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First published online April 20, 2007; 10.1104/pp.107.099002 Plant Physiology 144:952-960 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
White Leaf Sectors in yellow variegated2 Are Formed by Viable Cells with Undifferentiated Plastids1,[C],[W],[OA]Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710–0046, Japan
The yellow variegated2 (var2) is one of the best-characterized Arabidopsis (Arabidopsis thaliana) mutants showing leaf variegation. Leaf variegation of var2 results from the loss of an ATP-dependent metalloprotease, FtsH2, which is a major component of the FtsH heterocomplex in thylakoid membranes. While the functional role of FtsH2 in protein quality control has been extensively studied, the physiological state of plastids in white tissues of the var2 is not well characterized. Here we show that the white tissue in var2 is neither the result of photobleaching nor enhanced senescence. Visualization of plastids by plastid-targeted green fluorescent protein revealed that plastids in the white sector are distinct and have undifferentiated characteristics. The plastids are also distinct in that they contain large nucleoids, a complex structure of plastid DNA and proteins, that are typically found in undifferentiated plastids. Comparative analyses of protein profiles from green and white tissues suggested that the difference was observed in the proteins related to photosynthesis but not due to proteins of other organelles. Thus, cells in the white tissue are viable and their defect is limited to plastid function. The plastid accumulates normal levels of chloroplast transcripts, whereas a substantial repression of nuclear-encoded photosynthetic genes was evident in the white sector. Based upon these results, we inferred that the white sectors in var2 are made by viable cells that have plastids arrested in thylakoid formation. A proposed model to form the variegated sector in var2 is provided.
Leaf variegation is occasionally observed in higher plants. It raises a fundamental question of why and how two sectors containing different cell types, a green cell with normal-appearing chloroplasts and a white cell with abnormal plastids, can be formed in leaf tissue. Leaf variegation has been recognized as a genetic trait since early twentieth century (for review, see Kirk and Tilney-Bassett, 1978  ). Several recessive genes responsible for the variegation phenotype have been identified in model systems such as maize (Zea mays), Arabidopsis (Arabidopsis thaliana), and tomato (Lycopersicon esculentum; e.g. Han et al., 1992; Carol and Kuntz, 2001). Studies from these model systems have shown that leaf variegation has various causes. On the other hand, variegation is affected not only by leaf development but also in response to biotic and abiotic circumstances, resulting in the formation of complex patterns (for review, see Kirk and Tilney-Bassett, 1978; Sakamoto, 2003; Yu et al., 2007). Such unstable and unpredictable patterns for the appearance of white sectors hinders our ability to elucidate the physiological control of this phenomenon.
We have been studying yellow variegated1 (var1) and var2 in Arabidopsis as a model to study the mechanisms of leaf variegation. The responsible genes, VAR1 and VAR2, encode FtsH5 and FtsH2 metalloproteases, respectively (Chen et al., 2000
Mounting evidence indicates that FtsH plays a dual role in chloroplasts. First, given the fact that FtsH degrades D1 protein of PSII reaction center both in vivo and in vitro in a light-dependent manner (Lindahl et al., 2000
Despite these observations, the physiological properties of white sectors in var2 remain uncharacterized. It is apparent that white sectors do not simply represent dead tissues, as their phenotypes are apparently different from those of cell death mutants (e.g. Mach et al., 2001
White Sectors in Variegated Leaves Are Not Stained by Evans Blue
Histochemical staining was performed to determine whether white sectors of var2 contained necrotic tissue. Excised leaflets from var2-1 and wild-type plants were vacuum infiltrated with an Evans blue solution for specific staining of dead cells. With the exception for cut sites, no tissues were stained in either variegated var2-1 leaves nor wild type (Fig. 1
). When leaflets were used from an accelerated cell death mutant (accelerated cell death2 [acd2]; Mach et al., 2001
Plastids in the White Sectors Can Be Visualized by GFP We attempted to visualize chloroplasts of var2 with GFP analysis. A transgenic plant expressing a plastid-targeted L12-GFP protein (an N-terminal transit peptide from rice [Oryza sativa] ribosomal protein L12 was fused to GFP) was crossed to var2-1. Individuals expressing L12-GFP in var2-1 were obtained in the F2 population and F3 progeny was subjected to further analysis. Thin sections of leaf tissues of var2-1/L12-GFP showed that L12-GFP was expressed in both green and white sectors. Similar to wild type, overlapping detection of GFP signals with chlorophyll autofluorescence indicated that L12-GFP was localized in chloroplasts of green sectors (Fig. 2A ). We also detected L12-GFP signals representing plastids throughout the white sectors. Mesophyll protoplasts were prepared from var2-1/L12-GFP leaves to carefully observe L12-GFP. As shown in Figure 2B, organelle structures like undifferentiated plastids were detected in the white protoplasts. Their morphologies were variable but mostly smaller than the chloroplasts in green sectors, and occasionally contained stromule-like structures. Thus, cells in the white sectors are active and retain plastids with intact envelopes.
Mitochondria in the White Sectors Can Be Visualized by GFP
We also attempted to visualize mitochondria of var2 by using a mitochondria-targeted GFP. A transgenic plant expressing a mitochondria-targeted DIPS-GFP, containing N-terminal mitochondrial presequence from Arabidopsis mitochondrial ATP-
To observe the difference between white and green sectors, we next characterized their respective protein profiles. Green and white sectors were dissected from 6-week-old leaves (Fig. 3A
) and subjected to protein extraction and SDS-PAGE. The result shown in Figure 3B (proteins were loaded on equal fresh weight) displayed that white sectors appeared to contain less proteins than green sectors, but overall protein profiles were, if not all, similar. Immunoblot analyses confirmed that white sectors contain substantially reduced amounts of chloroplastic proteins, D1 (reaction center protein of PSII), PsbO (subunit of oxygen evolving system of PSII), and Lhcb1 (light-harvesting complex [LHC] proteins of PSII; Fig. 3C). The reduction of Rubisco large subunit and LHCII was also evident in the silver-stained SDS-PAGE (Fig. 3B, black arrowheads). Conversely, we noticed an increased accumulation of nonplastid proteins, Phb5 (mitochondrial prohibitin complex protein), BiP (endoplasmic reticulum luminal binding protein), and
We also compared chloroplastic protein accumulation between Col and green sectors of var2-1. Chloroplasts were purified by a Percoll step gradient from Col and var2-1 mature leaves and soluble proteins were prepared using phenol extraction and ammonium acetate precipitation. The extract was separated by two-dimensional electrophoresis (2-DE) gel as described in "Materials and Methods" and at least 70 protein spots were found on the gel after silver staining. Comparison of the gels from Col and the green sectors of var2-1, equally loaded based on chlorophyll content, did not result in the identification of differences in detectable spots (Supplemental Fig. S1). These data suggested that the accumulation of major soluble chloroplast proteins were indistinguishable between Col and green sectors.
The presented data so far implied that the difference between the green and white sectors is limited to the state of plastid development. We therefore examined the expression of photosynthetic genes, encoded both in chloroplast and nuclear genomes, by semiquantitative reverse transcription (RT)-PCR. Three types of photosynthetic genes, namely (1) nuclear genes, (2) plastid genes transcribed by a nuclear-encoded RNA polymerase (NEP), and (3) plastid genes transcribed by a plastid-encoded RNA polymerase (PEP) were selected as an index (Fig. 4
). In plastids, PEP is predominantly responsible for transcription of photosynthetic genes, whereas NEP mainly mediates the transcription of housekeeping genes (Kanamaru and Tanaka, 2004
We found that transcripts of nuclear-encoded genes (psbO and rbcS) decreased within white sectors. This is consistent with the result of protein accumulation (Fig. 3). In contrast, both NEP- and PEP-transcribed genes (accD and rpoB by NEP and psbA and rbcL by PEP) appeared to be transcribed and their transcripts stably accumulated in white tissues. Steady-state levels of the corresponding transcripts were comparable between Col, green, and white sectors. Given a very limited amount of D1 and large subunit of Rubisco in the white sectors, these proteins appeared to be regulated at the posttranscriptional level. A control experiment using actin2 showed that no significant difference was present for nonphotosynthetic nuclear genes (Fig. 4). Accumulation of psbA and rbcL mRNAs in the white plastids is consistent with our previous observation (Takechi et al., 2000 ), and demonstrates that the plastids are active. In contrast, the decrease of nuclear-encoded psbO and rbcS mRNAs suggested that a retrograde regulation occurs in the white sectors.
The presence of plastid mRNAs apparently indicated that plastid DNAs exist in the white sectors, but it remained unclear whether the quality and quantity of plastid DNAs are equivalent between the two sectors. To examine this, we first performed semiquantitative PCR to assess the plastid DNA level. Similar to the results obtained by RT-PCR, when primers specific for both nuclear and plastid genes were used, DNA levels were indistinguishable between Col, and green and white sectors of var-2-1 (Supplemental Fig. S2).
We considered that the observation of nucleoids was useful to know the state of plastids in the white sectors, since plastid nucleoids are subject to dynamic change during chloroplast development (Kuroiwa et al., 1981
To confirm whether the detected DAPI signals resulted from plastid nucleoids and to examine the plastid nucleoids in detail, we prepared protoplasts from var2-1/L12-GFP plants. The protoplasts were subjected to GFP detection, chlorophyll autofluorescence, and another DNA-specific fluorescent dye Hoechst 33342, which can stain DNA in living cells. Merged images of GFP and Hoechst 33342 signals showed that plastid nucleoids in the white cells were larger than those in green cells (Fig. 5B). Each nucleoid body in the green cells was detected as a small dot and single chloroplasts were observed to contain many nucleoids. Conversely, nucleoids in the white plastids consisted of a few large bodies that resembled those typically observed in undifferentiated plastids (Kuroiwa et al., 1981 ; Nemoto et al., 1988). Thus, the white sectors of var2 seem to contain cells with undifferentiated plastids throughout leaf development.
Detailed Characterization of the White Sectors in var2
Leaf variegation in var1 and var2 mutants, which is caused by the loss of FtsH2 (type B) or FtsH5 (type A), respectively, has been extensively studied by several laboratories. Characterization on two types of chloroplastic FtsHs, which are encoded by duplicated genes and whose expression is coordinately regulated, implicated that overall FtsH levels correlate with the degree of white sectors. These observations led to the threshold model that explains leaf variegation (Yu et al., 2004
Therefore, in this study we dissected and compared the green and white sectors of var2 morphologically and biochemically. First, we assumed that the white sectors are made by living cells that possess distinct plastid structures. Collective observations from Evans blue staining, visualization of plastids by GFP, and the accumulation of proteins, mRNAs, and DNAs supported this assumption. Second, we attempted to clarify that the white sectors are neither the consequence of photobleaching, senescence, nor cell death. In contrast to the necrotic tissue in coronatine-treated acd2, white sectors were never stained by Evans blue and therefore exclude the possibility that white cells are associated with enhanced senescence. In addition, light has no effect on Evans blue stain. This result is consistent with the observation by Zaltsman et al. (2005a)
It is noteworthy to recognize that photobleaching does not contribute to the formation of white sectors. We previously interpreted that the variegation of var2 was partly due to photoinhibition. This was concluded because FtsH plays a key role in the repair cycle of PSII and particularly in the quality control of D1 protein (Lindahl et al., 2000
Based on the proposed threshold model, the fate of chloroplast development in var2 may be explained as illustrated in Figure 6
. Differentiation of plastids into chloroplasts is light dependent. At a particular stage of leaf development, plastids are cell-autonomously destined to permit the formation of thylakoids. If sufficient FtsHs are provided at this stage, then the plastids enter this process and become normal-appearing chloroplasts. In contrast, insufficient FtsHs at this stage prevent plastids from developing thylakoid membranes. As notified by Zaltsman et al. (2005a)
Plastid nucleoids sometimes allow us to assess chloroplast status, because they vary in their size, number, and distribution in stroma in various developmental stages (Kuroiwa et al., 1981 ; Nemoto et al., 1988). A remarkable nucleoid structure detected in the white plastids, together with irregular plastid morphologies revealed by GFP, suggests that plastids in the white sectors are undifferentiated (Fig. 6). At an early stage of chloroplast formation from proplastids, nucleoids are large and attach to chloroplast envelope membranes, and the number of nucleoids increases. At a later developmental stage, the nucleoids attach to thylakoid membranes and constitute tight complexes with various proteins (for review, see Sato, 2001). In senescent leaves, nucleoids disappear in the initial stage of the chloroplast degradation process (Oldenburg and Bendich, 2004; Rowan et al., 2004). Although circumstantial, these results suggest that plastids from white sectors are different from known plastid types such as amyloplasts and etioplasts.
We previously revealed that chloroplast transcripts (psbA, rbcL, and atpB) accumulate normally regardless of the degree of variegation (Takechi et al., 2000 To further characterize the mechanism of leaf variegation, a molecule that accumulates preferentially in var2 needs to be identified. To date, a comparative analysis of protein profiles from green and white sectors failed to reveal any difference due to the normalization problem (as evidenced in Fig. 3 and unpublished data [Y. Kato and W. Sakamoto]). Additionally, the decisive role of FtsH in thylakoid formation also needs to be clarified. Although there are many causes for variegation, the physiological status in the white sectors may be similar to each other. Future studies on other variegated plants, as well as var2, may broaden our view on plastid differentiation.
Plant Materials and Growth Condition
Arabidopsis (Arabidopsis thaliana) Col ecotype was used as a wild-type plant in this study. Unless specifically noted, plants 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) Suc. Plants were maintained under 12-h light (approximately 60 µmol m–2 s–1) at a constant temperature of 22°C. For further analysis, plants were transferred to soil after 4 weeks and were maintained under 12-h light (100 µmol m–2 s–1) at a constant temperature of 22°C. A transgenic plant expressing a plastid-targeted GFP, L12-GFP, under the control of cauliflower mosaic virus 35S promoter, was kindly provided by Shinichi Arimura (Arimura et al., 1999
Excised leaflets from Col, var2-1, and acd2-2 mutants (6 weeks old under 12-h light, 100 µmol m–2 s–1) were vacuum infiltrated with 0.1% (w/v) Evans blue (purchased from Fluka) for 10 min. After staining, the leaves were washed three times with distilled water until they were fully decolorized. Leaves were subsequently observed with an Olympus SZ61 stereoscopic microscope. For the induction of cell death in acd2-2, 5 µL of 3 mM coronatine (Sigma-Aldrich) dissolved in methanol was spotted onto the leaf of acd2-2. Plants were maintained under 12-h light (100 µmol m–2 s–1) for 24 h after the induction of cell death.
Fully expanded leaves of Col and var2-1 (7–8 weeks old under 12-h light, 100 µmol m–2 s–1) were used for the preparation of protoplasts. Excised leaves were gently suspended in enzyme solution (0.1% [w/v] cellulase Onozuka R10 [Yakult], 0.05% [w/v] Pectolyase Y-23 [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 60g for 1 min and washed with ice-cold wash buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES pH 5.7). After centrifugation at 60g for 1 min, the isolated protoplasts were resuspended with 500 µL of wash buffer and used for microscopic observation.
Leaflets were harvested from 6-week-old Col and var2-1 plants, and green and white sectors of var2-1 were dissected. Samples were immediately frozen in liquid nitrogen and pulverized with a microtube homogenizer. Total proteins were extracted by adding 10 times volume of extraction buffer (125 mM Tris-Cl pH 6.8, 2% [w/v] SDS, 5% [v/v] glycerol, 5% [v/v] 2-mercaptoethanol, 0.005% [w/v] bromo phenol blue). After centrifugation at 14,000g for 10 min, equally loaded supernatants (based on fresh weights) were subjected to SDS-PAGE analysis. The resolved gels were stained using the PlusOne Silver Staining kit, protein (GE Healthcare), according to the manufacturer's instructions. For immunoblot analysis, total proteins were electroblotted onto polyvinylidene difluoride membrane (ATTO) after SDS-PAGE and membranes were blocked with 5% (w/v) skim milk in 50 mM sodium phosphate buffer (pH 7.5) containing 155 mM NaCl and 0.05% (v/v) Tween 20 (PBST buffer) for 1 h. After two washes with PBST buffer, the membranes were incubated with either anti-FtsH2 (dilution 1:5,000), anti-PsbO (dilution 1:5,000), anti-Lhcb1 (dilution 1:5,000), anti-D1 (dilution 1:5,000), anti-BiP (dilution 1:2,000; gift from Ikuko Hara-Nishimura; Hatano et al., 1997
Green and white sectors of var2-1 were dissected and total RNA was isolated from 4-week-old Col and the var2-1 mutant reverse transcribed with the QIAGEN RNeasy Plant Mini kit. Semiquantitative RT-PCR assays with gene-specific primers were performed using total RNA (final concentration 2 ng µL–1 to each RT-PCR reaction) as a template according to the methods of the QIAGEN one-step RT-PCR kit. RT was carried out at 50°C for 30 min, followed by an initial PCR activation step at 95°C for 15 min. PCR reactions consisted of a denaturation at 95°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 1 min (psbA-fw: 5'-GCAACTGGATAACTAGCACCGA-3', psbA-rv: 5'-GAGGGAAGTTGTGAGCATTACG-3', rbcL-fw: 5'-TCTTGGCAGCATTCCGAGTAAC-3', rbcL-rv: 5'-GAAAAAGATAACCGCGAGCACGA-3', psbO-fw: 5'-GAGCACCAAAGAGGCTAACG-3', psbO-rv: 5'-GGGTCATGAGCTTGGTGTTT-3', rbcS-fw: 5'-GTCGCTCCATTCACTGGTTT-3', rbcS-rv: 5'-ATCCATTTGTTGCGGAGAAG-3', accD-fw: 5'-TGTGGATTCAATGCGACAAT-3', accD-rv: 5'-TTTTGCGCAGAGTCAATACG-3', rpoB-fw: 5'-AACTCGCTTTGGGGAAAAAT-3', rpoB-rv: 5'-TGATCCTTTCAGGACCTTGG-3').
Thin segments of leaves from approximately 6-week-old var2-1 mutants were fixed in 2.5% (v/v) glutaraldehyde and stained with 1 µg mL–1 DAPI. To prepare Technovit sections, thin segments of the leaf tissues were fixed in 45% (v/v) ethanol, 5% (v/v) acetic acid, and 1.8% (v/v) formaldehyde, dehydrated by a dilution series of ethanol, and embedded in Technovit 7100 resin (Kulzer). Thin Technovit sections (3 µm) were subsequently stained with DAPI. We used the vital stain Hoechst 33342 (Invitrogen; final concentration 1 µg mL–1) for DNA staining in viable protoplast cells. Samples were visualized with an Olympus BX61 light microscope with a disc scan unit (Olympus) that was Col, and dissected green and white sectors of var2-1 were extracted in DNA extraction buffer (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA, 5% [w/v] SDS, pH 7.5). Samples were subsequently centrifuged at 15,000g for 10 min and nucleic acids in the supernatant were precipitated by an equal volume of isopropanol. Semiquantitative PCR assays were performed using the isolated DNAs (final concentration 2 ng µL–1 to each PCR reaction) as the template. Gene-specific primers and PCR reaction were described above.
The following materials are available in the online version of this article.
We would like to thank Shinichi Arimura for providing the L12GFP transgenic Arabidopsis. We would also like to thank Ikuko Hara-Nishimura for the anti-BiP antibody, and Rie Hijiya and Chieko Hattori for their technical assistance. Received March 7, 2007; accepted April 10, 2007; published April 20, 2007.
1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (grant no. 16085207) and by the Oohara Foundation (to W.S.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wataru Sakamoto (saka{at}rib.okayama-u.ac.jp).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.099002 * Corresponding author; e-mail saka{at}rib.okayama-u.ac.jp; fax 81–86–434–1206.
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ABSTRACT
RESULTS
subunit, was crossed to var2-1. Similar to the results from L12-GFP, DIPS-GFP was detected as rod- and occasionally string-like structures representing mitochondria in both green and white sectors. Mitochondrial morphologies detected by DIPS-GFP were indistinguishable between green and white sectors (
-tubulin in white sectors. This was most likely attributable to the fact that the blots were prepared by a fresh weight basis; since developed thylakoid membranes were lacking, white sectors contained a larger number of cells than the green cells. Likewise, several proteins that appeared to increase in the white sectors were detectable in the silver-stained gel (
