The Sauromatum guttatum alternative oxidase has been expressed in Schizosaccharomyces pombe under the control of the thiamine-repressible nmt1 promoter. Alternative oxidase protein and activity were detected both in spheroplasts and isolated mitochondria, indicating that the enzyme is expressed in a functional form and confers cyanide-resistant respiration to S. pombe, which is sensitive to inhibition by octyl-gallate. Protein import studies revealed that the precursor form of the alternative oxidase protein is efficiently imported into isolated mitochondria and processed to its mature form comparable to that observed with potato mitochondria. Western blot analysis and respiratory studies revealed that the alternative oxidase protein is expressed in the inner mitochondrial membrane in its reduced (active) form. Treatment of mitochondria with diamide and dithiothreitol resulted in interconversion of the reduced and oxidized species and modulation of respiratory activity. The addition of pyruvate did not effect either the respiratory rate or expression of the reduced species of the protein. To our knowledge this is the first time that the alternative oxidase has been effectively targeted to and integrated into the inner mitochondrial membrane of S. pombe, and we conclude that the expression of a single polypeptide is sufficient for alternative oxidase activity.

Changes in the level of the mitochondrial chaperone mtHSP70 have been investigated in pea (Pisum sativum) leaf mitochondria by Western blot analysis and quantified by scanning densitometry. As pea leaves develop (from 6 days to 30 days of age) the levels of mtHSP70 decrease. Analysis of the levels of the a subunit of the FxATPase show that the levels of this protein remain constant throughout the same developmental period, whereas the levels of the alternative oxidase increase. In vitro import of the alternative oxidase precursor protein into pea leaf mitochondria from day 6 to day 30 leaves and quantification by scanning densitometry indicates that protein import efficiency decreases with increasing maturity of the plant cell. Results are discussed in terms of how changing levels of the mtHSP70 chaperone, as a result of plant cell development, influence the efficiency of protein import.

The chloroplast psbD gene, which encodes the D2 subunit of photosystem II, is regulated by a blue light-responsive promoter (BLRP). We tested the ability of different regions of the barley (Hordeum vulagare) BLRP to drive transcription of the lacZ reporter gene in genomic transformants of Synechococcus sp. strain PCC7942. The barley BLRP was transcribed in Synechococcus from the same initiation sites that are used in plant chloroplasts in vivo. A region of the BLRP, residing between -83 and -112 bp upstream from the transcription initiation sites, functioned as a negative element in Synechococcus. Nucleotide sequences within this region are conserved among the psbD genes of several monocots and dicots, and with the nuclear negative regulatory element GT. Thus this new cis-element was designated Plastid GT, PGT. Proteins from chloroplasts of barley and Arabidopsis thaliana interacted with PGT in a sequence-specific and developmental-dependent manner. The DNA-protein complexes from Arabidopsis chloroplasts are composed of 60- and 38-kDa polypeptides. We postulate that GT and PGT have evolved in the nucleus and chloroplast, respectively, from a common ancestral regulatory element.

In eukaryotic cells, the role of mitochondria is to generate both ATP (through the process of oxidative phosphorylation) and carbon skeletons (via the Krebs cycle) for biosynthetic purposes. In non-green plant tissue ATP demand is met entirely through the mitochondrial ATP synthase; however, in photosynthetic tissue the situation is more complex. ATP demands in photosynthetic tissues can be satisfied either through the mitochondrial ATP synthase or through the process of photophosphorylation which allows ATP generated in the chloroplast stroma to be indirectly transferred to the cytosol [1,2]. In addition, in any plant organ there exists not only a range of cell types but also a range of cell ages [l]. It has been shown that the function of both mitochondria and chloroplasts is developmentally regulated and that distinct biochemical and morphological changes can be observed as the plant tissue develops and becomes more photosynthetically active [3]. These changes appear to be dependent upon a number of factors that can include cell maturity, tissue location and the interaction of a number of organelles, chiefly Abbreviation used: mtHSP70, matrix-located 70 kDa heat-shock protein.

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