a natural variation in pleiotropic developmental …a natural variation in pleiotropic developmental...
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A Natural Variation in PLEIOTROPIC DEVELOPMENTALDEFECTS Uncovers a Crucial Role for Chloroplast tRNAModification in Translation and Plant Development[OPEN]
Hui Liu,a Ding Ren,a,b,1 Ling Jiang,a Xiaojing Li,a Yuan Yao,a Limin Mi,a Wanli Chen,a Aowei Mo,a Ning Jiang,a
Jinshui Yang,a Peng Chen,c Hong Ma,d Xiaojin Luo,a,e,1 and Pingli Lua,b,1
a School of Life Sciences, Fudan University, Shanghai 200433, Chinab State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng 475004,Chinac Biomass and Bioenergy Research Centre, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan,430070, ChinadDepartment of Biology, Eberly College of Science, Huck Institutes of the Life Sciences, Pennsylvania State University, UniversityPark, Pennsylvania 16802eMOE Engineering Research Center of Gene Technology, Fudan University, Shanghai 200433, China
ORCID IDs: 0000-0002-9575-9401 (H.L.); 0000-0002-7805-1532 (D.R.); 0000-0002-6321-2911 (L.J.); 0000-0002-1254-9119 (X. Li);0000-0003-0502-3714 (Y.Y.); 0000-0002-8204-4108 (L.M.); 0000-0002-0743-2522 (W.C.); 0000-0002-7286-2211 (A.M.); 0000-0003-0664-6286 (N.J.); 0000-0002-2296-1465 (J.Y.); 0000-0002-3397-7235 (P.C.); 0000-0001-8717-4422 (H.M.); 0000-0003-0134-2208 (X.Luo); 0000-0002-3941-9955 (P.L.)
The modification of tRNA is important for accurate, efficient protein translation. A number of tRNA-modifying enzymes werefound to influence various developmental processes in distinct organisms. However, few genetic or molecular studies havefocused on genes encoding tRNA-modifying enzymes in green plant organelles. Here, we discovered that PDDOL, a naturalvariation allele of PLEIOTROPIC DEVELOPMENTAL DEFECTS (PDD), leads to pleiotropic developmental defects in a near-isogenic line (NIL) generated by introgressing the wild rice Oryza longistaminata into the rice (Oryza sativa) cv 187R. Map-based cloning revealed that PDD encodes an evolutionarily conserved tRNA-modifying GTPase belonging to the tRNAmodification E family. The function of PDD was further confirmed by genetic complementation experiments and mutantanalysis. PDD mRNA is primarily expressed in leaves, and PDD is localized to chloroplasts. Biochemical analyses indicatedthat PDD187R forms homodimers and has strong GTPase activity, whereas PDDOL fails to form homodimers and has weakGTPase activity. Liquid chromatography–coupled tandem quadrupole mass spectrometry revealed that PDD is associatedwith the 5-methylaminomethyl-2-thiouridine modification of chloroplast tRNA. Furthermore, compared to 187R, NIL-PDDOL
has severely reduced levels of proteins involved in photosynthesis and ribosome biogenesis but increased levels of plastid-encoded RNA polymerase subunits. Finally, we demonstrate that the defect due to PDDOL alters chloroplast gene expression,thereby affecting communication between the chloroplast and the nucleus.
INTRODUCTION
Rice (Oryza sativa) is one of the most important cropsworldwide, serving as amajor staple food crop for humans andas a model system to decipher the molecular mechanismsregulating plant development and growth. In addition to thetwo cultivated rice species, there are 21 wild species in theOryza genus (Vaughan et al., 2003), providing ample naturalgenetic resources for studying gene function and improving
agronomic traits in rice. Todate, studies using variouswild ricespecies as donors to create near-isogenic lines (NILs) viainterspecific introgression have identified a number of genesinvolved in establishing rice architecture, inflorescence for-mation, stress responses, and awn development (Jin et al.,2008; Zhu et al., 2013; Hirabayashi et al., 2015; Hua et al.,2015).Accurate and efficient protein translation is essential for en-
suring that proteins perform their normal functions in cells. tRNAis a vital component of the translation machinery, as it deliversthe corresponding amino acid to the elongating peptide chainbased on codon–anticodon recognition (Giegé et al., 2012). Tocorrectly decipher the genetic code, tRNA is extensively mod-ified at various sites (Björk and Hagervall, 2005). To date, >100modifications in tRNAs have been identified, most of whichcontribute to translational accuracy and efficiency (Björk et al.,1999; Gustilo et al., 2008; Cantara et al., 2011). For example, inyeast (Saccharomyces cerevisiae) and nematodes (Caeno-rhabditis elegans), themodification of uridine at position 34 (U34)
1 Address correspondence to [email protected]; [email protected] or [email protected] authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Pingli Lu ([email protected]; [email protected]) and Xiaojin Luo ([email protected]).[OPEN]Articles can be viewed without a subscription.1These authors contributed equally to this work.www.plantcell.org/cgi/doi/10.1105/tpc.19.00660
The Plant Cell, Vol. 32: 2345–2366, July 2020, www.plantcell.org ã 2020 ASPB.
in cytoplasmic tRNAs influences the translation rate andprotein aggregation (Nedialkova and Leidel, 2015). In miceand humans, the modification of adenosine at position 37 inmitochondrial tRNAs governs protein translation and is as-sociated with a mitochondrial-related disease (Wei et al.,2015). In rice, modifications of cytoplasmic pre-tRNAHis
regulate the translation efficiency of specific mRNAs (Chenet al., 2019).
Modifications of tRNAs are catalyzed by tRNA-modifying en-zymes, which are evolutionarily conserved in eukaryotic andprokaryotic organisms (Ferré-D’Amaré, 2003). In a given genome,;1 to 10% of genes are annotated as encoding tRNA-modifyingenzymes (El Yacoubi et al., 2012). Among these genes, membersof the tRNA modification E (TrmE) family are thought to directlymodify the fifth carbon atom of the wobble uridine (U34) in certaintRNAs, such as tRNALysUUU, tRNAGluUUC, tRNAGlnUUG, tRNA-LeuUAA, and tRNAArgUCU (Moukadiri et al., 2014). These tRNAs candecode two-family box triplets ending in A or G (Armengod et al.,2012). In Escherichia coli, TrmE modifies U34 in tRNA to 5-car-boxymethylaminomethyl-(2-thio)uridine, which can be de-carboxylated to 5-methylaminomethyl-(2-thio)uridine by TrmC incertain tRNAs (Cabedo et al., 1999). In yeast, the TrmE proteinMSS1 is localized to mitochondria and shares a similar functionwith TrmE in E. coli (Decoster et al., 1993). In human, althoughTrmE (GTPBP3) is also targeted tomitochondria, itmodifiesU34 intRNA to 5-taurinomethyl-(2-thio)uridine (Villarroya et al., 2008;Asano et al., 2018). Although several studies have suggested thatthese modifications can influence the accuracy of codon–anticodon recognition and/or reading frame maintenance duringtranslation (Yokoyama et al., 1985; Krüger et al., 1998; Brégeon
et al., 2001), strong genetic and molecular evidence for this hy-pothesis is lacking.Phylogenetic analysis revealed that TrmE proteins in green
plants form a single monophyletic group with homologs fromcyanobacteria, which are ancestors of chloroplasts (Suwastikaet al., 2014), suggesting that plant TrmE proteins might modifychloroplast tRNAs to ensure translation in chloroplasts. Thechloroplast, the site for photosynthesis, plays an important rolein plant growth (Waters and Langdale, 2009). This semi-autonomous organelle has its own genome and protein syn-thesis machinery. In general, the chloroplast genome encodes30 tRNAs and;80 unique proteins (Chumley et al., 2006). Threedecades ago, two-dimensional homochromatography and thinlayer chromatography revealed a few modified nucleotides inseveral chloroplast tRNAs in soybean (Glycine max), barley(Hordum vulgare), and the algaCodium fragile (Pillay et al., 1984;Schön et al., 1986; Francis et al., 1989). However, to date, fewstudies have focused on the functions of chloroplast tRNA-modifying enzymes, besides TADA, which is involved in thedeamination of adenine to inosine, affecting efficient chloroplasttranslation in Arabidopsis (Arabidopsis thaliana; Delannoy et al.,2009; Karcher and Bock, 2009).Here, taking advantage of natural genetic variation in the
Africanwild rice speciesOryza longistaminata, weuncovered thecrucial roles of thePLEIOTROPIC DEVELOPMENTALDEFECTS(PDD) gene in rice growth and development. We demonstratethat PDD is a conserved TrmE family protein localized to ricechloroplasts and that it participates in the modification ofchloroplast tRNAs, thereby affecting translation and plantdevelopment.
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RESULTS
NIL-PDDOL Shows Pleiotropic Developmental Defects
To take advantage of natural genetic variation to identify functionalgenes in rice, we constructed a set of NILs from backcross progeniesderived from a cross between rice cv 187R (O. sativa subsp indica) asthe recurrent parent and Africanwild rice speciesO. longistaminata asthe donor parent (Supplemental Figure 1A). In this population, weidentifiedaNILwithseverepleiotropicdevelopmentaldefects (detailedbelow).GeneticandphenotypicanalysesofF2progeniesderived fromF1 plants produced by crosses between this NIL and 187R revealedthat the defects were caused by a single recessive nuclear locus (F2segregation:134normalplants;39defectiveplants;3:1ratio:P50.46).Based on the phenotypic defects, we named the allele in the 187Rbackground PDD187R. The allele in O. longistaminata was namedPDDOL, and the NIL was named NIL-PDDOL.
Phenotypic analysis showed that, before the five-leaf stage,newly emerging leaves in NIL-PDDOL were albino and graduallyturned green but with an albino margin during seedling de-velopment (Figures 1A and 1B). However, after the five-leaf stage,newly formed leaves had a normal green color (Figure 1B;Supplemental Figure 1B). The photosynthetic pigment contentsweremuch lower inNIL-PDDOL than in187Rat the three-leaf stage(Figure 1C) but recovered to normal levels to those in 187R at laterstages of development (Supplemental Figure 1C). Given the closerelationship between photosynthetic pigments and chloroplastdevelopment, we compared the ultrastructures of chloroplasts in187R andNIL-PDDOL by transmission electronmicroscopy. 187Rleaf cells harbored normal chloroplasts with well-organized la-mellar structures and normally stacked grana and thylakoidmembranes (Figures1Dand1E).Bycontrast, cellswithin thewhitesectors of NIL-PDDOL were severely vacuolated and lacked well-organized lamellar structures (Figures 1F and 1G). However, cellsin the green sectors of NIL-PDDOL at later stages of developmenthad well-developed chloroplasts (Supplemental Figure 1D). Wealso compared the photosynthetic capacities of 187R and NIL-PDDOL. The Fv/Fm value, representing the maximum quantumyield of PSII photochemistry, was significantly lower inNIL-PDDOL
than in 187R at the three-leaf stage (Supplemental Figure 1E).Moreover, NIL-PDDOL plants showed sharply reduced plant
height and tiller number throughout their life cycles (Figures 1H to1J). NIL-PDDOL panicles were much shorter and had fewerbranches than 187R panicles (Figure 1K; Supplemental Figure 1F;Supplemental Figures 2A and 2B). All internodes were significantlyshorter inNIL-PDDOL than in187R (Figures1Kand1L).Microscopicobservation of internode cross sections revealed that the shortstature of NIL-PDDOL is caused by smaller cell size (Figure 1M).Additionally, although the grain size of NIL-PDDOL was similar tothatof187R (SupplementalFigure1G),NIL-PDDOLplantsdisplayedreducedgrainnumberperpanicle,seedsetting rate,and1000-grainweight (Supplemental Figures 2C to 2E), thereby leading to greatlyreduced yield per plant (Supplemental Figure 2F).
Map-Based Cloning and Identification of PDD
To identify the genecausing thepleiotropic defects ofNIL-PDDOL,we performed map-based cloning. Using 200 F2 plants derived
from a cross between NIL-PDDOL and 187R, the PDD locus wasinitially mapped to the long arm of chromosome 8 (Figure 2A). Tofinely map PDD, we crossed NIL-PDDOL with 93-11 (O. sativasubsp indica). By analyzing ;9000 BC1F2 plants, the PDD genewas further narrowed down to a 35-kb genomic region containingfive candidate genes (CGs) in the Nipponbare reference genome(Figure 2A). Among these genes, CG1 and CG3 are tandemlyduplicatedgenes.CG2,CG4, andCG5areannotatedasencodingan expressed protein, tRNA modification GTPase TrmE, andpeptidase, respectively (Supplemental Table 1). Based on ge-nome annotations and available mRNA expression data, wereasoned that CG4 or CG5 might be the responsible gene.We performed genetic complementation experiments to
identify which gene is indeed PDD. We generated two in-dependent binary constructs containing CG4 or CG5 genomicfragments with their own promoters from 187R (Figure 2B;Supplemental Figure 3D) and separately transformed theseconstructs into NIL-PDDOL. In total, we obtained 12 independentT0 lines for theCG4 construct and 10 independent T0 lines for theCG5 construct. The phenotypic defects of NIL-PDDOL were re-stored in transgenicplantsharboring theCG4 (LOC_Os08g31460)complementation construct (Figures 2C and 2D), including thepigment contents, tiller number, and plant height (SupplementalFigures 3A to 3C). By contrast, the construct harboring the CG5genomic fragment failed to rescue the developmental defects ofNIL-PDDOL (Supplemental Figure 3E). Therefore, the CG4 (LOC_Os08g31460) locus corresponds to PDD.To further confirm the function of PDD, we generated pdd
mutants via clustered regularly interspaced short palindromicrepeats (CRISPR)/ Cas9–mediated genome editing (Xie et al.,2015).Wedesigned twodistinct guideRNAs targeting thefirst andsecondexonsofPDD (Figure 3A). After transforming theconstructharboring the guideRNAs into 187R,we obtained 15 independentT0 lines. After genotyping, we identified five distinct genome-edited alleles at both sites in PDD in three T0 lines (pdd-1/1, pdd-2/pdd-3, and pdd-4/pdd-5; Figure 3A), which were predicted toproduce truncated proteins (Supplemental Figure 4). Un-fortunately, although the pdd-2/pdd-3 and pdd-4/pdd-5 plantslived for a short time, both died before the five-leaf stage, sug-gesting that the combined mutant alleles are too deleterious toallow survival to adulthood. However, in a population containing96 individual plants from the next generation of pdd-1/1, weobtained 4 pdd-1 homozygous plants that survived and com-pleted their life cycles.Genotyping revealed that pdd-1 contained a 1-bp T insertion in
the first exon and a 1-bp A insertion in the second exon of PDD,which were predicted to produce an mRNA with a prematuretermination codon (Figure 3A), potentially leading to the pro-duction of a truncated protein with only 35 amino acids from theNterminus (Supplemental Figure 4). To determine whether thesetwo small DNA polymorphisms resulted in nonsense-mediatedmRNA decay in pdd-1, we measured the mRNA levels of PDD in187R and pdd-1 via RT-qPCR. The level of PDD mRNA was notaffected in pdd-1 (Supplemental Figure 3F), implying that themutations in pdd-1 did not alter PDD mRNA abundance. Phe-notypic analysis showed that pdd-1 exhibited similar but moresevere developmental defects compared to NIL-PDDOL (Figures
Chloroplast tRNA Modification Affects Development 2347
Figure 1. Phenotypic Analysis of NIL-PDDOL.
(A) and (B)Phenotypes of 187R (left) andNIL-PDDOL seedlings (right) at the three-leaf stage (A) and five-leaf stage (B). Thewhite boxes above the seedlingsshowmagnifiedviewsof theareasof 187RandNIL-PDDOL leaves in the red rectangles. Bar in the toppanels of (A)and (B)52mm;bar in thebottompanel of(A) 5 2 cm; bar in the bottom panel of (B) 5 4 cm.(C) Pigment contents in 187R and NIL-PDDOL leaves at the three-leaf stage. Data represent mean6 SD from three independent biological replicates (fromdifferent seedling leaves). ***, P < 0.001 (Student’s t test). Chl a, chlorophyll a; Chl b, chlorophyll b.(D) to (G) Transmission electron microscopy images of cells from 187R (see [D] and [E]) and white leaves of NIL-PDDOL (see [F] and [G]) at the three-leafstage. The red arrow indicates normally stacked grana. Bar in (D) and (F) 5 1 mm; bar in (E) and (G) 5 200 nm.(H) Phenotypes of 187R (left) and NIL-PDDOL (right) at the mature stage. Bar 5 10 cm.(I) to (J)Plant height (I) and tiller number (J) in 187R andNIL-PDDOL. Before heading, plant height to the leaf tipwasmeasured. After heading, plant height tothe panicle tip was measured. The data are presented as mean 6 SD. ***, P < 0.001 (n 5 20 plants; Student’s t test).(K) Phenotypes of the stem structures of 187R (left) and NIL-PDDOL (right). The red arrows indicate the node position. Bar 5 10 cm.(L) Comparison of 187R (left) and NIL-PDDOL (right) panicles and internode length. The data are presented as mean 6 SD. ***, P < 0.001 (n 5 20 plants;Student’s t test).(M) Cross sections of the uppermost internodes of 187R and NIL-PDDOL. Bar 5 50 mm.
2348 The Plant Cell
3B to 3E; Supplemental Figures 2A to 2F), further confirming thatthe causal gene in NIL-PDDOL is PDD.
In addition, genetic analysis of a population of seeds derivedfromaheterozygouspdd-1/1plant revealed that theproportionofhomozygous individuals (;16%)wasmuchsmaller thanexpected(25%;P50.017), implying that a portion of homozygous embryosmight have failed to develop into mature seeds. Furthermore,comparative phenotypic analysis of seed germination and plantgrowth in 187R, NIL-PDDOL, and pdd-1 revealed that most pdd-1seeds germinated but failed to form seedlings, unlike 187R andNIL-PDDOL (Figures 3Fand3G), further demonstrating thatPDD is
essential for normal rice development. These results also indicatethat the natural variant in the Africanwild rice species is a relativelyweak allele.
PDD Encodes a Chloroplast-Localized TrmE Family Protein
In the rice genome, PDD is annotated as a tRNA-modifyingGTPase belonging to the TrmE family. Phylogenetic analysisshowed that most organisms from bacteria to humans containonly one TrmE gene (Supplemental Figure 5), implying that the
Figure 2. Map-Based Cloning and Characterization of the PDD Gene.
(A)Molecular cloning of thePDD gene. ThePDD locuswas narrowed down to a 35-kb genomic DNA region on the chromosome 8 (Chr.8), which containedfive CGs (CG1 to CG5). The number of recombinants identified is shown below each marker.(B) CG4 construct used for complementation. HPT, hygromycin phosphotransferase gene; LB, left border; NOS, nopaline synthase terminator; RB, rightborder.(C) and (D)Phenotypes of complementation lines (CP-1 andCP-2) at the seedling stage (C) and themature stage (D). Bar in (C)5 2 cm; bar in (D)5 20 cm.
Chloroplast tRNA Modification Affects Development 2349
copy number of TrmE tends to be under strict natural selectionduring evolution.
Bioinformation analysis of protein structure revealed that PDDpossesses a predicted chloroplast-targeting signal; one con-served N-terminal domain of;120 amino acid residues; a centralG domain of ;170 residues; and C-terminal region containinga highly conserved CxGK motif, where x represents any residue
(Figure 4A; Supplemental Figure 5). Multiple protein sequencealignment revealed two novel conserved regions (named R1 andR2) in the N-terminal domain of this protein (Figure 4A). The Gdomain contains four typical motifs (G1 to G4), which is charac-teristic of GTPases (Figure 4A). In addition, PDD is highly con-served among plant species, sharing >80% sequence identitywith homologs in Brachypodium distachyon, maize (Zea mays),
Figure 3. Genotypes and Phenotypes of Genome-Edited pdd Mutants.
(A) Genotypes of the pdd mutants obtained by CRISPR/Cas9.(B) and (C) Phenotypes of the pdd-1 mutant at the seeding stage (B) and the mature stage (C). Bar in (B) 5 2 cm; bar in (C) 5 20 cm.(D) and (E) Plant height (D) and tiller number (E) of 187R, NIL-PDDOL, and pdd-1. The data are shown as box-plot graphs. Lines across the box show themedian values. Bottom and top boxes indicate the 25th percentile to the 75th percentile. Whiskers represent the maximum and minimum values. ***, P <0.001 (n 5 20 plants; Student’s t test).(F) Seedling growth phenotypes of 187R, NIL-PDDOL, and pdd-1. Bar 5 5 cm.(G) Quantitative analysis of the proportions of seedlings from 96 187R, NIL-PDDOL, and pdd-1 seeds. The data are shown as mean 6 SD from threeindependent biological replicates. n.s., no significant difference; ***, P < 0.001 (Student’s t test).
2350 The Plant Cell
and sorghum (Sorghum bicolor) and 60.9% identity with its ho-molog in Arabidopsis (Supplemental Figure 6).
To explore the molecular function of PDD, we investigated itsmRNA expression pattern in various tissues of 187R plants byRT-qPCR. The PDD transcript was preferentially expressed ingreen tissues during development, including leaves at theseedling stage and leaves and sheaths at the tillering stage, butwith much lower expression in other tissues such as roots,stems, and panicles (Figure 4B). In addition, we generateda binary construct with the 2167-bp promoter region of PDD187R
driving the b-glucuronidase (GUS) reporter gene and used it totransform the japonica rice var Nipponbare. We detected GUSsignals in various tissues at different stages of development,such as leaves, the embryos of seeds, roots, panicles, andovaries (Figure 4C). To examine the subcellular localization ofPDD, we constructed the Pro35S:PDD187R-eGFP fusion vectorand introduced it into rice protoplasts. The GFP signals colo-calized with the autofluorescence signals from chlorophyll(Figure 4D), indicating that PDD is a chloroplast-localizedprotein.
Figure 4. PDD Encodes a Chloroplast-Localized TrmE Subfamily Protein.
(A) Schematic diagram of the structure of PDD and multiple sequence alignment of conserved motifs among PDD homologs. CTS, chloroplast-targetingsignal, M. musculus, Mus musculus (house mouse); S. pombe, Schizosaccharomyces pombe (fission yeast).(B) Expression analysis of PDD in various tissues by RT-qPCR. Data were normalized to ACTIN1 expression. The bars represent mean 6 SD of triplicateexperiments. L-S, leaves at the seedling stage; L-T, leaves at the tillering stage; P, panicle; R, root; S, stem;SH-S, sheath at the seedling stage;SH-T, sheathat the tillering stage.(C)GUSstaining of seeds at 1 d after germination (a), seedlings at the four-leaf stage (b), root (c),mature leaves (d), panicle (e), spikelet (f), andpistil (g). Bar in(a) and (f) 5 1 mm; bar in (b), (d), and (e) 5 1 cm; bar in (c) and (g) 5 200 mm.(D)Subcellular localization of PDD187R-eGFP in rice protoplasts. (Top) Negative control using empty vector. (Bottom) Localization of PDD187R-eGFP fusionprotein. Bar 5 10 mm.
Chloroplast tRNA Modification Affects Development 2351
Natural Variations in PDDOL Affect Homodimer Formationand GTPase Activity
To identify the genetic variations between PDD187R and PDDOL,we cloned and compared their genomic sequences, including theirpromoter regions. Sequence alignment revealed 468 poly-morphisms, including single-nucleotide polymorphisms (SNPs)and insertions/deletions, in this region between the two varieties(Table 1; Supplemental Figure 7). The protein-coding region inPDDOL contains one 6-bp deletion and 28 SNPs compared toPDD187R (Table 1; Supplemental Figure 7B). These DNA poly-morphisms are predicted to cause two amino acid deletions at thebeginningof theNterminusandfiveaminoacidalterationsalongtheentire protein region (Figure 5A; Supplemental Figure 7C). To de-termine whether the PDDOL version is defective in vivo, we gen-erated a binary construct containing the PDDOL coding sequencedriven by the 2167-bp native promoter from PDD187R (Figure 5B).After transforming the vector into NIL-PDDOL, we identified 16 in-dependent transgenic plants. All 16 plants displayed highly similarphenotypes to NIL-PDDOL plants (Figures 5C and 5D), indicatingthat PDDOL is a dysfunctional protein.
To investigate whether the variations in PDDOL affect its mRNAabundance,wemeasuredPDDexpression in187RandNIL-PDDOL
via RT-qPCR. The mRNA levels were higher in NIL-PDDOL than in187R (Supplemental Figure 3F), suggesting thatPDDOLmight havea stronger promoter or that its mRNA stability might be altered.Furthermore, given that there were two amino acid deletions andone amino acid substitution in the predicted chloroplast-targetingsignal, we investigated whether the subcellular localization of thisprotein is affected in NIL-PDDOL. When we introduced the Pro35S:PDDOL-eGFP construct into rice protoplasts, GFP signals still lo-calized to thechloroplast (Supplemental Figure8A), asobserved forPDD187R (Figure 4D), indicating that the chloroplast localization ofPDDOL was not affected by these changes.
The N-terminal domain primarily mediates the dimerization ofTrmE family members, and the G domain is responsible for GTPbinding and hydrolysis (Scrima et al., 2005). To determine whichdomain of PDDOL leads to the dysfunction of this protein, weexamined the dimerization and GTPase activity of PDD187R andPDDOL. A yeast two-hybrid (Y2H) assay revealed that PDD187R
successfully formedhomodimers, butPDDOLdidnot (Figure5E).Abimolecular fluorescence complementation (BiFC) analysis inwildtobacco (Nicotiana benthamiana) epidermal cells also showedthatPDD187R formeddimers in chloroplasts, further confirming thenotion that PDD is a chloroplast-localized protein. By contrast,BiFCshowed thatPDDOL lost theability to formdimers (Figure 5F).Finally, to determine whether theGTPase activity of PDDOL is alsoaffected, we expressed and purified both PDD187R and PDDOL inE. coli and examined their GTPase activities in vitro usingaGTPasecolorimetric assay.PDD187RhadstrongGTPaseactivity
(Figure 5G; Supplemental Figure 8B), but PDDOL had significantlylower GTPase activity than PDD187R (Figure 5G; SupplementalFigure 8B), implying that the GTPase activity of PDDOL is alsoimpaired. In summary, our results demonstrate that the variationsin PDDOL lead to both the inability of this protein to form homo-dimers and its reduced GTPase activity.
PDD Is Involved in the mnm5s2U Modification of ChloroplasttRNAs in Rice
Given the similar sequences and protein properties of PDD and itshomologs in theTrmEfamily (Figures4Aand5Eto5G),we reasonedthat PDD might modify tRNAs in rice chloroplasts. To explore thishypothesis, we performed liquid chromatography–coupled tan-dem quadrupole mass spectrometry (LC-MS/MS) to measurethe tRNA modifications in both 187R and NIL-PDDOL chlor-oplasts. First, we purified total tRNAs from chloroplasts isolatedfrom fresh leaves of 187R andNIL-PDDOL seedlings at the three-leaf stage. Subsequently, these tRNAs were completely di-gested to individual nucleosides for LC-MS/MSsample injection(Figure6A).Weexamined23distinct typesof tRNAmodificationsusingmultiple reactionmonitoringmode (Supplemental Table 2),including six types that were found to be associated with TrmEproteins in various species (Supplemental Figure 9). Our analysisidentified 18 modified nucleosides with well-separated, distinction peaks (Figures 6B to 6D). A comparison of the modificationlevels between 187R and NIL-PDDOL revealed no obvious dif-ferences in the relative quantities of 17 types of modifications(Figures 6E to 6G). However, 5-methylaminomethyl-2-thiour-idine (mnm5s2U) modification levels were significantly lower inNIL-PDDOL than in 187R (Figure 6G). We then examined themodification status of chloroplast tRNAs in transgenic com-plementation lines of NIL-PDDOL. The levels ofmodifiedmnm5s2
U nucleosides in these lines were fully recovered to that in 187R(Figure 6H). These results indicate that PDD is associated withthe mnm5s2U modification of chloroplast tRNA in rice.In addition, to investigate whether the tRNA modification is re-
covered in green leaves of NIL-PDDOL at later stages of de-velopment, we measured the levels of mnm5s2U in 187R and NIL-PDDOL chloroplasts isolated from seedlings at the six-leaf stage.The level of mnm5s2U in chloroplast tRNA was still lower in NIL-PDDOL than in 187R (Figure 6I), indicating that the level of themnm5
s2U modification is not recovered at later stages of development.
Levels of Chloroplast Proteins Associated withPhotosynthesis and Ribosome Biogenesis Are GreatlyReduced in NIL-PDDOL
Given that tRNAmodifications canaffect the fidelity andefficiencyof translation,wemeasured the levels of four keyproteins involved
Table 1. Sequence Polymorphisms between PDD187R and PDDOL
Sequence PDD187R (bp) PDDOL (bp) No. of Variations No. of SNPs No. of Insertions No. of Deletions
Promoter 2085 2809 286 249 25 12Genomic 4007 3771 182 159 15 8Coding 1659 1653 29 28 0 1
2352 The Plant Cell
in photosynthesis, including PSI subunit PsaB, PSII subunits D1and D2, and the large subunit of ribulose-1,5-bis-phosphatecarboxylase/oxygenase (Rubisco; RbcL), in 187R andNIL-PDDOL
leaves at the three-leaf stage by immunoblot analysis. In contrastto 187R, D1 and D2 were nearly absent in NIL-PDDOL, accom-panied by a striking decrease in RbcL andPsaB levels (Figure 7A).
These results indicate that proteins involved in both the light re-action and carbon fixation reaction of photosynthesis are greatlyaffected in NIL-PDDOL. Furthermore, we measured the levels ofproteins associated with the transcriptional and translationalsystems in chloroplasts, including subunits of plastid-encodedRNA polymerase (PEP; RpoA, RpoB, and RpoC2) and subunits of
Figure 5. Sequence and Functional Variation Analysis of PDD187R and PDDOL.
(A) Amino acid variations between PDD187R and PDDOL. CTS, chloroplast-targeting signal.(B) Diagram of the vector used for transformation. RB, right border; LB, left border; HPT, hygromycin phosphotransferase gene; NOS, nopaline synthaseterminator.(C) and (D) Phenotypes of transgenic lines (nos. 1 and 2) at the seedling stage (C) and the mature stage (D). Bar in (C) 5 2 cm; bar in (D) 5 10 cm.(E) Examination of homodimer formation of PDD187R and PDDOL by Y2H assay. AD, activation domain; BD, DNA binding domain.(F) Examination of homodimer formation of PDD187R and PDDOL by BiFC assay. Yellow fluorescent protein (YFP) signals were observed and imaged byconfocalmicroscopy. The left-most panels showbright-field images. The secondpanels showchlorophyll autofluorescence signals. The third panels showYFP signals. The last panels show merged chlorophyll autofluorescence images with YFP signals and bright-field images. Bar 5 20 mm.(G) Relative GTPase activity of PDD187R and PDDOL in vitro. The data are shown as mean6 SD from three independent biological replicates. ***, P < 0.001(Student’s t test).
Chloroplast tRNA Modification Affects Development 2353
Figure 6. Detection and Data Analysis of Chloroplast tRNA Modification Status.
(A) Scheme used for the chloroplast tRNA modification detection experiments.(B) to (D) LC-MS/MSchromatogram of the 18modified nucleosideswithwell-separated ion peaks.m1A, 1-methyladenosine; m2A, 2-methyladenosine;m6
A,N6-methyladenosine; D, dihydrouridine;m5U, 5-methyluridine; m1G, 1-methylguanosine; m2G,N2-methylguanosine; m7G, 7-methylguanosine; m5C, 5-methylcytidine; m2
2G, N2,N2-dimethylguanosine; C, pseudouridine; I, inosine; Am, 2’-O-methyladenosine; ncm5U, 5-carbamoylmethyluridine; t6A, N6
-threonylcarbamoyladenosine; Cm, 2’-O-methylcytidine; ac4C, N4-acetylcytidine.(E) to (G) Relative abundance of the modified nucleosides shown in abbreviated form in 187R and NIL-PDDOL. Data represent mean 6 SD from threeindependent biological replicates. *, P < 0.05 (Student’s t test). Cm, 2’-O-methylcytidine; m2A, 2-methyladenosine; Am, 2’-O-methyladenosine; m1G, 1-methylguanosine; m2G,N2-methylguanosine; ac4C,N4-acetylcytidine;C, pseudouridine; I, inosine; m5C, 5-methylcytidine; m1A, 1-methyladenosine; m2
2
G, N2,N2-dimethylguanosine; m7G, 7-methylguanosine; m5U, 5-methyluridine; D, dihydrouridine; ncm5U, 5-carbamoylmethyluridine; m6A, N6-methyl-adenosine; t6A, N6-threonylcarbamoyladenosine.
2354 The Plant Cell
the chloroplast ribosome (RPS2, RPS3, RPS12, RPL2, andRPL16), in both 187R andNIL-PDDOL. Unexpectedly, the levels ofRpoA, RpoB, and RpoC2 were higher in NIL-PDDOL than in 187R(Figure 7A), suggesting that the transcriptional system might beenhanced in NIL-PDDOL chloroplast. However, the protein levelsof the ribosome subunits were markedly reduced in NIL-PDDOL,especially RPS2, RPS3, and RPL2 (Figure 7A), indicating that theaccumulation of ribosome subunits is greatly impaired in NIL-PDDOL chloroplasts.
To determine whether the altered protein levels in NIL-PDDOL
were caused by corresponding changes in gene expression, wecompared the mRNA levels of genes encoding the above-mentioned proteins in 187R and NIL-PDDOL by RT-qPCR. ThemRNA levels of photosynthesis-related genes transcribed byPEP, including psaB,D1,D2, and rbcL, were significantly reducedinNIL-PDDOL (Figure7B).However, compared to187R, themRNAlevels of genes associated with the transcriptional and trans-lational machinery, such as rpoA, rpoB, rpoC2, rps2, rps3, rpl2,and rpl16, which are transcribed by nuclear-encoded RNApolymerase (NEP), were greatly increased in NIL-PDDOL
(Figure 7B).The observation that the accumulation of chloroplast ribosome
subunit proteins was reduced but their mRNA expression levelswere enhanced in NIL-PDDOL is similar to findings for severalmutants defective in chloroplast ribosomebiogenesis (Banget al.,2012; Wang et al., 2016; Zhang et al., 2017). Therefore, we ana-lyzed the composition and content of rRNAs in 187R and NIL-PDDOL using an Agilent 2100 Bioanalyzer. The chloroplast ribo-some has one 50S large subunit and one 30S small subunit,containing 23S, 16S, 5S, and 4.5S rRNAs and ribosomal proteins(Bieri et al., 2017).Both16Sand23S rRNA levelsweredramaticallylower in NIL-PDDOL than in 187R (Figure 7C), suggesting thatchloroplast ribosomebiogenesis is greatly affected inNIL-PDDOL.To investigate whether plastid-encoded protein levels recover atlater stages of development, we analyzed the levels of the above-mentioned proteins in the green leaves of both 187R and NIL-PDDOL plants at the six-leaf stage. The levels of these proteins inNIL-PDDOL almost fully recovered to those in 187R at this laterstage of development (Supplemental Figure 10).
The Balance of Chloroplast mRNA Transcription Is SeverelyDisturbed in NIL-PDDOL
To globally investigate the effects ofPDD onmRNA accumulationin chloroplasts, we compared the expression profiles of chloro-plast genes in 10-d-old (three-leaf stage) 187R and NIL-PDDOL
seedlings by high-throughput whole-transcriptome sequencing(RNA-seq) with three biological replicates (see Methods). Forty-eightchloroplastgenesexhibitedamore than twofolddifference inexpression levels (27 with reduced transcript levels; 21 with
increased transcript levels) in NIL-PDDOL compared to 187R(Figure 8; Supplemental Data Set 1). The differential expressionpatterns detected by RNA-seq were consistent with data forselected genes obtained by RT-qPCR (Figures 7B and 8), vali-dating the reliability of the RNA-seq data. Chloroplast genes canbe divided into three classes based on their transcription: class Igenes are predominantly transcribed by PEP, class II genes aretranscribed by both NEP and PEP, and class III genes are ex-clusively transcribed by NEP (Hajdukiewicz et al., 1997). In-terestingly, themRNA levels of class I genes were reduced in NIL-PDDOL, suchasgenesencodingPSI-relatedproteins,PSII-relatedproteins, and rbcL (Figure 8), indicating that PEP activity is de-fective in NIL-PDDOL. However, the expression levels of class IIIgenes, which encode the subunits of PEP and chloroplast ribo-somal proteins, were largely increased in NIL-PDDOL (Figure 8),indicating that the transcriptional activity mediated by NEP isenhanced in these plants.
Expression of ;4000 Nuclear Genes Is Altered in NIL-PDDOL
Chloroplast development is regulated by the coordinated ex-pression of both chloroplast and nuclear genes (Pogson andAlbrecht, 2011). Changes in developmental and/or gene ex-pression status in the chloroplast can evoke massive changes innucleargeneexpression (Chiet al., 2015). To investigate theglobalchanges in nuclear gene expression in NIL-PDDOL, we furtheranalyzed our RNA-seq data. The expression of 1520 nucleargenes was increased and the expression of 2377 nuclear geneswas reduced in NIL-PDDOL compared to 187R (P < 0.05; log2 (foldchange) > 1 or log2 (fold change) < –1; Figure 9A; SupplementalData Set 2).We further categorized the genes with differential expression
levels by gene ontology (GO) analysis using AgriGO(Supplemental Data Set 3; Tian et al., 2017), including genes in thebiological process, molecular function, and cellular componentcategories. Among the upregulated genes in NIL-PDDOL versus187R, biological processes involved in carbohydrate/poly-saccharide metabolism, response to stress, cellular nitrogencompound metabolism, and diterpenoid metabolism were sig-nificantly enriched (Figure 9B). In themolecular function category,GO terms hydrolase activity, peptidase inhibitor activity, andcation binding were the most highly enriched (Figure 9B). In thecellular component category, extracellular region, plastid, andapoplast were the most highly enriched (Figure 9B).Among the downregulated genes in NIL-PDDOL versus 187R,
the most highly enriched GO terms in the biological processcategory were photosynthesis, oxidation–reduction, ion trans-port, carbon fixation, and plant-type cell wall organization(Figure 9C). For the molecular function category, the most highlyenriched terms included oxidoreductase activity, tetrapyrrole (the
Figure 6. (continued).
(H)Relative abundance ofmnm5s2U in 187R,NIL-PDDOL, and the complementation lines (CP) chloroplast tRNA fromseedlings at the three-leaf stage. Datarepresent mean 6 SD from three independent biological replicates. n.s., no significant difference; *, P < 0.05 (Student’s t test).(I) Relative abundance of mnm5s2U in 187R and NIL-PDDOL chloroplast tRNA from seedlings at the six-leaf stage. Data represent mean 6 SD from threeindependent biological replicates. *, P < 0.05 (Student’s t test).
Chloroplast tRNA Modification Affects Development 2355
biochemical precursors of chlorophylls) binding, Rubisco activity,catalytic activity, and transporter activity (Figure9C). In thecellularcomponent category, the top five GO terms were thylakoid, cy-toplasmic vesicle, photosystem, chloroplast, and plastid(Figure 9C). These results suggest that the expression ofphotosynthesis-associated nuclear genes (PhANGs) is repressedin NIL-PDDOL. Furthermore, this analysis indicated that thePhANGs with reduced expression in NIL-PDDOL primarily par-ticipate in PSI and PSII, light harvesting, carbon fixation, andelectron carrier processes (Supplemental Data Set 4). In addition,the GO term plant-type cell wall organization included 16 genesencoding expansin family proteins (Supplemental Table 3), whichare closely associated with plant cell growth (Cosgrove, 2000).The expression of all 16 genes was greatly reduced in NIL-PDDOL
versus 187R, as revealed in a heatmap of the RNA-seq data(Figure 9D) and confirmed by RT-qPCR (Supplemental Figure 11);these results are consistent with the reduced cell size in NIL-PDDOL (Figure 1M).
DISCUSSION
The Chloroplast tRNA-Modifying Enzyme PDD Is Essentialfor Normal Development in Flowering Plants
tRNAs, which function as critical components of protein trans-lational systems in cells, are usually extensively modified atmultiple sites, ensuring their accurate performance. Many genes
Figure 7. Accumulation of Chloroplast Proteins Is Sharply Reduced in NIL-PDDOL.
(A) Immunoblot analysis of chloroplast proteins in 187R and NIL-PDDOL at the three-leaf stage. HSP90 was used as an internal control.(B) Expression analysis of chloroplast genes by RT-qPCR. Data were normalized to ACTIN1 expression. The bars represent mean 6 SD of triplicateexperiments. ***, P < 0.001 (Student’s t test).(C) rRNA analysis of 187 and NIL-PDDOL using an Agilent 2100 Bioanalyzer. FU, fluorescence units.
2356 The Plant Cell
have evolved encoding tRNA-modifying enzymes, occupying;1to 10% of a given genome (El Yacoubi et al., 2012). The loss offunction of certain cytoplasmic tRNA-modifying enzymes canresult in developmental defects, such as embryonic lethality inDrosophila melanogaster and mouse (Chen et al., 2009b; Walkeret al., 2011), neuronal dysfunction in C. elegans and human(Freude et al., 2004; Chen et al., 2009a), and defective growth (Huet al., 2010; Mehlgarten et al., 2010; Philipp et al., 2014; Jin et al.,2019) andstress responses (Zhouet al., 2013;Wanget al., 2017) inplants. In addition, mutations in genes encoding tRNA-modifyingenzymes localized to organelles can greatly affect various de-velopmental processes. For example, the knockout of the mousegene Cdk5rap1, encoding a mitochondrial tRNA-modifying en-zyme, led to respiratory defects and myopathy (Wei et al., 2015).However, few studies have focused on the biological functions oftRNA-modifying enzymes in chloroplasts, another important or-ganelle in green plants (Delannoy et al., 2009; Karcher and Bock,2009).
Here, we demonstrated the biological function of the nucleus-encoded tRNA-modifying enzyme PDD in rice, which is localizedto the chloroplast. We uncovered the natural allele PDDOL, whichwe integrated into rice cv 187R fromanAfricanwild rice bygeneticintrogression (Supplemental Figure 1A), leading to pleiotropicdevelopmental defects (Figure 1). Also, pdd-1 homozygotes andtwo heteroallelic combinations (pdd-2/pdd-3 and pdd-4/pdd-5),which were obtained by CRISPR/Cas9-based genome editing,showed poor survival due to severe genetic disruptions (Figure 3).These results suggest that PDD is essential for normal rice
development and that PDDOL is a weak allele. Considering thatthe wild rice parent O. longistaminata produces green leavesduring early development, we hypothesize that other mecha-nisms in O. longistaminata might exist that compensate for thedysfunction of PDDOL. Alternatively, it is also possible thatnucleo-cytoplasmic incompatibility between the introducedPDDOL and the native chloroplast genome contributes to thephenotypes of NIL-PDDOL.The CRISPR/Cas9-generated pdd-1 allele, a severely impaired
allele generated in this study, is predicted to produce a 35–aminoacid truncatedprotein (Supplemental Figure 4). Thepdd-2,pdd-4,and pdd-5 alleles are predicted to generate distinct peptideslacking half of the N-terminal domain and the entire G domain(Supplemental Figure 4), suggesting that both the N-terminal andG domains are essential for PDD function. By contrast, both thePDDOL and pdd-3 alleles encode nearly full-length PDD proteinssimilar to that in 187R, with a few amino acid substitutions ordeletions (Figure 5; Supplemental Figure 4). In detail, PDDOL
contains two amino acid deletions and five amino acid alterations,leading to two residue changes (Arg145Gly and Ala191Asp) in theN-terminal domain andoneaminoacid substitution (Glu376Gln) intheGdomain (Figure 5A). Thepdd-3 allele is predicted to generateaprotein lacking threeaminoacid residuesatpositions13, 14, and159 (Supplemental Figure 4). Protein sequence analysis revealedthat the Leu at position 159, alongwith the Ala at position 191, arehighly conserved in green plants and that theGlu at position 376 isrelatively conserved in most plants (Supplemental Figure 6).Therefore, we speculate that the inability of PDDOL to form
Figure 8. Differential Expression of Chloroplast-Encoded Genes in 187R and NIL-PDDOL.
The expression levels of the genes shown in magenta were detected by RT-qPCR, as shown in Figure 7B. FC, fold change.
Chloroplast tRNA Modification Affects Development 2357
homodimersmaybecausedbyanAla191Aspchangeand that theGlu at position 376 is important for normal GTPase activity.
In human and yeast, TrmE family proteins are targeted to mi-tochondria (Decoster et al., 1993; Villarroya et al., 2008). Here, weshowed that PDD localizes to chloroplasts in rice. These findingssuggest that TrmE family proteins predominantly function insemiautonomous organelles in eukaryotes. Chloroplasts andmitochondria are endosymbiotic organelles that were derivedfrom cyanobacterium and a-proteobacterium, respectively (Bockand Timmis, 2008). Phylogenetic analysis revealed that TrmEproteins in animals and fungi are grouped with a-proteobacteria,whereas TrmEs in green plants form a separate monophyleticclass with the cyanobacterial clade (Suwastika et al., 2014),
supporting the functional diversityofTrmEs indifferentorganelles.However, the genomes of the green plants analyzed in this studyonly contained TrmEs from cyanobacteria (Supplemental Fig-ure 5), suggesting that they have lost the mitochondrial copies ofTrmE during evolution. Indeed, when we cotransfected rice pro-toplasts with the mitochondrial marker F1-ATPase-g:RFP (Niwaet al., 1999) and Pro35S:PDD187R-eGFP, the PDD187R-eGFP fu-sion protein was capable of localizing to the mitochondria(Supplemental Figure 12). Therefore, perhaps cyanobacteria-derived TrmE has taken over the function of TrmE from the mi-tochondrial ancestor in greenplants. Themolecular andbiologicalfunctionsof TrmE in ricemitochondria shouldbecomprehensivelystudied in the future.
Figure 9. RNA-Seq Analysis of Nucleus-Encoded Genes in 10-d-Old 187R and NIL-PDDOL Seedlings.
(A) Number of differentially expressed genes in NIL-PDDOL compared to 187R (log2FC > 1 or log2FC < –1).(B) Gene ontology analysis of genes with increased expression levels in NIL-PDDOL compared to 187R.(C) Gene ontology analysis of genes with reduced expression levels in NIL-PDDOL compared to 187R.(D) Heatmap of expansin genes with reduced expression levels in NIL-PDDOL compared to 187R.
2358 The Plant Cell
tRNA Modification in Chloroplasts Might Be Required forAccurate and Efficient Translation of Various ProteinsAssociated with the PEP Transcriptional System andRibosome Biogenesis
TrmE proteins modify the wobble uridines in tRNAs that decodeNNA/NNG codons of the mixed codon family (Moukadiri et al.,2014). The rice chloroplast genome contains five tRNAs with thesefeatures, includingcp-tRNAArgUCU, cp-tRNAGluUUC, cp-tRNAGlnUUG
, cp-tRNALeuUAA, and cp-tRNALysUUU (Hiratsuka et al., 1989).Several decades ago, biochemical analysis indicated that thewobbleuridinesof cp-tRNAGluUUC inbarley aremodified tomnm5s2
U (Schön et al., 1986). Here, using LC-MS/MS and functional ge-netic analysis, wediscovered that theTrmEprotein PDD is involvedin themnm5s2Umodification in rice chloroplast tRNAs (Figure 6). Inaddition, based on studies in other species, the mnm5s2U modi-fication isonly thought tooccur inwobbleuridines (Armengodetal.,2014). Thesefindingssuggest thatPDDmight participate inwobbleuridine modifications in rice chloroplast tRNAs.
Modifications of tRNA anticodons, especially at the wobbleposition, play critical roles inmaintaining the fidelity and efficiencyof mRNA translation (Ranjan and Rodnina, 2016). The tRNAwobble uridine modification mediated by TrmE family proteins isthought to mainly prevent base pairing with C/U and/or preventribosomal frameshifting during translation, thereby maintainingthe accuracy of protein translation (Yokoyama et al., 1985; Bré-geon et al., 2001). Alternatively, tRNA modifications associatedwith TrmE family members can greatly influence the efficiency ofanticodons in reading cognate codons (Krüger et al., 1998; Jo-hansson et al., 2008; Westhof et al., 2014). For example, thehypomodified mt-tRNALeuUAA lacking 5-taurinomethyluridinefailed to decode the UUG codon efficiently (Kirino et al., 2004).In addition, superwobbling can occur in chloroplasts, in which theset of tRNAs is reduced (Alkatib et al., 2012). Several tRNAmodifications can prevent superwobbling (Suzuki et al., 2011).However, it is not known whether the mnm5s2U modification atU34 prohibits superwobbling in green plants.
Here, we found that the levels of PEP subunit proteins werehigher NIL-PDDOL than in 187R (Figure 7A). However, RNA-seqanalysis revealed that the expression levels of the genes that aretranscribedby thePEPsystemweregreatly reduced inNIL-PDDOL
(Figure 8), suggesting that PEP activity might be impaired in NIL-PDDOL, primarilydue to thedefect inPDD.Wespeculate thatsomePEP subunits might have reduced activities due to translationalerrors caused by superwobbling and/or frameshifting in NIL-PDDOLduring translation. Therefore, it is possible that themnm5s2
U modification is required to prevent superwobbling. In addition,the mRNA levels of chloroplast ribosome subunit genes weremuch higher in NIL-PDDOL than in 187R (Figure 7B). However, thelevels of the corresponding proteins were markedly reduced inNIL-PDDOL (Figure7A). Thesefindingssuggest that the translationefficiency of proteins involved in ribosome biogenesis is severelyaffected in NIL-PDDOL. In summary, we propose that PDD-mediated chloroplast tRNA modifications affect multiple as-pects of chloroplast translation.
Chloroplast Transcription and Translation Status inNIL-PDDOL Trigger Retrograde Signaling to Repress PhANGGene Expression
Chloroplast development and gene expression are under nuclearcontrol via anterograde signaling. A signaling systemderived fromchloroplasts named retrograde signaling also functions in plants;this system transmits information about the developmental andfunctional state of the chloroplast to the nucleus to regulate nu-clear geneexpression (Chan et al., 2016). The retrograde signalingsystem is a complex network containing multiple signals andpathways, including a class of signals triggered by chloroplastgene expression (Hernández-Verdeja and Strand, 2018). PhANGexpression is suppressed in mutants with defects in the chloro-plast transcriptional machinery, such as sig2 and sig6 (Woodsonet al., 2013). Treatment with the chloroplast translation inhibitorlincomycin also repressesPhANG expression (Susek et al., 1993).These findings indicate that the status of chloroplast transcriptionand translation can trigger retrograde signaling to repressPhANGexpression.In this study, we demonstrated that the functional defect of
PDDOL leads to a decrease in mnm5s2U tRNA modification, po-tentially impairing the translation process in chloroplasts (Figures6 and 7). RNA-seq and RT-qPCR revealed that the expression ofchloroplast-encoded genes is greatly affected in NIL-PDDOL, withmost PEP-transcribed genes showing reduced expression levelsand NEP-transcribed genes showing increased expression levels(Figures 7B and 8). These results indicate that PEP activity isimpaired and NEP activity is enhanced in NIL-PDDOL. Moreover,the expression levels of PhANG are significantly reduced in NIL-PDDOL (Supplemental Data Set 4). Based on these observations,we reasoned that in NIL-PDDOL, information about impairedtranscriptionand translation in thechloroplast is transmitted to thenucleus by retrograde signaling, causing the nucleus to increaseNEP activity in the chloroplast, thereby enhancing the expressionof genes related to the transcriptional and translational machineryin the chloroplast. On the other hand, the reduced expressionlevelsofPhANGandexpansingenes inNIL-PDDOLmight facilitateplant survival by allowing the cells to conserve energy by reducingplant growth.Taken together, we propose a working model for the molecular
mechanismunderlyingPDD function in 187Rand impairedPDDOL
function in NIL-PDDOL (Figure 10). According to our model, in187R, PDD187R modifies the uridines in chloroplast tRNAs intomnm5s2U, ensuring accurate and/or efficient translation in thechloroplast to promote normal plant development. In NIL-PDDOL,the defective function of PDDOL results in decreased chloroplasttRNA modification, consequently affecting the fidelity and/or ef-ficiency of translation in the chloroplast. Thus, aberrant chloro-plast translation leads to impaired ribosome biogenesis andreduced levels of photosynthetic proteins, resulting in the albinophenotype of NIL-PDDOL. In addition, in response to retrogradesignaling, the transcriptional levels ofPhANG andexpansin genesin the nucleus decrease in NIL-PDDOL, leading to smaller cell sizeand dwarf plant stature.
Chloroplast tRNA Modification Affects Development 2359
METHODS
Plant Materials and Growth Conditions
The F1 population generated by a cross between 187R (Oryza sativa subspindica) and African wild rice (Oryza longistaminata) was obtained fromRongbai Li. The plants were grown in a paddy field in Shanghai (31°279N,summer season, temperate climate), Taicang (31°459N, summer season,temperate climate), and Sanya (18°169N, winter season, subtropical cli-mate), China under local cultivation conditions. For experiments at theseedling stage, the plants were grown in a growth chamber at 30/26°Cunder a 10-h-light/14-h-dark cycle using one-half-strengthMurashige andSkoog medium as a nutrient source. An 18-W white light-emitting diode
full-spectrum grow light was used (color temperature value, 6500 K; lightintensity, 320 mmol photons m22 s21).
Photosynthetic Pigment Content and Fv/Fm Measurements
Chlorophyll and carotenoid contents were measured with a spectropho-tometer. Briefly, fresh leaves (;0.05 g) were collected and immersed in8 mL of chlorophyll extraction buffer (95% [v/v] ethanol: acetone:water 55:4:1) for 24 h in the dark with gentle shaking (Peng et al., 2012). Theabsorbance of the supernatants at 663, 645, and 470 nm was measuredwith a microplate spectrophotometer (Synergy II; BioTek). Each samplewas measured with three biological replicates from different seedlingleaves, and each biological replicate sample was measured three times
Figure 10. A Proposed Working Model of the Molecular Function of PDD.
In 187R, PDD187R correctly modifies the chloroplast tRNA’s U34 to mnm5s2U, ensuring accurate and/or efficient translation in the chloroplast to promotenormal plant development. InNIL-PDDOL, the functional defect of PDDOL results in reduced levels ofmnm5s2Umodification in chloroplast tRNAs, leading totranslational errors in the chloroplast. Aberrant chloroplast translation results in impaired ribosome biogenesis, reduced levels of photosynthetic proteins,and impaired PEP activity. The transcriptional levels of PhANG and expansin genes in the nucleus are reduced by retrograde signaling.
2360 The Plant Cell
using the same supernatant. For the Fv/Fm measurements, seedlings atthe three-leaf stage were dark adapted for at least 1 h. The Fv/Fm valueswere thendetermined using aportable photosynthesis system (LI-6400XT;LI-COR Biosciences) according to the manufacturer’s instructions.
Transmission Electron Microscopy
The third leaves from 187R and NIL-PDDOL plants were cut into smallpieces and fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer at4°C for 16 h. After three rinses with phosphate buffer, the samples wereincubated in 1% (w/v) osmic acid at room temperature for 5 h and washedwith phosphate buffer. The samples were dehydrated through an ethanolseries (30, 50, 70, 80, 90, 95, 100, and 100% [v/v]). The ethanol wassubsequently replaced by a series of Spurr’s resin dilutions (25, 50, 75, and100% [v/v]). Finally, the samples were embedded in Spurr’s resin at 60°Cfor 48 h. Ultrathin sections were cut with a diamond knife in a Leica UC7ultramicrotome and collected on copper grids. The sections were stainedwith uranyl acetate and observed under a Hitachi HT7700 transmissionelectron microscope.
Paraffin Sectioning and Observation of Cell Size
The uppermost internodes of 187R and NIL-PDDOL were fixed informaldehyde–acetic acid–ethanol solution for 24 to 48 h, dehydrated ina graded ethanol series, cleared with HistoChoice (1328-H103; Amresco),and embedded in Paraplast Plus (P3683; Sigma-Aldrich). Sections (10 mmin thickness) were obtained with a Leica RM2245 microtome, transferredonto poly-L-Lys–coated glass slides, deparaffinized with a HistoChoiceseries, and dehydrated through an ethanol series. The cell size wasphotographed by bright-field microscopy (Scope A1; Zeiss).
Seed Germination and Plant Growth Assay
Ninety-sixseedspersampleweresoaked inwater at30°C for2d,placedonwetfilter paper inaPetri dish, and incubatedat30°C for 2d.Theseedsweretransferred to agrowthchamber and incubatedunder standardconditions.After 10 d in the growth chamber, we counted the number of seedlings andcalculated the proportions of seedlings from 96 seeds. Each sample wasmeasured with three independent biological replicates.
Map-Based Cloning of PDD
Primary mapping was performed using 200 F2 plants derived by geneticcrosses between NIL-PDDOL and 187R. To finemap the PDD locus, 93-11was crossed with NIL-PDDOL. Recombinants were screened for theRM1309 and RM8264 markers using 4104 plants from the BC1F2 pop-ulation, and the PDD locus was ultimately mapped to the interval between420SNP and 480SNP using 4608 additional BC1F2 individuals.
Vector Construction and Plant Transformation
We created a complementation vector by amplifying a 6135-bp genomicDNA fragment of PDD including the 2096-bp promoter region from 187Rusing primers cPDD-F and cPDD-R. The DNA fragment was inserted intothe pCAMBIA 1304 vector using BamHI/KpnI sites. We constructeda vector by amplifying the 2096-bppromoter sequenceofPDD187Rand the1653-bp coding region sequence of PDDOL. The DNA fragments wereinserted into the pCAMBIA 1304 vector using HindIII/EcoRI sites. Therecombinant plasmid was introduced into NIL-PDDOL via Agrobacterium-mediated transformation (Hiei and Komari, 2008).
We used the CRISPR/Cas9 multiplex editing system to generategenome-edited mutants of PDD (Xie et al., 2015). The specific spacersequences were selected using the CRISPR-PLANT database (http://
www.genome.arizona.edu/crispr/; Xie et al., 2014). The construct wasgeneratedasdescribedbyXieet al. (2014). Thesynthesized fragmentswithtandemly arrayed tRNA-guide RNA architecture were inserted intopRGEB32. We confirmed the construct by sequencing and transformed itinto 187R via Agrobacterium-mediated transformation (Hiei and Komari,2008). Several rice transformations were conducted by the Hefei JianguBiotechnology.
Sequencing and Phylogenetic Analysis
Gene annotation was performed using the Michigan State University riceannotation database (http://rice.plantbiology.msu.edu/). Domain analysiswas performed using InterPro (http://www.ebi.ac.uk/interpro/search/sequence-search). The chloroplast-targeting signal was predicted usingChloroP 1.1 (http://www.cbs.dtu.dk/services/ChloroP/). Protein se-quences homologous to PDD were identified using BLASTP against theNational Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Multiple sequence alignments were generatedusingMEGAversion6.0, and thephylogenetic tree (Supplemental Figure 5)wasconstructedwithMEGAversion6.0using theneighbor-joiningmethodand default values. Bootstrap support values were calculated from 1000replicates and are listed on the branch nodes. The sequence alignmentsand tree files are provided as Supplemental Files 1 and 2, respectively.
RNA Extraction and RT-qPCR
TotalRNAwasextracted fromthesampleswithTRIzolReagent (15596018;Invitrogen) according to the manufacturer’s instructions. First-strandcDNA was synthesized from 2 mg of total RNA using a FastQuant RT kit(KR106; Tiangen). The cDNA used to detect the expression levels ofplastid-encoded genes was synthesized using random hexamer primers.RT-qPCR was conducted using SuperReal PreMix Plus (FP205; Tiangen)on a StepOnePlus real-time PCR system (Applied Biosystems). The PCRconditions were 95°C for 15min followed by 40 cycles of 95°C for 10 s and60°C for 30 s. The relative expression levels of the target RNAs werecalculated by the 2–DDCt method using ACTIN1 (LOC_Os03g50885) as aninternal control to normalize different samples. Each sample used threebiological replicates byextractingRNA fromdifferent batchesof seedlings.
Subcellular Localization
The coding sequences of PDD187R and PDDOL were amplified and clonedinto the N terminus of eGFP under the control of the cauliflower mosaicvirus 35S promoter using EcoRI/SalI sites (Tang et al., 2014). The re-combinant vector was transformed into rice protoplasts as previouslydescribed by Zhang et al. (2011). GFP fluorescence in the transformedprotoplasts was visualized under a confocal laser-scanning microscope(TCS SP8; Leica). F1-ATPase-g:RFP (Niwa et al., 1999) was used asa mitochondria localization marker.
Histochemical Staining of GUS Expression
The ProPDD187R:GUS vector was constructed by cloning the 2167-bppromoter region of PDD187R into the pCAMBIA1300 vector using HindIII/BamHI sites to driveGUS expression. The construct was transformed intojaponica rice var Nipponbare plants via Agrobacterium-mediated trans-formation. Various tissues of ProPDD187R:GUS T2 transgenic plants wereincubated in90%(v/v) acetoneon ice for 10min,washedseveral timeswithGUS staining buffer [10 mMEDTA, pH 8.0, 29 mMNa2HPO4, 21 mMNaH2
PO4, 1mMK4Fe(CN)6, 1mMK3Fe(CN)6, 0.1% (v/v) Triton X-100, 0.05% (w/v) 5-bromo-4-chloro-3-indolyl-b-D-glucuronide, and 20% (v/v) methanol],submerged in the staining buffer, and placed in a vacuum chamber for 20-min periods of vacuum infiltration. The samples were incubated overnight
Chloroplast tRNA Modification Affects Development 2361
in staining buffer at 37°C. Ethanol was used to remove the chlorophyll afterGUS staining.
Y2H Assay
Y2H assays were performed using the Matchmaker Gold Yeast Two-Hybrid system (630489; Clontech). The coding sequences of PDD187R
and PDDOL were cloned into pGBKT7 using EcoRI/SalI sites and clonedinto pGADT7 using EcoRI/BamHI sites. The constructs were transformedinto yeast strain Y2H gold (pGBKT7 constructs) or Y187 (pGADT7 con-structs) using the lithium acetate/polyethylene glycol method. Trans-formants were mated on 23 yeast peptone dextrose adenine liquidmedium for 24h, followedbyselection on synthetic dropoutmedium (SD)/-Trp-Leuplates for 36h. Transformantswere selectedonSD/-His-Ade-Trp-Leu plates to test for positive interactions. pGADT71pGBKT7wasused asthe negative control.
BiFC Assay
The full-length coding sequences of PDD from 187R and NIL-PDDOLwerecloned into pXY103/pXY104 using BamHI/SalI sites (Su et al., 2017). Theconstructs were transformed into Agrobacterium GV3101 cells. Trans-formants were harvested once the OD600 reached 2.0 and resuspended inMES/MgCl2/acetosyringone solution to a final OD600 of 1.0. Variouscombinations of cell suspensions were mixed at a 1:1 ratio and used toinfiltrate young Nicotiana benthamiana leaves. The leaves were excisedand visualized under a confocal microscope (TCS SP8; Leica) following48 h of incubation.
Protein Purification and GTPase Activity Assay
The coding sequences of PDD187R and PDDOL were cloned intoa modified pET28a vector with a His6-small ubiquitin-related modifier(SUMO) tag at theN terminus usingEcoRI/SalI sites (Yang et al., 2018).The plasmids were transformed into Escherichia coli strain C41 (DE3)for expression. A 20-mL aliquot of overnight bacterial cultures wasinoculated into 1 L of Luria-Bertani medium and cultured at 37°C.When theOD600 reached 0.6 to 0.8, protein expressionwas induced byadding 0.2 mM isopropyl b-D-thiogalactopyranoside, followed byincubation at 18°C for 18 h. The cells were harvested by centrifugationat 4500 rpm for 20 min and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 25 mM imidazole, pH 8.0). Followingdisruption in a JN-02C cell crusher, the cell debris was removed bycentrifugation (17,000 rpm) at 4°C for 1 h, and the supernatant wasloaded onto a nickel-chelating column. The protein with His6-SUMOtag was eluted from the column using elution buffer (20 mM Tris-HCl,pH 8.0, 500 mM NaCl, and 500 mM imidazole, pH 8.0). After overnighttreatment with SUMO protease Ulp1 at 4°C, the cleaved His6-SUMOtag was removed using a second nickel-chelating column. Finally,PDD187R and PDDOL were detected by SDS-PAGE and used in theGTPase activity assay.
TheGTPaseassaywasperformedusingacommercialGTPaseassaykit(602-0120; Innova Biosciences) following themanufacturer’s instructions,with minor modifications. Briefly, 200 mL of reaction buffer containing50 mM Tris-HCl, pH 7.5, 150 mM KCl, 2.5 mM MgCl2, 0.5 mM GTP, and100 mg of protein was incubated at 30°C for 10 min. After stopping thereaction byaddingGoldmix andstabilizer, theplatewas incubatedat roomtemperature for 30 min in the dark and the absorbance was read atawavelength of 650 nm.GFPproteinwas used as the negative control.Wecalculated relative enzyme activity as recommended.
Chloroplast Isolation, tRNA Extraction, and Purification
Chloroplasts were isolated from ;10-d-old 187R and NIL-PDDOL seed-lings using a modified Suc density gradient centrifugation method(Takamatsu et al., 2018). The entire processwascarriedat 4°C.Briefly, 20gof fresh leaves was cut into;1-cm pieces and homogenized in 400 mL ofisolation buffer (50 mM Tris-HCl, pH 8.0, 0.35 M Suc, 7 mM EDTA, 5 mMDTT, and0.1% [w/v] BSA). The homogenatewas filtered twice through twolayersofMiracloth (475855;Millipore). Thefiltratewascentrifugedat 1000gfor 10 min. After resuspending the pellet in 6 mL of isolation buffer, thesuspension was slowly layered onto a cushion of 20/45% (w/v) Suc in50 mM Tris-HCl, pH 8.0, 0.3 M sorbitol, and 7 mM EDTA. Followingcentrifugation at 2000g for 30min, the green layer at the 20%Sucand45%Suc interface was carefully collected, diluted by adding three volumes ofisolationbuffer, andcentrifugedat3000g for10min.RNAssmaller than200nucleotides were extracted from the chloroplast pellet using RNAiso forsmall RNA (9753A; Takara). The band containing tRNAs was sliced froma 15% (v/v) Tris–boric acid–EDTA-urea gel, and the tRNAswere recoveredusing a ZR small-RNA PAGE Recovery Kit (R1070; Zymo Research).
tRNA Digestion and LC-MS/MS Analysis
The nucleosideswere analyzed by LC-MS/MS as described byWang et al.(2017),withminormodifications.Briefly,;5mg tRNAwasdigestedwith2Uof P1 nuclease (N8630; Sigma-Aldrich) and 1.5 U of calf intestine alkalinephosphatase (CAP-111; Toyobo) in 20mMHepes-KOH,pH7.0, at 37°C for3 h. The samples were diluted in Milli-Q water (Synergy; Millipore) toa concentration of 15 ng/mL, and 10-mL sampleswere injected into the LC-MS/MS machine. All digested tRNA samples were analyzed using threebiological replicates from different batches of seedlings.
The LC-20A HPLC system with an Inertsil ODS-3 column (2.1 3 150mm, 5-mm particle size; Shimadzu) was used for nucleoside separation.The binary solvent system composed by 2 mM ammonium acetate (so-lution A) and methanol (solution B) was used as the mobile phase. Thegradientwasas follows:0 to10min, 0 to50%ofB;10 to13min, 50 to100%ofB; 13 to23min, 100%ofB; 23 to23.1min, 100 to5%ofB; 23.1 to 30min,5 to 0%of B. Solution A (100%)was applied for 10min to re-equilibrate thecolumn before the next sample was injected. An API 4000 Q-Trap massspectrometer (Applied Biosystems) was used for detection. Electrosprayionization mass spectrometry was conducted in positive ion mode. Mul-tiple reaction monitoring mode was used to determine parent-to-production transitions. The relative abundance of each modified nucleoside wascalculated by the ratio to the sum of A, U, C, and G.
Protein Extraction, SDS-PAGE, and Immunoblot Analysis
Total proteins were isolated from the leaves of ;10-d-old 187R and NIL-PDDOL seedlings. The tissueswere ground in liquid nitrogen and thawed inlysis buffer (7 M urea, 2 M thiourea, and 30 mM Tris). Cell debris was re-moved by centrifugation at 12,000g for 15 min at 4°C. Sample amountswere standardized by freshweight. Total proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted withvarious antibodies, and detected using Clarity Western ECL Substrate(1705060; Bio-Rad). Heat shock protein HSP90 was used as an internalreference (Li etal., 2011).Anti-PsaB (catalogno.AS10695;dilution1:1000),D1 (catalog no. AS05 084; dilution 1:1600), D2 (catalog no. AS06 146;dilution 1:5000), RbcL (catalog no. AS03 037; dilution 1:5000), and RPL2(catalog no. AS15 2876; dilution 1:3000) antibodies were purchased fromAgrisera (https://www.agrisera.com/). Anti-RpoA (catalog no. AbP80103-A-SE; dilution 1:1000), RpoB (catalog no. AbP80101-A-SE; dilution 1:1000), RpoC2 (catalog no. AbP80094-A-SE; dilution 1:500), RPS3 (catalogno. AbP80377-A-SE; dilution 1:500), and HSP90 (catalog no. AbM51099-31-PU; dilution 1:10,000) antibodies were obtained from BGI (http://www.proteomics.org.cn/). Anti-RPS2 (catalog no. PHY0427S; dilution 1:500),
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RPS12 (catalog no. PHY0434S; dilution 1:500), and RPL16 (catalog no.PHY0431S; dilution 1:500) antibodieswere obtained fromPhytoAB (http://www.phytoab.com/).
RNA-Seq and GO Enrichment Analysis
Total RNA was isolated from ;10-d-old 187R and NIL-PDDOL seedlings,each with three biological replicates from different batches of seedlings.Each samplewasquantified, and its qualitywaschecked in anAgilent 2100Bioanalyzer. The rRNA was removed from total RNA using a Ribo-ZerorRNA Removal Kit (Plant Leaf). The rRNA-depleted RNA was fragmentedand reverse transcribed. First-strand cDNAwas synthesized from theRNAusing random primers. Second-strand cDNA was synthesized usingSecond Strand Synthesis Enzyme Mix (including dACG-TP/dUTP). Thedouble-stranded cDNA was purified using AxyPrep Mag PCR Clean-up(Axygen) and treatedwith End Prep EnzymeMix to repair both ends and toadd a dA-tail in one reaction, followed by T-A ligation to add adaptors toboth ends. Size selection of adaptor-ligated DNA was performed usingAxyPrep Mag PCR Clean-up (Axygen), and ;360-bp fragments (with anapproximate insert size of 300 bp) were recovered. The library was con-structed and sequenced using the Illumina Hisequation 2000 system. Theraw sequencing data were collected and filtered. We obtained 95 millionreads each from187R andNIL-PDDOL for nucleus-encoded genes, and 33million reads each from 187R and NIL-PDDOL were obtained forchloroplast-encoded genes. The significance of differentially expressedgenes was determined using P-values < 0.05 and jlog2 (fold change)j > 1.GO analysis was performed using the AgriGO website (Tian et al., 2017).Rice chloroplast genes were identified by referring to the chloroplastgenome database (http://rocaplab.ocean.washington.edu/old_website/tools/cpbase/run).
Primer Sequences
The primers used in this study are listed in Supplemental Data Set 5.
Accession Numbers
Sequence data used to construct the phylogenetic tree were downloadfrom NCBI under the accession numbers listed in Supplemental Figure 5.Additional sequences of thePDDhomologs used in Supplemental Figure 6were download fromNCBI and EnsemblPlants (http://plants.ensembl.org/index.html) under the following accession numbers: XP_015648320 (O.sativa); XP_003574461 (B. distachyon); XP_002444316 (S. bicolor); XP_008677280 (Z. mays); TraesCS5B02G032900 (Triticum aestivum); XP_002301037 (Populus trichocarpa); XP_003522812 (G. max); NP_177924(Arabidopsis); XP_020521993 (Amborella trichopoda). The rawsequencingdata have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA602187.
Supplemental Data
Supplemental Figure 1. Construction of NIL-PDDOL and additionalphenotypic analysis of NIL-PDDOL.
Supplemental Figure 2. Quantitative analysis of the agronomic traitsof 187R, NIL-PDDOL, and pdd-1.
Supplemental Figure 3. Phenotypic analysis of transgenic comple-mentation lines harboring CG4 or CG5 and mRNA expression levels ofPDD in 187R, NIL-PDDOL, and pdd-1.
Supplemental Figure 4. Protein sequence alignment of PDD in 187Rand various genome-edited mutants.
Supplemental Figure 5. Phylogenetic analysis and protein structuresof TrmE family members.
Supplemental Figure 6. Protein sequence alignment of homologs ofPDD in various plant species.
Supplemental Figure 7. Alignment of the promoter sequences,coding sequences and protein sequences of PDD187R and PDDOL.
Supplemental Figure 8. Subcellular localization of PDDOL andGTPase colorimetric assay.
Supplemental Figure 9. Chemical structures of TrmE-related tRNAmodifications in E. coli and human mitochondria.
Supplemental Figure 10. Immunoblot analysis of chloroplast proteinsin 187R and NIL-PDDOL green leaves at the six-leaf stage (SupportsFigure 7).
Supplemental Figure 11. Expression analysis of expansin genes viaRT-qPCR.
Supplemental Figure 12. Subcellular localization of PDD187R-eGFP inmitochondria.
Supplemental Table 1. List of candidate genes in the mapped regionand their putative functions.
Supplemental Table 2. LC-MS/MS parameters for analysis ofmodified nucleosides.
Supplemental Table 3. Expansin genes with reduced expressionlevels in NIL-PDDOL compared to 187R.
Supplemental Data Set 1. Chloroplast genes with reduced expres-sion levels and increased expression levels in NIL-PDDOL comparedto 187R.
Supplemental Data Set 2. Nuclear genes with reduced expressionlevels and increased expression levels in NIL-PDDOL comparedto 187R.
Supplemental Data Set 3. GO analysis of nuclear genes with reducedor increased expression levels in NIL-PDDOL compared to 187R.
Supplemental Data Set 4. Nuclear genes involved in PSI and PSII,light harvesting, carbon fixation and electron carriers.
Supplemental Data Set 5. Primers used in this study.
Supplemental File 1. Sequence alignments of TrmE proteins.
Supplemental File 2. Phylogenetic tree file for TrmE proteins.
ACKNOWLEDGMENTS
We appreciate Rongbai Li (Guangxi University) for providing the F1 plantsgenerated from a cross between 187R (O. sativa subsp indica) and Africanwild rice (O. longistaminata). We thank JianhuaGan and Xi Chen (School ofLife Science, Fudan University) for help with protein purification. We aregrateful to Jirong Huang (College of Life and Environmental Sciences,Shanghai Normal University) for providing the antibodies for PsaB, D1, D2,and RbcL. We also thank the anonymous reviewers for their constructivecomments, which have significantly improved our article. This work wassupported by a grant from the National Natural Science Foundation ofChina (31770351 to P.L. and 31671655 to X. L.), the 111 Project (grantD16014), and start-up funding from Henan Provincial University to P.L.
AUTHOR CONTRIBUTIONS
H.L. and D.R. performed map-based cloning, most experiments and dataanalyses. L.J. and X. Li. generated the constructs for functional studies.Y.Y., L.M., W.C., and A.M. helped with the phenotypic analysis and
Chloroplast tRNA Modification Affects Development 2363
subcellular localization assays. N.J. performed bioinformatics analysis fortranscriptomesequencing.X.Luodeveloped the riceNILsandconstructedthe mapping population. P.C. assisted to detect and analyze tRNA mod-ifications via mass spectrometry. X. Luo, H.M., and J.Y. provided valuableadvice and supervised the study. P.L. conceived the study, designed theexperiments, interpreteddata, supervised theproject, andwrote thearticle.
Received September 12, 2019; revisedMarch 30, 2020; acceptedApril 15,2020; published April 23, 2020.
REFERENCES
Alkatib, S., Scharff, L.B., Rogalski, M., Fleischmann, T.T., Matthes,A., Seeger, S., Schöttler, M.A., Ruf, S., and Bock, R. (2012). Thecontributions of wobbling and superwobbling to the reading of thegenetic code. PLoS Genet. 8: e1003076.
Armengod, M.E., Meseguer, S., Villarroya, M., Prado, S.,Moukadiri, I., Ruiz-Partida, R., Garzón, M.J., Navarro-González, C., and Martínez-Zamora, A. (2014). Modification ofthe wobble uridine in bacterial and mitochondrial tRNAs readingNNA/NNG triplets of 2-codon boxes. RNA Biol. 11: 1495–1507.
Armengod, M.E., Moukadiri, I., Prado, S., Ruiz-Partida, R.,Benítez-Páez, A., Villarroya, M., Lomas, R., Garzón, M.J.,Martínez-Zamora, A., Meseguer, S., and Navarro-González, C.(2012). Enzymology of tRNA modification in the bacterial MnmEGpathway. Biochimie 94: 1510–1520.
Asano, K., et al. (2018). Metabolic and chemical regulation of tRNAmodification associated with taurine deficiency and human disease.Nucleic Acids Res. 46: 1565–1583.
Bang, W.Y., Chen, J., Jeong, I.S., Kim, S.W., Kim, C.W., Jung, H.S.,Lee, K.H., Kweon, H.S., Yoko, I., Shiina, T., and Bahk, J.D. (2012).Functional characterization of ObgC in ribosome biogenesis duringchloroplast development. Plant J. 71: 122–134.
Bieri, P., Leibundgut, M., Saurer, M., Boehringer, D., and Ban, N.(2017). The complete structure of the chloroplast 70S ribosome incomplex with translation factor pY. EMBO J. 36: 475–486.
Björk, G.R., Durand, J.M., Hagervall, T.G., Leipuvien _e, R.,Lundgren, H.K., Nilsson, K., Chen, P., Qian, Q., andUrbonavicius, J. (1999). Transfer RNA modification: Influence ontranslational frameshifting and metabolism. FEBS Lett. 452: 47–51.
Björk, G.R., and Hagervall, T.G. (2005). Transfer RNA modification.Ecosal Plus 1.
Bock, R., and Timmis, J.N. (2008). Reconstructing evolution: Genetransfer from plastids to the nucleus. BioEssays 30: 556–566.
Brégeon, D., Colot, V., Radman, M., and Taddei, F. (2001). Trans-lational misreading: A tRNA modification counteracts a 12 ribo-somal frameshift. Genes Dev. 15: 2295–2306.
Cabedo, H., Macián, F., Villarroya, M., Escudero, J.C., Martínez-Vicente, M., Knecht, E., and Armengod, M.E. (1999). The Es-cherichia coli trmE (mnmE ) gene, involved in tRNA modification,codes for an evolutionarily conserved GTPase with unusual bio-chemical properties. EMBO J. 18: 7063–7076.
Cantara, W.A., Crain, P.F., Rozenski, J., McCloskey, J.A., Harris,K.A., Zhang, X., Vendeix, F.A., Fabris, D., and Agris, P.F. (2011).The RNA modification database, RNAMDB: 2011 update. NucleicAcids Res. 39: D195–D201.
Chan, K.X., Phua, S.Y., Crisp, P., McQuinn, R., and Pogson, B.J.(2016). Learning the languages of the chloroplast: Retrograde sig-naling and beyond. Annu. Rev. Plant Biol. 67: 25–53.
Chen, C., Tuck, S., and Byström, A.S. (2009a). Defects in tRNAmodification associated with neurological and developmental
dysfunctions in Caenorhabditis elegans elongator mutants. PLoSGenet. 5: e1000561.
Chen, K., et al. (2019). Translational regulation of plant response tohigh temperature by a dual function tRNAHis guanylyltransferase inrice. Mol. Plant 12: 1123–1142.
Chen, Y.T., Hims, M.M., Shetty, R.S., Mull, J., Liu, L., Leyne, M.,and Slaugenhaupt, S.A. (2009b). Loss of mouse Ikbkap, a subunitof elongator, leads to transcriptional deficits and embryonic le-thality that can be rescued by human IKBKAP. Mol. Cell. Biol. 29:736–744.
Chi, W., Feng, P., Ma, J., and Zhang, L. (2015). Metabolites andchloroplast retrograde signaling. Curr. Opin. Plant Biol. 25: 32–38.
Chumley, T.W., Palmer, J.D., Mower, J.P., Fourcade, H.M., Calie,P.J., Boore, J.L., and Jansen, R.K. (2006). The complete chloro-plast genome sequence of Pelargonium 3 hortorum: Organizationand evolution of the largest and most highly rearranged chloroplastgenome of land plants. Mol. Biol. Evol. 23: 2175–2190.
Cosgrove, D.J. (2000). Loosening of plant cell walls by expansins.Nature 407: 321–326.
Decoster, E., Vassal, A., and Faye, G. (1993). MSS1, a nuclear-encoded mitochondrial GTPase involved in the expression of COX1subunit of cytochrome c oxidase. J. Mol. Biol. 232: 79–88.
Delannoy, E., Le Ret, M., Faivre-Nitschke, E., Estavillo, G.M.,Bergdoll, M., Taylor, N.L., Pogson, B.J., Small, I., Imbault, P.,and Gualberto, J.M. (2009). Arabidopsis tRNA adenosine de-aminase arginine edits the wobble nucleotide of chloroplast tRNAArg
(ACG) and is essential for efficient chloroplast translation. Plant Cell21: 2058–2071.
El Yacoubi, B., Bailly, M., and de Crécy-Lagard, V. (2012). Bio-synthesis and function of posttranscriptional modifications oftransfer RNAs. Annu. Rev. Genet. 46: 69–95.
Ferré-D’Amaré, A.R. (2003). RNA-modifying enzymes. Curr. Opin.Struct. Biol. 13: 49–55.
Francis, M.A., Suh, E.R., and Dudock, B.S. (1989). The nucleotidesequence and characterization of four chloroplast tRNAs from thealga codium fragile. J. Biol. Chem. 264: 17243–17249.
Freude, K., et al. (2004). Mutations in the FTSJ1 gene coding fora novel S-adenosylmethionine-binding protein cause nonsyndromicX-linked mental retardation. Am. J. Hum. Genet. 75: 305–309.
Giegé, R., Jühling, F., Pütz, J., Stadler, P., Sauter, C., and Florentz,C. (2012). Structure of transfer RNAs: similarity and variability. WileyInterdiscip. Rev. RNA 3: 37–61.
Gustilo, E.M., Vendeix, F.A., and Agris, P.F. (2008). tRNA’s mod-ifications bring order to gene expression. Curr. Opin. Microbiol. 11:134–140.
Hajdukiewicz, P.T., Allison, L.A., and Maliga, P. (1997). The twoRNA polymerases encoded by the nuclear and the plastid com-partments transcribe distinct groups of genes in tobacco plastids.EMBO J. 16: 4041–4048.
Hernández-Verdeja, T., and Strand, Å. (2018). Retrograde signalsnavigate the path to chloroplast development. Plant Physiol. 176:967–976.
Hiei, Y., and Komari, T. (2008). Agrobacterium-mediated trans-formation of rice using immature embryos or calli induced frommature seed. Nat. Protoc. 3: 824–834.
Hirabayashi, H., et al. (2015). qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigateheat stress damage at flowering in rice, O. sativa. J. Exp. Bot. 66:1227–1236.
Hiratsuka, J., et al. (1989). The complete sequence of the rice (Oryzasativa) chloroplast genome: intermolecular recombination betweendistinct tRNA genes accounts for a major plastid DNA inversionduring the evolution of the cereals. Mol. Gen. Genet. 217: 185–194.
2364 The Plant Cell
Hu, Z., Qin, Z., Wang, M., Xu, C., Feng, G., Liu, J., Meng, Z., and Hu,Y. (2010). The Arabidopsis SMO2, a homologue of yeast TRM112,modulates progression of cell division during organ growth. Plant J.61: 600–610.
Hua, L., et al. (2015). LABA1, a domestication gene associated withlong, barbed awns in wild rice. Plant Cell 27: 1875–1888.
Jin, J., Huang, W., Gao, J.P., Yang, J., Shi, M., Zhu, M.Z., Luo, D.,and Lin, H.X. (2008). Genetic control of rice plant architecture underdomestication. Nat. Genet. 40: 1365–1369.
Jin, X., Lv, Z., Gao, J., Zhang, R., Zheng, T., Yin, P., Li, D., Peng,L., Cao, X., Qin, Y., Persson, S., and Zheng, B., et al. (2019).AtTrm5a catalyses 1-methylguanosine and 1-methylinosineformation on tRNAs and is important for vegetative and re-productive growth in Arabidopsis thaliana. Nucleic Acids Res.47: 883–898.
Johansson, M.J., Esberg, A., Huang, B., Björk, G.R., and Byström,A.S. (2008). Eukaryotic wobble uridine modifications promotea functionally redundant decoding system. Mol. Cell. Biol. 28:3301–3312.
Karcher, D., and Bock, R. (2009). Identification of the chloroplastadenosine-to-inosine tRNA editing enzyme. RNA 15: 1251–1257.
Kirino, Y., Yasukawa, T., Ohta, S., Akira, S., Ishihara, K.,Watanabe, K., and Suzuki, T. (2004). Codon-specific trans-lational defect caused by a wobble modification deficiency in mu-tant tRNA from a human mitochondrial disease. Proc. Natl. Acad.Sci. USA 101: 15070–15075.
Krüger, M.K., Pedersen, S., Hagervall, T.G., and Sørensen, M.A.(1998). The modification of the wobble base of tRNAGlu modulatesthe translation rate of glutamic acid codons in vivo. J. Mol. Biol. 284:621–631.
Li, X., Bai, H., Wang, X., Li, L., Cao, Y., Wei, J., Liu, Y., Liu, L., Gong,X., Wu, L., Liu, S., and Liu, G. (2011). Identification and validation ofrice reference proteins for western blotting. J. Exp. Bot. 62:4763–4772.
Mehlgarten, C., Jablonowski, D., Wrackmeyer, U., Tschitschmann,S., Sondermann, D., Jäger, G., Gong, Z., Byström, A.S.,Schaffrath, R., and Breunig, K.D. (2010). Elongator function intRNA wobble uridine modification is conserved between yeast andplants. Mol. Microbiol. 76: 1082–1094.
Moukadiri, I., Garzón, M.J., Björk, G.R., and Armengod, M.E.(2014). The output of the tRNA modification pathways controlledby the Escherichia coli MnmEG and MnmC enzymes depends onthe growth conditions and the tRNA species. Nucleic Acids Res. 42:2602–2623.
Nedialkova, D.D., and Leidel, S.A. (2015). Optimization of codontranslation rates via tRNA modifications maintains proteome in-tegrity. Cell 161: 1606–1618.
Niwa, Y., Hirano, T., Yoshimoto, K., Shimizu, M., and Kobayashi, H.(1999). Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. Plant J.18: 455–463.
Peng, Y., Zhang, Y., Lv, J., Zhang, J., Li, P., Shi, X., Wang, Y.,Zhang, H., He, Z., and Teng, S. (2012). Characterization and finemapping of a novel rice albino mutant low temperature albino 1.J. Genet. Genomics 39: 385–396.
Philipp, M., John, F., and Ringli, C. (2014). The cytosolic thiour-idylase CTU2 of Arabidopsis thaliana is essential for post-transcriptional thiolation of tRNAs and influences root development.BMC Plant Biol. 14: 109.
Pillay, D.T., Guillemaut, P., and Weil, J.H. (1984). Nucleotide se-quences of three soybean chloroplast tRNAsLeu and re-examinationof bean chloroplast tRNA2
Leu sequence. Nucleic Acids Res. 12:2997–3001.
Pogson, B.J., and Albrecht, V. (2011). Genetic dissection of chloro-plast biogenesis and development: An overview. Plant Physiol. 155:1545–1551.
Ranjan, N., and Rodnina, M.V. (2016). tRNA wobble modificationsand protein homeostasis. Translation (Austin) 4: e1143076.
Schön, A., Krupp, G., Gough, S., Berry-Lowe, S., Kannangara,C.G., and Söll, D. (1986). The RNA required in the first step ofchlorophyll biosynthesis is a chloroplast glutamate tRNA. Nature322: 281–284.
Scrima, A., Vetter, I.R., Armengod, M.E., and Wittinghofer, A.(2005). The structure of the TrmE GTP-binding protein and its im-plications for tRNA modification. EMBO J. 24: 23–33.
Su, H., Cheng, Z., Huang, J., Lin, J., Copenhaver, G.P., Ma, H., andWang, Y. (2017). Arabidopsis RAD51, RAD51C and XRCC3 proteinsform a complex and facilitate RAD51 localization on chromosomesfor meiotic recombination. PLoS Genet. 13: e1006827.
Susek, R.E., Ausubel, F.M., and Chory, J. (1993). Signal transductionmutants of Arabidopsis uncouple nuclear CAB and RBCS geneexpression from chloroplast development. Cell 74: 787–799.
Suwastika, I.N., Denawa, M., Yomogihara, S., Im, C.H., Bang, W.Y.,Ohniwa, R.L., Bahk, J.D., Takeyasu, K., and Shiina, T. (2014).Evidence for lateral gene transfer (LGT) in the evolution ofeubacteria-derived small GTPases in plant organelles. Front. PlantSci 5: 678.
Suzuki, T., Nagao, A., and Suzuki, T. (2011). Human mitochondrialtRNAs: Biogenesis, function, structural aspects, and diseases.Annu. Rev. Genet. 45: 299–329.
Takamatsu, T., Baslam, M., Inomata, T., Oikawa, K., Itoh, K.,Ohnishi, T., Kinoshita, T., and Mitsui, T. (2018). Optimized methodof extracting rice chloroplast DNA for high-quality plastome re-sequencing and de novo assembly. Front. Plant Sci 9: 266.
Tang, H., Luo, D., Zhou, D., Zhang, Q., Tian, D., Zheng, X., Chen, L.,and Liu, Y.G. (2014). The rice restorer Rf4 for wild-abortive cyto-plasmic male sterility encodes a mitochondrial-localized PPR pro-tein that functions in reduction of WA352 transcripts. Mol. Plant 7:1497–1500.
Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., Xu, W., and Su, Z.(2017). agriGO v2.0: A GO analysis toolkit for the agriculturalcommunity, 2017 update. Nucleic Acids Res. 45 (W1): W122–W129.
Vaughan, D.A., Morishima, H., and Kadowaki, K. (2003). Diversity inthe Oryza genus. Curr. Opin. Plant Biol. 6: 139–146.
Villarroya, M., et al. (2008). Characterization of human GTPBP3,a GTP-binding protein involved in mitochondrial tRNA modification.Mol. Cell. Biol. 28: 7514–7531.
Walker, J., Kwon, S.Y., Badenhorst, P., East, P., McNeill, H., andSvejstrup, J.Q. (2011). Role of elongator subunit Elp3 in Drosophilamelanogaster larval development and immunity. Genetics 187:1067–1075.
Wang, Y., Li, D., Gao, J., Li, X., Zhang, R., Jin, X., Hu, Z., Zheng, B.,Persson, S., and Chen, P. (2017). The 29-O-methyladenosine nu-cleoside modification gene OsTRM13 positively regulates saltstress tolerance in rice. J. Exp. Bot. 68: 1479–1491.
Wang, Y., et al. (2016). WHITE PANICLE1, a Val-tRNA synthetaseregulating chloroplast ribosome biogenesis in rice, is essential forearly chloroplast development. Plant Physiol. 170: 2110–2123.
Waters, M.T., and Langdale, J.A. (2009). The making of a chloro-plast. EMBO J. 28: 2861–2873.
Wei, F.Y., et al. (2015). Cdk5rap1-mediated 2-methylthio modificationof mitochondrial tRNAs governs protein translation and contributesto myopathy in mice and humans. Cell Metab. 21: 428–442.
Westhof, E., Yusupov, M., and Yusupova, G. (2014). Recognition ofWatson-Crick base pairs: constraints and limits due to geometricselection and tautomerism. F1000Prime Rep. 6: 19.
Chloroplast tRNA Modification Affects Development 2365
Woodson, J.D., Perez-Ruiz, J.M., Schmitz, R.J., Ecker, J.R., andChory, J. (2013). Sigma factor-mediated plastid retrograde signalscontrol nuclear gene expression. Plant J. 73: 1–13.
Xie, K., Minkenberg, B., and Yang, Y. (2015). Boosting CRISPR/Cas9multiplex editing capability with the endogenous tRNA-processingsystem. Proc. Natl. Acad. Sci. USA 112: 3570–3575.
Xie, K., Zhang, J., and Yang, Y. (2014). Genome-wide prediction ofhighly specific guide RNA spacers for CRISPR-Cas9-mediatedgenome editing in model plants and major crops. Mol. Plant 7:923–926.
Yang, C., Wu, R., Liu, H., Chen, Y., Gao, Y., Chen, X., Li, Y., Ma, J.,Li, J., and Gan, J. (2018). Structural insights into DNA degradationby human mitochondrial nuclease MGME1. Nucleic Acids Res. 46:11075–11088.
Yokoyama, S., Watanabe, T., Murao, K., Ishikura, H., Yamaizumi,Z., Nishimura, S., and Miyazawa, T. (1985). Molecular mechanismof codon recognition by tRNA species with modified uridine in the
first position of the anticodon. Proc. Natl. Acad. Sci. USA 82:4905–4909.
Zhang, Y., Su, J., Duan, S., Ao, Y., Dai, J., Liu, J., Wang, P., Li, Y., Liu, B.,Feng, D., Wang, J., and Wang, H. (2011). A highly efficient rice greentissue protoplast system for transient gene expression and studyinglight/chloroplast-related processes. Plant Methods 7: 30.
Zhang, Z., Cui, X., Wang, Y., Wu, J., Gu, X., and Lu, T. (2017). TheRNA editing factor WSP1 is essential for chloroplast development inrice. Mol. Plant 10: 86–98.
Zhou, W., Karcher, D., and Bock, R. (2013). Importance of adeno-sine-to-inosine editing adjacent to the anticodon in an Arabidopsisalanine tRNA under environmental stress. Nucleic Acids Res. 41:3362–3372.
Zhu, Z., Tan, L., Fu, Y., Liu, F., Cai, H., Xie, D., Wu, F., Wu, J.,Matsumoto, T., and Sun, C. (2013). Genetic control of in-florescence architecture during rice domestication. Nat. Commun.4: 2200.
2366 The Plant Cell
DOI 10.1105/tpc.19.00660; originally published online April 23, 2020; 2020;32;2345-2366Plant Cell
Jinshui Yang, Peng Chen, Hong Ma, Xiaojin Luo and Pingli LuHui Liu, Ding Ren, Ling Jiang, Xiaojing Li, Yuan Yao, Limin Mi, Wanli Chen, Aowei Mo, Ning Jiang,
for Chloroplast tRNA Modification in Translation and Plant Development Uncovers a Crucial RolePLEIOTROPIC DEVELOPMENTAL DEFECTSA Natural Variation in
This information is current as of November 9, 2020
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References /content/32/7/2345.full.html#ref-list-1
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