Chloroplasts, endosymbiotic organelles in plant and algal cells, are essential for photosynthesis and metabolism. Their evolution involved extensive gene transfer to the nuclear genome, necessitating a complex system for protein import. This process involves coordinated gene expression, translation, and protein transport, utilizing multi-member protein complexes in the cytosol, envelope membranes, and stroma. The mechanism relies on chaperones, receptors, channels, and regulatory elements for accurate targeting, transport, and folding.

Cellular condensates are usually ribonucleoprotein assemblies with liquid– or solid–like properties. Because they lack a delineating membrane, the compositional determination of condensates is laborious. Here we set up a pipeline for proximity–biotinylation–dependent capture of RNA to investigate the RNA composition of the condensate in Arabidopsis known as the processing bodies (PBs). Using this pipeline together with in situ protein–protein interaction and RNA detection, in silico , and high–resolution imaging approaches, we studied PBs under normal and heat stress conditions. The composition of PBs in RNAs is much more dynamic than that of the total transcriptome. RNAs involved in cell wall development and regeneration, hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA binding proteins and liquid–to–solid phase transitions modulated specificity of RNA recruitment in PBs. Surprisingly, RNAs were sometimes recruited together with their encoded proteins. In PBs RNAs follow distinct fates, with small liquid-like PBs modulating RNA decay while larger ones storage. The size and properties of PBs are regulated by the actin polymerization cAMP receptor (SCAR)–WASP family verprolin homologous (WAVE) complex. SCAR/WAVE modulates signaling by shuttling RNAs between PBs and the translational machinery adjusting the ethylene signaling pathway. Heat stress leads to the storage of immunity–related RNAs in PBs by reducing PBs dynamics, suggesting why processes such as immunity malfunction under heat stress. In summary, we provide a method to identify RNAs in condensates which allowed us to reveal a mechanism for RNA fate regulation.

Chloroplasts are essential for energy production and metabolism in plants. Metabolites need to be transported across the two envelope membranes of the chloroplast. In contrast to the inner envelope, we know little about the properties of the outer envelope. We here summarize structural and functional insights on the prototype outer envelope channel OEP21 suggesting that these channels have a distinct level of selectivity, opposing the idea of acting as more simple and passive molecular sieves.

Triose phosphates (TPs) are the primary products of photosynthetic CO 2 fixation in chloroplasts, which need to be exported into the cytosol across the chloroplast inner envelope (IE) and outer envelope (OE) membranes to sustain plant growth. While transport across the IE is well understood, the mode of action of the transporters in the OE remains unclear. Here we present the high-resolution nuclear magnetic resonance (NMR) structure of the outer envelope protein 21 (OEP21) from garden pea, the main exit pore for TPs in C 3 plants. OEP21 is a cone-shaped β-barrel pore with a highly positively charged interior that enables binding and translocation of negatively charged metabolites in a competitive manner, up to a size of ~1 kDa. ATP stabilizes the channel and keeps it in an open state. Despite the broad substrate selectivity of OEP21, these results suggest that control of metabolite transport across the OE might be possible.

Male-sterile lines play important roles in plant breeding to obtain hybrid vigour. The male sterility Lembke (MSL) system is a thermosensitive genic male sterility system of Brassica napus and is one of the main systems used in European rapeseed breeding. Interestingly, the MSL system shows high similarity to the 9012AB breeding system from China, including the ability to revert to fertile in high temperature conditions. Here we demonstrate that the MSL system is regulated by the same restorer of fertility gene BnaC9-Tic40 as the 9012AB system, which is related to the translocon at the inner envelope membrane of chloroplasts 40 (TIC40) from Arabidopsis. The male sterility gene of the MSL system was also identified to encode a chloroplast-localized protein which we call BnChimera; this gene shows high sequence similarity to the sterility gene previously described for the 9012AB system. For the first time, a direct protein interaction between BnaC9-Tic40 and the BnChimera could be demonstrated. In addition, we identify the corresponding amino acids that mediate this interaction and suggest how BnaC9-Tic40 acts as the restorer of fertility. Using an RNA-seq approach, the effects of heat treatment on the male fertility restoration of the C545 MSL system line were investigated. These data demonstrate that many pollen developmental pathways are affected by higher temperatures. It is hypothesized that heat stress reverses the male sterility via a combination of slower production of cell wall precursors in plastids and a slower flower development, which ultimately results in fertile pollen. The potential breeding applications of these results are discussed regarding the use of the MSL system in producing thermotolerant fertile plants.

Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase-chorismate mutase-tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.

The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K+ transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1–YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.

Acclimation is an essential process in plants on many levels, but especially in chloroplasts under changing light conditions. It is partially known how the photosynthetic machinery reacts upon exposure to high light intensities, including rearrangement of numerous protein complexes. Since the majority of proteins residing within chloroplasts needs to be posttranslationally imported into the organelles, we endeavored to study how this important process is regulated upon subjecting plants from pea and Arabidopsis to high light. Our results reveal that acclimation takes place on the one hand in the cytosol by differential phosphorylation of preproteins and resulting from the altered expression of the responsible kinases, and on the other hand at the level of the translocation machineries in the outer (TOC) and inner (TIC) envelope membranes. Intriguingly, while phosphorylation is more pronounced under high light, import itself shows a lower efficiency, along with a reduced accumulation of the Toc receptor proteins Toc34 and Toc159.

Photosynthesis is limited by the slow relaxation of nonphotochemical quenching, which primarily dissipates excess absorbed light energy as heat. Because the heat dissipation process is proportional to light-driven thylakoid lumen acidification, manipulating thylakoid ion and proton flux via transport proteins could improve photosynthesis. However, an important aspect of the current understanding of the thylakoid ion transportome is inaccurate. Using fluorescent protein fusions, we show that the Arabidopsis (Arabidopsis thaliana) two-pore K+ channel TPK3, which had been reported to mediate thylakoid K+ flux, localizes to the tonoplast, not the thylakoid. The localization of TPK3 outside of the thylakoids is further supported by the absence of TPK3 in isolated thylakoids as well as the inability of isolated chloroplasts to import TPK3 protein. In line with the subcellular localization of TPK3 in the vacuole, we observed that photosynthesis in the Arabidopsis null mutant tpk3-1, which carries a transfer DNA insertion in the first exon, remains unaffected. To gain a comprehensive understanding of how thylakoid ion flux impacts photosynthetic efficiency under dynamic growth light regimes, we performed long-term photosynthesis imaging of established and newly isolated transthylakoid K+- and Cl--flux mutants. Our results underpin the importance of the thylakoid ion transport proteins potassium cation efflux antiporter KEA3 and voltage-dependent chloride channel VCCN1 and suggest that the activity of yet unknown K+ channel(s), but not TPK3, is critical for optimal photosynthesis in dynamic light environments.

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can - in contrast to fluorescent lights - be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Sucrose (Suc) is one of the most important types of sugars in plants, serving inter alia as a long-distance transport molecule, a carbon and energy storage compound, an osmotically active solute, and fuel for many anabolic reactions. Suc biosynthesis and degradation pathways are well known; however, the regulation of Suc intracellular distribution is poorly understood. In particular, the cellular function of chloroplast Suc reserves and the transporters involved in accumulating these substantial Suc levels remain uncharacterized. Here, we characterize the plastidic sugar transporter (pSuT) in Arabidopsis (Arabidopsis thaliana), which belongs to a subfamily of the monosaccharide transporter-like family. Transport analyses with yeast cells expressing a truncated, vacuole-targeted version of pSuT indicate that both glucose and Suc act as substrates, and nonaqueous fractionation supports a role for pSuT in Suc export from the chloroplast. The latter process is required for a correct transition from vegetative to reproductive growth and influences inflorescence architecture. Moreover, pSuT activity affects freezing-induced electrolyte release. These data further underline the central function of the chloroplast for plant development and the modulation of stress tolerance.

Chloroplasts are the characteristic endosymbiotic organelles of plant cells which during the course of evolution lost most of their genetic information to the nucleus. Thus, they critically depend on the host cell for allocation of nearly their complete protein supply. This includes gene expression, translation, protein targeting, and transport-all of which need to be tightly regulated and perfectly coordinated to accommodate the cells' needs. To this end, multiple signaling pathways have been implemented that interchange information between the different cellular compartments. One of the most complex and energy consuming processes is the translocation of chloroplast-destined proteins into their target organelle. It is a concerted effort from chaperones, receptor proteins, channels, and regulatory elements to ensure correct targeting, efficient transport, and subsequent folding. Although we have discovered and learned a lot about protein import into chloroplasts in the last decades, there are still many open questions and debates about the roles of individual proteins as well as the mechanistic details. In this review, I will summarize and discuss the published data with a focus on the translocation complex in the chloroplast inner envelope membrane.

Plastidic ferredoxin-NADP(+) oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of flavin adenine dinucleotide (FAD) dependent oxidoreductases. In this study, we show that the Arabidopsis thaliana At1g15140 [Ferredoxin-NADP(+) oxidoreductase-like (FNRL)] is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with k(cat)=3.2 +/- 0.2s(-1), K-m(NADPH)=1.6 +/- 0.3M and k(cat)/K-m(NADPH)=2.0 +/- 0.4M(-1)s(-1). Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1, which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. In addition, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded -sheet and one -helix, and a C-terminal NADP(+)-binding / domain with a five-stranded -sheet with a pair of -helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs.

Protein import into chloroplasts is an essential process for plant survival. Numerous studies in recent years have revealed many facts about the components involved and mechanistic features, but have also turned up some controversies. Especially the composition and function of the Tic (translocon at the inner envelope of chloroplasts) complex(es) are vividly debated. Whereas the importance of one central Tic component, Tic110, was generally accepted, its molecular task is still being controversially discussed. Quite recently, a new Tic translocon was proposed to represent the general import machinery with Tic20 and chloroplast encoded Ycf1 (=Tic214) as central components (Kikuchi et al., 2013aKikuchi S. Bedard J. Hirano M. Hirabayashi Y. Oishi M. Imai M. Takase M. Ide T. Nakai M. Uncovering the protein translocon at the chloroplast inner envelope membrane.Science. 2013; 339: 571-574Crossref PubMed Scopus (234) Google Scholar). According to this new model, the majority of imported proteins travel via Tic214/Tic20 into chloroplasts. However, a whole clade in the plant kingdom, the monocotyledonous plants, and several species from the dicotyledonous branch, are lacking Ycf1, suggesting that this protein cannot be a general import component when so many plants can do without it (de Vries et al., 2015de Vries J. Sousa F.L. Bölter B. Soll J. Gould S.B. YCF1: a green TIC?.Plant Cell. 2015; 27: 1827-1833Crossref PubMed Scopus (87) Google Scholar). In Arabidopsis, Ycf1 is an essential protein and thus analyzing its knockout mutants is not an option. There is, however, an elegant possibility to study plants in the absence of Ycf1 by growing them on media containing inhibitors of plastid translation. To this end, we grew two different ecotypes of Arabidopsis thaliana, Col-0 and Jl-3, on MS medium with or without spectinomycin, which is a specific inhibitor of plastid translation (Wirmer and Westhof, 2006Wirmer J. Westhof E. Molecular contacts between antibiotics and the 30S ribosomal particle.Methods Enzymol. 2006; 415: 180-202Crossref PubMed Scopus (62) Google Scholar) (Figure 1A ). In general, Col-0 plants without spectinomycin grew slightly faster than Jl-3 plants. On spectinomycin, however, JL-3 seedlings developed better and were less stressed than Col-0 as judged by the presence of anthocyanins in the leaves, which corresponds to the observations of Parker et al., 2014Parker N. Wang Y. Meinke D. Natural variation in sensitivity to a loss of chloroplast translation in Arabidopsis.Plant Physiol. 2014; 166: 2013-2027Crossref PubMed Scopus (36) Google Scholar. To analyze plastid protein content, total extract was isolated from equal amounts of plant material. Plants from plates without spectinomycin were harvested after 3 weeks, whereas those from plates containing spectinomycin needed to grow for 4–5 weeks until they reached a comparable size. Protein composition was expected to be very different, thus loading for the immuno blots was done according to a Coomassie stained gel (Figure 1B) rather than measuring total protein content. We did not aim for the results to be quantifiable, but rather to demonstrate the presence or absence of nuclear-encoded plastid proteins. The main proteins present in total extracts from green plants, the large (LSU) and small (SSU) subunits of Rubisco, as well as the light harvesting complex protein (LHC), are indicated in the figure and are missing in the white plants. Figure 1C–1G (C, outer envelope proteins; D, inner envelope proteins; E, stromal proteins; F, thylakoid proteins; G, cytosolic protein) show immuno blots probed with the indicated antibodies. Figure 1C clearly depicts that the outer envelope protein OEP16 is present in green as well as in white plants. This protein is inserted independent of the general import translocon and can consequently serve as an internal control (Pohlmeyer et al., 1997Pohlmeyer K. Soll J. Steinkamp T. Hinnah S. Wagner R. Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane.Proc. Natl. Acad. Sci. USA. 1997; 94: 9504-9509Crossref PubMed Scopus (101) Google Scholar). Toc75, however, was shown to depend on the general import machinery (Inoue and Keegstra, 2003Inoue K. Keegstra K. A polyglycine stretch is necessary for proper targeting of the protein translocation channel precursor to the outer envelope membrane of chloroplasts.Plant J. 2003; 34: 661-669Crossref PubMed Scopus (76) Google Scholar) and is equally present in green and in white plants (Figure 1D). Similarly, the highly abundant inner envelope protein Tic110 is efficiently imported into plastids. The same behavior can be observed for the inner envelope constituents Tic40, Iep37, and Fax1, all of which feature classical transit peptides and are imported by the general import pathway. Most importantly, however, this occurred in the absence of detectable amounts of Ycf1 (Figure 1D, upper panel). Strikingly, Tic20, which was isolated in a complex with Ycf1 (Kikuchi et al., 2013aKikuchi S. Bedard J. Hirano M. Hirabayashi Y. Oishi M. Imai M. Takase M. Ide T. Nakai M. Uncovering the protein translocon at the chloroplast inner envelope membrane.Science. 2013; 339: 571-574Crossref PubMed Scopus (234) Google Scholar), is also not detectable, suggesting that assembly of these two proteins is interdependent, and a feedback mechanism from the chloroplast to the nucleus and/or cytosol prevents synthesis or accumulation of Tic20. Tic62, which is a specific interaction partner of the FNR, is strongly reduced in white plants. The same holds true for FNR itself, which is a photosynthetic protein and clearly not needed in albinotic plants. As could already be deduced from the Coomassie stained gel, the small subunit of Rubisco is absent, most likely due to feedback from the plastid that the large subunit cannot be synthesized, and therefore no complex assembly can be achieved. The thylakoid proteins Cytf and D1 seem completely absent; D1 is plastid encoded and cannot be translated and therefore serves as an internal standard for the effectiveness of the spectinomycin treatment, whereas Cytf (PetA) can obviously not be integrated into any thylakoid membrane in the white plants. In the stroma, the housekeeping protein Ndpk2 is clearly present, whereas FBPase and GOGAT are very much reduced. Acc2, the large eukaryotic form of the fatty acid synthase, is not only present but seems upregulated in plants containing no detectable Ycf1. Since we used an anti-biotin serum for detection of Acc2, we could not unequivocally say that the band at 230 kDa represents Acc2 rather than Acc1, which is expressed in the cytosol, whereas Acc2 carries an N-terminal extension that could function as a chloroplast transit peptide (Parker et al., 2014Parker N. Wang Y. Meinke D. Natural variation in sensitivity to a loss of chloroplast translation in Arabidopsis.Plant Physiol. 2014; 166: 2013-2027Crossref PubMed Scopus (36) Google Scholar). To clarify this, plastids were isolated and evaluated in comparison with total extract (Figure 1H). The cytosolic control, Aha1 (Fellerer et al., 2011Fellerer C. Schweiger R. Schongruber K. Soll J. Schwenkert S. Cytosolic HSP90 cochaperones HOP and FKBP interact with freshly synthesized chloroplast preproteins of Arabidopsis.Mol. Plant. 2011; 4: 1133-1145Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), demonstrates the absence of cytosolic protein from the plastid preparation so that the band recognized by the biotin antibody can clearly be assigned to plastidial Acc2, which only appears when Ycf1 is below the detection limit. This suggests that Ycf1 could play a role in the assembly of plastid ACCase, which upon growth on spectinomycin lacks the essential plastid encoded subunit AccD (Sasaki et al., 1993Sasaki Y. Hakamada K. Suama Y. Nagano Y. Furusawa I. Matsuno R. Chloroplast-encoded protein as a subunit of acetyl-CoA carboxylase in pea plant.J. Biol. Chem. 1993; 268: 25118-25123PubMed Google Scholar). To complement for that loss, the second large eukaryotic isoform Acc2, which is usually very low abundant in comparison with the main isoform Acc1, could be upregulated and targeted to plastids, so that fatty acid synthesis can be performed. This assumption is supported by the somewhat increased presence of Fax1, a fatty acid transport protein mediating export of fatty acids (Li et al., 2015Li N. Gügel I.L. Giavalisco P. Zeisler V. Schreiber L. Soll J. Philippar K. FAX1, a novel membrane protein mediating plastid fatty acid export.PLoS Biol. 2015; 13: e1002053Crossref PubMed Scopus (127) Google Scholar). When we analyzed mRNA expression of selected genes, we found that the general response of Col-0 to spectinomycin is very different from Jl-3: Col-0 showed upregulated mRNA expression of the examined nuclear- and plastid-encoded genes, including ACC2, whereas JL-3 exhibited downregulated expression of the analyzed mRNAs with the notable exception of ACC2 (Supplemental Table 2). In contrast, the respective encoded proteins are either present in approximately equal amounts or absent in both ecotypes. Thus, RNA expression analysis is unsuitable to draw any conclusions about protein synthesis and their subsequent import. Our results show that Ycf1/Tic20 possibly cannot constitute the general import machinery, since in Arabidopsis plants with no detectable amounts of both proteins, import of plastid proteins is still maintained. In contrast to Ycf1/Tic20, Tic110 is found in the white plastids in approximately equal amounts compared with the normal green ones (Figure 1H), indicating that it could be indispensable for plastid import. This notion is supported by the observation that already the heterozygous tic110 mutant shows a clear growth phenotype (Kovacheva et al., 2005Kovacheva S. Bedard J. Patel R. Dudley P. Twell D. Rios G. Koncz C. Jarvis P. In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import.Plant J. 2005; 41: 412-428Crossref PubMed Scopus (156) Google Scholar). A function of Ycf1/Tic20 in import cannot be excluded at this point, but although both proteins are essential for plant growth, we can clearly conclude from the presented data that neither protein is mainly responsible for import of nuclear-encoded plastid proteins. During revision of this manuscript, Parker et al., 2016Parker N. Wang Y. Meinke D. Analysis of Arabidopsis accessions hypersensitive to a loss of chloroplast translation.Plant Physiol. 2016; 172: 1862-1875Crossref PubMed Scopus (19) Google Scholar reported that spectinomycin completely inhibits plastid translation and therefore Ycf1 synthesis (Parker et al., 2016Parker N. Wang Y. Meinke D. Analysis of Arabidopsis accessions hypersensitive to a loss of chloroplast translation.Plant Physiol. 2016; 172: 1862-1875Crossref PubMed Scopus (19) Google Scholar). In addition, Köhler et al., 2016Köhler D. Helm S. Agne B. Baginsky S. Importance of translocon subunit Tic56 for rRNA processing and chloroplast ribosome assembly.Plant Physiol. 2016; https://doi.org/10.1104/pp.16.01393Crossref Scopus (21) Google Scholar described mass spectrometric analyses of plants grown on spectinomycin, where ycf1 was non-detectable (Köhler et al., 2016Köhler D. Helm S. Agne B. Baginsky S. Importance of translocon subunit Tic56 for rRNA processing and chloroplast ribosome assembly.Plant Physiol. 2016; https://doi.org/10.1104/pp.16.01393Crossref Scopus (21) Google Scholar). Since mass spectrometry is much more sensitive than immuno blots, we could conclude that upon inhibition of plastid translation by spectinomycin Ycf1 is truly absent. Nevertheless, a number of imported plastid proteins were identified, confirming our immuno blot results and supporting the conclusion that plastid import is not dependent on Ycf1. This work was in part supported by the DFG through SFB-TR175 (projects B5 and B6) and the cluster of excellence CIPSM.

Comparative analyses of phenotypic and molecular traits of Arabidopsis thaliana grown under standardised conditions is still a challenge using climatic devices supplied with common light sources. These are in most cases fluorescent lights, which have several disadvantages such as heat production at higher light intensities, an invariable spectral output, and relatively rapid “ageing”. This results in non-desired variations of growth conditions and lowers the comparability of data acquired over extended time periods. In this study, we investigated the growth behaviour of Arabidopsis Col0 under different light conditions, applying fluorescent compared to LED lamps, and we conducted physiological as well as gene expression analyses. By changing the spectral composition and/or light intensity of LEDs we can clearly influence the growth behaviour of Arabidopsis and thereby study phenotypic attributes under very specific light conditions that are stable and reproducible, which is not necessarily given for fluorescent lamps. By using LED lights, we can also roughly mimic the sun light emission spectrum, enabling us to study plant growth in a more natural-like light set-up. We observed distinct growth behaviour under the different light regimes which was reflected by physiological properties of the plants. In conclusion, LEDs provide variable emission spectra for studying plant growth under defined, stable light conditions.

Chloroplasts originated from an endosymbiotic event in which a free-living cyanobacterium was engulfed by an ancestral eukaryotic host. During evolution the majority of the chloroplast genetic information was transferred to the host cell nucleus. As a consequence, proteins formerly encoded by the chloroplast genome are now translated in the cytosol and must be subsequently imported into the chloroplast. This process involves three steps: (i) cytosolic sorting procedures, (ii) binding to the designated receptor-equipped target organelle and (iii) the consecutive translocation process. During import, proteins have to overcome the two barriers of the chloroplast envelope, namely the outer envelope membrane (OEM) and the inner envelope membrane (IEM). In the majority of cases, this is facilitated by two distinct multiprotein complexes, located in the OEM and IEM, respectively, designated TOC and TIC. Plants are constantly exposed to fluctuating environmental conditions such as temperature and light and must therefore regulate protein composition within the chloroplast to ensure optimal functioning of elementary processes such as photosynthesis. In this review we will discuss the recent models of each individual import stage with regard to short-term strategies that plants might use to potentially acclimate to changes in their environmental conditions and preserve the chloroplast protein homeostasis.

Protein import into chloroplasts has been a focus of research for several decades. The first publications dealing with this fascinating topic appeared in the 1970s. From the initial realization that many plastid proteins are being encoded for in the nucleus and require transport into their target organelle to the identification of import components in the cytosol, chloroplast envelopes, and stroma, as well as elucidation of some mechanistic details, more fascinating aspects are still being unraveled. With this overview, we present a survey of the beginnings of chloroplast protein import research, the first steps on this winding road, and end with a glimpse into the future.