the functions of chloroplast glutamyl-trna intrnaglu in plastid translation and tetrapyrrole...

The Functions of Chloroplast Glutamyl-tRNA inTranslation and Tetrapyrrole Biosynthesis1[OPEN]

Shreya Agrawal,2 Daniel Karcher, Stephanie Ruf, and Ralph Bock3,4

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D–14476 Potsdam-Golm, Germany

ORCID IDs: 0000-0002-9469-5944 (S.R.); 0000-0001-7502-6940 (R.B.).

The chloroplast glutamyl-tRNA (tRNAGlu) is unique in that it has two entirely different functions. In addition to acting intranslation, it serves as the substrate of glutamyl-tRNA reductase (GluTR), the enzyme catalyzing the committed step in thetetrapyrrole biosynthetic pathway. How the tRNAGlu pool is distributed between the two pathways and whether tRNAGlu

allocation limits tetrapyrrole biosynthesis and/or protein biosynthesis remains poorly understood. We generated a series oftransplastomic tobacco (Nicotiana tabacum) plants to alter tRNAGlu expression levels and introduced a point mutation into theplastid trnE gene, which has been reported to uncouple protein biosynthesis from tetrapyrrole biosynthesis in chloroplasts of theprotist Euglena gracilis. We show that, rather than comparable uncoupling of the two pathways, the trnE mutation is lethal intobacco because it inhibits tRNA processing, thus preventing translation of Glu codons. Ectopic expression of the mutated trnEgene uncovered an unexpected inhibition of glutamyl-tRNA reductase by immature tRNAGlu. We further demonstrate thatwhereas overexpression of tRNAGlu does not affect tetrapyrrole biosynthesis, reduction of GluTR activity through inhibition bytRNAGlu precursors causes tetrapyrrole synthesis to become limiting in early plant development when active photosystembiogenesis provokes a high demand for de novo chlorophyll biosynthesis. Taken together, our findings provide insight intothe roles of tRNAGlu at the intersection of protein biosynthesis and tetrapyrrole biosynthesis.

The two DNA-containing organelles of plant cells,plastids (chloroplasts) and mitochondria, contain theirown protein synthesis machinery. In agreement withtheir endosymbiotic origin from bacteria, both organ-elles possess bacterial-type 70S ribosomes consisting ofa 30S and a 50S subunit (Tiller and Bock, 2014; Sun andZerges, 2015; Bieri et al., 2017; Zoschke and Bock, 2018;Waltz et al., 2019). Plastids also encode a complete set oftRNAs that is sufficient to decode all 64 triplets of thegenetic code (Alkatib et al., 2012b; Cognat et al., 2013).By contrast, plant mitochondria do not encode a fulltRNA set in their genome and depend on the import ofsome tRNA species from the cytosol (Salinas et al., 2006;Duchêne et al., 2009; Vinogradova et al., 2009), a path-way that is likely absent from plastids (Rogalski et al.,2008a; Alkatib et al., 2012a).

The chloroplast glutamyl-tRNA (tRNAGlu) isunique in that it has a second essential function. Inaddition to acting in plastid translation, it is also re-quired for tetrapyrrole biosynthesis. Tetrapyrroles aremacrocyclic molecules characterized by four pyrrolerings that are connected by methine bridges. Plantscontain four classes of tetrapyrroles (heme, chloro-phyll, siroheme, and phytochromobilin) that differ inconjugation state, side chains, and/or chelated ion.The universal precursor for the biosynthesis of alltetrapyrroles is 5-aminolevulinic acid (ALA). Thereare two alternative pathways for ALA synthesis, theC4 pathways (or Shemin pathway) and the C5 path-way. The C4 pathway exists in animals, fungi, andcertain groups of bacteria. It initiates with the con-densation of succinyl-CoA and Gly, a reaction that iscatalyzed by ALA synthetase, to form ALA. In eu-karyotes harboring the C4 pathway, ALA synthetasetypically is localized in the mitochondrial compart-ment. The C5 pathway depends on tRNAGlu and existsin plants, archaea, and some groups of bacteria. Inplants, the pathway is localized in plastids and uti-lizes charged plastid-encoded tRNAGlu as substrate,from which the enzyme glutamyl-tRNA reductase(GluTR) forms Glu-1-semialdehyde in the committedstep of the tetrapyrrole biosynthetic pathway. Glu-1-semialdehyde then undergoes an isomerization reac-tion catalyzed by the enzyme Glu-1-semialdehydeaminotransferase (GSAT) to form ALA (Grimm, 1998;Brzezowski et al., 2015; Wang and Grimm, 2015).Thus, tRNAGlu can enter one of two pathways in thechloroplast, namely protein biosynthesis or tetrapyr-role synthesis.

1This work was supported by the Deutsche Forschungsgemein-schaft (DFG; grant nos. BO 1482/17–1 and SFB–TR 175 to R.B.) andthe Max Planck Society.

2Present address: Section of Biomolecular Sciences, Department ofBiology, University of Copenhagen, DK–2200 Copenhagen N.,Denmark

3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ralph Bock ([email protected]).

S.A. performed the research; S.A., D.K., S.R., and R.B. designed theresearch and analyzed data; S.A. and R.B. wrote the article with inputfrom the other authors.

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https://orcid.org/0000-0002-9469-5944

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https://orcid.org/0000-0001-7502-6940

https://orcid.org/0000-0001-7502-6940

https://orcid.org/0000-0002-9469-5944

https://orcid.org/0000-0001-7502-6940

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http://dx.doi.org/10.13039/501100004189

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GluTR catalyzes the rate-limiting step in tetrapyrrolebiosynthesis and is a highly regulated enzyme (Richteret al., 2019). Tight regulation is essential, because manyproducts and intermediates of the pathway are highlytoxic when produced in excess and/or present as freecompounds. For example, free chlorophylls and theirprecursors are extremely phototoxic. ALA synthesisfrom tRNAGlu is regulated at multiple levels, including(1) biochemical feedback regulation by pathway pro-ducts, especially heme; (2) redox control of GluTR ac-tivity; (3) interactionwith a dedicated regulator protein,the GluTR-binding protein (GBP); (4) protein turnovervia recognition of the GluTR N terminus by the chlo-roplast Clp protease; and (5) control of enzyme activitythrough interaction with the protein FLUORESCENTIN BLUE LIGHT (FLU; Meskauskiene et al., 2001;Richter et al., 2019).

A C-to-U point mutation at nucleotide position 56 ofthe mature tRNAGlu (C56U) in plastids of the photo-synthetic protist Euglena gracilis is reported to uncoupletranslation from tetrapyrrole biosynthesis. The mutantstrain of E. gracilis that harbored the correspondingC56T mutation in the plastid gene for tRNAGlu (trnEgene) was orange in color and incapable of chlorophyllbiosynthesis, but was able to perform protein biosyn-thesis in plastids. Further investigation revealed thatthe point mutation in trnE likely impairs tRNAGlu

substrate recognition byGluTR (Stange-Thomann et al.,1994).

Since the trnE genes from E. gracilis and seed plantplastids share a high degree of homology, we hypoth-esized that the C56U mutation might have similar ef-fects in plants and also uncouple plastid translationfrom tetrapyrrole biosynthesis. An important differencebetween plants and E. gracilis is that E. gracilis possessesboth the C4 (Shemin pathway) and the C5 pathway forALA synthesis. Consequently, even if the C5 pathway isdysfunctional in E. gracilis, the cells should still be ableto synthesize some tetrapyrroles (especially heme,which is essential for cell survival) via the alternative C4pathway. By contrast, this is unlikely to be the case withplants, which have only the C5 pathway for synthesis ofall tetrapyrroles.

The aim of this study was to assess the role oftRNAGlu in plastid translation and tetrapyrrole bio-synthesis in seed plants. How the tRNAGlu pool isdistributed between the two pathways and whethertRNAGlu allocation limits tetrapyrrole biosynthesisand/or protein biosynthesis in plastids is currentlynot known. We therefore used plastid transformationin the model plant tobacco (Nicotiana tabacum) to (1)alter tRNAGlu expression levels, and (2) introduce theC56T mutation in an attempt to uncouple plastidtranslation from tetrapyrrole biosynthesis. We reportthat the C56T mutation, when introduced into theendogenous trnE gene in the tobacco chloroplast ge-nome, is lethal. We also show that ectopic expressionof a mutated trnE gene copy results in a pigment-deficientphenotype that, surprisingly, is due to impaired tRNAmaturation. Importantly, whereas accumulation of

immature tRNAGlu has no detectable effect on plastidtranslation, ALA synthesis rate is strongly diminished.Our findings indicate that immature tRNAGlu inhibitsGluTR at the level of enzyme activity. By contrast,overexpression of the wild-type tRNAGlu in chloro-plasts affects neither tetrapyrrole biosynthesis norprotein biosynthesis, suggesting that tetrapyrrole bio-synthesis in plants is not controlled at the level oftRNAGlu provision.

RESULTS

Introduction of the C56T Mutation and Generation ofTransplastomic Tobacco Lines with Altered ExpressionLevels of tRNAGlu

To dissect the functions of tRNAGlu in chloroplasttranslation and tetrapyrrole biosynthesis, four con-structs for stable transformation of the tobaccoplastid genome were designed (Fig. 1). VectorpEndWt (Fig. 1A) was constructed to test whetheroverexpression of tRNAGlu is possible and how thiswould affect plastid translation and tetrapyrrole bio-synthesis. To this end, the strongest known plastidpromoter, specifically the ribosomal RNA (rRNA)operon promoter (Nt Prrn), was placed upstream ofthe endogenous trnE promoter (Hanaoka et al., 2003).Transformation vector pEndMut is similar to pEndWtbut replaces the endogenous copy of trnE in the chlo-roplast genome with the mutated version harboring theC56T point mutation. Considering that (1) the pointmutation abolishes the C5 pathway in E. gracilis, and (2)plants do not have the alternative (Shemin) pathway forheme synthesis, this mutation could potentially lead toinviable plants. We therefore designed two additionalconstructs that left the endogenous trnE gene un-changed but introduced an additional gene copy into aneutral insertion site within a distant region of theplastid genome (Fig. 1B; Ruf et al., 2001; Wurbs et al.,2007). Vector pEctWt introduces an additional wild-type copy of trnE, whereas vector pEctMut introducesan additional trnE gene copy that carries the C56T pointmutation (Fig. 1B).

Using biolistic transformation and selection forspectinomycin resistance conferred by the chimericaadA gene (Svab and Maliga, 1993), putative trans-plastomic lines were obtained with all four con-structs. The transplastomic status of the lines waspreliminarily confirmed by PCR assays amplifyingthe aadA transgene, and positive lines were subjectedto additional rounds of regeneration and selection toenrich the transplastome and isolate homoplasmiclines (Maliga, 2004; Bock, 2015). Transplastomic lineswill subsequently be referred to as Nt-EndWt, Nt-EndMut, Nt-EctWt, and Nt-EctMut lines, respec-tively, with “End” indicating that the endogenoustrnE gene copy was manipulated and “Ect” indicat-ing that an ectopic copy of the trnE gene was intro-duced (Fig. 1).

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Lethality of the C56T Mutation in Tobacco

After three consecutive rounds of regeneration understringent antibiotic selection, the homoplasmic status ofthe transplastomic lines was assessed by DNA gel blotanalyses (Fig. 2). To this end, total DNA was extractedfrom transplastomic plants and digested with the re-striction enzymes BamHI (for Nt-EndWt and Nt-End-Mut) or BglII (forNt-EctWt andNt-EctMut) for analysisby restriction fragment length polymorphism (RFLP).After separation of DNA fragments by agarose gelelectrophoresis, the blotted digestion products weredetected by hybridization using either a trnE-trnD-trnYlocus-specific probe (expected fragment size was 5.8 kbin the transplastomic Nt-EndWt and Nt-EndMut lines

and 4.5 kb in the wild type; Fig. 2A) or a psaB-specificprobe (expected fragment size was 5.3 kb in the trans-plastomic Nt-EctWt and Nt-EctMut lines and 3.5 kb inthe wild type; Fig. 2B). Virtual absence of the wild-type-size fragment from the transplastomic samples wastaken as preliminary evidence of homoplasmy. Severalindependent homoplasmic Nt-EctWt, Nt-EctMut, andNt-EndWt lines were identified, transferred to thegreenhouse, and grown to maturity to obtain seeds.By contrast,Nt-EndMut plants, in which replacement

of the endogenous trnE with the mutated version wasattempted, were clearly not homoplasmic (Fig. 2A).Stable heteroplasmy in the presence of selection istypically associated with genetic changes that inacti-vate an essential gene function in the plastid genome

Figure 1. Physical maps of targetingregions in the tobacco plastid genome(ptDNA) and the modified regions inthe transplastomic mutants gener-ated by stable transformation of thechloroplast genome. A, Map of thetrnE-encoding region in the tobaccoplastid genome and changes intro-duced in Nt-EndWt and Nt-EndMuttransplastomic lines. Genes abovethe line are transcribed from left toright and genes below the line aretranscribed in the opposite direction.Relevant promoters are representedas green boxes, terminators (39 un-translated regions [UTRs]) as brownboxes, and the coding regions oftrnE and the selectable marker geneaadA as light-blue boxes. Note that inboth Nt-EndWt and Nt-EndMut trans-plastomic lines, trnE transcription isenhanced by the insertion of an addi-tional strong promoter (Nt Prrn) up-stream of the endogenous trnE genepromoter (PtrnE). The Nt-EndMut trans-plastomic lines additionally carry theC56T mutation in trnE. B, Maps of thetargeting region in the wild type(ptDNA) and the engineered regions inthe transplastomic Nt-EctWt and Nt-EctMut lines that ectopically over-express the wild-type tRNAGlu or themutated tRNAGlu sequence (harboringthe C56T mutation), respectively. Therecognition sites for digestion by re-striction endonucleases used for RFLPanalysis and the expected fragmentsizes are indicated. The hybridizationsites of radiolabeled probes for RFLPanalysis are shown as black horizontalbars. Cr PpsaA, psaA promoter fromChlamydomonas reinhardtii;Cr TatpB,39 UTR of the atpB gene from C. rein-hardtii; PtrnE, promoter of trnE in N.tabacum.

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tRNAGlu in Translation and Chlorophyll Synthesis



(Drescher et al., 2000; Shikanai et al., 2001; Kode et al.,2005; Rogalski et al., 2006). Moreover, in addition to theexpected bands for the wild-type genome and thetransformed genome, all lines displayed bands thatpresumably originated from undesired recombinationevents (Fig. 2A). Such unexpected hybridization pro-ducts are usually due to recombination between du-plicated expression elements that drive transgeneexpression as well as expression of the endogenousgene from which they were taken (Svab and Maliga,1993; Rogalski et al., 2006; Gray et al., 2009; Li et al.,2011). After multiple rounds of plant regeneration inthe presence of spectinomycin, a few homoplasmiclines were obtained. When these lines were tested forthe presence of the C56T point mutation in the trnEgene by sequencing of amplified PCR products, all ofthem were found to lack the mutation, whereas themutation was readily detectable in the heteroplasmiclines. Previous research had shown that deleteriousmutations that are present in a heteroplasmic state arefrequently eliminated by gene conversion (Khakhlovaand Bock, 2006). Occurrence of gene conversion inconjunction with segregation to homoplasmy stronglysuggests that the C56T mutation is lethal and remainsheteroplasmic until the mutation is eliminated throughgene conversion with a wild-type trnE copy.

Heteroplasmic Nt-EndMut plants displayed a char-acteristic leaf-loss phenotype in that the leaves showedvarious deformations and often lacked parts of the leafblade (Fig. 3A). Very similar phenotypes were described

previously for tobacco mutants with induced inactiva-tion of plastid translation (Ahlert et al., 2003) and trans-plastomic plants in which essential components of theplastid gene expression machinery, including essentialtRNA genes (Rogalski et al., 2008a; Alkatib et al., 2012a,2012b), were targeted by reverse genetics (Rogalski et al.,2008b; Fleischmann et al., 2011). In these plants, segre-gation to homoplasmy leads to the loss of plastid geneexpression, which in turn results in cell death. When thisoccurs early in leaf development, it can cause the loss ofan entire cell lineage, which then results in misshapenleaves with large sectors of the leaf blade missing.

Lethality of the C56T mutation in tobacco suggeststhat it is incompatible with tRNAGlu function in trans-lation or tetrapyrrole biosynthesis (or both). Since theNt-EndMut transplastomic plants were genetically un-stable, accumulated undesired recombination pro-ducts, and tended to lose the C56T point mutation intrnE, they were excluded from further analyses.

Phenotypes of Transplastomic Lines OverexpressingWild-Type or Mutant trnE Alleles

When grown in soil under standard greenhouseconditions, transplastomic Nt-EctWt and Nt-EndWtplants were indistinguishable from wild-type to-bacco plants (Fig. 3B). By contrast, transplastomicNt-EctMut plants were severely retarded in growth(by .2 weeks) compared to the wild type (Fig. 3B).Moreover, leaves ofNt-EctMut plants that developedat early stages of plant growth displayed a pale orvariegated phenotype. Interestingly, this phenotypewas absent from leaves that developed at later stages ofplant growth (Fig. 3B, bottom). The degree of chlorosisand the number of leaves showing the pale or varie-gated phenotypewas quite variable, even among plantsfrom the same transplastomic line. This suggests that,similar to the case for previously described variegationmutants (Miura et al., 2007; Wang et al., 2018), aphysiological threshold effect may be involved in thedevelopment of the phenotype.

Seeds were readily obtained from all three sets oftransplastomic lines. In order to ultimately confirmhomoplasmy of the lines and assess the phenotype atthe seedling stage under heterotrophic conditions,seeds were surface sterilized and germinated on syn-thetic (Suc-containing) culture media with or withoutspectinomycin (Fig. 4). Whereas wild-type seedlingswere clearly sensitive to spectinomycin (i.e. theybleached out and ceased to grow), the progenies of alltransplastomic lines showed uniform resistance to theantibiotic. Lack of segregation in the F1 generation isconsistent with the uniparental inheritance of theplastid genome in tobacco (Greiner et al., 2015) andprovides strong genetic evidence of homoplasmy of thetransplastome. Nt-EctMut seedlings were pale green,but their phenotype was clearly distinguishable fromspectinomycin-sensitive wild-type seedlings, whichwere completely white and showed arrested shoot and

Figure 2. RFLP analysis of transplastomic plants generated for over-expression of the wild-type trnE gene or the mutated trnE gene (givingrise to tRNAGlu_C56U) in tobacco chloroplasts. A, Southern blot anal-ysis ofNt-EndWt andNt-EndMut lines. Total DNAwas digestedwith therestriction enzyme BamHI and fragments were detected by hybridiza-tion with a radiolabeled trnE-trnD-trnY locus-specific probe (comparewith Fig. 1A). M, molecular weight marker. B, Analysis ofNt-EctWt andNt-EctMut lines. Total DNAwas digested with BglII and fragments weredetected by hybridization with the radiolabeled psaB-specific probeshown in Figure 1B.


Agrawal et al.



root growth. Also, the pale green appearance of Nt-EctMut seedlings was independent of the presence ofthe antibiotic (Fig. 4), confirming that these plants werealso homoplasmic.

Expression of tRNAGlu in Transplastomic Tobacco Plants

All constructs introduced into the plastid genomeshare the strong constitutive Nt Prrn promoter up-stream of trnE (Fig. 1). To determine whether thepresence of this additional promoter (in the Nt-EndWtlines) and/or the ectopic expression of an extra copy ofthe trnE gene (in the Nt-EctWt and Nt-EctMut lines)results in overexpression of tRNAGlu in transplastomicchloroplasts, northern blot experiments were con-ducted with total RNA samples extracted from twoindependent transplastomic lines for each of the con-structs. A radiolabeled oligonucleotide derived fromthe sequence of the mature tRNAGlu was used as thehybridization probe for detection of trnE-specific tran-scripts (Fig. 5).

Interestingly, provision of the endogenous trnE withan additional Nt Prrn promoter (Nt-EndWt plants) didnot lead to detectable overaccumulation of tRNAGlu

compared to the wild type (Nt-Wt; Fig. 5). By contrast,insertion of an additional copy of the trnE gene into theplastid genome resulted in a strong increase in tRNAGlu

abundance in transplastomic Nt-EctWt plants (Fig. 5).Surprisingly, transplastomic Nt-EctMut plants that ec-topically express an additional trnE gene carrying theC56Tmutation showed an additional hybridizing bandin the northern blot analysis. This band was substan-tially larger (;150–200 nucleotides) than that of themature tRNAGlu (76 nucleotides, including the 39 CCAend that is added enzymatically after 39 processing byribonuclease [RNase] Z). This bandwas absent from thewild type and all other transplastomic lines (which onlyaccumulated the mature tRNA) and could representincompletely processed tRNAGlu molecules (Fig. 5).This observation raised the interesting possibility thatthe C at position 56 of trnE plays a crucial role in thematuration of pre-tRNAGlu to produce functionaltRNAGlu. The nucleotide at position 56, although part of

Figure 3. Phenotypes of transplastomicplants generated in this study comparedto wild-type plants. A, Phenotype ofNt-EndMut transplastomic plants in tis-sue culture compared to a control plant(Nt-Wt). Shown at right are wild-typeleaves alongside examples of mis-shapen leaves that are typical of muta-tions in chloroplast genes encodingessential components of the translationmachinery (Ahlert et al., 2003; Rogalskiet al., 2006, 2008b; Alkatib et al.,2012a, 2012b; reviewed in Tiller andBock, 2014). Scale bars 5 1 cm. B,Phenotypes of Nt-EctWt, Nt-EctMut,and Nt-EndWt transplastomic plantsgrown in soil, showing 4-week-old (top;scale bar 5 5 cm) and 8-week-oldplants (bottom; scale bar 5 20 cm) ascompared to a control plant.





the loop region of the TCC arm, is known to form aWatson-Crick base pair with the nucleotide at position19. This tertiary interaction plays an important role intRNA elbow formation and is believed to be involved inthe recognition of the tRNA by several protein factors,including the 59 and 39 processing enzymes RNase Pand RNase Z (Zhang and Ferré-D’Amaré, 2016).

tRNAGlu Processing in Transplastomic Nt-EctMut Plants

To test the idea that the C-to-U substitution at posi-tion 56 in tRNAGlu results in impaired pre-tRNA pro-cessing, northern blot analyses were performed withhybridization probes specific to the 59 leader and 39trailer sequences of the pre-tRNAGlu. Bands weredetected exclusively in the Nt-EctMut plants (Fig. 6, Aand B) using both probes, suggesting that (1) tRNAprocessing in the wild-type and the transplastomic Nt-EndWt and Nt-EctWt plants is very efficient, sinceprecursors and processing intermediates are undetect-able; and (2) the C56U exchange affects maturation oftRNAGlu in transplastomicNt-EctMut plants at both the59 and 39 ends. The size of the incompletely processedtRNAGlu molecules was in the same range as the largerband detected in northern blots with the trnE probe(Fig. 5), although the leader and trailer probes alsodetected additional presumptive precursor RNAs ofeven larger size but much lower abundance (Fig. 6, Aand B).

To ultimately confirm the 59 and 39 processing defectsin the Nt-EctMut plants, RNA circularization experi-ments were undertaken. To this end, the regions cor-responding to the bands seen in the RNA gel blotexperiments were excised from urea-containing dena-turing polyacrylamide gels, self-ligated, and reversetranscribed into complementary DNA. SubsequentPCR amplification of the junction of the ligated terminiprecisely identified the 59 and 39 ends. When this assaywas performed with RNA samples from the wild type,the Nt-EndWt line, and the Nt-EctWt line, only fullyprocessed tRNAGlu molecules were detected (Fig. 6C).Interestingly, none of the processed tRNAGlu moleculesin the Nt-EctMut plants carried the point mutation, in-dicating that the faithfully processed tRNA moleculesoriginate exclusively from the resident trnE gene copy inthe plastid genome. Conversely, all incompletely pro-cessed tRNAGlu molecules carried the point mutation(Fig. 6C; Supplemental Fig. S1). Whereas all immaturetRNAmolecules had 39 extensions, only relatively few (2of 20 sequenced clones) carried 59 leader sequences

Figure 5. Northern blot analysis to compare transcript accumulationlevels and RNA processing of trnE in transplastomic Nt-EctWt, Nt-EctMut, and Nt-EndWt lines and wild-type (Nt-Wt) tobacco plants.One microgram of total cellular RNAwas electrophoretically separatedunder denaturing conditions and blotted. The blot was hybridized to aradiolabeled probe specific to the trnE gene (top). The ethidiumbromide(EtBr)-stained gel prior to blotting is shown as a control for equal loading(bottom). Dashes denote empty lanes. M, RNA size marker.

Figure 4. Seed assays to confirmhomoplasmy of transplastomic plants.Wild-type (Nt-Wt) seeds and T1 seedsfrom Nt-EctWt, Nt-EctMut, and Nt-EndWt were germinated on syntheticmedium in the presence of spectinomy-cin. The absence of antibiotic-sensitiveprogeny indicates the homoplasmicstate of all transplastomic lines. 1Spec,500mg L21 spectinomycin in the culturemedium; 2Spec, control with no spec-tinomycin in the culture medium. Scalebar5 2 cm.


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(Fig. 6C; Supplemental Fig. S1). This finding potentiallyindicates that the C56U mutation largely blocks precur-sor tRNAprocessing by the 39 processing enzyme RNaseZ (Stern et al., 2010; Stoppel and Meurer, 2012), whereas59 processing by the chloroplast form of RNase P(PRORP; Gutmann et al., 2012; Zhou et al., 2015) is lessseverely affected.

Ectopic Expression of Mutated tRNAGlu Affects NeitherAminoacylation of Mature tRNAGlu Nor Processing ofOther Plastid tRNAs

Having uncovered processing defects in the mutatedtRNAGlu (tRNAGlu_C56U), we next considered thepossibility that accumulation of immature tRNAGlu

molecules interferes with tRNA aminoacylation in theNt-EctMut plants, for example, by inhibiting the cor-responding aminoacyl-tRNA synthetase. We thereforedetermined the tRNA aminoacylation state by acidicurea PAGE of acylated versus deacylated tRNAs. Theseexperiments revealed that the aminoacylation state oftRNAGlu was unaffected in all transplastomic lines, in-cluding the Nt-EctMut lines ectopically expressing themutant tRNAGlu (Fig. 7A).Our sequencing analyses had indicated that the Nt-

EctMut lines efficiently processed the tRNAGlu mole-cules transcribed from the wild-type trnE gene copy,whereas processing of the mutated tRNAGlu_C56Uwasselectively impaired (Fig. 6C; Supplemental Fig. S1).This finding argues against the possibility that themutated tRNA interferes with the general activity ofPRORP and/or RNase Z in the chloroplast. To furtherverify this assumption, the processing status of twoother chloroplast tRNAs, namely tRNAPhe andtRNAArg, was investigated by northern blot analysis.As expected, only mature tRNAs could be detected inall transplastomic lines investigated (Fig. 7B), confirm-ing that accumulation of immature tRNAGlu_C56Udoes not generally interfere with the activity of PRORPand/or RNase Z in the Nt-EctMut lines.

Accumulation of Immature tRNAGlu_C56U Does NotAffect Chloroplast Protein Biosynthesis

Our analyses of tRNAprocessing and aminoacylationsuggested that processes upstream of tRNAGlu utiliza-tion in chloroplast translation and ALA synthesis areunlikely to be causally responsible for the mutant

Figure 6. Northern blot analysis to detect incompletely processedprecursors of tRNAGlu in transplastomic Nt-EctWt, Nt-EctMut, and Nt-EndWt lines and wild-type (Nt-Wt) tobacco plants. A and B, Detectionof trnE transcripts that remain unprocessed at their 59 end using the 59leader sequence of tRNAGlu as the radiolabeled probe (A), and trnEtranscripts that remain unprocessed at their 39 end using the 39 trailer

sequence of tRNAGlu as the probe (B). Samples of 3 mg total cellularRNA were electrophoretically separated under denaturing conditionsand blotted. Blots were hybridized to radiolabeled probes (top), and theethidium bromide (EtBr)-stained gels prior to blotting are shown ascontrols for equal loading (bottom). M, RNA size marker. C, Schematicrepresentation of the results from circular RT-PCR analysis used toidentify the termini of incompletely processed trnE transcripts. See textfor details. For sequences of individual clones from Nt-EctMut, seeSupplemental Figure S1.








phenotype observed in Nt-EctMut plants. Therefore,we subsequently examined whether expression of themutated trnE gene negatively affects chloroplast pro-tein biosynthesis.

Polysome analyses were performed to determinewhether the pale-leaf phenotype of Nt-EctMut plants(Fig. 3B) was the result of interference of tRNAGlu_C56Uwith the translation by chloroplast ribosomes. Recentwork has suggested that the abundance of plastid-encoded chlorophyll-binding proteins is adjusted tochlorophyll availability at the level of proteolysis ratherthan de novo synthesis (Zoschke et al., 2017). Conse-quently, any observable effect on plastid translation ratesshould be a direct consequence of the interaction of mu-tated tRNAGlu molecules with translating ribosomesrather than a secondary effect from the pigment defi-ciency of Nt-EctMut plants. Northern blot analysis ofRNAs extracted from fractionated polysome gradientsrevealed unaltered ribosome association of the mRNAsof two key genes involved in photosynthesis: rbcL (en-coding the large subunit of Rubisco) and psbD (encod-ing the D2 protein of the PSII reaction center; Fig. 8).These results indicate that accumulation of the mutatedtRNAGlu_C56U does not affect protein synthesis ratesin the chloroplast.

The Presence of Precursor tRNAGlu_C56U StronglyReduces ALA Synthesis Rates

Having excluded tRNAmetabolism, aminoacylation,and translation as possible causes of the mutant phe-notype ofNt-EctMut lines, the possibility remained thatthe accumulation of mutated and incompletely pro-cessed tRNAGlu molecules inhibits tetrapyrrole bio-synthesis. Such an inhibitory effect could come frombinding of immature tRNAGlu_C56U to GluTR, theenzyme that uses tRNAGlu as substrate for ALA syn-thesis. As nomature tRNAGlu_C56U accumulates and 39processing is completely blocked (Fig. 6C; SupplementalFig. S1), the tRNAGlu_C56U can neither undergo 39CCAaddition nor be charged with Glu. Thus, it seems clearthat tRNAGlu_C56U cannot serve as substrate to formGlu-1-semialdehyde in the committed step of tetrapyr-role biosynthesis. However, if the mutated and imma-ture tRNAmolecules would nonetheless bind to GluTR,they could either block the activity of the enzyme or,

Figure 7. Aminoacylation of processed tRNAGlu and unaffected pro-cessing of other plastid tRNAs in transplastomic Nt-EctWt, Nt-EctMut,and Nt-EndWt plants. A, Northern blot analysis to determine the ami-noacylation status of tRNAGlu in transplastomic Nt-EctWt, Nt-EctMut,and Nt-EndWt plants and wild-type tobacco (Nt-Wt). Samples of 2 mgtotal RNA (except forNt-EctWt, for which 500 ngwere loaded to correct

for its higher abundance; Fig. 5) isolated under acidic conditions waselectrophoretically separated in a 14%urea-containing polyacrylamidegel and blotted. The blot was hybridized to a radiolabeled trnE-specificprobe. Deacylated tRNAs were obtained by subjecting total RNA toalkaline pH. B, Northern blot analysis to investigate the processingstatus of two other plastid tRNAs. Samples of 1 mg total cellular RNAwere electrophoretically separated under denaturing conditions andblotted. Blots were hybridized to radiolabeled probes specific to eithertrnF (top) or trnR (bottom). The ethidium bromide (EtBr)-stained gelsprior to blotting are shown as control for equal loading. M, molecularweight marker.


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alternatively, destabilize the enzyme and condemn it torapid degradation.To investigate these possibilities, ALA synthesis rates

were measured in wild-type, Nt-EctWt, and Nt-EctMutplants. Although the transplastomic Nt-EctWt plantsstrongly overexpress tRNAGlu (Fig. 5), tRNA over-accumulation did not result in a significant stimulationof ALA synthesis (Fig. 9A), indicating that tRNAGlu

availability does not limit tetrapyrrole biosynthesis. Bycontrast, ALA synthesis was strongly reduced in Nt-EctMut plants, with synthesis rates reaching only;20% of those in wild-type and Nt-EctWt plants(Fig. 9A). This finding raises the interesting possibilitythat the ectopically expressed tRNAGlu_C56U inhibitsALA synthesis by GluTR.To examine whether tRNAGlu_C56U affects GluTR

accumulation (e.g. by forming a dead-end complex thattriggers proteolytic degradation of the enzyme), im-munoblot analysis with GluTR-specific antibodies wasperformed. These experiments revealed that GluTRprotein levels were very similar in all investigated plantlines (Fig. 9B), indicating that tRNAGlu_C56U does notexert its inhibitory action at the level of protein stability.Instead, this finding suggests that inhibition occurs atthe level of enzyme activity, presumably by the im-mature and uncharged tRNAGlu_C56U moleculesforming a stable dead-end complex with GluTR.Finally, we examined accumulation of two of the

end products of tetrapyrrole biosynthesis, chloro-phyll a and chlorophyll b, in the transplastomic lines(Supplemental Fig. S2). We particularly wanted to

know whether the green leaves appearing later in thedevelopment of Nt-EctMut plants (Fig. 3B) show fullrecovery at the level of chlorophyll accumulation.This was indeed the case for both chlorophyll a andchlorophyll b (Supplemental Fig. S2), suggesting thatrapid growth and high demand for chlorophyllsearly in development cause the pale-leaf phenotype,whereas the reduced demands later in developmentcan be fully satisfied by the reduced synthesis ca-pacity in Nt-EctMut plants. Also, none of the othertransplastomic lines displayed any alteration inchlorophyll accumulation (Supplemental Fig. S2),consistent with our finding that ALA synthesisrates are unaffected by overexpression of tRNAGlu

(Fig. 9A).

DISCUSSION

In the course of this work, we generated a set oftransplastomic tobacco plants to alter the expression ofchloroplast tRNAGlu and introduced a point mutationin the trnE gene, the latter of which had been describedin a spontaneous mutant of E. gracilis and reported touncouple plastid translation from tetrapyrrole biosyn-thesis (Stange-Thomann et al., 1994). The C56T muta-tion in the trnE gene of the Euglena mutant led toreduced accumulation of tRNAGlu, but the mutanttRNA appeared to be faithfully aminoacylated andthereforewas suggested to participate in plastid proteinbiosynthesis. By contrast, the mutant tRNAGlu did not

Figure 8. Analysis of plastid translation in transplastomic Nt-EctMut and Nt-EctWt plants. Polysomes extracted from samples of200 mg leaf tissue were separated by Suc density gradient centrifugation. RNAs isolated from different gradient fractions wereelectrophoretically separated under denaturing conditions and blotted. The wedges above the blots indicate the increasing Succoncentrations in the gradients. Blots were hybridized to radiolabeled probes specific to either rbcL or psbD transcripts. Theethidium bromide (EtBr)-stained gels prior to blotting are shown below each blot as a control for equal loading. A, Polysomeloading analysis to compare translation rates in transplastomic Nt-EctWt and Nt-EctMut lines and wild-type (Nt-Wt) tobaccoplants. B, Identification of polysome-containing gradient fractions by analysis of puromycin-treated samples. Puromycin is anantibiotic that causes premature translation termination, thus revealing the gradient fractions that contain untranslated mRNAs(including mRNAs present in ribonucleoprotein complexes other than polysomes).








support ALA synthesis by the C5 pathway (Stange-Thomann et al., 1994).

Our work reported here in the seed plant tobaccodid not confirm many of the findings reported for theEuglena mutant. Most importantly, when introducedinto the trnE gene of tobacco plastids, the C56T mu-tation was incompatible with cell survival. Trans-plastomic lines remained heteroplasmic and displayedthe malformed leaf phenotype that is typical of (het-eroplasmic) loss-of-function mutants of essentialgenes in the plastid genome (Figs. 2A and 3A). Nota-bly, this phenotype has been observed for all genesencoding essential components of the plastid transla-tional machinery when they are targeted by reversegenetics, including ribosomal proteins and tRNAs(Ahlert et al., 2003; Alkatib et al., 2012b; Tiller andBock, 2014; Zoschke and Bock, 2018). Our analysis oftRNAGlu_C56U expression in transplastomic plantsrevealed defective tRNA processing and a completeabsence of correctly processed tRNA molecules thatcould be aminoacylated and serve as substrates intranslation elongation (Fig. 6C; Supplemental Fig. S1).This finding suggests that the C56T mutation repre-sents a trnE loss-of-function allele, thus providing astraightforward explanation for the phenotype of theNt-EndMut plants (Fig. 3A). However, since the C5pathway is likely also essential in seed plants(Czarnecki et al., 2011a; Richter et al., 2019), the reasonwhy Nt-EndMut plants remain heteroplasmic and

segregation to homoplasmy causes cell death and leafmalformation could be twofold.

It is currently unclear why processed and amino-acylated tRNAGlu accumulated in the Euglena mutant.All sequenced Euglena strains contain a single copy ofthe trnE gene in their chloroplast genome. The tRNAprocessing machinery is highly conserved in that 59processing is conducted by RNase P and 39 processingis performed by RNase Z in nearly all organisms. Also,the tertiary interaction nucleotide C56 is involved in itsrole in tRNA elbow formation, and its involvement inthe interaction with the tRNA processing enzymes ap-pears well conserved (Zhang and Ferré-D’Amaré,2016). It therefore would be surprising if the samemutation completely blocks tRNA maturation in to-bacco plastids but allows for correct processing in Eu-glena plastids. In this context, it should be noted that thechloroplast genome of the Euglena mutant was notcharacterized beyond PCR amplification of the knowngenomic trnE locus. Thus, the presence of a second wildtype-like copy of the trnE gene that provides the func-tional tRNAGlu identified in the aminoacylation assays(Stange-Thomann et al., 1994), along with an unchar-acterized genetic defect that causes the chlorophylldeficiency, cannot be ruled out. Such gene duplicationcould be the result of a genomic rearrangement thatwent undetected in the studies conducted with themutant. It is noteworthy in this regard that gene du-plications in Euglena plastids are more stable than inseed-plant plastids. This is suggested, for example, bythe presence of the rRNA operon as tandem repeats(Hallick et al., 1993), and it may be due to the absence(or very low activity) of homologous recombination(Doetsch et al., 2001). Alternatively, it is possible thatthe correctly processed tRNAGlu is encoded in the nu-clear genome of Euglena and imported into plastids.However, whereas tRNA import is common in plantmitochondria (Duchêne et al., 2009), no direct evidencefor the presence of it in plastids has been obtained todate, although some indirect evidence comes from theexistence of highly reduced plastid genomes that do notencode a complete set of tRNAs (Morden et al., 1991;Delannoy et al., 2011). In view of all this, a thoroughreinvestigation of the Euglena mutant, if still available,would be very worthwhile.

In this study, we also tested two different approachestoward overexpression of the chloroplast tRNAGlu.Since tRNAGlu serves as substrate of the rate-limitinginitial reaction of tetrapyrrole biosynthesis (Brzezowskiet al., 2015; Richter et al., 2019), it was conceivable thattRNAGlu provision limits flux through the pathway. Infact, expression of tRNAGlu has been shown to be de-velopmentally regulated (Hanaoka et al., 2005). More-over, tRNAGlu was proposed to feedback-regulatechloroplast transcription by directly binding to one ofthe two RNA polymerases in plastids (Hanaoka et al.,2005). The two overexpression strategies we pursuedproduced transplastomic plants that were indistin-guishable from wild-type plants (Fig. 3B). Whereas in-sertion of an additional strong promoter upstream of

Figure 9. Analysis of ALA synthesis and GluTR accumulation levels intransplastomic Nt-EctWt and Nt-EctMut plants. A, Comparison of ALAsynthesis rates in Nt-EctWt, Nt-EctMut, and wild-type (Nt-Wt) tobaccoplants. Error bars represent the SD (n 5 3, Student’s t test, significantdifference to the wild type, ***P , 0.001). B, Immunoblot analysis todetermine GluTR levels in Nt-EctWt, Nt-EctMut, and wild-type plants.Samples of 20 mg of extracted total cellular protein were separated by10% SDS-PAGE, blotted, and hybridized to aGluTR-specific polyclonalantibody. Actin was analyzed as a control protein for equal loading.


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the trnE gene in its native genomic location did notresult in appreciable overaccumulation of tRNAGlu,integration of an extra gene copy into a distant genomiclocation resulted in strong overexpression of the tRNA(Fig. 5). However, this did not result in a measurable ef-fect on ALA synthesis rates (and chlorophyll accumula-tion) in the transplastomic Nt-EctWt plants (Fig. 9A;Supplemental Fig. S2), suggesting that tRNAGlu does notlimit tetrapyrrole biosynthesis, at least not under stan-dard growth conditions.Ectopic expression of tRNAGlu_C56U produced

plants that exhibit a strong pigment-deficient pheno-type at the seedling stage (Fig. 3B). Interestingly, as theplants matured, the mutant phenotype became lesssevere and the leaves appearing later in developmentbecame progressively greener until they finally werenearly indistinguishable fromwild-type leaves (Fig. 3B,lower, young Nt-EctMut leaves). Nt-EctMut plantsshowed unaltered expression of the endogenous trnEgene and accumulated wild-type levels of fully pro-cessed and faithfully aminoacylated tRNAGlu (Figs. 5and 7A). In addition, the plants accumulated the mu-tant tRNAGlu_C56U to a similar level; however, thistRNA remained unprocessed and therefore cannot beaminoacylated. These results suggest that the pigment-deficient mutant phenotype of the Nt-EctMut plants isthe result of a dominant-negative effect exerted by theectopic expression of tRNAGlu_C56U. We showed thatGluTR, the enzyme that utilizes tRNAGlu to synthesizeALA, accumulates to unaltered levels in Nt-EctMutplants. However, ALA synthesis rates were drasticallyreduced in the transplastomic plants (Fig. 9), indicatingthat the immature tRNAGlu_C56Umolecules inhibit theenzyme, presumably by binding to it and forming adead-end complex.Our data suggest that inhibition of GluTR activity to

;20% of the wild-type levels causes ALA synthesis tolimit biogenesis of the photosynthetic apparatus. Thisbottleneck is particularly severe in young developingplants that have high rates of photosystem biogenesis(Hojka et al., 2014; Schöttler and Tóth, 2014; Armarego-Marriott et al., 2019) and therefore a high demand forchlorophylls (Fig. 3B). By contrast, when growth de-celerates later in development, the low ALA synthesisrates are sufficient to satisfy the needs of the chloro-plast, resulting in a striking developmental gradient inthe leaf phenotypes. Interestingly, the pale leaves thatdeveloped under ALA limitation do not recover andnever regreen (Fig. 3B). This is likely due to loss of thephotosystem assembly capacity that is known to occuras leaves mature (Schöttler et al., 2011; Hojka et al.,2014; Schöttler and Tóth, 2014).The charging of tRNAGlu with the amino acid Glu

occurs by the two-step reaction typical of aminoacyl-tRNA synthetases. L-Glu is first activated by ATP toform glutamyl-AMP and then transferred to the ac-ceptor end (i.e. the 39 CCA end) of the tRNAGlu mole-cule. The aminoacylation of mature tRNAGlu wasvirtually complete in all transplastomic lines generatedin this study (Fig. 7A), suggesting that even strong

overexpression of the tRNA does not exceed thecapacity of the plastid tRNAGlu synthetase. Consis-tent with this finding, chloroplast translation wasunaffected in all lines (Fig. 8). As unprocessed anduncharged tRNAs are not taken into the ribosome,it is unsurprising that accumulation of immaturetRNAGlu_C56U does not interfere with translationelongation.In summary, our work here shows that the C56U

substitution in chloroplast tRNAGlu, unlike that previ-ously reported for Euglena (Stange-Thomann et al.,1994), does not uncouple plastid protein biosynthesisfrom tetrapyrrole biosynthesis in tobacco. Instead, theC56T substitution represents a lethal mutation. It in-hibits tRNA processing and in this way preventstranslation of Glu codons. Moreover, our work hasuncovered an unexpected inhibitory activity of unpro-cessed tRNAGlu on GluTR. Importantly, whereasoverexpression of tRNAGlu does not stimulate ALAbiosynthesis, reduction of GluTR activity through in-hibition by tRNAGlu precursors makes ALA synthesislimiting to an extent that chlorophyll biosynthesis inearly plant development cannot satisfy the demands ofphotosystem biogenesis.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Sterile tobacco (Nicotiana tabacum ‘Petit Havana’) plants were grown onMurashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing3% (w/v) Suc. The growth chambers had a light intensity of 50 mEm22 s21 anda photoperiod of 16 h light at 24°C and 8 h dark at 22°C. Soil-grown plants wereraised in growth chambers with a light intensity of 350 mE m22 s21 and aphotoperiod of 16 h light at 24°C and 8 h dark at 22°C.

Construction of Plastid Transformation Vectors

For construction of transformation vector pNt-EctWt (Fig. 1), PCR wasperformed to amplify (1) the Nt Prrn promoter using primer pair oSAA57_tr-nE_F3/oSAA58_ trnE_R3, (2) the trnE gene from the tobacco plastid genomeand its promoter using primer pair oSAA59_trnE_F4/oSAA65_ trnE_R8, and(3) the 39 untranslated region from the plastid rbcL gene of Chlamydomonasreinhardtii (Cr TrbcL; Lu et al., 2017) with primer pair oSAA64_trnE_F7/oSAA66_trnE_R7 (Supplemental Table S1). Overlap extension PCR was per-formed to generate the Nt Prrn-trnE-Cr TrbcL fusion product. The resultingamplification product was digested with the restriction enzymes SacI andHindIII and cloned into a modified pRB96 vector (Ruf et al., 2001; Wurbs et al.,2007) cut with SacI and HindIII.

For construction of transformation vector pNt-EctMut, site-directed muta-genesis was performed on the fusion product generated for construction of pNt-EctWt to introduce the desired pointmutation into trnE. To this end, PCRswereperformed with the Nt Prrn-trnE-Cr TrbcL fusion product as template using theprimer pairs oSAA57_trnE_ F3/oSAA62_trnE_ R5 and oSAA61_trnE_ F5/oSAA66 _trnE_R7 (Supplemental Table S1). Overlap extension PCR was per-formed to produce the Nt Prrn-trnE_C56U-Cr TrbcL fusion product. This am-plification product was cloned in the pRB96-derived plastid transformationvector as described above.

For construction of vectors pNt-EndWt and pNt-EndMut, an intermediatevector (pSAA26) was generated. To this end, PCRwas performed to amplify (1)an;1-kb region upstream of trnE using tobacco total DNA as template and theprimer pair oSAA53_trnE_F1 (introducing a SacI site)/oSAA54_ trnE_R1, and(2) an aadA expression cassette (Fig. 1A) with the primer pair oSAA55_trnE_F2/oSAA56_trnE_R2 (introducing a HindIII site; Supplemental Table S1). Overlapextension PCR was performed to generate a fusion product, which was









subsequently digested with SacI and HindIII and cloned into plasmid pBSKS(1) cut with SacI and HindIII.

For construction of pNt-EndWt, PCRs were performed to amplify (1) the NtPrrn sequence with primer pair oSAA57_trnE_F3 (introducing a HindIII site)/oSAA58_ trnE_R3, and (2) the trnE-trnY-trnD region of the tobacco plastidgenome and an ;500-bp region downstream with primer pair oSAA59_trnE_F4/oSAA60_trnE_R4 (introducing an XhoI site; Supplemental Table S1).Overlap extension PCR was performed to obtain a fusion product, which wasthen digested with HindIII and XhoI and cloned into pSAA26 cut with HindIIIand XhoI. pNt-EndMut was constructed via site-directed mutagenesis per-formed on the fusion product produced for construction of pNt-EndWt to in-troduce the desired point mutation into the trnE gene sequence. To this end,PCRs were performedwith the fusion product as template and the primer pairsoSAA57_trnE_F3 (introducing a HindIII site)/oSAA62_ trnE_R5 andoSAA61_trnE_F5/oSAA60_ trnE_R4 (introducing an XhoI site; SupplementalTable S1). Overlap extension PCR yielded a new fusion product, which wasdigested with HindIII and XhoI and cloned into pSAA26 cut with HindIIIand XhoI.

Plastid Transformation of Tobacco and Isolation ofTransplastomic Lines

Biolistic transformation of plastids was conducted according to publishedprotocols (Svab and Maliga, 1993). Briefly, young tobacco leaves from plantsgrown under sterile conditions were bombarded with plasmid DNA-coatedgold particles using the DuPont PDS-1000/He biolistic gun (Bio-Rad). Afterbombardment, leaves were cut into small pieces;53 5mm in size, placed ontoMS-based plant regeneration medium containing 500 mg L21 spectinomycin,and incubated for 2 to 3months. Primary spectinomycin-resistant shoots or calliwere subjected to two to three additional rounds of regeneration in the presenceof the antibiotic to enrich the transformed plastid genome and isolate homo-plasmic transplastomic lines (Bock, 2001). Finally, regenerated shoots wererooted and propagated on hormone-free MS medium containing 500 mg L21

spectinomycin, then transferred to soil and grown to maturity under standardgreenhouse conditions with a photoperiod of 16 h light at 25°C and 8 h darknessat 20°C.

Seed Assays

To test for maternal inheritance of the engineered plastid genomes, T1generation seeds from the transplastomic lineswere surface-sterilized and sownon MS medium containing 500 mg L21 spectinomycin. The presence of a ho-mogeneous antibiotic-resistance progeny confirmed homoplasmy of thetransformed chloroplast genome (Bock, 2001).

Isolation of Nucleic Acids and Gel Blot Analyses

Leaf tissue snap-frozen in liquid nitrogen was used for the isolation of nucleicacids. For extraction of total cellular DNA, a cetyltrimethylammoniumbromide-based method was used (Doyle and Doyle, 1990). Total plant RNA wasextracted using the peqGOLD TriFast reagent (Peqlab). For RFLP analysis,samples of 3 mg total DNAwere digestedwith appropriate restriction enzymes,separated in 1% (w/v) agarose gels by electrophoresis, and transferred ontoHybond nylon membranes (GE Healthcare) by capillary blotting. For northernblot analysis, total cellular RNA was electrophoretically separated in 2% (w/v)agarose gels containing 5% (w/v) formaldehyde, and blotted onto Hybondnylon membranes (GE Healthcare).

Probes for RFLP analysis were amplified by PCR using primers listed inSupplemental Table S1, followed by agarose gel electrophoresis and purifica-tion of the PCR products from excised gel slices using the NucleoSpin Extract IIkit (Macherey-Nagel). Purified probes were radiolabeled with [a-32P]dCTP byrandom priming using the Multiprime DNA labeling system (GE Healthcare).Antisense oligonucleotides (synthesized by Eurofins Genomics and listed inSupplemental Table S1) were used as probes for northern blot analyses. Forradioactive labeling, 20 pmol of the oligonucleotide were incubated with 10units of T4 polynucleotide kinase (New England Biolabs) and 30 mCi of [g-32P]ATP for 30min at 37°C to allow 59 end labeling. All hybridizations, except thosewith the trnE 59 leader and 39 trailer sequences as probes, were performed at65°C using standard protocols (Church and Gilbert, 1984). For probes derivedfrom trnE 59 leader and 39 trailer sequences, 55°C was used as the hybridization

temperature. Signals were analyzed using a Typhoon Trio1 variable modeimager (GE Healthcare).

Circular RT-PCR

Samples of 8 mg total RNA were electrophoretically separated in 8% (w/v)urea-containing polyacrylamide gels. Bands corresponding to the sizes ofprocessed and precursor tRNAs were cut out and treated with the enzymeRppH (New England Biolabs) to remove the pyrophosphate from the 59 end oftriphosphorylated RNAs. Subsequently, the monophosphorylated RNA mol-ecules were circularized using T4 RNA ligase (New England Biolabs), followedby reverse transcription with primer oSAA105_tRNA-E_R that anneals spe-cifically to the trnE gene sequence (Supplemental Table S1). RT-PCR wasperformed with complementary DNA as template and the primer pairoSAA101_tRNA-E_F/oSAA105_tRNA-E_R, followed by cloning of the am-plification products using the TOPO TA Cloning Kit (Invitrogen). The clonesobtained were analyzed by colony PCR and DNA sequencing.

tRNA Aminoacylation Assays

Aminoacylation of tRNAGlu was analyzed as described previously (Zhouet al., 2013). A specific hybridization probe for tRNAGlu was prepared asdescribed above.

Polysome Analysis

Polysome analyses and puromycin controls were performed essentially asdescribed previously (Barkan, 1998; Rogalski et al., 2008a). RNA was extractedfrom gradient fractions and the RNA pellet was resuspended in 30 mL RNase-free water. Aliquots of 5 mL per fraction were analyzed by northern blotting.

Determination of ALA Synthesis Rate andMeasurement ofChlorophyll Accumulation

ALA synthesis rates were determined according to published protocols(Czarnecki et al., 2011b). Chlorophyll concentrations were measured in frozentissue of defined fresh weight by extraction with 100% (v/v) acetone followingpublished procedures (Porra et al., 1989; Lu et al., 2017).

Protein Isolation and Immunoblot Analyses

Samples of 100mg ground frozen leaf tissue were used for extraction of totalcellular protein using a phenol-based method (Cahoon et al., 1992) with theminor modification that instead of phenyl-methanesulfonyl fluoride, cOmpleteProtease Inhibitor Cocktail (Roche) was added. The protein pellets were dis-solved in 1% (w/v) SDS and the protein concentrationwas determined with theBCA Protein Assay kit (Pierce Instruments). For immunoblot analysis, samplesof 20 mg protein were separated by 10% (w/v) SDS-PAGE and blotted ontopolyvinylidene difluoride membranes (GE Healthcare). Membranes weretreated with blocking buffer (13 Tris-buffered saline, 0.1% [v/v] Tween 20, and1% [w/v] bovine serum albumin) for 1 h and then incubated with a rabbitpolyclonal antiglutamyl-tRNA reductase antibody (Agrisera) for another hour.After incubation with antirabbit secondary antibody (Agrisera), immunode-tection was performed with the ECL Prime system (GE Healthcare). For actindetection, the membranes were stripped using Thermo Scientific Restore Pluswestern blot stripping buffer (Fisher Scientific) according to the manufacturer’sinstructions, followed by treatment with blocking buffer for 1 h, incubation inmouse polyclonal antiactin antibody (Sigma) for 1 h, incubation in antimousesecondary antibody (Sigma), and detection using the ECL Prime system (GEHealthcare).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession number NC_001879.2 (Nicotiana tabacum, completeplastid genome).


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Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Determination of the ends of incompletely pro-cessed trnE transcripts in the Nt-EctMut mutant.

Supplemental Figure S2. Quantification of chlorophyll a and b contents inmature transplastomic plants (12-leaf stage) grown under standardgreenhouse conditions in a diurnal cycle of 16 h light at 25°C and 8 hdarkness at 20°C.

Supplemental Table S1. List of synthetic oligonucleotides used as PCRprimers in this study.

ACKNOWLEDGMENTS

We thank Pierre Endries and Davina Störmer for help with tissue cultureand transformation, Dr. Alexander Hertle for microscopy, the Max-Planck-Institut für Molekulare Pflanzenphysiologie GreenTeam for plant cultivation,and Fabio Moratti and Dr. Reimo Zoschke for helpful discussion.

Received January 6, 2020; accepted January 31, 2020; published February 18,2020.

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the functions of chloroplast glutamyl-trna intrnaglu in plastid translation and tetrapyrrole...

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