| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online October 29, 2004; 10.1104/pp.104.049502 Plant Physiology 136:3524-3536 (2004) © 2004 American Society of Plant Biologists Molecular Physiological Analysis of the Two Plastidic ATP/ADP Transporters from Arabidopsis1,2Pflanzenphysiologie, Fachbereich Biologie, Universität Kaiserslautern, D–67663 Kaiserslautern, Germany (J.R., L.L., W.J., H.E.N.); and Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824–1312 (N.L.)
Arabidopsis (Arabidopsis thaliana) possesses two isoforms of plastidic ATP/ADP transporters (AtNTT1 and AtNTT2) exhibiting similar biochemical properties. To analyze the function of both isoforms on the molecular level, we examined the expression pattern of both genes by northern-blot analysis and promoter-  -glucuronidase fusions. AtNTT1 represents a sugar-induced gene mainly expressed in stem and roots, whereas AtNTT2 is expressed in several Arabidopsis tissues with highest accumulation in developing roots and young cotyledons. Developing lipid-storing seeds hardly contained AtNTT1 or -2 transcripts. The absence of a functional AtNTT1 gene affected plant development only slightly, whereas AtNTT2::T-DNA, AtNTT1-2::T-DNA, and RNA interference (RNAi) plants showed retarded plant development, mainly characterized by a reduced ability to generate primary roots and a delayed chlorophyll accumulation in seedlings. Electron microscopic examination of chloroplast substructure also revealed an impaired formation of thylakoids in RNAi seedlings. Moreover, RNAi- and AtNTT1-2::T-DNA plants showed reduced accumulation of the nuclear-encoded protein CP24 during deetiolation. Under short-day conditions reduced plastidic ATP import capacity correlates with a substantially reduced plant growth rate. This effect is absent under long-day conditions, strikingly indicating that nocturnal ATP import into chloroplasts is important. Plastidic ATP/ADP transport activity exerts significant control on lipid synthesis in developing Arabidopsis seeds. In total we made the surprising observation that plastidic ATP/ADP transport activity is not required to pass through the complete plant life cycle. However, plastidic ATP/ADP-transporter activity is required for both an undisturbed development of young tissues and a controlled cellular metabolism in mature leaves.
ATP represents the universal energy currency of all living cells. Due to both size and charge, adenylates do not cross biomembranes freely, making the involvement of highly specific transport proteins necessary. In eukaryotic cells mitochondria export ATP previously generated via oxidative phosphorylation at the matrix site in strict counter exchange to cytosolic ADP. The corresponding ADP/ATP carriers (AAC) function as dimers, comprising two identical subunits, each exhibiting six predicted transmembrane domains (Klingenberg, 1989  ). AAC proteins belong to the best characterized solute transporters and are the subject of numerous publications (Fiore et al., 1998).
We identified the plastidic ATP/ADP transporter as a second type of eukaryotic-adenylate carrier protein (Kampfenkel et al., 1995
The main function of plastidic ATP/ADP transporters is the supply of storage plastids with ATP (Schünemann et al., 1993
All orthologs of the plastidic ATP/ADP transporter, e.g. the two isoforms from Arabidopsis, a potato ortholog, or an ortholog from the primitive red alga Galderia sulfuralia, exhibit similar transport properties in respect to substrate specificity and substrate affinity (Möhlmann et al., 1998 To answer the questions of expression patterns and physiological implications we started a comprehensive approach and generated in total six independent transgenic Arabidopsis plants, namely AtNTT1- and AtNTT2-promoter::GUS lines, AtNTT1::T-DNA-, AtNTT2::T-DNA-, AtNTT1-2::T-DNA-, and RNA interference (RNAi) lines. A detailed molecular analysis revealed that AtNTT2 represents a widely expressed Arabidopsis gene, whereas AtNTT1 exhibits a spatial-expression pattern. The absence of AtNTT2 or the simultaneous absence of both transporters strongly affects plant development as revealed by analysis of root formation, chloroplast maturation, and plant growth rate. Inhibition of plastidic ATP/ADP-transporter activity also exerted substantial effect on lipid content in Arabidopsis seeds. Obviously, the continuous ATP supply into developing plastids from both young seedlings and embryo tissues and into mature chloroplast at night is required for a controlled plant development.
Expression Analysis of AtNTT1 and AtNTT2
Arabidopsis possesses two isoforms of the plastidic ATP/ADP transporter with similar biochemical transport properties. To reveal whether the presence of two independent transporter genes correlates with an organ- or development-specific expression pattern, we analyzed both the relative mRNA accumulation by northern-blot analysis and the promoter activity in transgenic plants carrying corresponding promoter- For reliable northern-blot analysis of isoform-specific mRNA accumulation it is required to use gene-specific probes. We generated probes specific for either AtNTT1- or AtNTT2 mRNA by using corresponding 3'-untranslated cDNA fragments (Fig. 1A). Although there is some minor cross hybridization, the probes used exhibited a sufficiently high specificity (Fig. 1A).
AtNTT1 mRNA accumulated strongest in root and stem tissue, and less in source leaves (Fig. 1B). In flowers and siliques the level of AtNTT1 mRNA was below or close, respectively, to the detection level (Fig. 1B). In contrast, AtNTT2 mRNA accumulated to similar amounts in roots, leaves, stem, and flower tissue (Fig. 1B). Similar to AtNTT1, the AtNTT2 mRNA was much less present in siliques (Fig. 1B). To gain first evidence on the expression pattern of both plastidic ATP/ADP-transporter genes during early germination, we monitored the relative mRNA abundance within the first 6 d of development. Within this time span Arabidopsis develops a primary root and gains photosynthetic competence as revealed by accumulation of both chlorophyll and chlorophylla/b-binding protein (CAB) mRNA (Fig. 1C). The level of AtNTT1 mRNA within this period of development remained close to the detection level without any substantial changes (Fig. 1C). This is different from the expression of AtNTT2, as latter mRNA strongly accumulated in days 1 and 2, and declined from day 3 to a level still above the AtNTT1 mRNA (Fig. 1C). To reveal whether plastidic ATP/ADP-transporter gene expression responds on altered sugar availabilities, we floated source leaf discs in either water (control) or 100 mM Glc or Suc (Fig. 1D). The incubation of leaf discs for 24 h in water did not alter the levels of AtNTT1 or AtNTT2 mRNA (Fig. 1D). In contrast, the presence of Glc or Suc strongly increased the accumulation of AtNTT1 mRNA but did not influence AtNTT2 mRNA concentration (Fig. 1D). To perform a second independent approach to study regulation of gene expression, we generated transgenic plants harboring either an AtNTT1-promoter::GUS- or AtNTT2-promoter::GUS gene, respectively. During the first 6 d of development the AtNTT1 promoter is hardly active (Fig. 2A). At day 2 AtNTT1 promoter activity is slightly detectable in the center of the primary root (Fig. 2A), and at day 6 cells comprising the vascular structures in photosynthesizing cotyledons exhibited AtNTT1 promoter activities (Fig. 2A). In contrast to this, AtNTT2 promoter activity is very high at day 1, especially in the root tip and developing cotyledons (Fig. 2A). At day 3, highest AtNTT2 promoter activity is detectable in the root hair zone and at the basis of cotyledons. At day 6, we still observed strong AtNTT2 promoter activity in the root and in rapidly developing secondary leaves (Fig. 2A), still representing strong sinks.
In source leaves, both promoters are barely active. AtNTT1 promoter-GUS activity is detectable only in the vascular bundles located at the edge of the leaf (Fig. 2B), whereas AtNTT2 promoter activity was hardly detectable in the leaf. This result does not necessary contradict the northern-blot analysis (Fig. 1B), as latter reflect the sum of AtNTT mRNA in total leaf tissue. In both flower tissue and developing siliques the AtNTT1 promoter activity is below the detection level (Fig. 2C). Petal crown leaves showed slight AtNTT2 promoter activity, which was, however similar to AtNTT1 promoter activity, nearly absent in developing siliques (Fig. 2C).
In the SALK library we identified a putative AtNTT1 knockout line exhibiting the T-DNA insertion in exon 1 (Fig. 3A). Corresponding heterozygous plants have been selfed to obtain homozygous mutants. By use of the gene-specific primers NTT1/1 and NTT1/2, we were able to amplify a PCR product of the expected size (about 2.4 kb) on wild-type DNA but not on DNA from AtNTT1::T-DNA plants (Fig. 3B).
The PCR product amplified on wild-type DNA has been sequenced to confirm the correct nucleotide sequence (data not shown). Using the primers NTT1/2 and left boarder (LB) we amplified a PCR product on DNA obtained from mutant plants but not from wild-type plants (Fig. 3B). The PCR product has been sequenced to confirm the insertion site (data not shown). To check that the T-DNA insertion into the AtNTT1 gene correlates with absence of the corresponding mRNA, we performed a reverse transcription (RT)-PCR analysis (Fig. 3C). As expected, we were able to demonstrate the presence AtNTT1 mRNA in wild-type leaf tissue but not in leaves from AtNTT1::T-DNA plants (Fig. 3C). Similarly, a northern-blot analysis demonstrated the presence of AtNTT1 mRNA in wild-type leaves but not in AtNTT1::T-DNA leaves (Fig. 3D, left section). Remarkably, the absence of AtNTT1 mRNA (Fig. 3, C and D) is not compensated by an increase of AtNTT2 mRNA (Fig. 3D, right section). A putative AtNTT2 knockout line was available in the GARLIC library carrying the T-DNA insertion in exon 2 (Fig. 4A). Heterozygous plants have been grown and selfed to obtain a homozygous knockout line. The combination of the gene-specific primers NTT2/2 and NTT2/4 allowed amplification of a PCR product of the expected size (about 2.4 kb) on genomic wild-type DNA but not on DNA from homozygous AtNTT2::T-DNA plants (Fig. 4B). The use of the gene-specific primer NTT2/2 and the LB primer allowed amplification of a fragment of about 1.3 kb on genomic DNA from AtNTT2:T-DNA plants but not in wild-type DNA (Fig. 4B). The PCR products have been sequenced to demonstrate that the correct DNA fragments have been amplified and to confirm the position of the T-DNA insertion (data not shown). To prove that the T-DNA insertion in the AtNTT2 gene correlates with the absence of the corresponding mRNA, we performed RT-PCR and northern-blot analysis. The gene-specific primers NTT2/2 and NTT2/4 allowed to amplify a PCR product of the expected size on cDNA prepared from wild-type leaf tissue but not on cDNA prepared from AtNTT2::T-DNA plants (Fig. 4C). Similar to this, the northern-blot analysis revealed the absence of AtNTT2 mRNA in the homozygous knockout plants but showed the presence of this mRNA in wild-type leaf tissue (Fig. 4D, left section).
Although both genes, AtNTT1 and AtNTT2, reside on chromosome 1, we crossed homozygous AtNTT1 and AtNTT2::T-DNA lines to receive null mutants (AtNTT1-2::T-DNA), lacking both functional plastidic ATP/ADP-transporter genes. Due to the relatively wide distance of both genes on chromosome 1, it appeared likely to receive null mutants due to crossover during meiosis. We screened about 100 independent plants and identified 5 plants lacking intact genes from both transporters. That these plants represent homozygous null mutants has been demonstrated by PCR on genomic DNA (Fig. 5, A and B). The primer combinations NTT1/1 and NTT1/2, and NTT2/2 and NTT2/4, respectively, allowed amplification of expected PCR products on genomic DNA from wild-type but not from AtNTT1-2::T-DNA plants (Fig. 5A). The use of the primer combinations NTT1/2 and LB, and NTT2/2 and LB, in contrast, allowed amplification of expected DNA fragments on DNA from homozygous AtNTT1-2::T-DNA plants but not on DNA isolated from wild-type plants (Fig. 5B).
To prove that putative effects connected with the absence of functional AtNTT1 or AtNTT2 genes, or which are present in the double-knockout line are really due to reduced levels of corresponding gene products, we created further transgenic plant lines exhibiting strongly reduced levels of both mRNA species due to an RNAi effect. For this, we cloned a 418-bp fragment from AtNTT1 (corresponding to base positions 1,006–1,424 in the AtNTT1 cDNA; Kampfenkel et al., 1995 ) in sense and antisense orientation into the Hannibal vector (Fig. 5C). This cDNA fragment of AtNTT1 exhibits 92% sequence identity to the corresponding cDNA domain in AtNTT2 (see Möhlmann et al., 1998) leading to the expectation that the final RNAi construct might reduce the levels of both mRNA species simultaneously. After transformation of Arabidopsis plants, we received various independent transgenic plants with strongly reduced levels of AtNTT1 and AtNTT2 mRNA (Fig. 5D). Especially in lines 9, 10, and 14, both mRNA species were below the detection level (Fig. 5D). The absence of highly specific antisera detecting plastidic ATP/ADP transport proteins so far prevents a quantification of the final NTT protein levels in RNAi lines. However, as all RNAi lines generally exhibited physiological characteristics similar to null mutants (see below), we assume that they contained very strongly reduced transport activities, usually named knock down mutants.
After 1 d of germination, Arabidopsis shows a primary root of about 3 to 4 mm length, exhibiting a root hair zone of about 1 mm following the root tip (Fig. 6A). Mutant plants lacking the functional transporter gene AtNTT1 showed a similar size and shape of the primary root as wild-type roots (Fig. 6A). In contrast, mutant plants lacking a functional AtNTT2 gene and RNAi line 10 exhibited less developed primary roots (Fig. 6A). RNAi lines 9 and 14 showed similarly reduced roots (data not shown). After 1 d of germination, AtNTT2::T-DNA plants and RNAi lines showed a substantially reduced number of rooted seedlings when compared to wild-type or AtNTT2::T-DNA plants (Fig. 6B).
To reveal whether plastidic ATP/ADP transporters are important for development of photosynthetically competent chloroplasts we analyzed chlorophyll accumulation within the first days of development. For this we germinated wild-type and mutant seeds for 5 d in a growth chamber under short-day conditions and analyzed the resulting chlorophyll content. Wild-type and AtNTT1::T-DNA seedlings showed similar levels of chlorophyll (Fig. 7, A and B). In contrast to this, knockout plants lacking a functional AtNTT2 gene; RNAi lines 10, 9, 14; and the null mutant showed a reduced seedling size (Fig. 7A; data not shown) and a strongly reduced average chlorophyll content (Fig. 7B; data not shown). Wild-type plants contained about 0.33 µg chlorophyll/plant, whereas chlorophyll in AtNTT2::T-DNA plants amounted to only 0.20 µg/plant (Fig. 7B). Both plants from RNAi line 10 and null mutants contained less than one-sixth of the chlorophyll present in wild-type seedlings (Fig. 7B).
To reveal whether the reduced chlorophyll level observed in some mutant lines is due to an impaired chlorophyll biosynthesis per se or might also correlate with alterations of the whole thylakoid system we examined the chloroplast ultrastructure by transmission electron microscopy. The ultrastructure of chloroplasts in 5-d-old wild-type plants exhibits a well-organized intraorganell membrane system, comprising grana and stroma thylakoids (Fig. 7C). In contrast, low-chlorophyll-containing chloroplasts from RNAi line 10 exhibited less thylakoids; especially the number of grana stacks appeared to be strongly reduced in this mutant (Fig. 7C). To complete our picture on the effects of altered NTT gene expression on chloroplast development, we additionally analyzed the accumulation of nuclear-encoded chloroplast protein during initiation of deetiolation. For this, we germinated wild-type, RNAi, and null mutant seedlings for 6 d in the dark and illuminated etiolated seedlings for 8 or 24 h (at 100 µmol quanta m2 s–1). Subsequently, the change in the chlorophyll content was monitored, and the accumulation of chlorophyll-binding protein CP24, as indicator for altered plastidic protein import/maturation capacity, was examined by western-blot analysis. After 6 d of dark incubation, the chlorophyll levels in wild-type and all mutant seedlings were similarly low and amounted to less than 0.01 mg/plant (Fig. 8A). After 8 and 24 h of illumination the chlorophyll level in wild-type leaves increased already to about 0.045 mg/g fresh weight (FW) and 0.160 mg/g FW, respectively (Fig. 8A). In contrast, RNAi lines 10, 9, 14, and the null mutant showed much less chlorophyll accumulation, amounting to only about 0.050 mg/g FW after 24 h of illumination (Fig. 8A; data no shown). This reveals that also during the sudden induction of deetiolation, mutant plants showed a reduced capacity for chlorophyll synthesis.
In none of the Arabidopsis lines analyzed was CP24 detectable after 6 d of dark germination (Fig. 8B). However, appreciable levels of CP24 were present in wild-type seedlings after already 8 h of light induction (Fig. 8B). CP24 levels in corresponding RNAi tissue (lines 10, 9, and 14) and null mutants were significantly lower than in wild-type tissue (Fig. 8B, and data not shown). After 24 h of light incubation, CP24 levels were high in wild-type tissue and still significantly lower in RNAi- (lines 10, 9, and 14) and null mutants (Fig. 8B; data not shown). Besides the direct import of ATP via a plastidic ATP/ADP transporter, plastids may regenerate endogenous ATP via glycolytic enzyme activities. To raise evidence on an up-regulation of plastidic glycolytic activity during deetiolation in mutants, we quantified the level of mRNAs encoding enzymes and transporters involved. For this we germinated wild-type or RNAi plants for 6 d in the dark and illuminated subsequently etiolated seedlings for 8 h before cDNA was prepared. Gene-specific primers were chosen to amplify about 500-bp fragments coding for either plastidic phosphoglycerate kinases 1 and 2; pyruvate kinases 1, 2, and 3; or plastidic triose P/P, Glc 6-P/P, phosphoenolpyruvate/P transporters 1 and 2, and xyluose 5-P/P transporter. We observed increased mRNA levels of plastidic PGK1, PK1, and PK3 in RNAi plants compared to wild-type plants (Fig. 9), whereas the mRNA levels of the PGK2, PK2, and all plastidic phosphate transporters have not been changed substantially in mutant tissues (Fig. 9).
Wild-type and AtNTT1::T-DNA plants grown for 50 d under short-day conditions exhibited an average rosette size of about 12 cm (Fig. 10A; data not shown). AtNTT2::T-DNA plants, RNAi, or null mutants showed, however, a strongly reduced average size of the leaf rosette, approaching only 6 and 3 cm on average (Fig. 10A; data not shown). Interestingly, under long-day conditions (16 h light/d), the growth difference between wild-type plants and null mutants is nearly abolished (Fig. 10B).
The observation that RNAi and null mutants exhibited severely impaired growth tempted us to study physiological and morphological changes in these mutants in more detail. As the impaired growth is due to processes connected to a reduced plastidic ATP supply under conditions of long-night phases we first focused on changes in starch levels at the end of the day and night phase. However, we did not observe specific changes in transitory starch metabolism in AtNTT1-; AtNTT2::T-DNA; RNAi lines 10, 9, and 14; or in null mutants when compared to wild-type leaves (plants were grown under short-day conditions). All plant lines exhibited starch contents equivalent to about 30 µmol C6/mg chlorophyll at the end of the day and about 7.5 µmol C6/mg chlorophyll at the end of the night.
To analyze the effect of reduced plastidic ATP/ADP-transporter activity on seed quality, we grew Arabidopsis wild-type and mutant plants under long-day conditions, a light period required for induction of flowering. Fully developed seeds from wild type, AtNTT1- and AtNTT2::T-DNA-, RNAi, and AtNTT1-2::T-DNA mutants were collected from opened siliques, and the seed weight, the lipid content, and the protein levels were quantified (Fig. 11).
Wild-type and AtNTT1::T-DNA seeds exhibited an average weight of 23 µg/seed (Fig. 11A). AtNTT2::T-DNA, RNAi, and AtNTT1-2::T-DNA seeds exhibited reduced average weights leading to 19, 20, and 18.5 µg/seed, respectively (Fig. 11A). Lipids represent the main storage product in Arabidopsis seeds. Both wild-type and AtNTT1::T-DNA seeds accumulated similar levels of storage lipids amounting to about 7.2 to 7.5 µg lipid/seed (Fig. 11B). In contrast, AtNTT2::T-DNA and RNAi seeds exhibited only 5.8 and 6.0 µg lipid/seed, respectively, and seeds from AtNTT1-2::T-DNA plants still showed only 4.5 µg lipid/seed (Fig. 11B). The protein in wild-type and AtNTT1::T-DNA seeds has been estimated to be about 4.8 µg/seed (Fig. 11C). AtNTT2::T-DNA, RNAi, and null mutants showed reduced protein levels approaching 3.3, 3.9, and 3.8 µg/seed, respectively (Fig. 11C).
Regulation of NTT Isoform Expression
ATP represents a uniquely important cellular-energy source and is required in most cell compartments to energize a wide number of anabolic and catabolic reactions. Up to now three structurally unrelated types of intracellular ATP transporters have been identified, namely the mitochondrial AAC proteins, the plastidic ATP/ADP-transporters NTT, and the peroxisomal ATP/AMP carrier found in yeast (Saccharomyces cerevisiae; Fiore et al., 1998
The observation that the Arabidopsis genome encodes two isoforms of plastidic ATP/ADP transporters with very similar biochemical properties (Möhlmann et al., 1998
AtNTT1 and AtNTT2 mRNA accumulate in photosynthesizing leaf and stem cells (Fig. 1B). We assume that the nocturnal ATP import into the chloroplasts is the main reason for expression of both plastidic ATP/ADP-transporter genes in photosynthesizing cells. This assumption is strengthened for the following reasons: (1) Heldt (1969)
The strong accumulation of AtNTT1 mRNA in leaf discs incubated on high Glc or Suc concentrations (Fig. 1D) indicates that this gene belongs to a large group of sugar up-regulated genes (Koch, 1996
Both the northern-blot analysis and the promoter-GUS analysis indicate that especially AtNTT2 expression is high in root tips and cotyledons of developing seedlings (Fig. 1C). This observation tempted us to study the effect of altered plastidic ATP/ADP-transporter activity on both root formation and establishment of photosynthetic competence (Figs. 6, A and B, 7, A–C, and 8). The deletion of a functional AtNTT1 gene in Arabidopsis (Fig. 3, A–C) does not result in an impaired root formation of young seedlings (Fig. 6, A and B), nor did it appear that chlorophyll accumulation or seedling development was affected (Fig. 7, A and B). This observation nicely correlates with the relatively low expression of AtNTT1 in corresponding tissues (Figs. 1C and 2A). In strong contrast, the absence of a functional AtNTT2 gene (Fig. 4, A–C) or the reduction of both mRNA species (AtNTT1 and AtNTT2) in RNAi mutants (Fig. 5D) led to a strongly decreased formation of primary roots in young seedlings (Fig. 6, A and B) and a retarded chlorophyll accumulation (Fig. 7B) corresponding to a reduced growth rate (Figs. 7A and 10A).
The impaired root development is most likely due to an inhibited rate of fatty-acid synthesis. In plants this process takes place exclusively in plastids, and in case of root plastids the process has been characterized to be strictly dependent upon ATP import rather than on internal ATP regeneration via glycolytic reactions (Kleppinger-Sparace et al., 1992
In the case of developing leaf tissue several processes are negatively affected by reduced plastidic ATP/ADP-transporter activity. First, the accumulation of chlorophyll is delayed in AtNTT2::T-DNA-, AtNTT1-2::T-DNA, and RNAi seedlings (Figs. 7A and 8A). Second, the generation of functional thylakoid structures is impaired in plants with strongly reduced plastidic ATP import capabilities (Fig. 7C); and third, the accumulation of nuclear-encoded proteins in developing-mutant chloroplasts is reduced (Fig. 8B). Both chlorophyll synthesis and protein import are dependent upon the presence of ATP at the stromal site (Soll and Tien, 1998
We showed in the past that starch accumulation in potato tubers is strongly affected by altering the plastidic ATP/ADP-transporter activity (Tjaden et al., 1998a As given in Figure 11, AtNTT1::T-DNA did not show altered seed weight, lipid, and protein content when compared to wild-type seeds, whereas AtNTT2::T-DNA seeds showed reduced weight, which correlates with reduced levels of lipids and storage protein (Fig. 11, A–C). Strongest reduction of the lipid content showed seeds generated from double-knockout mutants as these seeds contained only about 50% of the lipid content present in wild-type seeds (Fig. 11B). This result is surprising, because the expression level of NTT1 and NTT2 mRNA in developing siliques and seeds is remarkable low (Figs. 1B and 2C). Obviously, even low mRNA levels allow the maintenance of sufficient plastidic ATP import capacity.
It is important to note that the reduced seed oil phenotype is evident under long-day conditions, where the effects of gene knockout on whole-plant physiology, and hence maternal carbon supply to the embryo, were absent. These effects on storage product content are therefore likely to be specific to alterations to NNT gene expression in the seed. From this result we conclude that, similar to rapeseed and cauliflower (Brassica oleracea) inflorescence plastids (Möhlmann et al., 1994
However, Arabidopsis embryo plastids obviously possess, in addition to ATP/ADP-transporter proteins, endogenous sources for ATP regeneration because the absence of both transporter activities does not correlate with a total loss of storage product (Fig. 11B). In general two other metabolic pathways might allow stromal regeneration of ATP: First, chlorophyll-containing embryo plastids might regenerate ATP by photo-phosphorylation. Secondly, stromal-located glycolytic sequences might regenerate ATP at the enzymic steps catalyzed by phosphoglycerate kinase (PGK), or pyruvate kinase (PK). We would like to exclude the first possibility, since in case of rapeseed the light transmission into the developing seed tissue is supposed to be too low (Eastmond et al., 1996
These independent observations point to stromal glycolysis as the alternative source for endogenous ATP resynthesis. This assumption is substantiated by the demonstration that Glc 6-phosphate is a very suitable carbon precursor for fatty-acid synthesis in rapeseed plastids (Kang and Rawsthorne, 1996
Arabidopsis contains two isoforms of plastidic ATP/ADP transporter to allow an optimal spatially and developmentally regulated adaptation of gene expression. Surprisingly, Arabidopsis does not need plastidic ATP/ADP-transporter activity to pass through the complete developmental cycle. However, plastidic ATP/ADP-transporter activity is required for a controlled development of young tissues, especially shown for roots and cotyledons, and is required in mature chloroplasts at night. The absence of plastidic ATP import in developing embryo tissue correlates with a reduction of lipid accumulation, which however still occurs at appreciable levels. This observation points to an ATP regeneration by stromal-located glycolytic enzymes, which seems to participate on ATP provision.
AtNTT1 (AtNTT1::T-DNA) and AtNTT2 (AtNTT2::T-DNA) Knockout Mutant Plants The heterozygous AtNTT1::T-DNA mutant plant (Salk_013530) was provided by the SALK library. In that mutant the T-DNA is located in the first exon of AtNTT1 (locus At1g80300) on bp position 777. The heterozygous AtNTT2::T-DNA mutant (GARLIC_ 288_E08.b.1a.Lb3Fa) was provided by the Torrey Mesa Research Institute (San Diego). In that mutant the T-DNA is located in the second exon of AtNTT2 (locus At1g15500) on bp position 1,015. To confirm that we generated homozygous mutants after backcrossing, we used gene- and T-DNA-specific primers. For PCR on genomic DNA the following primers were used: NTT1/1(5'-TTTCTTCTGTGTATCTGCGGGAGAGAGTG-3'); NTT1/2 (5'-CTTTCTTTCCCCCCCAACAAAACCAAATA-3'); SALK_LB (5'-ACTCAACCCTATCTCGGGCTATTC-3'); NTT2/4 (5'-TCTCTTCTCCTCTCTACCCAGAGC-3'); NTT2/2 (5'-CCAAATCCCAAAACCCTTTTATTCATC-3'); and GARLIC_LB (5'-TAGCATCTGAATTTCATAACCAATCCGATACAC-3').
To generate double-knockout mutants (designated AtNTT1-2::T-DNA) lacking both functional plastidic ATP/ADP-transporter genes, homozygous AtNTT1::T-DNA and homozygous AtNTT2::T-DNA mutant plants were crossed. Although both genes reside on chromosome 1, we were able to identify double-knockout mutants by use of the primers given above.
Transgenic RNAi plants were generated to achieve strongly reduced mRNA-levels of both AtNTT1 and AtNTT2. For Arabidopsis (Arabidopsis thaliana) transformation the pART27 vector (Gleave, 1992
For the generation of promoter-GUS constructs the binary vector pGPTV (Becker et al., 1992
Gene-specific probes, each corresponding to the respective 3'untranslated region, were generated by PCR on cDNA. AtNTT1-specific probes were amplified with the following primers: At1oligo3sense, 5'-GGAGAAATCTGCTCC-3'; and At1oligo1anti, 5'-ACTTCAACGATACACACAAAGG-3'. AtNTT2-specific probes were amplified with the following primers: At2oligo3sense, 5'-ACTGGCATTTAGACG-3'; and At2oligo1anti, 5'-CTAGTTTGGTATTGG-3'. The PCR products were subsequently cloned into the pGEMTeasy vector. For northern-blot analysis the cloned fragments were excised, separated by gel electophoresis, gel purified, and radioactively labeled with [
Poly(A+) mRNA was isolated from rosette leaves or whole seedlings (0.1 g each) by use of Dynabeads (Dynal AS, Oslo) and converted to cDNA by reverse transcription (SuperscriptII, Invitrogen, Carlsbad, CA). For semiquantitative RT-PCR reactions were carried out with 1 µL template and 1 unit Taq-Polymerase in a total volume of 50 µL. PCR conditions were 3 min at 95°C, followed by 25 cycles of 30 s at 96°C, 30 s at 50°C, and 60 s at 72°C. Primers used for amplification are listed. Gene-specific primers were used to amplify PCR products of 450 to 550 bp. The following primers were used: pyruvate kinase I-fp, (At1g32440) 5'-GTGCGACTCTTCCATCCATT-3'; pyruvate kinase I-rp, (At1g32440) 5'-GGTTCTCAACGCCACAGTAT-3'; pyruvate kinase II-fp, (At3g22960) 5'-CAAGGCGCTCACGGTTCTAA-3'; pyruvate kinase-II-rp, (At3g22960) 5'-CATGTCCGAGACTGCGATCA-3'; pyruvate kinase III-fp, (At3g52920) 5'-CTCCTGAAGATGTGCCTAAC-3'; pyruvate kinase III-rp, (At3g52920) 5'-CCAGTCCTTCTCAGTGATTG-3'; phosphoglycerate kinase I-fp, (At1g56190) 5'-ACGATGGCGAAGAAGAGTGT-3'; phosphoglycerate kinase I-rp, (At1g56190) 5'-ACCACCAAGCAGAAGGATGT-3'; phosphoglycerate kinase II-fp, (At3g12780) 5'-CTACCGAAGGAGTCACTAAG-3'; phosphoglycerate kinase II-rp, (At3g12780) 5'-CTGAGTCTCCTCCTCCTATT-3'; phosphoenolpyruvate translocator I-fp, (At5g33320) 5'-CATCTTGCCGCTTGCTGTTGT-3'; phosphoenolpyruvate translocator I-rp, (At5g33320) 5'-TGGAAGCAGAGTGCAGCGATAA-3'; phosphoenolpyruvate translocator II-fp, (At3g01550) 5'-TCAGCGTTGAGCAGAAGAAG-3'; phosphoenolpyruvate translocator II-r, (At3g-01550) 5'-TCACCGAGCAAGAGAACAGA-3'; triose-phosphate translocator-fp, (At5g46110) 5'-CTGAAGGTGGAGATACCGCTG-3'; triose-phosphate translocator-rp, (At5g46110) 5'-GAGTGCGATGATGGAGATGTA-3'; Glc 6-phosphate translocator-fp, (At5g54800) 5'-TTCCATCGACGGAGCTTCCA-3'; Glc-6-phosphate translocator-rp, (At5g54800) 5'-ACGCAGGTTCACCACTCTTG-3'; xylulose-5-phosphate translocator-fp, (At5g17630) 5'-CCGTTGGCTCATCGGATTCAA-3'; xylulose-5-phosphate translocator-rp, (At5g17630) 5'-GCTCTGTAAGCTACGTTTAGA-3'; Actin-fp, 5'-TGTACGCCAGTGGTCGTACAACC-3'; and Actin-rp, 5'-GGAGCAAGAATGGAACCACCG-3'.
Wild-type and transgenic Arabidopsis plants (ecotype Columbia) were grown in a climate-controlled chamber on soil at 22°C and 100 µmol quanta m2 s–1. Prior to germination, seeds were incubated for 2 d in the dark at 4°C for imbibition (Weigel and Glazebrook, 2002
Total RNA was isolated from frozen tissue samples (liquid nitrogen) by using the Purescript extraction kit (Gentra Systems, North Minneapolis, MN), according to the manufacturer's instructions. For RNA gel-blot hybridization analysis, standard methods (Sambrook et al., 1989
Whole seedlings or tissue from transgenic plants were collected in glass scintillation vials, filled with ice-cold 90% acetone, and incubated for 20 min at room temperature. Subsequently, the samples were stained according to standard protocols (Weigel and Glazebrook, 2002
Accumulation of chlorophyll-binding protein CP24 during illumination was examined by western-blot analysis. Antibodies were kindly provided by Prof. R.B. Klösgen (Pflanzenphysiologie, Martin-Luther Universität Halle, Germany). Plant tissue (0.5 g) frozen in liquid nitrogen was homogenized in 250 µL buffer A (50 mM HEPES, 5 mM MgCl2, pH 7.5, 2% SDS, 1% Triton X-100, 15% glycerol, 1 mM EDTA, phenylmethylsulfonyl fluoride [PMSF] 1/100 [v/v]) at room temperature. SDS-PAGE, northern transfer, and immunodetection were conducted according to standard protocols.
For chloroplast ultrastructure analysis from wild-type and RNAi mutants cotyledons from 5-d-old seedlings, grown under short-day conditions, were used. The seedlings were fixed with solution 1 (3% [v/v] glutaraldehyd, 30 mM PIPES, pH 7.0) for 1 h and subsequently washed two times for 10 min in cacodylat buffer, pH 7.0 (50 mM sodium cacodylat, 6.4 mM HCl). The samples were post fixed in solution 2 (1% [w/v] osmium tetroxide, 50 mM sodium cacodylat, 6.4 mM HCl, pH 7.0) for 1 h and washed as described above. Subsequently, samples were incubated for 1 h in 0.5% uranylacetat, followed by a serial dehydration with 30%, 50%, 70%, 90%, and 100% (v/v) of acetone in water. The specimens were infiltrated with a series of 25%, 50%, 75%, and 100% Spurr (Ted Pella, Redding, CA) in acetone. After embedding in Spurr the blocks were sectioned and stained with 2% uranyl acetate and lead citrate before viewing in a transmission electron microscope (Zeiss, Oberkochen, Germany).
For lipid quantification, 0.1 g completely mature and air-dried seeds were homogenized in a mortar in liquid nitrogen. Subsequently, 1.5 mL isopropanol was added and further homogenized. The suspension was transferred into a 1.5-mL reaction tube and incubated for 12 h at 4°C on a laboratory shaker at 100 rpm. Subsequently, samples were centrifuged at 12,000g for 10 min and the supernatant was transferred into preweighted 1.5-mL reaction tube. Tubes were incubated at 60°C for 8 h to evaporate the isopropanol. Subsequently, total lipid was quantified gravitometrically. For seed protein quantification 0.1-g seeds were homogenized in a mortar at room temperature. Subsequently, 1,000 µL buffer medium 1 (50 mM HEPES, 5 mM MgCl2, pH 7.5, 1% Triton X-100, 15% glycerol, 2% SDS, 1 mM EDTA, PMSF, 1/100 [v/v]) was added and further homogenized. The suspension was transferred into 1.5-mL reaction tubes, and samples were centrifuged at 12,000g at room temperature for 10 min. The supernatant was transferred into new 1.5-mL reaction tubes, and proteins were quantified with bicinchoninic acid reagent (Pierce Chemical, Rockford, IL) according to manufacturer's instructions.
Chlorophyll quantification was carried out according to a standard protocol (Arnon, 1949
We thank Prof. R.B. Klösgen and Dr. M. Gutensohn (Martin-Luther Universität Halle, Germany) for kindly supplying CP24 antiserum. We are grateful to Dr. H. Fuge (Zellbiologie, Universität Kaiserslautern) for his support during electron microscopy. Received July 9, 2004; returned for revision September 10, 2004; accepted September 15, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft, Schwerpunktprogramm 1108 (Pflanzenmembrantransport). 
2 This paper is dedicated to a nestor of plant physiology and great biologist, Prof. Dr. Dr. hc Erwin Latzko (Kranzberg, Germany), on the occasion of his 80th birthday. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.049502. * Corresponding author; e-mail neuhaus{at}rhrk.uni-kl.de; fax 0631–205–2600.
Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1–15
Batz O, Scheibe R, Neuhaus HE (1992) Transport processes and corresponding changes in metabolite levels in relation to starch synthesis in barley (Hordeum vulgare L.) etioplasts. Plant Physiol 100: 184–190 Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20: 1195–1197[CrossRef][Web of Science][Medline] Buchanan BB, Gruissem W, Jones RL (2000) Biochemistry & Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD Drgon T, Sabova L, Nelson N, Kolarov J (1991) ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae. FEBS Lett 289: 159–162[CrossRef][Web of Science][Medline] Eastmond PJ, Kolacna L, Rawsthorne S (1996) Photosynthesis by developing embryos of oilseed rape (Brassica napus L.). J Exp Bot 47: 1763–1769
Eastmond PJ, Rawsthorne S (1998) Comparison of the metabolic properties of plastids isolated from developing leaves and embryos of Brassica napus L. J Exp Bot 49: 1105–1111 Fiore C, Trézéguet V, Le Saux A, Roux P, Dianoux AC, Noel F, Lauquin GJM, Brandolin G, Vignais PV (1998) The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects. Biochimie 80: 137–150[Medline]
Geigenberger P, Stamme C, Tjaden J, Schulze A, Quick WP, Betsche T, Kersting HJ, Neuhaus HE (2001) Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity. Plant Physiol 125: 1667–1678 Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20: 1203–1210[CrossRef][Web of Science][Medline] Heldt HW (1969) Adenine nucleotide translocation in spinach chloroplasts. FEBS Lett 5: 11–14[CrossRef][Web of Science][Medline]
Kalorov J, Kalorova N, Nelson N (1990) A third ADP/ATP translocator gene in yeast. J Biol Chem 265: 12711–12716 Kampfenkel K, Möhlmann T, Batz O, van Montagu M, Inzé D, Neuhaus HE (1995) Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants. FEBS Lett 374: 351–355[CrossRef][Web of Science][Medline] Kang F, Rawsthorne S (1994) Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.). Plant J 6: 795–805[CrossRef][Web of Science] Kang F, Rawsthorne S (1996) Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos. Planta 199: 321–327 Kleinig H, Liedvogel B (1980) Fatty acid synthesis by isolated chromoplasts from the daffodil: energy source and distribution patterns of the acids. Planta 150: 166–169[CrossRef]
Kleppinger-Sparace KF, Stahl RJ, Sparace SA (1992) Energy requirements for fatty acid synthesis and glycerolipid biosynthesis from acetate by isolated pea root plastids. Plant Physiol 98: 723–727 Klingenberg M (1989) Molecular aspects of adenine nucleotide carrier from mitochondria. Arch Biochem Biophys 270: 1–14[CrossRef][Web of Science][Medline] Koch KE (1996) Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 509–540[CrossRef][Web of Science] Kolarov J, Kolarova N, Nelson N (1990) A third ADP/ATP translocator gene in yeast. J Biol Chem 265: 12711–12716 Linka N, Hurka H, Lang BF, Burger G, Winkler HH, Stamme C, Urbany C, Seil I, Kusch J, Neuhaus HE (2003) Phylogenetic relationship of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes. Gene 306: 27–35[CrossRef][Web of Science][Medline] Möhlmann T, Scheibe R, Neuhaus HE (1994) Interaction between fatty-acid and starch synthesis in isolated amyloplasts from cauliflower floral buds. Planta 194: 492–497[CrossRef] Möhlmann T, Tjaden J, Schwöppe C, Winkler HH, Kampfenkel K, Neuhaus HE (1998) Occurrence of two plastidic ATP/ADP transporters in Arabidopsis thaliana L.: molecular characterisation and comparative structural analysis of similar ATP/ADP translocators from plastids and Rickettsia prowazekii. Eur J Biochem 252: 353–359[Web of Science][Medline] Murashige F, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Plant Physiol 15: 473–496[CrossRef] Neuhaus HE, Emes MJ (2000) Nonphotosynthetic metabolism in plastids. Annu Rev Plant Physiol Plant Mol Biol 51: 111–140[CrossRef][Web of Science] Nittyla T, Messerli G, Trevisan M, Chen J, Smith A, Zeeman SC (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 203: 87–89 Palmieri L, Rottensteiner H, Girzalsky W, Scarcia P, Palmieri F, Erdmann R (2002) Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter. EMBO J 20: 5049–5059[CrossRef][Web of Science] Quick WP, Scheibe R, Neuhaus HE (1995) Induction of hexose-phosphate translocator activity in spinach chloroplasts. Plant Physiol 109: 113–121[Abstract] Rawsthorne S (2002) Carbon flux and fatty acid synthesis in plants. Prog Lipid Res 41: 182–196[CrossRef][Web of Science][Medline] Robinson SP, Wiskich JT (1977) Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport. Biochim Biophys Acta 461: 131–140[Medline] Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Schünemann D, Borchert S, Flügge UI, Heldt HW (1993) ATP/ADP translocator from pea root plastids. Comparison with translocators from spinach chloroplasts and pea leaf mitochondria. Plant Physiol 103: 131–137[Abstract] Schwender J, Ohlrogge JB, Shachar-Hill Y (2004) A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos. J Biol Chem 278: 29442–29453 Soll J, Tien R (1998) Protein translocation into and across the chloroplastic envelope membranes. Plant Mol Biol 39: 191–207 Tjaden J, Möhlmann T, Kampfenkel K, Henrichs G, Neuhaus HE (1998a) Altered plastidic ATP/ADP-transporter activity influences potato (Solanum tuberosum) tuber morphology, yield and composition of tuber starch. Plant J 16: 531–540[CrossRef][Web of Science]
Tjaden J, Schwöppe C, Möhlmann T, Neuhaus HE (1998b) Expression of the plastidic ATP/ADP transporter gene in Escherichia coli leads to a functional adenine nucleotide transport system in the bacterial cytoplasmic membrane. J Biol Chem 273: 9630–9636 Tsang EWT, Yang J, Chang Q, Nowak G, Kolenosky A, McGregor DI, Keller WA (2003) Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene. Plant Mol Biol 51: 191–201[Medline]
Vigeolas H, van Dongen JT, Waldeck P, Hühn D, Geigenberger P (2003) Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of Brassica napus L. Plant Physiol 133: 2048–2060 Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Wesley SV, Helliwell CA, Smith SA, Wang NB, Rouse DT, Liu Q, Gooding PS, Sing SP, Abbott P, Stoutjesdijk PA, et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27: 581–590[CrossRef][Web of Science][Medline] Winkler HH, Neuhaus HE (1999) Non-mitochondrial ATP transport. Trends Biochem Sci 24: 64–68[CrossRef][Web of Science][Medline] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
32P]-dCTP by random priming, using the Ready To Go kit (Amersham-Pharmacia, Freiburg, Germany). 
