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• W2153507794 abstract "Article15 February 1998free access A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes Jeong Mee Park Jeong Mee Park Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Ja Hyang Cho Ja Hyang Cho Department of Clinical Anatomy, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Shin Gene Kang Shin Gene Kang Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Hyun Jung Jang Hyun Jung Jang Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Kyeong Tae Pih Kyeong Tae Pih Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Hai Lan Piao Hai Lan Piao Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Moo Je Cho Moo Je Cho Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Inhwan Hwang Corresponding Author Inhwan Hwang Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Jeong Mee Park Jeong Mee Park Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Ja Hyang Cho Ja Hyang Cho Department of Clinical Anatomy, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Shin Gene Kang Shin Gene Kang Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Hyun Jung Jang Hyun Jung Jang Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Kyeong Tae Pih Kyeong Tae Pih Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Hai Lan Piao Hai Lan Piao Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Moo Je Cho Moo Je Cho Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Inhwan Hwang Corresponding Author Inhwan Hwang Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea Search for more papers by this author Author Information Jeong Mee Park1, Ja Hyang Cho2, Shin Gene Kang3, Hyun Jung Jang3, Kyeong Tae Pih1, Hai Lan Piao3, Moo Je Cho1 and Inhwan Hwang 1,3 1Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701 Korea 2Department of Clinical Anatomy, Gyeongsang National University, Chinju, 660-701 Korea 3Department of Molecular Biology, Gyeongsang National University, Chinju, 660-701 Korea *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:859-867https://doi.org/10.1093/emboj/17.4.859 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by in vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts. In addition, the remaining chloroplasts appeared morphologically not fully developed. The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants. When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level. However, the mutant ADL1s were not detectable at the protein level by Western blot analysis. Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a post-transcriptional silencing effect of the transgenes. We propose, therefore, that ADLl is involved in the biogenesis of thylakoid membranes. Introduction Dynamin is a high molecular weight GTP-binding protein with an intrinsic GTPase activity. Recently many genes belonging to the dynamin family have been isolated from a variety of eukaryotic systems ranging from yeast to human (Shpetner and Vallee, 1989; Horisberger et al., 1990; Obar et al., 1990; Rothman et al., 1990; Chen et al., 1991; van der Bliek and Meyerowitz, 1991; Nakata et al., 1993; Dombrowski and Raikhel, 1995; Gu and Verma, 1996). As a result, it appears that these family members are quite diverse with regard to the degree of sequence similarity among them as well as the biological processes in which these proteins are thought to be involved. The primary structure of these proteins can be divided into two regions. The N-terminal region containing the GTP-binding motifs is highly conserved among these proteins, whereas the C-terminal half of the molecules shows varying degrees of sequence conservation. Thus, the members of the dynamin family can be divided into several subfamilies based on their sequence similarity. One of them is the dynamin subfamily consisting of mammalian dynamin isoforms, Dyn1, Dyn2, Dyn3 and Drosophila shibire (Obar et al., 1990; Chen et al., 1991; Nakata et al., 1993; Cook et al., 1994). Another subfamily is the Vps1p family which is composed of the yeast homologs Vps1p and Dnm1p (Rothman et al., 1990; Gammie et al., 1995). Two plant homologs, SDL of soybean and aG68 of Arabidopsis, can also be grouped into a separate subfamily (Dombrowski and Raikhel, 1995; Gu and Verma, 1996). In addition, there are two subfamilies, Mx (Aebi et al., 1989; Horisberger et al., 1990) and Mgm1p (Jones and Fangman, 1992), which are most distantly related to the dynamin family based on amino acid sequence similarity as well as proposed biological functions. Among all these proteins, dynamin 1, the brain-specific isoform, has been characterized most thoroughly (Robinson et al., 1993; Miki et al., 1994; Seedorf et al., 1994; Valee and Okamoto, 1995). Many studies in animal cells indicate that dynamin 1 is involved in endocytosis for neurotransmitter recycling in the brain, especially at the step of vesicle formation from invaginated plasma membranes (van der Bliek and Meyerowitz, 1991; Herskovits et al., 1993a; Robinson et al., 1993; Damke et al., 1994). Thus it has been postulated that dynamin 1 may function as a molecular switch in regulating endocytosis, because the GTPase activity of dynamin 1 can be modulated in various ways, such as by interaction with proteins containing SH3 domains, by a phosphorylation/dephosphorylation cycle, and by interaction with membranes (Gout et al., 1993; Herskovits et al., 1993b; Robinson et al., 1993; Miki et al., 1994; Seedorf et al., 1994). In yeast, the dynamin homolog Vps1p plays a different role. Here it has been shown to be involved in vacuole protein sorting (Rothman et al., 1990; Vater et al., 1992; Wilsbach and Payne, 1993). Another protein, Dnm1p, also a member of the Vps1p subfamily, is thought to be involved in pheromone-mediated endocytosis in yeast (Gammie et al., 1995). The plant homolog SDL is thought to be involved in yet another biological process (Gu and Verma, 1996, 1997). SDL is localized specifically at the cell division plate during the early stage of cell plate formation in dividing soybean root tip cells or tobacco suspension culture cells, as observed by in situ cytohistochemistry or in vivo targeting experiments, respectively. Based on this observation, Gu and Verma (1996, 1997) suggested that SDL may play a role in transporting materials needed for cell plate formation. As described above, the members of the dynamin family appear to be involved in very diverse biological processes. However, there is a common feature that is shared among most of these proteins; it is the involvement in trafficking of materials. As a means to investigate intracellular trafficking in plants, we decided to clone cDNAs encoding dynamin-like proteins in Arabidopsis. We isolated a cDNA clone, ADL1, that encodes a dynamin-like protein in Arabidopsis and found that the nucleotide sequence of this clone was identical to that of a previously published cDNA, aG68. We used the cloned cDNA to look into the biological role of the encoded protein, ADL1, in Arabidopsis. Here we present evidence that the Arabidopsis dynamin-like 1 (ADL1) protein encoded in the aG68 cDNA is localized in the thylakoid membranes in leaf tissue and that it possibly is involved in thylakoid membrane biogenesis during chloroplast development. Results Presence of ADL1 in chloroplasts In order to study intracellular trafficking in plants, we first attempted to isolate a cDNA encoding a dynamin homolog from Arabidopsis. Degenerate oligonucleotides corresponding to the conserved GTP-binding motif were used as probes for library screening (Hall and Brown, 1985; Ma et al., 1990). The cDNA clone, ADL1, isolated from an Arabidopsis leaf λgt11 cDNA library had exactly the same sequence as the Arabidopsis aG68 cDNA (Dombrowski and Raikhel, 1995). Using this cDNA, we investigated the biological role of the encoded protein, ADL1, in Arabidopsis. We first employed Western blot analysis using a polyclonal antibody raised against the N-terminal region (∼300 amino acid residues starting from the initiation codon) of ADL1. The Western blot analysis of cell extracts from leaf, root, flower and silique tissue showed that ADL1 was present in all tissues examined (data not shown). This result is in a good agreement with the Northern data of Dombrowski and Raikhel (1995) which showed that the aG68 gene was transcribed at high levels in leaf, root, flower and silique tissue. To characterize the protein further, we then examined the cellular localization of ADL1 in leaf tissue by Western blot analysis. The leaf tissue was homogenized gently and fractionated on a Percoll gradient. The top fraction of the Percoll gradient was fractionated further into supernatant and pellet fractions by ultracentrifugation. As shown in Figure 1A, the majority of ADL1 was co-purified with the chloroplasts (either intact or broken). Only a minor portion was present in the top fraction of the Percoll gradient. When the top fraction was fractionated again by ultracentrifugation, ADL1 stayed with the membranes in the pellet fraction. However, upon longer exposure, a weak band appeared consistently in the supernatant of the ultracentrifugation step, indicating that a very small amount of ADL1 may be present in a soluble form. Figure 1.Subcellular and suborganellar localization of ADL1 in leaf tissue. (A) Total leaf homogenate was fractionated into top, broken chloroplast and intact chloroplast fractions on a Percoll step gradient. The top fraction was fractionated further into supernatant and pellet fraction by ultracentrifugation. These fractions were examined for the presence of ADL1 by Western blot analysis using the anti-ADL1 antibody. Total, Top, Br. Chl. and Intact Chl. indicate total homogenate, top fraction of the Percoll gradient, broken chloroplasts and intact chloroplasts, respectively. (B and C) ADL1 in the thylakoid membranes was examined after additional fractionation of the intact chloroplasts into supernatant (stroma and envelope membranes) and pellet (thylakoids). Proteins were resolved by SDS-PAGE and stained with Coomassie brilliant blue (B) or subjected to immunostaining with the anti-ADL1 antibody (C). An identical blot was probed with the anti-OEP34 antibody as an outer envelope membrane marker. Download figure Download PowerPoint Since the chloroplast is composed of multiple suborganelles, we pursued the question of the localization of ADL1 within the chloroplast even further. The intact chloroplasts were lysed and subsequently fractionated into supernatant and pellet by low speed centrifugation. After this fractionation, the pellet contains thylakoid membranes and the supernatant contains chloroplast envelope membranes as well as soluble stromal proteins. The proteins in the supernatant and pellet were separated by SDS-PAGE and probed with the anti-ADL1 antibody. As shown in Figure 1C, the majority of ADL1 was present in the pellet containing the thylakoid membranes, and only a minor portion remained in the supernatant. When the supernatant was fractionated further into a soluble and a pellet fraction by ultracentrifugation, most of the ADL1 co-purified with the membranes in the pellet fraction (data not shown), indicating that the small amount of ADL1 in the supernatant of the low speed centrifugation is very likely attached to the membranes of the chloroplast envelope. As a control for the suborganellar fractionation of the chloroplast, an identical blot was probed with a polyclonal antibody raised against OEP34, a subunit of the chloroplast protein import complex localized on the outer membrane of the chloroplast (Seedorf et al., 1995). The antibody recognized a protein species in the supernatant as expected. Thus the control experiment confirms that there is very little, if any, contamination of chloroplast membranes in the thylakoid fraction. These results clearly establish that the majority of ADL1 in chloroplasts is present in the thylakoid membranes, although a minor portion may be present in the membranes of the chloroplast envelope. In order to understand better the association of ADL1 with the thylakoid membranes, we treated the thylakoid membrane fraction with high salt (1.0 M NaCl) or high pH (0.1 M Na2CO3, pH 11.5) and subsequently precipitated the thylakoid membranes by low speed centrifugation. The amount of ADL1 remaining in the pellet was then examined by Western blot analysis. As shown in Figure 2A, a large portion of ADL1 was washed away at high pH, indicating that ADL1 is a peripheral membrane protein. In another experiment, the thylakoid membrane fraction was digested with thermolysin in the presence or absence of Triton X-100 and then examined for the presence of ADL1 by Western blot analysis. As shown in Figure 2B, ADL1 was digested away under both conditions, suggesting that ADL1 is most likely located on the outward-facing side of the thylakoid membranes. Figure 2.Association of ADL1 with thylakoid membranes. (A) The thylakoid membrane fraction was treated with 1.0 M NaCl or 0.1 M Na2CO3 on ice for 30 min. Solubilized proteins were removed after centrifugation in a microcentrifuge. The pellet was washed with the buffer once and then reconstituted to the original volume. Proteins in equal volumes of samples were subjected to Western blot analysis. (B) The thylakoid fraction was incubated with thermolysin (100 μg/ml) in the presence or absence of Triton X-100. The remaining proteins were resolved by SDS-PAGE and probed with the anti-ADL1 antibody. Download figure Download PowerPoint Targeting of ADL1 into the chloroplast As an alternative approach to show the presence of ADL1 in the chloroplasts, we employed an in vivo targeting experiment using green fluorescent protein (GFP) tagging on ADL1 (Sheen et al., 1995; Davis and Viestra, 1996). We generated a fusion protein between ADL1 and smGFP (ADL1-smGFP). We also fused the transit peptide of the chlorophyll a/b-binding protein (Cab) to smGFP (Cab-smGFP) in order to use it as a control protein for chloroplast targeting (Karlin-Neumann and Tobin, 1986; Smeekens et al., 1986) (Figure 3A). DNA constructs encoding the two fusion proteins and smGFP were introduced into soybean suspension culture cells (soybean photoauxotrophic cell, SB-P) by the particle bombardment method (Takeuchi et al., 1992). Expression of the introduced genes was examined under a fluorescence microscope. As shown in Figure 3B, the majority of the ADL1-smGFP fusion protein was targeted to the chloroplast as indicated by the green fluorescent staining of the chloroplasts. The staining pattern was nearly identical to that of Cab-smGFP. In contrast to ADL1-smGFP and Cab-smGFP, smGFP remained in the cytosol as expected. In addition, there was a weak staining at the boundary of the cells transformed with the ADL1-smGFP fusion gene, suggesting that a minor portion of ADL1-smGFP might be targeted to the plasma membrane. Figure 3.Targeting of the ADL1-smGFP fusion protein to chloroplasts. (A) Schematic presentation of fusion constructs. a, b and c indicate ADL1-smGFP, Cab-smGFP and smGFP, respectively. (B) The soybean suspension cells were transformed with either ADL1-smGFP (panels A and B), Cab-smGFP (panels C and D) or smGFP (panels E and F). The left and right panels are fluorescent and light microscopic images of the cells, respectively. Bars indicate 12 μm. Download figure Download PowerPoint Immunogold labeling of ADL1 in thylakoid membranes To obtain direct evidence for the presence of ADL1 in the thylakoid membranes, we employed the immunogold labeling method using the polyclonal anti-ADL1 antibody. Four-week-old leaf tissue was fixed and processed for immunogold labeling. As shown in Figure 4, the immunogold label concentrated in the chloroplasts. The majority of the label was located in the thylakoid membranes, with only minor labeling of the membranes of the chloroplast envelope. Thus, the results of the immunogold labeling experiment are in agreement with those of the Western blot analysis described in Figure 1. Figure 4.Immunogold labeling of ADL1 in the thylakoid membranes. The sections prepared from leaf tissue were labeled with immunogold coated with the anti-ADL1 antibody (A, B and C). The control sample was treated with a polyclonal antibody against ARSK1 which is a root-specific protein (D). S indicates starch granules. The bars represent 1.2 μm (A), 0.15 μm (B) and 0.2 μm (C and D). Download figure Download PowerPoint Yellow leaf phenotype of the transgenic plants harboring deletion mutant genes To gain insight into the biological role of ADL1 in the chloroplasts, we decided to generate dominant-negative mutants by expressing mutant forms of the ADL1 cDNA in transgenic plants. The deletion mutant genes shown in Figure 5A were constructed, and transgenic plants were generated by the Agrobacterium-mediated transformation method (Valvekens et al., 1988). The deletion constructs were under the control of the 35S cauliflower mosaic virus (CaMV) promoter to obtain strong expression in leaf tissue. The T1 generation of the transformants was rescreened for kanamycin resistance. We then examined the transgenic plants visually for phenotypic changes. As shown in Figure 5B, one obvious phenotypic effect was manifested in the yellow leaves of the transgenic plants. The yellow leaf phenotype was reproducible in the T2 generation. The transgenic plants retained the phenotype when grown in soil, but at a somewhat reduced frequency. This phenotype was observed in many independent lines of transgenic plants derived from the same deletion mutant gene and from different deletion mutant genes, albeit that it differed in frequency and severity as shown in Table I. The results strongly suggest that the yellow leaf phenotype is caused by the introduction of the mutant gene. The variation in the frequency of the phenotype between the transgenic lines and between the plants with various deletion mutant genes is probably due to the randomness in their integration into the genome. Figure 5.Phenotype of transgenic plants. (A) Schematic diagrams of the deletion mutant genes of ADL1 cDNA. The three elements of the consensus GTP-binding motif are indicated by thick lines. (B) Phenotype of the T1 progeny of transgenic Arabidopsis plants. Transgenic plants containing pBI121 (a control transformation vector) (a) and ADL1del2 (b). Download figure Download PowerPoint Table 1. Phenotype analysis of transgenic plants Construct Line Total No. (T1) Phenotype Kmr Green Yellow Kms ADL1del1 7 18 5 8 5 8 16 5 4 7 10 12 3 7 2 15 14 11 0 3 ADL1del2 2 15 5 5 5 12 18 4 10 4 25 16 10 1 5 28 19 7 6 6 ADL1del4 1 14 3 6 5 13 13 7 1 5 14 15 2 9 4 15 11 5 3 3 ADL1ΔN 3 13 2 6 5 12 11 2 7 2 21 15 5 9 1 24 18 6 8 4 ADL1ΔC 1 12 6 4 2 2 13 3 4 6 5 8 4 2 2 19 17 5 7 5 Approximately 20 seeds from the T1 generation of each line indicated were sterilized and planted on MS plates containing kanamycin (50μg/ml). Plants were scored for kanamycin resistance and for the phenotype 3 weeks after planting. In search for the mechanism underlying the yellow leaf phenotype, we further characterized the transgenic plants. First, we examined the expression of the introduced deletion mutant genes by Northern blot analysis using total RNA obtained from the respective transgenic plants. The transgenic plants showed a high level of expression of the deletion mutant genes as expected. A typical result of Northern blot analysis is shown in Figure 6A. We also carried out Western blot analysis to measure the accumulation of the truncated proteins encoded by the deletion mutant genes. Surprisingly, we found no accumulation of any truncated form of ADL1, regardless of which deletion mutant gene had been introduced. An example of Western blot analysis is shown in Figure 6B. In order to rule out the possibility that a frameshift mutation occurred during the construction of the deletion mutant genes, we translated the deletion constructs in vitro using wheat germ extract and examined the translation products by Western blot analysis with the anti-ADL1 antibody. We observed that the deletion constructs not only yielded the expected truncated forms of ADL1, but also that the in vitro translated products were recognized by the anti-ADL1 antibody (data not shown). In contrast to the absence of accumulated truncated ADL1s, the transgenic plants showed various levels of endogenous ADL1 ranging from the control level to >10-fold reduced levels. The Western blot results thus stand in contrast to the Northern blot results. A possible explanation for this may be a post-transcriptional silencing effect by the introduced deletion mutant gene on the endogenous gene in the transgenic plants, in spite of our initial attempt to create a dominant-negative effect when introducing the deletion mutant genes. It has been shown before that gene silencing can occur either at the transcriptional level or at the post-transcriptional level (de Carvalho et al., 1992; Seo et al., 1995; Elmayan and Vaucheret, 1996). Figure 6.Northern and Western analysis of transgenic plants transformed with the ADL1del2 construct. (A) High level transcription of the ADL1del2 deletion mutant gene. Total RNA was isolated from transgenic plants containing the ADL1del2 deletion mutant gene and analyzed by Northern blot hybridization using the ADL1 cDNA as a probe. (B) Suppression of endogenous ADL1 by the introduced deletion mutant genes. Protein extracts were prepared from transgenic plants of the T1 generation. The level of endogenous ADL1 and deletion mutant ADL1del2 was examined by Western blot analysis with the anti-ADL1 antibody. The numbers denote independent lines of transgenic plants harboring ADL1del2. pBI121 indicates transgenic plants harboring a transformation vector, pBI121. Download figure Download PowerPoint Thylakoid membrane and thylakoid membrane protein reduction in the transgenic plants To address the phenotypic changes at the cellular level, sections of leaf tissue were examined under a microscope. In the leaves of the transgenic plants, the overall size of individual cells was slightly enlarged, while the number of cells was reduced. However, the most prominent changes in the transgenic plants occurred in the number and the morphology of the chloroplasts. The cells in the leaf tissue contained almost no or only very few chloroplasts. Furthermore, the remaining chloroplasts in the transgenic plants appeared to be not fully developed. We examined these chloroplasts with an electron microscope. As shown in Figure 7A and C, they show a marked reduction in the amount of thylakoid membranes without grana stacks. Instead the chloroplasts in the leaf cells of the transgenic plants had more lipid granules (plastoglobules, Pg) and were filled with small particulates. Usually the chloroplasts of a mature leaf cell exhibit thylakoid membranes that are organized into grana stacks at a high density, as shown in Figure 7B and D (Apel et al., 1983; Melis, 1984; Anderson and Beardall, 1991). We also addressed the changes in the level of thylakoid membrane polypeptides in the transgenic plants. Total cell protein was extracted from transgenic plants harboring pBI121 (a transformation vector) and the deletion mutant genes, and probed with polyclonal antibodies raised against maize proteins CP29, and the light-harvesting complex II (Bergantino et al., 1995). As shown in Figure 8, the amount of thylakoid membrane proteins such as CP29 and the LHCII complex was greatly reduced in the transgenic plants harboring the deletion mutant genes (ADL1del1 and ADL1del2) as compared with the control transgenic plants (pBI121) when equal amount of total protein was separated by SDS-PAGE. The level of these polypeptides correlated well with that of ADL1 in the transgenic plants. Figure 7.Comparison of chloroplast development in transgenic and wild-type plants. Leaf sections of transgenic and wild-type plants were examined by transmission electron microscopy. A transgenic plant with the yellow leaf phenotype (harboring ADL1del2) (A and C) and a wild-type plant (B and D). S, g and Pg indicate starch, grana stacks and plastoglobules (lipid granules). Bars represent 2.0 μm (A and B) and 0.1 μm (C and D). Download figure Download PowerPoint Figure 8.Accumulation of thylakoid membrane polypeptides in the transgenic plants. Total cell extracts were prepared from transgenic plants harboring the ADL1del1 (del1-7, del1-10) and ADL1del2 deletion mutant (del2-12, del2-25) genes as well as transgenic plants harboring a transformation vector, pBI121 (pBI121). Proteins (10 μg) were separated by 10% SDS-PAGE and stained with Coomassie brilliant blue to confirm equal loading (data not shown). For Western blot analysis, identical gels were blotted onto PVDF membranes. The blots were probed with antibodies raised against ADL1, CP29 (maize) and the light-harvesting complex II (maize, LHCII). Download figure Download PowerPoint Therefore, our results strongly suggest that the cells in the leaf tissue of transgenic plants are defective with regard to the accumulation of thylakoid membranes as well as thylakoid membrane proteins in their chloroplasts. In addition, there were other changes, such as a reduction in the volume of the cytosol as well as in the number of mitochondria; however, it is not clear whether these changes were the direct effect of the reduced ADL1 level or the indirect consequence of poor chloroplast development. Discussion In this study, we investigated the biological role of ADL1 in Arabidopsis. By subcellular localization studies of ADL1 using Arabidopsis leaf tissue, we found that a large portion of the ADL1 pool is present in chloroplasts. Subsequent suborganellar fractionation experiments revealed that the majority of the chloroplast ADL1 is associated with the thylakoid membranes. This finding was supported by two additional independent approaches. An ADL1-smGFP fusion protein was targeted to the chloroplasts when expressed transiently in soybean suspension cells, SB-P. The immunogold labeling studies, using the anti-ADL1 antibody, unequivocally demonstrated that the majority of the ADL1 in the chloroplasts is localized at the thylakoid membrane. As a means to elucidate the biological role of ADL1, we initially tried to generate dominant-negative mutants by expressing various deletion mutant genes in transgenic plants, since the protein seems to be composed of multiple domains and also exists in high molecular weight complexes in vivo (Park et al., 1997). The transgenic plants containing these deletion constructs had yellow leaves. Microscopic inspection of the yellow leaf phenotype at the cellular level showed that cells in the leaf tissue contain almost no or very few chloroplasts, which then appear not fully developed. Moreover, the chloroplasts as seen by electron microscopy contained a greatly reduced amount of thylakoid membranes. On the other hand, there was an increased number of lipid granules and an accumulation of small particulates. Also, the amount of thylakoid membrane polypeptides was greatly reduced in the transgenic plants showing the yellow leaf" @default.
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• W2153507794 title "A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes" @default.
• W2153507794 cites W1529429589 @default.
• W2153507794 cites W1565334821 @default.
• W2153507794 cites W1594530422 @default.
• W2153507794 cites W1668266668 @default.
• W2153507794 cites W1805954825 @default.
• W2153507794 cites W182091170 @default.
• W2153507794 cites W1955881543 @default.
• W2153507794 cites W1964520368 @default.
• W2153507794 cites W1967965496 @default.
• W2153507794 cites W1968242602 @default.
• W2153507794 cites W1969407643 @default.
• W2153507794 cites W1972370363 @default.
• W2153507794 cites W1975327433 @default.
• W2153507794 cites W1975542057 @default.
• W2153507794 cites W1976391336 @default.
• W2153507794 cites W1983322120 @default.
• W2153507794 cites W1986744284 @default.
• W2153507794 cites W1995172710 @default.
• W2153507794 cites W1999079263 @default.
• W2153507794 cites W2007405937 @default.
• W2153507794 cites W2007816311 @default.
• W2153507794 cites W2013767467 @default.
• W2153507794 cites W2031583450 @default.
• W2153507794 cites W2037852171 @default.
• W2153507794 cites W2037956598 @default.
• W2153507794 cites W2038255589 @default.
• W2153507794 cites W2040987875 @default.
• W2153507794 cites W2055473408 @default.
• W2153507794 cites W2070634601 @default.
• W2153507794 cites W2080087017 @default.
• W2153507794 cites W2082530736 @default.
• W2153507794 cites W2084285799 @default.
• W2153507794 cites W2092081030 @default.
• W2153507794 cites W2108264768 @default.
• W2153507794 cites W2112203177 @default.
• W2153507794 cites W2113946252 @default.
• W2153507794 cites W2149561709 @default.
• W2153507794 cites W2151345479 @default.
• W2153507794 cites W2157018895 @default.
• W2153507794 cites W2162092495 @default.
• W2153507794 cites W2409928594 @default.
• W2153507794 cites W267879079 @default.
• W2153507794 cites W341255601 @default.
• W2153507794 cites W50192545 @default.
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