Defective Etioplasts Observed in Variegation ... - Wiley Online Library

Journal of Integrative Plant Biology 2011, 53 (11): 846–857

Research Article

Defective Etioplasts Observed in Variegation Mutants May Reveal the Light-Independent Regulation of White/Yellow Sectors of Arabidopsis Leaves Wenjuan Wu† , Nabil Elsheery† , Qing Wei†,$ , Lingang Zhang# and Jirong Huang

∗

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200032, China $ Present address: Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 55905, USA # Present address: Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama, Japan † These authors contributed equally to this work. ∗ Corresponding author Tel(Fax): +86 21 5492 4145; E-mail: [email protected] Available online on 10 October 2011 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/j.1744-7909.2011.01079.x

Abstract Leaf variegation resulting from nuclear gene mutations has been used as a model system to elucidate the molecular mechanisms of chloroplast development. Since most variegation genes also function in photosynthesis, it remains unknown whether their roles in photosynthesis and chloroplast development are distinct. Here, using the variegation mutant thylakoid formation1 (thf1) we show that variegation formation is light independent. It was found that slow and uneven chloroplast development in thf1 can be attributed to defects in etioplast development in darkness. Ultrastructural analysis showed the coexistence of plastids with or without prolamellar bodies (PLB) in cells of thf1, but not of WT. Although THF1 mutation leads to significant decreases in the levels of Pchlide and Pchllide oxidoreductase (POR) expression, genetic and 5-aminolevulinic acid (ALA)-feeding analysis did not reveal Pchlide or POR to be critical factors for etioplast formation in thf1. Northern blot analysis showed that plastid gene expression is dramatically reduced in thf1 compared with that in WT, particularly in the dark. Our results also indicate that chlorophyll biosynthesis and expression of plastidic genes are coordinately suppressed in thf1. Based on these results, we propose a model to explain leaf variegation formation from the plastid development perspective. Keywords:

Arabidopsis; chloroplast; etioplast; THF1/Psb29; variegation;

Pchllide oxidoreductase;

Pchlide oxidoreductase;

chloroplast

development. Wu W, Elsheery N, Wei Q, Zhang L, Huang J (2011) Defective etioplasts observed in variegation mutants may reveal the light-independent regulation of white/yellow sectors of arabidopsis leaves. J. Integr. Plant Biol. 53(11), 846–857.

Introduction In addition to serving as a nutritional storage organ for young seedling growth, most cotyledons also function as a photosynthetic tissue supporting the transition from heterotrophy to autotrophy, which is particularly important for plants with small seeds such as Arabidopsis. Chloroplast development in cotyledons takes place twice. For example, Arabidopsis cotyledons turn green as early as 48 hours after fertilization, C

2011 Institute of Botany, Chinese Academy of Sciences

and remain green for more than 10 d, and then chloroplasts re-differentiate into non-photosynthetic plastids, known as eoplastids, during seed maturation (Mansfield and Briarty 1991); eoplastids turn into chloroplasts again during seedling establishment (Mansfield and Briarty 1992). The whole process of chloroplast development in cotyledons is usually divided into two stages under natural or controlled conditions. The first stage is etioplast formation from eoplasts in the dark. The unique structure of etioplasts is that of a prolamellar body (PLB)

Light-Independent Regulation of Leaf Variegation

which is formed by membrane aggregations of semi-crystalline lattices of branched tubules. PLB is primarily composed of lipids, protochlorophyllide (Pchlide), Pchlide oxidoreductase (POR) and NADPH in higher plants (Dehesh and Ryberg 1985). The second stage is chloroplast formation from etioplasts in light. At this stage, PLB turns into photosynthetically active thylakoids with the initiation of chlorophyll biosynthesis and photosynthetic complex assembly. The cotyledon has been extensively used to investigate molecular mechanism of chloroplast development in Arabidopsis (Waters and Langdale 2009). Thylakoid Formation1 (THF1), also named Psb29, which was originally identified in the isolated photosystem II (PSII) complexes from Synechocystis sp. PCC6803 (Kashino et al. 2002), is a highly conserved protein in all oxygenic photosynthetic organisms (Wang et al. 2004). Knockout of THF1 in Arabidopsis results in the variegation phenotype in both cotyledons and leaves, slower growth rates and increased sensitivity of PSII to high light (Wang et al. 2004; Keren et al. 2005), suggesting a role of THF1 in the biogenesis of PSII and thylakoids. Our previous work showed that leaf variegation of thf1 is genetically linked to the reduced level of FtsH complexes (Zhang et al. 2009), which are formed by two types of FtsH subunits, namely type A (FtsH1 and FtsH5/VAR1) and Type B (FtsH2/VAR2 and FtsH8) (Yu et al. 2004, 2005; Zaltsman et al. 2005a; Sakamoto 2006). However, how THF1 mutation leads to leaf variegation remains to be elucidated. To date, the molecular mechanism underlying leaf variegation has been extensively investigated in mutants such as var2 and immutans (im) (Aluru et al. 2006; Yu et al. 2007; Liu et al. 2010b). The severity of variegation of the mutants is always affected by developmental and environmental cues (Zaltsman et al. 2005b; Rosso et al. 2009). In the yellow/white sector, plastid differentiation is arrested at the early stage of thylakoid development (Kato et al. 2007; Sakamoto et al. 2009). Several hypotheses have been proposed to explain how a yellow/white sector arises in the same genetic background of var2 or im (Aluru et al. 2006; Yu et al. 2007; Liu et al. 2010b). One of the generally accepted hypotheses is that chloroplast development requires a threshold of FtsH activity (Yu et al. 2004, 2005). This hypothesis is supported by results from genetic screening for suppressor lines, most of which apparently exhibit a phenotype of delayed chloroplast development. The prolonged duration of plastid differentiation may lower the threshold of FtsH activity necessary for chloroplast development (Yu et al. 2007; Sakamoto et al. 2009). However, this hypothesis still lacks direct evidence because variegation formation in var2 is not dependent on its protease activity on degradation of the substrate D1 protein, which is a subunit of the PSII reaction center (Lindahl et al. 2000; Bailey et al. 2002; Sakamoto et al. 2003; Kato et al. 2009; Zhang et al. 2010). The second hypothesis is deduced from functional analysis of genes suppressing var2 leaf variegation. A number of mutations suppressing the

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var2 variegated-leaf phenotype directly or indirectly lead to a decrease in protein biosynthesis in plastids, indicating that the balance between protein biosynthesis and degradation is an important factor in the fate of chloroplast development (Miura et al. 2007; Yu et al. 2008; Liu et al. 2010a). Recently, the redox state of the stromal plastoquinone (PQ) pool has been proposed to play a key role in the goernance of leaf variegation via studying im-mediated chloroplast development (Rosso et al. 2009; Okegawa et al. 2010). The extent of leaf variegation is positively correlated with an increase in photooxidation, which leads to impaired biogenesis of thylakoid membranes under various environmental stresses (Rosso et al. 2009). This hypothesis is also consistent with the result that enhanced nonphotochemical quenching (NPQ) capacity alleviates photoinhibitory pressure and subsequently suppresses leaf variegation caused by a lack of VAR2 (Yu et al. 2011). Thus, it seems likely that leaf variegation is regulated by multiple mechanisms. In leaves, chloroplasts are developed directly from proplastids, similar to those in cotyledons during embryogenesis (Sakamoto et al. 2008; Tejos et al. 2010). Increasing evidence shows that the regulation of chloroplast development in cotyledons is different from that in leaves. A number of specific genes have been identified to control chloroplast development in leaves or cotyledons (Reiter et al. 1994; Waters et al. 2008; Ruppel et al. 2011). In contrast to var2 which is variegated only in leaves, thf1 displays the variegation phenotype in both cotyledons and leaves. Thus, investigation of THF1-regulated variegation may provide universal insights into molecular mechanism of chloroplast development. Here, we report that THF1 is involved in etioplast development from either proplastids or eoplasts in Arabidopsis.

Results Cotyledon greening of thf1 is slow and uneven To understand how THF1 regulates chloroplast development in cotyledons, we first characterized cotyledon greening of thf1 grown in either light or darkness followed by exposure to light. When seeds were germinated in light for 7 d (Figure 1A) or in darkness for 5 d and subsequently transferred to light for additional 2 d (Figure 1B), cotyledon greening of thf1 was obviously delayed and uneven compared with that of the wild type (WT) under both conditions. The chlorophyll fluorescence image also showed that chlorophyll autofluorescence emitted from thf1 cotyledons was not as uniform as that from WT (Figure 1B). These results indicate that THF1 mutation leads to slow and differential chloroplast development in cotyledons. To further investigate defective plastid differentiation in thf1 cotyledons, the ultrastructure of plastids was examined by transmission electron microscopy. In cotyledons of WT seedlings grown in darkness for 5 d and followed by

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exposure to light for 2 d, chloroplasts were cone shaped and contained thylakoid membranes and starch granules (Figure 1C), indicating that they are photosynthetically active. In contrast, we observed some cells containing developed and undeveloped plastids in thf1 (Figure 1C). In addition, chloroplasts were smaller and contained less thylakoid membranes in thf1 than in WT. Some plastids in thf1 contained the globular vacuolated membrane structure (Figure 1C), called plastidic vacuolated body (PVB), which is thought to be derived from PLB-like structure (Sakamoto et al. 2009). Here, we refer to these abnormal plastids as “veoplasts.” Thus, THF1 mutation severely inhibits chloroplast development in cotyledons.

Etioplast formation is defective in dark-grown thf1 seedlings

Figure 1. Characterization of the variegation phenotype of thf1 cotyledons. (A) Cotyledon greening of 7-d-old seedlings grown under the condition of 16 h light/8 h dark. Bar = 2 mm. (B) Cotyledon greening (left panel, Bar = 2 mm) and chlorophyll fluorescence (right panel, Bar = 200 µm) of 5-d-old dark-grown seedlings exposed to light for 2 d. (C) Ultrastructure of plastids from cotyledons of (B). Cells were shown to contain plastids with (black arrow) or without (white arrow) thylakoids in thf1 cotyledons. PVB, plastid vacuolated body.

To investigate when chloroplast development in thf1 was arrested, we examined ultrastructure of etioplasts in 5-d-old, darkgrown cotyledons. Electromicroscopy imaging showed that etioplasts from WT cotyledons had a typical, highly organized paracrystalline PLB structure (Figure 2A). In thf1 cotyledons, however, we observed various types of plastids that contain normal PLB (Figure 2B), less developed PLB (Figure 2C) and almost no PLB (Figure 2D). About 42.6% (29 of 68 plastids) of plastids had PLB, while the rest lacked any membranous structure. These results suggest that THF1 mutation blocks the differentiation of eoplasts into etioplasts in the dark. We then examined the effect of THF1 mutation on etioplast formation in leaves emerged during dark incubation. To obtain the samples, we first germinated seeds in light for 5 d and then transferred the young seedlings to darkness for 2 weeks, since true leaves will not emerge if seeds are germinated continuously in the dark. As shown in Figure 2E, PLB structure was well formed in WT. In thf1 however, in addition to well

Figure 2. Ultrastructure of plastids from etiolated seedlings. (A−D) A representative etioplast from 5-d-old etiolated cotyledons of WT (A) and thf1 (C, D). Bar = 500 nm. (E−J) A representative etioplast from WT (E), thf1 (F−H) and var2 (I−J) etiolated leaves, which emerged from 5-day-old light-grown seedlings two weeks after transfer to dark conditions. Bar = 1µm.


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developed plastids containing PLB similar to WT (Figure 2F), smaller plastids with less PLB (Figure 2G) and plastids lacking lamellae were observed (Figure 2H). It was estimated that 73% of plastids contained PLB structure in the dark-grown leaves of thf1. Taken together, we conclude that etioplast formation is inhibited in both cotyledons and leaves when thf1 is grown in the dark. To know whether etioplast formation was a common characteristic in leaf-variegated mutants, we examined plastid ultrastruture in the first pair of etiolated leaves of var2. The PLB structure was severely inhibited in var2 etioplasts (Figure 2I, J), indicating that etioplast formation is associated with leaf variegation.

Levels of Pchlide and POR are low in thf1 It is well documented that Pchlide and POR are essential for PLB formation in etioplasts (Butler and Briggs 1966; Klein and Schiff 1972; Franck et al. 2000). We thus analyzed Pchlide content and POR expression in 5-d-old dark-grown seedlings. As shown in Figure 3A, Pchlide content in thf1 was 3-fold less than that in WT, indicating it is possible that the biosynthetic pathway of Pchlide is blocked in thf1. Likewise, POR accumulation was also severely reduced in thf1 compared with WT (Figure 3B). Consistently, light-dependent chlorophyll biosynthesis was extremely slow in thf1 after 5-d-old etiolated seedlings were exposed to light (Figure 3C). Given that PORA is the predominantly expressed isoform in the etiolated seedlings among three PORs and that it is imported into plastids from the cytosol in a substrate (Pchlide)-dependent manner (Kim and Apel 2004), we therefore infer that Pchlide biosynthesis may be a limiting factor for chloroplast development in thf1.

Figure 3. Pchlide content and POR accumulation is low in thf1. (A) Relative Pchlide content in 5-d-old etiolated seedlings of thf1 and WT (Student’s t-test, ∗ P < 0.01); n = 3, error bars indicate ±SD. The data were calculated from the peak of the fluorescence value at 634 nm (insert) on equal fresh weight. (B) Western blot analysis of POR content in 5-d-old etiolated seedlings of thf1 and WT. The antibody recognizes both PORA and PORB proteins (above panel). The gel stained with Coomassie

Pchlide synthesis is not a limiting factor for leaf variegation in thf1 Considering δ-aminolevulinic acid (ALA) synthesis has been known as a limiting step in the Pchlide synthetic pathway, we analyzed ALA content in 5-d-old seedlings grown in either darkness or light. Our results showed that ALA content was significantly lower in thf1 than in WT under both conditions (Figure 4A, B). The thf1 mutant produced 74.8% and 28.6% of ALA content in WT in the dark and light, respectively. Thus, ALA synthesis is correlated with a decrease in Pchlide accumulation in thf1. To test if ALA is a limiting factor for Pchlide synthesis, we first applied a genetic approach to increase ALA content by introducing the flu locus into thf1; FLU negatively regulates ALA synthesis (Meskauskiene et al. 2001; Meskauskiene and Apel 2002). The ALA content in the thf1 flu double mutant increased to the same level as that in WT (Figure 4A). However, the level of Pchlide was still lower in thf1 flu than in WT, but dramatically higher than in thf1 (Figure 4C), suggesting that ALA is not

brilliant blue (CBB) was shown as a loading control (below panel). (C) Time course of chlorophyll accumulation upon illumination of 5d-old etiolated seedlings of thf1 and WT. The values are means ±SD (n = 3). Each replicate contains at least 30 seedlings. FW, fresh weight.

the only limiting step for Pchlide synthesis, and that Pchlide may also be regulated by other steps downstream of ALA in thf1. Phenotypic analysis also showed that 5-d-old etiolated seedlings of thf1 flu were able to survive upon exposure to light, whereas the flu etiolated seedlings died (Figure 4D), indicating that thf1 can suppress the flu phenotype in cell death via reduction of Pchlide accumulated in the dark. Consistently, the double mutant is able to grow under 16 h light/8 h dark photoperiod conditions (Figure 4E). However, thf1 flu seedlings turned green and grew more slowly than thf1 (Figure 4F), and still displayed the variegated phenotype. Thus, we conclude that Pchlide synthesis is not a key factor leading to leaf variegation of thf1.

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Figure 4. Analysis of ALA levels in seedlings of thf1 and WT. (A) ALA content in 5-d-old etiolated seedlings. (B) ALA content in 5-d-old light-grown seedlings. (C) Relative Pchlide content in 5-d-old etiolated seedlings of thf1,thf1 flu and WT. (D) Phenotypes of 5-d-old dark-grown seedlings exposed to light for 2 d. (E) Phenotypes of 7-d-old seedlings of thf1, thf1 flu, flu and WT with a 16-h-light/8-h-dark photoperiod. (F) The phenotype of 7-d-old thf1 and thf1 flu seedlings. Arrows indicate the first pair of true leaves. The values are means ± SD (n = 3). Each replicate contains at least 30 seedlings. ∗ indicates a significant (P < 0.01) difference between WT and a mutant.

Next, we enhanced the ALA level in 5-d-old etiolated seedlings by feeding exogenous ALA. As shown in Figure 5A, the rate of Pchlide synthesis in thf1 was almost the same as in WT after seedlings were fed with ALA, suggesting no significant difference in activity of enzymes required for Pchlide synthesis from ALA between thf1 and WT. However, when the ALA-

treated etiolated seedlings were transferred to light, we found that about half of Pchlide generated in thf1 was not catalyzed into Chlide while Pchlide produced in WT was completely photoconverted (Figure 5B). Taken together, the above results suggested that Pchlide synthesis should not constitute a limiting factor for chloroplast development in thf1.


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Figure 6. Effect of Pchlide accumulation on POR expression in WT and thf1 seedlings. (A) Western blot analysis of POR levels in WT, thf1, ALA-fed thf1 and thf1 flu seedlings. (B) The gel stained with Coomassie brilliant blue is shown as a Figure 5. Analysis of Pchlide accumulation in the ALA-feeding

loading control.

seedlings. (A) Time course of Pchlide accumulation in dark-grown thf1 and WT seedlings after ALA feeding. Data shown are the means ± SD

POR are not critical factors controlling etioplast formation in thf1.

(n = 3). (B) The relative room-temperature fluorescence spectra of ALA-fed seedlings upon exposure to light for 1 h.

Increased Pchlide content does not improve etioplast formation in thf1 To address whether differentiation of eoplasts into etioplasts was improved by increased levels of Pchlide, we examined the number of etioplasts formed in thf1 cotyledons treated with exogenous ALA. Our data showed that 42.6% of plastids contained PLB structure in the absence of ALA, as compared to 44.8% of plastids in the presence of ALA, indicating that etioplast development is not affected by ALA treatment in thf1. We also examined POR expression in ALA-treated or thf1 fluetiolated seedlings. Western blot analysis showed that POR accumulation was indeed enhanced in ALA-treated or thf1 fluetiolated seedlings compared with thf1, but still much less than that in WT (Figure 6). These results imply that Pchlide and/or

Expression of selected plastid genes is suppressed in thf1 We also investigated whether plastid gene expression was altered in thf1. Northern blot analysis showed that transcript levels of the examined plastid genes including rbcL (encoding the large subunit of Rubisco), psaA (encoding a core protein of photosystem I), Glu-tRNA (transfer RNA for glutamate), psbA (encoding the D1 protein of photosystem II) and psbB (encoding the CP47 protein of photosystem II) were significantly reduced in 5-d-old dark-grown seedlings of thf1 in comparison with those of WT and var2. However, transcripts of the nuclear genes such as RbcS (encoding the small subunit of Rubisco) were not affected by THF1 mutation. In addition, no significant difference in the gene expression was detected between thf1 and WT or var2 in 5-d-old, light-grown seedlings (Figure 7). These results indicate that THF1 plays a role in regulation of plastid gene expression either directly or indirectly particularly in the dark.

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Figure 7. Northern blotting analysis of plastid and nuclear gene expression.

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only in leaves, but not in cotyledons. It is not surprising that normal etioplasts are observed in var2 cotyledons. We therefore infer that the tissue examined by Chen et al. (2000) was the cotyledon. Consistently, our ultrastructural analysis revealed that etioplast formation is severely inhibited in the first pair of var2-etiolated leaves. In another variegation mutant im, which encodes a plastid terminal oxidase transferring electrons from plastoquinone (PQ) to oxygen to produce water, it has been reported that carotenoid biosynthesis is defective and its precursor phytoene is over-accumulated in the white sectors (Carol et al. 1999; Wu et al. 1999). Carotenoids have been demonstrated to play an important role in etioplast formation (Park et al. 2002). Carotenoid deficiency leads to a looser attachment of POR to the lipid phase and affects PLB formation (Denev et al. 2005). These results are consistent with data showing that etioplast formation is defective in etiolated seedlings of im (Aluru et al. 2001). Thus, we suggest that blocking etioplast formation may lead to defects in subsequent chloroplast development in variegated leaves.

Total RNA was isolated from 5-d-old seedlings grown in light or darkness. Two micrograms of total RNA were loaded in each lane. The bottom 25S rRNA stained with ethidium bromide is shown as a loading control.

Discussion Heterogeneity of plastid differentiation and leaf variegation In the present study, we characterized the process of chloroplast development in thf1 under light and dark conditions. Our results show that chloroplast development slows down in both cotyledons and leaves of thf1, and homogeneity of plastid differentiation is disturbed as well. Some plastids apparently lack the membranous structure and often contain PVB in cotyledons and leaves of both dark- and light-grown thf1 seedlings. These type of plastids have also been observed in the white sector of var2 and im mutants (Wetzel et al. 1994; Sakamoto et al. 2009). Here we name this type of plastid “veoplast.” The coexistence of etioplasts/chloroplasts and veoplasts in a cell may be a feature of variegated tissues. Etioplasts are regarded as an intermediate stage of chloroplast development (Solymosi and Schoefs 2010). However, whether etioplast formation is associated with the variegation phenotype remains unclear. Results derived from this study support the idea that etioplast formation is required for chloroplast development and is associated with formation of yellow/white sectors in leaf variegation mutants. However, it was reported that etioplasts in dark-grown var2 seedlings resemble those in WT (Chen et al. 2000). It should be noted that VAR2 mutation leads to defect in chloroplast development

Coordinated reduction in Pchlide synthesis and plastid gene expression in thf1 Etioplasts are characterized by a unique inner membrane network, namely PLB that is composed of various components required for thylakoid biogenesis upon exposure to light. These components include proteins, chlorophyll precursors, carotenoids and lipids. Among them, Pchlide and POR have been demonstrated to play an important role in regulation of PLB biogenesis. Our data reveal that PLB formation is tightly associated with levels of Pchlide and POR in darkgrown seedlings of thf1. However, evidence does not support the hypothesis that Pchlide and POR are the limiting factors to control the PLB structure of etioplastids in thf1. First, an increase in Pchlide contents and/or POR accumulation by knockout of FLU in thf1 cannot rescue the variegation phenotype of thf1. Second, feeding thf1 seedlings with exogenous ALA leads to a significant increase in the levels of Pchlide and POR simultaneously, but the number of etioplasts do not increase. Third, overexpressing POR in thf1 has no positive effect on chloroplast development (data not shown). Thus, the lower level of Pchlide in thf1 might be an active response to protect seedlings from photodamage in light. PLB formation may also depend on coordinated expression of nuclear and plastidic genes in addition to the above discussed important factors such as Pchlide, POR, carotenoids and glycolipids. It has been well documented that coordinated gene expression between nuclei and platids plays a critical role in chloroplast development and photosynthesis (Lopez-Juez and Pyke 2005; Pogson and Albrecht 2011). Recently, proteomic analysis of the highly purified PLB from wheat revealed that a number of proteins functioning in photosynthetic light


reaction, Calvin cycle and protein synthesis are already accumulated in PLB (Blomqvist et al. 2008). The presence of many thylakoid photosynthetic complexes such as ATPase, cytochrome b 6 f and FtsH protease were also detected in etioplasts of pea seedlings grown in the dark (Kanervo et al. 2008). Thus, it is proposed that PLBs are precursors of thylakoid membranes and facilitate a rapid formation of the photosynthetic membranes upon perception of light (Blomqvist et al. 2008). Our previous report showed that THF1 mutation leads to a decrease in transcriptional levels of many nuclear genes whose products are targeted to plastids in light-grown seedling (Zhang et al. 2009). In this study, we found that expression of plastid genes is severely reduced in thf1-etiolated seedlings compared with WT. The reduced plastid gene expression may result in defects in PLB formation in the etiolated thf1seedlings. Thus, THF1 plays multiple roles in chloroplast development. Another way in which THF1 may regulate PLB formation is through VAR2/VAR1 complexes. We previously reported that the THF1-mediated variegation phenotype is genetically attributed to FtsH protease, VAR1/FtsH5 and VAR2/FtsH2 (Zhang et al. 2009). Expression of both Type-A and Type-B FtsH subunits is significantly reduced in thf1. However, the molecular mechanism underlying THF1-reguated FtsH stability remains unclear. Interestingly, the FtsH complex consisting of VAR1 and VAR2 was detected in the etioplast and PLB, which is indicative of its function in the biogenesis of multiprotein complexes in the dark (Kanervo et al. 2008; Blomqvist et al. 2008). Consistently, several lines of evidence also support the observation that VAR2-mediated regulation of chloroplast development is independent of its protease activity for D1 degradation during PSII repair (Zaltsman et al. 2005b; Kato et al. 2009; Zhang et al. 2010). It will be interesting to discover the FtsH protease-targeting substrates in etioplast formation.

A model for leaf variegation Although several models have been proposed to explain formation of leaf variegation based on the physiological, genetic and molecular biological evidence derived from studies on var2 and im, it remains largely unknown how chloroplast development is blocked in the white sectors of variegated leaves at the molecular level. One of the central questions that remain to be answered is whether photooxidation is a key factor in determining chloroplast development in variegation mutants. Conflicting results have reported the effect of photooxidation on the degree of leaf variegation (Zaltsman et al. 2005b; Rosso et al. 2009). To address this critical question, it is important to distinguish a role of variegation-controlling genes in chloroplast development and photosynthesis. The etioplast representing an intermediate stage during chloroplast development can be used to study the role of a gene in chloroplast development. In this study, it is significant that etioplast formation is blocked in thf1 and var2 seedlings grown in the dark, indicating that the

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destiny of a plastid is determined as early as the time of etioplast formation. Thus, the function of THF1 or VAR2 in chloroplast development is distinguished from that in photosynthesis. As mentioned above, evidence is increasing to support that the role of VAR2 in chloroplast development is independent of its proteolytic activity in D1 degradation during PSII repair. It was reported that the leaf-variegation phenotype of var2 is rescued by a mutation in FUG1, a homologue to prokaryotic translation initiation factor 2 (cpIF2) located in plastids, but no difference in both impaired D1 degradation and photooxidative stress even under nonphotoinhibitory conditions was observed between var2 and var2 fug1 (Kato et al. 2009), indicating that leaf variegation is uncoupled to FtsH2 activity and photooxidation. Furthermore, it is shown that protease activity dead FtsH2 created by introduction of a point mutation at the catalytic center of the protease domain can complement the leaf-variegation phenotype of var2, indicating that chloroplast development is dispensability of its protease activity (Zhang et al. 2010). Consistently, proteomic analysis also revealed that D1 protein is absent whereas FtsH2-containing complex is accumulated in etioplasts, and that FtsH accumulation is reduced during de-etiolation (Kanervo et al. 2008). The way in which FtsH complexes affect PLB or subsequent thylakoid formation should be addressed in the future. To date, the generally-accepted hypothesis used to explain why a second mutation suppresses leaf variegation is that the threshold of FtsH for chloroplast development is lowered due to delayed chloroplast development (Sakamoto et al. 2009; Liu et al. 2010c). This hypothesis seems unlikely because chloroplast development in the first pair of leaves is actually faster in var2 suppressor lines than in the single mutant var2 (our unpublished data). The coexistence of chloroplasts or etioplasts and veoplasts in a cell of the variegated mutants prompts us to propose a new model for leaf variegation from the viewpoint of the organelle, in which the ratio of chloroplasts or etioplasts to veoplasts might determine a cell to be green or white/yellow. We propose that the veoplast is a middle stage in the course of chloroplast development, and can be developed into the chloroplast once the restriction is removed. Thus, it is important to find the critical factors suppressing formation of PLB or thylakoid membranes in veoplasts. Examination of plastid ultrastructure in the var2 suppressor lines may provide evidence on whether suppressor mutations promote chloroplast development by rescuing veoplastids or inhibiting veoplast formation.

Materials and Methods Plant materials and growth conditions Arabidopsis Columbia ecotype (Arabidopsis thaliana) was used as the wild type. The thf1 mutant was previously characterized (Huang et al. 2006). Seeds for flu mutant (kindly provided

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by Prof. Klaus Apel) were crossed with thf1. The thf1 flu double mutant was characterized by both plastid development deficiency and growth inhibition. Seeds were sterilized and sown on agar plates containing half Murashige and Skoog medium supplemented with 1% (w/v) sucrose under 16-h light (70 µmol m−2 s−1 ) at 22 ◦ C,or under darkness.

Measurement of chlorophyll, pchlide and ALA contents Chlorophyll was extracted with 80% acetone at 4 ◦ C. The content was determined with spectrophotometer (UNIC UV-2102 PCS, Shanghai, China) as described previously (Arnon 1949). For ALA content, 5-d-old etiolated or light-grown seedlings were incubated with 50 mM levulinic acid (Sigma-Aldrich, Germany) in 100 mM sodium phosphate buffer (pH 7.0) for 24 h in the dark or light, respectively. The samples were weighed and frozen in liquid nitrogen for further assay as described previously (McCormac and Terry 2002; Goslings et al. 2004). Pchlide was measured as described previously (Terry and Kendrick 1999; Masuda et al. 2003). Briefly, the extract was centrifuged at 13 000 r/min for 10 min and fluorescence between 550 and 700 nm were recorded in a 970-CRT spectrofluorometer with an excitation wavelength of 440 nm for Pchlide. Pchlide content was calculated from the fluorescence at 634 nm. The results were presented by relative fluorescence/µg.

Transmission electron microscopy and chlorophyll fluorescence observation Samples for electron microscopy observation included cotyledons of 5-d-old etiolated seedlings, cotyledons of the 5-d-old dark-grown seedlings followed by exposure to light for 2 d. We also examined plastid ultrastruture in the first pair of etiolated leaves which emerged during dark incubation (2 weeks) from 5-d-old light-grown seedlings. The materials were fixed and processed as previously described (Harris 1994), and examined with an H-7650 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan). Chlorophyll fluorescence was observed with a LSM 510 META laser scanning confocal microscope (ZEISS, Jena, Germany).

ALA feeding experiments For ALA feeding, 5-d-old dark-grown seedlings were incubated with ALA solution (10 mM ALA (Sigma-Aldrich), 5 mM MgCl 2 , 10 mM phosphate, pH 7.0) in darkness for the indicated time (Falbel and Staehelin 1996).

Western blot analysis Seedlings grown in the dark for 5 d and incubated with ALA for 8 hrs were sampled and ground in liquid nitrogen, then

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resuspended in 1 volume (w:v) of extraction buffer (20 mM Tris, 100 mM NaCl, 2.5 mM MgCl 2 , 1 mM EGTA, 1 mM dithiothreitol, 1% Triton X-100, pH 7.5 and 1:50 protease inhibitor cocktail [Sigma, St. Louis, Missouri]). Total protein was extracted by rocking at 4 ◦ C for 30 min. Samples were then centrifuged at 20 000 g for 20 min at 4 ◦ C. The supernatant was collected, and the total protein was quantified using a Bio-Rad protein assay kit. Protein samples (20 µg per well) were separated by 15% SDS-PAGE. For control, the gel was stained with Coomassie brilliant blue. For immunoblot analysis, proteins were transferred to Hybond-ECL Nitrocellulose membrane (Amersham Biosciences). Immunodetection of the protein gel blots was performed using the ECL plus Western blotting detection system (Amersham biosciences). Signals were detected with a medical film processor (SRX-101A, Konica, Minolta, Tokyo, Japan). Anti-POR antibodies were obtained from Agrisera (Sweden), and used at a dilution of 1: 2000. The level of POR expression was examined by Western blot with an antibody which can recognize all three isoforms in Arabidopsis. We detected one band for POR isoforms (Figure 2C), which is consistent with previous reports that PORA is the primary isoform expressed in darkness (Armstrong et al. 1995).

Northern blot analysis R

Total RNA was isolated using the RNAgents Total RNA Isolation System (Promega) in accordance with the manufacturer’s instructions. Two micrograms of RNA were loaded in each lane, separated on formaldehyde gels, then stained with ethidium bromide to confirm equal loading of RNA. Digoxingenin-labeled DNA probes with gene-specific primers were used in the hybridization solution. These primers are 5 -ATGGCTTCCTCTATGCTCTCTTCC-3 and 5 -TTGGTGGCTTGTAGGCAATGAAAC-3 for Rbcs; 5 -AGA GACTAAAGCAAGTGTTGGGTTC-3 and 5 -AATCAAGTCC ACCACGTAGACATTC-3 for rbcL; 5 -GCCCCCATCGTCTAG TGGTTCAG-3 and 5 -TACCCCCAGGGGAAGTCGAATC-3 for Glu-tRNA; 5 -CAATTGGCGCATTGGTCTTCGCAG 3 and 5 -GTGCTCGCTGTTTCACCAGGGGCTG-3 for and psaA; 5 -TTATCCATTTGTAGATGGAGCCTCA-3 5 -ATGACTGCAATTTTAGAGAGACGCG-3 for psbA; and 5 5 -ATACTGCTCTAGTTGCTGGTTGGGC-3 ATGGGAGTAGTTGCAGAACCATAC-3 for psbB. Northern blotting was conducted as described by Huang et al. (2000).

Acknowledgements We thank Prof. Klaus Apel for providing us the flu mutant, and Dr. Susheng Gan for discussions and comments on the manuscript. This work was supported by the Ministry of Science and Technology of China (2007CB108800 and 2009CB118504


to J. H.), Science and Technology Commission of Shanghai Municipality (09ZR1436300 to L. Z.), and National Special Grant for Transgenic Crops (2009ZX08009-081B to J. H.).

855

Franck F, Sperling U, Frick G, Pochert B, van Cleve B, Apel K, Armstrong GA (2000) Regulation of etioplast pigment–protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH: Protochloro-

Received 1 Sept. 2011

Accepted 27 Sept. 2011

phyllide oxidoreductases A and B. Plant Physiol. 124, 1678–1696. Goslings D, Meskauskiene R, Kim C, Lee KP, Nater M Apel K (2004) Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole

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