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First published online August 6, 2008; 10.1104/pp.108.126201 Plant Physiology 148:1106-1116 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Mitochondrial mRNA Polymorphisms in Different Arabidopsis Accessions1,[W],[OA]Institut Molekulare Botanik, Universität Ulm, D–89069 Ulm, Germany (J.F., A.H., C.J., S.T., M.S., B.W., S.B.); and Molekulare Genetik, Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, D–06466 Gatersleben, Germany (R.C.M.)
In our analysis of 5' and 3' end formation in plant mitochondria, we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis (Arabidopsis thaliana) accessions Columbia (Col), C24, and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2, no transcripts at all were detected in Ler. This and further analyses suggest that the atp6-2 gene arrangement is absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5' termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24, and Ler/C24 F1 hybrids revealed the differing transcript patterns of ccmC to be inherited maternally, suggesting these to arise from differences in the mitochondrial DNA. Biparental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB, and rpl5, indicating these differences to be caused by nuclear-encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible, as well as the mitochondrial cis-elements required, for 5' end generation of mitochondrial transcripts.
Mitochondrial genomes in higher plants are extraordinarily large (up to 2.4 Mb), but usually encode less than 60 genes and therefore have large noncoding regions (Unseld et al., 1997  ; Kubo et al., 2000; Notsu et al., 2002; Handa, 2003; Clifton et al., 2004; Satoh et al., 2004; Ogihara et al., 2005; Sugiyama et al., 2005). This is accompanied by high genome complexity provoked by frequent DNA recombinations and rearrangements (Mackenzie, 2007; Kubo and Newton, 2008). Furthermore, complex transcript patterns are typical for plant mitochondria. These are partially due to the presence of multiple promoters per transcription unit, but also arise from a variety of posttranscriptional RNA-processing events. These processes include, most notably, cis- and trans-splicing, RNA editing, and formation of secondary 5' and 3' termini (Gagliardi and Binder, 2007). Detailed investigations of these posttranscriptional processes are still at the beginning and up to now have been predominantly descriptive. Although functional studies using in vitro or in organello systems have allowed some insight into the mechanisms of RNA editing, splicing, or 3' end formation (Farré and Araya, 2001, 2002; Staudinger and Kempken, 2003; Takenaka and Brennicke, 2003; Perrin et al., 2004a, 2004b), still very little is known about the trans-factors active in these processes. One of the least explored maturation steps is the generation of mature 5' ends of mitochondrial mRNAs. No in vitro or in organello systems are presently available to investigate this process and both cis-elements as well as trans-factors are mostly unknown (Forner et al., 2007; Gagliardi and Binder, 2007).
Because the purification of proteins required for mitochondrial RNA (mtRNA) processing has proven to be very difficult, so far mainly genetic approaches have been used to study such factors. For instance, two 3' to 5' exoribonucleases involved in the generation of 3' ends (RNR1 and PNPase) have been characterized by reverse-genetics approaches and, very recently, a similar approach identified a protein required for trans-splicing of nad1 transcripts in Arabidopsis (Arabidopsis thaliana) mitochondria (Perrin et al., 2004a
To identify additional genes encoding components of the plant mitochondrial processing machinery, other approaches are required. One such alternative line of investigation is map-based cloning exploiting the natural genetic variation in Arabidopsis. This method has been successfully applied to identify genes involved in a number of physiological processes (Shindo et al., 2007 To investigate whether the natural genetic variation in different accessions of Arabidopsis also affects 5' and 3' end formation of mitochondrial steady-state mRNAs, we analyzed transcripts of all mitochondrial protein-coding genes in the accessions Col, C24, and Ler in this respect. We describe here several mitochondrial mRNA-length polymorphisms, which are consistently observed in all tissues investigated. This strongly suggests that they are independent from tissue-specific or developmental effects. The analysis of reciprocal F1 hybrids attributes the polymorphisms either to nuclear-encoded loci or to differences in mitochondrial DNAs (mtDNAs). This analysis is a very promising prerequisite for the identification of mitochondrial cis-elements and nuclear-encoded trans-factors involved in 5' end formation or mRNA stability.
Comparative Analysis of Mitochondrial Transcripts in Arabidopsis Accessions Col, Ler, and C24
As a prerequisite for the identification of mitochondrial cis-elements or nuclear-encoded trans-factors involved in mitochondrial mRNA 5' and 3' end formation, we systematically analyzed transcript termini of all mitochondrial protein-coding genes in Arabidopsis accessions Col, Ler, and C24. To this end, total RNA was prepared from above-ground parts of 14- to 17-d-old seedlings and the 5' and 3' termini of mitochondrial mRNAs were investigated by circularized RNA-reverse transcription-PCR (CR-RT-PCR) analyses with a set of primers successfully used for the mapping of mitochondrial mRNA termini in an Arabidopsis Col cell suspension culture (Forner et al., 2007
CR-RT-PCR product patterns differing between the accessions were found for transcripts from nad4, nad9, ccmB, rpl5, ccmC, atp6-2, and cox3 (Table I
; Fig. 1
). The latter has been described previously and is thus not included in this investigation (Forner et al., 2005
Likewise, products of identical sizes of about 580 bp were amplified from ccmC mRNAs for Col and Ler, whereas in C24 a 100-bp shorter product was obtained (Fig. 1E). For ccmB, two novel cDNA products were found in Ler (320 and 350 bp, respectively) in addition to the 260-bp product amplified from RNA of all three accessions (Fig. 1C).
The investigation of nad9 mRNAs yielded a complex picture. A CR-RT-PCR analysis with primers used in the examination of total RNA from the Col cell suspension culture repeatedly generated a weaker, but more focused product, within the expected size range in C24 in comparison to Col and Ler (data not shown). Investigation of these transcripts with a different 5' primer (PPCRrev:Atnad9-7), designed to generate smaller cDNA fragments, and an inspection of the products on high-resolution agarose gels, revealed a complex pattern. In Col and Ler, products with a predominant size of about 220 bp are seen (Fig. 1B). This CR-RT-PCR product corresponds to the respective product obtained with RNA from the Col tissue culture (Forner et al., 2007 A particular polymorphism in mRNA termini is observed for rpl5. Here, in all accessions investigated, the same PCR products of 640 and 580 bp, respectively, are observed (Fig. 1D). However, there are consistently reproducible differences in the abundances of these two cDNA products: Both are equally abundant in Ler, but the longer cDNA is predominant in Col, whereas the shorter amplification product is found to be dominant in C24.
From transcripts of the atp6-2 gene, no PCR products were obtained in Ler, indicating a complete absence of this gene in this accession at least in the genomic arrangement anticipated from the sequence of this gene in C24 (Unseld et al., 1997
For all other mitochondrial genes, no differences in the CR-RT-PCR product patterns were observed between the three accessions (data not shown). In addition, no differences were found between the results obtained from RNA isolated from green plants and the CR-RT-PCR-based mapping data with RNA from Col cell suspension culture, except for the above-described nad9 (Forner et al., 2007
To obtain detailed information about the observed polymorphisms, the PCR products with sizes deviating from the Col tissue culture studies were directly sequenced and/or sequenced after cloning (Forner et al., 2007 In Col and C24 plants, sequencing of the 430-bp PCR products identifies main 5' ends of the nad4 mRNAs at position –228, whereas in Ler the major 5' terminus of this transcript maps to position –390 in the 600-bp cDNA product (Table I; Supplemental Fig. S2). This latter end is also present in Col and C24, but represents an RNA of only minor abundance in these accessions. Particularly in C24, the –390 end is only observed in a CR-RT-PCR approach with nested primers located upstream of the –228 end (Supplemental Fig. S3). In contrast, the 5' mRNA end at position –228 is undetectable in Ler, even in a CR-RT-PCR approach specifically designed to detect this end (data not shown). This indicates a defect in the formation or stabilization of the nad4 transcript with the –228 5' end in Ler. Sequencing of the 470-bp product present in all three accessions revealed a 5' end at position –269 (data not shown).
Alternative ends are found for ccmC. In Col and Ler, the previously observed product of about 600 bp indicates the predominant mRNA to start 484/482 nucleotides upstream of the ATG codon (Forner et al., 2007
Sequence analysis of nad9 cDNA confirmed the complex pattern indicated by the 260- and 220-bp PCR products. A major 5' end is found at position –243 and a cluster of 5' ends is present around position –202, the latter being in accordance with previous mapping results (Forner et al., 2007 For the ccmB mRNA, a common 5' end at –140 is present in the 260-bp cDNA fragment amplified in all accessions investigated. Additional 5' termini at –200 and –231 were identified in the 320- and 350-bp products only in accession Ler (Supplemental Fig. S2). The sequences of the CR-RT-PCR products revealed no alternative 3' termini in any of these CR-RT-PCR polymorphisms. Thus, all transcript-size polymorphisms arise from alternative 5' ends. The results of these analyses are summarized in Table I.
To analyze the polymorphic ends by an independent experimental approach and to see whether they are found at major abundant transcripts, the nad4 and nad9 mRNAs were investigated by northern-blot and primer extension analyses. In total RNA preparations from Col and C24 seedlings, a nad4 probe detects a transcript of about 1,750 nucleotides as estimated in comparison to the cytoplasmic 18S rRNA (about 1,800 nucleotides) in the ethidium bromide-stained gel (Fig. 2A ; data not shown). A larger nad4 mRNA of approximately 1,900 nucleotides is detected in Ler total RNA. The sizes of the nad4 mRNA observed in the northern analysis are consistent with the data obtained by the CR-RT-PCR analyses (1,746 and 1,908 nucleotides).
The different nad4 mRNA 5' ends were also observed in a primer extension analysis of total RNA from seedlings (Fig. 2B). In Col and C24, extension of the primer Atnad4-2 yields a major cDNA of 350 nucleotides corresponding to the 5' terminus 228 nucleotides upstream of the translation start codon. In Ler, the main extension product is 520 nucleotides and detects the 5' mRNA end at –390. Longer expositions of the primer extension products also revealed the presence of the minor end at the –390 5' end in Col, whereas no signal corresponding to the –228 nad4 mRNA 5' end is visible in Ler. Faint signals are observed for the 5' terminus at position –269, confirming the CR-RT-PCR results (data not shown). In addition, a number of cDNAs with sizes around 460 nucleotides were seen in all accessions. Because we never observed any corresponding CR-RT-PCR products, these were considered to be false positive signals as frequently obtained in primer extension analyses by spurious priming events. In summary, the results of the northern, as well as of the primer extension analyses, confirm the nad4 mRNA transcript polymorphism between accessions Col/C24 and Ler with the absence of the –228 end in Ler and the concomitant appearance of the 5' terminus at position –390. In addition, the transcript 5' end polymorphism affects the abundant main steady-state mRNA species of nad4 in the different accessions (Figs. 1A and 2, A and B). For the nad9 transcripts, northern-blot analysis carried out with total RNA preparations from cell suspension cultures of Col, C24, and Ler and an nad9-specific probe spanning the complete reading frame revealed two about equally abundant major transcript species to be present in Col and Ler (Fig. 2C). The approximate sizes of these mRNA species (900 and 850 nucleotides) are in agreement with those calculated from the above-described mapping data (871 and 830 nucleotides). As expected from the CR-RT-PCR results, only the longer nad9 transcripts are seen in the northern analysis of C24 RNA. Thus, these two experimental approaches consistently indicate that the three accessions generate the main nad9 mRNAs with a 5' terminus at position –243, whereas mRNAs with equally prominent 5' ends around –202 are present only in Col and Ler and absent from C24. In addition, these experiments show that also in the Col (and Ler) cell suspension cultures the major nad9 mRNAs with 5' ends at –243 are present. Taken together, these results demonstrate that the 5' end polymorphisms observed in the CR-RT-PCR analysis affect the major nad4 and nad9 transcripts.
The differing transcript patterns in seedlings certainly reflect genetic differences between the three accessions. These might be attributed to accession-specific distinct expression patterns (of individual alleles, for instance) in certain tissues or developmental stages. To evaluate this possibility, we investigated the polymorphic transcript patterns in roots, flowers, and rosette leaves from about 3-week-old plants from Col, C24, and Ler, respectively. For all six mitochondrial genes, the CR-RT-PCR product patterns obtained in the RNA preparations from the various tissues indicate that the transcript ends are identical between these tissues and developmental stages (Figs. 1 and 3 ; Supplemental Fig. S4). Thus, the observed mtRNA polymorphisms between the accessions are not restricted to seedlings, but are also observed in other tissues. These results imply that they can be found throughout the entire plant and in all developmental stages.
Inheritance of the Transcript Polymorphisms in Reciprocal Crosses
The polymorphic transcript patterns could be caused by differing mitochondrial sequences or by differences in nuclear genes. In dicot plants, mtDNA is generally transmitted maternally, whereas the nuclear genome is inherited biparentally, with only rare exceptions from this rule having been reported recently for tobacco (Nicotiana tabacum; Svab and Maliga, 2007 For ccmC and atp6-2, the PCR product patterns and thus the transcripts differ between the reciprocal hybrids. The C24-specific short cDNA fragment (280 bp) indicative of the ccmC transcript with a 5' end at –390/–391 is only found in plants derived from crossings with C24 as female parent. Vice versa, the ccmC mRNAs with the further upstream-located 5' terminus at –482/–484 indicated by the presence of an approximately 370-bp PCR product are seen only in F1 plants with a Col or Ler female parent. This points to a clear maternal inheritance of the length of the ccmC transcript, as to be expected from a difference in the mitochondrial genome (Fig. 4A ). Likewise, the lack of atp6-2 mRNA-derived PCR products is restricted to F1 plants originating from a Ler female parent, whereas in F1 offspring derived from crossings with C24 and Col as maternal parent, the expected products are seen (Fig. 4B). Thus, the ccmC and atp6-2 mRNA polymorphisms are maternally inherited attributing them to differing cis-elements in the mtDNA (ccmC; see "Discussion") or to the absence of the respective locus (atp6-2). To test the presence of the atp6-2 gene, a Southern-blot analysis of total DNA from the three accessions was performed. A probe representing atp6-2-specific sequences hybridizes to a 1.6-kb fragment in Col and in C24, which is expected from the mtDNA sequence from C24 and the mtDNA copy sequence in chromosome 2 of Col. No hybridization is seen in Ler. Consistent with this result, a PCR analysis with oligonucleotides annealing to the 5' and 3' extremities of the atp6-2-specific part revealed the expected product of 250 bp only in C24 and Col, but not in Ler (Supplemental Fig. S5). These data strongly suggest that the atp6-2 gene in the version seen in C24 is not present in Ler.
Different modes of inheritance are observed for nad4, nad9, ccmB, and rpl5. For these genes, the CR-RT-PCR product patterns are identical between the respective reciprocal F1 hybrid plants. This strongly suggests that the transcript polymorphisms of these four mitochondrial genes are biparentally inherited, which is typical for nuclear genes. For the nad4 (–228), nad9 (–202), and ccmB (–200/–231) transcript 5' ends, a dominant-recessive mode of inheritance of the presence or absence of these termini is observed (Fig. 4, C–E). These ends are present in all F1 plants derived from crossings in which at least one of the parents shows these termini independent of the gender. For rpl5, the relative abundances of the two transcript species are distinctively shifted toward the –406 end dominating in all hybrids with a C24 parent and toward the –459 terminus in the Col/Ler crosses (Fig. 4F). Thus, the C24 allele seems to be dominant over the Col and Ler alleles, whereas the Col allele seems to be dominant over the respective gene version in Ler.
To obtain first estimations as to whether these four distinct nuclear-encoded transcript polymorphisms might be assigned to one nuclear locus or to different loci, the nad4, nad9, ccmB, and rpl5 transcript ends were investigated in additional accessions (Table II ).
In the additional accessions investigated, the nad4 –228 5' end is not detectable in Calver, Dijon G, and East Malling, whereas the ccmB 5' termini at –200/–231 are only observed in Dijon-1, Enkheim 1, and Pitztal-2. In contrast, a more balanced distribution is seen for the nad9 transcripts. The 5' termini at –202 are found in 11 accessions, but are not detectable in 16 others. For rpl5, the ratio of the –459 and the –406 5' mRNA ends is approximately 1:1 in more than one-half of the 23 accessions investigated for this trait. In five accessions, the –459 end dominates, and in three the –406 terminus is more abundant. Interestingly, the latter accessions are those that lack the nad4 –228 5' end. Because no correlation between the preference of the rpl5 –406 and the absence of the nad4 –228 ends is seen in C24 (nad4, –228; rpl5, –406 predominant) and Ler (nad4, –390; rpl5, –406/–459=1:1), the nuclear factors required for the accumulation of nad4 mRNAs with the –228 5' end and the preferential accumulation of an rpl5 transcript with the –406 5' terminus, respectively, are probably not identical. In summary, the uncorrelated distribution of the individual polymorphic transcript patterns in the various accessions suggests that the nuclear-encoded polymorphisms of the different mitochondrial transcripts can presumably be attributed to different loci rather than to different alleles of a single gene or locus.
Transcript End Polymorphisms in Different Arabidopsis Accessions
The systematic analysis of mitochondrial mRNAs in three Arabidopsis accessions revealed transcript polymorphisms for seven genes. The distinct transcripts found for cox3 had been analyzed in a separate report and were found to correlate with differences in the mtDNAs of the investigated accessions (Forner et al., 2005
The generation of the alternative ccmC 5' ends indicates that the 5' end region of this gene varies in the investigated accessions. A comparison of the mtDNA sequence from C24 and the mtDNA copy in chromosome 2 of Col revealed that the sequences immediately surrounding the –484/2 and 391/0 ccmC 5' ends are identical. However, a further upstream sequence stretch from positions –509 to –595 shows substantial differences between C24 and Col most likely also reflecting differences between the mtDNA of both accessions as seen for cox3 (Forner et al., 2005 The other 5' end polymorphisms identified in this study all originate from differences between the respective nuclear genomes. Sequences of mtDNA (obtained from amplification of the respective regions) and cDNA surrounding the polymorphic ends of nad4 in Ler and nad9 in Col did not reveal any differences, whereas a single nucleotide exchange is observed 497 nucleotides upstream of the ATG of the ccmB gene in Ler (data not shown). In addition, the comparison of the mtDNA sequence from C24 and the mtDNA copy in chromosome 2 of Col at the rpl5 5' ends revealed only one additional nucleotide in Col 486 nucleotides upstream of the 5' end located at position –459. For the mRNAs of these mitochondrial genes, most likely nuclear-encoded trans-factors are involved, the specific roles of the proteins responsible for the nad4, nad9, ccmB, and rpl5 mRNA polymorphisms presently remaining open. In theory, there are three potential functions. First, transcription initiation might be affected by the malfunction of a specific transcription factor. Second, 5' end processing may be impaired, for instance, by the restricted function of a processing factor. Third, the stability of certain mRNA species might be influenced. In the latter case, either increased or decreased stability could be the dominant trait. For nad9, any of three scenarios can explain the lack of the short mRNA in C24. However, because there is no promoter motif present at the 5' terminus of this mRNA, it seems rather unlikely that the absence of the short nad9 mRNA is due to a failure of transcription initiation. For nad4 mRNA 5' ends, a complex picture is observed. It seems clear that essential genetic information for the occurrence of a stable nad4 mRNA with the –228 5' terminus is missing in Ler. In turn, a slightly longer mRNA species with a further upstream-located end (–390) accumulates to substantial levels in this accession. Minor amounts of this latter RNA are also detectable in Col and C24. In addition, a minor 5' end at –269 detectable in all three accessions is increased in Ler. Although we cannot definitely exclude disturbed transcription initiation, this seems to be unlikely for two reasons: There is no promoter motif present at the –228 5' end of the short nad4 mRNA and abolished transcription initiation at –228 cannot reasonably explain the concomitant increase of the long nad4 transcript. Likewise, altered stability seems unlikely because this would require two parallel processes, the destabilization of the short RNA and an enhanced stability of the long nad4 mRNA with the –390 5' end. The variable amounts of the large nad4 mRNA with the –390 5' end rather suggest that this RNA species is an intermediate RNA, which is processed toward the (final) nad4 mRNA with the –228 5' end. In Col, a large portion of this intermediate RNA species with the –390 5' end is converted to RNA molecules with the –228 5' end. In C24, this processing is almost complete and the intermediate RNA with the –390 5' end is only detectable in a nested CR-RT-PCR approach. These quantitative differences are confirmed by the results of the primer extension analysis (Fig. 2B; data not shown). Thus, there are a defect allele in Ler and alleles of different strength for the generation of the short nad4 mRNA in Col and C24. This observation is in line with an impaired 5' end processing at position –228 in Ler. Alleles of different strength might also explain the polymorphism of rpl5 mRNAs. In all Arabidopsis accessions, RNAs with two different 5' ends (–459 and –406) are present, but in different relative quantities (Fig. 1D). In Col, the gene encoding a factor required for the accumulation of the short RNA seems to be encoded by a weak allele, so only a small amount of this mRNA is present. In contrast, in C24, the short mRNA dominates, suggesting that there is a strong allele present in this accession. The strength of the allele encoding the respective protein in Ler seems to be intermediate. Interestingly, the amount of the long rpl5 mRNA with the –459 end is inverse proportional to the accumulation of the short end. This suggests that the short mRNA is processed from the long RNA or that both mRNAs derive from the same precursor RNA. With the ccmB 5' ends, several explanations are possible for the observed pattern. It is possible that Ler contains a weak allele encoding a protein required for the generation of the ccmB RNA with the –140 5' terminus. This low-level processing leads indirectly to the accumulation of the longer mRNAs with ends at –200 and –231, respectively, although there seems to be no clear concomitant decrease of the ccmB mRNA with the –140 5' end (Fig. 1C). In addition, one would expect a recessive inheritance of the appearance of the Ler-specific transcripts. However, the analysis of the F1 hybrids suggests a dominant inheritance of the presence of these transcripts, which is consistent with the assumption that there is additional genetic information for the ccmB mRNA with ends at positions –200 and –231 present in Ler (Fig. 4E). This, in turn, might be explained by defect alleles present in Col and C24 or by a gain-of-function mutation in Ler. This gain of function may involve a de novo mutation of a transcription factor, a processing factor, or a stability factor with a new function probably without disabling the old intrinsic function.
Despite intensive research over several decades, most of the cis- and trans-components required for the generation of mature 5' and 3' ends and/or the stability of mitochondrial mRNAs of higher plants are still unknown. Many mRNA termini have been mapped (Kühn et al., 2005
Apart from atp6-2, all polymorphisms observed affect mitochondrial transcripts with different 5' termini, these polymorphisms being due to differences in mitochondrial or nuclear DNA. No alternative 3' end has been found so far. The reason for this bias is presently unknown, but there are several explanations. The number of accessions investigated might be too small. However, because it is broad enough to uncover several polymorphic 5' ends, 3' end polymorphisms must be at least comparatively rare. Another explanation could be that there are relatively few factors involved in the generation of many or all mature 3' ends. Consequently, lack or constricted function of one of these few factors might have fatal consequences for the generation of mature RNAs of several mitochondrial genes and thus for the plant. This would prevent an easy manifestation of respective variants in the gene pool.
In turn, there might be many specific factors for the accumulation of transcripts with different 5' termini, most of which may be needed only for the mRNA maturation of a single gene. The accumulation of a major transcript with a variant 5' end seems to have no consequences for the fitness of a plant, indicating that the mitochondrial translation machinery exhibits enough flexibility to translate such transcripts. Alternatively, plants are able to tolerate reduced accumulation of one such protein or even the complete loss of this protein (Gutierres et al., 1997
Plant Material, Plant Cultivation, and RNA Isolation Arabidopsis (Arabidopsis thaliana) plants from different accessions were raised in a growth chamber at 21°C, 50% relative humidity, and a light flux of 80 to 160 µmol m–2 s–1 under a 16-h-light/8-h-dark regime. Plants were grown on soil composed of 80% (v/v) Fruhstorfer Erde SoMi 537 Traysubstrat (Hawita Gruppe), 20% (v/v) vermiculite grain size 2 to 3 mm (Isola-Mineralwolle-Werke), and 1.5 g/L Osmocote Exact Mini fertilizer (Scotts Deutschland). The green parts of seedlings were harvested after 14 to 17 d. Flowers and roots were taken at various time points, but not before 21 d after sowing. Leaves were taken from 21-d-old plants, unless stated otherwise. Leaves from F1 plants were harvested from 3- to 4-week-old plants.
The Arabidopsis cell suspension cultures were grown in the dark at 23°C on a shaker (120 rpm). The C24 and the Col cell suspension cultures have been described previously (Forner et al., 2005 Plant material was immediately shock frozen in liquid nitrogen after harvesting. Subsequently, approximately 100 mg were ground by mortar and pestle and used for isolation of total RNA with the RNeasy plant mini kit following the manufacturer's instructions (Qiagen).
CR-RT-PCR analysis was carried out as described previously with the established primer pairs unless stated otherwise (Forner et al., 2007
PCR to verify the hybrid status of the F1 plants was carried out with GoTaq Flexi DNA polymerase under conditions recommended by the manufacturer (Promega). Templates were either residual DNA in total RNA preparations or total DNA prepared from the same plant material with the DNeasy plant mini kit (Qiagen) according to the instructions given in the manual. Approximately 1/30 of the RNA and 1/200 to 1/10 of the DNA preparations, respectively, was used per amplification reaction under standard conditions (Sambrook and Russel, 2001
Sequencing of PCR products was done commercially (4base Lab). The resulting sequences were analyzed using the BLAST tools at the National Center for Biotechnology Information server (McGinnis and Madden, 2004
Southern-blot and primer extension analyses, as well as basic methods of molecular biology, were carried out according to standard protocols (Sambrook and Russel, 2001 Northern-blot experiments were carried out using RNA denatured by glyoxal separated on agarose gels and blotted onto Duralon-UV or Hybond-XL membranes (Stratagene). Probes were radioactively labeled with the Rediprime II random-prime DNA labeling system (GE Healthcare). Signals were visualized by exposition to x-ray films. Agarose gels were photographed with a GeneFlash gel documentation system (Syngene).
The following materials are available in the online version of this article.
We are very grateful to Cornelia Guha for excellent technical assistance and thank all coworkers and students who directly or indirectly contributed to this project. F1 seeds were generated in the context of the GABI program, Creation of novel genetic variants of Arabidopsis. Received July 10, 2008; accepted August 4, 2008; published August 6, 2008.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. Bi 590/6–1, 6–2, and 10–1) and by a fellowship of the Studienstiftung des deutsche Volkes (to J.F.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Stefan Binder (stefan.binder{at}uni-ulm.de).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.126201 * Corresponding author; e-mail stefan.binder{at}uni-ulm.de.
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