The three desulfoglucosinolate sulfotransferase proteins in Arabidopsis have different substrate specificities and are differentially expressed

Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3'-phosphoadenosine 5'-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light/darkness cycle. High (500 microM) and low (50 microM) sulfate concentrations in the medium did not influence the levels of expression.     

Mitochondrial DNA variations and nuclear RFLPs reflect different genetic similarities among 23 Arabidopsis thaliana ecotypes

The mitochondrial genome of 23 Arabidopsis thaliana ecotypes was analysed by Southern hybridization in total cellular DNA. Firstly, the extent of divergence between the mitochondrial genomes in closely related lines of one plant species and secondly, the use of mitochondrial versus nuclear RFLPs to determine evolutionary relationships between Arabidopsis ecotype isolates was investigated. Highly divergent stoichiometries of alternative mitochondrial genome arrangements characterize individual ecotypes including the complete loss of a 5 kb region from ecotype Landsberg without apparent effect on plant viability. The genetic similarities between ecotypes suggested by mitochondrial genome arrangements differ from those deduced from 18 nuclear RFLP loci (CAPS markers). Similarity of nuclear RFLP patterns among the 23 Arabidopsis ecotypes neitehr correlates with their geographic origin nor with the observed mitochondrial genome arrangements. A promiscuous mitochondrial sequence insertion previously identified in ecotype Columbia is also found in the nuclear genomes of ecotypes Eifel, Enkheim and Hilversum. Two ecotypes (Eifel and Tabor) displaying identical RFLP patterns at all 18 nuclear loci show differences in both this sequence transfer and a mitochondrial DNA recombination event.     

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.     

Hypersensitive response in cucumber mosaic virus-inoculated Arabidopsis thaliana

The responses of 12 Arabidopsis ecotypes to cucumber mosaic virus strain Y (CMV(Y)) or strain O (CMV(O)) were characterized. Except for ecotype C24, all ecotypes were susceptible to both strains of CMV. In C24, CMV(O) multiplied systemically, but CMV(Y) did not spread systemically and induced only local necrotic spots in virus-inoculated leaves 21–27 h after inoculation. In CMV(Y)-inoculated C24 leaves, virus was confined to the inoculated leaves, and the amount of the pathogenesis-related-1 protein increased during the progress of local necrotic spot formation. These results indicate that C24 mounts a hypersensitive response (HR) to CMV(Y). By genetic analysis of crosses between C24 and ecotype Columbia or Landsberg (erecta) which are susceptible to CMV(Y) infection, the HR to CMV(Y) in C24 was found to be determined by a single major dominant gene whose function was influenced by a modifier gene from the Landsberg ecotype. Comparison of the responses between C24 leaves inoculated with pseudorecombinants of both strains of CMV suggested that the HR was controlled by CMV RNA3. The molecular interaction between the single major gene and CMV(Y) RNA3 is likely to induce the HR in CMV(Y)-inoculated C24.     

Growth and Dormancy in Norway Spruce Ecotypes (Picea abies) I. Interaction of Photoperiod and Temperature

Growth and dormancy as affected by photoperiod and temperature have been studied in Norway spruce ecotypes of different latitudinal and altitudinal origin. First-year seedlings were used. In all ecotypes apical growth cessation and terminal bud formation occurred within 2 weeks after exposure to SD at temperatures of 18 to 24°C. At lower temperatures or at near-critical photoperiods the response was delayed. The critical photoperiod for apical growth cessation varied from 21 hours in ecotype Steinkjer, Norway (64°N) to about 15 hours in ecotype Lankowitz, Austria (47°04′N). High-elevation ecotypes also had longer critical pholoperiods than low-elevation ecotypes from the same latitude. A detectable growth depression resulted from as little as 1 or 2 SDs of 10 hours, and with 4 or more SDs apical growth cessation took place. In contrast to the situation in the shoot, root growth was not affected by photoperiod. Accordingly, the top:root ratio is drastically affected by photoperiod. The critical photoperiod for cambial growth was shorter than that for apical growth in all ecotypes and cambial growth cessation was delayed for several weeks compared with cessation of apical growth. A transition to formation of late-wood tracheids with thick walls and narrow lumens took place upon exposure to SD. The photoperiodic effects were significantly modified by temperature, but the critical photoperiods were only slightly changed by temperature in the range of 12 to 24°C. However, a 10-hour “night” at 4°C caused growth cessation in continuous light in four ecotypes tested. Temperature optimum for apical growth under non-limiting photoperiods (24 hours) was 21°C in all ecotypes, but with little difference among 18,21 and 24°C. The Q₁₀ for apical growth was 3.5 in the temperature range 12 to 18°C. The growth potential as determined in 24-hour photoperiods was not significantly different among the various ecotypes except for one northern eco-type which was clearly inferior to the others. However, the growth of ecotype Steinkjer (64°N) was greatly suppressed even by the long midsummer days at 59°40′N, thus demonstrating the misleading impression one gets of the growth potential of northern ecotypes when they are moved southwards.     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

The multi-protein family of Arabidopsis sulphotransferases and their relatives in other plant species

All members of the sulphotransferase (SOT, EC 2.8.2.-) protein family use 3'-phosphoadenosine 5'-phosphosulphate (PAPS) as the sulphuryl donor and transfer the sulphonate group to an appropriate hydroxyl group of several classes of substrates. These enzymes have highly conserved domains and can be found in eubacteria and eukaryotes. In mammals, sulphate conjugation catalysed by SOTs constitutes an important reaction in the transformation of xenobiotics, and in the modulation of the biological activity of steroid hormones and neurotransmitters. In plants, sulphate-conjugation reactions seem to play an important role in plant growth, development, and adaptation to stress. To date only a few plant SOTs have been characterized in detail. The flavonol 3- and 4'-SOTs from Flaveria species (Asteraceae), which catalyse the sulphonation of flavonol aglycones and flavonol 3-sulphates, respectively, were the first plant SOTs for which cDNA clones were isolated. The plasma membrane associated gallic acid SOT of Mimosa pudica L. pulvini cells may be intrinsic to signalling events that modify the seismonastic response. In Brassica napus L. a SOT catalyses the O-sulphonation of brassinosteroids and thereby abolishes specifically the biological activity of 24-epibrassinolide. The fully sequenced genome of Arabidopsis thaliana Heynh. contains in total 18 genes that are likely to encode SOT proteins based on sequence similarities of the translated products with an average identity of 51.1%. So far only one SOT from A. thaliana (At5g07000) was functionally characterized: the protein was shown to catalyse the sulphonation of 12-hydroxyjasmonate and thereby inactivate excess jasmonic acid in plants. The substrates and, therefore, the physiological roles of SOTs are very diverse. By using the numerous informative databases and methods available for the model plant A. thaliana, the elucidation of the functional role of the SOT protein family will be accelerated.     

Enzymatic activity of the Arabidopsis sulfurtransferase resides in the C-terminal domain but is boosted by the N-terminal domain and the linker peptide in the full-length enzyme

Sulfurtransferases/rhodaneses are a group of enzymes widely distributed in plants, animals, and bacteria that catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. Sulfurtransferases (STs) consist of two globular domains of nearly identical size and conformation connected by a short linker sequence. In plant STs this linker sequence is exceptionally longer than in sequences from other species. The Arabidopsis ST1 protein (AJ131404) contains five cysteine residues: one residue is universally conserved in all STs and considered to be catalytically essential; a second one, closely located in the primary sequence, is conserved only in sequences from eukaryotic species. Of the remaining three cysteine residues two are conserved in the so far known plant STs and one is unique to the Arabidopsis ST1. The aim of our study was to investigate the role of the two-domain structure, of the unique plant linker sequence and of each cysteine residue. The N- and C-terminal domains of the Arabidopsis ST1, the full-length protein with a shortened linker sequence and several point-mutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The C-terminal domain itself displayed ST activity which could be increased by adding the separately prepared N-terminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wild-type activity, probably because of a drastically reduced protein stability. The replacement of each cysteine residue resulted in mutant forms which differed significantly in their stability, in the specific ST activities, and in their kinetic parameters which were determined for 3-mercaptopyruvate as well as thiosulfate as sulfur substrates: mutation of the putative active site cysteine (C332) essentially abolished activity; for C339 a crucial role at least for the turnover of thiosulfate could be identified.     

RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene,conferring resistance to cucumber mosaic virus requires salicylic acid,ethylene and a novel signal transduction mechanism

The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently.     

Assimilatory sulfate reduction in Escherichia coli: identification of the alternate cofactor for adenosine 3'-phosphate 5'-phosphosulfate reductase as glutaredoxin.

The alternate cofactor (7004 cofactor) for Escherichia coli adenosine 3'-phosphate 5'-phosphosulfate (PAPS) reductase originally discovered in an E. coli mutant (tsnC 7004) lacking thioredoxin activity has now been purified and characterized. The tryptic peptide map of the 7004 cofactor is totally different from that of thioredoxin, indicating that the two proteins are unrelated in their primary structure. The 7004 cofactor has an amino acid composition different from that of thioredoxin but similar to that of glutaredoxin, a protein required for the glutathione-dependent deoxyribonucleotide formation by ribonucleotide reductase. Thus, the 7004 cofactor could not be a mutated form of thioredoxin, as was suspected earlier. Thioredoxin but not glutaredoxin is a substrate for thioredoxin reductase, but both thioredoxin and glutaredoxin can catalyze the dithiothreitol- or glutathione-dependent reduction of PAPS. On a molar basis, the dithiothreitol-coupled cofactor activity of thioredoxin is three- to fourfold higher that that of glutaredoxin. Comparison of the cofactor activities in the glutathione-coupled and the dithiothreitol-coupled PAPS reductase reaction shows that the cofactor activity of thioredoxin in the glutathione-coupled reaction is only 23% of that observed in the dithiothreitol-coupled reaction. However, in the case of glutaredoxin, cofactor activities are approximately the same in both the dithiothreitol- and glutathione-coupled reactions.     

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.     

Identification of a tolerant locus on Arabidopsis thaliana to hypervirulent beet curly top virus CFH strain

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted boting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.     

Ecotypic differentiation of response to enhanced CO2 and temperature levels in Arabidopsis thaliana

Five ecotypes of Arabidopsis thaliana, from widely dispersed origins, were grown under combinations of ambient and elevated atmospheric CO₂ concentrations and ambient and elevated temperatures within solardomes. Total above-ground plant biomass was measured when the majority of plants across all ecotypes and treatments had formed seed pods. There were substantial differences in biomass between the ecotypes across all treatments. Temperature had no effect on biomass whilst CO₂ had a significant effect both alone and in interaction with ecotype. The CO₂ x ecotype interaction was mostly due to the enhancement of a single ecotype from the Cape Verde Islands.     

Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.     

A new Ac-like transposon of Arabidopsis is associated with a deletion of the RPS5 disease resistance gene

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4-11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA end-joining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.     

Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration.

We show that among ecotypes of Arabidopsis, there is considerable variation in their susceptibility to crown gall disease. Differences in susceptibility are heritable and, in one ecotype, segregate as a single major contributing locus. In several ecotypes, recalcitrance to tumorigenesis results from decreased binding of Agrobacterium to inoculated root explants. The recalcitrance of another ecotype occurs at a late step in T-DNA transfer. Transient expression of a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is deficient in crown gall tumorigenesis, transformation to kanamycin resistance, and stable GUS expression. This ecotype is also more sensitive to gamma radiation than is a susceptible ecotype. DNA gel blot analysis showed that after infection by Agrobacterium, less T-DNA was integrated into the genome of the recalcitrant ecotype than was integrated into the genome of a highly susceptible ecotype.     

Relative regeneration proficiency ofArabidopsis thaliana ecotypes

We have compared regeneration proficiency for cultured explants from different tissues of different ecotypes ofArabidopsis thaliana. Proficiency varies widely with both tissue and ecotype, and is highest when the flux of light during regeneration is low. Analysis of F1 hybrids suggests that high proficiency is dominant to low proficiency.     

RCH1, a locus in Arabidopsis that confers resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.     

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.