Dataset Information

Chloroplast-localized GUN1 contributes to the acquisition of basal thermotolerance in Arabidopsis thaliana.

ABSTRACT: Heat stress (HS) severely affects different cellular compartments operating in metabolic processes and represents a critical threat to plant growth and yield. Chloroplasts are crucial for heat stress response (HSR), signaling to the nucleus the environmental challenge and adjusting metabolic and biosynthetic functions accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein, has been recognized as one of the main players of chloroplast retrograde signaling. Here, we investigate HSR in Arabidopsis wild-type and gun1 plantlets subjected to 2 hours of HS at 45°C. In wild-type plants, Reactive Oxygen Species (ROS) accumulate promptly after HS, contributing to transiently oxidize the cellular environment and acting as signaling molecules. After 3 hours of physiological recovery at growth temperature (22°C), the induction of enzymatic and non-enzymatic antioxidants prevents oxidative damage. On the other hand, gun1 mutants fail to induce the oxidative burst immediately after HS and accumulate ROS and oxidative damage after 3 hours of recovery at 22°C, thus resulting in enhanced sensitivity to HS. These data suggest that GUN1 is required to oxidize the cellular environment, participating in the acquisition of basal thermotolerance through the redox-dependent plastid-to-nucleus communication.

SUBMITTER: Lasorella C

PROVIDER: S-EPMC9813751 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Chloroplast-localized GUN1 contributes to the acquisition of basal thermotolerance in <i>Arabidopsis thaliana</i>.

Lasorella Cecilia C Fortunato Stefania S Dipierro Nunzio N Jeran Nicolaj N Tadini Luca L Vita Federico F Pesaresi Paolo P de Pinto Maria Concetta MC

Frontiers in plant science 20221222

Heat stress (HS) severely affects different cellular compartments operating in metabolic processes and represents a critical threat to plant growth and yield. Chloroplasts are crucial for heat stress response (HSR), signaling to the nucleus the environmental challenge and adjusting metabolic and biosynthetic functions accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein, has been recognized as one of the main players of chloroplast retrograde signaling. Here, we investigate H ...[more]

PMID: 36618674

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Project description:BackgroundThe Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.Methodology/principal findingsTransient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.Conclusions/significanceWe show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.

Project description:Heat stress (HS) seriously restricts the growth and development of plants. When plants are exposed to extreme high temperature, the heat stress response (HSR) is activated to enable plants to survive. Sessile plants have evolved multiple strategies to sense and cope with HS. Previous studies have established that PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts as a key component in thermomorphogenesis; however, whether PIF4 regulates plant thermotolerance and the molecular mechanism linking this light transcriptional factor and HSR remain unclear. Here, we show that the overexpression of PIF4 indeed provides plants with a stronger basal thermotolerance and greatly improves the survival ability of Arabidopsis under severe HS. Via phylogenetic analysis, we identified two sets (six) of PIF4 homologs in wheat, and the expression patterns of the PIF4 homologs were conservatively induced by heat treatment in both wheat and Arabidopsis. Furthermore, the PIF4 protein was accumulated under heat stress and had an identical expression level. Additionally, we found that the core regulator of HSR, HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), was highly responsive to light and heat. Followed by promoter analysis and ChIP-qPCR, we further found that PIF4 can bind directly to the G-box motifs of the HSFA2 promoter. Via effector-reporter assays, we found that PIF4 binding could activate HSFA2 gene expression, thereby resulting in the activation of other HS-inducible genes, such as heat shock proteins. Finally, the overexpression of PIF4 led to a stronger basal thermotolerance under non-heat-treatment conditions, thereby resulting in an enhanced tolerance to severe heat stress. Taken together, our findings propose that PIF4 is linked to heat stress signaling by directly binding to the HSFA2 promoter and triggering the HSR at normal temperature conditions to promote the basal thermotolerance. These functions of PIF4 provide a candidate direction for breeding heat-resistant crop cultivars.

Dataset Information

Chloroplast-localized GUN1 contributes to the acquisition of basal thermotolerance in Arabidopsis thaliana.

Publications

Chloroplast-localized GUN1 contributes to the acquisition of basal thermotolerance in <i>Arabidopsis thaliana</i>.

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