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The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro.

ABSTRACT: Bacterial cell and chloroplast division are driven by a contractile "Z ring" composed of the tubulin-like cytoskeletal GTPase FtsZ. Unlike bacterial Z rings, which consist of a single FtsZ, the chloroplast Z ring in plants is composed of two FtsZ proteins, FtsZ1 and FtsZ2. Both are required for chloroplast division in vivo, but their biochemical relationship is poorly understood. We used GTPase assays, light scattering, transmission electron microscopy, and sedimentation assays to investigate the assembly behavior of purified Arabidopsis thaliana (At) FtsZ1 and AtFtsZ2 both individually and together. Both proteins exhibited GTPase activity. AtFtsZ2 assembled relatively quickly, forming protofilament bundles that were exceptionally stable, as indicated by their sustained assembly and slow disassembly. AtFtsZ1 did not form detectable protofilaments on its own. When mixed with AtFtsZ2, AtFtsZ1 reduced the extent and rate of AtFtsZ2 assembly, consistent with its previously demonstrated ability to promote protofilament subunit turnover in living cells. Mixing the two FtsZ proteins did not increase the overall GTPase activity, indicating that the effect of AtFtsZ1 on AtFtsZ2 assembly was not due to a stimulation of GTPase activity. However, the GTPase activity of AtFtsZ1 was required to reduce AtFtsZ2 assembly. Truncated forms of AtFtsZ1 and AtFtsZ2 consisting of only their conserved core regions largely recapitulated the behaviors of the full-length proteins. Our in vitro findings provide evidence that FtsZ1 counterbalances the stability of FtsZ2 filaments in the regulation of chloroplast Z-ring dynamics and suggest that restraining FtsZ2 self-assembly is a critical function of FtsZ1 in chloroplasts.

SUBMITTER: Porter KJ

PROVIDER: S-EPMC8142252 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 CTPS1-4 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 N- and C-terminal YFP-CTPS 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 YFP-tag location. However, when similar constructs were expressed in Saccharomyces cerevisiae, no filaments were observed, pointing to a requirement for organism-specific factors in vivo. Indications for filament assembly were also obtained in vitro when recombinant CTPS3 protein was incubated in the presence of CTP. T-DNA-insertion mutants in four CTPS loci revealed no apparent phenotypical alteration. In contrast, CTPS2 T-DNA-insertion 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.

Project description:Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops.

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The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro.

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