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Wataru Sakamoto1,∗, Yasuyuki Uno1,3, Quan Zhang2, Eiko Miura1, Yusuke Kato1 and Sodmergen2 1Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan 2Key Laboratory of Cell Proliferation and Differentiation (Ministry of Education), College of Life Sciences, Peking University, Beijing 100871, PR China Leaf variegation is seen in many ornamental plants and is often caused by a cell-lineage type formation of white sectors lacking functional chloroplasts. A mutant showing such leaf variegation is viable and is therefore suitable for studying chloroplast development. In this study, the formation of white sectors was temporally investigated in the Arabidopsis leaf-variegated mutant var2. Green sectors were found to emerge from white sectors after the formation of the first true leaf. Transmission electron microscopic examination of plastid ultrastructures confirmed that the peripheral zone in the var2 shoot meristem contained proplastids but lacked developing chloroplasts that were normally detected in wild type. These data suggest that chloroplast development proceeds very slowly in var2 variegated leaves. A notable feature in var2 is that the plastids in white sectors contain remarkable globular vacuolated membranes and prolamellar bodylike structures. Although defective plastids were hardly observed in shoot meristems, they began to accumulate during early leaf development. Consistent with these observations, large plastid nucleoids detected in white sectors by DNA-specific fluorescent dyes were characteristic of those found in proplastids and were clearly distinguished from those in chloroplasts. These results strongly imply that in white sectors, differentiation of plastids into chloroplasts is arrested at the early stage of thylakoid development. Interestingly, large plastid nucleoids were detected in variegated sectors from species other than Arabidopsis. Thus, plastids in variegated leaves appear to share a common feature and represent a novel plastid type. 3Present Keywords: Arabidopsis thaliana • Chloroplast development • Electron microscopy • Leaf variegation • Plastid nucleoid • Thylakoid membrane. Abbreviations: Col, Columbia; cpDNA, chloroplast DNA; DAPI, 4′,6-diamidino-2-phenylindolyle; FTL, first true leaf; MRL, mature rosette leaf; PEND, plastid envelope DNA binding; ptDNA, plastid DNA; PLB, prolamellar body; PVB, plastidic vacuolated body; SiR, sulfite reductase; SYBG, SYBR Green I; TEM, transmission electron microscopy; var1, yellow variegated 1; var2, yellow variegated 2. Special Issue – Regular Paper Arrested Differentiation of Proplastids into Chloroplasts in Variegated Leaves Characterized by Plastid Ultrastructure and Nucleoid Morphology Introduction Leaf variegation is defined as a patch of different colors in the same leaf and is often seen in a variety of ornamental plants. When the variegation pattern is identical in each leaf, it is likely formed by leaf tissues differentiating into specific cell types. This type of variegation is termed ‘figurative (or structural) variegation’ (Kirk and Tilney-Bassett 1978). On the other hand, non-identical variegation is termed ‘true variegation’ and is frequently caused by genetic mutations or by environmental stresses such as nutrient deficiency and pathogen infection. True variegation caused by genetic mutations sometimes follows cell lineage and is characterized by splashes of green/white sectors (Kirk and Tilney-Bassett 1978, Rodermel 2002). Other non-cell-lineage types show unequal variegation patterns such as irregularly shaped marginal sectors. Mutations that result in leaf variegation or stripes have been known to occur in many plant species for a long time. Such mutants are invaluable resources to address: Nanto Seeds Co., Kashihara, Nara, 634-0077 Japan. author: E-mail, [email protected]; Fax, +81-86-434-1206. ∗Corresponding Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2069 W. Sakamoto et al. functionally study chloroplast development (Sakamoto 2003, Aluru et al. 2006, Yu et al. 2007). Unlike albino mutants, which are completely defective in chloroplast development and thus do not grow into maturity, the formation of green/ white sectors allows us to simultaneously dissect both sectors. Chloroplast biogenesis is a complex process and various plastid types are known (Wise 2006, López-Juez 2007, Sakamoto et al. 2008). Plastids in white sectors may represent a novel plastid type. In the last decade, many variegation/stripe mutants have been characterized at the molecular level in model plant species including Arabidopsis (Carol and Kuntz 2001, Rodermel 2002, Sakamoto 2003, Aluru et al. 2006, Yu et al. 2007), maize (Han et al. 1992) and tomato (Keddie et al. 1996, Carol and Kuntz 2001). With the exception of mutations caused by transposons and organelle genomes (leading to a genetic mosaic), most of the mutants are recessive and are nuclear encoded. The recessive nature of variegation indicates that two plastid types separated in green/white sectors are formed in the same genetic background. Molecular characterization of corresponding genes in these mutants revealed that variegation is not caused by identical genes but rather by various redundant mechanisms associated with chloroplast function. While these studies demonstrate that a threshold of chloroplast factors exists and determines plastid differentiation into chloroplast in a cellautonomous manner, the precise mechanisms leading to the formation of variegated sectors still remain unclear. The permanent appearance of variegation sectors in these mutants also suggests that plastid differentiation into chloroplast is determined at a particular stage of leaf development and is not an irreversible event. In Arabidopsis, the yellow variegated2 (var2) mutant shows a variegation pattern typical of cell-lineage types and is thus an ideal model for studying chloroplast differentiation. It was originally reported along with var1 by Martínez-Zapater (1992) and mapped on chromosome 2. The gene responsible for variegation encodes FtsH2, one of the isoforms in the chloroplast FtsH, which is localized in the thylakoid membrane (Chen et al. 2000, Takechi et al. 2000). FtsH is an ATP-dependent metalloprotease that belongs to the AAA+ protein family (Neuwald et al. 1999, Ito and Akiyama 2005). It is one of the major prokaryotic proteases in chloroplasts and performs processive degradation to maintain functional chloroplasts (Adam et al. 2006, Sakamoto 2006). Plastidic FtsH forms a hetero-complex with two types of isoform that are functionally distinguishable: Type A includes FtsH1 and FtsH5, and Type B includes FtsH2 and FtsH8 (Yu et al. 2005, Zaltsman et al. 2005b). FtsH levels are coordinately regulated between the two types (Sakamoto et al. 2003, Yu et al. 2004), and we confirmed that leaf variegation in var1 (similar but not as intense as var2) is caused by the lack of FtsH5 (Sakamoto et al. 2002). 2070 The results in var1 and var2 appear to imply that a threshold of FtsH levels (or levels of other molecules regulated by FtsH) is an important functional constituent for proper chloroplast development. Meanwhile, studies on FtsH in Arabidopsis and Synechocystis demonstrated that FtsH is involved in avoiding photoinhibition by contributing to the light-dependent degradation of the Photosystem II (PSII) reaction center protein D1 (Nixon et al. 2005, Komenda et al. 2007, Kato and Sakamoto 2009). Thus, FtsH is currently proposed to play a dual role in chloroplasts; an unrelated role to variegation in the PSII repair cycle and a variegationrelated role in thylakoid formation. In addition, several transacting recessive mutations that suppressed leaf variegation in var2 have been identified. A majority of the suppressors appear to act on chloroplast protein synthesis (Miura et al. 2007, Yu et al. 2008). Based upon these findings, the balance between protein synthesis and degradation was proposed to mitigate the variegation phenotype. The accumulating amount of genetic evidence implied more complexity in the formation of variegation sectors. In contrast to the intensive genetic studies, little is known regarding how these abnormal plastids are formed along with leaf development. Variegation in var2 is only observed in true leaves but not in cotyledons. It is more severe in the first emerging leaves than in laterally developing leaves (Zaltsman et al. 2005a, Kato et al. 2007). In this study, plastid ultrastructures and nucleoids in var2 were deeply examined during the early stage of true leaf development. Our results demonstrate that white sectors contain a novel type of plastid that is likely formed as a result of the arrest at the early stage of chloroplast development. Results Characteristic features of plastids in var2 white sectors in mature leaves We first prepared ultrathin sections of mature rosette leaves (MRLs) at 30 d after sowing, and examined plastids in wild type (ecotype Columbia, Col) and var2 variegated sectors by transmission electron microscopy (TEM). Green sectors in var2 MRLs contain chloroplasts that were almost indistinguishable from those observed in wild type (Fig. 1A, B). In contrast, plastids in white sectors clearly lacked developed thylakoid membranes and granal stacks, but instead contained globular vacuolated membrane structures (termed hereafter as ‘plastidic vacuolated body’, PVB). They appeared to vary in size but were mostly globular and constituted of a single membrane (Fig. 1C, D, Supplementary Fig. S1). In addition to PVBs, we observed prolamellar body (PLB)-like structures in MRL plastids in white sectors (Fig. 1D, E). Subsequently to TEM analysis, we examined plastid nucleoids, plastid DNA (ptDNA)–protein complexes that are detectable with DNA fluorescence dyes such as Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors Fig. 1 Characteristic ultrastructures of plastids and their nucleoids observed in var2 mature leaves (30 d old). Lens-shaped chloroplasts detected in Col (A) and var2 green sectors (B), and a plastid detected in a var2 white sector (C–E). P, PVB. PLB, PLB-like structure. Plastid nucleoids detected by SYBG stain in Col (F) and var2 (G). In (G), the image was taken at the border between the green and white sectors. Areas corresponding to single chloroplasts or a plastid are enclosed by red or white circles, respectively. Signals corresponding to nuclei are indicated by yellow arrowheads. Bars, 1 µm in (A)–(E); 10 µm in (F) and (G). 4′,6-diamidino-2-phenylindolyle (DAPI) and SYBR Green I (SYBG) (Kuroiwa 1991) (Fig. 1F, G). The plastid nucleoids in white sectors were large and resembled those in proplastids (Miyamura et al. 1986, Miyamura et al. 1990, Fujie et al. 1994), suggesting that plastid development into chloroplasts is arrested in white sectors. The remarkable inner-membrane structures and nucleoid structures thus appeared to be characteristic of var2 white sectors. To examine these two features during an early stage of true leaf development, we focused on the first true leaf (FTL) and characterized sector formation in detail. Formation of green sectors in FTLs in var2 Previous reports by Chen et al. (1999) showed that the variegation pattern in var2 remained unchanged, when mature leaves were examined. Consistent with these reports, the variegation patterns that we observed in MRL remained unchanged at least for 10 d (Supplementary Fig. S2). When we observed expanding FTL in var2, however, we found that some green sectors emerged from the white sector (Fig. 2). FTL was highly variegated in var2 and often looked completely white. Photographs taken every day after the emergence of the FTL demonstrated that green sectors began to form at 7 d after sowing under our growth conditions. The sectors formed at this stage became clear as the leaf matured (at 9 d) and it appeared permanent in mature leaves (after 13 d). These findings indicate that at least in FTL, the differentiation of proplastids into chloroplasts occurs at approximately day 7. Plastid ultrastructure during variegation formation in the FTL in var2 To examine when abnormal plastids are detectable, ultrathin sections were prepared from 6-day-old seedlings (except for cotyledons and roots) and 8-day-old FTLs in Col and var2, and were examined by TEM (Figs. 3, 4). No green sectors were visible at this stage (Fig. 2), although cells that became green sectors could be included in the preparation. To follow chloroplast development, we first examined plastids at the peripheral zones of the shoot meristem. Cells in Col contained mostly globular chloroplasts of ∼2 µm with a limited number of thylakoids and no starch granules (Fig. 3A). Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2071 W. Sakamoto et al. Fig. 2 Appearance of the green sectors in TFL of var2 mutants. Photographs of identical plant at 7 d (A, E), 9 d (B, F), 11 d (C, G) and 13 d (D, H) after sowing. (E)–(H) are close-up images corresponding to (A)–(D), respectively. Bars, 2 mm. In contrast, cells at the peripheral zone in var2 contained smaller size (∼1 µm) proplastid-like plastids and only few detectable thylakoids (Fig. 4A). These results suggest that the formation of chloroplasts, represented by thylakoid development, proceeds slowly in var2. In general, a series of developing chloroplasts can be found by observing cells from the base to the tip of a single developing leaf (Leese and Leach 1976). In fact, the FTL in Col showed that chloroplasts became enlarged (from ∼2 to 8 µm) and lens shaped, contained an increased number of granal stacks and finally accumulated starch grains (Fig. 3B–E). When he FTL in var2 was examined, chloroplast development was found to be remarkably different. In var2, proplastids in shoot meristematic cells appeared to develop thylakoids along with leaf maturation (Fig. 4A, B). As aforementioned, however, this process was slow in comparison with that in Col. We also found that the middle to the tip parts of 6-day-old FTLs accumulated abnormal plastids that were characterized by irregularly shaped envelopes (Fig. 4C, D). No apparent PVBs and PLB-like structures were detectable in 6- and 8-day-old FTLs we observed. Because FTLs in var2 2072 are dominated by white sectors, most of the cells in FTL contain abnormal plastids (Fig. 4E). We rarely observed chloroplast-like plastids that retained thylakoids. These chloroplast-like plastids were found to coexist with abnormal plastids (not shown). Formation of abnormal plastids in white sectors Ultrastructures of plastids in Col and var2 during FTL development are summarized in Fig. 5. In Col, the formation of leaf primordia around the meristem is followed by initial thylakoid development. During this stage of development, chloroplasts are globular and thylakoids begin to develop. Subsequently, chloroplasts became oval and appeared to be distributed in proximity to plasma membranes. Afterwards, thylakoid membranes constituted granal networks and mature chloroplasts accumulated large starch bodies. In contrast to the normal chloroplast development as described above, var2 contained many proplastids within the peripheral zones of the shoot meristem. Overall, we observed distinct plastid development in var2 as schematically presented in Fig. 6. Three features characteristic of var2 were detected. Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors Fig. 3 TEM analysis of chloroplasts in Col. Chloroplasts in 6-day-old plants at the shoot meristem (A), the basal part of FTL (B), the middle part of FTL (C) and the tip part of FTL (D). Chloroplasts in 8-day-old plants at the tip part of FTL (E). Bar, 2 µm. Fig. 4 TEM analysis of plastids in var2. Plastids (indicated by red arrowheads) in 6-day-old plants at the shoot meristem (A), the basal part of FTL (B), the middle part of FTL (C) and the tip part of FTL (D). Plastids in 8-day-old plants at the tip part of FTL (E). Bar, 2 µm. Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2073 W. Sakamoto et al. 8 days 6 days Base Tip Middle var2 Col Meristem 1µm Fig. 5 Summarized plastid ultrastructures during leaf development in Col and var2 leaves. Representative images of plastids in 6-day-old (meristem, base, middle, tip parts of FTL) and 8-day-old (tip part of FTL) plants are shown. In var2, two types of plastid representing abnormal plastids and chloroplasts that likely form green sectors afterwards (after 6 d, middle part) are indicated as separate rows. First, we found that abnormal plastids started to accumulate from the tip to the middle part of the FTL at 6 d. These data suggested that this is the stage where cells are destined to undergo normal chloroplast differentiation or a possible arrest of thylakoid development (feature 1, Fig. 6). Secondly, cells in green sectors form normal chloroplasts, but this process occurs at a much slower rate than the normal chloroplast development in Col (feature 2, Fig. 6). However, chloroplast development in var2 appears to proceed into maturity, since green sectors in mature leaves contain fully developed chloroplasts. Finally, abnormal plastids accumulate PVBs and PLB-like structures instead of thylakoids, but these bodies were not formed during an early step of 2074 leaf development and concomitant formation of abnormal plastids (feature 3, Fig. 6). Formation of plastid nucleoids during leaf development The morphologies of plastid nucleoids, as detected by DAPI staining of thin sections, are known to change along with chloroplast development (Lawrence and Possingham 1986, Miyamura et al. 1990, Rowan et al. 2004). These changes are also well correlated with thylakoid development and granal stacking. Because the observation of plastid nucleoids in var2 MRL indicated that white sectors contained nucleoids that were larger and less abundant than those observed in Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors Col (3) var2 (1) (2) Fig. 6 A schematic representation of the green/white sector formation in var2. Temporal chloroplast development in Col FTL is indicated from left to right. Green bars represent the degree of grana formation. Three features characteristic of var2 are indicated by numbers. Feature 1 is the point when the abnormal plastids start to accumulate. This point corresponds to 6 day after sowing. Feature 2 is the fact that chloroplast development, as represented by granal stacking, proceeds very slowly in var2 compared with Col. Feature 3 is the formation of PVBs in plastids. PVB is predominantly seen in MRL, but not at the early step of leaf development in FTL. Fig. 7 Morphological change in plastid nucleoids during leaf development as observed by SYBG-stained thin sections of FTL in Col (A–D) and var2 (E–H). Cells in 8-day-old plants at the basal part of FTL (A, E), the middle part of FTL (B, F) and the tip part of FTL (C, G). Cells in 10-day-old plants at the tip part of FTL (D, H). Note that fluorescent signals in each image were incorporated by a digital camera at the identical time setting. Signals corresponding to nuclei are indicated by yellow arrowheads. Areas corresponding to single chloroplasts or plastids are enclosed by red circles. Bar, 10 µm. green sectors (Fig. 1G), we characterized plastid nucleoids during leaf development in Col and var2 FTLs in this study. We fixed Col and var2 seedlings at 6 or 8 d from which roots and cotyledons were removed (Supplementary Fig. S3A, B). Fixed tissues were embedded into Technovit resin and a cross-section of FTL was stained with SYBG to visualize plastid nucleoids (Supplementary Fig. S3C). In Col, developing chloroplasts at the tip and the middle parts of the FTL contained a few large nucleoids that were frequently observed as surrounding the envelope (Fig. 7A, B). As chloroplasts matured and contained granal stacks (tip part of 8-day-old leaf), nucleoids became smaller in size and more abundant (Fig. 7C, D). The nucleoids appeared dense (images in Fig. 7 were recoded by the same time length) and Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2075 W. Sakamoto et al. were dispersed in the stroma. In some instances, nucleoids were observed in proximity to the exterior of thylakoid membranes. In var2, however, it was evident that abnormal plastids contained nucleoids whose structures differed from those in Col. Throughout leaf development, plastids retained nucleoids that appeared at the initial stage. These nucleoids did not become smaller in size and did not disperse within plastids and rather remained as large aggregates (Fig. 7E–H). In some leaf tips at 6 or 8 d, prominent large signals were occasionally detected. These results are consistent with our TEM observation, which revealed that thylakoid formation is strongly impaired in the plastids of white sectors, suggesting that abnormal plastids resulted from an arrest of proper chloroplast development. ptDNA levels in the white sectors Since our cytological observations were not quantitative, we assumed that the structural change in plastid nucleoids did not accompany an overall decrease in ptDNA amounts. In fact, our previous semi-quantitative PCR analysis did not reveal significant changes in ptDNA level between green and white sectors of var2 (Kato et al. 2007). To examine ptDNA levels carefully, we separately isolated single green and white protoplasts from both sectors and performed quantitative PCR (Fig. 8A). Protoplasts from white and green cells were clearly distinguishable from each other due to the presence of chlorophyll auto-fluorescence (Fig. 8B–E). The number of chloroplasts per protoplast was not significantly different between Col and var2 green sectors (Supplementary Fig. S4). Single protoplasts from Col were also subjected to our PCR analysis. The results using two primer sets, detecting ptDNAs corresponding to psbA or ndhA, showed that relative ptDNA levels were not significantly different between Col, and green and white sectors in var2 (Fig. 8F). We also measured the area size of protoplasts and found that cells in white sectors are smaller than those in green sectors and F psbA 2 Relative cpDNA amount 1 ndhA 2 1 Col var2 green var2 white Fig. 8 Estimation of cpDNA levels in protoplasts derived from green and white cells. (A) Capture of protoplasts with a glass capillary (denoted by red arrowhead, bar = 200 µm). Protoplasts from green sectors (B, C) and from white sectors (D, E) are shown. Images captured by bright field (B, D) and by G filter set (C, E, detecting chlorophyll autofluorescence) are shown (bar, 10 µm). (F) CpDNA levels estimated by real-time PCR analysis using a single protoplast from Col, var2 green and white sectors (n = 10). Results from two gene-specific primers (psbA and ndhA) are shown. 2076 Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors Ipomoea nil Oryza sativa Artemisia princeps Pampan. Felicia amelloides Habenaria radiata Ficus benzyamina Alternanthera ficoidea Arabis glabra Fig. 9 Plastid nucleoids detected by DAPI-stained thin sections from variegated leaves. Images of eight species were shown along with the photograph of each corresponding variegated leaf. For each species, representative images from green sectors (left) and white sectors (right) are shown (bars, 10 µm). Note that fluorescent signals in each image were incorporated by a digital camera at the identical time setting. Signals corresponding to nuclei are indicated by yellow arrowheads. Areas corresponding to single chloroplasts or plastids are enclosed by red and white circles, respectively. Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2077 W. Sakamoto et al. Col (Supplementary Fig. S5). As a result, our quantitative PCR with single protoplasts supported our previous observation that larger plastid nucleoids in white sectors do not reflect an altered amount of ptDNA per cell. Enlarged plastid nucleoids are commonly found in variegated leaves We assumed that enlarged plastid nucleoids are characteristic of undifferentiated plastids lacking thylakoid membranes and that white sectors in variegated leaves in species other than Arabidopsis may contain similar plastid nucleoids. To test this, mature variegated leaves from eight species were selected to examine plastid nucleoids with DAPI staining. The pattern and degree of variegation in these species were variable. Specifically, Ipomoea, Oryza, Artemisia and Alternanthera species showed cell-lineage type variegation and other species showed non-cell-lineage type variegation. Except for the Oryza stripe mutant, it was unclear whether leaf variegation is caused by a mutation. Nevertheless, variegated sectors were permanently observed in each species under our growth conditions in a greenhouse (Fig. 9, pictures of leaves). Leaf tissues containing both green and white sectors were fixed and embedded in Technovit resin and thin sections were stained with DAPI to visualize plastid nucleoids. We set the time period unchanged when recording images with fluorescence microscopy and the images from both sectors were compared (Fig. 9). In all species, green sectors were found to contain small/dense DAPI signals that were dispersed within stroma (Fig. 9, red circles in left panels). In strong contrast however, white sectors contained enlarged DAPI signals that were brighter and resembled those observed in var2 white sectors (Fig. 9, white circles in right panels). Similar to those in var2, the enlarged nucleoids detected in cell-lineage type species (e.g. Artemisia and Ipomoea) were located along with the envelope. Given that enlarged nucleoids represent plastids with undifferentiated thylakoids, these results suggest that abnormal plastids in white sectors of variegated leaves generally contain plastids with arrested thylakoid formation. Discussion Formation of green and white sectors in var2 Although plastids in white sectors of variegated leaves have been previously observed in many species (Kirk and Tilney-Bassett 1978), there has not been a systematic analysis to examine at what stage abnormal plastids arise. In this study, we focused on FTLs of Arabidopsis Col and var2, and characterized sector formation during leaf development by observing plastid ultrastructure and nucleoid morphology. Detailed observations of white sectors demonstrated that the decision for a leaf cell to undergo normal chloroplast 2078 differentiation is made at a very early stage of leaf development. The FTL in var2 is dominated by white sectors. In the shoot meristem, Col contained many small globular chloroplasts with thylakoids and a few granal stacks, whereas the var2 mutant contained proplastids and a limited number of thylakoids with no stacking. Thus, one notable feature of var2 that was revealed in this study was a clear delay in chloroplast development. Given the observation that the var2 shoot apical meristem predominantly contained proplastids, we conclude that proplastids are normal in shoot meristems and have a potential to become functional chloroplasts. It should be noted that prior to the appearance of green sectors, the FTL appeared yellow at 7 d, an observation that perhaps indicates that proplastids can initiate chloroplast differentiation at the very initial step (Fig. 2E). The green sectors became more evident as the yellow color somewhat disappeared (Fig. 2F). Collectively, all of these data support our hypothesis that chloroplast development is initiated but arrested at the initial step. The delayed emergence of green sectors that we observed in Fig. 2 has not been previously reported in var2, because previous studies focused on expanded leaves. In this study, we showed that expanding FTL contains a patch of the cells that escape from the arrest and undergo chloroplast development (Fig. 2F–H). Namely, the gradual appearance of green patches in white sectors indicates that chloroplast development proceeds very slowly in var2. Variegation is more severe in the FTL than in the second and subsequent leaves, partly because other FtsHs than FtsH2 are upregulated in the second and subsequent leaves in var2 (Zaltsman et al. 2005a). Increased FtsH levels in the second and subsequent leaves may be one of the reasons that FTL only showed a marked emergence of green sectors. The delay of chloroplast development in var2 can be connected to the suppressor mutations in which leaf variegation was rescued (Park and Rodermel 2004, Miura et al. 2007, Yu et al. 2008, Zhang et al. 2009). One possibility is that a suppressor mutation can delay overall chloroplast development in the var2 background. As a consequence, the threshold level of FtsH (or other factors regulated by FtsH) was mitigated, thereby allowing most of the mesophyll cells to contain chloroplasts of normal appearance. In this scenario, it is possible that any mutations delaying chloroplast differentiation may also act as a suppressor. It also implies that the balance between the leaf maturation process and chloroplast development, rather than the balance between protein synthesis and degradation in chloroplasts, is important for proper chloroplast formation. The other important finding from our TEM analysis pertains to PVBs and PLB-like structures that were found in abnormal plastids. Because the PVBs were predominantly detected in MRL white sectors, we predicted that PVBs arise during leaf development. Unexpectedly, their precursor-like Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors structures were hardly detectable in the early stage of FTL development, suggesting that PVB formation is very slow or predominantly proceeds in mature leaves. An alternative possibility is that apparent PVBs are not formed in FTL. This possibility seems unlikely but not completely ruled out, since we did not examine FTL at 30 d. Although the origin of PVB remains unclear, the co-existence of PLB-like structures raises a possibility that PVBs are derived from PLB-like structures. The PLB is a particular type of membrane structure in etioplasts and is thought to represent a novel type of thylakoid under dark conditions (Kirk and Tilney-Bassett 1978, Wise 2006). It is reasonable to assume that the abnormal plastids in FTL are unable to stay arrested in development and show characteristics similar to etioplasts by forming PLBs (Park et al. 2002, Philippar et al. 2007). Supporting this observation is the previous study by Chen et al. (2000) that demonstrated that etioplast formation in cotyledons is comparable between Col and var2. It is possible that the white plastids in MRL perhaps have a potential to develop but they fail to form a functional thylakoid network from PLB-like structures due to the lack of photosynthetic pigments and other protein components. As a result, these defective plastids form PVBs instead. Thus, our observations demonstrate that plastids in the white sectors of variegated leaves represent a novel plastid type. Membrane structures like PVBs in a mutant defective in chloroplast development have been previously reported in other studies (Motohashi et al. 2001, Abdelkader et al. 2007, Okegawa et al. 2007). On the other hand, we did not detect invaginated membrane structures along with the inner envelope, which were reported in several mutants defective in thylakoid formation (Kroll et al. 2001, Kobayashi et al. 2007). We do not assume that PVBs directly derive from inner envelope. Nucleoid structures in variegated leaves Similar to our TEM analysis, our initial characterization of plastid nucleoids was carried out only in MRL. Therefore we decided to observe plastid nucleoids during FTL development in this study. As previously reported, a series of sections in 6- and 8-day-old FTLs demonstrated that plastid nucleoids follow the typical morphological alteration (Kuroiwa 1991, Fujie et al. 1994, Sakamoto et al. 2008). Immature chloroplasts tend to contain nucleoids that are large, less packed and dispersed along with the inner envelope. As the chloroplasts mature with many granal stacks, the nucleoids appeared to move in stroma and they become small and packed (Fig. 7A–D). It is evident that in var2, the prominent large SYBG signals remain in abnormal plastids of white sectors (Fig. 7E–H). These results are consistent with our previous observation and support our hypothesis that thylakoid development is arrested in white sectors. DNA compaction in nucleoids was suggested to affect gene expression (Sekine et al. 2002). In contrast, our previous study indicated that expression of plastid genes is not significantly altered between green and white sectors in var2 (Kato et al. 2007). It thus appears that the altered nucleoid morphologies in var2 white sectors do not affect plastid gene expression at the transcript level. Some chloroplast proteins were biochemically identified to interact with ptDNAs and constitute plastid nucleoids (Sato et al. 2003, Sakai et al. 2004, Sakamoto et al. 2008). Those include, for example, plastid envelope DNA-binding protein (PEND; Sato et al. 1998) and sulfite reductase (SiR; Sato et al. 2001), MFP1 (Jeong et al. 2003) and CND41 (Murakami et al. 2000) whose precise roles in nucleoid formation and gene expression remain unclear. PEND is located in the inner envelope and was shown to anchor ptDNAs to the plastid envelope. A study using truncated PEND–green fluorescent protein showed that nucleoids change their morphology within different tissues (Terasawa and Sato 2005). Large plastid nucleoids detected in white sectors may be associated with PEND since they are located along with the envelope. DAPI-stained plastid nucleoids were also studied previously by Fujie et al. (1994). Plastid nucleoids detected in 6- and 8-day-old Col FTL in this study were similar to those reported in the previous studies. Plastid nucleoids detected in white tissues resemble those from non-photosynthetic tissues such as roots and petals (Fujie et al. 1993). Once again, these data indicate that nucleoid morphologies are well correlated with the development of thylakoid membranes. Although our focus was on nucleoid morphology between the green and white sectors in var2, it appeared to accompany quantitative differences in DNA signals. To clarify this question, we carefully estimated ptDNA levels using single protoplasts. However, no significant differences were detected between Col, white and green sectors. This result was consistent with our previous semi-quantitative PCR analysis (Kato et al. 2007) and confirms that alteration of nucleoid morphologies do not represent ptDNA amounts. Regarding chloroplast DNA (cpDNA) amounts during leaf development, contradictory results were reported from different laboratories (Rowan and Bendich 2009). Chloroplasts in mature leaves from Arabidopsis were shown to contain a substantially reduced level of ptDNAs based on fluorescence detection and quantitative RT-PCR (Rowan et al. 2004). On the other hand, cpDNA levels were reported to remain unchanged based on Southern blot analysis (Li et al. 2006, Zoschke et al. 2007). Given that the var2 white sectors contain plastids whose development into chloroplasts is arrested at the initial step, our data indirectly indicate that ptDNA amounts do not significantly change throughout chloroplast development. It is possible, however, that ptDNAs were unchanged between the two sectors because the number of plastids decreased in white sectors. These data also suggest that each plastid in the white sectors indeed contained a larger amount of DNA than chloroplasts in green sectors. Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2079 W. Sakamoto et al. We were unable to exclude this possibility since the plastid number in white cells was not determined. Besides the quantitative change of ptDNA amounts, we raised a possibility in this study that many variegated sectors, other than var2 in Arabidopsis, display large plastid nucleoids due to the impairment of normal thylakoid development. The examination of variegated leaves from different species, including both cell-lineage and non-cell-lineage types, demonstrated that white sectors contain large nucleoids as expected. It is unclear whether variegated leaves examined in this study were due to a genetic deficiency (except for rice) or from physiological effects. In addition, cell area size, number of chloroplasts per cell and the size of chloroplasts varied among these species. Nevertheless, white sectors always tended to be abundant with large nucleoids that were clearly distinguishable from those in the green sectors. Our results indicate that despite various patterns found among different variegated leaves, white sectors have common features that represent an arrest in thylakoid differentiation. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col) was used as a wild-type plant in this study. For observing leaf variegation in var2, the var2-1 allele was used (with the exception that photos shown in Supplementary Fig. S2 used var2-6). This var2-1 allele possesses a nonsense mutation at Gln597 (Sakamoto et al. 2004). Sterilized seeds were germinated and grown on 0.7% (w/v) agar plates containing Murashige and Skoog medium supplemented with Gamborg’s vitamins (Sigma-Aldrich) and 1.5% (w/v) sucrose. After stratification for 2 or 3 d at 4°C under darkness, plates were maintained under 12 h light (approximately 100 µ mol/m2/s at a constant temperature at 22°C. For further analysis, plants were transferred to soil after 4–5 weeks. Additional sources for variegated plants used in Fig. 9 (with the exception of the rice stripe mutant) were purchased from a local nursery and subsequently grown in a greenhouse at the Research Institute for Bioresources, Okayama University. The rice stripe mutant (st-5, mutation mapped on chromosome 4) was generously provided by Dr Masahiko Maekawa (Okayama University). Transmission electron microscopy Leaf samples for TEM were prepared as follows. For 6-day-old FTLs, cotyledons and roots were removed from plate-grown seedlings and the whole remaining leaf tissues were fixed. For 8-day-old FTLs, only middle and tip regions from the FTLs were excised and fixed for microscopic observation. For 30-day-old MRLs, a 1 × 1 mm region of leaves was excised and fixed. Leaf samples were fixed in 4% (v/v) glutaraldehyde in 2080 cacodylate buffer (pH 7.4) for at least 24 h at room temperature. After four separate rinses with cacodylate buffer, the samples were treated with 2% (w/v) osmium tetraoxide for 4–6 h at room temperature. After four separate rinses with cacodylate buffer, the samples were stained by 2% (w/v) uranyl acetate in 50% (v/v) ethanol for 1 h at room temperature. Samples were further dehydrated with a graded ethanol series [50%, 70%, 85%, 95%, 100% (v/v)]. Ethanol was subsequently replaced by a series of Spurr’s resin dilutions [25%, 50%, 75%, 100% (v/v)] and the resin was hardened for 16 h at 65°C. Ultrathin sections (60–70 nm) were made with an Ultracut N (Reichert-Nissei). Thin sections were stained with 0.25% (w/v) lead citrate and examined with a JEM-1010 JEOL transmission electron microscope (at the College of Life Sciences, Peking University. Technovit thin sections and staining with SYBG and DAPI For preparation of Arabidopsis sections, cotyledons and roots were removed from 6- and 8-day-old seedlings under a dissection microscope (as illustrated in Supplementary Fig. S3). The leaf tissues were fixed in FAA solution [5% (v/v) Formalin, 5% (v/v), acetic acid, 45% (v/v) ethanol] for at least 3 h or stored at 4°C. For other plant species, a 1 × 2 mm region of leaves containing green and white sectors was excised prior to fixation. Fixed tissues were pre-stained by 2.5% (w/v) safranin and washed in 0.1 M phosphate-buffered saline (PBS pH 7.4). The tissues were dehydrated by a graded series of ethanol dilutions (50%, 70%, 80%, 90%, 95%, 99.5%, 100% (v/v). Ethanol was replaced by a series of Technovit7100 resin dilutions [25%, 50%, 74%, 100% (v/v)]. The samples were kept overnight at room temperature and the resin was finally hardened overnight at 4°C. Thin sections (0.9 µm) were stained with 1 µg/ml DAPI (Invitrogen) or 1 µg/ml SYBRG (Invitrogen) and examined by an Olympus AX-84-FLBD fluorescent microscope (U-MWU2 filter set for DAPI and U-MW13 for SYBG). Images were recorded with a DP71 digital camera (Olympus). Images from green and white sectors were recorded with identical time settings. Protoplast preparation and manipulation Mesophyll protoplasts were prepared as previously described in Kato et al. (2007). Leaves from Col and var2-1 plants were cut into small pieces and were gently suspended in enzyme solution [0.1% (w/v) cellulose Onozuka R10 (Yakult), 0.05% (w/v) pectolyase Y23 (Kyowa Chemical Products), 400 mM mannitol, 10 mM CaCl2, 20 mM KCl, 5 mM EGTA, 20 mM MES, pH 5.7]. After incubation at room temperature for 1 h, protoplasts were collected by centrifugation at 60 × g for 1 min. The isolated protoplasts were then resuspended in wash buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7). For PCR analysis, protoplasts were placed on a slide glass and were detected via inverse Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors microscopy (Olympus CKX41N-31PH). Single protoplasts were collected into a glass capillary (pore size 100 µm) by using a pressure-controlled PicoPipet device (Alatair Corp). Protoplasts from green or white sectors in var2 were easily distinguishable due to the presence or absence of chloroplasts, respectively. Protoplasts from white sectors contained abnormal plastids that were detectable under bright-field microscopy, and were separated by protoplasts derived from epidermal cells. Protoplasts were carefully transferred into a PCR tube, suspended in 9.2 µl of sterilized water, sonicated in a water bath for 1 min (Type UR-20P; Tomy Seiko) and stored at 4°C until further use. For estimating the area of protoplasts, they were suspended in wash buffer and observed by fluorescence microscopy. Images of protoplasts from Col, green and white sectors in var2 were recorded by digital camera and the area for each protoplast was calculated with Metamorph software (Molecular Devices). For counting chloroplast numbers, protoplasts were fixed in 0.02% (v/v) glutaraldehyde and immediately squashed on a slide glass by gently pressing down on the cover glass. Images of broken protoplasts with intact chloroplasts were recorded by a digital camera and were then used to count chloroplasts. Real-time PCR analysis The entire protoplast suspension in a PCR tube was used for RT-PCR reactions (SYBR Premix ExTaq; Takara). RT-PCR was performed in 20 µl of a reaction mixture as recommended by the supplier. PCR cycles were set to 50 under the follow conditions: 94°C for 13 s, 55°C for 15 s and 72°C for 20 s. LightCycler Software Version 4.0 (Roche Diagnostics) was used to quantify DNA levels. For detecting cpDNA, two sets of primers, detecting the region corresponding to psbA and ndhA were used: psbA-F (5′-TTGCGGTCAATAAGGT AGGG-3′), psbA-R (5′-TAGAGAATTTGTGCGCTTGG-3′), ndhA-F (5′-TGAGATCCGCTAAAACAAGG-3′), ndhA-R (5′CTAGCCGATGGGACAAAA-3′) Supplementary data Supplementary data are available at PCP online. Funding The Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (No. 16085207 to W.S.), the Oohara Foundation (to W.S.), the Asahi Glass Foundation (to W.S.), the Novartis Foundation (to W.S.) and the National Natural Science Foundation of China for a Creative Research Group Program (No. 30421004 to Sod.). Eiko Miura is a postdoctoral fellow supported by the Japan Society for the Promotion of Science. 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