Photosynth Res DOI 10.1007/s11120-015-0131-z

REGULAR PAPER

Purine biosynthetic enzyme ATase2 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis Zhipan Yang1 • Zengzhen Shang1,2 • Lei Wang1,2 • Qingtao Lu1 • Xiaogang Wen1 Wei Chi1 • Lixin Zhang1 • Congming Lu1

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Received: 28 January 2015 / Accepted: 24 March 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract To investigate the molecular mechanism of chloroplast biogenesis and development, we characterized an Arabidopsis mutant (dg169, delayed greening 169) which showed growth retardation and delayed greening phenotype in leaves. Newly emerged chlorotic leaves recovered gradually with leaf development in the mutant, and the mature leaves showed similar phenotype to those of wild-typewild-type plants. Compared with wild-type, the chloroplasts were oval-shaped and smaller and the thylakoid membranes were less abundant in yellow section of young leaves of dg169. In addition, the functions of photosystem II (PSII) and photosystem I (PSI) were also impaired. Furthermore, the amount of core subunits of PSII and PSI, as well as PSII and PSI complexes reduced in yellow section of young leaves of dg169. Map-based positional cloning identified that phenotype of dg169 was attributed to a point mutation of ATase2 which converts the conserved Ile-155 residue to Asn. ATase2 catalyzes the first step of de novo purine biosynthesis. This mutation resulted in impaired purine synthesis and a significant decrease in ATP, ADP, GTP and GDP contents. The analysis

Electronic supplementary material The online version of this article (doi:10.1007/s11120-015-0131-z) contains supplementary material, which is available to authorized users. & Congming Lu [email protected] Zhipan Yang [email protected] 1

Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

2

University of Chinese Academy of Sciences, Beijing 100049, China

of ATase2-GFP protein fusion showed that ATase2 was localized to nucleoid of chloroplasts. Our results further demonstrated that the levels of PEP-dependent transcripts in yellow section of young leaves of dg169 were decreased while NEP-dependent and both PEP- and NEP-dependent transcripts and chloroplast DNA replications were increased. The results in this study suggest that ATase2 plays an essential role in early chloroplast development through maintaining PEP function. Keywords Arabidopsis  ATase2  Chloroplast development  Chloroplast gene expression  Purine biosynthesis

Introduction The chloroplast is the special organelle of green plants. It is the site not only for photosynthesis, but also the production of many essential metabolites, such as amino acids, fatty acids, purine and pyrimidine nucleotides, plant hormones, and vitamins (Neuhaus and Emes 2000). The formation of normal chloroplasts is indispensable for plant growth and development. Chloroplast biogenesis and development are affected by many factors, such as chloroplast gene transcription and translation, nuclear gene transcription, chloroplast protein import and processing, retrograde signaling, chloroplast division, as well as environmental factors (Pogson and Albrecht 2011). In plastids, transcription is mediated by two types of RNA polymerase: plastid-encoded RNA polymerases (PEPs) and nuclear-encoded RNA polymerases (NEPs) (Hajdukiewicz et al. 1997; Pfalz et al. 2006; Pfalz and Pfannschmidt 2013). PEPs, containing four core subunits (a, b, b0 , b00 ) encoded by plastid genes, rpoA, rpoB, rpoC1

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and rpoC2, respectively, are responsible for transcription of photosynthesis genes in chloroplasts. NEPs are responsible for transcription of housekeeping genes encoding PEPs core subunits and ribosomal proteins (Hajdukiewicz et al. 1997; Bruce Cahoon and Stern 2001). Many studies have shown that PEP-dependent transcription plays an important role in chloroplast development. The decrease in PEP activity often leads to impaired chloroplast development (Pfalz and Pfannschmidt 2013). Many attempts have been made to identify nuclear-encoded protein factors involved in the regulation of PEPdependent transcription. By using different biochemical purification procedures, distinct RNA polymerase complexes were isolated from chloroplasts (Pfalz and Pfannschmidt 2013). Basically, three major types of plastid RNA polymerase preparations can be distinguished: nucleoids, the insoluble RNA polymerase preparation called plastid transcriptionally active chromosome (pTAC), and the soluble RNA polymerase preparation. Many nuclearencoded proteins were found in these complexes. The pTAC preparations are purer than those of nucleoids with respect to protein composition. At least 35 proteins have been identified in pTACs of Arabidopsis and mustard (Sinapis alba) (Pfalz et al. 2006). Some pTACs such as pTAC2, pTAC6, pTAC7, pTAC12, and pTAC14 have been found to be necessary for sustaining PEP-dependent transcriptional activity. Mutation of pTAC2, pTAC6, pTAC7, pTAC12, and pTAC14 leads to the decrease in PEP activity and impaired chloroplast development (Pfalz et al. 2006; Gao et al. 2011; Yu et al. 2013). In addition, some protein factors found in nucleoids are also involved in the regulation of PEP-dependent transcription. For example, mutation of MurE, FSD2, FSD3, FLN1/FLN2, and TrxZ results in decreased PEP activity and deficient chloroplast development (Pfalz et al. 2006; Garcia et al. 2008; Myouga et al. 2008; Gilkerson et al. 2012; Arsova et al. 2010). Recently, we have found that small heat shock protein HSP21 is also localized to nucleoids. It interacts with pTAC5 and is essential for PEP transcription activity and chloroplast development in Arabidopsis under heat stress conditions (Zhong et al. 2013). In addition, sigma factors have been identified to mediate the PEP transcription initiation in response to developmental and environmental cues precisely (Allison 2000; Schweer et al. 2010; LerbsMache 2011). We have previously shown that DELAYED GREENING1 is required for PEP-dependent gene expression during early chloroplast development by interacting with sigma factor 6 (Chi et al. 2008, 2010). Purine is essential component of purine nucleotides, which are not only subunits of nucleic acids and major energy donor but also precursors of B-class vitamins, coenzymes, and plant hormones (Senecoff et al. 1996; Herz et al. 2000; Hanson and Gregory 2002; Smith and Atkins

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2002; Zrenner et al. 2006). Therefore, purine synthesis is crucial for plant growth, development, and metabolism. De novo synthesis pathway and salvage pathway are responsible for purine production in plants. The de novo synthesis pathway is located in the plastid while the salvage pathway is located in the cytoplasm. De novo synthesis pathway is the major purine synthesis pathway in plants (Senecoff et al. 1996). There are fourteen enzymatic reactions for the de novo purine biosynthetic pathway. ATase (glutamine phosphoribosyl pyrophosphate amidotransferase) is the key enzyme that catalyzes the first step for the de novo synthesis pathway (Smith and Atkins 2002). In Arabidopsis, there are three isoenzymes of ATase, ATase1, 2, and 3. ATase2 is the main isoform expressing in leaves and critical for plant growth and development (Hung et al. 2004; Woo et al. 2011). An Arabidopsis knock out mutant of ATase2, atd2, showed strong growth retardation and leaf chlorosis (van der Graaff et al. 2004). The chloroplast was defective in the proper formation of the organized thylakoid membrane structure and exhibited a vesiculation of membranes in the atd2 mutant. The growth of atd2 under a varying range of light intensities showed that under low light conditions the leaves remained green, suggesting that the defect in chloroplast development is caused by photo-oxidative damage. Hung et al. (2004) acquired another null mutant of ATase2, cia1, by screening mutants defective in protein import into chloroplasts. They also observed leaf chlorosis in the cia1 mutant and proposed that a decrease in energy charge may result in a decrease in growth and chloroplast protein import significantly. In addition, a point-mutation of ATase2 gene in alx13 which converts valine-364 to methionine also resulted in growth retardation and variegation phenotype, but not as irregular chlorosis in atd2 and cia1, yellow or white chlorotic sectors in alx13 developing in the lateral regions of the leaf blade, whereas medial leaf regions exhibited normal green pigmentation (Woo et al. 2011). Detailed analysis indicated that there is no correspondence between ROS accumulation and chlorosis in alx13 mutants. Instead, a combined effect of direct light exposure and light-influenced systemic signals is in concert with impaired chloroplast development. (Woo et al. 2011). Although the chloroplast development deficiency in atase2 mutants was observed for a long time, the exact mechanism linking de novo purine biosynthesis to chloroplast biogenesis remains unclear. To investigate the molecular mechanism of chloroplast development, we have screened a number of Arabidopsis chloroplast developmental mutants. Here we characterized an ATase2 mutant dg169 (delayed greening 169), which was due to a missense mutation that converts conserved Ile155 to Asn in ATase2. Our results showed that the mutant showed delayed greening phenotype and the mutation led to

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impaired chloroplast development. Thylakoid membranes were less abundant in yellow section of young leaves of the mutant than in those of wild-type. More importantly, our results showed that there was a significant decrease in PEPdependent transcription in yellow section of young leaves of dg169. Our results suggest that ATase2 plays an important role in early chloroplast development through maintaining PEP function.

Methods and materials Plant material and growth conditions The seeds of wild-typewild-type and mutant plants were surface sterilized and sown on Murashige and Skoog (MS) medium containing 3 % sucrose and 0.7 % agar. To ensure synchronized germination, the seeds were incubated in darkness for 48 h at 4 °C after sowing. The plants were grown at 22 °C under short-day conditions (12 h of light/12 h of dark) with a photon flux density of 100 lmol m-2 s-1. After 10 days growth on plates, the seedlings were transferred to soil and cultured in a growth chamber with same condition. Measurements of leaf area were performed with the standard protocols by an LI-3000 A (LI-Cor). Chlorophyll contents were determined in 80 % (v/v) acetone according to Porra et al. (1989). Leaves from 3-week-old plants were used for analyses of chloroplast ultrastructure, chlorophyll fluorescence, light-induced P700 absorbance changes at 820 nm, immunoblot, BN-PAGE, and nucleotide contents. Transmission electron microscopy Transmission electron microscopy was performed basically according to our previous study (Peng et al. 2006). Leaves from 3-week-old plants grown in soil were collected. The samples were observed with a transmission electron microscope (JEM-1230; JEOL). Chlorophyll (Chl) fluorescence analysis Chl fluorescence was measured using a PAM-2000 portable chlorophyll fluorometer (Heinz Walz, Germany) according to our previous study (Zhang et al. 2011). After a dark adaptation period of 30 min, minimum fluorescence (Fo) was determined by a weak red light. Maximum fluorescence of dark-adapted state (Fm) was measured during a subsequent saturating pulse of white light (8000 lmol m-2 s-1 for 0.8 s). The leaf was then continuously illuminated with actinic light at an intensity of 100 lmol m-2 s-1 (which is equivalent of growth light intensity) for about 5 min. The

steady-state fluorescence (Fs) was thereafter recorded and a second saturating pulse of white light (8000 lmol m-2 s-1 for 0.8 s) was imposed to determine the maximum fluorescence level in the light-adapted state (F0 m). The actinic light was removed and the minimal fluorescence level in the lightadapted state (F0 o) was determined by illuminating the leaf with a 3 s pulse of far-red light. Using above fluorescence parameters, we calculated: (1) the maximal efficiency of PSII photochemistry in the dark-adapted state, Fv/Fm = (Fm - Fo)/Fm; (2) the actual PSII efficiency, UPSII = (F0 m - Fs)/F0 m; (3) the photochemical quenching coefficient, qP = (F0 m - Fs)/(F0 m - F0 o). (4) the non-photochemical quenching, NPQ = Fm/F0 m - 1. The light-induced P700 absorbance changes at 820 nm were measured using the PAM-101 fluorometer connected to an emitter-detector unit ED 800T (Walz) as described by Meurer et al. (1996). Absorbance changes induced by saturating far-red light were used to estimate the photochemical capacity of PSI. Chl fluorescence relaxation kinetics The decay of Chl a fluorescence yield after a single turnover flash was measured with a double-modulation fluorescence fluorometer (model FL-200, Photon Systems Instruments, Brno, Czech Republic) according to our previous study (Zhang et al. 2011). The instrument contained red LEDs for both actinic (20 ls) and measuring (2.5 ls) flashes, and was used in the time range of 100 ls–100 s. Before measurements, the leaves were dark adapted for 30 min in the presence or absence of 50 lM DCMU. The intensity of the measuring flashes was set at a value that was low enough to avoid reduction of QA in the presence of DCMU. SDS-PAGE, immunoblot, and BN-PAGE analysis For immunoblot analysis, total proteins were extracted from the samples as described by Martinez-Garcia et al. (1999). Total leaf proteins were separated using 15 % SDS polyacrylamide gels containing 6 M urea. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Amersham Biosciences, USA). The membranes were incubated with specific primary antibodies, and the signals from secondary conjugated antibodies were detected using the enhanced chemiluminescence method. X-ray films were scanned and analyzed using ImageMasterTM 2D Platinum software. BN-PAGE was performed using acrylamide gels with linear 6–12 % gradients basically according to our previous study (Peng et al. 2006).

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Map-based cloning and complementation

Subcellular localization of ATase2

The dg169 mutant was crossed to Landsberg erecta and the resulting F1 heterozygous plants were selfed to generate a large F2 mapping population. Homozygous F2 mutant plants were subjected to PCR analysis using specific SSLP (simple sequence length polymorphism) molecular markers. At last, candidate genes with predicted chloroplast transit peptides were sequenced. To confirm the mutation, a complementation experiment was performed as following. Full-length ATase2 coding sequence was amplified with primers 50 -GGATCCATGGCG GCCACCTCTAG-30 and 50 -GGTAACCTACCGTACCCA ACCTCC-30 , and subcloned into the pCAMBIA1301 vector under the control of the cauliflower mosaic virus 35S promoter. The constructs were then transformed into Agrobacterium tumefaciens strain GV3101pMP90 and introduced into the dg169 mutant plants using the floral dip method (Clough and Bent 1998). Transformed plants were selected on MS medium containing 40 mg/L hygromycin. Resistant plants were transferred to soil and grown in a growth chamber to produce seeds.

Full-length CDS of the ATase2 and pTAC5 were cloned into p221-GFP and p221-RFP vectors (for primers used, see Online Resource ESM 1). The resulting fusion constructions and the control vectors were transformed into protoplasts of Arabidopsis. GFP and RFP fluorescence was observed by confocal scanning microscopy (LSM 510 Meta; Zeiss). 488 nm laser line and 505–530 nm band pass filter were used for GFP excitation and emission respectively. RFP was excited with the 543 nm laser line and detected using 600–630 nm band pass filter. Chlorophyll auto-fluorescence was excited with the 488 nm laser line, and was detected using a 650 nm band pass filter.

Nucleotide measurements The leaves (400 mg) of wild-typewild-type and mutant plants were frozen immediately in liquid nitrogen and homogenized into powder. Nucleotides were extracted by the trichloroacetic acid (TCA) method described by Hajirezaei et al. (2003). The nucleotide content in the TCA extracts was measured by HPLC using a Waters e2695 separation module HPLC system fitted with a 100 9 4.6 mm Partisil 5 SAX RAC II anion-exchange column (Waterman) and detected by absorption at OD254. Nucleotides were identified and quantified by comparison with respective nucleotide standards as described by Geigenberger et al. (1997).

RT-PCR, quantitative PCR, and RNA gel blot To examine the expression of chloroplast genes, RT-PCR and quantitative real-time RT-PCR analyses were performed according to our previous study (Chi et al. 2008). The amplification of Actin was used as an internal control for normalization. For primers used, see Online Resource ESM 1. For RNA gel blot analysis, total RNAs were fractionated with formaldehyde denaturing 1.2 % agrose gel and transferred onto nylon membranes as described by Sambrook and Russell (2001). The membranes were probed with 32P-labeled probes that were prepared as described by Yu et al. (2008). Quantitative PCR was used to determine the ratios of nuclear to chloroplast genome in wild-typewild-type and mutant plants as described by Garton et al. (2007). Genomic DNA was extracted from the rosette leaves of shortday-grown plants using Plant DNAzolÒ Reagent (Invitrogen). Primers for At4g04930 and ArthCp025, the unique sequence of the nuclear and chloroplast genomes, were listed in the Online Resource ESM 1.

Antiserum production

Results

For the production of polyclonal antibodies against ATase2, the nucleotide sequences encoding the N-terminus of ATase2 (amino acids 91–350 corresponding to nucleotide positions 271–1050 bp of the ATase2 gene) were amplified by PCR using the primers 50 -GGATCCGGAATCTACGGTGACTCAG-30 and 50 -CTCGAGTCCAAAGACATGCCTAGACT-30 . The resulting DNA fragment was cleaved with BamH I and Xho I and fused in frame with the N-terminal His affinity tag of pET28a (Novagen), and the resulting plasmids were transformed into Escherichia coli strain BL21 (DE3). The fusion protein was purified on a nickel-nitrilotriacetic acid agarose resin matrix and raised in rabbit with purified antigen.

dg169 exhibited delayed greening phenotype in a developmentally regulated manner

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The dg169 mutant was screened from a collection of pSKI015 T-DNA mutagenized Arabidopsis (ecotype Columbia) lines from the ABRC (http://arbc.osu.edu/). The mutant showed normal developed cotyledons. However, the true leaves exhibited delayed greening phenotype in a developmentally regulated manner as shown in Fig. 1a. The new emerged true leaves were yellowish. With the development, the distal end of leaves became greening. The mature leaves could recover the green color uniformly, but the chlorophyll content is still lower than that of wild-

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Chloroplast development was impaired in dg169 To investigate the effects of the DG169 mutation on chloroplast development, we observed the ultrastructure of chloroplasts in three-week-old wild-typewild-type and mutant plants by transmission electron microscopy (Fig. 2). In wild-type, chloroplasts in young and mature leaves showed lens shape and had well-structured thylakoid membranes (Fig. 2a). However, the chloroplast of yellow section in young leaves in the mutant was oval-shaped and smaller than those in wild-type (Fig. 2b). In addition, the thylakoid membrane organization in the chloroplast of young leaves in the mutant was disturbed. The thylakoid membranes were much less abundant compared to those of wild-type and no grana structure was observed. In contrast, the thylakoid membrane organization and abundance in the chloroplast of mature leaves of the mutant were comparable to those in wild-type (Fig. 2c). It should be noted that the thylakoid membrane organization and abundance in the chloroplast of green section in young leaves were similar to those of mature leaves in the mutant (data not shown). PSII and PSI functions were impaired in dg169

Fig. 1 Phenotypes of the dg169 mutant. a Seven-day-old seedlings grown on the MS plate (upper) and 3-week-old seedlings grown in soil (lower). b Chlorophyll contents of wild-type (wt), dg169 and complemented plants of dg169. In dg169, chlorophyll contents in mature leaves and the yellow section of young leaves were measured separately. Values shown are averages ± SD of three independent experiments. c Growth kinetics of wild-type (wt), dg169 and complemented plants of dg169. Values shown are averages ± SD of six independent samples

type (Fig. 1b). Compared to wild-type, the vegetative growth of the mutant was retarded significantly and bolting and flowering of the mutant were also delayed (Fig 1c). However, the growth of the mutant was almost comparable to that of wild-type at the later stage of reproduction growth.

Noninvasive fluorometric analyses were performed to investigate if and how PSII function is affected in the mutant (Fig. 3a; Table 1). Chlorophyll fluorescence induction kinetics curve and the parameters related to PSII photochemistry of mature leaves of dg169 were similar to those of the wild-type. In contrast, we observed a significant increase in Fo level and corresponding decrease in Fv/Fo in yellow section of young leaves of the mutant (Fig. 3a). The fluorescence yield in yellow section of young leaves of the mutant went below the Fo level when stimulated by continuous actinic light and couldn’t recover to Fo finally. In addition, there was a significant decrease in Fv/Fm and UPSII in yellow section of young leaves of the mutant. Values of Fv/Fm and UPSII in yellow section of young leaves of the mutant were about 68 and 63 % of those of wild-typewild-type plants, respectively. qP was not strongly affected in yellow section of young leaves of the mutant compared with that in wild-typewild-type plants, indicating that the electrons released by PSII could be transported through the electron transport chain and the subsequent photosynthetic electron chain was functional (Schult et al. 2007). However, NPQ value had an obvious increase in yellow section of young leaves of the mutant compared to that in wild-type, indicating that a non-photochemical quenching process developed during the energy dissipation in the mutants. These results suggest that the function of PSII was impaired in the mutant. We further investigated how the acceptor and donor side functions of PSII were affected in the mutant through the

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Photosynth Res Fig. 2 Transmission electron microscopic images of chloroplasts in mature leaves of wild-type (a), yellow section of young leaves of dg169 (b), mature leaves of dg169 (c), and mature leaves of complemented plants of dg169 (d). Chloroplasts in the yellow section of young leaves and mature leaves of dg169 showed different developmental status (bars 1 lm)

Fig. 3 Noninvasive fluorometric analyses in mature leaves of wildtype (wt), mature leaves and the yellow section of young leaves of the dg169 mutant, and mature leaves of complemented plants of dg169. Three-week-old seedlings were used. a Chlorophyll fluorescence induction kinetics. The minimal level of fluorescence (Fo) of darkadapted whole plants with all PSII reaction centers open was determined under a weak measuring light (650 nm, 0.8 lmol m-2 s-1). The Fm level with all PSII reaction centers closed was

determined by a saturating pulse of white light (8000 lmol m-2 s-1 for 0.8 s). The ratios of variable to maximum fluorescence, reflecting the maximum photochemical efficiency of PSII, were calculated from Fv/Fm = (Fm - Fo)/Fm. B P700 redox kinetics. The oxidation of P700 was investigated by measuring absorbance changes of P700 at 820 nm induced by far-red light (720 nm). AL, actinic light (100 lmol m-2 s-1); FR, far-red light

measurements of the flash-induced increase and subsequent relaxation of fluorescence yield after single flash excitation (Fig. 4; Table 2). In the absence of DCMU, chlorophyll fluorescence decay kinetics in yellow section of young

leaves of dg169 changed more profoundly than that of mature leaves compared to that in wild-type. The relaxation of the flash induced increase in variable Chl fluorescence yield can be resolved into three phases (Fig. 4; Table 2). The fast

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Photosynth Res Table 1 Parameters related to the functions of PSII and PSI in wild-type and dg169 plants. The yellow section of young leaves of the dg169 mutant was used

Wild-type

Dg169 young

Dg169 mature

Dg169 complemented

Fv/Fm

0.82 ± 0.01

0.56 ± 0.08

0.79 ± 0.02

0.82 ± 0.02

UPSII

0.71 ± 0.05

0.45 ± 0.06

0.66 ± 0.04

0.70 ± 0.03

qP

0.89 ± 0.06

0.97 ± 0.05

0.87 ± 0.04

0.88 ± 0.05

NPQ

0.17 ± 0.03

0.46 ± 0.03

0.19 ± 0.05

0.18 ± 0.01

27 ± 2

73 ± 3

DA820max ð%Þ

100

103 ± 3

Mean ± SD values were calculated from three independent experiments

Fig. 4 Fluorescence decays induced by single-turnover flash in leaves of 3-week-old wild-type (wt) and dg169 plants. The yellow section of young leaves of the dg169 mutant was used. A The curves were the actual data of the fluorescence signals in the absence of 50 lM DCMU. b The curves were normalized relative to the total

variable fluorescence in the absence of 50 lM DCMU. c The curves were the actual data of the fluorescence signals in the presence of 50 lM DCMU. d The curves were normalized relative to the total variable fluorescence in the presence of 50 lM DCMU

phase and middle phase are related to the electron transfer from Q A to QB in PSII reaction centers having an occupied or empty QB pocket at the time of flashing and the slow phase reflects the reoxidation of Q A via recombination with the donor-side components. Compared to wild-type, the fast phase of the mutant showed increase in decay half time but a decrease in the amplitude. The T1/2 of the fast phase increased from 133.8 ls in wild-type to 157.3 and 189.9 ls in mature leaves and yellow section of young leaves of dg169.

The amplitude of the fast phase decreased from 43.3 % in wild-type to 38.9 and 15.7 % (Table 2). For the middle phase, both the decay half time and the amplitude decreased in dg169 compared to that of wild-type. The T1/2 of the middle phase decreased from 1.48 ms in wild-type to 1.01 ms and 0.84 ms and the amplitude of the middle phase decreased from 20.70 % in wild-type to 15.21 and 17.27 % in mature leaves and yellow section of young leaves of dg169 (Table 2). The slow phase demonstrated a reverse

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Photosynth Res Table 2 Parameters of decay kinetics of flash-induced variable fluorescence in wildtype and dg169 plants

Total amplitude (%)

Fast phase T1/2 (ls) [A(%)]

Middle phase T1/2 (ms) [A(%)]

Slow phase T1/2 (s) [A(%)]

100a

133.8 ± 15.2

1.48 ± 0.14

1.31 ± 0.17

(43.3 ± 3.2)b

(20.70 ± 1.89)

(36.03 ± 3.29)

Without DCMU Wild-type dg169 young dg169 mature

96 ± 3 98 ± 2

189.9 ± 17.8

0.84 ± 0.07

0.65 ± 0.03

(15.7 ± 2.1)

(17.27 ± 2.23)

(67.01 ± 5.62)

157.3 ± 19.5

1.01 ± 0.15

1.02 ± 0.05

(38.9 ± 2.8)

(15.21 ± 1.76)

(45.83 ± 3.96)

With DCMU Wild-type

100

- (0)

- (0)

0.71 ± 0.06 (100)

dg169 young

100

- (0)

- (0)

0.68 ± 0.05 (100)

dg169 mature

100

- (0)

- (0)

0.69 ± 0.02 (100)

The yellow section of young leaves of the dg169 mutant was used The relaxation of the flash-induced fluorescence yield was measured without or with 50 lM DCMU. Mean ± SD values were calculated from four to six independent experiments a

Values represent the amplitude of total variable fluorescence as a percentage of that in wild-type plants

b

Values in parentheses are relative amplitude as a percentage of total variable fluorescence obtained from wild-type and dg169 plants

variation in the decay half time and amplitude in dg169 compared with wild-typewild-type plants. The T1/2 of the slow phase decreased from 1.31 s in wild-type to 1.02 s and 0.65 s whereas the amplitude of the slow phase increased from 36.03 % in wild-type to 45.83 and 67.01 % in mature leaves and yellow section of young leaves of dg169, respectively (Table 2). DCMU blocks the electron transfer from Q A to QB. In the presence of DCMU, the fluorescence relaxation was dominated by a slow component in wild-type and mutant plants (Fig. 4; Table 2). There was no significant change in the time constant for this slow component between wild-type and mutant plants. These results suggest that electron transfer of acceptor side of PSII in yellow section of young leaves of dg169 was impaired whereas electron transfer of donor-side is not affected. The functional state of the PSI can be assessed by measuring the absorbance changes around 820 nm (Meurer et al. 1996). Compared to that in wild-type, there was about a 70 % decrease in an absorbance change of P700 in yellow section of young leaves of dg169 (Fig. 3b; Table 1). In mature leaves of dg169, absorbance of 820 nm recovered to a large extent, but there was still approximately 30 % decrease in an absorbance change of P700 compared to that in mature leaves of wild-type plants (Fig. 3b; Table 1). Protein contents of PSII and PSI were reduced in dg169 The impaired functions of PSII and PSI in yellow section of young leaves of the mutant may be associated with the altered protein levels in PSII and PSI complexes (Peng

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et al. 2006; Liu et al. 2012). To examine the changes in the steady state levels of PSII and PSI complexes in dg169, total proteins were isolated from yellow section of young leaves and mature leaves from 3-week-old seedlings of dg169 and wild-type and immunoblot analysis was carried out (Fig. 5a). The levels of PSII and PSI components were reduced noticeably in yellow section of young leaves of dg169. Among them, plastid-encoded core proteins D1, D2, CP47, CP43, and PsaA/B were significantly reduced to 10–20 % of those of the wild-type. The levels of nuclearencoded PSII components, such as LHCII, and PsbO, the contents of proteins related to electron transfer, such as cyt f, FNR, and b-subunit of ATP synthase, as well as plastidencoded Rubisco large subunit, showed a 50 % decrease in yellow section of young leaves of dg169. One exception is nuclear-encoded Rubisco activase (RCA), no decrease in protein content was observed in yellow section of young leaves of dg169, suggesting expression of RCA is regulated by a relative independent pathway. In mature leaves of dg169, contents of proteins mentioned above recovered to a large extent and came near to those in wild-type. Chlorophyll-protein complexes solubilized from thylakoid membranes using dodecyl-b-D-maltopyranoside (DM) were analyzed by Blue Native gel (BN-PAGE) (Fig. 5b). When equal amounts of chlorophyll were loaded, the native thylakoid membrane complexes of wild-type were separated into seven major bands that represent PSII supercomplexes (band I), monomeric PSI and dimeric PSII (band II), monomeric PSII (band III), LHCII assembly (band IV), CP43-free PSII (band V), trimeric LHCII (band VI), and monomeric LHCII (band VII) (Sun et al. 2010).

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Fig. 5 Analysis of chloroplast proteins of 3-week-old wild-type (wt) and dg169. Proteins from the yellow section of young leaves and mature leaves of dg169 were compared to those of wild-type, respectively. a Immunodetection of chloroplast proteins. Total leaf proteins were separated by SDS-urea-PAGE, and the blots were probed with specific antibodies. b BN gel analysis of thylakoid membrane protein complexes. Thylakoid membranes (10 lg chlorophyll) from wild-type and dg169 mutant leaves were solubilized with 1 % dodecyl-b-D-maltoside and separated by BN gel electrophoresis. The positions of protein complexes representing PSII supercomplexes (Band I, PSII SC), monomeric PSI and dimeric PSII (band II, PSI-M,

and PSII-D), monomeric PSII (band III, PSII), light harvesting complex II assembly (band IV, LHCII), CP43-free PSII (band V, CP43-PSII), trimeric LHCII (band VI, LHCII-T), and monomeric LHCII (band VII, LHCII-M) are indicated. C BN-PAGE-separated thylakoid protein complexes in a single lane were subsequently separated in a second dimension by 15 % SDS-urea-PAGE gel and stained with Coomassie brilliant blue. Names of the proteins resolved by the second-dimension SDS-PAGE are indicated by arrows. Three independent experiments were performed, and one representative experiment is presented

Similar results were obtained from the mature leaves of mutants. However, the amount of thylakoid membrane complexes in yellow section of young leaves of dg169 showed a significant change. There was a decrease in the

amounts of PSII supercomplexes (band I), monomeric PSI and dimeric PSII (band II), monomeric PSII (band III), LHCII assembly (band IV), CP43-free PSII (band V), and trimeric LHCII (band VI) in the dg169. However, the

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monomeric LHCII (band VII) increased significantly in yellow section of young leaves of dg169. Analyses of the SDS-PAGE gels followed by the BN-PAGE showed that there was a significant decrease in the amounts of the PSII and PSI core subunits D1, D2, CP47, CP43, and PsaA/B but an increase in the amount of the monomeric LHCII in yellow section of young leaves of dg169 (Fig. 5c). dg169 was resulted from a point mutation in ATase2 encoding gene At4g34740 We could not get useful information of the mutation site by tail PCR. Thus, positional cloning techniques were employed to identify the dg169 mutation. After hybridization to Landsberg erecta ecotype, all of the F1 progenies showed wild-type phenotype. Genetic analysis showed that among 1732 F2 progenies, 425 plants presented delayed greening phenotype. The ratio of F2 segregation was 3.075, indicating that the mutation was a single recessive mutation. Map-based cloning by using simple sequence length polymorphism (SSLP) molecular markers displayed that the mutation located in chromosome 4. Further analysis indicated that the mutation was in a 280-kb region between BAC clones F10M10 and M4E13 (see Online Resource ESM 2). Sequencing results showed that there is a single base-pair T464A mutation in At4g34740 gene which converts the conserved Ile-155 residue to Asn (see Online Resource ESM 2). To confirm this result, a complementation experiment was carried out using full-length At4g34740 gene of wild-type under the control of the cauliflower mosaic virus 35S promoter. Multiple independent transgenic plants were obtained and they showed normal wild-type phenotype and chlorophyll fluorescence parameters (Figs. 1, 2, 3). Thus, it could be confirmed that phenotype of the dg169 mutant was due to the point mutation in At4g34740 At4g34740 encodes glutamine phosphoribosyl pyrophosphate amidotransferase (ATase2). ATase2 catalyzes the first committed step in de novo purine biosynthesis, which converts 5-phosphoribosyl-1-pyrophosphate to phosphoribosylamin. Nucleotide measurements showed that there was about 25–50 % decrease in ATP, ADP, GTP, and GDP contents in the dg169 mutant compared to that in wild-type (Fig. 6). These results suggest that mutation of conserved Ile-155 in ATase2 affected the de novo synthesis of purines. Sublocalization of ATase2 Previous study has identified that ATase2 is localized to the stroma of chloroplasts (Hung et al. 2004). Our results also showed that ATase2 is localized to the stroma of chloroplasts (Fig. 7a). To further investigate the sublocalization

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Fig. 6 Purine nucleotide levels in wild-type (wt), dg169 and complemented plants of dg169. The yellow section of young leaves of the dg169 mutant was used. Nucleotides were extracted by TCA method and measured by HPLC. Values shown are average of three independent experiments. Error bars indicate SD

of ATase2, ATase2-GFP protein fusion was introduced into Arabidopsis protoplasts and GFP fluorescence was found to be localized to chloroplasts (Fig. 7b). The fluorescence pattern of ATase2 inside chloroplasts resembled the appearance of nucleoids (Zhong et al. 2013). To further confirm if ATase2 is localized to nucleoids, we further examined whether ATase2-GFP was colocalized with red fluorescent protein (RFP) fused with pTAC5, a well-characterized protein localized in nucleoids (Zhong et al. 2013). The fluorescence signal overlay of ATase2-GFP and pTAC5-RFP in chloroplast nucleoids further indicated that ATase2 and pTAC5 colocalized in chloroplast nucleoids (Fig. 7c). Transcript accumulation of plastid-encoded genes altered in dg169 Since there was a significant decrease in the levels of plastid and nuclear-encoded proteins in dg169 compared with that in the wild-type (Fig. 5a), we investigated

Photosynth Res b Fig. 7 Subcellular localization of the ATase2. a Immunolocalization

of ATase2. Intact chloroplasts were isolated from the leaves of wildtype seedlings and then separated into thylakoid membrane and stroma fractions. Polyclonal antisera were used against ATase2, the integral membrane protein D1, Tic40, and the abundant stroma protein ribulose biphosphate carboxylase large subunit (RbcL). b Localization of ATase2 protein within the chloroplast by GFP assay. p221-GFP, control with empty vector; Mit, control with mitochondrial localization signal of FRO1; Chl, control with the transit peptide of the ribulose bisphosphate carboxylase small subunit; Nuc, control with nuclear localization signal of fibrillarin; ATase2GFP, ATase2-GFP fusion construction. C Colocalization of ATase2GFP (green) and pTAC5-RFP (red) fluorescence within chloroplast nucleoids. To discriminate the RFP fluorescence and chlorophyll auto-fluorescence, the latter was displayed as purple color

both PEP and NEP-dependent genes (class II); LhcII, Rca, and PsbO were selected as representatives of nuclear-encoded genes. Our results showed that transcript levels of class I genes were down-regulated significantly while transcript levels of class II and class III genes were upregulated in yellow section of young leaves of the mutant (Fig. 8). These results suggest that PEP-dependent transcription in chloroplasts is impaired in the dg169 mutant. Expression of nuclear-encoded gene whose products are targeted to chloroplasts displayed different changes. Gene encoding Rubisco activase (Rca) which related to dark reaction was unchanged in the dg169 mutant compared with that in wild-type whereas genes encoding PSII components (PsbO and LhcII) were down-regulated (Fig. 8). This could be a secondary effect of reduction of PSII core components. These results suggest that PEP activity is decreased in the dg169 mutant. Plastid-encoded rRNAs were comprised of 23S, 16S, 5S, and 4.5S rRNA (Tiller and Bock 2014). They were arranged in single operons (rrn operons) and transcribed by both PEP and NEP (Fig. 9a). The RNA gel blot analyses by using gene specific probes showed that there was a significant decrease in the levels of chloroplast rRNAs in yellow section of young leaves in the mutant when equal amount of total RNA were loaded (Fig. 9b). However, the amount of nuclear encoded 25S and 18S rRNAs was increased in dg169 (Fig. 9c). Chloroplast DNA replication was not impaired in dg169 whether such a decrease was associated with the expression of plastid- and nuclear-encoded genes. We used semiquantitative RT-PCR and quantitative real-time PCR methods to analyze the levels of transcripts that encode several chloroplast proteins transcribed by PEP and/or NEP in wild-type and mutant plants. psaA, psbA, psbB, psbC, psbD, and rbcL were selected as PEP-dependent genes (class I); accD, rpoA, and rpoB were selected as NEPdependent genes (class III); clpP and atpB were chosen as

Purine nucleotides are also the substrates for DNA synthesis. The reduced purine levels may affect plastid genome replication and division and have an effect on chloroplast development. To investigate whether chloroplast DNA replication was affected in dg169, the ratio of chloroplast genome to nuclear genome was investigated by quantitative PCR method as described by Garton et al. (2007). We observed that the ratio of chloroplast genome

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Photosynth Res Fig. 8 Differential accumulation of chloroplast and nuclear genes mRNA in mature leaves of wild-type (wt) and the yellow section of young leaves of dg169. a RT-PCR analyses of chloroplast and nuclear gene expression. Total RNAs were isolated from young leaves of wild-type and dg169 and were reverse transcribed to cDNA using random hexamer primers. The expression of chloroplast and nuclear genes was determined using semiquantitative PCR with gene specific primers. b Qunantitative-PCR analyses of chloroplast and nuclear gene expression. The log2 (mu/wt, mutant/wild-type) values were calculated and normalized by using Actin as a reference. I. PEP-dependent chloroplast genes; II. NEP-dependent chloroplast genes; III. Both PEP and NEP dependent chloroplast genes. Values shown are average of three independent replicates. Error bars indicate SD

to nuclear genome was increased in yellow section of young leaves of dg169 (Fig. 10). These results suggest that the impaired chloroplast development in dg169 may be not associated with chloroplast DNA replication.

Discussion Arabidopsis pigment deficiency mutants are often an important tool to investigate the molecular mechanism of chloroplast development. In this study, we characterized a

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delayed greening mutant dg169, which is due to a missense mutation that converts conserved Ile-155 to Asn in ATase2. The most distinct characteristics of dg169 were the retarded greening of true leaves. The young leaves were initially chlorotic, then gradually greened during development in the dg169 mutant, and the mature and green leaves showed similar phenotype to those of wild-type plants (Fig. 1). The delayed greening phenotype in the dg169 mutant in this study was not observed in the knock-out mutants of ATase2 which show an irregular chlorosis phenotype as reported previously (Hung et al. 2004; van

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Fig. 9 Chloroplast rRNA profile in mature leaves of wild-type (wt), the yellow section of young leaves and mature leaves of dg169. a Schema of chloroplast rrn operon in Arabidopsis. The four genes for chloroplast rRNAs (23S, 16S, 5S, and 4.5S) as well as two tRNA genes (tRNA-I and tRNA-A) are arranged in one operon (rrn operon). They are transcribed as a large polycistronic RNA and then were processed to yield the mature rRNA and tRNA species. b Accumulation of individual chloroplast rRNAs. Northern blot analyses were

Fig. 10 Ratios of chloroplast genomes per nuclear genome in mature leaves of wild-type (wt), the yellow section of young leaves and mature leaves of dg169. Values are average of three independent experiments. Error bars indicate SD

der Graaff et al. 2004). Thus, our results suggest that ATase2 is required for the early chloroplast development. To investigate how chloroplast development is affected in

conducted using equal amounts of total RNAs (5 lg) from leaves of 3-week-old wild-type (wt) and dg169. Full-length sequences of four rRNAs were used as probes for RNA gel blot analyses. c Total RNA profile of wild-type (wt) and dg169. Equal amounts of leaf RNA (5 lg) were separated by employing formaldehyde denaturing agarose gel electrophoresis and stained by ethidium bromide

the dg169 mutant, we examined the changes in ultrastructure of chloroplasts in wild-type and mutant plants. Our results showed that the size and shape of chloroplasts as well as the formation of thylakoid membranes in the yellow section of young leaves in dg169 were disturbed severely. There were no grana thylakoid membranes, but only a few of stroma thylakoid membranes were observed (Fig. 2). In addition, we also examined the changes in the functions of PSII and PSI in the yellow section of the mutant. Our results showed that there was a significant decrease in Fv/Fm and UPSII in the mutant, suggesting that the PSII function was severely impaired (Fig. 3a; Table 1). The analysis of chlorophyll fluorescence decay kinetics indicates that electron transfer of acceptor side of PSII was impaired whereas electron transfer of donor-side was not affected (Fig. 4; Table 2). A significant decrease in an absorbance change of P700 suggests that the PSI function was also severely impaired in young leaves of the mutant (Fig. 3b; Table 1). Furthermore, we examined whether the impaired thylakoid membrane biogenesis in the yellow section of young leaves in the mutant was related to less accumulation of PSII and PSI complexes. Our results show

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that there was a significant decrease in the contents of the core subunits of PSII and PSI as well as the functional PSII and PSI complexes in the yellow section of young leaves in the mutant (Fig. 5). Although the chloroplast development deficiency in the atase2 mutant was observed for a long time, the exact mechanism linking atase2 mutation to chloroplast development remains unclear. Defect in protein import into chloroplasts and a combined effect of direct light exposure and light-influenced systemic signals have been proposed previously (Hung et al. 2004; Woo et al. 2011). It has also been proposed that RNA synthesis could be reduced by a block in purine nucleotide synthesis in atase2 mutants, which may offer another mechanism whereby loss of ATase function leads to the observed physiological symptoms (Walsh et al. 2007). Our results showed that there was a significant decrease in the levels of of PSII and PSI core subunit proteins in the yellow section of young leaves of dg169 (Fig. 5). Such a decrease may be due to the reduction of corresponding transcripts (Fig. 8). More importantly, our results showed that there was a significant decrease in PEP activity in the yellow section of young leaves of dg169 (Fig. 8). Previous studies have demonstrated that Drpo mutants that do not accumulated PEP show impaired chloroplast development (Serino and Maliga 1998; Krause et al. 2000). In addition, ptac mutants and mutants with lesions in PEP function also show retarded chloroplast development (Pfalz et al. 2006; Chi et al. 2008; Myouga et al. 2008; Arsova et al. 2010; Gao et al. 2011; Gilkerson et al. 2012; Yagi et al. 2012; Yu et al. 2013; Zhong et al. 2013). Moreover, several studies have shown that the decrease in PEP activity resulted in the delayed greening phenotype (Ishizaki et al. 2005; Chi et al. 2008, 2010). Therefore, our results suggest that the delayed greening in the dg169 mutant may be associated with the decreased PEP activity and that ATase2 plays an important role in maintaining the PEP transcription activity. How ATase2 regulates PEP-dependent transcription? Our results showed that the mutation of ATase2 resulted in the decrease in the levels of purine nucleotides in the yellow section of young leaves of dg169 (Fig. 6). Purine nucleotides are substrates for nucleic acid synthesis. Thus, the decrease in the levels of purine nucleotides will results in the decrease in the transcription of chloroplast genes. In chloroplasts, transcription is mediated by PEPs and NEPs. PEP transcribes many photosynthesis genes whereas NEP transcribes a few house-keeping genes (Hajdukiewicz et al. 1997; Bruce Cahoon and Stern 2001). During early chloroplast development, PEP activity increases tremendously while NEP activity remains low (Hanaoka et al. 2005). Our results show that there was a significant decrease in PEP activity but an increase in NEP activity in the yellow section of young leaves of dg169 (Fig. 8). These

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results suggest that the decrease in the levels of purine nucleotides in dg169 may not provide sufficient material for tremendously increased PEP activity and thus result in a significant decrease in PEP activity in early chloroplast development. On the other hand, the limited decrease in the levels of purine nucleotides in dg169 may be sufficient for sustaining NEP activity and therefore an increase in NEP activity was observed in dg169. PEP is multisubunit polymerase. In addition to the plastid-encode catalytic core subunits, a number of nuclearencoded polypeptides, such as pTACs and proteins located in nucleoids, have been shown to be associated with PEP and may regulate its activity. Mutants of these PEP regulators often showed albino, chlorosis, or delayed greening phenotype (Pfalz et al. 2006; Gao et al. 2011; Yu et al. 2013). We found that ATase2 is also localized to nucleoids (Fig. 7). Thus, it’s possible that ATase2 may act as a PEP regulator and is involved in the regulation of PEP transcription machinery during early stages of chloroplast development and the decrease of PEP-dependent transcripts in dg169 might be due to impairment of the PEP transcription machinery. It’s also possible that the reduced level of PEP-dependent transcripts in the dg169 mutant is due to the decreased stability of the PEP-dependent transcripts. It has been reported that some nuclear-encoded proteins are important to stabilize the mRNA transcripts in chloroplast (Boudreau et al. 2000; Meurer et al. 2002). Deletion of these proteins leads to the less accumulation of corresponding transcripts. Since there is no known domain for RNA binding in ATase2, it may be involved in this process by interacting with other proteins which have RNA binding domain, such as PPR proteins. As discussed above, our results suggest that the delayed greening phenotype in the dg169 mutant is associated with decreased transcription, i.e. decreased PEP activity. However, recent studies have suggested that reduced translation in the chloroplast may also lead to the delayed greening phenotype. It has been reported that knockdown of RH22, a DEAD RNA helicase which was involved in the chloroplast ribosomal assembly in Arabidopsis, resulted in the phenotype of delayed greening (Chi et al. 2012). It has also been reported that SUPPRESSOR OF VARIEGATION3 (SVR3) gene encodes a putative TypA-like translation elongation factor. The svr3 mutant showed uniformly pale green leaves under normal temperature conditions (22 °C). However, the svr3 mutant showed a delayed greening phenotype under chilling stress conditions (8 °C) that is due to abnormal chloroplast rRNA processing and chloroplast protein accumulation (Liu et al. 2010). We observed the dramatic decrease in plastid-encoded rRNAs in yellow section of young leaves of dg169 (Fig. 9b) that may lead to a decrease in translation in the chloroplast. Therefore, the delayed greening phenotype in the dg169

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mutant may be also associated with decreased translation in the chloroplast. However, the definite function of ATase2 in chloroplast gene expression and its possible targets remain to be investigated further. Acknowledgments This work was supported by the State Key Basic Research and Development Plan of China (2015CB150105) and the Key Research Programme of the Chinese Academy of Sciences (KGZD-EW-T05).

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