An analysis of nuclear and plastome gene expression revealed significant defects in plastid ribosomal RNA processing in kea1kea2 mutant lines. This likely results from altered stromal ion concentrations inhibiting the function of nuclear-encoded chloroplast RNA binding proteins involved in plastid gene expression. This defect coincided with decreased steady-state levels of photosynthesis-related proteins, and lower translation rates in the stroma. We also discovered that plastid-to-nucleus retrograde signaling protein Genomes Uncoupled 1 (GUN1) was essential to seedling survival in kea1kea2. Loss of GUN1 in the kea1kea2 background resulted in higher expression of many nuclear-encoded photosynthesis-associated genes which are normally suppressed in response to disruption of PGE. In summary, ionome-induced impairment of plastid gene expression and subsequent retrograde signaling to suppress nuclear gene expression culminates in the virescent phenotype displayed by the kea1kea2 mutants. These findings underscore the importance of ion transporters in chloroplast development
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Characterization of filamentâforming CTP synthases from Arabidopsis thaliana
Summary
Cytidine triphosphate (CTP) is essential for DNA, RNA and phospholipid biosynthesis. De novo synthesis is catalyzed by CTP synthases (CTPS). Arabidopsis encodes five CTPS isoforms that unanimously share conserved motifs found across kingdoms, suggesting all five are functional enzymes. Whereas CTPS1â4 are expressed throughout Arabidopsis tissues, CTPS5 reveals exclusive expression in developing embryos. CTPS activity and substrates affinities were determined for a representative plant enzyme on purified recombinant CTPS3 protein. As demonstrated in model organisms such as yeast, fruit fly and mammals, CTPS show the capacity to assemble into large filaments called cytoophidia. Transient expression of Nâ and Câterminal YFPâCTPS fusion proteins in Nicotiana benthamiana allowed to monitor such filament formation. Interestingly, CTPS1 and 2 always appeared as soluble proteins, whereas filaments were observed for CTPS3, 4 and 5 independent of the YFPâtag location. However, when similar constructs were expressed in Saccharomyces cerevisiae, no filaments were observed, pointing to a requirement for organismâspecific factors in vivo. Indications for filament assembly were also obtained in vitro when recombinant CTPS3 protein was incubated in the presence of CTP. TâDNAâinsertion mutants in four CTPS loci revealed no apparent phenotypical alteration. In contrast, CTPS2 TâDNAâinsertion mutants did not produce homozygous progenies. An initial characterization of the CTPS protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, Drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTPS activity.
Significance Statement
An initial characterization of the CTP synthase protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTP Synthase activity