Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OFPOR1 Plays a Role in Stabilization ofLight-Dependent Protochlorophyllide Oxidoreductasein Nicotiana benthamiana and ArabidopsisC W

Jae-Yong Lee,a,1 Ho-Seok Lee,a,1 Ji-Young Song,b Young Jun Jung,c Steffen Reinbothe,d Youn-Il Park,b

Sang Yeol Lee,c and Hyun-Sook Paia,2

a Department of Systems Biology, Yonsei University, Seoul 120-749, KoreabDepartment of Biological Science and Graduate School of Analytical Science and Technology, Chungnam National University,Daejeon 305-764, KoreacDivision of Applied Life Sciences, Gyeongsang National University, Jinju 660-701, KoreadBiologie Environnementale et Systémique, Université Joseph Fourier LBFA, BP53F 38041 Grenoble cedex 9 France

Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide(Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we reportthat Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential proteinfor chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domainand three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms inchloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished PORprotein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants underlight conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a resultimpairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore,in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-lightconditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and theubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in theregulation of POR.

INTRODUCTION

Chaperones play essential roles in the regulation of protein ac-tivity and interactions under changing environments. In chloro-plasts of oxygenic photosynthetic organisms, chaperones suchas heat shock proteins, Hsp70, Hsp90, and Hsp60, and pro-teases, including Caseinolytic protease, Filamentous tempera-ture sensitive H, and Degradation of periplasmic proteinparticipate in protein folding/unfolding, translocation, assembly/disassembly of protein complexes, and degradation (Mulo et al.,2008; Nordhues et al., 2010). In addition to these conventionalchaperones and proteases, many chloroplast proteins play spe-cific roles in the assembly of protein complexes and in the main-tenance of functional protein conformations within chloroplasts.For example, LOW PHOTOSYSTEM II ACCUMULATION1 (LPA1)

and Albino (Alb) family proteins are involved in assembly ofthe photosystems in Arabidopsis thaliana and Chlamydomonasreinhardtii, respectively (Göhre et al., 2006; Peng et al., 2006).cpSRP43, a targeting factor in the chloroplast signal-recognitionparticle, functions as a specific molecular chaperone of the light-harvesting chlorophyll a,b– binding protein (CAB) to prevent itsaggregation (Falk and Sinning, 2010). C. reinhardtii Calvin CycleProtein12 (CP12) protects glyceraldehyde-3-phosphate de-hydrogenase (GAPDH) from denaturation and aggregation asa permanent chaperone-like protein in chloroplasts (Erales et al.,2009).In angiosperms, the chlorophyll biosynthetic pathway consists

of a complex series of reactions catalyzed by multiple nuclear-encoded enzymes (Masuda and Fujita, 2008). The reduction ofprotochlorophyllide (Pchlide) to chlorophyllide via the trans addi-tion of hydrogen across the C17-C18 double bond of the D-ring ofPchlide is a key regulatory step in chlorophyll biosynthesis and iscatalyzed by two distinct enzymes, the light-dependent Pchlideoxidoreductase (POR) and the light-independent (dark-operative)Pchlide oxidoreductase (DPOR) (Masuda and Takamiya, 2004;Reinbothe et al., 2010). Both enzymes are widely distributedin oxygenic photosynthetic organisms, but angiosperms haveevolved to have only POR. Due to the strict light dependenceof POR for catalysis, chlorophyll biosynthesis is arrested at the

1 These authors contributed equally to this work.2 Address correspondence to hspai@yonsei.ac.kr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Hyun-Sook Pai (hspai@yonsei.ac.kr).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.113.111096

The Plant Cell, Vol. 25: 3944–3960, October 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

Pchlide stage in dark-grown angiosperm seedlings; instead, thehighly accumulated POR, together with Pchlide, NADPH, lipids,and carotenoids, form a paracrystalline membrane structure,known as the prolamellar body (PLB), within etioplasts (Masudaand Takamiya, 2004; Reinbothe et al., 2010). Illumination trig-gers rapid degradation of POR proteins and breakdown of PLBs,in parallel with chlorophyll synthesis and chloroplast biogenesis(Reinbothe et al., 1995; Masuda and Takamiya, 2004; Reinbotheet al., 2010).

The x-ray crystal structure of POR has not been determinedat present. However, homology modeling suggests that thestructure of POR is similar to those of members of the short-chain alcohol dehydrogenase family with characteristics ofa globular and soluble protein (Heyes and Hunter, 2005; Sytinaet al., 2008). Nevertheless, an important feature of POR as-sembly is its association with plastid membranes: In chloro-plasts, POR is associated with the thylakoid and envelopemembranes as a peripheral protein, whereas in etioplasts, PORforms large aggregates as the PLB (Masuda and Takamiya,2004; Reinbothe et al., 2010). The mechanism of membranebinding of POR despite its lack of predictable transmembraneregions has not yet been revealed. Catalytic mechanisms ofPOR were studied using ultrafast pump-probe absorption dif-ference spectroscopy (Heyes and Hunter, 2005; Sytina et al.,2008). The results indicate that the light-driven activation of PORis critically required before catalysis: The reaction is triggered bylight absorption of the POR-Pchlide complex followed by light-independent steps to produce chlorophyllide.

POR genes are ubiquitously found in oxygenic photosyntheticorganisms, such as cyanobacteria, green algae, mosses, gym-nosperms, and angiosperms, and share a high degree of se-quence similarity (Masuda and Takamiya, 2004). There arevarying numbers of POR genes depending on the species:Among angiosperms, tobacco (Nicotiana tabacum) has two PORgenes, POR1 and POR2 (Masuda et al., 2002), whereas Arabi-dopsis has three POR genes, PORA, PORB, and PORC, whichshow different expression profiles (Frick et al., 2003; Masudaet al., 2003; Masuda and Takamiya, 2004; Paddock et al., 2010).Arabidopsis PORA mRNA and protein are both detected abun-dantly in etiolated seedlings, but their levels dramatically de-crease upon illumination. PORB mRNA and protein are presentin both etiolated seedlings and light-grown plants, whereas themRNA and protein levels of PORC are very low in etiolatedseedlings but are induced by light. During the last two decades,the information known about POR has dramatically increasedwith regard to its in vivo function, catalytic mechanism, locali-zation, evolution, and gene expression pattern. However, it re-mains unknown how POR is protected from photooxidativedamage, considering that POR is a light-activated enzyme(Heyes and Hunter, 2005; Sytina et al., 2008) and that its sub-strate and product are photosensitizers (Reinbothe et al., 1996;Sperling et al., 1997; Buhr et al., 2008).

Cell growth defect factor1 (Cdf1; At5g23040) was identified bya yeast genetic screen for the isolation of Arabidopsis celldeath–related genes (Kawai-Yamada et al., 2005). Cdf1 over-expression resulted in apoptosis-like cell death in yeast, ac-companied by excessive accumulation of reactive oxygenspecies (ROS), although Cdf1 function in planta has not been

characterized (Kawai-Yamada et al., 2005). During a virus-induced gene silencing (VIGS)–based screening of chloroplastdevelopment genes (Cho et al., 2004; Jeon et al., 2012), wefound that silencing of Nicotiana benthamiana Cdf1 causesa significant reduction in POR protein accumulation. Here, wereport that Cdf1, designated herein as chaperone-like protein ofPOR1 (CPP1), plays a crucial role in chlorophyll synthesis andchloroplast biogenesis by regulating the stability and function ofPOR.

RESULTS

Cdf1/CPP1 Is a Membrane Protein with a J-Like Domain

Cdf1/CPP1 from Arabidopsis (At-CPP1) and N. benthamiana(Nb-CPP1) is predicted to have a chloroplast transit peptide,a J-like (JL) domain, and three transmembrane domains (seeSupplemental Figures 1A and 1C online). The J-domain is foundin DnaJ/Hsp40 cochaperones that function as partners of thehighly conserved Hsp70; it is required for both interaction withHsp70 and stimulation of Hsp70 ATPase activity (Qiu et al.,2006; Rajan and D’Silva, 2009). CPP1 homologs are ubiqui-tously present in oxygenic photosynthetic organisms that havePOR, including cyanobacteria, green algae, mosses, gymno-sperms, and angiosperms (see Supplemental Figures 1B and 1Cand Supplemental Data Set 1 online). The JL domains ofNb-CPP1, At-CPP1, and slr1918 (the CPP1 homolog of Syn-echocystis sp PCC6803) are similar in structure to the J-domainof Tid1 (a human mitochondrial homolog of bacterial DnaJ co-chaperones; Lo et al., 2004) based on computational models,albeit with low sequence similarity (see Supplemental Figure 2online). Interestingly, the JL domain of CPP1 lacks a highlyconserved tripeptide of His, Pro, and Asp (the HPD motif) of theJ-domain, which is required for its interaction with Hsp70 (Qiuet al., 2006; Rajan and D’Silva, 2009) (see Supplemental Figures1C and 2 online).

VIGS of Nb-CPP1 Results in Defective ChloroplastBiogenesis and Chlorophyll Synthesis

We examined the in vivo effects of CPP1 deficiency inN. benthamiana and Arabidopsis using VIGS and a dexamethasone(DEX)–inducible RNA interference (RNAi) strategy. Our analysessuggest that a null mutation of CPP1 is embryo lethal in Arabi-dopsis (see Supplemental Figure 3 online). VIGS of Nb-CPP1in N. benthamiana resulted in leaf yellowing, similar to thephenotypes from VIGS-mediated double silencing ofN. benthamiana POR1 (Nb-POR1) and Nb-POR2 (Figures 1Aand 1C). VIGS of either Nb-POR1 or Nb-POR2 alone caused novisible phenotype. Real-time quantitative RT-PCR showed si-lencing of Nb-CPP1 in tobacco rattle virus (TRV):NbCPP1 lines,but not in TRV:NbPOR1/2 lines, compared with the TRV control(Figure 1B). Semiquantitative RT-PCR showed silencing of bothNb-POR1 and Nb-POR2 in TRV:NbPOR1/2 double-silencedlines compared with the TRV control (Figure 1D). Differentialinterference contrast and confocal laser scanning microscopyrevealed that the average number and size of chloroplasts in the

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3945

Figure 1. VIGS of Nb-CPP1 and Nb-POR1/2.

(A) For VIGS of Nb-CPP1, a 726-bp Nb-CPP1 cDNA fragment was cloned into the TRV-based VIGS vector pTV00. To simultaneously silence Nb-POR1and Nb-POR2 using VIGS, a 402-bp Nb-POR1 and a 400-bp Nb-POR2 cDNA fragment were cloned together into pTV00. aa, amino acids.(B) The amount of endogenous Nb-CPP1 mRNA in TRV:NbCPP1 and TRV:NbPOR1/2 VIGS lines was measured by real-time quantitative RT-PCR toconfirm gene silencing. b-Tubulin mRNA levels were used as a control. Data points represent means 6 SD of three experiments. Asterisks denote thestatistical significance of the differences between the samples. **P # 0.01.(C) VIGS of Nb-CPP1 and Nb-POR1/2 both resulted in leaf yellowing phenotypes compared with the TRV control. VIGS of either Nb-POR1 or Nb-POR2alone did not result in any visible effects. The plants were photographed 20 d after infiltration.(D) The amounts of endogenous Nb-POR1 and Nb-POR2mRNAs in the TRV:NbPOR1/2 double-silenced lines were measured by RT-PCR. Actin mRNAlevels were used as a control.

3946 The Plant Cell

yellow sector of TRV:NbCPP1 leaves were reduced to ;31.2and ;66.9% of the TRV control, respectively (Figure 1E; seeSupplemental Figure 4 online). Chlorophyll autofluorescence ofthe TRV:NbCPP1 chloroplasts diminished to ;4.9% of thecontrol chloroplasts (Figure 1E). Total chlorophyll content in theyellow sectors of the TRV:NbCPP1 leaves was also reduced to;15.4% of levels in the TRV control, suggesting defectivechlorophyll synthesis (Figure 1F). Transmission electron microscopy(TEM) of the spongy mesophyll cells in the yellow sectors revealedsignificantly decreased chloroplast numbers and abnormal chloro-plast morphology in Nb-CPP1 VIGS plants (Figure 1G). The af-fected chloroplasts were small and elongated and contained poorlydeveloped thylakoids. Taken together, these results demonstratethat Nb-CPP1 deficiency impairs chlorophyll synthesis and chlo-roplast biogenesis.

Silencing of CPP1 Results in Photobleaching in Arabidopsis

The DEX-inducible At-CPP1 RNAi seedlings were germinatedand grown on medium containing ethanol (2DEX) or 10 mMDEX. Under low-light conditions, (+)DEX RNAi seedlings werephotobleached in the cotyledons, accompanied by excessiveaccumulation of ROS, visualized by nitro blue tetrazolium (NBT)staining (Figures 2A and 2B; see Supplemental Figure 5 online).NBT forms a dark-blue formazan precipitate when in contactwith superoxide radicals (Bielski et al., 1980). Under high-lightconditions, growth of (+)DEX RNAi seedlings was stunted, withexcessive production of anthocyanin pigments in cotyledons(Figure 2A; see Supplemental Figure 5 online). When the At-CPP1RNAi plants were grown on soil and sprayed with DEX, the plantsdeveloped leaf yellowing with growth retardation (Figure 2C).Real-time quantitative RT-PCR revealed silencing of At-CPP1upon DEX spraying (Figure 2D).

CPP1 Is Localized in Chloroplast Membranes

To determine the subcellular localization of CPP1, green fluo-rescent protein (GFP) fusion constructs of Nb-CPP1 and At-CPP1 were transiently expressed in N. benthamiana leavesusing agroinfiltration (Voinnet et al., 2003). Confocal laser scan-ning microscopy using protoplasts isolated from the infiltratedleaves revealed that green fluorescent signals of AtCPP1:GFPwere detected in the chloroplasts, but not in mitochondria, visu-alized by tetramethyl rhodamine methyl ester (TMRM) staining(Figure 3A). In yeast, At-CPP1 (previously named Cdf1) was lo-calized to mitochondria, and its overexpression caused Bax-likelethality (Kawai-Yamada et al., 2005). Green fluorescent signals of

NbCPP1:GFP fusion proteins were detected in chloroplasts, anda GFP fusion protein of Nb-POR1 (NbPOR1:GFP) also showeda similar localization pattern in chloroplasts (Figure 3B). To de-termine the subplastidic localization of CPP1, immunoblottingwas performed with chloroplast fractions purified fromN. benthamiana leaves. Immunoblotting using anti-CPP1 andanti-POR antibodies detected CPP1 and POR proteins ofN. benthamiana, respectively, in both thylakoid and envelopemembrane fractions (Figure 3C). As a control for fractionation, thecorresponding antibodies detected rbcL, Tic40, and D1 in thestromal, envelope, and thylakoid fractions, respectively. Locali-zation of CPP1 in plastid membranes was confirmed by proteo-mics studies in Arabidopsis, maize (Zea mays), and cauliflower(Brassica oleracea ssp botrytis) (Bräutigam and Weber, 2009;Ferro et al., 2010; Huang et al., 2013). POR proteins were pre-viously detected in both stromal thylakoid and envelope mem-branes in chloroplasts of light-adapted plants, similar to the duallocalization of several enzymes catalyzing the final steps ofchlorophyll biosynthesis (Barthélemy et al., 2000; Masuda andFujita, 2008).

CPP1 Interacts with POR Isoforms

To determine whether CPP1 and POR interact, we first usedbimolecular fluorescence complementation (BiFC). CPP1 andPOR isoforms of Arabidopsis and N. benthamiana were ex-pressed in combination as yellow fluorescent protein (YFP)N andYFPC fusion proteins in N. benthamiana leaves by agro-infiltration. Confocal laser scanning microscopy of the meso-phyll cells of the infiltrated leaves (Figure 4A) and theirprotoplasts (see Supplemental Figure 6A online) revealed strongYFP fluorescence within chloroplasts, indicating an interactionbetween CPP1 and POR proteins regardless of the species oforigin. However, BiFC of At-CPP1 with Arabidopsis CHL27 (forMg2+-protoporphyrin IX monomethylester oxidative cyclase),CHLM (for Mg2+-protoporphyrin IX methyl transferase), andGENOMES UNCOUPLED4 (GUN4) and BiFC of Nb-CPP1 withN. benthamiana CHLI2 (for Mg-chelatase subunit I isoform 2) allresulted in no fluorescence, suggesting a lack of interaction(Figure 4A; see Supplemental Figure 6B online). Next, we per-formed coimmunoprecipitation assays (Figure 4B). A fusionprotein between Nb-POR1 and the hemagglutinin tag (NbPOR1:HA) and NbPOR2:HA were transiently expressed using agro-infiltration and then immunoprecipitated from n-dodesyl-b-D-maltoside–solubilized chloroplast membranes using anti-HAantibodies. The immunoprecipitates contained the coexpressedNbCPP1:Flag proteins, suggesting interaction of Nb-CPP1 with

Figure 1. (continued).

(E) Chlorophyll autofluorescence of chloroplasts (pseudo-colored blue) in leaf protoplasts of the TRV control and TRV:NbCPP1 lines was visualized (left)and quantified (right) by confocal laser scanning microscopy. Differential interference contrast (DIC) images are also shown. Yellow/white sectors ofTRV:NbCPP1 leaves were used for the analyses. Data are expressed as mean 6 SD of 30 individual protoplasts.(F) Quantification of total chlorophyll, chlorophyll a, and chlorophyll b in TRV and TRV:NbCPP1 leaves. FW, fresh weight.(G) Light micrographs of leaf sections (a and e) and transmission electron micrographs of leaf mesophyll cells (b and f) and chloroplasts (c, d, g, and h)from the TRV control (a to d) and TRV:NbCPP1 lines (e to h). c, chloroplasts. Bars = 100 mm in (a) and (e), 5 mm in (b) and (f), 1 mm in (c), (d), and (g), and2 mm in (h).

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3947

both Nb-POR1 and Nb-POR2 in vivo (Figure 4B). However,HA fusions of N. benthamiana GUN4 (Nb-GUN4) or CHLI2(Nb-CHLI2) could not be coimmunoprecipitated by NbCPP1:Flag, consistent with the BiFC results (see Supplemental Figure7 online). For in vitro binding assays, purified maltose bindingprotein (MBP)–fused Nb-CPP1 and slr1918 (the CPP1 homolog

of Synechocystis sp PCC6803) were immobilized on MBP resinand incubated with lysates of Escherichia coli cells expressingHis tag–fused Nb-POR1 or Nb-GUN4 proteins (Figure 4C). Afterextensive washing of the resin, bound proteins were eluted andsubjected to immunoblotting with anti-MBP and anti-His anti-bodies. Resin-bound MBP:NbCPP1 and MBP:slr1918, but not

Figure 2. Analyses of Arabidopsis DEX-Inducible At-CPP1 RNAi Lines.

(A) Phenotypes of DEX-inducible At-CPP1 RNAi seedlings. The RNAiseedlings (line #1) were germinated and grown under low light (20 mmolm22 s21) or high light (200 mmol m22 s21) on medium containing ethanol(2DEX) or DEX.(B) NBT staining to visualize superoxide production in the RNAi seed-lings (lines #1 and #4) grown under low light (20 mmol m22 s21). Thecotyledons of the seedlings grown on medium with DEX exhibiteda strong blue color indicating excessive production of superoxide.(C) Leaf yellowing following DEX treatment. RNAi seedlings (line #1) weregrown for 2 weeks on soil and then sprayed with ethanol (2DEX) or30 mM DEX for 10 d. Leaf morphology of the RNAi plants following 10 d ofethanol or DEX spraying is also shown.(D) Real-time quantitative RT-PCR analysis with the RNAi seedlings (line#1) confirmed silencing of At-CPP1 upon spraying with DEX (0, 4, and8 d). Data points represent means 6 SD of three experiments. Asterisksdenote the statistical significance of the differences between the samples.**P # 0.01.

Figure 3. Chloroplast Localization of CPP1.

(A) Chloroplast localization of AtCPP1:GFP. AtCPP1:GFP fusion proteinwas transiently expressed in N. benthamiana leaves by agroinfiltrationand observed by confocal laser scanning microscopy. Mitochondriawere visualized by TMRM staining. Chlorophyll autofluorescence ispseudo-colored blue.(B) Chloroplast localization of NbCPP1:GFP and NbPOR1:GFP. NbCPP1:GFP fusion protein was transiently expressed in N. benthamiana leaves byagroinfiltration and observed by confocal laser scanning microscopy (top).After agroinfiltration of the N. benthamiana POR1 (NbPOR1):GFP con-struct, protoplasts were isolated and observed by confocal microscopy(bottom). Green fluorescent signals of NbCPP1:GFP and NbPOR1:GFPwere detected in the chloroplasts.(C) Immunoblotting showing localization of Nb-CPP1 in chloroplastenvelope and thylakoid membranes. As a control, specific antibodiesdetected rbcL, Tic40, and D1 in the stromal, envelope, and thylakoidfractions, respectively.

3948 The Plant Cell

MBP alone, could pull down NbPOR1:His in vitro, suggesting aninteraction between the CPP1 homologs and POR (Figure 4C).However, MBP:NbCPP1 could not pull down NbGUN4:His(Figure 4C). Collectively, these results support that CPP1 andPOR interact with each other in chloroplast membranes.

CPP1 Deficiency Leads to Reduced Accumulation of PORProteins in Light-Grown Plants

Immunoblotting demonstrated that steady state POR proteinlevels in pale-green sectors of the leaves were significantly re-duced in TRV:NbCPP1 VIGS N. benthamiana plants and TRV:NbPOR1/2 plants (for double silencing of POR1 and POR2)compared with control TRV or TRV:NbGyrB (for DNA gyrasesubunit B) lines, whereas the levels of ATP synthase b-subunit(atpB), CAB, D1, and chloroplast HSP70 (cpHSP70) remainedconstant (Figure 5A). The VIGS control TRV:NbGyrB lines aresilenced for chloroplast-targeted GyrB of N. benthamiana andshow leaf yellowing (Cho et al., 2004). Thus, CPP1 deficiencyresulted in reduced POR protein accumulation in N. benthamiana.In Arabidopsis, silencing of CPP1 by DEX spraying decreasedPOR protein levels in seedlings from four independent DEX-inducible CPP1 RNAi lines compared with control samples (seeSupplemental Figure 8 online). Total POR protein levels and CPP1protein levels progressively decreased in the Arabidopsis RNAiseedlings following 0 to 8 d of DEX spraying, whereas the levels ofother control chloroplast proteins were unchanged (Figure 5C).The transcript levels of Arabidopsis PORB and PORC, which areexpressed under light conditions (Frick et al., 2003; Masuda et al.,2003), as well as the levels of CHLH (for Mg-chelatase subunit H)and CHL27 remained unaffected by CPP1 silencing, whereasCPP1 was silenced by RNAi (Figure 5D). These results sug-gest that CPP1 is required for POR protein accumulation inchloroplasts.We next examined whether CPP1 depletion leads to accu-

mulation of the POR substrate Pchlide caused by POR de-ficiency by measuring fluorescence emission spectrum (Figure5B; see Supplemental Figure 9 online). In N. benthamiana, bothTRV:NbCPP1 and TRV:NbPOR1/2 VIGS leaves accumulatedhigh levels of the POR substrate Pchlide, consistent with PORdeficiency in those lines, while the Pchlide peaks were muchsmaller in the TRV and TRV:NbGyrB control lines (Figure 5B).Pchlide accumulation was also observed in Arabidopsis CPP1RNAi seedlings upon DEX spraying, albeit weakly (see SupplementalFigure 9 online).To determine whether CPP1 is involved in the import of POR,

we performed chloroplast import assays (see SupplementalFigure 10 online). First, NbPOR1:GFP was expressed in leavesof TRV control and TRV:NbCPP1 VIGS N. benthamiana plants byagroinfiltration. Confocal laser scanning microscopy revealedthat NbPOR1:GFP was targeted normally to the chloroplasts inboth TRV and TRV:NbCPP1 lines, suggesting that Nb-CPP1deficiency did not significantly affect the import of Nb-POR1 into

Figure 4. Interactions of CPP1 with POR Isoforms in Chloroplasts.

(A) BiFC-visualized interactions between CPP1 and POR isoforms ofN. benthamiana and Arabidopsis. CHL27 encodes Mg2+-protoporphyrinIX monomethylester oxidative cyclase involved in chlorophyll biosynthesis.YN, YFPN; YC, YFPC.(B) Coimmunoprecipitation of NbCPP1:Flag and NbPOR:HA proteins.After agroinfiltration, the chloroplast membrane fraction was immuno-precipitated (IP) with anti-HA antibodies, and the coimmunoprecipitateswere detected by immunoblotting (IB) using anti-Flag antibodies.

(C) In vitro binding assays showing interactions of MBP:NbCPP1 andMBP:slr1918 with NbPOR1:His, but not with NbGUN4:His. Slr1918 is theCPP1 homolog of Synechocystis sp PCC6803.

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3949

chloroplasts (see Supplemental Figure 10A online). Next, PORC:Flag was expressed in the leaves of TRV and TRV:NbCPP1plants by agroinfiltration. Immunoblotting of the leaf proteinextracts using anti-Flag antibodies detected only the pro-teolytically processed, mature form of PORC, but not its pre-cursor form, in both TRV and TRV:NbCPP1 lines, suggesting

normal import of PORC into chloroplasts (see SupplementalFigure 10B online). Finally, the PORC:Flag construct wastransformed into Arabidopsis protoplasts isolated from the At-CPP1 RNAi plants that were sprayed with ethanol (2DEX) orDEX for 6 d. Immunoblotting of the protoplast protein extractsusing anti-Flag antibodies detected only the mature form of

Figure 5. Reduced POR Stability in CPP1-Silenced Plants.

(A) Immunoblotting of leaf protein extracts from the N. benthamiana VIGS lines. Pale-green sectors were used for the VIGS lines that show leafyellowing.(B) Fluorescence emission spectra showing relative fluorescence of Pchlide (fluorescence emission maximum at 636 nm) in the VIGS plants.(C) Immunoblotting to examine time-dependent regulation of CPP1 and POR protein levels in DEX-inducible At-CPP1 RNAi Arabidopsis seedlings (line#1) after 0 to 8 d of ethanol (2DEX) or DEX spraying. GluTR, glutamyl-tRNA reductase.(D) RT-PCR in At-CPP1 RNAi seedlings (line #1) after 0, 4, or 8 d of DEX spraying. The transcript level of UBC10 was used as a control.(E) Susceptibility of POR proteins to photooxidative stress. Wild-type (WT) N. benthamiana plants grown under normal light conditions (60 mmol m22 s21)were transferred to high-light intensity (500 mmol m22 s21) and incubated for 0 to 72 h. Phenotypes of the plants at 0 (HL-0 h) and 72 h of high-lighttreatment (HL-72 h) are shown. Leaf protein extracts prepared from the high-light-treated plants at various time points were subjected to immuno-blotting using various antibodies. At 72 h of treatment, green (g), pale-green (pg), and yellow leaves (y) were collected for immunoblotting.

3950 The Plant Cell

PORC in both (2)DEX and (+)DEX samples, while the total PORprotein level was reduced (see Supplemental Figure 10C online).These results suggest that chloroplast import of PORC wasunaffected by At-CPP1 silencing. Taken together, these resultssuggest that CPP1 is required for POR stability rather than forimport of POR into chloroplasts.

Consistent with the protective function of CPP1 for POR, wefound that POR proteins were susceptible to photooxidativestress (Figure 5E). Wild-type N. benthamiana plants in the eight-leaf stage grown under normal light conditions (60 mmol m22 s21)were transferred to high-light intensity (500 mmol m22 s21) andincubated for 0 to 72 h. The plants did not exhibit any visiblesymptoms until 48 h of treatment, but after 72 h showed a leafyellowing phenotype, particularly in older leaves (Figure 5E).Immunoblotting revealed that Nb-POR protein accumulationprogressively decreased until 48 h, despite the lack of leaf yel-lowing, while N. benthamiana CPP1, cpHSP70, atpB, and D1protein levels remained constant throughout the period (Figure5E). When immunoblotting was performed with green, pale-green, and yellow leaves collected from the plants at 72 h oftreatment, CPP1, cpHSP70, and atpB protein levels were higherin green leaves than in pale-green or yellow leaves. POR proteinaccumulation was greatly reduced in yellow leaves at 72 h ofhigh-light treatment (Figure 5E). However, we did not observeany increases in POR protein amounts in insoluble fractions,suggesting that POR proteins are degraded before beingaggregated under high-light conditions. Interestingly, werepeatedly observed that D1 proteins disappeared in greenleaves but were present in pale-green and yellow leaves at72 h. This may be due to the fact that green leaves were lo-cated in the upper part of the plants and thus were exposed tohigher light intensity. It has been reported that D1 proteins areeasily degraded under high light conditions (Aro et al., 1993).Taken together, these results suggest that Nb-POR protein issensitive to photooxidative stress.

CPP1 Deficiency Leads to Reduced Accumulation of PORProteins in Etioplasts and Delayed Greeningupon Illumination

To study the effects of CPP1 silencing during transition fromdark to light, At-CPP1 RNAi seedlings were grown in the dark for5 d and then transferred to light for 4 or 24 h on medium with orwithout DEX. When grown on medium with DEX, RNAi seedlingsexhibited delayed greening and anthocyanin accumulation uponillumination (Figure 6A). Real-time quantitative RT-PCR showsthat CPP1 was silenced in the DEX-inducible At-CPP1 RNAiseedlings upon DEX treatment during dark-to-light transition(Figure 6B, bottom). This analysis also showed that the CPP1transcript level increased upon illumination (Figure 6B, bottom),consistent with an increase in CPP1 protein levels upon illumi-nation in wild-type seedlings (Figure 6B, top). Immunoblottingrevealed that illumination caused rapid degradation of PORproteins, accompanied by accumulation of CAB, D1, and rbcL inwild-type, (2)DEX, and (+)DEX RNAi seedlings (Figure 6C; seeSupplemental Figure 11 online), consistent with the previousreports (Masuda and Takamiya, 2004; Reinbothe et al., 2010).Compared with (2)DEX and wild-type seedlings, dark-grown

seedlings treated with DEX had less POR accumulation inetioplasts and delayed accumulation of the photosynthesis-related proteins upon illumination, consistent with the delayedgreening phenotype. Transcription profiles demonstrated noeffect of DEX on expression profiles of PORA, PORB, PORC,CHLH, and CHL27 (Figure 6D). PORA and PORB were down-regulated by light, while PORC, CHLH, and CHL27 were inducedby light.TEM observation revealed that the cotyledons of etiolated (2)

DEX RNAi seedlings displayed normal etioplasts with extensivePLBs and unstacked prothylakoids (Figure 6E). The PLB dis-appeared or shrank after 4 h of illumination, followed by for-mation of grana stacks after 24 h. Etioplasts of DEX-treatedRNAi seedlings had either smaller PLBs or no PLB, and bothphenotypes were simultaneously observed in a single cotyledoncell. Approximately 48.4% of (+)DEX etioplasts lacked a PLB,while only 10% of (2)DEX etioplasts lacked a PLB (seeSupplemental Figure 12A online). Analyses of TEM pictures ofthe cotyledon sections using Image J software demonstratedthat the average ratio of PLB area to etioplast area was 0.47 in(2)DEX and 0.22 in (+)DEX etioplasts, suggesting reduced PLBsize in CPP1-deficient etioplasts (see Supplemental Figure 12Bonline). Furthermore, the thylakoid systems in DEX-treatedetioplasts developed poorly even after 24 h of illuminationand many plastids were degenerating by that time (Figure 6E).Dark-grown Arabidopsis seedlings that have defects in PORaccumulation in etioplasts due to downregulation of POR geneexpression show defective PLB formation (Sperling et al., 1997,1998; Franck et al., 2000). These results suggest that CPP1modulates POR protein accumulation and thereby affects PLBformation in etioplasts. Thus, CPP1 appears to play a crucialrole in regulating POR function during dark-to-light transition aswell as in light-adapted conditions.

CPP1 Can Stabilize POR Proteins with ItsChaperone Activity

We next examined whether CPP1 has chaperone activity forPOR by assessing its ability to prevent heat-induced de-naturation and aggregation of POR proteins in vitro. To useNbPOR1:His as a substrate for chaperone assay, we firstdemonstrated that purified NbPOR1:His and Nb-POR1 proteinswithout a His tag (prepared by treatment with tobacco etch virus[TEV] protease) exhibited a similar pattern of protein aggregationin response to heat, and aggregation of Nb-POR proteins(without tag) was almost completely inhibited by MBP:NbCPP1(see Supplemental Figure 13A online). Addition of purified MBP:NbCPP1 proteins suppressed thermal aggregation of NbPOR1:His in a concentration-dependent manner: A molar ratio of 2:1(chaperone to substrate) completely suppressed aggregation(Figure 7A), whereas addition of MBP, glutathione S-transferase(GST), or MBP:NbRae1 fusion protein as a control had no effect(Figure 7F; see Supplemental Figure 13B online). Nb-Rae1 isa nuclear envelope protein of N. benthamiana. MBP:slr1918 alsoexhibited chaperone activity for NbPOR1:His, albeit weakly(Figure 7B). Nb-CPP1 lacking the JL domain (MBP:NbCPP1DJL)failed to prevent NbPOR1:His aggregation, indicating that the JLdomain is required for chaperone activity (Figure 7C). However,

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3951

Figure 6. Analyses of the DEX-Inducible At-CPP1 RNAi Lines during the Transition from Dark to Light Conditions.

(A) Phenotypes of the seedlings. RNAi seedlings were dark grown or grown under dark conditions and then transferred to light (80 mmol m22 s21) for4 or 24 h on medium with or without DEX.(B) Immunoblotting and real-time quantitative RT-PCR. Immunoblotting was performed with wild-type (WT) seedlings during the transition from dark (D)to light conditions (for 4 and 24 h) (top). At-CPP1 mRNA levels in the RNAi seedlings upon DEX treatment during the dark-to-light transition weremeasured by real-time quantitative RT-PCR and compared with the level in (2)DEX samples grown in the dark (bottom). Asterisks denote the statisticalsignificance of the differences between the samples. **P # 0.01.(C) Immunoblotting during the transition from dark to light conditions (for 4 and 24 h). Due to a short exposure, only light-induced POR degradation wasshown.(D) RT-PCR. At-CPP1 was silenced in DEX-treated RNAi seedlings without affecting the transcript profiles of PORA, PORB, PORC, CHLH, and CHL27.UBC10 was used as a control.(E) TEM of ultrastructural changes of the cotyledon etioplasts during the dark-to-light transition. The arrow indicates degenerating plastids.P, prolamellar bodies. Bars = 1 mm in (a), (c), and (d) and 0.5 mm in (b), (e), (f), (g), and (h).

3952 The Plant Cell

the slr1918 JL domain alone [20(Met)-81(Lys); slr1918-JL] ex-hibited no chaperone activity for NbPOR1:His, suggesting thatother regions of CPP1 are required for the activity (Figure 7G).These regions may play a role in maintaining the proper con-formation of CPP1 for its chaperone activity in vitro. The trans-membrane domain deletion mutants of Nb-CPP1 and slr1918could not be expressed in E. coli. GST-fused N. benthamianachloroplast HSP70 (Schroda et al., 1999; Su and Li, 2010) (GST:

NbcpHSP70) also exhibited chaperone activity for NbPOR1:His(Figure 7D), but there was no significant synergism when MBP:NbCPP1 and GST:NbcpHSP70 were combined in the presenceor absence of ATP (Figure 7E; see Supplemental Figure 13Conline). Thermal aggregation of NbPOR2:His was also sup-pressed by MBP:NbCPP1 (see Supplemental Figures 13D and13E online). Taken together, these results indicate that CPP1has holdase chaperone activity for POR proteins and that both

Figure 7. Chaperone Activity of CPP1 for POR.

Absorbance at 340 nm (A340) was measured at 2-min intervals to quantify turbidity. Thermal aggregation of NbPOR1:His (1.6 mM) was examined for20 min at 43°C with increasing amounts of MBP:NbCPP1 (A), MBP:slr1918 (the CPP1 homolog of Synechocystis sp PCC6803) (B), MBP:NbCPP1ΔJL(C), and GST:NbcpHSP70 (D). In addition, indicated amounts of MBP:NbCPP1 were combined with GST:NbcpHSP70 (0.16 mM) (E). MBP (1.6 mM) orGST (1.6 mM) alone did not show any chaperone activity for NbPOR1:His (F). Thermal aggregation of NbPOR1:His (1.6 mM) was examined for 20 min at43°C with MBP:slr1918 or with different concentrations of slr1918-JL (without tag), which contains only the JL domain (G). Aggregation of NbPOR1:His(1.6 mM) in response to 1 mM H2O2 was examined for 20 min at 30°C with different amounts of MBP:NbCPP1 (H) and MBP:slr1918 (I). Addition of water(DW) instead of 1 mM H2O2 did not result in aggregation of NbPOR1:His.[See online article for color version of this figure.]

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3953

the JL domain and the transmembrane domains are required forfull activity.

We next tested whether CPP1 can prevent POR denaturation/aggregation induced by oxidative stress in vitro. NbPOR1:Hisproteins were aggregated at 30°C upon exposure to 1 mM hy-drogen peroxide (H2O2) but not to water as a control, and additionof purified MBP:NbCPP1 proteins suppressed aggregation ofNbPOR1:His in a concentration-dependent manner (Figure 7H).Addition of MBP:slr1918 also protected NbPOR1:His from H2O2-induced denaturation and aggregation (Figure 7I). Addition of GSTas a control did not affect aggregation of NbPOR1:His in re-sponse to H2O2 (see Supplemental Figure 13F online). In addition,real-time quantitative RT-PCR analysis suggested that Nb-CPP1mRNAs in leaves of N. benthamiana plants (eight-leaf stage) were;5.0- and 1.9-fold more abundant than Nb-POR1 and Nb-POR2mRNAs, respectively (see Supplemental Figure 14 online). Col-lectively, these results support our proposal that CPP1 stabilizesPOR proteins against photooxidative stress in chloroplasts.

Conserved CPP1 Functions in Cyanobacteria

In cyanobacteria, Pchlide reduction in the dark exclusively de-pends on the light-independent DPOR (Muraki et al., 2010), butthe contribution of POR increases with light intensity; under highintensity, Pchlide is exclusively reduced by POR (Fujita et al.,1998; Yamazaki et al., 2006). Cyanobacterial POR also seems tofunction as a scavenger for Pchlide, which can cause photo-oxidative damage upon illumination (Fujita et al., 1998). Syn-echocystis sp PCC6803 has a single CPP1 homolog, slr1918(see Supplemental Figure 1C online). Wild-type and slr1918knockout mutant Synechocystis strains grown under dim light(10 mmol m22 s21) were transferred to three different lightconditions and incubated for 1 to 3 d under photoautotrophicconditions. Under low-light intensity (20 mmol m22 s21), both thewild type and slr1918 mutant strain grew well and synthesizedchlorophyll normally (Figure 8A). However, under higher lightintensity (100 and 150 mmol m22 s21), cell growth and chloro-phyll synthesis were inhibited more strongly in the mutant thanin the wild type. Immunoblotting revealed that the wild type andslr1918 mutant strains showed similar patterns of light-inducedaccumulation of POR proteins under low-light intensity (Figure8B). However, under high-light intensity, much less POR proteinaccumulated in the mutant than in the wild type. Therefore, thePOR-stabilizing function of slr1918 may be required for Pchlidereduction under high-light conditions when DPOR activity issuppressed by high oxygen evolution (Fujita et al., 1998;Masuda and Takamiya, 2004; Yamazaki et al., 2006). By con-trast, protein expression patterns of atpB, GluTR, rbcL, D1,and HSP70 were similar in both strains under different lightconditions.

DISCUSSION

In this study, we identified Cdf1/CPP1 as a critical regulator ofPOR stability in angiosperms and cyanobacteria. Most of thechlorophyll intermediates, including Pchlide, are strong photo-sensitizers that can produce ROS upon photoexcitation, causingoxidative damage and cell death when present in excess (Ledford

and Niyogi, 2005; Li et al., 2009). Photoexcitation of Pchlidemolecules produces singlet oxygen, H2O2, superoxide, and hy-droxyl radical (Meskauskiene et al., 2001; Hideg et al., 2010).Oxidative stresses cause oxidation of amino acid residues, par-ticularly Met and Cys, leading to protein destabilization anddenaturation (Berlett and Stadtman, 1997; Stadtman, 2006). It isknown that the POR enzyme is activated by light and that itssubstrate and product are photosensitizers, but it remains unclearhow POR is protected from photooxidative damage. We dem-onstrated in this study that CPP1 deficiency results in diminishedPOR protein accumulation and defective chlorophyll synthesisand that CPP1 has holdase chaperone activity for POR proteins invitro in response to heat or H2O2. We also demonstrated that PORproteins are indeed susceptible to photooxidative stress in vivo.These data suggest that CPP1 may regulate POR stability inchloroplasts by preventing denaturation and aggregation of PORwith its chaperone activity. In the absence of CPP1, POR may besusceptible to denaturation due to photooxidation, leading to itsdegradation by chloroplast proteases. Depletion of POR may thenresult in accumulation of its substrate Pchlide, the photoexcitationof which causes excessive accumulation of ROS. Being localizedin chloroplast membranes with multiple transmembrane domains,CPP1 may also contribute to anchoring of POR proteins onthe thylakoid and envelope membranes through protein–proteininteractions.

Figure 8. Conserved Function of slr1918, the CPP1 Homolog of Syn-echocystis sp PCC6803, for POR Protein Accumulation and ChlorophyllSynthesis.

(A) The wild-type (wt) and slr1918 knockout mutant (m) strains weregrown under dim light (10 mmol m22 s21) and transferred to light withhigher intensity for 3 d.(B) The wild-type and slr1918 mutant strains were incubated under dif-ferent light intensity for 1 to 3 d before being subjected to immuno-blotting. Equal amounts of proteins were loaded in each lane.

3954 The Plant Cell

There are quite a few examples of protective mechanismsagainst photooxidative stress in chloroplasts. Chloroplast Hsp70chaperone, its partner DnaJ/Hsp40, HSP21, DegP, and Fila-mentous temperature sensitive H proteases, and auxiliaryproteins, such as LPA1/REP27 and TLP18.3, play roles inphotoprotection and repair of photosystem II (Mulo et al., 2008;Nordhues et al., 2010). NADPH-dependent thioredoxin reductaseand 2-Cys peroxiredoxins, which constitute a chloroplast H2O2-scavenging system, are involved in the protection of CHL27(Stenbaek et al., 2008). Interestingly, LIL3, a light-harvesting-likeprotein, interacts with geranylgeranyl reductase (GGR) and isrequired for stable accumulation of GGR for chlorophyll bio-synthesis, suggesting that LIL3 stabilizes GGR in plastidmembranes (Tanaka et al., 2010). LIL3 was also proposed tofunction as a membrane anchor of GGR that lacks predictabletransmembrane regions. In addition, CP12, a small protein pres-ent in the chloroplasts of most photosynthetic organisms, formsa stable complex with GAPDH and functions as its specificchaperone-like protein to prevent aggregation and inactivation ofthe enzyme (Erales et al., 2009). CPP1 may represent anotherexample of a specific protein stabilization mechanism in chloro-plasts. Unlike most chaperones that transiently bind to denaturedproteins, CPP1 appears to form an interaction with the native formof POR isoforms. Thus, similar to the function of CP12 forGAPDH, CPP1 may closely associate with POR and stabilize PORprotein structure using its chaperone activity in ROS-rich envi-ronments. Although we mainly tested the relationship betweenCPP1 and POR enzymes in this study, there is a possibility thatCPP1 has other substrates/interaction partners within chloroplast,which will be a subject of our future study. In addition, we cannotexclude the possibility that CPP1 may be involved in normalmaturation and activation of POR in addition to stabilizing theprotein against oxidative stresses.

Interestingly, CPP1 deficiency resulted in defective greeningof the etiolated seedlings upon illumination in addition to itseffect in light-adapted plants. CPP1 depletion in etiolatedseedlings led to reduced POR protein levels, reduced PLB for-mation, and delayed photomorphogenesis. These phenotypesare reminiscent of the phenotypes of POR-deficient plantsgenerated by various means. When grown under continuous far-red light, Arabidopsis wild-type etiolated seedlings are depletedof POR proteins, accompanied by the loss of PLB and they failto green upon transfer to white light due to photooxidativedamage (Sperling et al., 1997). The Arabidopsis cop1 photo-morphogenetic mutant also lacks PORA and PLB in etiolatedseedlings, and the susceptibility of the mutant to photooxidativedamage can be rescued by overexpression of either PORA orPORB (Sperling et al., 1998). Defective PLB formation is alsoobserved in dark-grown Arabidopsis POR antisense seedlings,in which both PORA and PORB are downregulated (Francket al., 2000). These results demonstrated that the total PORprotein levels closely correlate with PLB formation in etioplastsand the subsequent light-induced chloroplast development. Ourresults show that CPP1 is required for POR accumulation andPLB formation in etioplasts, and its deficiency disrupts biogenesisof thylakoid membranes and chloroplasts upon illumination.Within etioplasts, a ternary complex of POR-NADPH-Pchlide isthe main protein component of PLB that displays a regular lattice-

like membrane structure (Masuda and Takamiya, 2004; Reinbotheet al., 2010). The proteomics during the transition of etioplasts tochloroplasts shows that a battery of proteins involved in anti-oxidation and detoxification, such as ascorbate peroxidases,glutathione reductase, and PsbS, are highly accumulated inetioplasts, suggesting the importance of defense against oxida-tive stress in etioplasts (Kanervo et al., 2008). Currently, it remainsunclear how CPP1 contributes to POR accumulation in etioplasts.There is a possibility that CPP1 plays a role in POR protection inetioplasts from oxidative stress, similar to its role in light-adaptedplants. Alternatively, CPP1 may play a role in the assembly and/orstability of the POR complex in etioplasts. Further study will berequired to determine how CPP1 modulates POR accumulation inetioplasts.POR catalyzes sequential hydride and proton transfers in the

photoexcited and ground states, respectively (Menon et al.,2009; Heyes et al., 2011). The conserved Tyr and Lys residues inthe active site of POR are involved in the formation of ternaryenzyme-substrate complexes. Furthermore, the Tyr residuestabilizes the Pchlide excited state, promoting hydride transferfrom NADPH to Pchlide, and participates in proton transfer(Menon et al., 2009). It has been generally thought that PORevolved from cyanobacteria, and the evolution of POR reflectsthe transformation from anoxygenic to oxygenic photosynthesis(Reinbothe et al., 1996, 2010; Masuda and Takamiya, 2004).When the catalytic mechanism was compared between cyano-bacterial and plant POR, the light-driven hydride transfer stepwas found to be conserved among all POR enzymes, but theproton transfer step is variable, suggesting that functional ad-aptation and optimization occurred during the evolution of POR(Heyes et al., 2011). CPP1 homologs are ubiquitously foundin oxygenic photosynthetic organisms that possess POR,including cyanobacteria, green algae, mosses, gymnosperms,and angiosperms. These CPP homologs possess the conservedprotein structure that consists of a JL domain and multipletransmembrane domains. We demonstrated that the cyano-bacterial CPP1 homolog (slr1918) critically regulates POR sta-bility and chlorophyll synthesis in Synechocystis under high-lightconditions when oxygen-sensitive DPOR activity is repressed.The results suggest that the chaperone-like function of CPP1 isrequired regardless of the differences in catalytic structure be-tween cyanobacterial and eukaryotic POR. The conserved CPP1function in cyanobacteria and the ubiquitous presence of CPP1in oxygenic photosynthetic organisms suggest the essential roleof CPP1 for POR function in chlorophyll synthesis.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana (ecotype Columbia-0) and Nicotiana benthamianaplants were grown in a growth room at 23°C under a 16-h-light/8-h-darkcycle. The T-DNA insertion mutant of Arabidopsis CPP1 (GK-852F12-025757) was obtained from GABI-Kat (www.gabi-kat.de/).

Cyanobacterial Strains and Culture Conditions

The motile Synechocystis sp PCC 6803 wild-type strain and the slr1918knockout mutant strain were grown on 1% BlueGreen-11 agar plates

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3955

buffered with 10 mM TES-potassium hydroxide (KOH), pH 8.0, at 28°Cunder continuous white light (10 mmol m22 s21) as described (Songet al., 2011), before exposure to increased irradiance (20, 100, and150 mmol m22 s21) for 3 d. Chloramphenicol (20 mg mL21) was addedto the growth medium for the slr1918 mutant that contains a geneconferring chloramphenicol resistance.

Phylogenetic Analysis

The CPP1 homologous sequences from N. benthamiana, grape (Vitisvinifera), soybean (Glycine max), Populus trichocarpa, Arabidopsis, rice(Oryza sativa), maize (Zea mays), Physcomitrella patens, wheat (Triticumaestivum), Synechocystis sp PCC 6803, Chlamydomonas reinhardtii, andPicea sitchensiswere aligned using the ClustalW program (http://www.ch.embnet.org/software/ClustalW.html). Mega phylogenetic trees for thesedata were analyzed using the MEGA 5.2.2 program (http://www.megasoftware.net/). The maximum likelihood tree was generated withthe following parameters: Poisson model, uniform rates, completedeletion, and bootstrap (2000 replicates).

Gene Cloning

The initial VIGS screening was performed with a partial Nb-CPP1 cDNA of726 bp. To obtain the full-length Nb-CPP1 cDNA, 59-rapid amplification ofcDNA ends (RACE) PCR was performed with mRNAs isolated fromN. benthamiana seedlings using the SMART RACE cDNA amplification kit(BD Biosciences) according to the manufacturer’s manual. The gene-specific primers used for 59-RACE PCR were NbCPP1-Gsp(1)/(2). Lists ofprimers and accession numbers of the genes used in this study are shownin Supplemental Tables 1 and 2 online, respectively. Information onvarious constructs and cloning methods is available in SupplementalTable 3 online.

VIGS in N. benthamiana

A 726-bpNb-CPP1 cDNA fragment was amplified by PCR and cloned intothe pTV00 vector containing part of the TRV genome, using the BamHIand ApaI sites. The Nb-POR1 and Nb-POR2 cDNA fragments wereamplified by PCR with NbPOR1-VIGS(F)/(R) and NbPOR2-VIGS(F)/(R)primers, respectively. To achieve cosilencing of Nb-POR1 and Nb-POR2,the PCR product of Nb-POR1 (402 bp) was first cloned into pTV00 vectorusing the BamHI and ApaI sites, followed by the cloning of the Nb-POR2fragment (400 bp) into the same plasmid using BamHI and SpeI sites.VIGS was performed as described (Cho et al., 2004; Ahn et al., 2011; Jeonet al., 2012). All primers used for cloning are listed in Supplemental Table 1online.

Generation of Arabidopsis DEX-Inducible At-CPP1 RNAi Lines

A 400-bp At-CPP1 cDNA fragment was amplified by PCR using AtCPP1-sense(F)/(R) primers containing XhoI andClaI sites for the sense constructand AtCPP1-antisense(F)/(R) primers containing SpeI and EcoRI sites forthe antisense construct. Using these constructs, DEX-inducible At-CPP1RNAi transgenic Arabidopsis lines were generated as described (Ahnet al., 2011). For induction of RNAi, the transgenic seedlings were grownon medium containing 10 mM DEX in ethanol (0.033%). Alternatively, theRNAi seedlings were sprayed with 30 mM DEX in ethanol (0.033%) andTween 20 (0.01% [w/v]).

Agrobacterium tumefaciens–Mediated Transient Expression

Agroinfiltration was performed as described (Voinnet et al., 2003).Agrobacterium C58C1 cultures containing expression constructs wereadjusted to OD600 = 0.6 in MES buffer (10 mM MES, pH 7.5, and 10 mM

MgSO4). The suspension was incubated with acetosyringone for 2 to3 h at a final concentration of 150 mM and infiltrated into leaves ofN. benthamiana plants. In all infiltration experiments,AgrobacteriumC58C1carrying the 35S-p19 construct was coinfiltrated to achievemaximum levelsof protein expression as described (Voinnet et al., 2003). Expressed proteinswere analyzed 24 to 48 h after infiltration.

RT-PCR Analyses

RT-PCR was performed with total RNA isolated from leaves of VIGS andRNAi lines with 15 to 35 cycles of amplification as described (Ahn et al.,2011). To measure the transcript levels in N. benthamiana, the followingprimers were used: CPP1, NbCPP1(F)/(R); POR1, NbPOR1(F)/(R); POR2,NbPOR2(F)/(R); and actin, Nbactin(F)/(R). To measure the transcript levelsin Arabidopsis, the following primers were used: CPP1, AtCPP1(F)/(R);PORA, PORA(F)/(R); PORB, PORB(F)/(R); PORC, PORC(F)/(R); CHLH,CHLH(F)/(R); CHL27, CHL27(F)/(R); and UBC10, UBC10(F)/(R).

Real-Time Quantitative RT-PCR

Real-time quantitative PCR was performed as described (Ahn et al., 2011)using the following primers: Nb-CPP1, NbCPP1-qRT-PCR(F)/(R); Nb-b-tubulin, b-tubulin-qRT-PCR(F)/(R); At-CPP1, AtCPP1-qRT-PCR(F)/(R);and At-UBC10, UBC10-qRT-PCR(F)/(R).

Analyses of Chlorophyll Precursors

Chlorophyll precursors were extracted from N. benthamiana VIGS lines asdescribed (Terry and Kendrick, 1999). Spectrofluorometry was performedusing a fluorescence spectrophotometer (Hitachi F-2000) at an excitationwavelength of 440 nm and an emission wavelength of 600 to 700 nm asdescribed (Terry and Kendrick, 1999).

Measurement of Chlorophyll Contents

Yellow sectors of the fourth leaf above the infiltrated leaf were collectedfrom the VIGS plants. Chlorophyll concentration per unit fresh weight wascalculated as previously described (Pattanayak et al., 2005).

TEM

Tissue sectioning and TEM were performed as described (Jeon et al.,2012), using the fourth or fifth leaf above the infiltrated leaf of theN. benthamiana VIGS lines 20 d after infiltration and the cotyledons of theetiolated Arabidopsis At-CPP1 RNAi seedlings grown on medium with orwithout DEX for 5 d.

NBT Staining

To detect the production of superoxide radicals in situ, NBT staining wasperformed as described (Bielski et al., 1980; Wohlgemuth et al., 2002).

Preparation of Chloroplast Fractions

From chloroplasts isolated from N. benthamiana leaves, stroma, enve-lope, and thylakoid membranes were fractionated as described (Kwonand Cho, 2008).

Subcellular Localization and BiFC

cDNAs of Nb-CPP1, Nb-POR1, and At-CPP1 corresponding to the full-length protein were cloned into the pCambia-GFP plasmid to generateGFP fusion proteins. EcoRI and NcoI sites were used for Nb-CPP1 andNb-POR1 cDNAs, and BamHI and BglII sites were used for the At-CPP1

3956 The Plant Cell

cDNA. The recombinant plasmids or the control vector were introducedinto N. benthamiana leaves by agroinfiltration. Expression of the GFPfusion proteins was monitored 24 h after transformation using a confocallaser scanning microscope (Zeiss LSM510) as previously described (Choet al., 2004). For visualization of mitochondria, TMRM staining wasperformed, and TMRM fluorescence was detected by confocal laserscanningmicroscopy as described (Cho et al., 2004). For BiFC, the codingregions of Nb-CPP1 and At-CPP1 were amplified by PCR and cloned intothe pSPYNE vector containing the N-terminal region (amino acid residues1 to 155) of the YFP, resulting in pSPYNE-NbCPP1 and pSPYNE-AtCPP1,respectively. N. benthamiana POR1, POR2, PORA, PORB, and PORCcDNAs were cloned into pSPYCE vector containing the C-terminal regionof YFP (residues 156 to 239). Agrobacteria containing the pSPYNE andpSPYCE fusion constructs were agroinfiltrated together into leaves of3-week-oldN. benthamiana plants as described (Walter et al., 2004). After48 h, leaf discs or protoplasts were generated from the leaves, and theYFP signal was detected by confocal laser scanning microscopy andfluorescent microscopy.

Coimmunoprecipitation

NbCPP1:Flag and NbPOR1:HA or NbCPP1:Flag and NbPOR2:HA fusionproteins were coexpressed inN. benthamiana leaves by agroinfiltration. Inaddition, NbCPP1:Flag and NbGUN4:HA or NbCPP1:Flag and NbCHLI2:HA fusion proteins were coexpressed by agroinfiltration as a control. After48 h of incubation, chloroplast membranes were prepared from the leavesand solubilized using the solubilization buffer containing n-dodesyl b-D-maltoside as described (Sun et al., 2010). The solubilized membranefraction was incubated with 2 mg of anti-HA antibody (Applied BiologicalMaterials) for 4 h at 4°C, followed by addition of 10 mL of ProteinA-Sepharose beads (Fast Flow; Amersham Pharmacia) for a further 3-hincubation. After washing, the sepharose beads were resuspended in 23SDS sample buffer and boiled, and the resin was precipitated by briefcentrifugation. The supernatant was subjected to SDS-PAGE and immu-noblotting using the monoclonal anti-Flag antibody (1:10,000; Sigma-Aldrich)and monoclonal anti-HA antibody (1:5000; Applied Biological Materials).

Purification of Recombinant Proteins

To purify MBP:NbCPP1 and MBP:NbCPP1DJL, the Nb-CPP1 cDNAfragments corresponding to the amino acid residues 61(Trp)-252(Lys) and112(Lys)-252(Lys), respectively, were amplified by PCR and cloned intopMAL c2 vector (New England Biolabs). To purify MBP:slr1918, the full-length coding region of the slr1918 gene was cloned into pMAL c2 vector.To purify GST:NbcpHSP70, the Nb-cpHSP70 cDNA fragment corre-sponding to the amino acid residues 62(Pro)-707(Lys) was cloned intopGEX-4T-1 vector (Amersham Pharmacia). The MBP and GST fusionproteins were purified following the manufacturer’s instructions usingMBP Excellose resin and Glutathione Excellose resin, respectively (Bio-progen). To purify NbPOR1:His and NbPOR2:His, the Nb-POR1 and Nb-POR2 cDNA fragments corresponding to the amino acid residues 63(Ala)-396(Leu) and 66(Ile)-399(Ala), respectively, were amplified by PCR and clonedinto the pET-29a vector (Novagen). The 63His fusion proteinswere purifiedusing Nickel-nitrilotriacetic acid agarose (Qiagen) following the manu-facturer’s instructions. To purify Nb-POR1 without epitope tag, the Nb-POR1 cDNA fragment described abovewas cloned into amodifiedpET-28avector carrying six N-terminal His residues and a TEV cleavage site (Koet al., 2008). After purification of the protein using Nickel-nitrilotriacetic acidagarose (Qiagen), His tag was removed by TEV protease as described (Koet al., 2008). To purify slr1918-JL without tag, the slr1918 gene fragmentcorresponding to the amino acid residues 20(Met)-81(Lys) was cloned intothe modified pET-28a vector, and protein purification and His tag removalwere performed as described above.

In Vitro Binding Assay

MBP, MBP:NbCPP1, and MBP:slr1918 proteins immobilized on MBPExcellose resin (Takara) were incubated with Escherichia coli lysatesexpressing NbPOR1:His or NbGUN4:His in buffer (50 mMHEPES, pH 7.5,200 mM NaCl, and 10% glycerol) for 2 h at room temperature. Afterextensive washing of the resin, bound proteins were eluted with SDSsample buffer, and 25% of the eluted proteins were subjected toimmunoblotting.

Immunoblot Analyses

Protein extracts (30 µg) were subjected to SDS-PAGE and immuno-blotting as described (Ahn et al., 2011; Jeon et al., 2012). Anti-CPP1antibodies were generated in rabbits against an oligopeptide, ASEEEI-WASRNFLL, that corresponds to amino acid residues of At-CPP1 atpositions 79 to 92 using the antibody production services of Cosmo-genetech. Immunoblotting was performed using mouse monoclonalantibodies against the HA tag (1:10,000 dilution; Applied BiologicalMaterials), the His tag (1:5000 dilution; Applied Biological Materials), andthe Flag tag (1:10,000; Sigma-Aldrich), using rabbit polyclonal antibodiesagainst CPP1 (1:2000; Cosmogenetech), using rabbit polyclonal anti-bodies against POR, rbcL, D1, atpB, psaA, cpHsp70, and GluTR (1:5000,1:10,000, 1:5000, 1:10,000, 1:5000, 1:10,000, and 1:5000 dilution, re-spectively; Agrisera), or using goat polyclonal antibodies against CAB(1:5000; Santa Cruz Biotechnology). The membranes were then treatedwith horseradish peroxidase–conjugated goat anti-mouse IgG antibodies(1:10,000; Invitrogen), goat anti-rabbit antibodies (1:10,000; Invitrogen), ordonkey anti-goat antibodies (1:10,000; Santa Cruz Biotechnology), re-spectively. Signals were detected on x-ray film (Kodak) using an ECLchemiluminescence kit (ELPIS-Biotech).

POR Import into Chloroplasts

POR import into chloroplasts was analyzed as described (Lee et al., 2008).Flag-tagged PORC proteins were transiently expressed in leaves of TRVcontrol and TRV:NbCPP1 VIGS lines. After 16 h of incubation, totalproteins were extracted from the leaves and subjected to SDS-PAGE andimmunoblotting with anti-Flag antibody (1:10,000; Sigma-Aldrich) todetect expressed PORC proteins. In vitro transcription and translation ofthe precursor form of PORC was performed using a wheat germ extractsystem (Promega) according to the manufacturer’s instructions.

Measurement of Chaperone Activity

NbPOR1:His (1.6 mM) was incubated in 50 mM HEPES-KOH, pH 8.0,buffer at 43°C with various concentrations of MBP:NbCPP1, MBP:NbCPP1ΔJL, MBP:slr1918, slr1918ΔTM, or GST:NbcpHSP70. In addi-tion, NbPOR1:His (1.6 mM) was incubated with both MBP:NbCPP1 (atvarious concentrations) and GST:NbcpHSP70 (0.16 mM) to observe anysynergistic effects on chaperone activity. NbPOR2:His (1.6 mM) was in-cubated in 50 mM HEPES-KOH, pH 8.0, buffer at 45°C with variousconcentrations of MBP:NbCPP1. Aggregation of NbPOR1:His (1.6 mM) inresponse to 1 mM H2O2 or water was examined for 20 min at 30°C withdifferent amounts of MBP:NbCPP1 and MBP:slr1918. Aggregation ofNbPOR1:His or NbPOR2:His induced by heat or H2O2 was determined bymonitoring the turbidity increase (A340) at 2-min intervals using a temperature-controlled spectrophotometer (DU800; Beckman) as described (Parket al., 2009).

Generation of the slr1918 Knockout Mutant Strain of Cyanobacteria

DNA constructs for generating the slr1918 knockout mutant were pre-pared using a fusion PCR cloning strategy (Wang et al., 2002). Briefly,

CPP1 Regulates Protochlorophyllide Oxidoreductase Stability 3957

fragment A including the slr1918 59-upstream flanking region and theslr1918 start codon was amplified by PCR with slr1918-fA(F)/(R) primersusing genomic DNA as template. Fragment B, including the slr1918 stopcodon and the 39-downstream region, was amplified by PCR usingslr1918-fB(F)/(R) primers. The overlapping ends of fragment A, thechloramphenicol resistance cassette, and fragment B were annealed at55°C for three-piece PCR fusion as described (Lee et al., 2007). Theamplified DNA fragment was then ligated into the blunt-end cloningvector TOP Cloner (Enzynomics). The recombinant plasmid was trans-formed into Synechocystis wild-type strains as described previously (Leeet al., 2007).

Statistical Analyses

Two-tailed Student’s t tests were performed using theMinitab 16 programto determine the statistical differences between the samples.

Accession Numbers

Accession numbers for the genes used in this study are shown inSupplemental Table 2 online.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Protein Structure, Phylogenetic Tree, andSequence Alignment of CPP1.

Supplemental Figure 2. Computational Modeling of the CPP1 J-LikeDomain.

Supplemental Figure 3. An Embryo-Lethal Phenotype of the Arabi-dopsis cpp1 Null Mutant.

Supplemental Figure 4. Chloroplast Defects in TRV:NbCPP1 VIGSLines.

Supplemental Figure 5. Light Sensitivity of the DEX-InducibleAt-CPP1 RNAi Seedlings.

Supplemental Figure 6. Bimolecular Fluorescence ComplementationAnalyses.

Supplemental Figure 7. Control Coimmunoprecipitation Experiments.

Supplemental Figure 8. Stability of POR in Different Arabidopsis DEX-Inducible At-CPP1 RNAi Lines.

Supplemental Figure 9. Fluorescence Emission Spectrum ShowingRelative Fluorescence of Pchlide (Fluorescence Emission Maximum at636 nm).

Supplemental Figure 10. Analyses of POR Import into Chloroplasts.

Supplemental Figure 11. POR Protein Levels during Dark-to-LightTransition in the Wild-Type Arabidopsis Plants.

Supplemental Figure 12. Quantification of Prolamellar Bodies inEtioplasts of DEX-Inducible At-CPP1 RNAi Seedlings.

Supplemental Figure 13. Control Experiments for Chaperone Assay.

Supplemental Figure 14. Relative mRNA Levels of Nb-CPP1, Nb-POR1,and Nb-POR2.

Supplemental Table 1. List of Primers Used in This Study.

Supplemental Table 2. Accession Numbers.

Supplemental Table 3. Constructs.

Supplemental Data Set 1. Sequence Alignments Used for thePhylogenetic Analysis Shown in Supplemental Figure 1B.

ACKNOWLEDGMENTS

We thank Teh-hui Kao (Penn State University) and Chanhong Kim (BoyceThompson Institute) for helpful discussions. We also thank Hee-KyungAhn (Yonsei University, Korea) for article editing and Hyun-Soo Cho andKuklae Kim (Yonsei University, Korea) for slr1918-JL proteins. Thisresearch was supported by the the Next-Generation BioGreen 21Program (Plant Molecular Breeding Center [No. PJ009079] and SystemsSynthetic Agrobiotech Center [No. PJ008214]; Rural Development &Administration) of Korea.

AUTHOR CONTRIBUTIONS

J.-Y.L. and H.-S.L. performed most of the experiments and analyzed theresults. Y.J.J. and S.Y.L. helped to set up the chaperone assay. J.-Y.S. andY.-I.P. generated the slr1918 knockout mutant strain of Synechocystis spPCC6803. Y.-I.P., S.R., and S.Y.L. discussed the results and commentedon the article. H.-S.P. designed the experiments and wrote the article.

Received February 27, 2013; revised September 11, 2013; acceptedSeptember 30, 2013; published October 22, 2013.

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