Rerouting of ribosomal proteins into splicing in plant organelles.

Production and expression of RNA requires the action of multiple RNA-binding proteins (RBPs). New RBPs are most often created by novel combinations of dedicated RNA-binding modules. However, recruiting existing genes to create new RBPs is also an important evolutionary strategy. In this report, we analyzed the eight-member uL18 ribosomal protein family in Arabidopsis uL18 proteins share a short structurally conserved domain that binds the 5S ribosomal RNA (rRNA) and allows its incorporation into ribosomes. Our results indicate that Arabidopsis uL18-Like proteins are targeted to either mitochondria or chloroplasts. While two members of the family are found in organelle ribosomes, we show here that two uL18-type proteins function as factors necessary for the splicing of certain mitochondrial and plastid group II introns. These two proteins do not cosediment with mitochondrial or plastid ribosomes but instead associate with the introns whose splicing they promote. Our study thus reveals that the RNA-binding capacity of uL18 ribosomal proteins has been repurposed to create factors that facilitate the splicing of organellar introns.

Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self-sufficient for weeks.

During the life cycle of plants, seedlings are considered vulnerable because they are at the interface between the highly stress tolerant seed embryos and the established plant, and must develop rapidly, often in a challenging environment, with limited access to nutrients and light. Using a simple experimental system, whereby the seedling stage of Arabidopsis is considerably prolonged by nutrient starvation, we analysed the physiology and metabolism of seedlings maintained in such conditions up to 4 weeks. Although development was arrested at the cotyledon stage, there was no sign of senescence and seedlings remained viable for weeks, yielding normal plants after transplantation. Photosynthetic activity compensated for respiratory carbon losses, and energy dissipation by photorespiration and alternative oxidase appeared important. Photosynthates were essentially stored as organic acids, while the pool of free amino acids remained stable. Seedlings lost the capacity to store lipids in cytosolic lipid droplets, but developed large plastoglobuli. Arabidopsis seedlings arrested in their development because of mineral starvation displayed therefore a remarkable resilience, using their metabolic and physiological plasticity to maintain a steady state for weeks, allowing resumption of development when favourable conditions ensue.

The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins.

The small heat shock proteins (sHSPs) are molecular chaperones that share an alpha-crystallin domain but display a high diversity of sequence, expression, and localization. They are especially prominent in plants, populating most cellular compartments. In pea, mitochondrial HSP22 is induced by heat or oxidative stress in leaves but also strongly accumulates during seed development. The molecular function of HSP22 was addressed by studying the effect of temperature on its structural properties and chaperone effects using a recombinant or native protein. Overexpression of HSP22 significantly increased bacterial thermotolerance. The secondary structure of the recombinant protein was not affected by temperature in contrast with its quaternary structure. The purified protein formed large polydisperse oligomers that dissociated upon heating (42 °C) into smaller species (mainly monomers). The recombinant protein appeared thermosoluble but precipitated with thermosensitive proteins upon heat stress in assays either with single protein clients or within complex extracts. As shown by in vitro protection assays, HSP22 at high molar ratio could partly prevent the heat aggregation of rhodanese but not of malate dehydrogenase. HSP22 appears as a holdase that could possibly prevent the aggregation of some proteins while co-precipitating with others to facilitate their subsequent refolding by disaggregases or clearance by proteases.

Decoding the Divergent Subcellular Location of Two Highly Similar Paralogous LEA Proteins.

Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA) from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement), we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene.

Measuring CO2 and HCO3- permeabilities of isolated chloroplasts using a MIMS-18O approach.

To support photosynthetic CO2 fixation by Rubisco, the chloroplast must be fed with inorganic carbon in the form of CO2 or bicarbonate. However, the mechanisms allowing the rapid passage of this gas and this charged molecule through the bounding membranes of the chloroplast envelope are not yet completely elucidated. We describe here a method allowing us to measure the permeability of these two molecules through the chloroplast envelope using a membrane inlet mass spectrometer and 18O-labelled inorganic carbon. We established that the internal stromal carbonic anhydrase activity is not limiting for this technique, and precisely measured the chloroplast surface area and permeability values for CO2 and bicarbonate. This was performed on chloroplasts from several plant species, with values ranging from 2.3 × 10-4 m s-1 to 8 × 10-4 m s-1 permeability for CO2 and 1 × 10-8 m s-1 for bicarbonate. We were able to apply our method to chloroplasts from an Arabidopsis aquaporin mutant, and this showed that CO2 permeability was reduced 50% in the mutant compared with the wild-type reference.

Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency.

The pyrenoid of the unicellular green alga Chlamydomonas reinhardtii is a microcompartment situated in the centre of the cup-shaped chloroplast, containing up to 90% of cellular Rubisco. Traversed by a network of dense, knotted thylakoid tubules, the pyrenoid has been proposed to influence thylakoid biogenesis and ultrastructure. Mutants that are unable to assemble a pyrenoid matrix, due to expressing a vascular plant version of the Rubisco small subunit, exhibit severe growth and photosynthetic defects and have an ineffective carbon-concentrating mechanism (CCM). The present study set out to determine the cause of photosynthetic limitation in these pyrenoid-less lines. We tested whether electron transport and light use were compromised as a direct structural consequence of pyrenoid loss or as a metabolic effect downstream of lower CCM activity and resulting CO2 limitation. Thylakoid organization was unchanged in the mutants, including the retention of intrapyrenoid-type thylakoid tubules, and photosynthetic limitations associated with the absence of the pyrenoid were rescued by exposing cells to elevated CO2 levels. These results demonstrate that Rubisco aggregation in the pyrenoid functions as an essential element for CO2 delivery as part of the CCM, and does not play other roles in maintenance of photosynthetic membrane energetics.

Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii.

During oxygenic photosynthesis, metabolic reactions of CO2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)-mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand.

Symbiodinium transcriptome and global responses of cells to immediate changes in light intensity when grown under autotrophic or mixotrophic conditions.

Symbiosis between unicellular dinoflagellates (genus Symbiodinium) and their cnidarian hosts (e.g. corals, sea anemones) is the foundation of coral reef ecosystems. Dysfunction of this symbiosis under changing environmental conditions has led to global reef decline. Little information is known about Symbiodinium gene expression and mechanisms by which light impacts host-symbiont associations. To address these issues, we generated a transcriptome from axenic Symbiodinium strain SSB01. Here we report features of the transcriptome, including occurrence and length distribution of spliced leader sequences, the functional landscape of encoded proteins and the impact of light on gene expression. Expression of many Symbiodinium genes appears to be significantly impacted by light. Transcript encoding cryptochrome 2 declined in high light while some transcripts for Regulators of Chromatin Condensation (RCC1) declined in the dark. We also identified a transcript encoding a light harvesting AcpPC protein with homology to Chlamydomonas LHCSR2. The level of this transcript increased in high light autotrophic conditions, suggesting that it is involved in photo-protection and the dissipation of excess absorbed light energy. The most extensive changes in transcript abundances occurred when the algae were transferred from low light to darkness. Interestingly, transcripts encoding several cell adhesion proteins rapidly declined following movement of cultures to the dark, which correlated with a dramatic change in cell surface morphology, likely reflecting the complexity of the extracellular matrix. Thus, light-sensitive cell adhesion proteins may play a role in establishing surface architecture, which may in turn alter interactions between the endosymbiont and its host.

Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance.

LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms.

Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii.

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H2 production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H2 photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H2 production.

The Arabidopsis leaf quantitative atlas: a cellular and subcellular mapping through unified data integration.

Quantitative analyses and models are required to connect a plant's cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels.

Tailoring confocal microscopy for real-time analysis of photosynthesis at single-cell resolution.

Photoautotrophs' environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single-cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology based on the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developing tissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.

Improving photosynthetic efficiency toward food security: Strategies, advances, and perspectives.

Photosynthesis in crops and natural vegetation allows light energy to be converted into chemical energy and thus forms the foundation for almost all terrestrial trophic networks on Earth. The efficiency of photosynthetic energy conversion plays a crucial role in determining the portion of incident solar radiation that can be used to generate plant biomass throughout a growth season. Consequently, alongside the factors such as resource availability, crop management, crop selection, maintenance costs, and intrinsic yield potential, photosynthetic energy use efficiency significantly influences crop yield. Photosynthetic efficiency is relevant to sustainability and food security because it affects water use efficiency, nutrient use efficiency, and land use efficiency. This review focuses specifically on the potential for improvements in photosynthetic efficiency to drive a sustainable increase in crop yields. We discuss bypassing photorespiration, enhancing light use efficiency, harnessing natural variation in photosynthetic parameters for breeding purposes, and adopting new-to-nature approaches that show promise for achieving unprecedented gains in photosynthetic efficiency.

Photosynthetic Light Harvesting and Thylakoid Organization in a CRISPR/Cas9 Arabidopsis Thaliana LHCB1 Knockout Mutant.

Light absorbed by chlorophylls of Photosystems II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is trimeric LHCII. In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of LHCII. In absence of LHCB1, the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss, leading to a pale green phenotype and growth delay. The Photosystem II absorption cross-section was smaller, while the Photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring Photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by lower Photosystem I over Photosystem II reaction center ratio and by the dephosphorylation of LHCII and Photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knockout lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state.

Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic alpha-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.

Auxiliary electron transport pathways in chloroplasts of microalgae.

Microalgae are photosynthetic organisms which cover an extraordinary phylogenic diversity and have colonized extremely diverse habitats. Adaptation to contrasted environments in terms of light and nutrient's availabilities has been possible through a high flexibility of the photosynthetic machinery. Indeed, optimal functioning of photosynthesis in changing environments requires a fine tuning between the conversion of light energy by photosystems and its use by metabolic reaction, a particularly important parameter being the balance between phosphorylating (ATP) and reducing (NADPH) power supplies. In addition to the main route of electrons operating during oxygenic photosynthesis, called linear electron flow or Z scheme, auxiliary routes of electron transfer in interaction with the main pathway have been described. These reactions which include non-photochemical reduction of intersystem electron carriers, cyclic electron flow around PSI, oxidation by molecular O(2) of the PQ pool or of the PSI electron acceptors, participate in the flexibility of photosynthesis by avoiding over-reduction of electron carriers and modulating the NADPH/ATP ratio depending on the metabolic demand. Forward or reverse genetic approaches performed in model organisms such as Arabidopsis thaliana for higher plants, Chlamydomonas reinhardtii for green algae and Synechocystis for cyanobacteria allowed identifying molecular components involved in these auxiliary electron transport pathways, including Ndh-1, Ndh-2, PGR5, PGRL1, PTOX and flavodiiron proteins. In this article, we discuss the diversity of auxiliary routes of electron transport in microalgae, with particular focus in the presence of these components in the microalgal genomes recently sequenced. We discuss how these auxiliary mechanisms of electron transport may have contributed to the adaptation of microalgal photosynthesis to diverse and changing environments.

Coral bleaching independent of photosynthetic activity.

The global decline of reef-building corals is due in part to the loss of algal symbionts, or "bleaching," during the increasingly frequent periods of high seawater temperatures. During bleaching, endosymbiotic dinoflagellate algae (Symbiodinium spp.) either are lost from the animal tissue or lose their photosynthetic pigments, resulting in host mortality if the Symbiodinium populations fail to recover. The >1,000 studies of the causes of heat-induced bleaching have focused overwhelmingly on the consequences of damage to algal photosynthetic processes, and the prevailing model for bleaching invokes a light-dependent generation of toxic reactive oxygen species (ROS) by heat-damaged chloroplasts as the primary trigger. However, the precise mechanisms of bleaching remain unknown, and there is evidence for involvement of multiple cellular processes. In this study, we asked the simple question of whether bleaching can be triggered by heat in the dark, in the absence of photosynthetically derived ROS. We used both the sea anemone model system Aiptasia and several species of reef-building corals to demonstrate that symbiont loss can occur rapidly during heat stress in complete darkness. Furthermore, we observed damage to the photosynthetic apparatus under these conditions in both Aiptasia endosymbionts and cultured Symbiodinium. These results do not directly contradict the view that light-stimulated ROS production is important in bleaching, but they do show that there must be another pathway leading to bleaching. Elucidation of this pathway should help to clarify bleaching mechanisms under the more usual conditions of heat stress in the light.

Hydrogen production in Chlamydomonas: photosystem II-dependent and -independent pathways differ in their requirement for starch metabolism.

Under sulfur deprivation conditions, the green alga Chlamydomonas reinhardtii produces hydrogen in the light in a sustainable manner thanks to the contribution of two pathways, direct and indirect. In the direct pathway, photosystem II (PSII) supplies electrons to hydrogenase through the photosynthetic electron transport chain, while in the indirect pathway, hydrogen is produced in the absence of PSII through a photosystem I-dependent process. Starch metabolism has been proposed to contribute to both pathways by feeding respiration and maintaining anoxia during the direct pathway and by supplying reductants to the plastoquinone pool during the indirect pathway. At variance with this scheme, we report that a mutant lacking starch (defective for sta6) produces similar hydrogen amounts as the parental strain in conditions of sulfur deprivation. However, when PSII is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, conditions where hydrogen is produced by the indirect pathway, hydrogen production is strongly reduced in the starch-deficient mutant. We conclude that starch breakdown contributes to the indirect pathway by feeding electrons to the plastoquinone pool but is dispensable for operation of the direct pathway that prevails in the absence of DCMU. While hydrogenase induction was strongly impaired in the starch-deficient mutant under dark anaerobic conditions, wild-type-like induction was observed in the light. Because this light-driven hydrogenase induction is DCMU insensitive and strongly inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, we conclude that this process is regulated by the proton gradient generated by cyclic electron flow around PSI.

Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation.

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.

Functional divergence of former alleles in an ancient asexual invertebrate.

Theory suggests it should be difficult for asexual organisms to adapt to a changing environment because genetic diversity can only arise from mutations accumulating within direct antecedents and not through sexual exchange. In an asexual microinvertebrate, the bdelloid rotifer, we have observed a mechanism by which such organisms could acquire the diversity needed for adaptation. Gene copies most likely representing former alleles have diverged in function so that the proteins they encode play complementary roles in survival of dry conditions. One protein prevents desiccation-sensitive enzymes from aggregating during drying, whereas its counterpart does not have this activity, but is able to associate with phospholipid bilayers and is potentially involved in maintenance of membrane integrity. The functional divergence of former alleles observed here suggests that adoption of asexual reproduction could itself be an evolutionary mechanism for the generation of diversity.