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.

Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

A bipartite chromatophore transit peptide and N-terminal processing of protein in the Paulinella chromatophore

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium ~100 million years ago, independently from plastids in plants and algae. Despite its more recent origin, at least one third of the chromatophore proteome consists of nucleus-encoded proteins that are imported by an unknown mechanism across the chromatophore double envelope membranes. Chromatophore-targeted proteins fall into two classes. Proteins exceeding 250 amino acids carry a conserved N-terminal sequence extension, termed the 'chromatophore transit peptide' (crTP), that is presumably involved in guiding these proteins into the chromatophore. Short imported proteins do not carry discernable targeting signals. To explore whether the import of protein is accompanied by their N-terminal processing, here we used a mass spectrometry-based approach to determine protein N-termini in Paulinella chromatophora and identified N-termini of 208 chromatophore-localized proteins. Our study revealed extensive N-terminal modifications by acetylation and proteolytic processing in both, the nucleus and chromatophore-encoded fraction of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Our results imply that the crTP mediates trafficking through the Golgi, is bipartite and surprisingly only the N-terminal third ('part 1') becomes cleaved upon import, whereas the rest ('part 2') remains at the mature proteins. In contrast, short imported proteins remain largely unprocessed. Finally, this work sheds light on N-terminal processing of proteins encoded in an evolutionary-early-stage photosynthetic organelle and suggests host-derived post-translationally acting factors involved in dynamic regulation of the chromatophore-encoded chromatophore proteome.

Characterization and Dynamics of Intracellular Gene Transfer in Plastid Genomes of Viola (Violaceae) and Order Malpighiales

Functional gene transfer from organelles to the nucleus, known as intracellular gene transfer (IGT), is an ongoing process in flowering plants. The complete plastid genomes (plastomes) of two Ulleung island endemic violets, Viola ulleungdoensis and V. woosanensis, were characterized, revealing a lack of the plastid-encoded infA, rpl32, and rps16 genes. In addition, functional replacement of the three plastid-encoded genes in the nucleus was confirmed within the genus Viola and the order Malpighiales. Three strategies for the acquisition of a novel transit peptide for successful IGT were identified in the genus Viola. Nuclear INFA acquired a novel transit peptide with very low identity between these proteins, whereas the nuclear RPL32 gene acquired an existing transit peptide via fusion with the nuclear-encoded plastid-targeted SOD gene (Cu-Zn superoxide dismutase superfamily) as one exon, and translated both proteins in the cytosol using alternative mRNA splicing. Nuclear RPS16 contains an internal transit peptide without an N-terminal extension. Gene loss or pseudogenization of the plastid-borne rpl32 and rps16 loci was inferred to occur in the common ancestor of the genus Viola based on an infrageneric phylogenetic framework in Korea. Although infA was lost in the common ancestor of the order Malpighiales, the rpl32 and rps16 genes were lost multiple times independently within the order. Our current study sheds additional light on plastid genome composition and IGT mechanisms in the violet genus and in the order Malpighiales.

HSPs under Abiotic Stresses

Different organisms respond to the altered environmental conditions by different ways. Heat shock proteins’ (HSPs) production is one among the different defense mechanisms which defend the photosystem II and thylokoid membrane in plants. There are different types of HSPs based on their size, that is, high molecular weight (60–100 kDa) and low molecular weight heat shock proteins (15–30 kDa). Small HSPs are further classified based on their localization and role in different sub-cellular organelles. Cp-sHSPs are the chloroplast-specific small HSPs that protect the photosystem II and thylokoid membrane. A model to control the Cp-sHSPs in Chenopodium album has been put forward in this chapter. According to this model, Cp-sHSPs of Chenopodium album are created in cytoplasm and are moved toward chloroplast. The transit peptide is removed on reaching to the target sub-cellular organelle, that is, chloroplast and the premature Cp-sHSPs are converted into mature ones which have multiple roles under different abiotic stress conditions.

Chaperone-like protein DAY plays critical roles in photomorphogenesis

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARKANDYELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features

In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

Translocation of chloroplast NPR1 to the nucleus in retrograde signaling for adaptive response to salt stress in tobacco

Chloroplasts play a pivotal role in biotic and abiotic stress responses, accompanying changes in the cell reduction/oxidation (redox) state. Chloroplasts are an endosymbiotic organelle that sends retrograde signals to the nucleus to integrate with environmental changes. This study showed that salt stress causes the rapid accumulation of the nonexpressor of pathogenesis-related genes 1 (NPR1) protein, a redox-sensitive transcription coactivator that elicits many tolerance responses in chloroplasts and the nucleus. The transiently accumulated chloroplast NPR1 protein was translocated to the nucleus in a redox-dependent manner under salinity stress. In addition, immunoblotting and fluorescence image analysis showed that chloroplast-targeted NPR1-GFP fused with cTP (chloroplast transit peptide from RbcS) was localized in the nucleus during the responses to salt stress. Chloroplast functionality was essential for retrograde translocation, in which the stomules and cytoplasmic vesicles participated. Treatments with H2O2 and an ethylene precursor enhanced this retrograde translocation. Compared to each wild-type plant, retrograde signaling-related gene expression was severely impaired in the npr1-1 mutant in Arabidopsis, but enhanced transiently in the NPR1-Ox transgenic tobacco line. Therefore, NPR1 might be a retrograde signaling hub that improves a plant's adaptability to changing environments.

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development

ABSTRACTThe Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7 or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.HIGHLIGHTThe nucleus-localized CCT domain proteins CIA2 and CIL in Arabidopsis and the homologous chloroplast-localized HvCMF3 and HvCMF7 in barley retained partially overlapping functions in chloroplast development.