RADA proteins are conserved in plants and structurally similar to bacterial RadA

The Arabidopsis (Arabidopsis thaliana) genome was screened for orthologs of RadA and the At5g50340 gene was identified as coding for a protein remarkably similar to bacterial RadA/Sms (45% similarity), and therefore named RADA. Phylogenetic analysis showed that RADA is present in all groups of the green lineage, including land plants, green and red algae, as well as in brown algae, diatoms and in several organisms of the Stramenopile group that are not photosynthetic, such as the water mold Phytophtora infestans (Fig 1A). However, no RADA ortholog was found in animals or in yeast. While it is probable that plants inherited RADA from the prokaryote ancestor of mitochondria or chloroplasts, the high conservation of the sequences did not allow us to infer whether the ancestor was a proteobacterial or a cyanobacterial gene. Sequence alignments (Figs 1B and S1) showed that plant RADA all share the important functional motifs described for bacterial RadA/Sms [23,27]. A three-dimensional model of Arabidopsis RADA was built (Fig 1C), based on the known structure of Streptococcus pneumoniae RadA [27]. The modeling further confirmed the very high conservation in structure of plant RADA as compared to protobacteria RadA. The RadA structure comprises an N-terminal C4 zinc-finger, required for the interaction with RecA [29], and two main domains: a RecA-like ATPase domain and a Lon protease-like domain. The RecA-like ATPase domain comprises the Walker A and B motifs, and a RadA-specific KNRFG sequence (Fig 1B). In bacteria, the Walker A and KNRFG motifs are indispensable for the branch migration function of the protein, and are also involved in the DNA-binding and helicase activities of RadA [23,27]. Walker A and KNRFG mutants are dominant negative, interfering with the function of the wild-type protein. The structural similarity between RadA and RecA suggests functional similarities, but while RecA specialized in the recognition of homologous sequences and strand invasion, RadA rather evolved for driving branch migration. Finally, the C-terminal P-domain of RadA is similar to the Lon protease-like domain of RecA, but the residues involved in protease activity are not conserved and no protease activity could be detected [30]. This domain is actually involved in DNA binding and it works as a scaffold for the protein architecture, promoting its oligomerization as hexameric rings [27,30].

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Fig 1. RADA is an ortholog of bacterial RadA that is present in all plant lineages.

(A) Phenogram tree showing that genes coding for RadA-like proteins are found in all bacteria, in land plants, green, brown and red algae, diatoms and other organisms of the Stramenopile group. Bootstrap support values are indicated, and branches with values below 70% were collapsed. The scale bar indicates nucleotide substitutions per site. (B) The modular structure of plant RADA is similar to the one from bacteria, with a N-terminal zinc-finger (C4), a helicase domain and a Lon-protease-like domain (H and P domains, respectively). Plant precursor proteins have an N-terminal extension containing an organellar targeting sequence (OTS). (C) Model of Arabidopsis RADA superposed on the known structure of S. pneumoniae RadA (in gray) (Marie et al., 2017 [27]). The color code of relevant domains is as in (B). A bound ADP (stick representation) and the Mg²⁺ ion (yellow ball) are shown.

https://doi.org/10.1371/journal.pgen.1010202.g001

Arabidopsis RADA is targeted to both mitochondria and chloroplasts

Plant RADA sequences have non-conserved N-terminal extensions predicted to be organellar targeting peptides. We confirmed this by expression of N-terminal fusions to GFP in transgenic Arabidopsis plants. Both the N-terminal sequence of RADA as well as the full protein could target GFP to both chloroplasts and mitochondria, as shown by colocalization with the autofluorescence of chlorophyll and with MitoTracker (Fig 2A and 2B). In chloroplasts, the protein localized mainly in discrete speckles that seem to be nucleoids, according to colocalization with the nucleoid marker PEND:dsRED (Fig 2A and 2C). It is likely that in mitochondria, RADA also localizes in nucleoids. Thus, RADA is a dually targeted organellar protein, like several other factors involved in the maintenance of organellar genomes [6]. Among those factors, MutS-like MSH1 was described as having a spatially regulated localization to plastids within the epidermis and vascular parenchyma [31]. We also found RADA:GFP mainly accumulated in the epidermis and vascular tissue of the Arabidopsis rosette leaves (S2A and S2B Fig). However, we observed the same spatial distribution with the fusion comprising only the N-terminus of RADA and GFP (S2C Fig). Because this N-terminal signaling peptide is not conserved among plant RADA sequences, the differential spatial localization is apparently not linked to any RADA-specific functions. A previous report described that a rice RADA ortholog is targeted to the nucleus [32]. This was inferred from immunodetection with an antibody raised against the recombinant protein. However, our observations of RADA:GFP did not give any hint that Arabidopsis RADA is also targeted to the nucleus (Figs 2 and S2).

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Fig 2. Arabidopsis RADA is targeted to both chloroplasts and mitochondria.

(A and B) RADA:GFP constitutively expressed in Arabidopsis transgenic plants is double targeted to both chloroplasts (colocalization with autofluorescence of chlorophyll) and into mitochondria (characteristic cytosolic speckles and colocalization with red fluorescence of MitoTracker). The image corresponds to epidermal mesophyll cells from the abaxial side of rosette leaves. In chloroplasts RADA:GFP is predominant in dots (insert in A) that could be nucleoids according to colocalization with plastidial nucleoid marker PEND:dsRED (C).

https://doi.org/10.1371/journal.pgen.1010202.g002

Expression of a promoter:GUS fusion construct revealed higher GUS expression in young shoots, in sepals and in the stigma (S3A Fig). These results are in line with expression data available at Genevestigator (https://genevestigator.com), indicating preferential expression of RADA in hypocotyls and in the shoot apex (S3B Fig).

Plant RADA is an ssDNA-binding protein that stimulates branch migration in strand-exchange reactions

An Arabidopsis RADA recombinant protein was expressed in bacteria, fused to an N-terminal His-tag. A Walker A-deficient mutant protein (K201A) was also prepared. The mutation of the equivalent position in Escherichia coli RadA abolished DNA-dependent ATPase activity and generated a dominant negative radA allele [23,25]. According to the structure of the protein bound to ADP, the mutation abolishes ATPase activity but it does not affect ATP or ADP binding [27]. Both WT RADA and K201A could be expressed and purified as soluble proteins. By gel filtration they resolved as two peaks of high molecular weight (S4A and S4B Fig), indicating different degrees of oligomerization. Dynamic light scattering showed that the smaller size oligomer was constituted by a monodispersed particle of about 340 kDa, consistent with a hexameric RADA complex (S4C Fig). EMSA experiments showed that both peak fractions could bind to an ssDNA oligonucleotide (S4D Fig). The purified proteins were tested in EMSA experiments, using constant probe concentrations and increasing concentrations of recombinant protein. Different DNA structures were tested as substrates, including ssDNA, dsDNA, fork-like structures and double-stranded molecules containing 5’ or 3’ ssDNA overhangs. Arabidopsis RADA could bind to all structures containing ssDNA regions. It could also bind dsDNA, but with much less affinity (Fig 3A). In our experimental conditions we found that high molecular weight complexes could be formed, which could be resolved in 4.5% polyacrylamide gels and at low voltage. These higher molecular weight complexes were promoted by the presence of ATP or ADP and could correspond to the polymerization of RADA on ssDNA, forming nucleofilaments (Fig 3B). We did not see significant differences in binding with the K201A mutant protein, which seemed to bind ssDNA with equivalent affinity as the WT protein. The sequence specificity of RADA binding to ssDNA was tested in competition experiments. In these experiments, poly-dT, poly-dC and poly-dG could compete binding as efficiently as the homologous probe, while poly-dA was a less efficient competitor (Fig 3C). Thus, Arabidopsis RADA binds preferentially to ssDNA, with little sequence specificity.

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Fig 3. RADA preferentially binds ssDNA.

(A) EMSA experiments showing that RADA binds to any ssDNA-containing DNA structure with higher affinity than to dsDNA. Lower and higher molecular weight complexes are indicated by white and black arrowheads respectively. A predominant band seen with the 5’-overhang probe is artefactual, already present in the absence of protein. (B) Analysis on low concentration gel (4.5% as compared to 8% in A) resolves the high-molecular weight RADA:ssDNA filament, which is promoted by ATP or ADP (1 mM). The K201A mutant protein binds ssDNA with an affinity comparable to the RADA WT protein. Increasing concentrations of RADA or of K201A used in (A) and (B) are indicated by the gray triangles. (C) Competition experiments. A 30-mer ssDNA oligonucleotide (7x[AGTC]AG) was used as probe in EMSA experiments with recombinant RADA and sequence specificity was tested by competition, with increasing concentrations of the cold homologous oligonucleotide or with 30-oligomers (poly-dA, pol-dT, poly-dC and poly-dG). Quantification of the results is shown on the right. Only poly-dA showed reduced competition for RADA binding to ssDNA.

https://doi.org/10.1371/journal.pgen.1010202.g003

The branch migration activity of plant RADA was tested using an in vitro strand-exchange reaction. In this test, bacterial RecA in the presence of ATP and of SSB initiates the invasion of dsDNA by homologous ssDNA and promotes branch migration till the final heteroduplex product is completed (Fig 4A). In the presence of plant RADA the branched intermediates were resolved faster, leading to the earlier appearance of the final nicked double-stranded circular product (Fig 4A). The faster resolution of recombination intermediates was reproducibly observed in six independent experiments (Fig 4B). We also tested whether RADA can promote branch migration independently from the presence of RecA. For that, assays were arrested by freezing at a time point (7 min) when there was already accumulation of branched intermediates, but no final heteroduplex product was visible (Fig 4C left panel). The reaction mix was deproteinated and the purified nucleic acids added to new reaction mixes, in the presence or absence of RADA. In the absence of both RecA and RADA the branched intermediates could not spontaneously evolve and remained stable (Fig 4C middle panel). However, in the presence of RADA they were converted to the final product, showing that plant RADA alone can promote branch migration (Fig 4C right panel). This departs from what was described for bacterial RadA, whose function seems to depend from its interaction with RecA. However, RADA is not a recombinase redundant with RecA, because RADA alone in the absence of RecA is not able to initiate strand-invasion (Fig 4D). Finally, in the same experimental conditions, the K201A mutant protein was unable to promote branch migration, and rather behaved like a dominant-negative, completely inhibiting the reaction (Fig 4E). The incapacity of the K201A mutant to migrate along the heteroduplexes probably blocks the activity of RecA. This result is consistent with the dominant negative effect of the equivalent mutation in E. coli RadA [25].

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Fig 4. Branch-migration activities of RADA.

(A) Recombinant RADA accelerates branch-migration of DNA heteroduplexes initiated by RecA, in an in vitro reaction. An explanation of the different substrates and products is shown. (B) Ratio of final product as compared to the initial linear dsDNA substrate in 6 independent experiments, showing that in the presence of RADA there is faster resolution of branched intermediates. (C) Experiment showing that RADA alone can finalize branch-migration initiated by RecA: a reaction at t = 7 min was arrested by deproteination (left panel) and the DNA purified. Without further addition of RecA or RADA proteins there is no spontaneous progression of the reaction (middle panel), but added RADA can alone resolve the recombination intermediates into the final product (right panel). (D) RADA alone cannot initiate strand invasion. (E) Mutation of the ATPase Walker domain of RADA (K201A) inhibits the reaction. (F) Complementation of an E. coli radA mutant (ΔRadA) for growth in the presence of genotoxic ciprofloxacin (CIP). Arabidopsis RADA (At-RADA) complements the mutation as efficiently as the bacterial protein (Ec-RadA) cloned in the same expression vector. Serial dilutions of the cell suspensions were spotted and are indicated in the x-axis.

https://doi.org/10.1371/journal.pgen.1010202.g004

Plant RADA can complement bacterial RadA in the repair of DNA damage

We tested the ability of plant RADA to complement E. coli RadA in the repair of genotoxic stress-induced DNA lesions. A radA785(del)::kan strain was used for complementation assays, and Arabidopsis RADA or E. coli RadA were expressed from the low-copy number plasmid pACYC. As previously described by others, we found the radA strain to be little affected by genotoxic treatments as compared to WT [24,25]. The conditions we found best to test complementation were in the presence of ciprofloxacin, an inhibitor of gyrase that induces DNA DSBs. In a spot assay, the growth of RadA-deficient cells was much reduced as compared to WT. This ciprofloxacin-triggered growth defect could be complemented by the expression of Arabidopsis RADA, as efficiently as by the expression of bacterial RadA cloned in the same expression vector (Fig 4F). Therefore, plant RADA can functionally substitute bacterial RadA in the repair of DNA damages induced by ciprofloxacin, in agreement with the similar in vitro activities of the bacterial and plant proteins.

Arabidopsis plants deficient in RADA are severely affected in their development and fertility

Two Arabidopsis T-DNA lines (radA-1 and radA-2) could be validated, with T-DNA insertions in exons 8 and 5 respectively and both in the Col-0 accession background (Fig 5A). Homozygous plants from both mutant lines displayed severe developmental defects such as retarded growth and distorted leaves showing chlorotic sectors (Fig 5B). The phenotype was fully penetrant, with all homozygous mutants displaying this phenotype, although some plants were more severely affected than others. To fully confirm that the observed phenotypes were because of a deficiency in RADA, radA-1 plants were complemented with the WT RADA gene expressed under its own promoter. Heterozygous radA-1 plants were transformed and homozygous radA-1 plants that also contained WT RADA as a transgene were segregated in the T2 generation. They were phenotypically normal (Fig 5B, right), confirming the complementation and the linkage of the growth defects to RADA loss of function.

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Fig 5. Arabidopsis radA mutants and phenotypes.

(A) Schematic representation of the Arabidopsis RADA gene. Coding sequences are in black and 5’- and 3’-UTRs are in gray. The position of the T-DNA insertions in radA mutant lines is shown. (B) radA plants show severe growth retardation, with distorted leaves presenting chlorotic sectors. These phenotypes can be complemented by expression of HA-tagged RADA (RADA:HA) under control of the endogenous RADA promoter. (C) Detail of mutant phenotypes as compared to WT, showing deformed leaves, root shortening and flower stems with very small radA siliques that mostly contain aborted seeds. Most of the few seeds produced are non-viable. Flowers are also deformed, and visible and SEM images show that in radA no pollen binds to the papillae of the stigma. Alexander staining of pollen in radA anthers as compared to WT shows little pollen production and an abundance of small and aberrant pollen grains (indicated by arrows).

https://doi.org/10.1371/journal.pgen.1010202.g005

Details of radA plants phenotypes are shown in Fig 5C. As compared to WT the mutants have small and distorted leaves, short roots, very small siliques mostly containing aborted seeds, and most of the few seeds produced are non-viable. Flowers are also distorted, and visible and SEM images show that virtually no pollen binds to the papillae of the stigma. That could be because papillae cells are modified and unable to bind pollen, or because the stigma develops and is receptive before pollen maturation. Pollen stained positive by Alexander staining, suggesting that it is viable. That was confirmed by the successful transmission of the radA allele in crosses using radA pollen. However, pollen production was much reduced compared to WT, and many pollen grains were of aberrant size and shape. To test whether radA female gametes are viable, emasculated flowers from WT or radA were pollinated with WT pollen and ovules were observed before and after pollination (S5 Fig). The mature unfertilized radA ovules looked morphologically normal. Three days after pollination virtually all WT ovules were fertilized and showed a developing embryo, but only 1/6th of the radA ovules had developing embryos. No elongation of the pollinated pistils was observed even at seven days after pollination, suggesting that normal pollen could not fertilize the apparently normal radA ovules, potentially because of a deficiency in pollen germination on the stigma of radA plants. Thus, the partial sterility of radA could be due to both male and female defects.

Because RADA is targeted to mitochondria and plastids, we observed if the organelles are phenotypically normal in radA mesophyll cells, by transmission electron microscopy (S6 Fig). The TEM images showed that in radA cells, chloroplasts are morphologically normal and indistinguishable from those of WT leaf cells. On the other hand, mitochondria looked enlarged in size and less electron dense than those from WT, suggesting that it is the mitochondrion that is predominantly affected in radA. Bigger mitochondria were also reported in other mutants impaired in mitochondrial gene expression or genome maintenance, such RNAi lines affected in the expression of organellar RNase P and in mutants of SHOT1 that is apparently involved in the organization of mitochondrial nucleoids [33,34].

radA mutants are affected in the stability of the mitochondrial genome

Several Arabidopsis mutants affected in mtDNA recombination functions (ex: msh1, osb1, recA2, recA3, recG1) have mitogenome instability because of increased ectopic recombination across IRs [6,35,36]. The radA mutants were thus also tested for such molecular phenotype. Illumina sequence of the DNA extracted from a pool of radA plants revealed a coverage profile of the mtDNA displaying dramatic changes in the relative stoichiometry of mtDNA sequences, as compared to the mostly homogeneous coverage of the mtDNA in WT (Fig 6A). Several of those regions are flanked by the large repeats LR1 or LR2, and by pairs of directly oriented IRs (ex, pair of repeats A [556 bp], F [350 bp], L [249 bp] and EE [127 bp]), suggesting that the process at play is the looping out of circular subgenomes by recombination, followed by their autonomous replication (Fig 6A).

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Fig 6. Changes in mtDNA sequences stoichiometry in radA mutants because of increased recombination across intermediate size repeats.

(A) Comparison of Illumina sequence coverage of the mtDNA in radA-1 as compared to WT. Coordinates are those of the Col-0 mtDNA sequence. The position of the mtDNA large repeats LR1 and LR2 and of relevant IRs are shown above. Regions with changed stoichiometry that are flanked by repeat pairs are shadowed. (B) Picture of severely affected (radA-1#1 and radA-2#1) and mildly affected (radA-1#2 and radA-2#2) seedlings and scanning of their mtDNA for changes in relative copy numbers of the different mtDNA regions. Sequences spaced 5–10 kb apart on the mtDNA were quantified by qPCR. Regions with changed stoichiometry flanked by repeat pairs are shadowed as in A. (C) Accumulation of recombination products across repeats L, F or EE in radA seedlings. The scheme above shows the qPCR relative quantification of parental sequences 1/1 and 2/2 and of the corresponding alternative arrangements 1/2 and 2/1. Results are in a log2 scale and error bars correspond to SD values from three technical replicates.

https://doi.org/10.1371/journal.pgen.1010202.g006

To assess if these changes in mtDNA stability correlate with the developmental phenotype, we analyzed individual plants (Fig 6B). Four seedlings grown in vitro were selected, according to the severity of the growth defects, and the relative copy number of the different mtDNA regions quantified by qPCR, using a set of primer pairs spaced about 5 kb apart across the genome, as described previously [17]. Changes in the relative stoichiometry of mtDNA sequences were observed in all plants, mostly overlapping with the ones identified by DNAseq (Fig 6B). However, the amplitude of the changes was higher in the severely affected plants (radA-1#1 and radA-2#1) than in the mildly affected ones (radA-1#2 and radA-2#2). As compared to WT, an increase in copy number of large genomic regions could be observed, as high as seven-fold. We tested by qPCR the accumulation of alternative conformations resulting from recombination across repeats F, L or EE and, as expected, in all plants there was a significant increase in several of such products versus WT levels (Fig 6C), with a significantly higher accumulation in the more affected plants than in the mildly affected ones. Recombination resulted in the asymmetrical accumulation of mainly one of the two possible products, which could be because of alternative mtDNA repair by the break-induced replication (BIR) pathway [6,7]. Big differences in the relative accumulation of recombination products were seen, depending on the pair of repeats analyzed. However, these values are misleading because they are the ratio to the basal levels that exist in WT plants. Thus, a 30-fold increase in recombination product L-1/2 might be equivalent in absolute copy number to a 1000-fold increase in product EE-2/1, because the former is already quite abundant in WT Col-0 and easily detected by hybridization, while the latter is virtually absent in WT [17,35].

Because RADA is also targeted to chloroplasts, the plastidial DNA (cpDNA) of radA plants was likewise scanned for changes in sequence stoichiometry. No changes were detected between mutant and WT (S7A Fig). We also checked whether radA plants have increased rearrangements of the cpDNA by recombination involving microhomologies, as described for why1why3 and why1why3reca1 Arabidopsis mutants and for maize cptK1 [37–39]. For that we developed a pipeline for identification of Illumina reads corresponding to cpDNA rearrangements, using soft-clipping information (S7C Fig). The radA sequence libraries contained an equivalent proportion of rearrangements as compared to WT (S7B Fig), less than one event per cpDNA copy, i.e. too low to be responsible for the severe growth defects of radA plants.

The full-length RADA gene expressed under its own promoter can fully complement the radA growth defects (Fig 5B). To further explore possible effects of RADA on cpDNA maintenance, we tested hemicomplementation of the radA mutant with constructions allowing specific targeting of RADA either to mitochondria (AOX1:RADA) or to the chloroplasts (RBCS:RADA) (S8A Fig). These targeting sequences have been shown to be sufficient for specific and efficient organellar targeting [40]. Recently, it was reported that the RBCS targeting sequence might leak into mitochondria in conditions of overexpression [41]. However, such problem shouldn’t affect our experiments, using the endogenous RADA promoter. The constructs were introduced in heterozygous radA-1 plants, and homozygous mutants containing the hemicomplementation constructs were selected (hereafter referred to as radA AOX1:RADA and radA RBCS:RADA respectively, S8B Fig). None of the hemicomplementation constructs could achieve complete complementation of the radA growth defect phenotype. We could not compare the transgenes expression levels, as the HA-tagged transgene-deriving proteins could not be detected in total plant extracts from either leaves or inflorescences. Nevertheless, radA AOX1:RADA plants were less affected in growth than radA RBCS:RADA plants and did not present the deformed leaves phenotype observed in non-complemented radA plants. Analysis of the recombination involving pair of repeats L also revealed decreased accumulation of alternative product L-1/2 in radA AOX1:RADA as compared to radA-1 or to radA RBCS:RADA plants (S8C Fig). Taken together, DNAseq and hemicomplementation results support the hypothesis that the severe growth phenotype of radA plants results from defects in mtDNA maintenance, and that the loss of RADA has no significant effects on the stability of the cpDNA.

RADA is the predominant branch migration pathway of plant organelles

In bacteria, the radA mutation is highly synergistic with recG [23,24]. We have therefore tested the epistatic relationship between Arabidopsis RADA and RECG1, the Arabidopsis ortholog of bacterial RecG. In our study we used the recG1-2 mutant that is a true knock-out of RECG1 [17]. To compare all mutant plants at the first homozygous generation, heterozygous recG1-2 plants were crossed with radA-1 heterozygous plants used as pollen donor. In the segregating F2 population, we obtained WT, recG1-2, radA-1 and recG1-2 radA-1 double homozygous mutants (Fig 7A). In this cross, plants inherited the organellar genomes of accession Ws, which is the genetic background of recG1-2. As previously described, the recG1-2 single mutants were phenotypically normal [17]. The radA–1 single mutant developed the same growth defects observed in the Col-0 background. Surprisingly, the recG1-2 radA-1 double mutants were as severely affected in growth as the radA-1 single mutant, with no evidence for an additive effect of the two mutations (Fig 7A). A negative synergistic epistatic relation was expected if both genes were involved in alternative, partially redundant pathways, as in bacteria. Since recG1 radA double mutants are as severely affected in growth as radA plants, RADA is apparently the predominant branch migration pathway in plant organelles.

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Fig 7. Synergistic effect of radA with recA3 but not with recG1 is consistent with a role in the RECA2-dependent pathway.

(A) Crosses of recG1-2 and radA-1 (pollen donor) showed that double homozygote recG1 radA plants are as affected in development as simple homozygote radA plants. Contrarily, double homozygote recA3 radA do not grow roots and are lethal at the seedling stage, a phenotype similar to the one of recA2. The scale bar is 1 mm. (B) qPCR scanning of relative copy numbers of the different mtDNA regions, as described in Fig 6, in pools of recA3, recA2 and recA3 radA seedlings. Results were compared to those of WT seedling of same size, and are represented in log2 scale. (C) RADA interacts with RECA2, but not with RECA1 or with RECA3, on a yeast two-hybrid assay. Serial dilutions of cell suspensions were spotted. BD and AD are the binding and activating domains of GAL4, respectively. Replicate experiments with two independent yeast colonies co-transformed with both plasmids (#1 and #2) confirmed this result, and controls with empty vectors showed that there was no autoactivation by either BD:RADA or AD:RECA2 (lower panel). The interaction between the F-box protein EBF2 and EIN3 was used as positive control.

https://doi.org/10.1371/journal.pgen.1010202.g007

RADA is a player in the RECA2-dependent mitochondrial pathway, but apparently not in the one that relies on RECA3

The mtDNA recombination pathways depend on two RecA-like recombinases, RECA2 that is essential and RECA3 that is dispensable [15]. It was shown that recG1 is synergistic with recA3 [17], suggesting that RECG1-dependent branch migration is only important in one of the alternative pathways. Therefore, we also tested the epistatic relationship between radA and recA3. Heterozygous recA3-2 and radA-1 (both in Col-0 background) were crossed, but no double homozygous mutants could be retrieved from F2 plants growing on soil. Seeds from sesquimutant recA3-/- radA+/- were germinated in vitro and seedlings were genotyped, revealing that recA3 radA double homozygous mutants can germinate, but are unable to grow roots and to expand their cotyledons (Fig 7A). This seedling lethal phenotype was similar to the one observed for recA2 [15]. The effects of recA2, recA3, and recA3 radA mutations on the stability of the mtDNA were analyzed, as described for radA in Fig 6B. This was done on recA3 and recA3 radA first-generation homozygous mutant plants segregated from the same crosses, and compared to WT seedlings of same size. The molecular analysis showed that mtDNA stability was greatly affected in recA3 radA. While segregating recA3 single mutant plants showed only mild effects on the stoichiometry of mtDNA sequences, in recA3 radA plants there were dramatic changes, some regions being more than 20-fold reduced or increased as compared to neighboring sequences (Fig 7B). The magnitude of the changes was higher than the one observed for highly affected radA plants of third homozygote generation (Fig 6B). The profiles were equivalent to the one obtained for recA2 plants, with both recA2 and recA3 radA showing dramatic reduction of the regions between repeats L-2 and A-2 and between large repeat LR1-2 and repeat L-1. Because these sequences are duplicated in the nuclear genome of Col-0 [42], most probably the reduction observed corresponds to a complete loss from the mtDNA. Concordant with this assumption, a small sequence (278 bp) unique to the mtDNA downstream LR1-2 is absent in both recA2 and recA3 radA. However, these lost sequences do not contain any known essential gene. As previously found for recA2 [15], the copy number of the cpDNA was also not significantly affected in recA3 radA (S9 Fig). Thus, the seedling lethality of recA3 radA and of recA2 correlate with massive problems in the stoichiometric replication and segregation of the mtDNA.

The synergistic effect of the radA and recA3 mutations suggests that the two factors intervene in alternative pathways, with RADA predominantly acting in the essential RECA2-dependent recombination pathways, explaining why the loss of both RADA and RECA3 phenocopies the loss of RECA2. In bacteria, the function of RadA involves direct interaction with RecA. We have therefore tested if plant RADA has affinity to any of the three organellar RECA proteins of Arabidopsis. RECA1, RECA2 and RECA3 were tested by yeast two-hybrid for interaction with RADA, either fused to the binding or activating domains of GAL4 (BD and AD respectively). We observed interaction between BD:RADA and AD:RECA2, but not with AD:RECA1 or AD:RECA3 (Fig 7C, upper panel). Replicate experiments confirmed this result, and controls with empty vectors showed that there was no autoactivation by either BD:RADA or AD:RECA2 (lower panel). Thus, the apparent interaction between RADA and RECA2 but not with RECA1 or RECA3 further supports that branch-migration by RADA is predominantly important in the mitochondrial RECA2-dependent recombination pathway, but apparently not in the RECA3-dependent one, or in plastidial recombination that relies on RECA1.

radA-induced mtDNA instability has no apparent negative effects on the accumulation of mitochondrial transcripts

The instability of the mtDNA in radA plants correlated with the severity of the growth defect phenotypes, but in no case we did find a significant reduction in the copy number of mtDNA genes, potentially linking phenotype to a defect in gene expression. The relative abundance of the mitochondrial gene transcripts and rRNAs was quantified by RT-qPCR, but no apparent defect in mtDNA gene expression was found. Rather, for most transcripts an increased accumulation was observed, as compared to WT plants of the same size (Fig 8A), up to 8-fold in the case of the rps4 transcript. To test whether radA plants are deficient in the assembly of OXPHOS complexes, these were analyzed by Blue-Native gel electrophoresis. However, no visible defects were detected, neither on Coomassie stained complexes nor by activity staining of Complex I and immunodetection of Complex III, which revealed complexes of the same size and abundance (Fig 8B).

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Fig 8. Accumulation of mitochondrial transcripts in radA.

(A) Representative mitochondrial transcripts were quantified by RT-qPCR, from the RNA of 10-day-old seedlings grown in vitro, and normalized against a set of nuclear housekeeping genes. Results are on a log2 scale and are the mean from four biological replicates (two pools of radA-1 and two pools of radA-2 seedlings) with the corresponding SD error bars. The asterisks indicate significance of p<0.01 by Student’s t test. (B) Blue-Native gel analysis of purified mitochondria from WT and radA-1 plants. 80 μg of proteins were loaded in each well, and run in parallel with size markers (High Molecular Weight Calibration Kit, GE Healthcare). The identifiable OXPHOS complexes (CI, CII, CIV and CV) are indicated. Coomassie staining, complex I activity staining and immunodetection of complex III, with a cytochrome c1-specific antibody, revealed no apparent deficiency in the assembly of OXPHOS complexes in radA. The white arrowheads indicate green-yellow bands that were already visible before staining and likely corresponded to contaminating plastidial complexes. The black arrowhead indicates the complex I activity band.

https://doi.org/10.1371/journal.pgen.1010202.g008

Instability of the mtDNA in radA mutants affects cell cycle progression

Scanning electron microscopy (SEM) showed that radA epidermal leaf cells were much enlarged as compared to cells of WT leaves (Fig 9A). The radA leaves also had significantly fewer stomata (384.mm^-2 in radA-1 versus 779.mm^-2 in WT, χ² test = 8.5E-14). This suggested an inhibition of cell division in radA. To test such a possibility, nuclear DNA ploidy levels were measured by flow cytometry, in the fully developed first true leaves of 20-day-old radA and WT plants (Fig 9B and 9C). radA leaves displayed a higher proportion of 4C and 8C nuclei than WT leaves, and an important reduction in 16C nuclei, suggesting that endoreduplication is inhibited in radA. In all radA samples, an accumulation of intermediate peaks (ex. 8-16C) was observed, but not in the WT, also suggesting a blockage of cell cycle progression in S phase, or an induction of programmed cell death. To further explore whether mtDNA instability in radA impacts cell cycle progression, we quantified nuclear DNA replication in root tips, using the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU). In radA, the proportion of EdU-positive nuclei was significantly reduced (Fig 9D). Counting of mitotic events in root tips also showed much reduced numbers of cells undergoing mitosis in radA (Fig 9E).

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Fig 9. Cell cycle progression is impaired in radA plants.

(A) Scanning electron microscopy images showing much larger epidermal cells and fewer stomata in radA leaves. Scale bar is 20 μm. (B) Flow-cytometry profiles of WT and radA. The DNA content of nuclei extracted from the first true leaves of 20-day-old plants was analyzed. (C) Ploidy distribution, showing decreased endoreduplication in the mutant, with an increased proportion of 4C and 8C nuclei and a decreased proportion of 16C nuclei. Values are the mean ± SD from 4 experiments for WT and from six experiments for radA (n>20 000 nuclei). (D) Decreased DNA synthesis in the nuclei of radA root tip cells, as evaluated by the ratio between EdU positive and DAPI positive cells. (E) Decreased number of cells undergoing mitosis. Significances were calculated by Student’s t test. (F) RT-qPCR analysis of the expression of a set of cell cycle related genes in 10-day-old WT and radA seedlings, revealing significant activation of SMR5 and ANAC085. The data is represented in a log2 scale and is the mean ± SD of three biological replicates. (G) NBT (nitro blue tetrazolium) staining for O^2-, in plants of equivalent size grown under the same conditions, showing accumulation of ROS in radA plants. Scale bar is 1 cm. (H) The expression of several cell cycle-related genes is suppressed by the ROS quencher ascorbic acid (1 mM), both in WT and in radA 10-day-old seedlings.

https://doi.org/10.1371/journal.pgen.1010202.g009

Cell cycle checkpoints adjust cellular proliferation to changing growth conditions, via the inhibition of the main cell cycle controllers that include cyclin-dependent kinases (CDKs) [43]. In plants, SMR genes encode inhibitors of CDK-cyclin complexes that are transcriptionally induced in response to a variety of stress conditions, integrating environmental and metabolic signals with cell cycle control [44–46]. Interestingly, chloroplastic defects have been shown to induce cell cycle arrest through the induction of SMR5 and SMR7 [37,46]. How these genes are activated remains to be fully elucidated, but the mechanism could involve the activation of the DNA damage response (DDR). This signaling cascade is controlled by the ATM and ATR kinases [47] that phosphorylate the SOG1 transcription factor, leading to the up-regulation of thousands of target genes, including SMR5 and SMR7 [48,49]. We have therefore tested the expression of several cell cycle markers and of several genes that are transcriptionally activated as part of the DDR. These included 14 SMR genes, selected cell cycle-related genes and the NAC-type transcription factors ANAC044 and ANAC085 that inhibit cell cycle progression in response to DNA damage through their activation by SOG1, but also in response to heat stress through a SOG1-independent pathway [50]. Among the 14 SMR genes tested we found strong induction of SMR5 in radA (Fig 9F). But expression of SMR7, whose activation has been described as associated with that of SMR5 in the case of chloroplastic defects [44,46], remained unchanged. ANAC085 was strongly activated in radA, more than 30-fold as compared to WT, as well as ANAC044, but to a much lower degree (Fig 9F). We also observed a low-level induction of the DDR genes BRCA1 and RAD51, and of the CYCB1;1 gene encoding a cyclin associated with G2 arrest and DSB repair [51].

SMR5 is a cyclin-dependent kinase inhibitor that is induced by different conditions leading to oxidative stress [52]. ROS-dependent transcriptional activation of SMR5 and of SMR7 was confirmed in several ROS-inducing conditions [44]. We have therefore tested whether the induction of SMR5 in radA mutants could be due to the accumulation of ROS. Whole rosettes of WT and radA plants of the same size were stained with nitro blue tetrazolium (NBT) to reveal O^2-, and a much higher accumulation of ROS was indeed observed in radA plants (Fig 9G). To confirm the effect of ROS on the activation of cell cycle regulators, radA and WT plants were grown in the presence of the ROS quencher ascorbic acid, and the expression of selected genes tested. In both WT and radA seedlings, the expression of SMR5, ANAC044, ANAC085 and CDKB1;2 was significantly reduced, as compared to plants grown in the absence of ascorbate, up to 8-fold in the case of SMR5 (Fig 9H). Thus, a component of the radA growth phenotype is apparently a retrograde response that activates cell cycle regulators to inhibit cell proliferation. This could be because of the release of ROS as a consequence of mtDNA instability.

Plant materials

Arabidopsis T-DNA insertion mutant lines, all in the Col-0 background, were obtained from the Nottingham Arabidopsis Stock Centre (radA-1: SALK_097880, radA-2: WiscDsLoxHs058_03D). Seeds were stratified for 3 days at 4°C and plants were grown on soil or on half-strength MS medium (Duchefa) supplemented with 1% (w/v) sucrose, at 22°C. Plant genotypes were determined by PCR using gene and T-DNA specific primers. DNA was extracted using the cetyltrimethylammonium bromide method. RNA was extracted using TRI Reagent (Molecular Research Centre, Inc.). For mutant complementation, the WT RADA gene and promoter sequence was cloned in binary vector pGWB613, fused to a C-terminal 3xHA tag, and used to transform heterozygous radA–1 plants. Expression of the transgene in the T1 transformants was monitored by western-blot with a HA-specific antibody. For hemicomplementation, i) the promoter sequence and 5’ UTR of RADA, ii) the N-terminal sequence (49 codons) of soybean alternative oxidase 1 (AOX1; NM_001249237) or the N-terminal sequence (80 codons) of the small subunit of ribulose bisphosphate carboxylase (RBSC; At1g67090), and iii) the RADA sequence minus its first 86 codons fused to a C-terminal HA epitope and Nos terminator, were assembled together by MultiSite Gateway cloning in vector pB7m34GW,0 (https://gatewayvectors.vib.be/), giving constructs AOX1:RADA and RBCS:RADA respectively. These constructs were used to transform heterozygous radA-1 plants by floral dip. For promoter:GUS fusion expression analysis, the RADA promoter and 5’-UTR (1114 bp) was cloned in vector pMDC162 and the construction was introduced into Arabidopsis Col-0 plants.

Intracellular localization

The cDNA sequence coding the N-terminal domain of RADA (first 174 codons) was cloned into the binary vector pBIN+, to obtain the N-RADA:GFP construct. For full protein fusion to GFP (RADA:GFP), the gene sequence and 5’-UTR (from -156 to 4172 relative to the start codon) was cloned in vector pUBC-paGFP-Dest (http://n2t.net/addgene:105107). Arabidopsis Col-0 plants were transformed by the floral dip method and leaves of selected transformants were observed on a Zeiss LSM700 confocal microscope. The fluorescence of GFP and chlorophyll was observed at 505 to 540 nm and beyond 650 nm, respectively. For mitochondrial colocalization, leaves were infiltrated with a 1/1000 dilution of MitoTraker orange (Thermo Fisher Scientific) solution. Excitation was at 555 nm and observation at 560–615 nm. Nucleoid colocalization was tested by biolistic co-transfection in Nicotiana benthamiana epidermal leaf cells with a PEND:dsRED construct [62].

Isolation of mitochondria and Blue Native PAGE

Arabidopsis mitochondria were prepared from the aerial part of three-week old plants grown on soil, by differential centrifugation and step density gradients as described previously [60]. Arabidopsis mitochondrial complexes were solubilized with 1.5% dodecylmaltoside and analyzed on BN-PAGE 4–13% (w/v gels, as described [63]. Blue staining and in gel NADH dehydrogenase activity were performed as described [64].

In vitro strand exchange reaction and DNA Binding Assays

Recombination assays were performed with single-strand linear ΦX174 virion DNA and double strand circular ΦX174 RFI DNA (New England Biolabs) linearized with PstI, in 20 mM Tris-acetate pH 7.4, 12.5 mM phosphocreatine, 10 U/mL creatine kinase, 3 mM ammonium glutamate, 1 mM dithiothreitol, 2% glycerol and 11 mM magnesium acetate. In our conditions, 20.1 μM (in nucleotides) linear ssDNA, 6.7 μM RecA (New England Biolabs), 2 μM RADA are incubated with buffer for 8 min at 37°C. Then, 20.1 μM (in nucleotides) linear dsDNA is added and the whole reaction is incubated for 5 min at 37°C. Finally, strand exchange is initiated by adding 3 mM ATP and 3.1 μM SSB (Merck). Aliquots are stopped at indicated times by addition of 12 μM EDTA and 0.8% SDS. Strand exchange products were analyzed on 0.8% agarose gels run at 4°C and after migration visualized by ethidium bromide staining. For reactions terminated in the absence of RecA, the RecA-initiated strand exchange reaction was stopped at the indicated time and DNA was deproteinized by phenol-chloroform extraction followed by ethanol precipitation. The DNA pellet was solubilized in reaction buffer, 2 μM RADA was added and the reaction was further incubated at 37°C for the indicated time, before quenching with 12 μM EDTA and 0.8% SDS.

For electrophoretic mobility shift assays (EMSA) the purified recombinant protein (50–500 fmol according to the experiment) was incubated with oligonucleotide probes (0.01 pmol-10 fmol) 5’-radiolabeled with [γ-³²P]ATP (5000 Ci/mmol; PerkinElmer Life Science). Different dsDNA structures were prepared by annealing the radiolabeled sense oligonucleotides with a twofold excess of unlabeled complementary oligonucleotide and purified on non-denaturing polyacrylamide gels. The binding reactions were performed in 20 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 10% glycerol, 1 mM DTT and protease inhibitors (Complete-EDTA; Roche Molecular Biochemicals), incubated at 20°C for 20 min and run on 8 or 4.5% polyacrylamide gels in Tris-Borate-EDTA buffer at 4°C. For competition assays, labeled probe and unlabeled competitor were added simultaneously to the reaction mixture. Radiolabelled bands were revealed using a Typhoon phosphorimager (GE Healthcare Life Sciences).

Recombinant proteins

The Arabidopsis RADA cDNA sequence minus first 48 codons was cloned in bacterial expression vector pET28a, fused to an N-terminal His-tag. A mutant was prepared to express a Walker A-deficient protein (K201A). The corresponding constructs pET28-RADA and pET28A-RADA[K201A] were used to express recombinant proteins in E. coli Rosetta 2 (DE3) pLysS (Novagen). The recombinant RADA and RADA[K201A] proteins were affinity purified in a precalibrated HisTrap FF Crude (GE Healthcare Life Sciences) column run at 0.5 mL/min, washed with 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 50 mM imidazole and eluted with a 50–500 mM imidazole gradient. The recombinant protein fractions were further purified by gel filtration on Superdex S200 columns and aliquots were flash frozen in liquid nitrogen and stored at -80°C. RADA concentration was determined by spectrophotometry (extinction coefficient 42,440 M^-1 cm^-1).

Bacterial complementation

The BW25113 radA+ and JW4352 radA785(del)::kan strains were used for complementation assays. The Arabidopsis RADA cDNA (sequence coding for amino acids 137 to 627) or the E. coli RadA/Sms sequence were cloned between the PstI and BamHI restriction sites of the pACYCLacZ vector [15] under the control of the lac promoter. Both constructs were introduced in the JW4352 strain. The pACYCLacZ empty vector was introduced in the BW25113 and JW4352 strains as control. Bacteria were grown in LB supplemented with 10 μg/mL chloramphenicol till OD_600nm = 0.4 before addition of 2.5 mM IPTG. At OD_600nm = 1.2 bacteria were diluted 10⁴ fold and grown on LB agar plates supplemented with 15 nM ciprofloxacin, 2 mM IPTG and 10 μg/mL chloramphenicol.

Yeast two-hybrid assays

The cDNA sequences of Arabidopsis RADA, RECA1, RECA2 and RECA3 minus the first 44, 51, 38 and 40 codons, respectively, were cloned into pGADgwT7-AD and pGBKgwT7-BD vectors containing the GAL4 activation and binding domains (AD and BD respectively). The yeast strain AH109 was co-transformed with different combinations of AD and BD vectors and transformants were selected on synthetic defined (SD)/-Leu/-Trp (SD-LW) medium for 4 days at 30°C. A single colony for each combination was inoculated in liquid SD-LW medium and grown over-night. Cultures were adjusted to an OD_600nm of 0.5 and serial dilutions were plated on SD-LW and SD/-Leu/-Trp/-His (SD-LWH) media. As positive control we tested the known interaction between the F-box protein EBF2 and EIN3 [65].

qPCR and RT-qPCR analysis

qPCR experiments were performed in a LightCycler480 (Roche) in a total volume of 6 μL containing 0.5 mM of each specific primer and 3 μL of SYBR Green I Master Mix (Roche Applied Science). The second derivative maximum method was used to determine Cp values and PCR efficiencies were determined using LinRegPCR software (http://LinRegPCR.nl). Three technical replicates were performed for each experiment. Results of qPCR and RT-qPCR analysis were standardized as previously described [17]. The recently corrected Arabidopsis Col-0 mtDNA sequence was taken as reference [66]. Quantification of mtDNA and cpDNA copy numbers used a set of primer pairs located along the organellar genomes, as described previously [17,39]. Results were normalized against the UBQ10 (At4G05320) and ACT1 (At2G37620) nuclear genes. The accumulation of ectopic HR products involving IRs was quantified using primers flanking each pair of repeats, as described [15]. The COX2 (AtMG00160) and 18S rRNA (AtMG01390) mitochondrial genes and the 16S rRNA (AtCG00920) chloroplast gene were used for normalization. For RT-qPCR experiments, 5 μg of RNA were depleted from contaminating DNA by treatment with RQ1 RNase-free DNase (Promega) and reverse-transcribed with Superscript IV Reverse Transcriptase (Thermo Fisher Scientific) using random hexamers. The GAPDH (At1G13440) and ACT2 (At3G18780) transcripts were used as standards.

Sequencing

Total leaf DNA of WT and radA-1 plants was quantified with a QuBit Fluorometer (Life Technologies) and libraries were prepared with the Nextera Flex Library kit, according to manufacturer’s recommendations (Illumina) using 100 ng of each DNA sample. Final libraries were quantified, checked on a Bioanalyzer 2100 (Agilent) and sequenced on a Illumina Miseq system (2 × 150 paired end reads).

Flow cytometry and EdU staining

Nuclear DNA content was measured in leaves of 20-d-old seedlings, using the CyStain UV Precise P Kit (Partec) according to the manufacturer’s instructions. Nuclei were released in nuclei extraction buffer (Partec) by chopping with a razor blade, stained with 4’,6-diamidino-2-phenylindole (DAPI) buffer and filtered through a 30 μM Celltrics mesh (Partec). Between 20,000 and 30,000 isolated nuclei were used for each ploidy level measurement using the Attune Cytometer and the Attune Cytometer software (Life Technologies). At least four independent biological replicates were analyzed. EdU staining was as described [67]. For each root tip (n>10), the number of mitotic events was counted directly under the microscope.

Bioinformatics analysis

Sequence alignments were constructed with T-Coffe implemented in the Macvector package. Targeting predictions were tested using the SUBA web site (http://suba.plantenergy.uwa.edu.au). For phylogenetic analysis, sequences were aligned with T-Coffe implemented in the Macvector package with default parameters (Myers-Miller dynamic algorithm, gap open penalty of -50 and gap extension penalty of -50). A consensus maximum likelihood tree was built with IQ-TREE [68], from 1000 bootstrap iterations (http://iqtree.cibiv.univie.ac.at). Graphical representation and edition of the tree were performed with TreeDyn (v198.3). The Arabidopsis RADA structure was modeled on the structure of RadA from Streptococcus pneumoniae (pdb: 5LKM), using Modeller (http://salilab.org/modeller/about_modeller.html). For Illumina sequence analysis of the mtDNA or cpDNA, reads were aligned against the Arabidopsis reference genomes using Burrows-Wheeler Aligner [69] with default parameters and filtered to keep only reads mapping to the mtDNA or cpDNA. The coverage was extracted with Bedtool genomecov [70] and coverage positions were rounded down to the upper kb. The coverage of each 1 kb-range was normalized relative to 1,000,000 total Arabidopsis reads. For analysis of rearranged sequences based on soft-clipping information, reads pairing to the mtDNA or cpDNA were filtered to only keep those showing a soft-clipping sequence (threshold 20 nucleotides) and no indel (looking for the presence of a S in the CIGAR string without any I,D and H). The soft-clipping sequences were then extracted with SE-MEI/extractSoftclipped (github.com/dpryan79/SE-MEI) and aligned using bowtie2 [71] against the cpDNA or mtDNA with "—no-mixed" parameters. The positions of the soft-clipping sequences and of their relative reads were rounded down to the upper kb to analyze the location of the rearrangement. Those corresponding to the isomerization that results from the recombination involving the cpDNA large inverted repeats were filtered out. The pipeline is described in detail in https://github.com/ARNTET/Plant_organellar_DNA_recombination.

Accession numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: RADA, At5g50340; RECG1, At2g01440; RECA2, At2g19490; RECA3, At3g10140; ANAC044, At3g01600; ANAC085, At5g14490; KNOLLE, At1g08560; CDKB1;2, At2g38620; EHD2, At4g05520; PLE, At5g51600; MYB3R3, At3g09370; MYB3R4, At5g11510; CYCD3;1, At4g34160; CYCD3;2, At5g67260; CYCD3;3, At3g50070; MCM2, At1g44900; MCM3, At5g46280; PCNA1, At1g07370; PCNA2, At2g29270; CDT1a, At2g31270; CycB1;1, At4g37490; WEE1, At1g02970; BRCA1, At4g21070; RAD51, At5g20850; XRI1, At5g48720; SIM, At5g04470; SMR1, At3g10525; SMR2, At1g08180; SMR3, At5g02420; SMR4, At5g02220; SMR5, At1g07500; SMR6, At5g40460; SMR7, At3g27630; SMR8, At1g10690; SMR9, At1g51355; SMR10, At2g28870; SMR11, At2g28330; SMR12, At2g37610; SMR13, At5g59360; AOX1, NM_001249237; RBCS, At1g67090.