Catherine Colas des Francs-Small

Rampant Gene Loss in the Underground Orchid Rhizanthella gardneri Highlights Evolutionary Constraints on Plastid Genomes

Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ∼110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.

Identification of a Pentatricopeptide Repeat Protein Implicated in Splicing of Intron 1 of Mitochondrial nad7 Transcripts

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.

The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana

We have identified a mitochondrial protein (RUG3) that is required for accumulation of mitochondrial respiratory chain complex I. RUG3 is related to human REGULATOR OF CHROMOSOME CONDENSATION 1 (RCC1) and Arabidopsis UV-B RESISTANCE 8 (UVR8). Although the family of RCC1-like proteins in Arabidopsis has over 20 members, UVR8 is the sole plant representative of this family to have been functionally characterized. Mitochondria from Arabidopsis plants lacking a functional RUG3 gene showed greatly reduced complex I abundance and activity. In contrast, accumulation of complexes III, IV and V of the oxidative phosphorylation system and the capacity for succinate-dependent respiration were unaffected. A comprehensive study of processes contributing to complex I biogenesis in rug3 mutants revealed that RUG3 is required for efficient splicing of the nad2 mRNA, which encodes a complex I subunit. A comparison of the formation of complex I assembly intermediates between rug3 and wild type mitochondria indicated that NAD2 enters the assembly pathway at an early stage. Remarkably, rug3 mutants displayed increased capacities for import of nucleus-encoded mitochondrial proteins into the organelle and showed moderately increased mitochondrial transcript levels. This observation is consistent with global transcript changes indicating enhanced mitochondrial biogenesis in the rug3 mutant in response to the complex I defect.

The Pentatricopeptide Repeat Protein OTP87 Is Essential for RNA Editing of nad7 and atp1 Transcripts in Arabidopsis Mitochondria

In plant organelles, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in RNA of both mitochondria and plastids, altering the information encoded by the gene. The cytidine to be edited is determined by a cis-element surrounding the editing site that is specifically recognized and bound by a trans-acting factor. All the trans-acting editing factors identified so far in plant organelles are members of a large protein family, the pentatricopeptide repeat (PPR) proteins. We have identified the Organelle Transcript Processing 87 (OTP87) gene, which is required for RNA editing of the nad7-C24 and atp1-C1178 sites in Arabidopsis mitochondria. OTP87 encodes an E-subclass PPR protein with an unusually short E-domain. The recombinant protein expressed in Escherichia coli specifically binds to RNAs comprising 30 nucleotides upstream and 10 nucleotides downstream of the nad7-C24 and atp1-C1178 editing sites. The loss-of-function of OTP87 results in small plants with growth and developmental delays. In the otp87 mutant, the amount of assembled respiratory complex V (ATP synthase) is highly reduced compared with the wild type suggesting that the amino acid alteration in ATP1 caused by loss of editing at the atp1-C1178 site affects complex V assembly in mitochondria.

nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.

The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria

Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3′-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3′ untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3′ end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria.

A PORR domain protein required for rpl2 and ccmFC intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria

Angiosperm mitochondria encode approximately 20 group II introns, which interrupt genes involved in the biogenesis and function of the respiratory chain. Nucleus-encoded splicing factors have been identified for approximately half of these introns. The splicing factors derive from several protein families defined by atypical RNA binding domains that function primarily in organelles. We show here that the Arabidopsis protein WTF9 is essential for the splicing of group II introns in two mitochondrial genes for which splicing factors had not previously been identified: rpl2 and ccmF(C). WTF9 harbors a recently recognized RNA binding domain, the PORR domain, which was originally characterized in the chloroplast splicing factor WTF1. These findings show that the PORR domain family also functions in plant mitochondria, and highlight the parallels between the machineries for group II intron splicing in plant mitochondria and chloroplasts. In addition, we used the splicing defects in wtf9 mutants as a means to functionally characterize the mitochondrial rpl2 and ccmF(C) genes. Loss of ccmF(C) expression correlates with the loss of cytochromes c and c₁, confirming a role for ccmF(C) in cytochrome biogenesis. By contrast, our results strongly suggest that splicing is not essential for the function of the mitochondrial rpl2 gene, and imply that the Rpl2 fragment encoded by rpl2 exon 1 functions in concert with a nuclear gene product that provides the remainder of this essential ribosomal protein in trans.

Group II intron splicing factors in plant mitochondria

Group II introns are large catalytic RNAs (ribozymes) which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA) in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has been retained in the mtDNA of various angiosperms: the matR gene encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4). Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4), which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory splicing cofactors.

Mutations in an Arabidopsis Mitochondrial Transcription Termination Factor–Related Protein Enhance Thermotolerance in the Absence of the Major Molecular Chaperone HSP101

Heat shock protein101 (HSP101) is essential for rescue of proteins damaged by heat stress. However, reduced function of a protein involved in mitochondrial gene expression was found, unlike many mitochondrial mutations, to reduce oxidative stress and allow survival of heat stress in the absence of HSP101. Understanding control of oxidative stress by mitochondria is important to engineering plants with stress tolerance. The molecular chaperone heat shock protein101 (HSP101) is required for acquired thermotolerance in plants and other organisms. To identify factors that interact with HSP101 or that are involved in thermotolerance, we screened for extragenic suppressors of a dominant-negative allele of Arabidopsis thaliana HSP101, hot1-4. One suppressor, shot1 (for suppressor of hot1-4 1), encodes a mitochondrial transcription termination factor (mTERF)–related protein, one of 35 Arabidopsis mTERFs about which there is limited functional data. Missense (shot1-1) and T-DNA insertion (shot1-2) mutants suppress the hot1-4 heat-hypersensitive phenotype. Furthermore, shot1-2 suppresses other heat-sensitive mutants, and shot1-2 alone is more heat tolerant than the wild type. SHOT1 resides in mitochondria, indicating it functions independently of cytosolic/nuclear HSP101. Microarray analysis suggests altered mitochondrial function and/or retrograde signaling in shot1-2 increases transcripts of other HSPs and alters expression of redox-related genes. Reduced oxidative damage is the likely cause of shot1 thermotolerance, indicating HSP101 repairs protein oxidative damage and/or reduced oxidative damage allows recovery in the absence of HSP101. Changes in organelle-encoded transcripts in shot1 demonstrate that SHOT1 is involved in organelle gene regulation. The heat tolerance of shot1 emphasizes the importance of mitochondria in stress tolerance, and defining its function may provide insights into control of oxidative damage for engineering stress-resistant plants.

The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I

Two splicing factors of the multipartite mitochondrial nad5 transcript are pentatricopeptide repeat proteins. Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two pentatricopeptide repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far.