Alejandro Araya

cis Recognition Elements in Plant Mitochondrion RNA Editing

ABSTRACT RNA editing in higher plant mitochondria modifies mRNA sequences by means of C-to-U conversions at highly specific sites. To determine thecis elements involved in recognition of an editing site in plant mitochondria, deletion and site-directed mutation constructs containing the cognate cox II mitochondrial gene were introduced into purified mitochondria by electroporation. The RNA editing status was analyzed for precursor and spliced transcripts from the test construct. We found that only a restricted number of nucleotides in the vicinity of the target C residue were necessary for recognition by the editing machinery and that the nearest neighbor 3′ residues were crucial for the editing process. We provide evidence that two functionally distinguishable sequences can be defined: the 16-nucleotide 5′ region, which can be replaced with the same region from another editing site, and a 6-nucleotide 3′ region specific to the editing site. The latter region may play a role in positioning the actual editing residue.

Nuclear SDH2-1 and SDH2-2 genes, encoding the iron-sulfur subunit of mitochondrial complex II in Arabidopsis, have distinct cell-specific expression patterns and promoter activities.

Three different nuclear genes encode the essential iron-sulfur subunit of mitochondrial complex II (succinate dehydrogenase) in Arabidopsis (Arabidopsis thaliana), raising interesting questions about their origin and function. To find clues about their role, we have undertaken a detailed analysis of their expression. Two genes (SDH2-1 and SDH2-2) that likely arose via a relatively recent duplication event are expressed in all organs from adult plants, whereas transcripts from the third gene (SDH2-3) were not detected. The tissue- and cell-specific expression of SDH2-1 and SDH2-2 was investigated by in situ hybridization. In flowers, both genes are regulated in a similar way. Enhanced expression was observed in floral meristems and sex organ primordia at early stages of development. As flowers develop, SDH2-1 and SDH2-2 transcripts accumulate in anthers, particularly in the tapetum, pollen mother cells, and microspores, in agreement with an essential role of mitochondria during anther development. Interestingly, in contrast to the situation in flowers, only SDH2-2 appears to be expressed at a significant level in root tips. Strong labeling was observed in all cell layers of the root meristematic zone, and a cell-specific pattern of expression was found with increasing distance from the root tip, as cells attain their differentiated state. Analysis of transgenic Arabidopsis plants carrying SDH2-1 and SDH2-2 promoters fused to the β-glucuronidase reporter gene indicate that both promoters have similar activities in flowers, driving enhanced expression in anthers and/or pollen, and that only the SDH2-2 promoter is active in root tips. These β-glucuronidase staining patterns parallel those obtained by in situ hybridization, suggesting transcriptional regulation of these genes. Progressive deletions of the promoters identified regions important for SDH2-1 expression in anthers and/or pollen and for SDH2-2 expression in anthers and/or pollen and root tips. Interestingly, regions driving enhanced expression in anthers are differently located in the two promoters.

The four subunits of mitochondrial respiratory complex II are encoded by multiple nuclear genes and targeted to mitochondria in Arabidopsis thaliana

Mitochondrial respiratory complex II contains four subunits: a flavoprotein (SDH1), an iron-sulphur subunit (SDH2) and two membrane anchor subunits (SDH3 and SDH4). We have found that in Arabidopsis thaliana SDH1 and SDH3 are encoded by two, and SDH4 by one nuclear genes, respectively. All these encoded polypeptides are found to be imported into isolated plant mitochondria. While both SDH1 proteins are highly conserved when compared to their counterparts in other organisms, SDH3 and SDH4 share little similarity with non-plant homologues. Expression of SDH1-1, SDH3 and SDH4 genes was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the second SDH1 gene (SDH1-2) is expressed at a low level.

A nuclear gene encoding the iron-sulfur subunit of mitochondrial complex II is regulated by B3 domain transcription factors during seed development in Arabidopsis.

Mitochondrial complex II (succinate dehydrogenase) is part of the tricarboxylic acid cycle and the respiratory chain. Three nuclear genes encode its essential iron-sulfur subunit in Arabidopsis (Arabidopsis thaliana). One of them, SUCCINATE DEHYDROGENASE2-3 (SDH2-3), is specifically expressed in the embryo during seed maturation, suggesting that SDH2-3 may have a role as the complex II iron-sulfur subunit during embryo maturation and/or germination. Here, we present data demonstrating that three abscisic acid-responsive elements and one RY-like enhancer element, present in the SDH2-3 promoter, are involved in embryo-specific SDH2-3 transcriptional regulation. Furthermore, we show that ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON2, three key B3 domain transcription factors involved in gene expression during seed maturation, control SDH2-3 expression. Whereas ABI3 and FUS3 interact with the RY element in the SDH2-3 promoter, the abscisic acid-responsive elements are shown to be a target for bZIP53, a member of the basic leucine zipper (bZIP) family of transcription factors. We show that group S1 bZIP53 protein binds the promoter as a heterodimer with group C bZIP10 or bZIP25. To the best of our knowledge, the SDH2-3 promoter is the first embryo-specific promoter characterized for a mitochondrial respiratory complex protein. Characterization of succinate dehydrogenase activity in embryos from two homozygous sdh2-3 mutant lines permits us to conclude that SDH2-3 is the major iron-sulfur subunit of mature embryo complex II. Finally, the absence of SDH2-3 in mutant seeds slows down their germination, pointing to a role of SDH2-3-containing complex II at an early step of germination.

Editing status of mat-r transcripts in mitochondria from two plant species: C-to-U changes occur in putative functional RT and maturase domains

Abstract The intronic mat-r ORF encodes a protein with significant homology to retroviral reverse transcriptases. Here, we describe the nucleotide sequence of potato mat-r and study the editing status of mat-r transcripts in two systems, potato and wheat, where the mat-r ORF is part of the trans-introns but in two different configurations relative to nad1 exons d and e. In potato and wheat, 13 and 15 C-to-U transitions respectively were observed. Most transcripts were partially edited, but potato transcripts were edited more efficiently than wheat transcripts. As in functional mitochondrial genes, RNA editing increased the similarity between plant mat-r proteins and their homologous non-plant counterparts. Interestingly, editing of mat-r was clustered in the reverse-transcriptase (RT) and the maturase (X) domains, two well defined regions having known functions in other systems. These results, together with the integrity and sequence conservation of mat-r, strongly suggest that the encoded protein plays a functional role in plant mitochondria.

Primer tRNATrp enhances the inhibition of avian myeloblastosis virus reverse transcriptase by pyridoxal-5′-phosphate

Abstract In the presence of tRNA Trp the inhibition of avian myeloblastosis virus reverse transcriptase by pyridoxal-5′-phosphate is greatly enhanced. This effect of tRNA is specific. While tRNA beef Trp (the primer of the in vitro DNA synthesis with 35 S viral RNA as template) gives the maximal effect, tRNA beef Val will not affect the enzyme activity. In the presence of tRNA beef Trp two additional lysines are titrated with pyridoxal-5′-phosphate as measured by reduction of the enzyme-pyridoxal-5′-phosphate complex with tritiated NaBH 4 . The effect of tRNA is dependent on the presence of the β subunit of avian myeloblastosis virus reverse transcriptase, since the enzymatic activity of the α subunit, although inhibited by pyridoxal-5′-phosphate, is not affected by tRNA.

Inheritance of an induced male-sterile trait in transgenic plants expressing an engineered unedited atp9 mitochondrial gene

Abstractu2002Flowers of tobacco transformed with the genomic (unedited) coding sequence of the mitochondrial atp9 gene (u-atp9), fused to the yeast coxIV mitochondrial targeting pre-sequence, exhibited either semi-fertile or male-sterile phenotypes. The inheritance of the induced male-sterile trait, was first investigated on a population of 25 plants of the R0 generation and then on R1, F1 and R2 progeny of the lines H2.11 and H2.16. The analyses showed that the male-sterile character was generally inherited as a Mendelian trait for one or two loci, and that u-atp9 is involved in the male-sterile phenotype and is stably inherited in the progeny of the original transformants. Molecular investigations performed on the plants of the R0, R1 and R2 generations confirmed the genetic analyses. The final proof of the involvement of u-atp9 in the emergence of the male-sterile trait was provided from experiments dealing with the restoration of male fertility. By using antisense RNA technology, we showed that crossing male-sterile plants containing the u-atp9 transgene with transgenic plants containing the same gene in antisense orientation (as-atp9) produces F1 progeny restored to male fertility. The high expression of as-atp9 causes a dramatic reduction of u-atp9 transcript levels and suppresses their deleterious effects, resulting in normal flower development and seed production.