analysis-protein-import-into-chloroplasts-isolated-from-stressed

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...

University of Oxford

In higher plants, class I KNOTTED1-like homeobox (KNOX) gene suppression and leaf polarity establishment are two processes crucial for leaf morphogenesis. The Arabidopsis genes, ASYMMETRIC LEAVES1 and 2 (AS1 and AS2), are required for repressing the class I KNOX genes and promoting leaf adaxial cell fates. In addition, the RNA-DEPENDENT RNA POLYMERASE6 (RDR6) gene acts synergistically with AS1 and AS2 to specify the adaxial polarity and repress the KNOX genes in leaves. It is known that RDR6 is one of the key components in plant post-transcriptional gene silencing (PTGS), and is likely to function with other silencing components in a genetic pathway in regulating leaf patterning. Here we report phenotypic analyses of double mutants combining as1 or as2 with other mutations relating to different RNA silencing pathways. We show that plants carrying rdr6, suppressor of gene silencing3 (sgs3) or zippy (zip, also called ago7) in combination with as1 or as2 demonstrate severe morphological defects, and the double mutant plants are generally similar to one another. Detailed phenotypic and molecular analyses reveal that leaves of rdr6 as2(1), sgs3 as2(1) and zip as2(1) all show an abnormal adaxial identity, and contain high levels of microRNA165/166 and FILAMENTOUS FLOWER (FIL) transcripts. These results suggest that RDR6, SGS3 and AGO7 act in the same pathway, which genetically interacts with the AS1-AS2 pathway for leaf development. The RDR6-SGS3-AGO7 pathway was previously identified as regulating the plant vegetative phase change. Our results reveal a new function of the pathway, which is also required for normal leaf morphogenesis.

Genetic interaction between the AS1-AS2 and RDR6-SGS3-AGO7 pathways for leaf morphogenesis.

During leaf development, establishment of adaxial-abaxial polarity is essential for normal leaf morphogenesis. This process is known to be strictly regulated by several putative transcription factors, microRNA165/166 (miR165/166), a trans-acting short-interfering RNA (tasiR-ARF), as well as proteins involved in RNA silencing. Among the putative transcription factor genes, ASYMMETRIC LEAVES1 and 2 (AS1 and 2) facilitate the specification of leaf adaxial identity; however, the mechanism by which AS1 and AS2 cooperate with other leaf polarity components remains largely undetermined. In the current study, we characterized the phenotype of mutants by combining as1 and as2 with mutations of several key transcription factors. Our data showed that double mutant plants carrying as1/as2 and rev, phb or phv enhanced as1/as2 defects by producing more severely abaxialized leaves. In contrast, triple mutants, obtained by combining as1/as2 with double mutant filamentous flower yabby3 (fil yab3) or kanadi1 kanadi2 (kan1 kan2), exhibited additive phenotypes. Additionally, while leaves of rev as2 contained high levels of FIL transcripts, only slightly elevated miR165/166 levels were noted, indicating that FIL and miR165/166 act in parallel in leaf patterning. Moreover, 35S::MIR165a/rev as2 transgenic plants resulted in a more severe abaxialized leaf phenotype than the rev and as2 single mutant plants transformed with the same 35S::MIR165a fusion. Genetic interactions between the key regulators during leaf patterning are discussed.

Genetic interactions between leaf polarity-controlling genes and ASYMMETRIC LEAVES1 and 2 in Arabidopsis leaf patterning.

Establishing abaxial-adaxial polarity is central to leaf morphogenesis and function. Groups of genes that encode different components for leaf patterning have been identified in recent years. These include transcriptional factors, small RNAs, 26S proteasome and components required for post-transcriptional gene silencing and chromatin remodeling, showing a complex regulatory network and indicating that the regulation occurs at different levels. In this work, we report the identification and characterization of asymmetric leaves1/2 enhancer5 (ae5) and ae6 mutants. These two mutants had a phenotype of abnormal leaf patterning, with the abaxial mesophyll features appearing in the adaxial mesophyll domain, and double mutants ae5 as1/2 and ae6 as1/2 producing severely abaxialized leaves. AE5 and AE6 encode the ribosomal large subunit proteins RPL28A and RPL5A, respectively, and mutations in two other ribosomal protein genes, RPL5B and RPL24B, resulted in plant phenotypes similar to those of ae5 and ae6. Because these four ribosomal proteins are located in distinct sites in the ribosomal large subunit, we propose that the conserved translational function of the ribosome may be required for regulating key components during leaf patterning. Collectively, our data indicate that specific ribosome subunit-mediated translational control is essential in leaf polarity establishment.

Ribosomal proteins promote leaf adaxial identity.

Leaf morphogenesis requires the establishment of adaxial-abaxial polarity in emerging leaf primordia, and a number of genes participating in this process have been identified in recent years. We previously reported that the 26S proteasome is important in specifying the leaf adaxial fate. More recently, two papers from separate researches showed that several genes encoding ribosomal large subunit proteins also play an important role in leaf adaxial-abaxial patterning. Here we show that plants with a single mutation in the genes encoding either 26S proteasome subunits or ribosomal proteins shared similar abnormalities in some leaves, with an outgrowth formed on the distal part of the leaf abaxial side. Plants harboring these 26S proteasome or ribosome mutations in combination with an additional mutation asymmetric leaves1 or 2 (as1 or as2) demonstrated severely defective leaves, and the phenotypes of these double mutants were very similar. Because activities of the 26S proteasome and ribosome both affect the level of functional proteins, the recent findings suggest that a previously unrecognized regulation, the protein level regulation, is critical in normal leaf patterning. A regulatory model for the 26S proteasome and ribosome actions in leaf patterning is discussed.

A model for the 26S proteasome and ribosome actions in leaf polarity formation.

The SPAD-502 meter is a hand-held device that is widely used for the rapid, accurate and non-destructive measurement of leaf chlorophyll concentrations. It has been employed extensively in both research and agricultural applications, with a range of different plant species. However, its utility has not been fully exploited in relation to the most intensively studied model organism for plant science research, Arabidopsis thaliana. Measurements with the SPAD-502 meter produce relative SPAD meter values that are proportional to the amount of chlorophyll present in the leaf. In order to convert these values into absolute units of chlorophyll concentration, calibration curves must be derived and utilized. Here, we present calibration equations for Arabidopsis that can be used to convert SPAD values into total chlorophyll per unit leaf area (nmol/cm(2); R(2) = 0.9960) or per unit fresh weight of leaf tissue (nmol/mg; R(2) = 0.9809). These relationships were derived using a series of Arabidopsis chloroplast biogenesis mutants that exhibit chlorophyll deficiencies of varying severity, and were verified by the subsequent analysis of senescent or light-stressed leaves. Our results revealed that the converted SPAD values differ from photometric measurements of solvent-extracted chlorophyll by just ~6% on average.

Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana.

Two different, essential Omp85 (Outer membrane protein, 85 kD)-related proteins exist in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD), encoded by atTOC75-III; and OEP80 (Outer Envelope Protein, 80 kD), encoded by AtOEP80/atTOC75-V. The atToc75-III protein is closely related to the originally identified pea (Pisum sativum) Toc75 protein, and it forms a preprotein translocation channel during chloroplast import; the AtOEP80 protein is considerably more divergent from pea Toc75, and its role is unknown. As knockout mutations for atTOC75-III and AtOEP80 are embryo lethal, we employed a dexamethasone-inducible RNA interference strategy (using the pOpOff2 vector) to conduct in vivo studies on the roles of these two proteins in older, postembryonic plants. We conducted comparative studies on plants silenced for atToc75-III (atToc75-III↓) or AtOEP80 (AtOEP80↓), as well as additional studies on a stable, atToc75-III missense allele (toc75-III-3/modifier of altered response to gravity1), and our results indicated that both proteins are important for chloroplast biogenesis at postembryonic stages of development. Moreover, both are important for photosynthetic and nonphotosynthetic development, albeit to different degrees: atToc75-III↓ phenotypes were considerably more severe than those of AtOEP80↓. Qualitative similarity between the atToc75-III↓ and AtOEP80↓ phenotypes may be linked to deficiencies in atToc75-III and other TOC proteins in AtOEP80↓ plants. Detailed analysis of atToc75-III↓ plants, by electron microscopy, immunoblotting, quantitative proteomics, and protein import assays, indicated that these plants are defective in relation to the biogenesis of both photosynthetic and nonphotosynthetic plastids and preproteins, confirming the earlier hypothesis that atToc75-III functions promiscuously in different substrate-specific import pathways.

In vivo analyses of the roles of essential Omp85-related proteins in the chloroplast outer envelope membrane.

The Toc75 and OEP80 proteins reside in the chloroplast outer envelope membrane. Both are members of the Omp85 superfamily of β-barrel proteins, and both are essential in Arabidopsis plants with important roles throughout development. Toc75 forms the translocation channel of the TOC complex, which is responsible for importing nucleus-encoded proteins into chloroplasts, while the function of OEP80 remains uncertain. Deficiency of Toc75 in plants that have artificially reduced OEP80 levels suggests that the latter may be involved in the biogenesis of β-barrel proteins, in similar fashion to Omp85-related proteins in other systems. To elucidate the evolutionary relationship between the two proteins, we conducted a phylogenetic analysis using 48 sequences from diverse species. This indicated that Toc75 and OEP80 belong to sister groups in the Omp85 superfamily, and originate from a gene duplication in an ancient eukaryotic organism > 1.2 billion years ago. Our analysis also supports the notion that the Toc75 family has undergone a phase of neofunctionalization to accommodate the organelle's newly acquired need to import proteins.

Neofunctionalization within the Omp85 protein superfamily during chloroplast evolution.

Development of chloroplasts and other plastids depends on the import of thousands of nucleus-encoded proteins from the cytosol. Import is initiated by TOC (translocon at the outer envelope of chloroplasts) complexes in the plastid outer membrane that incorporate multiple, client-specific receptors. Modulation of import is thought to control the plastid's proteome, developmental fate, and functions. Using forward genetics, we identified Arabidopsis SP1, which encodes a RING-type ubiquitin E3 ligase of the chloroplast outer membrane. The SP1 protein associated with TOC complexes and mediated ubiquitination of TOC components, promoting their degradation. Mutant sp1 plants performed developmental transitions that involve plastid proteome changes inefficiently, indicating a requirement for reorganization of the TOC machinery. Thus, the ubiquitin-proteasome system acts on plastids to control their development.

Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system.

The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation >400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development.

Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts.

Recent work has revealed that mitochondria and chloroplasts are subject to direct control by the ubiquitin-proteasome system (UPS). Ubiquitin E3 ligases are present at the outer membrane of both organelles where they mediate ubiquitination and turnover of other organellar proteins. Both organelles exhibit remarkable structural dynamism and UPS control is particularly concerned with these properties. In mitochondria, the UPS targets factors involved in organellar fission and fusion, with significant impacts upon organellar morphology, mitophagy, and apoptosis. In chloroplasts (and other plastids), the UPS targets components of the protein import machinery, facilitating reorganization of the organellar proteome to determine organellar development and functions. Acquisition of such regulatory control during evolution is perhaps linked to the dynamic characteristics of the two organelles, which are not paralleled in their prokaryotic relatives. Here we discuss our current understanding of the role of the UPS in the regulation of endosymbiotic organelles.

Dynamic regulation of endosymbiotic organelles by ubiquitination.

In a recently published work, we identified the first chloroplast-localized E3 ligase, and showed that it degrades TOC translocon proteins by the ubiquitin-proteasome system (UPS). This regulation was found to be critical for plastid developmental transitions, such as the conversion of etioplasts to chloroplasts. Here we discuss the importance of SP1 for plants under different circumstances, and speculate about SP1 function and its possible application in agriculture. We anticipate that further work in this area may reveal additional roles of the UPS system, through action on other chloroplast OEPs with different roles in plastid biogenesis and function.

The ubiquitin-proteasome system regulates chloroplast biogenesis.

Although the single-nucleotide polymorphism (SNP) rs12979860 in the IL28B gene is a better predictor of sustained virological response to treatment of chronic hepatitis C (CHC) than other baseline factors, some CHC patients with the favorable C allele cannot achieve a sustained virological response when treated with peginterferon plus ribavirin. The aim of this study was to examine baseline factors as predictors of rapid virological response (RVR) and complete early virological response (cEVR) to peginterferon alfa-2a plus ribavirin treatment in Chinese CHC patients with hepatitis C virus (HCV) genotype 1b, with emphasis on the difference between the rs129860 CC and CT/TT genotypes. A total of 337 treatment-naïve patients participated in this study. All patients were treated with peginterferon alfa-2a plus ribavirin at standard dosage. Serum samples from all patients were collected at baseline, week 4, and week 12 for testing of laboratory parameters, and IL28B genotypes were determined. Multivariate analysis showed that among rs12979860 CC genotype patients, glucose level and aspartate amino transferase (AST) activity were inversely associated with RVR, while abnormal platelet count and allergy inversely associated with cEVR. Among rs12979860 CT genotype patients, age below 40 years and short infection duration were associated with RVR, while age below 40 years, short infection duration, high body mass index (BMI), and no history of allergies were associated with cEVR. The baseline factors associated with the response to CHC treatment may depend on the IL28B genotype. Refinement of the baseline predictors based on IL28B genotypes may be useful for management of HCV infection.

Baseline factors associated with treatment response in patients infected with hepatitis C virus 1b by stratification of IL28B polymorphisms.

Plastids, such as chloroplasts, are widely distributed endosymbiotic organelles in plants and algae. Apart from their well-known functions in photosynthesis, they have roles in processes as diverse as signal sensing, fruit ripening, and seed development. As most plastid proteins are produced in the cytosol, plastids have developed dedicated translocon machineries for protein import, comprising the TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes. Multiple lines of evidence reveal that protein import via the TOC complex is actively regulated, based on the specific interplay between distinct receptor isoforms and diverse client proteins. In this review, we summarize recent advances in our understanding of protein import regulation, particularly in relation to control by the ubiquitin-proteasome system (UPS), and how such regulation changes plastid development. The diversity of plastid import receptors (and of corresponding preprotein substrates) has a determining role in plastid differentiation and interconversion. The controllable turnover of TOC components by the UPS influences the developmental fate of plastids, which is fundamentally linked to plant development. Understanding the mechanisms by which plastid protein import is controlled is critical to the development of breakthrough approaches to increase the yield, quality and stress tolerance of important crop plants, which are highly dependent on plastid development. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Functions of plastid protein import and the ubiquitin-proteasome system in plastid development.

Chloroplasts are the organelles responsible for photosynthesis in plants [1, 2]. The chloroplast proteome comprises ∼3,000 different proteins, including components of the photosynthetic apparatus, which are highly abundant. Most chloroplast proteins are nucleus-encoded and imported following synthesis in the cytosol. Such import is mediated by multiprotein complexes in the envelope membranes that surround each organelle [3, 4]. The translocon at the outer envelope membrane of chloroplasts (TOC) mediates client protein recognition and early stages of import. The TOC apparatus is regulated by the ubiquitin-proteasome system (UPS) in a process controlled by the envelope-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) [5, 6]. Previous work showed that SP1-mediated regulation of chloroplast protein import contributes to the organellar proteome changes that occur during plant development (e.g., during de-etiolation). Here, we reveal a critical role for SP1 in plant responses to abiotic stress, which is a major and increasing cause of agricultural yield losses globally [7]. Arabidopsis plants lacking SP1 are hypersensitive to salt, osmotic, and oxidative stresses, whereas plants overexpressing SP1 are considerably more stress tolerant than wild-type. We present evidence that SP1 acts to deplete the TOC apparatus under stress conditions to limit the import of photosynthetic apparatus components, which may attenuate photosynthetic activity and reduce the potential for reactive oxygen species production and photo-oxidative damage. Our results indicate that chloroplast protein import is responsive to environmental cues, enabling dynamic regulation of the organellar proteome, and suggest new approaches for improving stress tolerance in crops.

Regulation of Chloroplast Protein Import by the Ubiquitin E3 Ligase SP1 Is Important for Stress Tolerance in Plants.

Diverse proteolytic pathways regulate chloroplasts. Recent work has revealed significant new roles for chloroplast ubiquitination in stress adaptation, involving targeted protein removal through the ubiquitin-proteasome system, or selective, whole-chloroplast degradation.

Article	Year
Genetic interaction between the AS1-AS2 and RDR6-SGS3-AGO7 pathways for leaf morphogenesis. Plant & cell physiology\| PubMed ID: 16699177	2006
Genetic interactions between leaf polarity-controlling genes and ASYMMETRIC LEAVES1 and 2 in Arabidopsis leaf patterning. Plant & cell physiology\| PubMed ID: 17395603	2007
Ribosomal proteins promote leaf adaxial identity. Development (Cambridge, England)\| PubMed ID: 18305007	2008
A model for the 26S proteasome and ribosome actions in leaf polarity formation. Plant signaling & behavior\| PubMed ID: 19704563	2008
Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana. Photosynthesis research\| PubMed ID: 21188527	2011
In vivo analyses of the roles of essential Omp85-related proteins in the chloroplast outer envelope membrane. Plant physiology\| PubMed ID: 21757633	2011
Neofunctionalization within the Omp85 protein superfamily during chloroplast evolution. Plant signaling & behavior\| PubMed ID: 22307047	2012
Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science (New York, N.Y.)\| PubMed ID: 23118188	2012
Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts. PloS one\| PubMed ID: 23675512	2013
Dynamic regulation of endosymbiotic organelles by ubiquitination. Trends in cell biology\| PubMed ID: 23706390	2013
The ubiquitin-proteasome system regulates chloroplast biogenesis. Communicative & integrative biology\| PubMed ID: 23749079	2013
Baseline factors associated with treatment response in patients infected with hepatitis C virus 1b by stratification of IL28B polymorphisms. Archives of virology\| PubMed ID: 25687192	2015
Functions of plastid protein import and the ubiquitin-proteasome system in plastid development. Biochimica et biophysica acta\| PubMed ID: 25762164	2015
Regulation of Chloroplast Protein Import by the Ubiquitin E3 Ligase SP1 Is Important for Stress Tolerance in Plants. Current biology : CB\| PubMed ID: 26387714	2015
Plant Signaling: Ubiquitin Pulls the Trigger on Chloroplast Degradation. Current biology : CB\| PubMed ID: 26766233	2016

Qihua Ling

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