Arabidopsis chlorophyll biosynthesis: an essential balance between the methylerythritol phosphate and tetrapyrrole pathways.

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)-derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis.

Nitric oxide accumulation in Arabidopsis is independent of NOA1 in the presence of sucrose.

Nitric oxide signals diverse responses in animals and plants. Whereas nitric oxide synthesis mechanisms in animals are well understood, how nitric oxide is synthesized and regulated in plants remains controversial. NOA1 is a circularly permuted GTPase that is important for chloroplast function and is implicated in nitric oxide synthesis. However, the reported consequences of a null mutation in NOA1 are inconsistent. Whereas some studies indicate that the noa1 mutant has severe reductions in nitric oxide accumulation, others report that nitric oxide levels are indistinguishable between noa1 and the wild type. Here, we identify a correlation between the reported ability of noa1 to accumulate nitric oxide with growth on sucrose-supplemented media. We report that noa1 accumulates both basal and salicylic acid-induced nitric oxide only when grown on media containing sucrose. In contrast, nitric oxide accumulation in wild type is largely insensitive to sucrose supplementation. When grown in the absence of sucrose, noa1 has low fumarate, pale green leaves, slow growth and reduced chlorophyll content. These phenotypes are consistent with a defect in chloroplast-derived photosynthate production and are largely rescued by sucrose supplementation. We conclude that NOA1 has a primary role in chloroplast function and that its effects on the accumulation of nitric oxide are likely to be indirect.

Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function.

BACKGROUND: The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli. These subunits are homologous to components of multi-subunit Na(+)/H(+) antiporters, and so are implicated in proton translocation. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen site-specific mutations were constructed at two corresponding positions in each of the three subunits. Two positions were selected in each subunit: L_K169, M_K173, N_K158 and L_Q236, M_H241, N_H224. Membrane vesicles were prepared from all of the resulting mutant strains, and were assayed for deamino-NADH oxidase activity, proton translocation, ferricyanide reductase activity, and sensitivity to capsaicin. Corresponding mutations in the three subunits were found to have very similar effects on all activities measured. In addition, the effect of adding exogenous decylubiquinone on these activities was tested. 50 µM decylubiquinone stimulated both deamino-NADH oxidase activity and proton translocation by wild type membrane vesicles, but was inhibitory towards the same activities by membrane vesicles bearing the lysine substitution at the L236/M241/N224 positions. CONCLUSIONS/SIGNIFICANCE: The results show a close correlation with reduced activity among the corresponding mutations, and provide evidence that the L, M, and N subunits have a common role in Complex I.