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• W2022083147 abstract "Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants. Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants. Aminoacyl-tRNA synthetases (ARSs) 5The abbreviations used are:ARSsaminoacyl-tRNA synthetasesERSglutamyl-tRNA synthetaseSRSseryl-tRNA synthetaseIRSisoleucyl-tRNA synthetasec-IRScytoplasmic isoleucyl-tRNA synthetaseRubiscoribulose-bisphosphate carboxylase/oxygenaseVIGSvirus-induced gene silencingRTreverse transcriptionGFPgreen fluorescent proteinRFPred fluorescent proteinTMRMtetramethylrhodamine methyl esteraaamino acid(s)MGMitoTracker Green FMc-IRScytosolic form of IleRSDAPI4′,6-diamidino-2-phenylindolecp-DNAchloroplast DNATRVtobacco rattle virusPFGEpulse-field gel electrophoresis. play a critical role in protein synthesis by catalyzing the addition of amino acids to their cognate tRNAs (1Ibba M. Soll D. Annu. Rev. Biochem. 2000; 69: 617-650Crossref PubMed Scopus (1115) Google Scholar, 2Ribas de Pouplana L. Schimmel P. Trends Biochem. Sci. 2001; 26: 591-596Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The specificity of aminoacyl-tRNA synthesis in pairing the appropriate tRNAs and amino acids is a key determinant in faithful transmission of genetic information. Despite the crucial function of ARSs in protein synthesis, there are only limited reports on function and regulation of the ARS genes and proteins in plants (3Browning K.S. Plant Mol. Biol. 1996; 32: 107-144Crossref PubMed Scopus (187) Google Scholar). Protein synthesis in plants takes place in the cytosol, mitochondria, and chloroplasts, and these compartments do not require full sets of unique ARSs encoded by separate nuclear genes. In general, plant ARSs are classified into two groups based on their substrate specificity: the cytosolic enzymes that most efficiently aminoacylate plant or yeast cytosolic tRNAs, and the organellar enzymes that aminoacylate organelle or Escherichia coli tRNAs (4Steimetz A. Weil J.H. Methods Enzymol. 1986; 118: 212-231Crossref Scopus (28) Google Scholar). aminoacyl-tRNA synthetases glutamyl-tRNA synthetase seryl-tRNA synthetase isoleucyl-tRNA synthetase cytoplasmic isoleucyl-tRNA synthetase ribulose-bisphosphate carboxylase/oxygenase virus-induced gene silencing reverse transcription green fluorescent protein red fluorescent protein tetramethylrhodamine methyl ester amino acid(s) MitoTracker Green FM cytosolic form of IleRS 4′,6-diamidino-2-phenylindole chloroplast DNA tobacco rattle virus pulse-field gel electrophoresis. Recent research has uncovered an extensive degree of sharing of ARS isoforms between compartments, between cytosol and mitochondria or between plastids and mitochondria (5Small I. Akashi K. Chapron A. Dietrich A. Duchene A.M. Lancelin D. Maréchal-Drouard L. Menad B. Mireau H. Moudden Y. Ovesna J. Peeters N. Sakamoto W. Souciet G. Wintz H. J. Hered. 1999; 90: 333-337Crossref Scopus (21) Google Scholar). For example, the cytosolic and mitochondrial leucyl-tRNA synthetases of Phaseolus vulgaris aminoacylate cytosolic and mitochondrial tRNAsLeu with equal efficiency but do not aminoacylate chloroplast and E. coli tRNAsLeu (6Guillemaut P. Steinmetz A. Burkard G. Weil J.H. Biochim. Biophys. Acta. 1975; 378: 64-72Crossref PubMed Scopus (36) Google Scholar). In addition, AlaRS, ValRS, and ThrRS of Arabidopsis are dual targeted to both the cytosol and mitochondria, and the same gene encodes both the mitochondrial and the cytosolic enzyme in each case (7Mireau H. Lancelin D. Small I.D. Plant Cell. 1996; 8: 1027-1039Crossref PubMed Scopus (68) Google Scholar, 8Souciet G. Menand B. Ovesna J. Cosset A. Dietrich A. Wintz H. Eur. J. Biochem. 1999; 266: 848-854Crossref PubMed Scopus (55) Google Scholar). Dual targeting to mitochondria and chloroplasts has also been observed for HisRS (9Akashi K. Grandjean O. Small I. FEBS Lett. 1998; 431: 39-44Crossref PubMed Scopus (69) Google Scholar), MetRS (10Peeters N.M. Chapron A. Giritch A. Grandjean O. Lancelin D. Lhomme T. Vivrel A. Small I. J. Mol. Evol. 2000; 50: 413-423Crossref PubMed Scopus (72) Google Scholar, 11Menand B. Maréchal-Drouard L. Sakamoto W. Dietrich A. Wintz H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11014-11019Crossref PubMed Scopus (80) Google Scholar), AsnRS (10Peeters N.M. Chapron A. Giritch A. Grandjean O. Lancelin D. Lhomme T. Vivrel A. Small I. J. Mol. Evol. 2000; 50: 413-423Crossref PubMed Scopus (72) Google Scholar), CysRS (10Peeters N.M. Chapron A. Giritch A. Grandjean O. Lancelin D. Lhomme T. Vivrel A. Small I. J. Mol. Evol. 2000; 50: 413-423Crossref PubMed Scopus (72) Google Scholar), and one of the GlyRS enzymes (12Duchene A.-M. Peeters N. Dietrich A. Cosset A. Small I.D. Wintz H. J. Biol. Chem. 2001; 276: 15275-15283Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) in Arabidopsis. Phenotypes of several mutants with defects in ARS gene structure or expression have been characterized in Arabidopsis. A tDNA insertion mutant of the cytosolic and mitochondrial AlaRS showed an embryonic lethal phenotype (13Ge S.J. Yao X.L. Yang Z.X. Zhu Z.P. Cell Res. 1998; 8: 119-134Crossref PubMed Scopus (13) Google Scholar). Embryo development of this mutant was arrested at the globular stage with altered patterns of cell division and differentiation. Altered expression of a cytosolic ValRS in plant tissues also caused severe defects in early embryogenesis in the twn2 mutant in Arabidopsis (14Zhang J.Z. Somerville C.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7349-7355Crossref PubMed Scopus (93) Google Scholar). In this mutant, development of apical cells is arrested at a very early stage, and the basal cells proliferate abnormally, giving rise to multiple embryos. Taken together, these results show that cytosolic ARS activity is indispensable in plant cell viability and development. To date, there has been only one report regarding the function of an organelle-specific ARS (15Uwer U. Willmitzer L. Altmann T. Plant Cell. 1998; 10: 1277-1294Crossref PubMed Scopus (92) Google Scholar). The insertion mutation of the organellar GlyRS gene was lethal, arresting embryo growth between the globular and heart stages of embryo development in Arabidopsis. An N-terminal fragment of the GlyRS protein was able to direct a marker protein into both chloroplasts and mitochondria (12Duchene A.-M. Peeters N. Dietrich A. Cosset A. Small I.D. Wintz H. J. Biol. Chem. 2001; 276: 15275-15283Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Thus, normal organellar ARS function is obviously essential for plant embryogenesis. However, there has been no report directly demonstrating how loss of organellar ARS function affects the development and physiology of chloroplasts and mitochondria, and how it in turn affects plant cells in which the organelles reside. In this study, we investigated cellular functions of organellar ARSs by generating reduced-expression mutants using virus-induced gene silencing (VIGS), thereby circumventing the embryonic-lethal phenotypes associated with the complete loss of ARS function. VIGS of NbERS and NbSRS, encoding the Nicotiana benthamiana organellar forms of GluRS and SerRS, respectively, severely affected the numbers, morphology, and physiology of both chloroplasts and mitochondria. These data are consistent with the import of the Arabidopsis orthologs of NbERS and NbSRS into both chloroplasts and mitochondria and suggest that NbERS and NbSRS are responsible for the GluRS and SerRS activity required for translation in both organelles. Virus-induced Gene Silencing—Various cDNA fragments of NbERS and NbSRS were PCR-amplified and cloned into the pTV00 vector containing a part of the TRV genome (16Ratcliff F. Martin-Hernandez A.M. Baulcombe D.C. Plant J. 2001; 25: 237-245Crossref PubMed Scopus (738) Google Scholar) using BamHI and ApaI sites. VIGS was carried out as described (16Ratcliff F. Martin-Hernandez A.M. Baulcombe D.C. Plant J. 2001; 25: 237-245Crossref PubMed Scopus (738) Google Scholar, 17Ahn J.W. Kim M. Kim G.T. Lim J.H. Pai H.-S. Plant J. 2004; 38: 969-981Crossref PubMed Scopus (65) Google Scholar, 18Cho H.S. Lee S.S. Kim K.D. Kim S.J. Hwang I. Lim J.S. Park Y.I. Pai H.-S. Plant Cell. 2004; 16: 2665-2682Crossref PubMed Scopus (74) Google Scholar). The fourth leaf above the infiltrated leaf was used for RT-PCR and cytological analyses. RT-PCR—Semiquantitative RT-PCR was performed with 5 μg of total RNA, isolated from the fourth leaf above the infiltrated leaf, as described previously (17Ahn J.W. Kim M. Kim G.T. Lim J.H. Pai H.-S. Plant J. 2004; 38: 969-981Crossref PubMed Scopus (65) Google Scholar). To detect the NbERS transcripts, the ERS-A (5′-ATGCCTCACTTTGCGCAT-3′ and 5′-TGCATCCTTCCACCTTTC-3′) and the ERS-B (5′-GTACCTCAAGCTGGGTTT-3′ and 5′-GCAAGTGGCAGTAAGATA-3′) primer sets were used. To detect the NbSRS transcripts, the SRS-A (5′-ACAGCTTATCAAAGCCGT-3′ and 5′-TAAACGTCTGTCCCATGT-3′) and the SRS-B (5′-ATGTTCATACTGTGCCGA-3′ and 5′-ATTGCTGACCTCACCATA-3′) primer sets were used. To detect the NbIRS transcripts, the IRS-A (5′-GGCAGTAAACCGCATTTC-3′ and 5′-CAACCCCCTTTCATCCAT-3′) and IRS-B (5′-ATCTCAGGTGGAGATTCT-3′ and 5′-CATTGTGCTATAGCCTCT-3′) primer sets were used. The primers used to detect nuclear genes were: for rbcS, 5′-CCTTGACATCACTTCCATT-3′ and 5′-AGCCCTCTGGCTTGTAGG-3′; for Lhcb,5′-TGAAGGATATAGGGTTGGTG-3′ and 5′-GGGTCATTAATCTGGTCAAA-3′; for FtsZ,5′-TCTGCTGCCTGTTCCCCCAA-3′ and 5′-AGCAACTCTGGACCCTCTCA-3′; for chalcone synthase (CHS), 5′-GAATACATGGCTCCTTCT-3′ and 5′-CCCAGGCCCAAATCCAAA-3′; for ChlD,5′-TTGGACGTATCATGATTGTTGC-3′ and 5′-CTATGACGAGGAGAGACATTCC-3′; for ChlH,5′-CACCCTTTGGCTCCTTGTATGT-3′ and 5′-CCATGATCACAGCCACATAGTG-3′; for SGT, 5′-AGAACGCTGAGCTTTTCG-3′ and 5′-CAGCAGATCCTTGATAGG-3′; for SKP,5′-GTAGAAGAGTCAGTTGCC-3′ and 5′-TCAGCAGACTATTGACGC-3′; for pyruvate dehydrogenase E1α (PDHE-1α), 5′-GGGTCATGGGATTGTTGG-3′ and 5′-GCAGGATCAGACATGGAG-3′; for malate dehydrogenase (MDH), 5′-ACCCCTGGTGTTGCCGCT-3′ and 5′-CCATCTTGGGTTCGCTTG-3′; for actin, 5′-GCCACACTGTCCCAATTTATGA-3′ and 5′-GAAGCCAAAATAGAACCTCCAA-3′. Subcellular Localization of AtERS and AtSRS—The AtERS cDNA fragments corresponding to the N-terminal 70 amino acids (residues 1-70) and the full-length sequence of 570 amino acids (residues 1-570) were cloned into the 326-GFP (green fluorescent protein) plasmid (19Lee Y.J. Kim D.H. Kim Y.-W. Hwang I. Plant Cell. 2001; 13: 2175-2190Crossref PubMed Scopus (187) Google Scholar) using BamHI sites to generate AtERS-GFP fusion proteins. The AtSRS cDNA fragments corresponding to the N-terminal 90 amino acids (residues 1-90) and the full-length sequence of 514 amino acids (residues 1-514) were cloned into the same vector for the AtSRS-GFP fusion proteins. The various GFP fusion constructs and the F1ATPase-γ-RFP fusion construct were introduced into protoplasts prepared from Arabidopsis seedlings by polyethylene glycol-mediated transformation (19Lee Y.J. Kim D.H. Kim Y.-W. Hwang I. Plant Cell. 2001; 13: 2175-2190Crossref PubMed Scopus (187) Google Scholar). Expression of the fusion constructs was monitored at 24 h after transformation by confocal laser scanning microscopy, as described previously (18Cho H.S. Lee S.S. Kim K.D. Kim S.J. Hwang I. Lim J.S. Park Y.I. Pai H.-S. Plant Cell. 2004; 16: 2665-2682Crossref PubMed Scopus (74) Google Scholar). Measurement of Chlorophyll Content—The fourth leaf above the infiltrated leaf was collected from the VIGS plants and boiled in 95% ethanol at 80 °C for 30 min to extract chlorophyll. Chlorophyll concentration per unit fresh weight was calculated as described before (20Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4743) Google Scholar). Analysis of Starch Content—Leaves were harvested from the VIGS lines at 20 days after infiltration, and bleached in 80% (v/v) ethanol. After rinsing with double-distilled water, the leaves were stained with Lugol's iodine staining reagent (Sigma) and briefly destained with water. Histochemical Analyses—Tissue sectioning and microscopy were carried out as described previously (17Ahn J.W. Kim M. Kim G.T. Lim J.H. Pai H.-S. Plant J. 2004; 38: 969-981Crossref PubMed Scopus (65) Google Scholar), using the fourth leaf above the infiltrated leaf from the VIGS lines. Confocal Laser Scanning Microscopy—For TMRM (tetramethylrhodamine methyl ester, Molecular Probes) staining of mitochondria, TMRM was added into the leaf protoplasts at the final concentration of 200 nm. After incubation for 1-2 min at 25 °C, protoplasts were transferred to wells on microscope slides and examined by confocal microscopy (Carl Zeiss LSM 510) with a BP560-615 (543 nm excitation, 560-615 nm emission) optical filter to visualize the red fluorescent probe. Quantitative images were captured, and data were analyzed using the LSM 510 software (version 2.8). Chlorophyll autofluorescence was observed using an LP650 (excitation 488 nm, emission 650 nm) optical filter. MitoTracker Green FM (Molecular Probes) staining of protoplasts and 4′,6-diamidino-2-phenylindole staining of chloroplasts were carried out, as described previously (18Cho H.S. Lee S.S. Kim K.D. Kim S.J. Hwang I. Lim J.S. Park Y.I. Pai H.-S. Plant Cell. 2004; 16: 2665-2682Crossref PubMed Scopus (74) Google Scholar). PFGE Analysis—Protoplasts were prepared from the fourth to sixth leaves above the infiltrated leaf of the VIGS lines and counted. Roughly equal numbers of protoplasts from the VIGS lines were embedded, and pulse-field gel electrophoresis was carried out as described (18Cho H.S. Lee S.S. Kim K.D. Kim S.J. Hwang I. Lim J.S. Park Y.I. Pai H.-S. Plant Cell. 2004; 16: 2665-2682Crossref PubMed Scopus (74) Google Scholar, 21Lilly J.W. Havey M.J. Jackson S.A. Jiang J. Plant Cell. 2001; 13: 245-254Crossref PubMed Scopus (89) Google Scholar). The rbcL cDNA was used as a probe. Virus-induced Gene Silencing of the NbERS and NbSRS Caused Severe Leaf-yellowing Phenotypes—Functional genomics has been carried out in N. benthamiana using TRV-based virus-induced gene silencing (VIGS) to assess functions of signaling genes and genes that likely cause embryo or seedling lethality when their expression is suppressed. VIGS is based on the phenomenon that gene expression is suppressed in a sequence-specific manner by infection with viral vectors carrying host genes (22Burch-Smith T.M. Anderson J.C. Martin G.B. Dinesh-Kumar S.P. Plant J. 2004; 39: 734-746Crossref PubMed Scopus (554) Google Scholar) and has been proved to be a powerful tool to analyze embryoor seedling-lethal genes (17Ahn J.W. Kim M. Kim G.T. Lim J.H. Pai H.-S. Plant J. 2004; 38: 969-981Crossref PubMed Scopus (65) Google Scholar, 18Cho H.S. Lee S.S. Kim K.D. Kim S.J. Hwang I. Lim J.S. Park Y.I. Pai H.-S. Plant Cell. 2004; 16: 2665-2682Crossref PubMed Scopus (74) Google Scholar). Using a VIGS screen, we found that gene silencing of the N. benthamiana NbERS and NbSRS genes, encoding isoforms of GluRS and SerRS, respectively, causes severe leaf yellowing and abnormal leaf morphology, while maintaining almost normal plant growth. The full-length NbERS cDNA was obtained by 5′-rapid amplification of cDNA ends. The full-length NbSRS cDNA was obtained by PCR based on the sequence in the TIGR data base (accession number TC8134). NbERS and NbSRS encode polypeptides of 569 and 506 amino acids, respectively, corresponding to theoretical molecular masses of 63410.40 and 56266.37 Da. The NbERS and NbSRS genes exhibited high sequence similarity to the corresponding genes of prokaryotes, particularly cyanobacterial species, indicating that they encode an organellar isoform of GluRS and SerRS. The Arabidopsis data base contains one sequence encoding an organellar GluRS (GenBank™ accession number NP_201210), designated AtERS, and a sequence for an organellar SerRS (AAL16281), designated AtSRS. When the amino acid sequences of the organellar ERS and SRS genes from plants were aligned with the corresponding genes from prokaryotes, a notable difference between the plant proteins and the corresponding bacterial proteins is that plant proteins contain a long extension at their N terminus, which has features of chloroplast and mitochondria targeting signals (23Peeters N. Small I. Biochim. Biophys. Acta. 2001; 1541: 54-65Crossref PubMed Scopus (207) Google Scholar). Suppression of the NbERS and NbSRS Transcripts by VIGS—To induce gene silencing of NbERS and NbSRS, we cloned different fragments of the NbERS and NbSRS cDNAs into the TRV-based VIGS vector pTV00 (16Ratcliff F. Martin-Hernandez A.M. Baulcombe D.C. Plant J. 2001; 25: 237-245Crossref PubMed Scopus (738) Google Scholar), and infiltrated N. benthamiana plants with Agrobacterium containing each plasmid (Fig. 1, A and B). TRV:ERS(1) and TRV: ERS(2) contain the N-terminal 333 bp and the C-terminal 383 bp of the NbERS cDNA, respectively, whereas TRV:ERS(3) contains a 1.5-kb cDNA fragment. Similarly, TRV:SRS(2) and TRV:SRS(3) contains the N-terminal 330 bp and the C-terminal 373 bp of the NbSRS cDNA, respectively, whereas TRV:SRS(1) contains a 1.2-kb cDNA fragment. VIGS with these constructs all resulted in a similar yellowing phenotype in newly emerged leaves, whereas overall plant growth was normal (Fig. 1, A and B). During flower formation, the sepals became yellow in the ERS plants (Fig. 1A). However, except that, flower development was normal in both ERS and SRS VIGS lines (data not shown). In contrast, VIGS of NbIRS, which encodes an organellar form of IleRS resulted in only a mild leaf-yellowing phenotype, when a 1.5-kb cDNA fragment was used for VIGS (Fig. 1C). As a control, we also carried out VIGS of c-IRS, which encodes a cytosolic form of IleRS, and it resulted in arrested plant growth and severe developmental abnormalities in newly emerged leaves, distinct from the VIGS phenotypes of the organellar ARSs (Fig. 1D). Effects of gene silencing on the amounts of endogenous NbERS and NbSRS mRNAs were examined by semiquantitative RT-PCR (Fig. 1, E and F). RT-PCR using the ERS-A primers (indicated in Fig. 1A) produced significantly reduced amounts of PCR products in the yellow sectors of the leaves from the TRV:ERS(2) line, indicating that the endogenous level of the NbERS transcripts is greatly reduced in these plants (Fig. 1E). The same primers detected high levels of viral genomic transcripts containing the N-terminal region of NbERS in the TRV: ERS(1) and TRV:ERS(3) lines. Similarly, RT-PCR using the ERS-B primers (indicated in Fig. 1A) produced significantly reduced amounts of PCR products in the yellow sectors of leaves from the TRV:ERS(1) line, whereas the same primers detected high levels of viral genomic transcripts containing the C-terminal region of NbERS in the TRV:ERS(2) and TRV:ERS(3) lines. In all of the ERS VIGS lines, the transcript level of NbSRS, NbIRS, and actin remained constant (Fig. 1E). Silencing of NbSRS was also examined by semiquantitative RT-PCR (Fig. 1F). RT-PCR using the SRS-A and SRS-B primers produced significantly reduced amounts of PCR products in yellow sectors of leaves from the TRV:SRS(3) and TRV:SRS(2) lines, respectively, indicating that the endogenous level of the NbSRS transcripts is significantly reduced in those plants. In all of the SRS VIGS lines, the transcript levels of NbERS, NbIRS, and actin stayed constant (Fig. 1F). These results clearly show that VIGS using the NbERS and NbSRS cDNA fragments specifically suppresses expression of these genes. The transcripts levels of NbERS and NbSRS in TRV were identical to those in wild-type, indicating that TRV infection did not affect the expression of NbERS and NbSRS (supplementary Fig. S1A). Although VIGS of NbIRS caused only mild phenotypes compared with silencing of NbERS and NbSRS, the degree of gene silencing of NbIRS in its VIGS lines was similar to that of NbERS and NbSRS, at least at the mRNA level (supplementary Fig. S1B). These data suggest that each organellar ARS may have a distinct threshold level required for normal organelle function. Expression Patterns of NbERS and NbSRS—To examine tissue-specific expression patterns of NbERS and NbSRS, semiquantitative RTPCR analyses were carried out using the NbERS- and NbSRS-specific primer sets. RT-PCR products of NbERS and NbSRS were detected in all of the tissues examined, including roots, stems, mature leaves, young leaves, flower buds, and open flowers in N. benthamiana, with somewhat higher levels of the transcripts in young tissues such as young leaves and flower buds (Fig. 2A). The light-dependent gene expression of NbERS and NbSRS was examined by semiquantitative RT-PCR (Fig. 2B). N. benthamiana seedlings were grown for 7 days on MS media under normal light conditions (16 h light/8 h dark), under dark conditions, or under dark conditions followed by a transfer to light for 1 h. The NbERS and NbSRS mRNA levels were lower in dark-grown seedlings than light-grown seedlings, but exposure to light for 1 h after the dark-grown period increased the NbERS and NbSRS mRNA levels to that of light-grown seedlings (Fig. 2B). As a positive control, NbGyrB expression in response to light was also monitored. NbGyrB encodes subunit B of DNA gyrase, which plays a critical role in chloroplast DNA metabolism, and is induced by light (18Cho H.S. Lee S.S. Kim K.D. Kim S.J. Hwang I. Lim J.S. Park Y.I. Pai H.-S. Plant Cell. 2004; 16: 2665-2682Crossref PubMed Scopus (74) Google Scholar). These results indicate that expression of NbERS and NbSRS is stimulated by light. Dual Targeting of AtERS and AtSRS to Chloroplasts and Mitochondria—We examined the subcellular localization of AtERS and AtSRS (Fig. 3). Both AtERS and AtSRS contain a long extension at their N terminus, which contains features of dual targeting signals to chloroplasts and mitochondria (23Peeters N. Small I. Biochim. Biophys. Acta. 2001; 1541: 54-65Crossref PubMed Scopus (207) Google Scholar). We generated fusion proteins in which the N-terminal regions or the full-length proteins of AtERS and AtSRS were fused to GFP. For AtERS, the N-terminal sequence of 70 aa (Met-1 to Gly-70, M1G70) and the full-length sequence of 570 aa (Met-1 to Thr-570, M1T570) were used. For AtSRS, the N-terminal sequence of 90 aa (Met-1 to Ala-90, M1A90) and the full-length sequence of 514 aa (Met-1 to Lys-514, M1K514) were used. These DNA constructs encoding different forms of AtERS-GFP or AtSRS-GFP fusion proteins under the control of the CaMV35S promoter were introduced into protoplasts isolated from Arabidopsis seedlings. To track mitochondria, a DNA construct encoding an F1ATPase-γ-RFP fusion protein of the mitochondrial F1ATPase-γ subunit (19Lee Y.J. Kim D.H. Kim Y.-W. Hwang I. Plant Cell. 2001; 13: 2175-2190Crossref PubMed Scopus (187) Google Scholar), and red fluorescent protein (RFP) was cotransformed into the protoplasts. After incubation at 25 °C for 24 h, expression of the introduced genes was examined by confocal laser scanning microscopy with different filters to capture images of GFP, RFP, and autofluorescence of chlorophyll. For both AtERS(M1G70)-GFP- and AtERS(M1T570)-GFP-transformed protoplasts, the green fluorescent signal completely overlapped with both autofluorescence of chloroplasts and the red fluorescent signal of F1ATPase-γ-RFP, indicating that the AtERS protein was targeted to both chloroplasts and mitochondria (Fig. 3A). Similarly, both AtSRS(M1A90)-GFP- and AtSRS(M1K514)-GFP-transformed protoplasts exhibited overlapping fluorescent signals from GFP, RFP, and chlorophyll autofluorescence, indicating dual targeting of AtSRS (Fig. 3B). These results demonstrate that the N-terminal extension of AtERS and AtSRS contains a signal for transport of the protein to both chloroplasts and mitochondria. Numbers of Chloroplasts and Mitochondria—Protoplasts were generated from yellow sectors of the leaves from the TRV:ERS and TRV: SRS lines, and examined by confocal laser scanning microscopy (Fig. 4A). As controls, protoplasts from the TRV were also observed. In both TRV:ERS and TRV:SRS, almost all of the protoplasts, mostly originated from leaf mesophyll cells, exhibited drastically reduced numbers of chloroplasts. The average number of chloroplasts in the TRV:ERS and TRV:SRS lines was about 27 and 35% of the TRV control, respectively (Fig. 4B). Chlorophyll content of the leaves was consistently much lower in the VIGS lines than the TRV controls (Fig. 4, C and G). Furthermore, chloroplasts from the TRV:ERS and TRV:SRS lines were significantly smaller than TRV controls (Fig. 4D) and measured ∼31 and 55% of the average control diameter, respectively. Some of the chloroplasts in the TRV:ERS and TRV:SRS lines were dumbbell-shaped, indicating disrupted chloroplast division (results not shown). The chloroplast numbers and diameter, chlorophyll autofluorescence, and total chlorophyll contents in TRV were very similar to those in wild-type (supplementary Fig. 2, A-D). Mitochondria in leaf protoplasts isolated from the TRV control, TRV: ERS, and TRV:SRS lines were examined by TMRM and MitoTracker Green FM (MG) fluorescent probes (Fig. 4, A, E, and F). TMRM is a lipophilic cation that accumulates in mitochondria in proportion to the mitochondrial membrane potential (24Zhang H. Huang H.M. Carson R.C. Mahmood J. Thomas H.M. Gibson G.E. Anal. Biochem. 2001; 298: 170-180Crossref PubMed Scopus (77) Google Scholar), and a drop in the membrane potential leads to a decrease in fluorescence. The average TMRM fluorescence of protoplasts from TRV:ERS and TRV:SRS leaves was ∼9 and 25% of TRV controls, respectively (Fig. 4E). This decrease in fluorescence could be due to either reduced mitochondrial numbers or altered membrane potential of mitochondria in the protoplasts. MitoTracker Green FM (MG) accumulates in mitochondria regardless of the mitochondrial membrane potential, and is widely used to determine mitochondrial mass (25Oubrahim H. Stadtman E.R. Chock P.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9505-9510Crossref PubMed Scopus (99) Google Scholar). The MG fluorescence of the protoplasts from TRV: ERS and TRV:SRS leaves, ∼8 and 11%, respectively, was significantly lower than that of the TRV control (Fig. 4, A and F). This indicates that mitochondrial numbers and/or mass are reduced in the ERS and SRS VIGS lines. The TMRM and MG fluorescence of mitochondria in TRV were very similar to those in wild-type (supplementary Fig. S2, E and F). Ultrastructural Analysis of Chloroplasts—Transmission electron microscopy of transverse leaf sections of the TRV:ERS and TRV:SRS VIGS lines showed a large difference in the chloroplast number and morphology compared with TRV controls (Fig. 5), whereas the leaf cell structure" @default.
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• W2022083147 title "Inactivation of Organellar Glutamyl- and Seryl-tRNA Synthetases Leads to Developmental Arrest of Chloroplasts and Mitochondria in Higher Plants" @default.
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