Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.

The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The improvement of CRISPR-based technologies efficiency will enable the implementation of genome editing tools in a variety of crop plants, as well as accelerate progress in basic research and molecular breeding.

The OPR Protein MTHI1 Controls the Expression of Two Different Subunits of ATP Synthase CFo in Chlamydomonas reinhardtii.

In the green alga Chlamydomonas (Chlamydomonas r einhardtii), chloroplast gene expression is tightly regulated posttranscriptionally by gene-specific trans-acting protein factors. Here, we report the identification of the octotricopeptide repeat protein MTHI1, which is critical for the biogenesis of chloroplast ATP synthase oligomycin-sensitive chloroplast coupling factor. Unlike most trans-acting factors characterized so far in Chlamydomonas, which control the expression of a single gene, MTHI1 targets two distinct transcripts: it is required for the accumulation and translation of atpH mRNA, encoding a subunit of the selective proton channel, but it also enhances the translation of atpI mRNA, which encodes the other subunit of the channel. MTHI1 targets the 5' untranslated regions of both the atpH and atpI genes. Coimmunoprecipitation and small RNA sequencing revealed that MTHI1 binds specifically a sequence highly conserved among Chlorophyceae and the Ulvale clade of Ulvophyceae at the 5' end of triphosphorylated atpH mRNA. A very similar sequence, located ∼60 nucleotides upstream of the atpI initiation codon, was also found in some Chlorophyceae and Ulvale algae species and is essential for atpI mRNA translation in Chlamydomonas. Such a dual-targeted trans-acting factor provides a means to coregulate the expression of the two proton hemi-channels.

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression.

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H2 O2 , during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod-like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock-out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane-enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress.

Substrates of the chloroplast small heat shock proteins 22E/F point to thermolability as a regulative switch for heat acclimation in Chlamydomonas reinhardtii.

KEY MESSAGE: We have identified 39 proteins that interact directly or indirectly with high confidence with chloroplast HSP22E/F under heat stress thus revealing chloroplast processes affected by heat. Under conditions promoting protein unfolding, small heat shock proteins (sHsps) prevent the irreversible aggregation of unfolding proteins by integrating into forming aggregates. Aggregates containing sHsps facilitate the access of Hsp70 and ClpB/Hsp104 chaperones, which in ATP-dependent reactions disentangle individual proteins from the aggregates and assist in their refolding to the native state. Chlamydomonas reinhardtii encodes eight different sHsps (HSP22A to H). The goal of this work was to identify chloroplast-targeted sHsps in Chlamydomonas and to obtain a comprehensive list of the substrates with which they interact during heat stress in order to understand which chloroplast processes are disturbed under heat stress. We show that HSP22E and HSP22F are major chloroplast-targeted sHsps that have emerged from a recent gene duplication event resulting from the ongoing diversification of sHsps in the Volvocales. HSP22E/F strongly accumulate during heat stress and form high molecular mass complexes. Using differential immunoprecipitation, mass spectrometry and a stringent filtering algorithm we identified 39 proteins that with high-confidence interact directly or indirectly with HSP22E/F under heat stress. We propose that the apparent thermolability of several of these proteins might be a desired trait as part of a mechanism enabling Chlamydomonas chloroplasts to rapidly react to thermal stress.

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii.

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat-stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat-induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat-induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii.

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.

In vitro characterization of bacterial and chloroplast Hsp70 systems reveals an evolutionary optimization of the co-chaperones for their Hsp70 partner.

The chloroplast Hsp70 (heat-shock protein of 70 kDa) system involved in protein folding in Chlamydomonas reinhardtii consists of HSP70B, the DnaJ homologue CDJ1 and the GrpE-type nucleotide-exchange factor CGE1. The finding that HSP70B needs to be co-expressed with HEP2 (Hsp70 escort protein 2) to become functional allowed the reconstitution of the chloroplast Hsp70 system in vitro and comparison with the homologous Escherichia coli system. Both systems support luciferase refolding and display ATPase and holdase activities. Steady-state activities are low and strongly stimulated by the co-chaperones, whose concentrations need to be balanced to optimally support luciferase refolding. Although the co-chaperones of either system generally stimulate ATPase and folding-assistance activities of the other, luciferase refolding is reduced ~10-fold and <2-fold if either Hsp70 is supplemented with the foreign DnaJ and GrpE protein respectively, suggesting an evolutionary specialization of the co-chaperones for their Hsp70 partner. Distinct features are that HSP70B's steady-state ATPase exhibits ~20-fold higher values for Vmax and Km and that the HSP70B system displays a ~6-fold higher folding assistance on denatured luciferase. Although truncating up to 16 N-terminal amino acids of CGE1 does not affect HSP70B's general ATPase and folding-assistance activities in the physiological temperature range, further deletions hampering dimerization of CGE1 via its N-terminal coiled coil do.

A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystem biogenesis?

The vesicle inducing protein in plastids (VIPP1) is an essential protein for the biogenesis of thylakoids in modern cyanobacteria, algae, and plants. Although its exact function is still not clear, recent work has provided important hints to its mode of action. We believe that these data are consistent with a structural role of VIPP1 within thylakoid centers, which are considered as sites from which thylakoid membranes emerge and at which the biogenesis at least of photosystem II is thought to occur. Here we present a model that may serve as starting point for future research.

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas.

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.