Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

Functional Organization of Sequence Motifs in Diverse Transit Peptides of Chloroplast Proteins

Although the chloroplasts in plants are characterized by an inherent genome, the chloroplast proteome is composed of proteins encoded by not only the chloroplast genome but also the nuclear genome. Nuclear-encoded chloroplast proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the chloroplasts. In the latter process, an N-terminal cleavable transit peptide serves as a targeting signal required for the import of nuclear-encoded chloroplast interior proteins. This import process is mediated via an interaction between the sequence motifs in transit peptides and the components of the TOC/TIC (translocon at the outer/inner envelope of chloroplasts) translocons. Despite a considerable diversity in primary structures, several common features have been identified among transit peptides, including N-terminal moderate hydrophobicity, multiple proline residues dispersed throughout the transit peptide, preferential usage of basic residues over acidic residues, and an absence of N-terminal arginine residues. In this review, we will recapitulate and discuss recent progress in our current understanding of the functional organization of sequence elements commonly present in diverse transit peptides, which are essential for the multi-step import of chloroplast proteins.

Current Understanding of Temperature Stress-Responsive Chloroplast FtsH Metalloproteases

Low and high temperatures are life-threatening stress factors, diminishing plant productivity. One of the earliest responses of plants to stress is a rapid burst of reactive oxygen species (ROS) in chloroplasts. Widespread efforts over the past decade shed new light on the chloroplast as an environmental sensor, translating the environmental fluctuation into varying physiological responses by utilizing distinct retrograde (chloroplast-to-nucleus) signals. Recent studies have unveiled that chloroplasts mediate a similar unfolded/misfolded/damaged protein response (cpUPR) as observed in the endoplasmic reticulum and mitochondria. Although observing cpUPR is not surprising since the chloroplast is a prime organelle producing harmful ROS, the intertwined relationship among ROS, protein damage, and chloroplast protein quality controls (cpPQCs) with retrograde signaling has recently been reported. This finding also gives rise to critical attention on chloroplast proteins involved in cpPQCs, ROS detoxifiers, transcription/translation, import of precursor proteins, and assembly/maturation, the deficiency of which compromises chloroplast protein homeostasis (proteostasis). Any perturbation in the protein may require readjustment of proteostasis by transmitting retrograde signal(s) to the nucleus, whose genome encodes most of the chloroplast proteins involved in proteostasis. This review focuses on recent findings on cpUPR and chloroplast-targeted FILAMENTOUS TEMPERATURE-SENSITIVE H proteases involved in cpPQC and retrograde signaling and their impacts on plant responses to temperature stress.

Beyond RuBisCO: Convergent molecular evolution of multiple chloroplast genes in C4 plants

BackgroundThe recurrent evolution of the C4 photosynthetic pathway in angiosperms represents one of the most extraordinary examples of convergent evolution of a complex trait. Comparative genomic analyses have unveiled some of the molecular changes associated with the C4 pathway. For instance, several key enzymes involved in the transition from C3 to C4 photosynthesis have been found to share convergent amino acid replacements along C4 lineages. However, the extent of convergent replacements potentially associated with the emergence of C4 plants remains to be fully assessed. Here, we introduced a robust empirical approach to test molecular convergence along a phylogeny including multiple C3 and C4 taxa. By analyzing proteins encoded by chloroplast genes, we tested if convergent replacements occurred more frequently than expected in C4 lineages compared to C3 lineages. Furthermore, we sought to determine if convergent evolution occurred in multiple chloroplast proteins beside the well-known case of the large RuBisCO subunit encoded by the chloroplast gene rbcL.MethodsOur study was based on the comparative analysis of 43 C4 and 21 C3 grass species belonging to the PACMAD clade, a focal taxonomic group in many investigations of C4 evolution. We first used protein sequences of 67 orthologous chloroplast genes to build an accurate phylogeny of these species. Then, we inferred amino acid replacements along 13 C4 lineages and 9 C3 lineages using reconstructed protein sequences of their ancestral branches, corresponding to the most recent common ancestor of each lineage. Pairwise comparisons between ancestral branches allowed us to identify both convergent and divergent amino acid replacements between C4-C4, C3-C3 and C3-C4 lineages.ResultsThe reconstructed phylogenetic tree of 64 PACMAD grasses was characterized by strong supports in all nodes used for analyses of convergence. We identified 217 convergent replacements and 201 divergent replacements in 45/67 chloroplast proteins in both C4 and C3 ancestral branches. Pairs of C4-C4 ancestral branches showed higher levels of convergent replacements than C3-C3 and C3-C4 pairs. Furthermore, we found that more proteins shared unique convergent replacements in C4 lineages, with both RbcL and RpoC1 (the RNA polymerase beta’ subunit 1) showing a significantly higher convergent/divergent replacements ratio in C4 branches. Notably, significantly more C4-C4 pairs of ancestral branches showed higher numbers of convergent vs. divergent replacements than C3-C3 and C3-C4 pairs. Our results demonstrated that, in the PACMAD clade, C4 grasses experienced higher levels of molecular convergence than C3 species across multiple chloroplast genes. These findings have important implications for both our understanding of the evolution of photosynthesis and the goal of engineering improved crop varieties that integrates components of the C4 pathway.

Are plastocyanin and ferredoxin specific electron carriers or generic redox capacitors? Classical and murburn perspectives on two chloroplast proteins

Within the context of light reaction of photosynthesis, the structure-function correlations of the chloroplast proteins of plastocyanin and ferredoxins (Fd) are analyzed via two perspectives: 1) The Z-scheme, which considers PC/Fd as specific affinity binding-based electron-relay agents, thereby deterministically linking the functions of Cytochrome b6f (Cyt. b6f) and Photosystem I (PS I) to NADP+ reduction by Fd:NADPH oxidoreductase (FNR) via protein-protein contacts and 2) The murburn explanation for oxygenic photophosphorylation, which deems PC/Fd as generic ‘redox capacitors’, temporally accepting and releasing one-electron equivalents in reaction milieu. Amino acid residues located on the surface loci of key patches of PC/Fd vary in electrostatic/contour (topography) signatures. Crystal structures of four different complexes each of cyt.f-PC and Fd-FNR show little conservation in the contact-surfaces, thereby discrediting ‘affinity binding-based electron transfers (ET)’ as an evolutionary logic. Further, thermodynamic and kinetic data on wildtype and mutant proteins interactions do not align well with model 1. Furthermore, micromolar physiological concentrations of PC (when Kd values 100 μM) and the non-conducive architecture of chloroplasts render the classical model untenable. In the 2nd model, PC is optional and higher concentrations of PC (sought by model 1) could inhibit ET, quite like the role of cytochrome c of mitochondria and cytochrome b5 of cytoplasmic microsomes. Also, PC is found in both lumen and stroma, and plants lacking PC survive and grow. Thus, evidence from structure, interactive dynamics with redox partners and physiological implications of PC/Fd supports the murburn perspective that these proteins serve as generic redox-capacitors in chloroplasts.

Translocation of Drought-Responsive Proteins from the Chloroplasts

Some chloroplast proteins are known to serve as messengers to transmit retrograde signals from chloroplasts to the nuclei in response to environmental stresses. However, whether particular chloroplast proteins respond to drought stress and serve as messengers for retrograde signal transduction are unclear. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) to monitor the proteomic changes in tobacco (Nicotiana benthamiana) treated with drought stress/re-watering. We identified 3936 and 1087 differentially accumulated total leaf and chloroplast proteins, respectively, which were grouped into 16 categories. Among these, one particular category of proteins, that includes carbonic anhydrase 1 (CA1), exhibited a great decline in chloroplasts, but a remarkable increase in leaves under drought stress. The subcellular localizations of CA1 proteins from moss (Physcomitrella patens), Arabidopsis thaliana and rice (Oryza sativa) in P. patens protoplasts consistently showed that CA1 proteins gradually diminished within chloroplasts but increasingly accumulated in the cytosol under osmotic stress treatment, suggesting that they could be translocated from chloroplasts to the cytosol and act as a signal messenger from the chloroplast. Our results thus highlight the potential importance of chloroplast proteins in retrograde signaling pathways and provide a set of candidate proteins for further research.