Pre‐protein translocation into chloroplasts is accomplished by two distinct translocation machineries in the outer and inner envelope, respectively. We have isolated the translocon at the inner envelope membrane (Tic complex) by blue‐native PAGE and describe a new Tic subunit, Tic62. Tic62, together with Tic110 and Tic55, forms a core translocation unit. The N‐terminus of Tic62 shows strong homologies to NAD(H) dehydrogenases in eukaryotes and to Ycf39‐like proteins present in cyanobacteria and non‐green algae. The stromal‐facing C‐terminus of Tic62 contains a novel, repetitive module that interacts with a ferredoxin‐NAD(P)+ oxidoreductase. Ferredoxin‐NAD(P)+ oxidoreductase catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P). Substrates that interfere with either NAD binding, such as deamino‐NAD, or influence the ratio of NAD(P)/NAD(P)H, such as ruthenium hexamine trichloride, modulate the import characteristics of leaf‐specific ferredoxin‐NAD(P)+ oxidoreductase isologues differently. We conclude that the Tic complex can regulate protein import into chloroplasts by sensing and reacting to the redox state of the organelle.

The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time-and concentration-dependent manner. Neither Gln nor L-methionine sulfoximine-induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).

SummaryAlthough soil contains only traces of soluble carbohydrates, plant roots take up glucose and sucrose efficiently when supplied in artificial media. Soluble carbohydrates and other small metabolites found in soil are in part products from exudation from plant roots. The molecular nature of the transporters for uptake and exudation is unknown. Here, fluorescence resonance energy transfer (FRET) glucose and sucrose sensors were used to characterize accumulation and elimination of glucose and sucrose in Arabidopsis roots tips. Using an improved image acquisition set-up, FRET responses to perfusion with carbohydrates were detectable in roots within less than 10 sec and over a wide concentration range. Accumulation was fully reversible within 10-180 sec after glucose or sucrose had been withdrawn; elimination may be caused by metabolism and/or efflux. The rate of elimination was unaffected by pre-incubation with high concentrations of glucose, suggesting that elimination is not due to accumulation in a short-term buffer such as the vacuole. Glucose and sucrose accumulation was insensitive to protonophores, was comparable in media differing in potassium levels, and was similar at pH 5.8, 6.8 and 7.8, suggesting that both influx and efflux may be mediated by protonindependent transport systems. High-resolution expression mapping in root tips showed that only a few proton-dependent transport of the STP (Sugar Transport Protein) and SUT/SUC (Sucrose Transporter/Carrier) families are expressed in the external cell layers of root tips. The root expression maps may help to pinpoint candidate genes for uptake and release of carbohydrates from roots.