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Functional modulation of LHCSR1 protein from Physcomitrella patens by zeaxanthin binding and low pH.

ABSTRACT: Light harvesting for oxygenic photosynthesis is regulated to prevent the formation of harmful photoproducts by activation of photoprotective mechanisms safely dissipating the energy absorbed in excess. Lumen acidification is the trigger for the formation of quenching states in pigment binding complexes. With the aim to uncover the photoprotective functional states responsible for excess energy dissipation in green algae and mosses, we compared the fluorescence dynamic properties of the light-harvesting complex stress-related (LHCSR1) protein, which is essential for fast and reversible regulation of light use efficiency in lower plants, as compared to the major LHCII antenna protein, which mainly fulfills light harvesting function. Both LHCII and LHCSR1 had a chlorophyll fluorescence yield and lifetime strongly dependent on detergent concentration but the transition from long- to short-living states was far more complete and fast in the latter. Low pH and zeaxanthin binding enhanced the relative amplitude of quenched states in LHCSR1, which were characterized by the presence of 80 ps fluorescence decay components with a red-shifted emission spectrum. We suggest that energy dissipation occurs in the chloroplast by the activation of 80 ps quenching sites in LHCSR1 which spill over excitons from the photosystem II antenna system.

SUBMITTER: Pinnola A

PROVIDER: S-EPMC5593824 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Functional modulation of LHCSR1 protein from Physcomitrella patens by zeaxanthin binding and low pH.

Pinnola Alberta A Ballottari Matteo M Bargigia Ilaria I Alcocer Marcelo M D'Andrea Cosimo C Cerullo Giulio G Bassi Roberto R

Scientific reports 20170911 1

Light harvesting for oxygenic photosynthesis is regulated to prevent the formation of harmful photoproducts by activation of photoprotective mechanisms safely dissipating the energy absorbed in excess. Lumen acidification is the trigger for the formation of quenching states in pigment binding complexes. With the aim to uncover the photoprotective functional states responsible for excess energy dissipation in green algae and mosses, we compared the fluorescence dynamic properties of the light-har ...[more]

PMID: 28894198

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Project description:BackgroundPlant small heat shock proteins (sHsps) accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, in vivo evidence for the involvement of sHsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriate mutants in specific sHsp genes.ResultsIn this study we characterized the function of a sHsp in abiotic stress tolerance in the moss Physcomitrella patens, a model for primitive land plants. Using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from P. patens encoding a 16.4 kDa cytosolic class II sHsp. PpHsp16.4 was also induced by salicylic acid, dithiothreitol (DTT) and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. Expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. PpHsp16.4 is encoded by two identical genes arranged in tandem in the genome. Targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. In vivo localization studies revealed that PpHsp16.4 localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth.ConclusionsWe identified a member of the class II sHsp family that showed hormonal and abiotic stress gene regulation. Induction of the gene by DTT treatment suggests that damaged proteins may act as signals for the stress-induction of PpHsp16.4. The product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. Our study provides the first direct genetic evidence for a role of a sHsp in osmotic and salt stress tolerance, and supports a function for this protein particularly during the stress recovery stage of P. patens.

Project description:BackgroundThe moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2.ResultsOur results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis.ConclusionsOur study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.

Dataset Information

Functional modulation of LHCSR1 protein from Physcomitrella patens by zeaxanthin binding and low pH.

Publications

Functional modulation of LHCSR1 protein from Physcomitrella patens by zeaxanthin binding and low pH.

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