Molecular Plant 7, 1586–1590, October 2014
LETTER TO THE EDITOR
Ferredoxin:Thioredoxin Reductase Is Required for Proper Chloroplast Development and Is Involved in the Regulation of Plastid Gene Expression in Arabidopsis thaliana Dear Editor, In oxygenic photosynthetic organisms, carbon metabolism is regulated in a light-dependent manner by a distinctive ferredoxin/thioredoxin (Fd/TRX) redox system, in which Fd:TRX reductase (FTR) acts as the central redox transmitter to catalyze the reduction of TRX, with photosynthetic electrons coming from Fd (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). Recently, there is increasing evidence to suggest the diverse functions of TRX in chloroplast development, which requires lightinduced transcription and translation of nuclear and plastid genes, biosynthesis of lipids and pigments, assembly of photosynthetic pigment–protein complexes, etc. (Pogson and Albrecht, 2011). F- and m-type TRXs (TRX m and TRX f) in pea, and NADPH-dependent thioredoxin reductase C (NTRC) in Arabidopsis, participate in the redox control of multiple enzymes in chlorophyll biosynthesis (Luo et al., 2012; Richter et al., 2013). The recently identified z-type TRX (TRX z) plays an essential role in the redox regulation of PEP (plastid-encoded RNA polymerase)-dependent plastid gene expression (Arsova et al., 2010; Schröter et al., 2010). Three TRX m isoforms in Arabidopsis could modulate the redox status of the intermolecular disulfide bonds in photosystem II (PSII) inner antennal protein CP47 to assists the assembly of CP47 into PSII (Wang et al., 2013). However, the physiological significance of FTR in chloroplast development remains to be addressed. Biochemical characteristics and regulatory functions of FTR have been widely investigated in various organisms (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). However, genetic approaches to characterize FTR in vivo have rarely been successful, most likely due to lethality in the FTR knockout mutants or to functional redundancy within the Fd/TRX redox system. FTR is a heterodimer with a variable subunit (FTRv) and a catalytic subunit (FTRc). There are two FTRv isoforms in Arabidopsis, and the single FTRv mutant plants appear normal until exposure to oxidative stress, implying a functional complementation between the two FTRv isoforms (Keryer et al., 2004). In contrast, the Arabidopsis genome has a unique FTRc subunit that catalyzes the reduction of the conserved dithiol group in TRX (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). To avoid the lethality of FTR knockout
mutants and to address the physiological function of FTR in vivo, we used the virus-induced gene silencing (VIGS) system to silence the FTRc gene in Arabidopsis (Burch-Smith et al., 2006). Three weeks after infection, the inner young leaves of VIGS–FTRc plants exhibited a sectored chlorotic leaf phenotype (Figure 1A and 1B). The sectors near the petioles of young leaves in VIGS–FTRc plants appeared conspicuously chlorotic, and we called the yellow parts of VIGS–FTRc leaves VIGS–FTRc-Y (Figure 1A and 1B). Interestingly, the VIGS–FTRc-Y leaves gradually turned green as the plants matured; thus, the distal sectors away from the petioles in young leaves and adult leaves in VIGS–FTRc plants (called the green parts of VIGS–FTRc leaves, VIGS–FTRc-G) were not different from leaves in the control VIGS–GFP plants (Figure 1A and 1B). Immunoblot analyses showed FTRc proteins were undetectable in both the VIGS–FTRc-G and VIGS– FTRc-Y leaves (Figure 1C), indicating that the FTRc gene is efficiently silenced in both VIGS–FTRc-G and VIGS–FTRc-Y leaves. Moreover, a severely reduced level of FTRc in VIGS– FTRc plants was reflected in photosynthetic performance. The photochemical activities of two photosystems and CO2 assimilation rates were significantly reduced in VIGS–FTRc plants when compared with VIGS–GFP plants (Figure 1E and 1F, and Supplemental Table 1). Consistently, the obviously higher PSII excitation pressure (1-qP) and non-photochemical quenching (NPQ) were observed in VIGS–FTRc plants than that in VIGS–GFP plants (Figure 1F and 1G). Apart from its role in the regulation of carbon fixation, the Fd/TRX redox system is also involved in antioxidant defense in chloroplast (Dietz and Pfannschmidt, 2011). We wondered whether the delayed-greening leaf phenotype in FTRc-silenced plants could be attributed to an imbalance of ROS levels. Results of histochemical staining and relative expression of antioxidant genes indicated an enhanced radical-scavenging capacity in the FTRc-silenced plants (Supplemental Figure 1).
© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu069, Advance Access publication 2 June 2014 Received 4 April 2014; accepted 26 May 2014
Molecular Plant
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Figure 1 FTR Is Required for PEP-Dependent Plastid Gene Expression at the Early Stages of Chloroplast Development. (A) Identification and characterization of VIGS–FTRc Arabidopsis plants. Phenotypes of VIGS–GFP (a negative control for the effect of the virus infection) and VIGS–FTRc plants. Plants were observed 3 weeks after infiltration. Three biological replicates were performed, and similar results were obtained. (B) Representative leaves from VIGS plants. (C) Immunodetection of FTRc in VIGS–FTRc plants. Aliquots of 50 μg (one-half VIGS–GFP), 100 μg (VIGS–GFP), and 100 μg (VIGS–FTRc) of total leaf proteins were loaded on the gels. A CBB (Coomassie brilliant blue)-stained gel of the samples is shown below to provide an estimate of gel loading. Three biological replicates were performed, and similar results were obtained. (D–G) Photosynthetic electron transfer properties of VIGS–FTRc Arabidopsis plants. Chlorophyll fluorescence parameters were examined in VIGS–FTRc plants under different actinic light intensities, including the quantum yield of PSI [D, Y(I)], quantum yield of PSII [E, Y(I)], excitation pressure of PSII (F, 1-qP), and non-photochemical quenching (G, NPQ). The data represent means ± SD of three biological replicates. (H) Transmission electron microscope images of chloroplast ultrastructures in VIGS–GFP (upper panel), VIGS–FTRc-G (middle panel), and VIGS–FTRc (lower panel); leaves were taken 3 weeks after infiltration. The images in the left panels show light micrographs of whole chloroplasts, which were observed further by electron microscopy in the magnified images in the right panels. Scale bars are indicated. Two biological replicates were performed, and similar results were obtained.
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Letter to the Editor
Taken together, these results indicate that FTRc is required for leaf greening, particularly in young leaves. To assess whether the silencing of FTRc causes ultrastructural changes in chloroplasts, chloroplast morphology in the VIGS–FTRc leaves was observed by transmission electron microscopy. In comparison with chloroplasts from VIGS–GFP leaves and VIGS–FTRc-G leaves, chloroplasts from VIGS–FTRc-Y leaves were not fully developed, and the stroma contained only rudimentary thylakoid lamellae (Figure 1H). This observation could be supported by the organization of thylakoid protein complexes in these thylakoids. The organization of PSI (photosystem I) and PSII complexes in VIGS–FTRc-Y thylakoids was drastically disrupted, in that almost no PSII supercomplexes, PSII monomers, and PSI monomers were detected in VIGS–FTRc-Y thylakoids (Figure 1I). Moreover, the relative amounts of photosystem subunits in these photosystem complexes were greatly reduced in VIGS–FTRc-Y thylakoids (Figure 1J– 1L). Collectively, these results suggest that FTR probably participates in the regulation of chloroplast development. Due to their endosymbiotic origin, plastids possess their own genome, the plastome, which encodes the complete machinery for transcription, RNA metabolism, translation, etc. (Pfalz and Pfannschmidt, 2013). Although the plastomes of higher plants contain only about 100–120 genes, intricate regulation of plastid gene expression is a prerequisite for proper chloroplast development (Pfalz and Pfannschmidt, 2013). The plastid transcription-regulation system relies on both PEP and NEP (the nuclearencoded plastid RNA polymerase; Pfalz and Pfannschmidt, 2013). During the early stages of chloroplast development, proper transcription of photosynthesis-related genes is dependent on PEP activity (Pfalz and Pfannschmidt, 2013).
Molecular Plant Various mutants or transgenic plants with defects in PEP activity, such as virescent or delayed-greening mutants, exhibit VIGS–FTRc-like sectored leaf chlorosis (Pfalz and Pfannschmidt, 2013). However, FTR is not known as a DNA binding protein, and not to be identified as the intrinsic subunits of PEP complex (Schröter et al., 2010); it appears probably that FTR indirectly participates in the regulation of PEP-dependent gene expression by reducing TRX. Global proteomic analyses demonstrated that TRX z and its potential target proteins, two fructokinase-like proteins (FLN1 and FLN2), are intrinsic subunits of the PEP complex (Schröter et al., 2010). Moreover, trx z mutant and FLNssilenced plants exhibit drastically decreased PEP-dependent plastid gene expression (Arsova et al., 2010). The observations that partial FTRc proteins could interact with TRX z proteins (Supplemental Figure 2), but also to associate with plastid nucleoids, in which plastid gene expression takes place (Figure 1M), support the hypothesis that FTR probably participate in the regulation of PEP activity. To further confirm the potential role of FTR in the regulation of plastid gene expression, the transcription of plastid-encoded genes in the VIGS–FTRc plants was analyzed by quantitative real-time RT–PCR. Intriguingly, the expression pattern of plastid genes in VIGS–FTRc-Y leaves resembles that of the ∆rpo mutant and other mutants that lack a functional PEP (Pfalz and Pfannschmidt, 2013); all of these show a down-regulation of class I plastid genes transcribed by PEP, whereas the expression of class II genes transcribed by both PEP and NEP, and class III genes transcribed exclusively by NEP is up-regulated (Figure 1N). To further validate impaired PEP-dependent plastid gene expression in FTRc-silenced plants, the steady-state levels of thylakoid membrane proteins were analyzed in the
(I) BN–PAGE analyses of thylakoid chlorophyll–protein complexes. Equal amounts of thylakoid membranes (7 μg chlorophyll) from VIGS– FTRc and VIGS–GFP plants were solubilized with 1% DM (w/v) and separated by BN–PAGE. Assignments of the thylakoid membrane macromolecular protein complexes indicated at right were identified. (J–L) 2D BN/SDS–PAGE fractionation of thylakoid protein complexes. Individual lanes from the BN–PAGE gels in (I) were subjected to second-dimension SDS–urea–PAGE followed by Coomassie brilliant blue staining. Identities of the relevant proteins are indicated by arrows. Three biological replicates were performed, and similar results were obtained. (M) Co-localization of FTRc:YFP fusion proteins (yellow) and PEND:CFP (blue) with chloroplast nucleoids. PEND (plastid envelop DNA binding) is a well characterized plastid DNA binding protein. Bars = 5 μm. Three biological replicates were performed, and similar results were obtained. (N) Changes in transcript abundance of plastid-encoded genes in VIGS–FTRc plants. The log2 (VIGS–FTRc/VIGS–GFP) value is given, where 3.32 corresponds to a 10-fold up-regulation, and –3.32 corresponds to a 10-fold down-regulation, in two different sectors in VIGS–FTRc plants relative to VIGS–GFP plants. I, class I genes; II, class II genes; III, class III genes; gray bars, VIGS–FTRc-G; dark gray bars, VIGS–FTRc-Y. The UBQ4 mRNA level was used as a reference. The data represent means ± SD of three biological replicates. Significant differences between VIGS–FTRc-G or VIGS–FTRc-Y leaves and VIGS–GFP leaves were calculated using Student’s t-test and are indicated by * P ≤ 0.05 and ** P ≤ 0.01. (O) Immunoblot analysis of thylakoid membrane proteins in the green and yellow sectors of VIGS–FTRc leaves. Aliquots of 2.5 μg (onequarter VIGS–GFP), 5 μg (one-half VIGS–GFP), 10 μg (VIGS–GFP), and 10 μg (VIGS–FTRc) of total thylakoid proteins were loaded on the gels. Designations of thylakoid membrane protein complexes and their diagnostic components are labeled on the left. Three biological replicates were performed, and similar results were obtained.
Molecular Plant FTRc-silenced plants by immunoblot analyses. Consistently with the plastid gene expression analysis (Figure 1N), the relative amounts of PSI and PSII subunits in VIGS–FTRc-Y thylakoids were obviously reduced, whereas the accumulation of photosynthetic proteins in VIGS–FTRc-G thylakoids was comparable to that in VIGS–GFP thylakoids (Figure 1O and Supplemental Figure 3). It is widely accepted that light-driven redox control plays an essential role in the regulation of PEP-dependent plastid gene expression (Pfannschmidt and Liere, 2005; Dietz and Pfannschmidt, 2011). In particular, TRX z has been viewed as an essential redox regulator of FLNs to control PEP activity (Arsova et al., 2010; Schröter et al., 2010). However, to our surprise, genetic studies in vivo exhibited that PEP function in trx z and fln1 mutants could be greatly restored by complementation with redox inactive TRX zC106S and FLN1C105/106A protein variants, respectively (Wimmelbacher and Bornke, 2014). These observations open the wide discussion about redox control of plastid gene expression (Pfannschmidt and Liere, 2005; Dietz and Pfannschmidt, 2011). Although no consistent observations have been made concerning whether FTR is able to reduce TRX z (Chibani et al., 2011; Bohrer et al., 2012), the delayedgreening leaf phenotype (Figure 1A and 1B), impaired chloroplast development (Figure 1H), protein interaction of FTRc with TRX z in plastid nucleoids (Figure 1M and Supplemental Figure 2), and dramatically reduced PEPdependent gene expression in the FTRc-silenced plants (Figure 1N) provide physiological evidence to support the hypothesis that FTR and TRX z act together in the regulation of PEP function during early stages of chloroplast development. An intriguing question concerns the mechanisms underlying the sectored chlorosis observed in young leaves in FTRc-silenced plants. Considering the same genotype in different sectors of VIGS–FTRc leaves (Figure 1C), two potential explanations can be envisioned. The first is that FTR only functions in chloroplast development in young leaves. However, the FTRc gene exhibits stable transcriptional activity with increasing leaf age (Supplemental Figure 4). Second, it is possible that one or more alternative TRX-reducing systems are able to compensate for a lack of FTR in the green sectors of young leaves and in adult leaves in VIGS–FTRc plants. In linear photosynthetic electron transfer, Fd:NADP+ oxidoreductase (FNR) transfers electrons from Fd to NADP+ for the production of NADPH (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). Mass spectrometry analysis identified a soluble subunit of FNR in PEP complex (Schröter et al., 2010). Considering the fact that NTRC allows for reduction of disulfides at the expense of NADPH as electron donor, the possibility that NTRC participates in the regulation of the PEP enzyme via redox regulating PEP subunits cannot be excluded. To further understand the role of TRX-mediated thiol modification in the regulation of PEP activity, it is worth investigating the
Letter to the Editor
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involvement of the FNR and NTRC redox transmitters in the regulation of plastid gene expression in future studies.
SUPPLEMENTARY DATA Supplementary data are available at Molecular Plant Online.
FUNDING This work was supported by grants from the National Natural Science Foundation of China (31171173 and 31371237), the Guangdong Provincial Natural Science Foundation of China (S2012010010533), and the Fundamental Research Funds for the Central Universities (14lgzd04).
Acknowledgments We thank Yule Liu (Tsinghua University, China) for the kind gift of TRV-based VIGS vectors, Weihua Wu (China Agricultural University) for providing BiFC vectors, and Lianwei Peng (Institute of Botany, The Chinese Academy of Sciences) for assistance in 2D BN/SDS–PAGE. No conflict of interest declared.
Peng Wang2, Jun Liu2, Bing Liu, Qingen Da, Dongru Feng, Jianbin Su, Yang Zhang, Jinfa Wang, and Hong-Bin Wang1 State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yatsen University, 510275 Guangzhou, People’s Republic of China 1 To whom correspondence should be addressed. E-mail [email protected], tel. +86-20-84039179, fax +86-20-84039179. 2 These authors contributed equally to this work.
References Arsova, B., Hoja, U., Wimmelbacher, M., Greiner, E., Ustun, S., Melzer, M., Petersen, K., Lein, W., and Bornke, F. (2010). Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana. Plant Cell. 22, 1498–1515. Bohrer, A.S., Massot, V., Innocenti, G., Reichheld, J.P., IssakidisBourguet, E., and Vanacker, H. (2012). New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis. J. Exp. Bot. 63, 6315–6323.
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Burch-Smith, T.M., Schiff, M., Liu, Y., and Dinesh-Kumar, S.P. (2006). Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. 142, 21–27.
Pogson, B.J., and Albrecht, V. (2011). Genetic dissection of chloroplast biogenesis and development: an overview. Plant Physiol. 155, 1545–1551.
Chibani, K., Tarrago, L., Schurmann, P., Jacquot, J.P., and Rouhier, N. (2011). Biochemical properties of poplar thioredoxin z. FEBS Lett. 585, 1077–1081.
Richter, A.S., Peter, E., Rothbart, M., Schlicke, H., Toivola, J., Rintamaki, E., and Grimm, B. (2013). Posttranslational influence of NADPH-dependent thioredoxin reductase C on enzymes in tetrapyrrole synthesis. Plant Physiol. 162, 63–73.
Dietz, K.J., and Pfannschmidt, T. (2011). Novel regulators in photosynthetic redox control of plant metabolism and gene expression. Plant Physiol. 155, 1477–1485. Keryer, E., Collin, V., Lavergne, D., Lemaire, S., and IssakidisBourguet, E. (2004). Characterization of Arabidopsis mutants for the variable subunit of ferredoxin:thioredoxin reductase. Photosynth. Res. 79, 265–274. Luo, T., Fan, T., Liu, Y., Rothbart, M., Yu, J., Zhou, S., Grimm, B., and Luo, M. (2012). Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox-mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants. Plant Physiol. 159, 118–130. Pfalz, J., and Pfannschmidt, T. (2013). Essential nucleoid proteins in early chloroplast development. Trends Plant Sci. 18, 186–194. Pfannschmidt, T., and Liere, K. (2005). Redox regulation and modification of proteins controlling chloroplast gene expression. Antioxid. Redox Signal. 7, 607–618.
Schröter, Y., Steiner, S., Matthai, K., and Pfannschmidt, T. (2010). Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression. Proteomics. 10, 2191–2204. Schürmann, P., and Buchanan, B.B. (2008). The ferredoxin/thioredoxin system of oxygenic photosynthesis. Antioxid. Redox Signal. 10, 1235–1274. Wang, P., Liu, J., Liu, B., Feng, D., Da, Q., Shu, S., Su, J., Zhang, Y., Wang, J., and Wang, H.B. (2013). Evidence for a role of chloroplastic m-type thioredoxins in the biogenesis of photosystem II in Arabidopsis. Plant Physiol. 163, 1710–1728. Wimmelbacher, M., and Bornke, F. (2014). Redox activity of thioredoxin z and fructokinase-like protein 1 is dispensable for autotrophic growth of Arabidopsis thaliana. J. Exp. Bot. 65, 2405–2413.
LETTER TO THE EDITOR
Ferredoxin:Thioredoxin Reductase Is Required for Proper Chloroplast Development and Is Involved in the Regulation of Plastid Gene Expression in Arabidopsis thaliana Dear Editor, In oxygenic photosynthetic organisms, carbon metabolism is regulated in a light-dependent manner by a distinctive ferredoxin/thioredoxin (Fd/TRX) redox system, in which Fd:TRX reductase (FTR) acts as the central redox transmitter to catalyze the reduction of TRX, with photosynthetic electrons coming from Fd (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). Recently, there is increasing evidence to suggest the diverse functions of TRX in chloroplast development, which requires lightinduced transcription and translation of nuclear and plastid genes, biosynthesis of lipids and pigments, assembly of photosynthetic pigment–protein complexes, etc. (Pogson and Albrecht, 2011). F- and m-type TRXs (TRX m and TRX f) in pea, and NADPH-dependent thioredoxin reductase C (NTRC) in Arabidopsis, participate in the redox control of multiple enzymes in chlorophyll biosynthesis (Luo et al., 2012; Richter et al., 2013). The recently identified z-type TRX (TRX z) plays an essential role in the redox regulation of PEP (plastid-encoded RNA polymerase)-dependent plastid gene expression (Arsova et al., 2010; Schröter et al., 2010). Three TRX m isoforms in Arabidopsis could modulate the redox status of the intermolecular disulfide bonds in photosystem II (PSII) inner antennal protein CP47 to assists the assembly of CP47 into PSII (Wang et al., 2013). However, the physiological significance of FTR in chloroplast development remains to be addressed. Biochemical characteristics and regulatory functions of FTR have been widely investigated in various organisms (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). However, genetic approaches to characterize FTR in vivo have rarely been successful, most likely due to lethality in the FTR knockout mutants or to functional redundancy within the Fd/TRX redox system. FTR is a heterodimer with a variable subunit (FTRv) and a catalytic subunit (FTRc). There are two FTRv isoforms in Arabidopsis, and the single FTRv mutant plants appear normal until exposure to oxidative stress, implying a functional complementation between the two FTRv isoforms (Keryer et al., 2004). In contrast, the Arabidopsis genome has a unique FTRc subunit that catalyzes the reduction of the conserved dithiol group in TRX (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). To avoid the lethality of FTR knockout
mutants and to address the physiological function of FTR in vivo, we used the virus-induced gene silencing (VIGS) system to silence the FTRc gene in Arabidopsis (Burch-Smith et al., 2006). Three weeks after infection, the inner young leaves of VIGS–FTRc plants exhibited a sectored chlorotic leaf phenotype (Figure 1A and 1B). The sectors near the petioles of young leaves in VIGS–FTRc plants appeared conspicuously chlorotic, and we called the yellow parts of VIGS–FTRc leaves VIGS–FTRc-Y (Figure 1A and 1B). Interestingly, the VIGS–FTRc-Y leaves gradually turned green as the plants matured; thus, the distal sectors away from the petioles in young leaves and adult leaves in VIGS–FTRc plants (called the green parts of VIGS–FTRc leaves, VIGS–FTRc-G) were not different from leaves in the control VIGS–GFP plants (Figure 1A and 1B). Immunoblot analyses showed FTRc proteins were undetectable in both the VIGS–FTRc-G and VIGS– FTRc-Y leaves (Figure 1C), indicating that the FTRc gene is efficiently silenced in both VIGS–FTRc-G and VIGS–FTRc-Y leaves. Moreover, a severely reduced level of FTRc in VIGS– FTRc plants was reflected in photosynthetic performance. The photochemical activities of two photosystems and CO2 assimilation rates were significantly reduced in VIGS–FTRc plants when compared with VIGS–GFP plants (Figure 1E and 1F, and Supplemental Table 1). Consistently, the obviously higher PSII excitation pressure (1-qP) and non-photochemical quenching (NPQ) were observed in VIGS–FTRc plants than that in VIGS–GFP plants (Figure 1F and 1G). Apart from its role in the regulation of carbon fixation, the Fd/TRX redox system is also involved in antioxidant defense in chloroplast (Dietz and Pfannschmidt, 2011). We wondered whether the delayed-greening leaf phenotype in FTRc-silenced plants could be attributed to an imbalance of ROS levels. Results of histochemical staining and relative expression of antioxidant genes indicated an enhanced radical-scavenging capacity in the FTRc-silenced plants (Supplemental Figure 1).
© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu069, Advance Access publication 2 June 2014 Received 4 April 2014; accepted 26 May 2014
Molecular Plant
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Figure 1 FTR Is Required for PEP-Dependent Plastid Gene Expression at the Early Stages of Chloroplast Development. (A) Identification and characterization of VIGS–FTRc Arabidopsis plants. Phenotypes of VIGS–GFP (a negative control for the effect of the virus infection) and VIGS–FTRc plants. Plants were observed 3 weeks after infiltration. Three biological replicates were performed, and similar results were obtained. (B) Representative leaves from VIGS plants. (C) Immunodetection of FTRc in VIGS–FTRc plants. Aliquots of 50 μg (one-half VIGS–GFP), 100 μg (VIGS–GFP), and 100 μg (VIGS–FTRc) of total leaf proteins were loaded on the gels. A CBB (Coomassie brilliant blue)-stained gel of the samples is shown below to provide an estimate of gel loading. Three biological replicates were performed, and similar results were obtained. (D–G) Photosynthetic electron transfer properties of VIGS–FTRc Arabidopsis plants. Chlorophyll fluorescence parameters were examined in VIGS–FTRc plants under different actinic light intensities, including the quantum yield of PSI [D, Y(I)], quantum yield of PSII [E, Y(I)], excitation pressure of PSII (F, 1-qP), and non-photochemical quenching (G, NPQ). The data represent means ± SD of three biological replicates. (H) Transmission electron microscope images of chloroplast ultrastructures in VIGS–GFP (upper panel), VIGS–FTRc-G (middle panel), and VIGS–FTRc (lower panel); leaves were taken 3 weeks after infiltration. The images in the left panels show light micrographs of whole chloroplasts, which were observed further by electron microscopy in the magnified images in the right panels. Scale bars are indicated. Two biological replicates were performed, and similar results were obtained.
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Taken together, these results indicate that FTRc is required for leaf greening, particularly in young leaves. To assess whether the silencing of FTRc causes ultrastructural changes in chloroplasts, chloroplast morphology in the VIGS–FTRc leaves was observed by transmission electron microscopy. In comparison with chloroplasts from VIGS–GFP leaves and VIGS–FTRc-G leaves, chloroplasts from VIGS–FTRc-Y leaves were not fully developed, and the stroma contained only rudimentary thylakoid lamellae (Figure 1H). This observation could be supported by the organization of thylakoid protein complexes in these thylakoids. The organization of PSI (photosystem I) and PSII complexes in VIGS–FTRc-Y thylakoids was drastically disrupted, in that almost no PSII supercomplexes, PSII monomers, and PSI monomers were detected in VIGS–FTRc-Y thylakoids (Figure 1I). Moreover, the relative amounts of photosystem subunits in these photosystem complexes were greatly reduced in VIGS–FTRc-Y thylakoids (Figure 1J– 1L). Collectively, these results suggest that FTR probably participates in the regulation of chloroplast development. Due to their endosymbiotic origin, plastids possess their own genome, the plastome, which encodes the complete machinery for transcription, RNA metabolism, translation, etc. (Pfalz and Pfannschmidt, 2013). Although the plastomes of higher plants contain only about 100–120 genes, intricate regulation of plastid gene expression is a prerequisite for proper chloroplast development (Pfalz and Pfannschmidt, 2013). The plastid transcription-regulation system relies on both PEP and NEP (the nuclearencoded plastid RNA polymerase; Pfalz and Pfannschmidt, 2013). During the early stages of chloroplast development, proper transcription of photosynthesis-related genes is dependent on PEP activity (Pfalz and Pfannschmidt, 2013).
Molecular Plant Various mutants or transgenic plants with defects in PEP activity, such as virescent or delayed-greening mutants, exhibit VIGS–FTRc-like sectored leaf chlorosis (Pfalz and Pfannschmidt, 2013). However, FTR is not known as a DNA binding protein, and not to be identified as the intrinsic subunits of PEP complex (Schröter et al., 2010); it appears probably that FTR indirectly participates in the regulation of PEP-dependent gene expression by reducing TRX. Global proteomic analyses demonstrated that TRX z and its potential target proteins, two fructokinase-like proteins (FLN1 and FLN2), are intrinsic subunits of the PEP complex (Schröter et al., 2010). Moreover, trx z mutant and FLNssilenced plants exhibit drastically decreased PEP-dependent plastid gene expression (Arsova et al., 2010). The observations that partial FTRc proteins could interact with TRX z proteins (Supplemental Figure 2), but also to associate with plastid nucleoids, in which plastid gene expression takes place (Figure 1M), support the hypothesis that FTR probably participate in the regulation of PEP activity. To further confirm the potential role of FTR in the regulation of plastid gene expression, the transcription of plastid-encoded genes in the VIGS–FTRc plants was analyzed by quantitative real-time RT–PCR. Intriguingly, the expression pattern of plastid genes in VIGS–FTRc-Y leaves resembles that of the ∆rpo mutant and other mutants that lack a functional PEP (Pfalz and Pfannschmidt, 2013); all of these show a down-regulation of class I plastid genes transcribed by PEP, whereas the expression of class II genes transcribed by both PEP and NEP, and class III genes transcribed exclusively by NEP is up-regulated (Figure 1N). To further validate impaired PEP-dependent plastid gene expression in FTRc-silenced plants, the steady-state levels of thylakoid membrane proteins were analyzed in the
(I) BN–PAGE analyses of thylakoid chlorophyll–protein complexes. Equal amounts of thylakoid membranes (7 μg chlorophyll) from VIGS– FTRc and VIGS–GFP plants were solubilized with 1% DM (w/v) and separated by BN–PAGE. Assignments of the thylakoid membrane macromolecular protein complexes indicated at right were identified. (J–L) 2D BN/SDS–PAGE fractionation of thylakoid protein complexes. Individual lanes from the BN–PAGE gels in (I) were subjected to second-dimension SDS–urea–PAGE followed by Coomassie brilliant blue staining. Identities of the relevant proteins are indicated by arrows. Three biological replicates were performed, and similar results were obtained. (M) Co-localization of FTRc:YFP fusion proteins (yellow) and PEND:CFP (blue) with chloroplast nucleoids. PEND (plastid envelop DNA binding) is a well characterized plastid DNA binding protein. Bars = 5 μm. Three biological replicates were performed, and similar results were obtained. (N) Changes in transcript abundance of plastid-encoded genes in VIGS–FTRc plants. The log2 (VIGS–FTRc/VIGS–GFP) value is given, where 3.32 corresponds to a 10-fold up-regulation, and –3.32 corresponds to a 10-fold down-regulation, in two different sectors in VIGS–FTRc plants relative to VIGS–GFP plants. I, class I genes; II, class II genes; III, class III genes; gray bars, VIGS–FTRc-G; dark gray bars, VIGS–FTRc-Y. The UBQ4 mRNA level was used as a reference. The data represent means ± SD of three biological replicates. Significant differences between VIGS–FTRc-G or VIGS–FTRc-Y leaves and VIGS–GFP leaves were calculated using Student’s t-test and are indicated by * P ≤ 0.05 and ** P ≤ 0.01. (O) Immunoblot analysis of thylakoid membrane proteins in the green and yellow sectors of VIGS–FTRc leaves. Aliquots of 2.5 μg (onequarter VIGS–GFP), 5 μg (one-half VIGS–GFP), 10 μg (VIGS–GFP), and 10 μg (VIGS–FTRc) of total thylakoid proteins were loaded on the gels. Designations of thylakoid membrane protein complexes and their diagnostic components are labeled on the left. Three biological replicates were performed, and similar results were obtained.
Molecular Plant FTRc-silenced plants by immunoblot analyses. Consistently with the plastid gene expression analysis (Figure 1N), the relative amounts of PSI and PSII subunits in VIGS–FTRc-Y thylakoids were obviously reduced, whereas the accumulation of photosynthetic proteins in VIGS–FTRc-G thylakoids was comparable to that in VIGS–GFP thylakoids (Figure 1O and Supplemental Figure 3). It is widely accepted that light-driven redox control plays an essential role in the regulation of PEP-dependent plastid gene expression (Pfannschmidt and Liere, 2005; Dietz and Pfannschmidt, 2011). In particular, TRX z has been viewed as an essential redox regulator of FLNs to control PEP activity (Arsova et al., 2010; Schröter et al., 2010). However, to our surprise, genetic studies in vivo exhibited that PEP function in trx z and fln1 mutants could be greatly restored by complementation with redox inactive TRX zC106S and FLN1C105/106A protein variants, respectively (Wimmelbacher and Bornke, 2014). These observations open the wide discussion about redox control of plastid gene expression (Pfannschmidt and Liere, 2005; Dietz and Pfannschmidt, 2011). Although no consistent observations have been made concerning whether FTR is able to reduce TRX z (Chibani et al., 2011; Bohrer et al., 2012), the delayedgreening leaf phenotype (Figure 1A and 1B), impaired chloroplast development (Figure 1H), protein interaction of FTRc with TRX z in plastid nucleoids (Figure 1M and Supplemental Figure 2), and dramatically reduced PEPdependent gene expression in the FTRc-silenced plants (Figure 1N) provide physiological evidence to support the hypothesis that FTR and TRX z act together in the regulation of PEP function during early stages of chloroplast development. An intriguing question concerns the mechanisms underlying the sectored chlorosis observed in young leaves in FTRc-silenced plants. Considering the same genotype in different sectors of VIGS–FTRc leaves (Figure 1C), two potential explanations can be envisioned. The first is that FTR only functions in chloroplast development in young leaves. However, the FTRc gene exhibits stable transcriptional activity with increasing leaf age (Supplemental Figure 4). Second, it is possible that one or more alternative TRX-reducing systems are able to compensate for a lack of FTR in the green sectors of young leaves and in adult leaves in VIGS–FTRc plants. In linear photosynthetic electron transfer, Fd:NADP+ oxidoreductase (FNR) transfers electrons from Fd to NADP+ for the production of NADPH (Schürmann and Buchanan, 2008; Dietz and Pfannschmidt, 2011). Mass spectrometry analysis identified a soluble subunit of FNR in PEP complex (Schröter et al., 2010). Considering the fact that NTRC allows for reduction of disulfides at the expense of NADPH as electron donor, the possibility that NTRC participates in the regulation of the PEP enzyme via redox regulating PEP subunits cannot be excluded. To further understand the role of TRX-mediated thiol modification in the regulation of PEP activity, it is worth investigating the
Letter to the Editor
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involvement of the FNR and NTRC redox transmitters in the regulation of plastid gene expression in future studies.
SUPPLEMENTARY DATA Supplementary data are available at Molecular Plant Online.
FUNDING This work was supported by grants from the National Natural Science Foundation of China (31171173 and 31371237), the Guangdong Provincial Natural Science Foundation of China (S2012010010533), and the Fundamental Research Funds for the Central Universities (14lgzd04).
Acknowledgments We thank Yule Liu (Tsinghua University, China) for the kind gift of TRV-based VIGS vectors, Weihua Wu (China Agricultural University) for providing BiFC vectors, and Lianwei Peng (Institute of Botany, The Chinese Academy of Sciences) for assistance in 2D BN/SDS–PAGE. No conflict of interest declared.
Peng Wang2, Jun Liu2, Bing Liu, Qingen Da, Dongru Feng, Jianbin Su, Yang Zhang, Jinfa Wang, and Hong-Bin Wang1 State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yatsen University, 510275 Guangzhou, People’s Republic of China 1 To whom correspondence should be addressed. E-mail [email protected], tel. +86-20-84039179, fax +86-20-84039179. 2 These authors contributed equally to this work.
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