A pathogenic non coding RNA that replicates and accumulates in chloroplasts traffics to this organelle through a nuclear-dependent step

Viroids belonging to the family Avsunviroidae are the only functional RNAs known to traffic selectively into chloroplasts. Subcellular targeting is a critical step in guaranteeing their access to the machineries involved in their replication. However, the host mechanisms exploited by these non coding pathogenic RNAs to be selectively imported into chloroplasts are poorly understood. Recently, we provide evidence supporting the idea that the Avsunviroidae have evolved to subvert a signaling mechanism between the nucleus and chloroplasts to regulate their differential compartmentalization into the chloroplast of infected cells. Here, we discuss our model and previous observations that provide biological relevance to our hypothesis.     

The discovery of plastid-to-nucleus retrograde signaling—a personal perspective

DNA and machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is relatively small, that most genes for chloroplast-localized proteins reside in the nucleus and that chloroplast membranes, ribosomes, and protein complexes are composed of proteins encoded in both the chloroplast and the nuclear genome. This situation has made the existence of mechanisms highly probable that coordinate the gene expression in plastids and nucleus. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided by studies on plastid ribosome deficient mutants with reduced amounts and/or activities of nuclear-encoded chloroplast proteins including the small subunit of Rubisco, ferredoxin NADP+ reductase, and enzymes of the Calvin cycle. This review describes first models of plastid-to-nucleus signaling and their discovery. Today, many plastid signals are known. They do not only balance gene expression in chloroplasts and nucleus during developmental processes but are also generated in response to environmental changes sensed by the organelles.     

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis.

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.     

Implication of chlorophyll biosynthesis on chloroplast-to-nucleus retrograde signaling

The biogenesis and function of chloroplast are controlled both by anterograde mechanisms involving nuclear-encoded proteins targeted to chloroplast and by retrograde signals from plastid to nucleus contributing to regulation of nuclear gene expression. A number of experimental evidences support the implication of chlorophyll biosynthesis intermediates on the retrograde signaling, albeit an earlier-postulated direct link between accumulation of chlorophyll intermediates and changes in nuclear gene expression has recently been challenged. By characterization of Arabidopsis mutants lacking the chloroplast localized NADPH-thioredoxin reductase (NTRC) we have recently proposed that imbalanced activity of chlorophyll biosynthesis in developing cells modifies the chloroplast signals leading to alterations in nuclear gene expression. These signals appear to initiate from temporal perturbations in the flux through the pathway from protoporphyrin to protochlorophyllide rather than from the accumulation of a single intermediate of the tetrapyr-role pathway.Key words: chloroplast biogenesis, NADPH-thioredoxin reductase, porphyrins, ROS, signaling, tetrapyrrole, thioredoxinOrchestrated regulation of gene expression in the nucleus and plastids is crucial for the proper biogenesis of the organelle during the development and for the acclimation of plants to environmental cues. Multiple potential candidates for initiating plastidial signals have been recognized, including intermediates of the tetrapyrrole biosynthetic pathway, redox state of chloroplast electron transfer components and reactive oxygen species (ROS). These multiple signaling pathways are likely to interact with each others, resulting in a complex signaling network between plastid and nucleus (reviewed in ref. ¹).     

Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins.

Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.     

Substitution of the gene for chloroplast RPS16 was assisted by generation of a dual targeting signal

Organelle (mitochondria and chloroplasts in plants) genomes lost a large number of genes after endosymbiosis occurred. Even after this major gene loss, organelle genomes still lose their own genes, even those that are essential, via gene transfer to the nucleus and gene substitution of either different organelle origin or de novo genes. Gene transfer and substitution events are important processes in the evolution of the eukaryotic cell. Gene loss is an ongoing process in the mitochondria and chloroplasts of higher plants. The gene for ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher plants but not in Medicago truncatula and Populus alba. Here, we show that these 2 species have compensated for loss of the rps16 from the chloroplast genome by having a mitochondrial rps16 that can target the chloroplasts as well as mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa, whose chloroplast genomes encode the rps16, we show that the product of the mitochondrial rps16 has dual targeting ability. These results suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts emerged before the divergence of monocots and dicots (140-150 MYA). The gene substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher plants is the first report about ongoing gene substitution by dual targeting and provides evidence for an intermediate stage in the formation of this heterogeneous organelle.     

Genetic analysis of chloroplast biogenesis in higher plants

The biogenesis of chloroplasts is genetically complex, involving hundreds of genes distributed between the nucleus and organelle. In higher plants, developmental parameters confer an added layer of complexity upon the genetic control of chloroplast biogenesis: the properties of plastids differ dramatically between different cell types. While the biochemistry and structure of different plastid types have been described in detail, factors that determine the timing and localization of chloroplast development and that mediate chloroplast assembly have remained elusive. To identify nuclear genes that play novel roles in chloroplast biogenesis, we are exploiting nuclear mutations that block the accumulation of subsets of chloroplast proteins. Detailed study of the mutant phenotypes provides clues concerning the primary defect in each mutant. Mutants with defects in chloroplast translation and mRNA metabolism have been identified. Other mutants defective in the accumulation of multiple thylakoid complexes show no apparent defect in the synthesis of the missing proteins. These may identify factors involved in the integration of proteins into the thylakoid membrane and their assembly into functional complexes.     

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions.     

Chloroplasts are partially mobilized to the vacuole by autophagy

Excluding the central vacuole, chloroplasts constitute the largest compartment within the leaf cells of plants and contain approximately 80 percent of the total leaf nitrogen, mainly as proteins. Much of this nitrogen is allocated to the carbon-fixing enzyme in photosynthesis, Rubisco. During senescence, plants can mobilize nitrogen from chloroplasts in older leaves to other organs, such as developing seeds. Whereas bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have recently demonstrated that stroma-targeted green fluorescent protein (GFP), DsRed, and GFP-labeled Rubisco can be mobilized to the vacuole of living cells via Rubisco-containing bodies, in an ATG gene-dependent manner. Our results indicate the presence of a specific autophagic pathway for chloroplast stromal proteins, which does not cause chloroplast lysis. Here, we also discuss the involvement of stroma-filled tubules, stromules, which are important for the structural flexibility of the organelle, on the autophagic transfer of stromal proteins to the vacuole.     

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.     

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.     

Novel nuclear-encoded subunits of the chloroplast NAD(P)H dehydrogenase complex

The NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic electron transfer in higher plant chloroplasts and is crucial for plant responses to environmental stress. Chloroplast NDH complex is a close relative to cyanobacterial NDH-1L complex, and all fifteen subunits so far identified in NDH-1L have homologs in the chloroplast NDH complex. Here we report on the identification of two nuclear-encoded proteins NDH48 and NDH45 in higher plant chloroplasts and show their intimate association with the NDH complex. These two membrane proteins are shown to interact with each other and with the NDH complex enriched in stroma thylakoids. Moreover, the deficiency of either the NDH45 protein or the NDH48 protein in respective mutant plants leads to severe defects in both the accumulation and the function of the NDH complex. The NDH48 and NDH45 proteins are not components of the hydrophilic connecting domain of the NDH complex but are strongly attached to the hydrophobic membrane domain. We conclude that NDH48 and NDH45 are novel nuclear-encoded subunits of the chloroplast NDH complex and crucial both for the stable structure and function of the NDH complex.     

Calcium regulation of chloroplast protein import

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling system.     

The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus-encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non-photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc-Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.