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Mitochondrial DNA in the T male-sterile cytoplasm (cms-T) of maize contains an open reading frame (ORF 13) associated with the T type of sterility. Antibodies raised to a chemically synthesized oligopeptide corresponding to ORF 13 were used to establish the expression of a 13-kDa protein from this reading frame. The 13-kDa polypeptide is synthesized uniquely in cms-T maize and purifies with the membrane fraction of T mitochondria. We assign the symbol urpf3-T to designate this mitochondrial gene. Presence of the nuclear restorer gene Rfl in cms-T plants results in a decrease in abundance of 13-kDa protein and alteration in the transcripts of urfl3-T.Cytoplasmic male sterility (cms) in higher plants represents one of the few well-characterized examples of heritable variability transmitted through the cytoplasm. Pollen production is aborted in cms plants, yet female fertility is unaffected. In maize (Zea mays L.), the trait has been used extensively in the commercial production of hybrid seed as a means of preventing self-fertilization. The three major malesterile cytoplasms of maize-S, C, and T-are distinguished according to the pattern of fertility restoration by nuclear restorer genes. The S and C cytoplasms require a single dominant restorer gene for fertility restoration-RJ3 and Rf4, respectively-whereas two restorer genes, Rfl and Rf2, are necessary to restore T cytoplasm cms (cms-T) plants to male fertility (1-4).In maize, abundant evidence indicates that changes in organization and expression of the mitochondrial genome are responsible for the cms trait (for review, see ref. 5). Maize plants carrying the T cytoplasm are also distinguished by an apparently inseparable association between male sterility and susceptibility to the fungal pathogen Bipolaris maydis, race T (6-8). This pathogen produces a host-specific toxin that affects the permeability of cms-T mitochondria and promotes the uncoupling of oxidative phosphorylation and the leakage of NAD+ and Ca2l (9-11). In contrast, mitochondria from male fertile (N), cms-C, and cms-S cytoplasms are insensitive to the toxin.Protein synthesis studies of isolated mitochondria have revealed differences between the protein products of T versus N mitochondria (12). In particular, a 13-kDa polypeptide is observed in T mitochondria that is absent in N, and a 21-kDa protein is synthesized in N mitochondria that is not detected in T. The 13-kDa protein is further characterized by a dramatically reduced abundance in cms-T plants that are restored to fertility by nuclear restorer genes Rfl and Rf2 (13).We have previously reported the identification of a highly rearranged DNA sequence, designated TURF 2H3, unique to the T cytoplasm of maize and associated with this type of sterility (14). TURF 2H3 contains two major open reading frames, ORF 13 and ORF 25, which may encode polypeptides of 13 and 25 kDa, respectively. The organization and transcription of ORF 13, so far as we know, are unique to the T cytoplasm of maize. Significantly, ORF 13 transcripts are modified...

The endoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Delta12-desaturase) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues of plants. Little is known, however, concerning the post-transcriptional mechanisms that regulate the activity of this important enzyme. The soybean genome possesses two seed-specific isoforms of FAD2, designated FAD2-1A and FAD2-1B, which differ at only 24 amino acid residues. Expression studies in yeast revealed that the FAD2-1A isoform is more unstable than FAD2-1B, particularly when cultures were maintained at elevated growth temperatures. Analysis of chimeric FAD2-1 constructs led to the identification of two domains that appear to be important in mediating the temperature-dependent instability of the FAD2-1A isoform. The enhanced degradation of FAD2-1A at high growth temperatures was partially abrogated by treating the cultures with the 26S proteasome-specific inhibitor MG132, and by expressing the FAD2-1A cDNA in yeast strains devoid of certain ubiquitin-conjugating activities, suggesting a role for ubiquitination and the 26S proteasome in protein turnover. In addition, phosphorylation state-specific antipeptide antibodies demonstrated that the Serine-185 of FAD2-1 sequences is phosphorylated during soybean seed development. Expression studies of phosphopeptide mimic mutations in yeast suggest that phosphorylation may downregulate enzyme activity. Collectively, the results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.

Nornicotine is a secondary tobacco alkaloid that is produced by the N-demethylation of nicotine. Nornicotine production and accumulation in tobacco are undesirable because nornicotine serves as the precursor in the synthesis of the well characterized carcinogen N-nitrosonornicotine during the curing and processing of tobacco. Although nornicotine is typically a minor alkaloid in tobacco plants, in many tobacco populations a high percentage of individuals can be found that convert a substantial proportion of the nicotine to nornicotine during leaf senescence and curing. We used a microarray-based strategy to identify genes that are differentially regulated between closely related tobacco lines that accumulate either nicotine (nonconverters) or nornicotine (converters) as the predominant alkaloid in the cured leaf. These experiments led to the identification of a small number of closely related cytochrome P450 genes, designated the CYP82E2 family, whose collective transcript levels were consistently higher in converter versus nonconverter tobacco lines. RNA interference-induced silencing of the CYP82E2 gene family suppressed the synthesis of nornicotine in strong converter plants to levels similar to that observed in nonconverter individuals. Although each of the six identified members of the P450 family share >90% nucleotide sequence identity, sense expression of three selected isoforms revealed that only one (CYP82E4v1) was involved in the conversion of nicotine to nornicotine. Yeast expression analysis revealed that CYP82E4v1 functions as a nicotine demethylase. Identification of the gene(s) responsible for nicotine demethylation provides a potentially powerful tool toward efforts to minimize nornicotine levels, and thereby N-nitrosonornicotine formation, in tobacco products.NЈ-nitrosonornicotine ͉ N-demethylation ͉ tobacco ͉ alkaloid ͉ tobacco-specific nitrosamines