Chloroplast Protein Research Articles

Calmodulin is involved in the dual subcellular location of two chloroplast proteins

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

The zeaxanthin epoxidase is degraded along with the D1 protein during photoinhibition of photosystem II.

The xanthophyll zeaxanthin is synthesized in chloroplasts upon high light exposure of plants and serves central photoprotective functions. The reconversion of zeaxanthin to violaxanthin is catalyzed by the zeaxanthin epoxidase (ZEP). ZEP shows highest activity after short and moderate high light periods, but becomes gradually down‐regulated in response to increasing high light stress along with down‐regulation of photosystem II (PSII) activity. ZEP activity and ZEP protein levels were studied in response to high light stress in four plant species: Arabidopsis thaliana, Pisum sativum, Nicotiana benthamiana and Spinacia oleracea. In all species, ZEP protein was degraded during photoinhibition of PSII in parallel with the D1 protein of PSII. In the presence of streptomycin, an inhibitor of chloroplast protein synthesis, photoinhibition of PSII and ZEP activity as well as degradation of D1 and ZEP protein was strongly increased, indicating a close correlation of ZEP regulation with PSII photoinhibition and repair. The concomitant high light‐induced inactivation/degradation of ZEP and D1 prevents the reconversion of zeaxanthin during photoinhibition and repair of PSII. This regulation of ZEP activity supports a coordinated degradation of D1 and ZEP during photoinhibition/repair of PSII and an essential photoprotective function of zeaxanthin during the PSII repair cycle.

Iris yellow spot virus–induced chloroplast malformation results in male sterility

Iris yellow spot virus (IYSV) is one of the most devastating viral pathogens, which causes high economic losses in the onion yield. Physiological and genetic changes are associated with the appearance of chlorotic symptom in the infected plants. IYSV-N gene sequence analysis revealed that it shared sequence identity of 99% with other Egyptian isolates, at both genomic and proteomic levels. In addition, N protein sequence with computational examination indicated many motifs involved and played different roles in the virus activity and its regulation and stability were detected. In the Differential Display-Polymerase Chain Reaction (DD-PCR) study, a highly up-regulated gene at 15 days post-biological IYSV inoculation (dpi) was selected for sequencing. Based on the sequencing results that showed the identified gene was coding for a chloroplast-related gene, degenerate specific primers were designed for Real-Time PCR analysis. A significant change in the transcription level of the chloroplast-related gene after 15 dpi suggested that some IYSV proteins interact and/or regulate with chloroplast proteins and this finding supports the DD-PCR results. At 20 dpi, the ultrathin sections showed that IYSV infection caused many dramatic chloroplasts malformations. The malformation appeared as chloroplast broken envelope with the presence of numerous spherical particles inside it and chloroplasts with long stromule. Our findings indicated that IYSV interrupts normal chloroplast functions, as a part of the onion defence response, however many crucial factors remain to be elucidated and further studies are needed at both biological and molecular levels.

Melatonin modifies the expression of the genes for nuclear- and plastid-encoded chloroplast proteins in detached Arabidopsis leaves exposed to photooxidative stress

Melatonin, a potent regulator during plant development and stress responses, affects diverse plastid-related processes. However, its role in the regulation of plastid gene expression is largely unknown. In this study, exogenous melatonin was shown to reduce the negative influence of excess light by increasing the efficiency of the photosystems and rearranging the expression of chloroplast- and nuclear-encoded genes in detached Arabidopsis leaves. The positive effects of melatonin predominantly occurred at lower concentrations, while high doses had an inhibitory effect. The impact of melatonin was not straightforward. It mainly influenced the expression of the genes encoding the chloroplast transcription machinery and housekeeping genes involved in maintaining transcriptional activity and the functional state of chloroplasts. Despite the fact that melatonin treatment improved photosynthetic parameters, the levels of photosynthesis gene transcripts and photosynthetic proteins remained practically unaltered suggesting that melatonin impact on photosynthetic apparatus which would allow the balancing of chloroplast functions with stress responses is highly complicated.

Does the stromal concentration of Pi control chloroplast ATP synthase protein amount in contrasting growth environments?

ABSTRACTChanges in the growth environment can generate imbalances in chloroplast photosynthetic metabolism. Under water deficit, stomatal closure limits CO2 availability such that the production of ATP and NADPH by the thylakoid membrane-localized electron transport chain may not match the consumption of these energy intermediates by the stroma-localized Calvin-Benson cycle, thus challenging energy balance. Alternatively, in an elevated CO2 atmosphere, carbon fixation by the Calvin-Benson cycle may outpace the activity of downstream carbohydrate-utilizing processes, thus challenging carbon balance. Our previous studies have shown that, in both of the above scenarios, a mitochondrial alternative oxidase contributes to maintaining energy or carbon balance, highlighting the importance of photosynthesis-respiration interactions in optimizing photosynthesis in different growth environments. In these previous studies, we observed aberrant amounts of chloroplast ATP synthase protein across the different transgenic plant lines and growth conditions, compared to wild-type. Based on these observations, we develop here the hypothesis that an important determinant of chloroplast ATP synthase protein amount is the stromal concentration of inorganic phosphate. ATP synthase is a master regulator of photosynthesis. Coarse control of ATP synthase protein amount by the stromal inorganic phosphate status could provide a means to coordinate the electron transport and carbon fixation reactions of photosynthesis.

Cold-modulated small proteins abundance in winter triticale (x Triticosecale, Wittm.) seedlings tolerant to the pink snow mould (Microdochium nivale, Samuels and Hallett) infection.

Two winter triticale (x Triticosecale Wittmack) model cultivars: Hewo (tolerant to pink snow mould) and Magnat (sensitive) were used to test the effect of cold-hardening (4 weeks at 4°C) on soluble ≤50 kDa protein profiles of the seedling leaves. The presence and abundance of individual proteins were analysed via two-dimensional gel electrophoresis (2-DE) and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). Up to now, no proteomics analysis of triticale response to hardening has been performed. Thus, the present paper is the first in the series describing the obtained results. In our experiments, the exposure to the low temperature-induced only quantitative changes in the leaves of both cultivars, causing either an increase or decrease of 4-50 kDa protein abundance. Among proteins which were cold-accumulated in cv. Hewo's leaves, we identified two thioredoxin peroxidases (chloroplastic thiol-specific antioxidant proteins) as well as mitochondrial- β-ATP synthase subunit and ADP-binding resistance protein. On the contrary, in hardened seedlings of this genotype, we observed the decreased level of chloroplastic RuBisCO small subunit PW9 and epidermal peroxidase 10. Simultaneous SELDI-TOF analysis revealed several low mass proteins better represented in cold-hardened plants of tolerant genotype in comparison to the sensitive one and the impact of both genotype and temperature on their level. Based on those results, we suggest that indicated proteins might be potential candidates for molecular markers of cold-induced snow mould resistance of winter triticale and their role is worth to be investigated in the further inoculation experiments.

Manipulation of Plastidial Protein Quality Control Components as a New Strategy to Improve Carotenoid Contents in Tomato Fruit.

Carotenoids such as β-carotene (pro-vitamin A) and lycopene accumulate at high levels during tomato (Solanum lycopersicum L.) fruit ripening, contributing to the characteristic color and nutritional quality of ripe tomatoes. Besides their role as pigments in chromoplast-harboring tissues such as ripe fruits, carotenoids are important for photosynthesis and photoprotection in the chloroplasts of photosynthetic tissues. Interestingly, recent work in Arabidopsis thaliana (L.) Heynh. has unveiled a critical role of chloroplast protein quality control components in the regulation of carotenoid biosynthesis. The accumulation (i.e. degradation rate) and activity (i.e. folding status) of phytoene synthase (PSY) and other Arabidopsis biosynthetic enzymes is modulated by chaperones such as Orange (OR) and Hsp70 in coordination with the stromal Clp protease complex. OR and Clp protease were recently shown to also influence PSY stability and carotenoid accumulation in tomato. Here we show how manipulating the levels of plastid-localized Hsp70 in transgenic tomato plants can also impact the accumulation of carotenoids in ripe fruit. The resulting carotenoid profile and chromoplast ultrastructure, however, are different from those obtained in tomatoes from transgenic lines with increased OR activity. These results suggest that different chaperone families target different processes related to carotenoid metabolism and accumulation during tomato ripening. We further discuss other possible targets for future manipulation in tomato based on the knowledge acquired in Arabidopsis.

Chilli leaf curl virus infection downregulates the expression of the genes encoding chloroplast proteins and stress-related proteins.

Virus infection alters the expression of several host genes involved in various cellular and biological processes in plants. Most of the studies performed till now have mainly focused on genes which are up-regulated and later projected them as probable stress tolerant/susceptible genes. Nevertheless, genes which are down-regulated during plant-virus interaction could also play a critical role on disease development as well as in combating the virus infection. Hence, to identify such down-regulated genes and pathway, we performed reverse suppression subtractive hybridization in Capsicum annuum var. Punjab Lal following Chilli leaf curl virus (ChiLCV) infection. The screening and further processing suggested that majority of the genes (approximately 35% ESTs) showed homology with the genes encoding chloroplast proteins and 16% genes involved in the biotic and abiotic stress response. Additionally, we identified several genes, functionally known to be involved in metabolic processes, protein synthesis and degradation, ribosomal proteins, energy production, DNA replication and transcription, and transporters. We also found 3% transcripts which did not show homology with any known genes. The redundancy analysis revealed the maximum percentage of chlorophyll a-b binding protein (15/96) and auxin-binding proteins (13/96). We developed a protein interactome network to characterise the relationships between proteins and pathway involved during the ChiLCV infection. We identified that the most of the interaction occurs either among the chloroplast proteins (Arabidopsis proteins interactive map) or biotic and abiotic stress responsive proteins (Solanum lycopersicum interactome). Taken together, our study provides the first transcriptome and protein interactome of the down-regulated genes during C. annuum-ChiLCV interaction. These resources could be exploited in deciphering the steps involved in the process of virus infection.

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Chloroplast proteins involved in drought stress response in selected cultivars of common bean (Phaseolus vulgaris L.).

One of the major cell organelles, whose functions are affected during drought stress are chloroplasts. In this study, chloroplast proteome under drought was studied in two cultivars of common bean (Phaseolus vulgaris L), Tiber and more sensitive to drought, Starozagorski čern, which were subjected to drought for 6 and 13days. A comparative proteomic analysis with 2D-DIGE was performed on the isolated chloroplast proteins from leaves. Together, 44 proteins with changed abundance between control and stressed plants were identified with LC-MS/MS from both cultivars. The majority of the identified proteins were involved in photosynthetic processes. The results showed a decrease in abundance in different structure components of photosystem I and II, and ATP synthase, which may indicate a suppression of light-dependent reactions by drought stress. Similar proteomic response for both cultivars after 6 and 13days of drought was observed. Proteins with contrasting abundance patterns between the cultivars or proteins specific for only one cultivar, such as ferredoxin-NADP reductase, photosystem II stability/assembly factor HCF136, curvature thylakoid protein 1B, and plastidial membrane protein porin were pointed out as major identified proteins revealing differential abundance between the cultivars. Taken together, our results provide insight into the molecular response of chloroplasts in common bean under drought stress, whereas conclusions about the tolerance mechanisms require further studies.

Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

The thioredoxin (Trx) redox state sensor protein can visualize Trx activities in the light/dark response in chloroplasts

Thiol-based redox regulation via ferredoxin-thioredoxin (Trx) reductase/Trx controls various functions in chloroplasts in response to light/dark changes. Trx is a key factor of this regulatory system, and five Trx subtypes, including 10 isoforms, have been identified as chloroplast-localized forms in Arabidopsis thaliana These subtypes display distinct target selectivity, and, consequently, they form a complicated redox regulation network in chloroplasts. In this study, we developed a FRET-based sensor protein by combining CFP, YFP, and the N-terminal region of CP12, a redox-sensitive regulatory and Trx-targeted protein in chloroplasts. This sensor protein enabled us to monitor the redox change of chloroplast thioredoxin in vivo, and we therefore designated this protein "change in redox state of Trx" (CROST). Using CP12 isoforms, we successfully prepared two types of CROST sensors that displayed different affinities for two major chloroplast Trx isoforms (f-type and m-type). These sensor proteins helped unravel the real-time redox dynamics of Trx molecules in chloroplasts during the light/dark transition.

The chloroplast proteome response to drought stress in cassava leaves

Cassava is an important tropical crop with strong resistance to drought stress. The chloroplast, the site of photosynthesis, is sensitive to stress, and the drought-response proteins in cassava chloroplasts are worthy of investigation. In this study, cassava leaves were collected for ultra-structure observation from plants subjected to different drought stress conditions. Our results showed that drought stress can promote starch accumulation in cassava chloroplasts. To evaluate changes in chloroplast proteins under different drought conditions, two-dimensional electrophoresis was performed using purified chloroplasts, which resulted in the identification of 26 unique chloroplast proteins responsive to drought stress. These drought-responsive proteins are predominantly related to photosynthesis, carbon and nitrogen metabolism, and amino acid metabolism. Among them, most photosynthesis-related proteins are downregulated, with decreases in photosynthetic parameters upon drought stress. Several proteins associated with carbon and nitrogen metabolism, including rubisco and carbonic anhydrase, were upregulated, which might promote drought tolerance in cassava by enhancing the carbohydrate conversion efficiency and protecting the plant from oxidative stress. Our proteomic data not only provide insight into the complement of proteins in cassava chloroplasts but also further our overall understanding of drought-responsive proteins in cassava chloroplasts.

EIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function.

The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 x i4g2, and i4g1 x i4g2 x i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 x i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.

Molecular Evolution and Functional Analysis of Rubredoxin-Like Proteins in Plants.

Rubredoxins are a class of iron-containing proteins that play an important role in the reduction of superoxide in some anaerobic bacteria and also act as electron carriers in many biochemical processes. Unlike the more widely studied about rubredoxin proteins in anaerobic bacteria, very few researches about the function of rubredoxins have been proceeded in plants. Previous studies indicated that rubredoxins in A. thaliana may play a critical role in responding to oxidative stress. In order to identify more rubredoxins in plants that maybe have similar functions as the rubredoxin-like protein of A. thaliana, we identified and analyzed plant rubredoxin proteins using bioinformatics-based methods. Totally, 66 candidate rubredoxin proteins were identified based on public databases, exhibiting lengths of 187–360 amino acids with molecular weights of 19.856–37.117 kDa. The results of subcellular localization showed that these candidate rubredoxins were localized to the chloroplast, which might be consistent with the fact that rubredoxins were predominantly expressed in leaves. Analyses of conserved motifs indicated that these candidate rubredoxins contained rubredoxin and PDZ domains. The expression patterns of rubredoxins in glycophyte and halophytic plant under salt/drought stress revealed that rubredoxin is one of the important stress response proteins. Finally, the coexpression network of rubredoxin in Arabidopsis thaliana under abiotic was extracted from ATTED-II to explore the function and regulation relationship of rubredoxin in Arabidopsis thaliana. Our results showed that putative rubredoxin proteins containing PDZ and rubredoxin domains, localized to the chloroplast, may act with other proteins in chloroplast to responses to abiotic stress in higher plants. These findings might provide value inference to promote the development of plant tolerance to some abiotic stresses and other economically important crops.

Unraveling Hidden Components of the Chloroplast Envelope Proteome: Opportunities and Limits of Better MS Sensitivity

The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

English

A glasshouse experiment was conducted to compare the performance of a stay-green QTL introgression line (RSG 03123) and its parental lines (B35, a stay-green and R16, a high yielding cultivar with rapid senescence) after flowering, when water-limited (WL) or well-watered (WW). Flowering time and plant height were similar in RSG 03123 and the recurrent parent, R16. Under WW conditions, retention of green leaf area (GLA) and chlorophyll was similar in RSG 03123 and R16, whereas under WL conditions RSG 03123 retained significantly (P<0.05) more GLA. The rate of senescence was also lower in RSG 03123 as compared to R16 under WL conditions. B35 displayed a delayed onset of senescence under both WW and WL conditions while both R16 and RSG 01323 displayed an earlier onset of senescence under WL conditions. Photochemical efficiency of photosystem II (ΦPSII), gas exchange characteristics, and total soluble carbohydrates contents, in both leaf and stem, were higher in RSG 03123 than in R16 under WL conditions. These parameters were enhanced in RSG 03123 due to higher retention of chloroplast proteins, such as light-harvesting chlorophyll binding protein complex of PSII (LHCPII), oxygen-evolving complex 33 kDa (OEC33), phosphoenolpyruvate carboxylase (PEPC) and the large subunit of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The stay-green characteristics of RSG 03123 were intermediate between R16 and B35; nonetheless, the results show that stay-green QTL are functional during senescence and improve tolerance to water limitation after flowering. Key words: Stay-green QTL, introgression, drought, photosynthesis.

Proteomic and lipidomic analyses of the Arabidopsis atg5 autophagy mutant reveal major changes in endoplasmic reticulum and peroxisome metabolisms and in lipid composition.

Autophagy is a universal mechanism in eukaryotic cells that facilitates the degradation of unwanted cell constituents and is essential for cell homeostasis and nutrient recycling. The salicylic acid-independent effects of autophagy defects on leaf metabolism were determined through large-scale proteomic and lipidomic analyses of atg5 and atg5/sid2 mutants under different nitrogen and sulfur growth conditions. Results revealed that irrespective of the growth conditions, plants carrying the atg5 mutation presented all the characteristics of endoplasmic reticulum (ER) stress. Increases in peroxisome and ER proteins involved in very long chain fatty acid synthesis and β-oxidation indicated strong modifications of lipid metabolism. Lipidomic analyses revealed changes in the concentrations of sphingolipids, phospholipids and galactolipids. Significant accumulations of phospholipids and ceramides and changes in GIPCs (glycosyl-inositol-phosphoryl-ceramides) in atg5 mutants indicated large modifications in endomembrane-lipid and especially plasma membrane-lipid composition. Decreases in chloroplast proteins and galactolipids in atg5 under low nutrient conditions, indicated that chloroplasts were used as lipid reservoirs for β-oxidation in atg5 mutants. In conclusion, this report demonstrates the strong impact of autophagy defect on ER stress and reveals the role of autophagy in the control of plant lipid metabolism and catabolism, influencing both lipid homeostasis and endomembrane composition.

Chloroplast Protein Degradation in Senescing Leaves: Proteases and Lytic Compartments.

Leaf senescence is characterized by massive degradation of chloroplast proteins, yet the protease(s) involved is(are) not completely known. Increased expression and/or activities of serine, cysteine, aspartic, and metalloproteases were detected in senescing leaves, but these studies have not provided information on the identities of the proteases responsible for chloroplast protein breakdown. Silencing some senescence-associated proteases has delayed progression of senescence symptoms, yet it is still unclear if these proteases are directly involved in chloroplast protein breakdown. At least four cellular pathways involved in the traffic of chloroplast proteins for degradation outside the chloroplast have been described (i.e., “Rubisco-containing bodies,” “senescence-associated vacuoles,” “ATI1-plastid associated bodies,” and “CV-containing vesicles”), which differ in their dependence on the autophagic machinery, and the identity of the proteins transported and/or degraded. Finding out the proteases involved in, for example, the degradation of Rubisco, may require piling up mutations in several senescence-associated proteases. Alternatively, targeting a proteinaceous protein inhibitor to chloroplasts may allow the inhibitor to reach “Rubisco-containing bodies,” “senescence-associated vacuoles,” “ATI1-plastid associated bodies,” and “CV-containing vesicles” in essentially the way as chloroplast-targeted fluorescent proteins re-localize to these vesicular structures. This might help to reduce proteolytic activity, thereby reducing or slowing down plastid protein degradation during senescence.

Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus.

Chloroplasts evolved from a cyanobacterial endosymbiont that resided within a eukaryotic cell. Due to their prokaryotic heritage, chloroplast outer membranes contain transmembrane β-barrel proteins. While most chloroplast proteins use N-terminal transit peptides to enter the chloroplasts through the translocons at the outer and inner chloroplast envelope membranes (TOC/TIC), only one β-barrel protein, Toc75, has been shown to use this pathway. The route other β-barrel proteins use has remained unresolved. Here we use in vitro pea (Pisum sativum) chloroplast import assays and transient expression in Nicotiana benthamiana to address this. We show that a paralog of Toc75, outer envelope protein 80 kD (OEP80), also uses a transit peptide but has a distinct envelope sorting signal. Our results additionally indicate that β-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.