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1.  Comparative transcriptome analysis of green/white variegated sectors in Arabidopsis yellow variegated2: responses to oxidative and other stresses in white sectors 
Journal of Experimental Botany  2010;61(9):2433-2445.
The yellow variegated2 (var2) mutant in Arabidopsis thaliana has been studied as a typical leaf-variegated mutant whose defect results from the lack of FtsH2 metalloprotease in chloroplasts. The var2 green sectors suffer from photo-oxidative stress and accumulate high levels of reactive oxygen species (ROS) because of compromised Photosystem II repair. This study investigated and compared microarray-based expression profiles of green and white sectors of var2 leaves. Results suggest that ROS that accumulate in chloroplasts of var2 green sectors do not cause much significant change in the transcriptional profile related to ROS signalling and scavenging. By contrast, transcriptome in the white sectors apparently differs from those in the green sectors and wild type. Numerous genes related to photosynthesis and chloroplast functions were repressed in the white sectors. Furthermore, many genes related to oxidative stress were up-regulated. Among them, ROS scavenging genes were specifically examined, such as Cu/Zn superoxide dismutase 2 (CSD2), that were apparently up-regulated in white but not in the green sectors. Up-regulation of CSD2 appears to be partly attributable to the lack of a microRNA (miR398) in the white sectors. It was concluded that the white sectors exhibit a response to oxidative and other stresses, including CSD2 up-regulation, which might be commonly found in tissues with abnormal chloroplast differentiation.
PMCID: PMC2877895  PMID: 20400527
Arabidopsis; chloroplasts; miR398; plastids; reactive oxygen species (ROS); superoxide dismutase (SOD); yellow variegated2 (var2)
2.  DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants 
Journal of Experimental Botany  2013;65(1):117-130.
Chloroplast-localized DER (Double Era-like GTPase) contains two consecutive GTP-binding domains, each of which possesses GTPase activity. DER binds to 23S and 16S ribosomal RNAs, and plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants
This study investigated protein characteristics and physiological functions of DER (Double Era-like GTPase) of higher plants. Nicotiana benthamiana DER (NbDER) contained two tandemly repeated GTP-binding domains (GD) and a C-terminal domain (CTD) that was similar to the K-homology domain involved in RNA binding. Both GDs possessed GTPase activity and contributed to the maximum GTPase activity of NbDER. NbDER fused to green fluorescent protein was localized primarily to chloroplast nucleoids. Arabidopsis der null mutants exhibited an embryonic lethal phenotype, indicating an essential function of DER during plant embryogenesis. Virus-induced gene silencing of NbDER resulted in a leaf-yellowing phenotype caused by disrupted chloroplast biogenesis. NbDER was associated primarily with the chloroplast 50S ribosomal subunit in vivo, and both the CTD and the two GD contributed to the association. Recombinant proteins of NbDER and its CTD could bind to 23S and 16S ribosomal RNAs in vitro. Depletion of NbDER impaired processing of plastid-encoded ribosomal RNAs, resulting in accumulation of the precursor rRNAs in the chloroplasts. NbDER-deficient chloroplasts contained significantly reduced levels of mature 23S and 16S rRNAs and diverse mRNAs in the polysomal fractions, suggesting decreased translation in chloroplasts. These results suggest that DER is involved in chloroplast rRNA processing and ribosome biogenesis in higher plants.
PMCID: PMC3883289  PMID: 24272962
Chloroplast abnormality; Nicotiana benthamiana; ribosomal RNA processing; ribosome association; RNA binding; virus-induced gene silencing.
3.  Leaf Variegation in the Rice zebra2 Mutant Is Caused by Photoperiodic Accumulation of Tetra-Cis-Lycopene and Singlet Oxygen 
Molecules and Cells  2011;33(1):87-97.
In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O2−], hydrogen peroxide [H2O2], and singlet oxygen [1O2]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with 1O2 levels generated during the light period, which consequently altered the expression of 1O2-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive 1O2 under natural day-night conditions.
PMCID: PMC3887748  PMID: 22134723
carotenoid isomerase; rice; singlet oxygen; tetra-cis-lycopene; zebra2
4.  Reactive oxygen species derived from impaired quality control of photosystem II are irrelevant to plasma-membrane NADPH oxidases 
Plant Signaling & Behavior  2010;5(3):264-266.
Protein quality control plays an important role in the photosynthetic apparatus because its components receive excess light energy and are susceptible to photooxidative damage. In chloroplasts, photodamage is targeted to the D1 protein of Photosystem II (PSII). The coordinated PSII repair cycle (PSII disassembly, D1 degradation and synthesis, and PSII reassembly) is necessary to mitigate photoinhibition. A thylakoid protease FtsH, which is formed predominantly as a heteromeric complex with two isoforms of FtsH2 and FtsH5 in Arabidopsis, is the major protease involved in PSII repair. A mutant lacking FtsH2 (termed var2) shows compromised D1 degradation. Furthermore, var2 accumulates high levels of chloroplastic reactive oxygen species (cpROS), reflecting photooxidative stress without functional PSII repair. To examine if the cpROS produced in var2 are connected to a ROS signaling pathway mediated by plasma membrane NADPH oxidase (encoded by AtRbohD or AtRbohF), we generated mutants in which either Rboh gene was inactivated under var2 background. Lack of NADPH oxidases had little or no impact on cpROS accumulation. It seems unlikely that cpROS in var2 activate plasma membrane NADPH oxidases to enhance ROS production and the signaling pathway. Mutants that are defective in PSII repair might be valuable for investigating cpROS and their physiological roles.
PMCID: PMC2881273  PMID: 20023407
reactive oxygen species (ROS); photosystem II repair cycle; chloroplast; FtsH; NADPH oxidase; D1 protein; protein turnover
5.  Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta “Gold Standard” Leaves 
Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.
PMCID: PMC4813207  PMID: 27005614
Hosta “Gold Standard”; proteomics; variegated leaves; leaf color; excess nitrogen fertilization
6.  RNR1, a 3′–5′ exoribonuclease belonging to the RNR superfamily, catalyzes 3′ maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana  
Nucleic Acids Research  2005;33(8):2751-2763.
Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.
PMCID: PMC1110743  PMID: 15891117
7.  The rice ALS3 encoding a novel pentatricopeptide repeat protein is required for chloroplast development and seedling growth 
Rice  2015;8:17.
Pentatricopeptide repeat (PPR) proteins play essential roles in modulating the expression of organelle genes and have expanded greatly in higher plants. However, molecular mechanisms of most rice PPR genes remain unclear.
In this study, a new rice PPR mutant, asl3 (albinoseedlinglethality3) exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered photosynthetic-pigment and chloroplast development. Map-based cloning showed that ASL3 encodes a novel rice PPR protein with 10 tandem PPR motifs, which localizes to the chloroplast. ASL3 showed tissue-specific expression, as it was highly expressed in the chlorenchyma, but expressed at much lower levels in roots and panicles. RNAi of ASL3 confirmed that ASL3 plays an essential role in the early development and chloroplast development in rice. Moreover, expression analysis revealed that the asl3 mutation severely affected the transcriptional levels of important genes associated with plastid translation machinery and photosynthesis, which may impair photosynthesis and finally led to the seedling death in asl3 mutant. These results evidenced the important role of ASL3 in the early development of rice, especially chloroplast development.
The ASL3 gene encoded a novel chloroplast-targeted PPR protein with 10 tandem PPR motifs in rice. Disruption of the ASL3 would lead to a defective chloroplast and seedling lethality, and affected expression levels of genes associated with chloroplast development and photosynthesis at early leaf stage of rice.
Electronic supplementary material
The online version of this article (doi:10.1186/s12284-015-0050-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4390607  PMID: 25859292
Albino; Chloroplast development; Lethality; Rice; Pentatricopeptide repeat (PPR) proteins
8.  Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, differentially expressed in variegated Epipremnum aureum ‘Golden Pothos’ is achieved through a unique method of comparative study using tissue regenerated plants 
Journal of Experimental Botany  2010;61(5):1483-1493.
Variegated plants provide a valuable tool for studying chloroplast biogenesis by allowing direct comparison between green and white/yellow sectors within the same leaf. While variegated plants are abundant in nature, the mechanism of leaf variegation remains largely unknown. Current studies are limited to a few mutants in model plant species, and are complicated by the potential for cross-contamination during dissection of leaf tissue into contrasting sectors. To overcome these obstacles, an alternative approach was explored using tissue-culture techniques to regenerate plantlets from unique sectors. Stable green and pale yellow plants were developed from a naturally variegated Epipremnum aureum ‘Golden Pothos’. By comparing the gene expression between green and pale yellow plants using suppression subtractive hybridization in conjunction with homologous sequence search, nine down-regulated and 18 up-regulated genes were identified in pale yellow plants. Transcript abundance for EaZIP (Epipremnum aureum leucine zipper), a nuclear gene homologue of tobacco NTZIP and Arabidopsis CHL27, was reduced more than 4000-fold in qRT-PCR analysis. EaZIP encodes the Mg-protoporphyrin IX monomethyl ester cyclase, one of the key enzymes in the chlorophyll biosynthesis pathway. Examination of EaZIP expression in naturally variegated ‘Golden Pothos’ confirmed that EaZIP transcript levels were correlated with leaf chlorophyll contents, suggesting that this gene plays a major role in the loss of chlorophyll in the pale yellow sectors of E. aureum ‘Golden Pothos’. This study further suggests that tissue-culture regeneration of plantlets from different coloured sectors of variegated leaves can be used to investigate the underlying mechanisms of variegation.
PMCID: PMC2914579  PMID: 20167611
Golden Pothos; Mg-protoporphyrin IX monomethyl ester cyclase; tissue culture; transcript abundance; variegation formation
9.  Natural foliar variegation without costs? The case of Begonia 
Annals of Botany  2012;109(6):1065-1074.
Background and Aims
Foliar variegation is recognized as arising from two major mechanisms: leaf structure and pigment-related variegation. Begonia has species with a variety of natural foliar variegation patterns, providing diverse examples of this phenomenon. The aims of this work are to elucidate the mechanisms underlying different foliar variegation patterns in Begonia and to determine their physiological consequences.
Six species and one cultivar of Begonia were investigated. Light and electron microscopy revealed the leaf structure and ultrastructure of chloroplasts in green and light areas of variegated leaves. Maximum quantum yields of photosystem II were measured by chlorophyll fluorescence. Comparison with a cultivar of Ficus revealed key features distinguishing variegation mechanisms.
Key Results
Intercellular space above the chlorenchyma is the mechanism of variegation in these Begonia. This intercellular space can be located (a) below the adaxial epidermis or (b) below the adaxial water storage tissue (the first report for any taxa), creating light areas on a leaf. In addition, chlorenchyma cell shape and chloroplast distribution within chlorenchyma cells differ between light and green areas. Chloroplasts from both areas showed dense stacking of grana and stroma thylakoid membranes. The maximum quantum yield did not differ significantly between these areas, suggesting minimal loss of function with variegation. However, the absence of chloroplasts in light areas of leaves in the Ficus cultivar led to an extremely low quantum yield.
Variegation in these Begonia is structural, where light areas are created by internal reflection between air spaces and cells in a leaf. Two forms of air space structural variegation occur, distinguished by the location of the air spaces. Both forms may have a common origin in development where dermal tissue becomes loosely connected to mesophyll. Photosynthetic functioning is retained in light areas, and these areas do not include primary veins, potentially limiting the costs of variegation.
PMCID: PMC3336944  PMID: 22362664
Begonia; chlorenchyma; chlorophyll fluorescence; chloroplast; Ficus pumila ‘Sonny’; intercellular space; internal reflection; ultrastructure; variegation
10.  Arabidopsis ANGULATA10 is required for thylakoid biogenesis and mesophyll development 
Journal of Experimental Botany  2014;65(9):2391-2404.
This study characterized ANU10, a novel chloroplast protein encoded by a nuclear gene in Arabidopsis, which influences leaf and chloroplast shape and is required for thylakoid stacking and grana formation.
The chloroplasts of land plants contain internal membrane systems, the thylakoids, which are arranged in stacks called grana. Because grana have not been found in Cyanobacteria, the evolutionary origin of genes controlling the structural and functional diversification of thylakoidal membranes in land plants remains unclear. The angulata10-1 (anu10-1) mutant, which exhibits pale-green rosettes, reduced growth, and deficient leaf lateral expansion, resulting in the presence of prominent marginal teeth, was isolated. Palisade cells in anu10-1 are larger and less packed than in the wild type, giving rise to large intercellular spaces. The ANU10 gene encodes a protein of unknown function that localizes to both chloroplasts and amyloplasts. In chloroplasts, ANU10 associates with thylakoidal membranes. Mutant anu10-1 chloroplasts accumulate H2O2, and have reduced levels of chlorophyll and carotenoids. Moreover, these chloroplasts are small and abnormally shaped, thylakoidal membranes are less abundant, and their grana are absent due to impaired thylakoid stacking in the anu10-1 mutant. Because the trimeric light-harvesting complex II (LHCII) has been reported to be required for thylakoid stacking, its levels were determined in anu10-1 thylakoids and they were found to be reduced. Together, the data point to a requirement for ANU10 for chloroplast and mesophyll development.
PMCID: PMC4036511  PMID: 24663344
Arabidopsis thaliana; chloroplast; grana; LHCII trimers;  mesophyll development; thylakoid biogenesis; thylakoid stacking.
11.  Establishment of the chloroplast genetic system in rice during early leaf development and at low temperatures 
Chloroplasts are the central nodes of the metabolic network in leaf cells of higher plants, and the conversion of proplastids into chloroplasts is tightly coupled to leaf development. During early leaf development, the structure and function of the chloroplasts differ greatly from those in a mature leaf, suggesting the existence of a stage-specific mechanism regulating chloroplast development during this period. Here, we discuss the identification of the genes affected in low temperature-conditional mutants of rice (Oryza sativa). These genes encode factors involved in chloroplast rRNA regulation (NUS1), and nucleotide metabolism in mitochondria, chloroplasts, and cytosol (V2, V3, ST1). These genes are all preferentially expressed in the early leaf developmental stage P4, and depleting them causes altered chloroplast transcription and translation, and ultimately leaf chlorosis. Therefore, it is suggested that regulation of cellular nucleotide pools and nucleotide metabolism is indispensable for chloroplast development under low temperatures at this stage. This review summarizes the current understanding of these factors and discusses their roles in chloroplast biogenesis.
PMCID: PMC4127815  PMID: 25157260
chloroplast transcription; translation; Oryza sativa; low temperature; nucleotide metabolism
12.  Cosuppression of the chloroplast localized molecular chaperone HSP90.5 impairs plant development and chloroplast biogenesis in Arabidopsis 
BMC Research Notes  2014;7:643.
HSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast. However, how these processes are controlled by HSP90 is not well understood. To understand the role of HSP90.5 in chloroplast function and biogenesis, in this study, we generated transgenic Arabidopsis plants that overexpress a C-terminally FLAG-tagged HSP90.5. By characterizing three HSP90.5 cosuppression lines, we demonstrated the essential role of HSP90.5 in plant growth and chloroplast biogenesis.
Immunoblotting and quantitative PCR analyses revealed three independent HSP90.5 cosuppressing transgenic lines. All three cosuppression lines displayed a certain degree of variegated phenotype in photosynthetic tissues, and the cosuppression did not affect the expression of cytosolic HSP90 isoforms. HSP90.5 cosuppression was shown to be developmentally regulated and occurred mostly at late developmental stage in adult leaves and inflorescence tissues. HSP90.5 cosuppression also caused significantly reduced rosette leaf growth, transient starch storage, but did not affect rosette leaf initiation or inflorescence production, although the fertility was reduced. Isolation of chloroplasts and size exclusion chromatography analysis indicated that the FLAG at the HSP90.5 C-terminus does not affect its proper chloroplast localization and dimerization. Finally, transmission electron microscopy indicated that chloroplast development in HSP90.5 cosuppression leaves was significantly impaired and the integrity of chloroplast is highly correlated to the expression level of HSP90.5.
We thoroughly characterized three HSP90.5 cosuppression lines, and demonstrated that properly controlled expression of HSP90.5 is required for plant growth and development in many tissues, and especially essential for chloroplast thylakoid formation. Since the homozygote of HSP90.5 knockout mutant is embryonically lethal, this study provides transgenic lines that mimic the conditional knockout line or siRNA line of the essential HSP90.5 gene in Arabidopsis.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-643) contains supplementary material, which is available to authorized users.
PMCID: PMC4168064  PMID: 25216779
Molecular chaperone HSP90; Chloroplast biogenesis; Transgene-induced gene silencing; Cosuppression; Transgenic plants; Protein folding; FLAG-tag
13.  Short non-coding RNA fragments accumulating in chloroplasts: footprints of RNA binding proteins? 
Nucleic Acids Research  2011;40(7):3106-3116.
Chloroplast RNA metabolism is controlled and excecuted by hundreds of nuclear-encoded, chloroplast-localized RNA binding proteins. Contrary to the nucleo-cytosolic compartment or bacteria, there is little evidence for non-coding RNAs that play a role as riboregulators of chloroplasts. We mined deep-sequencing datasets to identify short (16–28 nt) RNAs in the chloroplast genome and found 50 abundant small RNAs (sRNAs) represented by multiple, in some cases, thousands of sequencing reads, whereas reads are in general absent from the surrounding sequence space. Other than sRNAs representing the most highly abundant mRNAs, tRNAs and rRNAs, most sRNAs are located in non-coding regions and many are found a short distance upstream of start codons. By transcript end mapping we show that the 5′ and 3′ termini of chloroplast RNAs coincide with the ends of sRNAs. Sequences of sRNAs identified in Arabidopsis are conserved between different angiosperm species and in several cases, we identified putative orthologs in rice deep sequencing datasets. Recently, it was suggested that small chloroplast RNA fragments could result from the protective action of pentatricopeptide repeat (PPR) proteins against exonucleases, i.e. footprints of RNA binding proteins. Our data support this scenario on a transcriptome-wide level and suggest that a large number of sRNAs are in fact remnants of PPR protein targets.
PMCID: PMC3326302  PMID: 22139936
14.  Mediated Plastid RNA Editing in Plant Immunity 
PLoS Pathogens  2013;9(10):e1003713.
Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes.
Author Summary
Plastids originated from cyanobacteria that were incorporated into the eukaryotic cell through an endosymbiotic relationship. During the gradual evolution from endosymbiont to organelle, most genes of the cyanobacterial genome were transferred to the nuclear genome. Therefore, plastid biogenesis and function relies on nuclear gene expression and the import of these gene products into plastids, with the molecular dialogue between these two plant cell compartments therefore needing a precise coordination. Nuclei-to-chloroplast communication, and vice versa, are thus regulated through anterograde and retrograde signaling pathways, respectively. Post-transcriptional RNA editing of plastid RNAs by nuclear encoded regulatory proteins, such as pentatricopetide repeat (PPRs) proteins, represents one of such mechanisms of control. Through the characterization of the nuclear-encoded OCP3 protein, previously found to function as a disease resistance regulator in Arabidopsis, we have discovered a pathogen-sensitive editing-mediated control of the plastidial NDH complex involved in cyclic electron flow (CEF) around photosystem I. This led us to find that different PPRs controlling editing extent of transcripts for plastidial NDH complex are modulated by pathogenic cues. Our results thus represent the first series of evidence indicating engagement of chloroplast RNA editing and chloroplast NDH activity in plant immunity.
PMCID: PMC3814343  PMID: 24204264
15.  Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation 
Molecular Plant  2009;2(6):1233-1246.
In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant (mex1), which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme (DPE1) results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.
PMCID: PMC2782796  PMID: 19946617
Carbohydrate metabolism; photosynthesis; senescence; chloroplast biology; Arabidopsis; autophagy
16.  CpLEPA Is Critical for Chloroplast Protein Synthesis Under Suboptimal Conditions in Arabidopsis thaliana 
PLoS ONE  2012;7(11):e49746.
LEPA is one of the most conserved translation factors and is found from bacteria to higher plants. However, the physiological function of the chloroplast LEPA homolog in higher plants remains unknown. Herein, we demonstrate the physiological role of cpLEPA in enabling efficient photosynthesis in higher plants. The cplepa-1 mutant displays slightly high chlorophyll fluorescence and pale green phenotypes under normal growth conditions. The growth of the cplepa-1 mutant is reduced when grown on soil, and greater reduction is observed under intense light illumination. Photosynthetic activity is impaired in the cplepa-1 mutants, which is reflected in the decreased steady-state levels of chloroplast proteins. In vivo protein labeling experiments explained the decrease in the steady-state levels of chloroplast proteins. An abnormal association of the chloroplast-encoded mRNAs with ribosomes suggests that the protein synthesis deficiencies in cplepa-1 are due to defects in translation initiation in the chloroplasts. The cpLEPA protein appears to be an essential translation factor that promotes the efficiency of chloroplast protein synthesis.
PMCID: PMC3499520  PMID: 23166764
17.  A Zinc Finger Motif-Containing Protein Is Essential for Chloroplast RNA Editing 
PLoS Genetics  2015;11(3):e1005028.
C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.
Author Summary
Transcripts encoding chloroplast and mitochondrial proteins of flowering plants are profoundly affected by RNA editing. In Arabidopsis, over 600 genomically-encoded Cs are modified to Us in organelle transcripts, altering the encoded amino acids and creating stop and start codons. Pentatricopeptide proteins are known to bind to cis-elements near C targets of editing and chloroplast RNA editing also requires members of two additional protein families. Nevertheless, not all protein components of the editosome have been identified. We now report the discovery of a member of fourth gene family essential for chloroplast RNA editing: OZ1, member of a family of Arabidopsis RanBP2-type zinc finger proteins. Identifying all of the proteins in the RNA editosome is critical for understanding the mechanism behind the remarkable specificity of C-to-U editing.
PMCID: PMC4359148  PMID: 25768119
18.  Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae). 
Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes (30 encoding plastid proteins) and a red or blue phycobiliprotein antenna pigment, and the chromobiotes (heterokonts and haptophytes), which lost phycobilins and evolved the brown carotenoid fucoxanthin that colours brown seaweeds, diatoms and haptophytes. Chromobiotes transferred the 30 genes to the nucleus and lost the NM genome and nuclear-pore complexes, but retained its membrane as the periplastid reticulum (PPR), putatively the phospholipid factory of the periplastid space (former algal cytoplasm), as did the ancestral alveolate independently. The chlorarachnean NM has three minute chromosomes bearing approximately 300 genes riddled with pygmy introns. I propose that the periplastid membrane (PPM, the former algal plasma membrane) of chromalveolates, and possibly chlorarachneans, grows by fusion of vesicles emanating from the NM envelope or PPR. Dinoflagellates and euglenoids independently lost the PPM and PPR (after diverging from Sporozoa and chlorarachneans, respectively) and evolved triple chloroplast envelopes comprising the original plant double envelope and an extra outermost membrane, the EM, derived from the perialgal vacuole. In all metaalgae most chloroplast proteins are coded by nuclear genes and enter the chloroplast by using bipartite targeting sequences--an upstream signal sequence for entering the ER and a downstream chloroplast transit sequence. I present a new theory for the four-fold diversification of the chloroplast OM protein translocon following its insertion into the PPM to facilitate protein translocation across it (of both periplastid and plastid proteins). I discuss evidence from genome sequencing and other sources on the contrasting modes of protein targeting, cellular integration, and evolution of these two major lineages of eukaryote "cells within cells". They also provide powerful evidence for natural selection's effectiveness in eliminating most functionless DNA and therefore of a universally useful non-genic function for nuclear non-coding DNA, i.e. most DNA in the biosphere, and dramatic examples of genomic reduction. I briefly argue that chloroplast replacement in dinoflagellates, which happened at least twice, may have been evolutionarily easier than secondary symbiogenesis because parts of the chromalveolate protein-targeting machinery could have helped enslave the foreign plastids.
PMCID: PMC1693104  PMID: 12594921
19.  Chloroplasts autophagy during senescence of individually darkened leaves 
Plant Signaling & Behavior  2009;4(6):565-567.
We recently reported that autophagy plays a role in chloroplasts degradation in individually-darkened senescing leaves. Chloroplasts contain approximately 80% of total leaf nitrogen, mainly as photosynthetic proteins, predominantly ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco). During leaf senescence, chloroplast proteins are degraded as a major source of nitrogen for new growth. Concomitantly, while decreasing in size, chloroplasts undergo transformation to non-photosynthetic gerontoplasts. Likewise, over time the population of chloroplasts (gerontoplasts) in mesophyll cells also decreases. While bulk degradation of the cytosol and organelles is mediated by autophagy, the role of chloroplast degradation is still unclear. In our latest study, we darkened individual leaves to observe chloroplast autophagy during accelerated senescence. At the end of the treatment period chloroplasts were much smaller in wild-type than in the autophagy defective mutant, atg4a4b-1, with the number of chloroplasts decreasing only in wild-type. Visualizing the chloroplast fractions accumulated in the vacuole, we concluded that chloroplasts were degraded by two different pathways, one was partial degradation by small vesicles containing only stromal-component (Rubisco containing bodies; RCBs) and the other was whole chloroplast degradation. Together, these pathways may explain the morphological attenuation of chloroplasts during leaf senescence and describe the fate of chloroplasts.
PMCID: PMC2688315  PMID: 19816145
Arabidopsis; autophagy; chloroplast; dark treatment; leaf senescence; nutrients recycling
20.  The Rice TCM5 Gene Encoding a Novel Deg Protease Protein is Essential for Chloroplast Development under High Temperatures 
Rice  2016;9:13.
High temperature affects a broad spectrum of cellular components and metabolism in plants. The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms. Deg proteases are required for the survival of Escherichia coli at high temperatures. However, it is still unclear whether rice Deg proteases are required for chloroplast development under high temperatures.
In this study, we reported the first rice deg mutant tcm5 (thermo-sensitive chlorophyll-deficient mutant 5) that has an albino phenotype, defective chloroplasts and could not survive after the 4–5 leaf seedling stage when grown at high temperature (32 °C). However, when grown at low temperatures (20 °C), tcm5 has a normal phenotype. Map-based cloning showed that TCM5 encoding a chloroplast-targeted Deg protease protein. The TCM5 transcripts were highly expressed in all green tissues and undetectable in other tissues, showing the tissue-specific expression. In tcm5 mutants grown at high temperatures, the transcript levels of certain genes associated with chloroplast development especially PSII-associated genes were severely affected, but recovered to normal levels at low temperatures. These results showed important role of TCM5 for chloroplast development under high temperatures.
The TCM5 encodes chloroplast-targeted Deg protease protein which is important for chloroplast development and the maintenance of PSII function and its disruption would lead to a defective chloroplast and affected expression levels of genes associated with chloroplast development and photosynthesis at early rice seedling stage under high temperatures.
Electronic supplementary material
The online version of this article (doi:10.1186/s12284-016-0086-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4801845  PMID: 27000876
Chloroplast development; Deg protease; Photosystem; Rice
21.  The tetratricopeptide repeat-containing protein slow green1 is required for chloroplast development in Arabidopsis  
Journal of Experimental Botany  2014;65(4):1111-1123.
A chloroplast-localized tetratricopeptide repeat-containing protein, SG1, was identified through a slow-greening mutant in Arabidopsis. SG1 is required for proplastid to chloroplast transition and its mutation disrupted the transcriptions of chloroplast-related genes. It also genetically interacts with GUN1 or GUN4.
A new gene, SG1, was identified in a slow-greening mutant (sg1) isolated from an ethylmethanesulphonate-mutagenized population of Arabidopsis thaliana. The newly formed leaves of sg1 were initially albino, but gradually became pale green. After 3 weeks, the leaves of the mutant were as green as those of the wild-type plants. Transmission electron microscopic observations revealed that the mutant displayed delayed proplastid to chloroplast transition. The results of map-based cloning showed that SG1 encodes a chloroplast-localized tetratricopeptide repeat-containing protein. Quantitative real-time reverse transcription–PCR data demonstrated the presence of SG1 gene expression in all tissues, particularly young green tissues. The sg1 mutation disrupted the expression levels of several genes associated with chloroplast development, photosynthesis, and chlorophyll biosynthesis. The results of genetic analysis indicated that gun1 and gun4 partially restored the expression patterns of the previously detected chloroplast-associated genes, thereby ameliorating the slow-greening phenotype of sg1. Taken together, the results suggest that the newly identified protein, SG1, is required for chloroplast development in Arabidopsis.
PMCID: PMC3935568  PMID: 24420572
Albino; Arabidopsis thaliana; chloroplast development; proplastid to chloroplast transition; slow greening; tetratricopeptide repeat-containing protein.
22.  Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth 
BMC Plant Biology  2010;10:213.
The roles of non-coding RNAs in regulating gene expression have been extensively studied in both prokaryotes and eukaryotes, however few reports exist as to their roles in organellar gene regulation. Evidence for accumulation of natural antisense RNAs (asRNAs) in chloroplasts comes from the expressed sequence tag database and cDNA libraries, while functional data have been largely obtained from artificial asRNAs. In this study, we used Nicotiana tabacum to investigate the effect on sense strand transcripts of overexpressing a natural chloroplast asRNA, AS5, which is complementary to the region which encodes the 5S rRNA and tRNAArg.
AS5-overexpressing (AS5ox) plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. Analysis of AS5 transcripts revealed four distinct species in wild-type (WT) and AS5ox plants, and additional AS5ox-specific products. Of the corresponding sense strand transcripts, tRNAArg overaccumulated several-fold in transgenic plants whereas 5S rRNA was unaffected. However, run-on transcription showed that the 5S-trnR region was transcribed four-fold more in the AS5ox plants compared to WT, indicating that overexpression of AS5 was associated with decreased stability of 5S rRNA. In addition, polysome analysis of the transformants showed less 5S rRNA and rbcL mRNA associated with ribosomes.
Our results suggest that AS5 can modulate 5S rRNA levels, giving it the potential to affect Chloroplast translation and plant growth. More globally, overexpression of asRNAs via chloroplast transformation may be a useful strategy for defining their functions.
PMCID: PMC3017836  PMID: 20920268
23.  FtsHi4 Is Essential for Embryogenesis Due to Its Influence on Chloroplast Development in Arabidopsis 
PLoS ONE  2014;9(6):e99741.
Chloroplast formation is associated with embryo development and seedling growth. However, the relationship between chloroplast differentiation and embryo development remains unclear. Five FtsHi genes that encode proteins with high similarity to FtsH proteins, but lack Zn2+-binding motifs, are present in the Arabidopsis genome. In this study, we showed that T-DNA insertion mutations in the Arabidopsis FtsHi4 gene resulted in embryo arrest at the globular-to-heart–shaped transition stage. Transmission electron microscopic analyses revealed abnormal plastid differentiation with a severe defect in thylakoid formation in the mutant embryos. Immunocytological studies demonstrated that FtsHi4 localized in chloroplasts as a thylakoid membrane-associated protein, supporting its essential role in thylakoid membrane formation. We further showed that FtsHi4 forms protein complexes, and that there was a significant reduction in the accumulation of D2 and PsbO (two photosystem II proteins) in mutant ovules. The role of FtsHi4 in chloroplast development was confirmed using an RNA-interfering approach. Additionally, mutations in other FtsHi genes including FtsHi1, FtsHi2, and FtsHi5 caused phenotypic abnormalities similar to ftshi4 with respect to plastid differentiation during embryogenesis. Taken together, our data suggest that FtsHi4, together with FtsHi1, FtsHi2, and FtsHi5 are essential for chloroplast development in Arabidopsis.
PMCID: PMC4070914  PMID: 24964212
24.  AtPPR2, an Arabidopsis pentatricopeptide repeat protein, binds to plastid 23S rRNA and plays an important role in the first mitotic division during gametogenesis and in cell proliferation during embryogenesis 
Pentatricopeptide repeat (PPR) proteins are mainly involved in regulating post-transcriptional processes in mitochondria and plastids, including chloroplasts. Mutations in the Arabidopsis PPR2 gene have previously been found to cause defects in seed development and reduced transmission through male and female gametophytes. However, the exact function of AtPPR2 has not been defined. We found that a loss-of-function mutation of AtPPR2 leads to arrest of the first mitotic division during both male and female gametogenesis. In addition, the Atppr2 mutation causes delayed embryogenesis, leading to embryonic lethality. Mutation in emb2750, which appears to be a weak mutant allele of the AtPPR2 locus, also results in defective seeds. However, a majority of emb2750 seeds were able to germinate, but their cotyledons were albino and often deformed, and growth of the emb2750 seedlings were arrested after germination. AtPPR2 is mainly expressed in plant parts that undergo cell division, and AtPPR2 protein was localized to chloroplasts. RNA immunoprecipitation and protein gel mobility shift assays showed that AtPPR2 binds to plastid 23S rRNA. Our study adds to a growing body of evidence that plastids and/or chloroplasts play a key role in cell division. AtPPR2 may modulate the translational process to fine-tune plastid function, thereby regulating cell division.
PMCID: PMC3214271  PMID: 21435048
AtPPR2; pentatricopeptide repeat proteins; gametogenesis; embryogenesis; cell division; plastid
25.  PPR proteins of green algae 
RNA Biology  2013;10(9):1526-1542.
Using the repeat finding algorithm FT-Rep, we have identified 154 pentatricopeptide repeat (PPR) proteins in nine fully sequenced genomes from green algae (with a total of 1201 repeats) and grouped them in 47 orthologous groups. All data are available in a database, PPRdb, accessible online at Based on phylogenetic trees generated from the repeats, we propose evolutionary scenarios for PPR proteins. Two PPRs are clearly conserved in the entire green lineage: MRL1 is a stabilization factor for the rbcL mRNA, while HCF152 binds in plants to the psbH-petB intergenic region. MCA1 (the stabilization factor for petA) and PPR7 (a short PPR also acting on chloroplast mRNAs) are conserved across the entire Chlorophyta. The other PPRs are clade-specific, with evidence for gene losses, duplications, and horizontal transfer. In some PPR proteins, an additional domain found at the C terminus provides clues as to possible functions. PPR19 and PPR26 possess a methyltransferase_4 domain suggesting involvement in RNA guanosine methylation. PPR18 contains a C-terminal CBS domain, similar to the CBSPPR1 protein found in nucleoids. PPR16, PPR29, PPR37, and PPR38 harbor a SmR (MutS-related) domain similar to that found in land plants pTAC2, GUN1, and SVR7. The PPR-cyclins PPR3, PPR4, and PPR6, in addition, contain a cyclin domain C-terminal to their SmR domain. PPR31 is an unusual PPR-cyclin containing at its N terminus an OctotricoPeptide Repeat (OPR) and a RAP domain. We consider the possibility that PPR proteins with a SmR domain can introduce single-stranded nicks in the plastid chromosome.
PMCID: PMC3858436  PMID: 24021981
pentatricopeptide repeat; green algae; chloroplast; mitochondrion; evolution; cyclin; small MutS-related; tRNA methyltransferase

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