SummaryIron is an important nutrient in N 2 -®xing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N 2 -®xing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identi®ed Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen ®xation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 con®rmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55 Fe(II) uptake in the ferrous iron transport de®cient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen ®xation is discussed.
We have investigated the expression and regulatory properties of the two alternative oxidase (Aox) proteins that are expressed in tomato (Lycopersicon esculentum L. Mill cv. Sweetie) after storage of green fruit at 4 degrees C. Four Aox genes were identified in the tomato genome, of which two (LeAox1a and LeAox1b) were demonstrated to be expressed in cold-treated fruit. The activity and regulatory properties of LeAox1a and LeAox1b were assayed after expression of each protein in yeast cells (Saccharomyces cerevisiae), proving that each is an active Aox protein. The LeAox1b protein was shown to have altered regulatory properties due to the substitution of a Ser for the highly conserved Cys(I) residue. LeAox1b could not form inactive disulfide-linked dimers and was activated by succinate instead of pyruvate. This is the first example of a dicot species expressing a natural Cys(I)/Ser isoform. The implications of the existence and expression of such Aox isoforms is discussed in the light of the hypothesised role for Aox in plant metabolism.
The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5?7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4?5) conditions.
The Australian grass subtribe Neurachninae contains closely related species that use C 3 , C 4 , and C 2 photosynthesis. To gain insight into the evolution of C 4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C 3 -C 4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO 2 ) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO 2 is concentrated in C 2 and C 4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C 3 Neurachninae species with C * values of 56-61 mmol mol 21 , whereas twothirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C 2 phenotype with C * of 44 mmol mol 21 . Increased investment of GLDP in MS tissue of the C 2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C 4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO 2 in MS tissues. 566
Cent re fo r Rhizobium Studies, Division Of Science, M u rdoc h U n ive rsity, Murdoch, Perth, Western Australia 61 50 Cells of Sinorhizobium meliloti WSM419 showed an adaptive acid-tolerance response when grown a t pH 58 instead of pH 70. Increasing concentrations of calcium in the exposure medium significantly decreased the death rate of WSM419 cells under conditions of acid stress (pH 4-0). The effect of calcium on survival at pH 4.0 however, appears unconnected to exopolysaccharide (EPS), since a strain with a mutation in exoY (Rm0540) responded to calcium in the exposure medium in the same way as its wild-type parent (RmZOlI). The concentration of calcium in the growth medium also affected subsequent survival a t pH 4.0, and the effect varied with pH. In cells grown at pH 58, higher calcium concentrations also markedly increased the rate of synthesis of EPS; this was not seen in cells grown a t pH 7.0. 'H NMR spectra for isolated EPS from WSM419 cultures grown a t pH 5.8 and pH 7-0 showed that low pH markedly lowered the degree of substitution with acetyl and pyruvyl groups, but not the degree of substitution with succinyl groups; calcium concentration did not affect the pattern of substitution a t either pH. For EPS to be involved in the effect of calcium concentration in the growth medium on survival would imply a deleterious effect of the EPS produced at low pH.
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