Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (E_GSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in E_GSH and H₂O₂ levels with the genetically encoded biosensors Grx1-roGFP2 (for E_GSH) and roGFP2-Orp1 (for H₂O₂) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Summary: In natural ecosystems, plants compete for space, nutrients and light. The optically dense canopies limit the penetration of photosynthetically active radiation and light often becomes a growth‐limiting factor for the understory. The reduced availability of photons in the lower leaf layers is also a major constraint for yield potential in canopies of crop monocultures. Traditionally, crop breeding has selected traits related to plant architecture and nutrient assimilation rather than light use efficiency. Leaf optical density is primarily determined by tissue morphology and by the foliar concentration of photosynthetic pigments (chlorophylls and carotenoids). Most pigment molecules are bound to light‐harvesting antenna proteins in the chloroplast thylakoid membranes, where they serve photon capture and excitation energy transfer toward reaction centers of photosystems. Engineering the abundance and composition of antenna proteins has been suggested as a strategy to improve light distribution within canopies and reduce the gap between theoretical and field productivity. Since the assembly of the photosynthetic antennas relies on several coordinated biological processes, many genetic targets are available for modulating cellular chlorophyll levels. In this review, we outline the rationale behind the advantages of developing pale green phenotypes and describe possible approaches toward engineering light‐harvesting systems.

Photosynthetic microbes are gaining increasing attention as heterologous hosts for the light-driven, low-cost production of high-value recombinant proteins. Recent advances in the manipulation of unicellular algal genomes offer the opportunity to establish engineered strains as safe and viable alternatives to conventional heterotrophic expression systems, including for their use in the feed, food, and biopharmaceutical industries. Due to the relatively small size of their genomes, algal chloroplasts are excellent targets for synthetic biology approaches, and are convenient subcellular sites for the compartmentalized accumulation and storage of products. Different classes of recombinant proteins, including enzymes and peptides with therapeutical applications, have been successfully expressed in the plastid of the model organism Chlamydomonas reinhardtii, and of a few other species, highlighting the emerging potential of transplastomic algal biotechnology. In this review, we provide a unified view on the state-of-the-art tools that are available to introduce protein-encoding transgenes in microalgal plastids, and discuss the main (bio)technological bottlenecks that still need to be addressed to develop robust and sustainable green cell biofactories.

Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana, CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca²⁺-dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.

Heterologous expression of the NAD⁺-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD⁺/NADP⁺ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP⁺ and NAD⁺ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance.

From sea shores to the abysses of the deep ocean, marine ecosystems have provided humanity with valuable medicinal resources. The use of marine organisms is discussed in ancient pharmacopoeias of different times and geographic regions and is still deeply rooted in traditional medicine. Thanks to present-day, large-scale bioprospecting and rigorous screening for bioactive metabolites, the ocean is coming back as an untapped resource of natural compounds with therapeutic potential. This renewed interest in marine drugs is propelled by a burgeoning research field investigating the molecular mechanisms by which newly identified compounds intervene in the pathophysiology of human diseases. Of great clinical relevance are molecules endowed with anti-inflammatory and immunomodulatory properties with emerging applications in the management of chronic inflammatory disorders, autoimmune diseases, and cancer. Here, we review the historical development of marine pharmacology in the Eastern and Western worlds and describe the status of marine drug discovery. Finally, we discuss the importance of conducting sustainable exploitation of marine resources through biotechnology.