Two NADPH: Protochlorophyllide Oxidoreductase (POR) Isoforms Play Distinct Roles in Environmental Adaptation in Rice
© The Author(s). 2017
Received: 16 November 2016
Accepted: 29 December 2016
Published: 11 January 2017
NADPH: protochlorophyllide oxidoreductase (POR) is an essential enzyme that catalyzes the photoreduction of protochlorophyllide to chlorophyllide, which is ultimately converted to chlorophyll in developing leaves. Rice has two POR isoforms, OsPORA and OsPORB. OsPORA is expressed in the dark during early leaf development; OsPORB is expressed throughout leaf development regardless of light conditions. The faded green leaf (fgl) is a loss-of-function osporB mutant that displays necrotic lesions and variegation in the leaves due to destabilized grana thylakoids, and has increased numbers of plastoglobules in the chloroplasts. To investigate whether the function of OsPORA can complement that of OsPORB, we constitutively overexpressed OsPORA in fgl mutant.
In the 35S:OsPORA/fgl (termed OPAO) transgenic plants, the necrotic lesions of the mutant disappeared and the levels of photosynthetic pigments and proteins, as well as plastid structure, were recovered in developing leaves under natural long days in the paddy field and under short days in an artificially controlled growth room. Under constant light conditions, however, total chlorophyll and carotenoid levels in the developing leaves of OPAO plants were lower than those of wild type. Moreover, the OPAO plants exhibited mild defects in mature leaves beginning at the early reproductive stage in the paddy field.
The physiological function of OsPORB in response to constant light or during reproductive growth cannot be completely replaced by constitutive activity of OsPORA, although the biochemical functions of OsPORA and OsPORB are redundant. Therefore, we suggest that the two OsPORs have differentiated over the course of evolution, playing distinct roles in the adaptation of rice to the environment.
KeywordsRice Faded green leaf OsPORA OsPORB Chlorophyll synthesis
In angiosperms, chlorophyll (Chl) absorbs light and imparts its energy to other components of the electron transport chain during photosynthesis (Grossman et al. 1995; Barber et al. 2000). Chl is an essential compound in higher plants. However, the intermediate compounds in Chl synthesis can bind to oxygen molecules, leading to the production of reactive oxygen species (ROS) including singlet oxygen radicals (op den Camp et al. 2003). The accumulation of ROS accelerates cellular signaling pathways or oxidizes cellular elements, leading to photo-oxidative damage and cell death (Kim et al. 2008). Therefore, Chl synthesis must be precisely controlled during chloroplast development (Sakuraba et al. 2013). During Chl synthesis, NADPH: protochlorophyllide oxidoreductases (PORs) catalyze the light-dependent conversion of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a critical intermediate step in the process (Virgin et al. 1963; Henningsen 1970; Griffiths 1978; Masuda and Takamiya 2004).
In angiosperms, during skotomorphogenesis in the dark, PORs combine with NADPH and Pchlide to produce a ternary complex that makes up the main protein component of the prolamellar body (PLB) in the etioplast (Schoefs and Franck 2003; Paddock et al. 2012). The PLB, which has a lattice-like structure, also contains Chl precursors, carotenoids, and lipids (Rosinski and Rosen 1972; Selstam and Sandelius 1984). The NADPH: POR: Pchlide ternary complex reduces the D-ring in Pchlide to produce Chlide, which is esterified and modified to create Chl a and Chl b in a light-dependent manner (Apel et al. 1980; Lebedev and Timko 1998; Heyes and Hunter 2005). Through the process of light-induced greening, the PLB in the etioplast collapses and the grana thylakoids emerge, resulting in the formation of chloroplasts (Virgin et al. 1963; Henningsen 1970). This photomorphogenesis process in higher plants begins with the visible accumulation of Chl, which is a consequence of the functions of the NADPH: POR: Pchlide ternary complex (Solymosi et al. 2007). At the same time, the transition from etioplasts containing PLBs to chloroplasts with mature thylakoids is closely associated with the role of POR in Chl synthesis (Solymosi et al. 2007). Thus, POR is ultimately involved in the formation of thylakoid membranes in higher plants (Forreiter et al. 1991).
Many gymnosperms, algae, and cyanobacteria can synthesize photosynthetically competent chloroplasts, even in the dark, because they contain two different enzymes, light-dependent POR (LPOR) and dark-operative POR (DPOR) (Forreiter and Apel 1993; Shui et al. 2009); LPOR requires light for its function, but DPOR can function in the absence of light. By contrast, angiosperms possess only LPORs; the functional deficiency of DPOR in higher plants may be related to the adaptation of specific POR regulatory mechanisms (Masuda and Takamiya 2004; Paddock et al. 2010). In addition, phylogenic analysis suggests that gene duplication might have resulted in the formation of POR families (Masuda and Takamiya 2004).
The roles of PORs have been well-studied in angiosperms, including Arabidopsis thaliana (Armstrong et al. 1995; Benli et al. 1991; Oosawa et al. 2000), Nicotiana tabacum (Zavaleta-Mancera et al. 1999; Masuda et al. 2002), Zea mays (Millerd and McWilliam 1968; Hopkins 1982; Hopkins and Elfman 1984), Avena sativa (Darrah et al. 1990), Hordeum vulgare (Schulz et al. 1989; Holtorf et al. 1995), Triticum aestivum (Teakle and Griffiths 1993), Cucumis sativus (Yoshida et al. 1995; Fusada et al. 2000), Amaranthus tricolor (Iwamoto et al. 2001), Brassica oleracea (Solymosi et al. 2004), and Pisum sativum (Spano et al. 1992). Three POR genes (AtPORA, AtPORB, and AtPORC) have been identified in Arabidopsis (Armstrong et al. 1995; Oosawa et al. 2000). Whereas barley and tobacco have two isoforms of POR (Holtorf et al. 1995; Masuda et al. 2002), cucumber and pea possess only a single POR (Spano et al. 1992; Fusada et al. 2000).
The biological functions of PORs in barley and Arabidopsis are well known, as these plants are representative monocots and dicots, respectively. The two POR isoforms in barley are distinct, although the enzymatic activities of HvPORA and HvPORB in vitro are similar (Holtorf et al. 1995). For example, HvPORA is mainly expressed in etiolated seedlings and is downregulated by illumination, but HvPORB is constitutively expressed during leaf development, regardless of light conditions (Holtorf et al. 1995). In Arabidopsis, the functions and expression patterns of AtPORA and AtPORB are analogous to those of the two PORs in barley (Armstrong et al. 1995; Runge et al. 1996). However, AtPORC is upregulated in the light and downregulated in the dark (Oosawa et al. 2000; Su et al. 2001). Moreover, AtPORC expression strongly increases under high light conditions, indicating that AtPORC activity is crucial for protecting Chl from breakdown under excessive light during plant development (Masuda et al. 2003).
Rice has two POR isoforms, OsPORA and OsPORB. A mutation in OsPORB (osporB) was first identified in a study of the faded green leaf (fgl) mutant, which displays necrotic lesions and variegation during leaf development (Sakuraba et al. 2013). The defects in fgl mutant are strongly associated with the reduced expression of OsPORA and the lack of OsPORB activity in developing leaves (Sakuraba et al. 2013). Also, the formation of ROS, which is induced by the accumulation of non-photoactive Pchlide, causes the necrotic phenotype in the fgl mutant leaves (Chakraborty and Tripathy 1992; op den Camp et al. 2003; Sakuraba et al. 2013). The biological functions of OsPORA and OsPORB are highly similar to those of HvPORA and HvPORB, respectively (Sakuraba et al. 2013). In addition, OsPORB is upregulated under high light treatment, suggesting that the function of OsPORB overlaps with that of AtPORC in Chl synthesis and maintenance (Sakuraba et al. 2013).
Although some species, including cucumber and pea, have only one POR, many photosynthetic plants have two or three isoforms of POR. Therefore, por mutants must be identified and characterized in every angiosperm to explore the biological diversity of POR members in detail, for example by studies of Arabidopsis POR isoforms (Frick et al. 2003; Masuda et al. 2003; Paddock et al. 2012; Sakuraba et al. 2013). In addition to studying por mutants, the functional redundancy of PORs has been examined by producing genetically modified Arabidopsis plants. Constitutive overexpression of AtPORA can restore the phenotypes of the atporB atporC double mutant (Paddock et al. 2010). In the current study, based on this finding, we explored whether OsPORA can fully substitute for OsPORB by producing transgenic rice plants constitutively overexpressing OsPORA in the fgl mutant background. We analyzed the transgenic plants under various growth conditions, finding that the function of OsPORB can be replaced by constitutively expressing OsPORA during vegetative growth under normal long days in paddy field conditions, but not under excessive light conditions or during reproductive growth. We discuss the physiological and developmental differentiation of the two rice PORs.
Constitutive Overexpression of OsPORA in fgl Mutant Background Rescues Chl Synthesis and Leaf Development under Natural Field Conditions
OsPORA Overexpression Restores Photosynthetic Proteins and Pigments and Inhibits ROS Production in fgl Mutant
Next, we measured photosynthetic pigments in the leaf tissues of WT, fgl, and OPAO plants. Total Chl concentrations increased and the Chl a/b ratio decreased in the three OPAO lines compared to fgl mutant (Fig. 1e and f). The Chl level in OPAO line #11 was similar to that of WT, indicating that constitutive expression of OsPORA fully recovered Chl synthesis in fgl mutant background. Moreover, the total carotenoid content was fully recovered in line #11 (Fig. 1g). Similarly, the photosynthetic protein levels were higher in the OPAO lines than in fgl mutant (Fig. 1h). Thus, the levels of Chl synthesis and the formation of the photosynthetic apparatuses in the OPAO lines were nearly proportional to the expression levels of OsPORA. These results strongly suggest that OsPORA can substitute for OsPORB in Chl synthesis if OsPORA is constitutively expressed in developing leaves under natural growth conditions.
Yellow/white leaf variegation in fgl mutant is induced by excessive accumulation of ROS such as singlet oxygen (1O2) (Sakuraba et al. 2013). We thus examined the levels of singlet oxygen in the OPAO lines. In contrast to fgl mutant, singlet oxygen did not accumulate in the three OPAO lines (Fig. 1i), suggesting that the recovery of the necrotic lesion phenotype in these lines is related to the decrease in ROS.
OPAO Plants Exhibit Normal Chloroplast and Etioplast Structure
Overexpression of OsPORA Restores Normal Total and Photoactive Pchlide Levels
OPAO Plants Show Partially Rescued Chl and Carotenoid Contents under Constant Light Conditions
Next, we analyzed the phenotypes of the other OPAO lines (line #2 and #27; Fig. 1) in more detail. The three OPAO lines had different levels of OsPORA mRNA (Fig. 1d and Additional file 2: Figure S2) and displayed dark (line #11), intermediate (line #2), and light green (line #27) leaf color (Fig. 4b). In line #27, which had the lowest OsPORA mRNA level among OPAO lines, the leaf color and photosynthetic protein levels were similar to those of fgl mutant (Fig. 4b and c). We also examined total Chl contents, Chl a/b ratios, and total carotenoid levels in leaves (Fig. 4d-f). In contrast to the results under natural conditions, the total Chl and carotenoid contents in the three OPAO lines did not recover to WT levels (Fig. 4d and f). In particular, the photosynthetic pigment levels in line #27 were similar to those in fgl mutant, indicating that OsPORA expression in line #27 is below the threshold level required to recover Chl synthesis in the fgl mutant background (Fig. 4d-f). Interestingly, in line #11, with the highest OsPORA mRNA level, the Chl and carotenoid levels did not fully recover to WT levels (Fig. 4d and f). We then performed phenotypic characterization of line #11 plants grown under short day (SD) conditions compared to CL conditions (Additional file 5: Figure S5). Under SD conditions, all leaf-associated phenotypes of line #11 completely recovered to those of WT, in contrast to the results under CL conditions (Additional file 5: Figure S5). This result strongly suggests that Chl synthesis in the absence of OsPORB cannot be completely restored during early development under CL conditions, even though OsPORA mRNA levels are extraordinarily high.
Abnormally high levels of ROS cause the lesion formation and leaf variegation phenotypes in fgl mutant (Sakuraba et al. 2013). Some leaves in line #27 showed variegation and contained ROS such as hydrogen peroxide (H2O2) and superoxide anion radicals (O2 ·-), as was observed in the leaf tissues of fgl, but this was not the case in lines #11 and #2 (Fig. 4g and h). The leaf variegation of line #27 under CL conditions is likely caused by ROS accumulation to levels corresponding to those found in fgl mutants.
OsPORA Expression in OPAO Plants is Consistent under CL and SD Conditions
Recovery of the Expression of Photosynthesis- and Chl Synthesis-Related Genes in the OPAO Lines
Compromised Rescue of OPAO Plants under Natural Field Conditions Beginning at the Early Reproductive Phase
In the model dicot plant Arabidopsis, three homologous POR genes encode structurally similar enzymes that are synthesized as pro-proteins and modified in the plastid (Franck et al. 2000; Frick et al. 2003; Masuda et al. 2003; Paddock et al. 2010; Reinbothe et al. 2015). Even though the sequences of PORs are highly conserved, the mature POR isoforms play somewhat distinct roles during development due to their dissimilar expression patterns (Franck et al. 2000; Frick et al. 2003; Masuda et al. 2003; Paddock et al. 2010; Paddock et al. 2012). Rice contains two POR proteins, OsPORA and OsPORB, which have distinct functions during leaf growth. For example, OsPORA functions in the early stage of leaf development, while OsPORB functions throughout development (Sakuraba et al. 2013). In the current study, by producing transgenic rice plants overexpressing OsPORA in the fgl mutant background, we found that the functional deficiency of OsPORB could be overcome by the ectopic expression of OsPORA. Under CL conditions, however, the Chl contents of the OPAO lines during early development were lower than those of WT (Fig. 4d), in contrast to the results obtained under SD conditions in growth chambers (Additional file 5: Figure S5e) or natural long day conditions in the greenhouse (Fig. 1e). Because the expression levels of OsPORA mRNA and protein in OPAO line #11 were highly upregulated under both SD and CL conditions (Fig. 5b and c), the partial rescue of the mutant phenotype under CL conditions might be related to the lack of OsPORB, indicating that functional variation exists between OsPORA and OsPORB.
The Arabidopsis porA mutants show severe deficiencies in photoautotrophic development, as well as diminished photoactive Pchlide conversion and reduced prolamellar body volume during skotomorphogenesis (Paddock et al. 2012). In addition, the elimination of AtPORA activity during photomorphogenesis results in reduced total Chl contents and abnormal plant growth, although AtPORA is transiently expressed during illumination (Armstrong et al. 1995; Paddock et al. 2012). However, the function of OsPORA in rice is still unclear, since no reports about osporA-deficient mutants are currently available. The rice fgl mutant shows milder defects than Arabidopsis atporB atporC double mutants (Frick et al. 2003; Sakuraba et al. 2013). Because OsPORA is highly expressed in developing leaves at the early seedling stage, the function of OsPORA is considered to be essential for Chl synthesis in rice leaves (Sakuraba et al. 2013). Our observation of OPAO lines suggests that OsPORA functions in Chl synthesis in leaves, even after early development (Fig. 1 and Additional file 3: Figure S3). Our findings imply that the role of OsPORA in rice development is generally controlled by its expression pattern. However, OPAO T2 plants grown under natural field conditions exhibited partially recovered phenotypes in leaf blades beginning at the booting stage (before the heading stage), i.e., approximately 90 days after sowing (Fig. 7a). Finally, the leaf phenotype of the OPAO lines was intermediate between WT and fgl mutant (Fig. 7d-g), suggesting that beginning at the reproductive phase, Chl synthesis is not completely controlled only by OsPORA, although OsPORA expression levels are quite high. Previous and current findings suggest that the difference between the activities of OsPORA and OsPORB might at least be partially attributed to the different spatial and temporal expression patterns of the two OsPORs, as well as their differing protein functions.
A reduction in PLB size leads to reduced total Pchlide levels (Franck et al. 2000; Sakuraba et al. 2013). The PLB size in the OPAO lines was recovered to that of WT, leading to an increase in total Pchlide contents (Fig. 2d-g and 3a). In addition to increased total Pchlide levels, photoactive Pchlide in the OPAO lines accumulated to WT levels (Fig. 3b). This result indicates that although maintaining threshold levels of Pchlide formation in etiolated seedlings appears to require OsPORB activity, ectopic expression of OsPORA is sufficient for rescuing total and photoactive Pchlide levels in fgl mutant. In other words, normal levels of OsPORA in the absence of OsPORB activity are not sufficient for the formation of enough Pchlide to maintain persistent green coloration in mature leaves. Thus, the enzymatic functions of the two OsPORs are redundant during the juvenile growth stage. Moreover, POR-mediated conversion from Pchlide to Chlide is completely light dependent (Griffiths 1978). Pchlide bound to POR is referred to as “photoactive”, whereas Pchlide that is not bound by POR, which is referred to as “non-photoactive”, readily functions as a photosensitizer to produce singlet oxygen, thereby inducing photo-oxidative damage (Matringe et al. 1989; Reinbothe et al. 1996; Sperling et al. 1997; Mock et al. 1998; Heyes and Hunter 2005). In addition, HvPORA plays a photo-protective role during greening through the reconstitution of the light-harvesting POR: Pchlide complex (Buhr et al. 2008). The leaf variegation in fgl mutant might be caused by photosensitization induced by the accumulation of high levels of non-photoactive Pchlide (Sakuraba et al. 2013). Therefore, the rescue of necrotic lesions in the OPAO lines is closely related to the disappearance of ROS, including singlet oxygen, generated from non-photoactive Pchlide (Figs. 1i and 4g-h). In addition, ROS accumulation in fgl chloroplasts likely provokes the downregulation of Chl synthesis and photosynthetic genes in the nucleus via retrograde signaling from the chloroplast (Gadjev et al. 2006; Sakuraba et al. 2013). Indeed, the transcript levels of photosynthesis-associated genes (Lhcb1 and Lhcb4), as well as upstream (GSAT and CHLH) and downstream (DVR) genes of POR, were rescued in OPAO line #11 (Fig. 6). These results suggest that the recovery of necrotic lesions in the OPAO lines is caused by the disappearance of ROS derived from the reduction in non-photoactive Pchlide levels due to OsPORA overexpression, which upregulates these genes, thereby leading to the removal of oxidative damage.
OsPORA and OsPORB protein levels are directly proportional to their transcript levels, suggesting that OsPOR activity is mainly controlled at the transcriptional level (Sakuraba et al. 2013). In particular, the levels of OsPORA mRNA and protein are rapidly downregulated under long periods and high levels of illumination (Sakuraba et al. 2013). Similarly, Arabidopsis PORA (AtPORA) mRNA is barely detectable in leaf tissues of plants grown in the light, in contrast to AtPORB and AtPORC mRNA levels (Armstrong et al. 1995; Su et al. 2001). In the OPAO lines, we predicted that OsPORA would be constitutively expressed throughout development. As expected, OsPORA transcript levels in OPAO line #11 were consistent under both SD and CL conditions (Fig. 5b). Moreover, OsPORA protein levels did not differ between SD and CL conditions (Fig. 5c), indicating that excessive light does not affect the protein stability of OsPORA. However, Chl synthesis in the OPAO plants was not completely restored under CL conditions (Fig. 6d), although the levels of OsPORA protein remained high independent of light period. Hence, the compromised recovery of leaf greenness in the OPAO lines under CL conditions may be due to the weakening of OsPORA activity upon exposure to excessive light, and thus, the overall POR activity drops below the threshold level, which is not sufficient for producing Pchlide for Chl synthesis in the absence of light-stable OsPORB activity.
Many studies have investigated the possibility of the functional redundancy of the POR isoforms, as their exact functions in plant development are currently unclear, although their sequences are highly conserved (Sperling et al. 1998; Franck et al. 2000; Masuda et al. 2003; Frick et al. 2003; Paddock et al. 2010; Paddock et al. 2012). The amino acid sequences of OsPORA and OsPORB, the two POR isoforms in rice, are remarkably similar, and POR enzymes are highly conserved among angiosperms (Sakuraba et al. 2013). Because of this sequence conservation, we hypothesized that the two rice POR proteins are functionally equivalent, despite their distinct expression patterns. The Arabidopsis atporB atpocC double mutants are totally recovered by ectopic expression of AtPORA (Paddock et al. 2010). In the current study, however, overexpressing OsPORA in plants lacking OsPORB gave incomplete rescue of leaves, but only under CL conditions at the early vegetative stage and beginning at the early reproductive phase under natural field conditions (Figs. 4 and 7). Both lesion formation and degreening in the fgl mutant under natural field conditions (beginning at the tips of older leaves) are caused by the suppression of OsPORA expression, resulting in the complete absence of POR activity (Sakuraba et al. 2013). In this study, however, we found that in developing leaves under excessive light conditions and in mature leaves beginning at the reproductive stage, even high levels of OsPORA mRNA and protein failed to fully substitute for OsPORB activity, which is required for normal levels of Chl synthesis.
OsPORs, which are essential for Chl synthesis, are present in two isoforms in rice, but the biological diversity of their roles has been unclear. We generated transgenic plants to investigate the functional differences between OsPORA and OsPORB under various growth conditions. Although the constant presence of OsPORA activity restored the leaf phenotype of fgl mutant, the function of OsPORB was not fully replaced by OsPORA activity under certain growth conditions. Taken together, our findings indicate that the functional differentiation of the two rice PORs is closely associated with maintaining the photosynthetic activity of leaves until senescence. Thus, we propose that the two OsPORs have differentiated during evolution to play distinct roles in the adaptation of rice to the environment.
Plant Materials and Growth Conditions
The single recessive fgl mutant was previously generated from japonica rice cultivar ‘Kinmaze’ by methyl nitrosourea (MNU) mutagenesis as previously described (Iwata and Omura 1975). For phenotypic characterization, plants were grown under natural long days (~14 h light/day) in a paddy field (Suwon, Republic of Korea, 37° N latitude), greenhouse (Seoul, Republic of Korea), and growth chambers. The chamber conditions were: 24 h light at 30 °C for CL and 10 h light at 30 °C/14 h dark at 24 °C for SD with 70% relative humidity. Light-emitting diodes (LEDs) were used and the average photon flux density was 200 μmol m−2 s−1.
Vector Construction and Rice Transformation
OsPORA cDNA was obtained by reverse-transcription polymerase chain reaction (RT-PCR) with total RNA extracted from the leaves of japonica rice cultivar ‘Nipponbare’ using gene-specific primers (Additional file 6: Table S1). OsPORA was ligated into the pCR8/GW/TOPO plasmid (Invitrogen). To overexpress OsPORA in fgl mutant, full-length OsPORA cDNA was subcloned into the pMDC32 Gateway binary vector containing the cauliflower mosaic virus 35S promoter (Curtis and Grossniklaus 2003). The recombinant plasmid was introduced into calli generated from mature embryos from fgl mutant seeds through Agrobacterium (strain EHA105)-mediated transformation (Jeon et al. 2000). Transgenic plants were grown in Murashige and Skoog medium for 2 weeks and transferred to soil. Vector insertion in the transgenic plants was confirmed by PCR with primers for the pMDC32 vector and OsPORA fragment (Additional file 6: Table S1).
Measurement of Photosynthetic Pigments
To evaluate total chlorophyll and carotenoid concentrations, pigments were extracted from leaves with 80% acetone. Chlorophyll and carotenoid levels were measured with a UV/VIS spectrophotometer (BioTek) as described previously (Lichtenthaler 1987). Total Pchlide was extracted from leaf blades with 80% acetone containing 0.1 N ammonium hydroxide at 4 °C. Total Pchlide was measured in etiolated seedlings grown in the dark for 10 days. Photoactive Pchlide levels were calculated based on the difference between total Pchlide and non-photoactive Pchlide, which was determined in illuminated etiolated seedlings. Pchlide and photoactive Pchlide concentrations were calculated using a Cary Eclipse fluorescence spectrophotometer (Varian) or UV/VIS spectrophotometer (BioTek). The slit widths of both excitation and emission in the fluorescence spectrophotometer were set at 10 nm, and the excitation and emission wavelengths were 433 nm and 634 nm, respectively. The values from fluorescence spectrophotometry were calibrated using an absorption coefficient of 30.4 mM−1 cm−1 (Brouers and Michel-Wolwertz 1983; Sperling et al. 1998). UV/VIS spectrophotometry was performed as described previously (Brouers and Michel-Wolwertz, 1983). All experiments were performed as previously described (Anderson and Boardman 1963; Brouers and Michel-Wolwertz 1983; Sperling et al. 1998; Masuda et al. 2003; Paddock et al. 2010; Paddock et al. 2012).
Transmission Electron Microscopy (TEM) Analysis
Leaf samples for TEM analysis were harvested from 3-month-old plants grown in the greenhouse and from 10-day-old dark-grown etiolated seedlings. Whole tissue preparation was carried out as described previously (Park et al. 2007). Segments of leaf tissues were fixed in modified Karnovsky’s fixative (2% paraformaldehyde, 2% glutaraldehyde, and 50 mM sodium cacodylate buffer, pH 7.2) and washed three times with 50 mM sodium cacodylate buffer, pH 7.2, at 4 °C for 10 min. The samples were post fixed with 1% osmium tetroxide in 50 mM sodium cacodylate buffer, pH 7.2, at 4 °C for 2 h and briefly washed twice with distilled water at 25 °C. The samples were en bloc stained in 0.5% uranyl acetate at 4 °C for a minimum of 30 min, dehydrated in a gradient series of ethanol and propylene oxide, and embedded in Spurr’s resin. After polymerization at 70 °C for 24 h, the sections were sliced to 60 nm with an ultramicrotome (MT–X; RMC) and stained with 2% uranyl acetate for 5 min and Reynolds’ lead citrate for 2 min at 25 °C. The processed samples were then examined under a JEM-1010 EX electron microscope (JEOL).
Detection of singlet oxygen was conducted as previously described with some modifications (Sakuraba et al. 2013). For singlet oxygen staining, 3-month-old leaves were infiltrated in the dark with a solution of 200 μM Singlet Oxygen Sensor Green reagent (SOSG, Invitrogen) in 50 mM sodium potassium buffer (pH 7.5). After 30 min incubation, the leaf discs were washed once with 50 mM sodium potassium buffer and three times in distilled water. The fluorescence emission of SOSG was detected by confocal laser scanning microscopy (LSM510, Carl Zeiss). The excitation and emission wavelengths were 480 nm and 520 nm, respectively. Detection of hydrogen peroxide and superoxide by 3,3-diaminobendizine (DAB) and nitroblue tetrazolium chloride (NBT) staining, respectively, was performed as previously described (Han et al. 2012).
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot Analysis
Photosynthesis-related and OsPOR proteins were detected as previously described, with some modifications (Han et al. 2012; Kwon et al. 2015a). Leaf tissue (10 mg) was homogenized in 100 μl of SDS sample buffer (50 mM Tris, pH 6.8, 2 mM EDTA, 10% w/v glycerol, 2% SDS, and 6% β-mercaptoethanol), and the extracted proteins were denatured at 100 °C for 3 min, resolved by SDS-PAGE, and immunoblotted. All antibodies were obtained from Agrisera. Rubisco large subunit (RbcL) was visualized by staining with Coomassie Brilliant Blue reagent (Sigma-Aldrich).
RT-PCR and Quantitative Reverse-transcription PCR (qRT-RCR)
RT-PCR and qRT-PCR were carried out as previously described with slight modifications (Kwon et al. 2015b). Total RNA was extracted from leaves using a Total RNA Extraction Kit (MGmed, Seoul, Republic of Korea). First-strand cDNA was synthesized from 2 μg total RNA using oligo(dT)15 primer and M-MLV reverse transcriptase (Promega). The transcript levels of genes were detected using gene-specific primers (Additional file 6: Table S1). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control (Additional file 6: Table S1). The 20 μl reactions included 2 μl of 0.5 μM primer, 2 μl of cDNA mixture, and 10 μl of 2X GoTaq qPCR Master Mix (Promega). PCR was performed with a Light Cycler 2.0 instrument (Roche) using the following program: 94 °C for 2 min, 40 cycles of 94 °C for 15 s and 60 °C for 1 min.
Reactive oxygen species
NADPH: protochlorophyllide oxidoreductase
- fgl :
faded green leaf
Transmission electron microscopy
Singlet oxygen sensor green
Nitroblue tetrazolium chloride
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Rubisco large subunit
Glyceraldehyde phosphate dehydrogenase
Mg-chelatase H subunit
3,8–divinyl chlorophyllide a 8–vinyl reductase
light-harvesting Chl a/b-binding protein of photosystem II
light-harvesting Chl a/b-binding protein of photosystem I
Photosystem II protein C
Photosystem I protein A
Days after sowing
We thank Yong-Jae Kim at the National Center for Inter-University Research Facilities (NCIRF) for technical assistance with CLSM analysis and Jihyeon Choi for technical support with the Cary Eclipse fluorescence spectrophotometer. This work was carried out with the support of the Cooperative Research Program for Agriculture & Technology Development (Project No. PJ011063), Rural Development Administration, Republic of Korea. Choon-Tak Kwon was supported by a postdoctoral fellowship from the Basic Science Research Program (NRF-2015R1A6A3A01057535) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education.
CTK and SHK designed and performed all experiments. NCP designed and supervised the project. CTK, SHK, and NCP wrote and edited the manuscript. GS assisted in analyzing the phenotypes of transgenic plants grown under various conditions. DK helped generate the transgenic plants and analyze the phenotypes of the transgenic lines. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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