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First published online October 29, 2004; 10.1104/pp.104.049841 Plant Physiology 136:3594-3604 (2004) © 2004 American Society of Plant Biologists Deletion of the Chloroplast-Localized Thylakoid Formation1 Gene Product in Arabidopsis Leads to Deficient Thylakoid Formation and Variegated Leaves1Department of Plant Pathology (Q.W., R.W.S., K.L.K.) and Department of Biological Sciences (A.K., R.L.H.), University of Arkansas, Fayetteville, Arkansas 72701; and Department of Biology (J.H.) and Departments of Biology and Pharmacology (A.M.J.), University of North Carolina, Chapel Hill, North Carolina 27599
Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. The Arabidopsis (Arabidopsis thaliana) Thylakoid formation1 (Thf1) gene product is shown here to control an important step required for the normal organization of these vesicles into mature thylakoid stacks and ultimately for leaf development. The Arabidopsis Thf1 gene encodes an imported chloroplast protein, as shown by in vitro import and localization of a Thf1-green fluorescent protein fusion product in transgenic plants. This gene is conserved in oxygenic photoautotrophs ranging from cyanobacteria to flowering land plants. Transcript levels for Thf1 are induced in the light and decrease under dark conditions, paralleling profiles of light-regulated nuclear genes involved in chloroplast function. Disruption of the Thf1 gene via T-DNA insertion results in plants that are severely stunted with variegated leaf patterns. Nongreen sectors of variegated leaves lacking Thf1 expression contain plastids that accumulate membrane vesicles on the interior and lack organized thylakoid structures. Green sectors of Thf1-disrupted leaves contain some chloroplasts that form organized thylakoid membranes, indicating that an inefficient compensatory mechanism supports thylakoid formation in the absence of Thf1. Genetic complementation of a Thf1 knockout line confirms the role of this gene in chloroplast and leaf development. Transgenic plants expressing the Thf1 gene in antisense orientation are stunted with altered thylakoid organization, especially in young seedlings. The data indicate that the Thf1 gene product plays a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis.
The development of normal chloroplasts is a crucial step in the survival of photosynthetic eukaryotes. Maturation and activity of the chloroplast is highly dependent on its relationship with the nucleus, as the bulk of proteins that function in the chloroplast are encoded in the nucleus and are posttranslationally imported from the cytoplasm (Keegstra and Cline, 1999  ). Lesions in genes encoding plastid-localized proteins can have severe effects on the many metabolic processes that occur there and ultimately can prevent normal plant growth and development. Demonstrating the overriding importance of chloroplast proteins, the majority of mutations in a screen of mutagenized Arabidopsis (Arabidopsis thaliana), designed to identify genes important for seedling viability, appeared to affect chloroplast formation or function (Budziszewski et al., 2001). Environmental conditions, especially light, also play important roles in triggering chloroplast development and orchestrating gene expression required for plastid function (Anderson, 1986). In spite of the dependence of plants on the photosynthesis occurring on chloroplast thylakoid membranes, there is much to be learned regarding the mechanisms by which these internal membranes originate and how the photosynthetic machinery is assembled.
When green tissues develop in the presence of light, chloroplasts are normally formed from undifferentiated proplastids lacking internal membranes. As cells and organelles divide, new thylakoid membranes must be synthesized and organized along with the light-gathering and photosynthetic protein components (for review, see Vothknecht and Westhoff, 2001
Several nuclear- and plastid-encoded Arabidopsis genes have been identified that can ultimately affect thylakoid formation or chloroplast development. However, it remains to be determined whether the actions of these genes lead directly or indirectly through alteration of important metabolic processes, to plastid malformation. The apparent function of the individual proteins encoded by these genes can vary widely. For example, impairment of plastid RNA polymerases can lead to altered chloroplasts and plants with pale or variegated leaves (De Santis-Maciossek et al., 1999
Deletion of the Arabidopsis vesicle-inducing protein (VIPP1) shows that it is involved in the formation of vesicles leading to thylakoid biogenesis. Mutation of VIPP1 leads to pale-green plants that cannot grow as photoautotrophs and contain plastids that do not form the vesicles that are thought to be necessary for thylakoid formation (Kroll et al., 2001 Through characterization of a T-DNA-tagged mutant line of Arabidopsis and production of transgenic lines, we have identified a nuclear-encoded gene important for the normal development of thylakoid membrane stacks and ultimately for leaf growth and development.
Thf1 Is Light Regulated and Encodes a Chloroplast-Localized Protein
A cDNA clone for an apparent ortholog of the Arabidopsis Thylakoid formation1 (Thf1) gene was originally isolated from potato (Solanum tuberosum; accession no. AY342161) and identified as a light-regulated gene (R.W. Sullivan and K.L. Korth, unpublished data). The apparent ubiquitous nature of this gene in plants, along with the presence of a conserved strong coiled-coil motif, made it an interesting candidate for further study. A cDNA encoding Arabidopsis Thf1 was amplified using reverse-transcription (RT)-PCR based on the sequence of a full-length cDNA listed in the GenBank database (accession no. AY087394). Analysis of the Arabidopsis genome indicates that this is a single-copy gene without a closely related sequence in the remainder of the genome. The predicted amino acid sequence encoded by Thf1 suggests the presence of a chloroplast transit signal at the amino terminus and a coiled-coil domain near the carboxyl terminus in a hydrophilic protein of 33.8 kD (Fig. 1A). The predicted size of a fully processed Thf1 protein in chloroplasts is 26.8 kD. A search of the GenBank database with the predicted amino acid sequence yielded several full-length entries with a high degree of similarity to Thf1. With one exception, all of the sequences similar to Thf1 were found in photosynthetic organisms, ranging from cyanobacteria to land plants. A predicted protein with 48.9% similarity to the Thf1 sequence in the overlapping region (excluding the chloroplast transit signal) has also been identified in a large, double-stranded DNA virus (Van Etten, 2003
The presence of a putative transit signal on Thf1 led us to examine intact chloroplasts for the ability to import the protein. Radiolabeled Thf1 was efficiently imported into chloroplasts as judged by protease protection of the chloroplast-localized protein. Furthermore, the labeled Thf1 was processed to the predicted size (26.8 kD) after cleavage of the transit peptide (Fig. 2A). Fractionation of protease-treated chloroplasts containing imported Thf1 showed that the protein is localized to both soluble stroma and membranes. Addition of the protease thermolysin to membrane fractions results in the degradation of the bulk of Thf1, indicating that it is most likely bound at the periphery and not integrated into the lipid bilayer. This result is consistent with the absence of a predicted transmembrane region in the Thf1 sequence. However, a weak signal for Thf1 remains in the thermolysin-treated thylakoid fraction (Fig. 2A), suggesting that Thf1 is partially protected from protease treatment. Localization of chloroplast proteins cab80 (light-harvesting complex protein [LHCP]; Cashmore, 1984 ) and p36 (Schnell et al., 1990) was used as a measure of the effectiveness of fractionation. The LHCP protein is known to integrate into thylakoids and is only partially accessible to added protease (Fig. 2B). The p36 phosphate translocator protein is localized to the chloroplast inner envelope membrane, and the localization patterns for both imported LHCP (Fig. 2B) and p36 (Fig. 2C) confirm that there was no contamination of soluble fractions with either thylakoid or envelope membrane fragments. Both of the control proteins migrated on the gel at the predicted positions; the size of the processed LHCP protein is 27 kD and of the processed p36 protein is 36 kD.
Fusion of the Thf1 protein at the carboxyl terminus with green fluorescent protein (GFP) verified chloroplast localization of the protein in planta (Fig. 3). To confirm the subcellular localization of Thf1 in plastids or chloroplasts, we made the construct 35S:Thf1-GFP and transformed it into suspension cells and plants. Thf1-GFP was localized in plastids and stromules, which seem to connect plastids together (Fig. 3A). Some stromules extended to the plasma membrane, indicating that communication might take place between the plasma membrane and plastids. In mesophyll cells, Thf1-GFP was distributed throughout the chloroplasts but predominantly in uneven patterns around the periphery, perhaps associated with the chloroplast envelope (Fig. 3B). This result is consistent with the above in vitro assay. These data showed that Thf1 was localized not only in plastids and chloroplasts but also in stromules.
Transcripts for Thf1 accumulate in leaves of plants grown under normal long daylight (16 h)/dark (8 h) conditions. Leaves collected 4 h into the light cycle contain abundant transcripts, whereas transcript levels are greatly reduced in leaves from plants placed in complete darkness for 24 h (Fig. 4). Transcript levels return to normal within 6 h after plants are returned to normal light levels. Alterations in Thf1 transcript levels closely parallel those of a characterized light-regulated nuclear gene encoding a chloroplast protein, Lhca1 (Kim et al., 1999 ), in response to light conditions (Fig. 4).
A T-DNA-Tagged Thf1 Mutant Is Stunted with Variegated Leaves An Arabidopsis line with a potential T-DNA insertion in the Thf1 gene (SALK_094925) was acquired after a search of the Salk Institute Genomic Analysis Laboratory Arabidopsis gene-mapping database (http://signal.salk.edu/cgi-bin/tdnaexpress). Seedlings were screened via PCR for the presence of a T-DNA insertion in chromosome 2 within the gene designated At2g20890. Amplification of a specific band in several individual plants confirmed incorporation of the T-DNA in the Thf1 gene (data not shown). We found no insertions in another plant line (SALK_094926) listed as having a T-DNA insertion at the same location. Sequencing of the PCR products showed that the insertion was at nucleotide position 299 in the Thf1 cDNA, near the 3' end of exon 1 (Fig. 5A). With the exception of one plant, individuals that grew to maturity from the SALK_094925 seed stock were indistinguishable from wild-type Arabidopsis ecotype Columbia plants grown alongside. Among the plants of normal appearance, there was a mixture of PCR-positive and -negative individuals, indicative of segregation of the T-DNA tag within the population. One plant, designated 25-7, was severely stunted with leaves containing green and yellow/white sectors (Fig. 5 B and C). Young seedlings of this line, less than 10 d old, were yellow, but, as the plants aged, they eventually began to develop interspersed green patches. Although the timing was delayed, line 25-7 flowered and formed viable seed. When reciprocal crosses were made between line 25-7 and wild-type Columbia plants, the resulting F1 progeny all grew normally and were indistinguishable from the wild-type parent (Fig. 5D). Segregation patterns in subsequent generations showed that the mutation leading to the abnormal leaf phenotype behaved as a single, recessive gene.
Genomic DNA-blot analysis confirmed that line 25-7 contained a unique restriction enzyme pattern when hybridized with a radiolabeled Thf1 cDNA and compared to the other lines (data not shown). The DNA restriction profiles were consistent with line 25-7 being homozygous for the T-DNA insertion, whereas other PCR-positive lines appeared to be heterozygous and PCR-negative lines contained no T-DNA insertion. Segregation patterns of the yellowed/stunted phenotype in seedlings derived by selfing PCR-positive lines also indicate that 25-7 is homozygous for the T-DNA insertion and that the other original lines are heterozygous. Even upon prolonged exposure of leaf RNA blots, transcripts hybridizing to Thf1 appear to be completely absent in line 25-7, whereas they are found at normal levels in the other Arabidopsis lines tested (Fig. 5E). The profiles of transcripts for other light-inducible nuclear-encoded genes encoding chloroplast proteins such as LhcA1 are not altered in 25-7, indicating that the deficiency of Thf1 mRNA is not due to a lesion affecting transcript accumulation for this class of genes. Leaves containing a mixture of green and yellow sectors were used for RNA extractions; the results therefore show that, even in normal-appearing green sectors of line 25-7, there is no Thf1 expression.
Ultrastructural examination via transmission electron microscopy (TEM) shows that some chloroplasts from green sectors of leaves in line 25-7 appear to have normal thylakoid development in terms of abundance and structure. However, many plastids in the same tissue had reduced amounts of thylakoid stacking, and distances between grana stacks were greater than in wild-type chloroplasts (Fig. 6 A and B, ). Although a few chloroplasts of relatively normal appearance were found, most plastids in yellow/white sectors of line 25-7 lacked thylakoid membranes, grana, or starch granules. However, the envelope membrane structure, size, and general shape of these plastids appear to be normal (Fig. 6C). These plastids accumulated numerous membrane vesicles of varying sizes in the interior soluble region (Fig. 6D), but the vesicles do not coalesce to form thylakoids.
Transgenic Lines Confirm the Role of Thf1 in Chloroplast and Leaf Development The Thf1 cDNA was integrated into the Arabidopsis genome in both sense and antisense orientations under the control of a cauliflower mosaic virus 35S promoter using Agrobacterium-mediated transformation. This allowed us to test whether changing levels of Thf1 expression leads to altered leaf and/or chloroplast development. Progeny of selfed, transformed lines expressing the antisense Thf1 grew more slowly than offspring of plants transformed with either the sense construct or the binary vector alone (Fig. 7, A–D). Although they were initially smaller, Thf1 antisense lines in short daylight (12 h) ultimately grew to nearly equal size of the sense lines or vector-transformed controls. We observed no differences in plant growth or development when comparing the progeny of independent selfed lines transformed with either the sense construct or the vector alone. When antisense plants were grown under long daylight conditions (16 h), their leaves had a more wrinkled appearance, and although they flowered, they were smaller than control lines and formed less siliques (Fig. 7D). The overall size of Thf1 antisense lines was reduced compared to vector-transformed control lines, as indicated by smaller rosettes over the life of the plant (Fig. 7E). Unlike the homozygous knockout mutant line 25-7, the antisense lines did not always display yellow-sectored leaves, and when variegation appeared, the phenotype was not as pronounced as in line 25-7. The level of Thf1 protein is severely reduced in antisense chloroplasts, as indicated by immunoblot analysis (Fig. 7F). The time to bolting was also delayed in the antisense lines. The vector-transformed control lines all bolted within 41 d after planting, whereas not all antisense lines bolted until 52 d. The plants containing the antisense construct eventually developed normal flowers and viable seeds.
When the knockout line 25-7 was transformed with the Thf1 sense construct, the resulting lines grew normally (Fig. 8A) and transgenic lines accumulated high levels of Thf1 transcripts (Fig. 8B). This complementation confirms that disruption of the Thf1 gene leads to the severe phenotype observed in line 25-7.
Analysis of chloroplasts via TEM of transgenic lines suggests that thylakoid organization is severely affected in young plants transformed with the Thf1 antisense construct. Although loosely organized thylakoid membranes seem to form in 10-d-old antisense-construct seedlings, the chloroplasts lack normal grana stacks when compared to chloroplasts from control lines of the same age (Fig. 9 A and B). Abundant membranes and plastoglobules accumulate in these chloroplasts, but the membrane matrix is severely disrupted, giving the interior a sponge-like appearance. As the plants age, chloroplasts in antisense lines take on a more normal appearance (Fig. 9, C–E). However, as in chloroplasts from the green sectors of the 25-7 knockout line, in antisense lines the grana stacks appear shorter and there is less starch granule accumulation. As in line 25-7 tissues, the structure and size of other cell components and organelles such as mitochondria appear normal in the sense and antisense lines.
Through analysis of T-DNA-tagged Arabidopsis mutants and transgenic plants, we have determined a role in chloroplast and leaf development for the previously undefined Arabidopsis Thf1 gene. A conserved form of Thf1 appears to be present in all plants and cyanobacteria species that were examined via gene database searches, suggesting that it has a vital function in photoautotrophs. A highly similar form of the sequence is also present in a large double-stranded DNA virus that infects freshwater green algae. It is possible that this viral form of the gene was transferred from a photosynthetic organism to the viral genome. Although the gene is well conserved among cyanobacteria, examination of cyanobacterial genomes (http://www.kazusa.or.jp/cyano/cyano.html) does not indicate that it is part of an operon of known function. There are abundant examples of genes of cyanobacterial origin being ultimately encoded in the nuclear genome of plants (Martin et al., 1998 ), and it is likely that transfer of the Thf1 gene occurred from the chloroplast genome to the nuclear genome.
There is mounting evidence that a dynamic system of vesicle transport is involved in the biogenesis of thylakoids in mature chloroplasts (Westphal et al., 2001
The characteristics of phenotype in Thf1 knockout lines and Thf1 antisense lines in several ways parallel those of other known mutants of chloroplast development. The variegated leaf phenotype of Thf1 knockout lines can be observed in several characterized Arabidopsis mutants. For example, lesions in several chloroplast RNA polymerase genes lead to variegated leaves and the accumulation of vesicles in plastids of young leaf tissue (De Santis-Maciossek et al., 1999
There are examples to suggest that variegation and vesicle formation characteristics are not necessarily a general response to interference with chloroplast function. A severe decrease of the light-harvesting complex of PSII, brought about via antisense-mediated decrease in the light-harvesting complex of PSII subunits, did not result in altered chloroplast formation, and thylakoid stacks readily formed in such plants (Andersson et al., 2003
In the case of Thf1 antisense lines, it is likely that low amounts of Thf1 protein are produced. This explains the observed differences in phenotype between the severely stunted, variegated knockout line 25-7 (Fig. 5) and the antisense lines (Fig. 7). As we have also observed of the Thf1 antisense lines, there are several reports of chloroplast-linked mutations that are most pronounced in the early stages of plant development but that are eventually overcome as plants mature. In low-light conditions, immutans leaves are not variegated, but the slow-growing mutants do eventually reach the same size as wild-type plants (Aluru et al., 2001
It is difficult to predict the function of Thf1 based solely on the derived sequence, although the prediction of a strong coiled-coil motif might provide some clues as to a role for the protein. Coiled coils are most often associated with peptide oligomerization and can be found in proteins with a range of functions (Burkhard et al., 2001
It has been suggested that SNARE proteins play a critical role in virtually all examples of eukaryotic vesicle fusion that are known (Rizo and Südhof, 2002
Comparisons of the Arabidopsis Thf1 sequence with genomes of other organisms offer us a seeming paradox. Cyanobacteria clearly contain sequences that are similar to Arabidopsis Thf1, yet these species might lack a vesicle transport system (Westphal et al., 2003
Plant Growth and Maintenance
Arabidopsis (Arabidopsis thaliana) seeds were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH). Plants were maintained and seeds were collected and treated, essentially as described (Weigel and Glazebrook, 2002 Rosette growth was determined by measuring at the maximum diameter. Ten plants of each genotype were measured daily and were no longer measured after they started to bolt.
The Arabidopsis Thf1 cDNA was amplified via RT-PCR from total RNA of Arabidopsis (ecotype Columbia) using an oligo(dT) primer and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) for first-strand synthesis. Amplification was carried out with specific primers (forward primer, 5'-ctcacttcaaggtaccgatggctgc-3'; reverse primer, 5'-catatacacgtctagaatgttcttag-3') based on the GenBank accession AY062799. Products of the RT-PCR were cloned into a plasmid vector, and identification of the cloned cDNA was confirmed via DNA sequencing.
The predicted Thf1 protein was analyzed for the presence of a putative chloroplast transit signal using the ChloroP program (Emanuelsson et al., 1999 The predicted amino acid sequence encoded by Arabidopsis Thf1 was aligned with similar full-length sequences, identified in a BLAST search, using the Clustal program within the GCG sequence analysis package. Amino acid sequence distances were calculated using Protdist and an unrooted tree was constructed via the unweighted pair group method with arithmetic mean (UPGMA) method in the NEIGHBOR program; both programs are contained in PHYLIP version 3.5c (http://evolution.genetics.washington.edu/phylip/html).
Total RNA was isolated from leaves frozen in liquid nitrogen and extracted with TriReagent (Molecular Research Center, Cincinnati) following the manufacturer's directions. RNA was separated on 1% agarose formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled Thf1 cDNA as a probe using established methods (Sambrook et al., 1989
For in vitro chloroplast import assays, the Thf1 cDNA was first cloned into the pGEM4z plasmid vector for protein expression. Linearized DNA was used in an in vitro transcription/wheat germ translation system in the presence of 35S-labeled Met to produce radiolabeled protein. Import of radiolabeled proteins into isolated pea (Pisum sativum) chloroplasts, subfractionation of chloroplasts, gel electrophoresis, and radiography were carried out as described (Henry et al., 1994
The open reading frame of AtThf1 (At2g20890) was first cloned into the entry vector pENTR/SD/-TOPO (Invitrogen). For expression of the GFP-tagged Thf1 in plant cells, the open reading frame of AtThf1 was recombined into Gateway destination vectors pGWB5 (Research Institute of Molecular Genetics, Shimane, Japan). Stable transformed suspension cells and transgenic plants (Ferrando et al., 2000
The T-DNA-tagged mutant of Arabidopsis (Alonso et al., 2003
For plant transformation, the Arabidopsis Thf1 cDNA was synthesized via RT-PCR from total RNA as described above. The sense fragment used for overexpression was amplified with the forward and reverse primers described above for cDNA amplification. The inverted Thf1 fragment used in an antisense construct was amplified with a forward primer (5'-ctcacttctagatagcgatggctgc-3'; introduced XbaI site in bold) and a reverse primer (5'-ggttatatagggtacctcccag-3'; introduced KpnI site in bold). The sense and antisense constructs in a binary vector were generated by directional ligation of the amplified inserts into a modified pCAMBIA2300 vector (GenBank accession no. AF234315) containing a 35S promoter of cauliflower mosaic virus (provided by Y. Yang, University of Arkansas, Fayetteville, AR). The resulting constructs were introduced into Agrobacterium tumefaciens strain GV3010 via electroporation. Plants were transformed using a floral dip method (Clough and Bent, 1998
Fresh material from rosette leaves was prepared and viewed via electron microscopy as described by Kim and Fulton (1984)
Fully expanded rosette leaf tissue (5g) was homogenized using polytron (Brinkman model PT 10-35) in ice-cold extraction buffer (20 mL) containing 0.35 M sorbitol, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.1% BSA (w/v), and 0.1% Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY087394.
We thank Sandy Goeke for acquiring the TEM images. We thank Yinong Yang for providing the binary vector, K.S. Kim for help with interpreting electron microscopy images, and the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. The LhcA1 clone was kindly provided by Dr. N.E. Hoffman. Received July 22, 2004; returned for revision September 1, 2004; accepted September 7, 2004.
1 This work was supported in part by the Arkansas Rice Research and Promotion Board (grants to K.L.K.), by the National Institutes of Health COBRE Program of the National Center for Research Resources (grant no. P20–RR15569 to R.L.H.), by the Department of Energy (grant no. DE–FG02–01ER15161 to R.L.H.), by the National Institute of General Medical Sciences (grant no. GM65989–01 to A.M.J.), and by the National Science Foundation (grant no. MCB–0209711 to A.M.J.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.049841. * Corresponding author; e-mail kkorth{at}uark.edu; fax 479–575–2771.
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ABSTRACT
RESULTS
-SNAP (accession no. 
-mercaptoethanol (v/v). All steps were carried out at 4°C. Cheesecloth-filtered homogenate was centrifuged at 2,000g for 20 min. The pellet was gently resuspended in 2 mL of extraction buffer and centrifuged again at 2,000g for 15 min. The final pellet was mixed with 2 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 20 µL of a 100x protease inhibitor cocktail). The chloroplast extract was treated via sonication and insoluble debris was removed by centrifugation at 10,000g for 15 min. Protein concentration was determined using the Bio-Rad (Hercules, CA) protein assay. All SDS-PAGE and immunoblots were carried out as described (Harlow and Lane, 1988
