One of the largest contributions to biologically available nitrogen comes from the reduction of N 2 to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N 2 fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N 2 fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N 2 fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis.mutualism ͉ nitrogen fixation ͉ peas ͉ symbiosis T he largest input of available nitrogen in the biosphere comes from biological reduction of atmospheric N 2 to ammonium (1). Most of this comes from legume-Rhizobium symbioses, arising from infection of host plants and resulting in root structures called nodules (2). These symbioses are initiated by plant-released flavonoids and related compounds, which elicit synthesis of lipochitooligosaccharide Nod factors by rhizobia. Bacteria are trapped by curling root hairs that they enter via infection threads. These grow into the root cortex, into a zone of newly induced meristematic cells forming the origin of the nodule. Bacteria are released from infection threads by endocytosis and are surrounded by a plant-derived symbiosome membrane. In nodules of galegoid legumes (a clade in the subfamily Papilionoideae, such as Medicago, Pisum, or Vicia), bacteria undergo dramatic increases in size, shape, and DNA content (3) before they start to reduce N 2 . Plants provide differentiated bacteria (bacteroids) with dicarboxylic acids, which energize N 2 reduction to ammonium for secretion back to the plant (4).A simple exchange of dicarboxylates and ammonium is the classical model of nutrient exchange in nodules, but amino acid transport by bacteroids has also been shown to be essential (5). Rhizobium leguminosarum mutated in 2 broad-specificity amino acid ABC transporters (Aap and Bra) formed N 2 -fixing pea bacteroids that appeared morphologically normal in electron micrographs,...

SummaryPlant shoots have thick, polylamellate outer epidermal walls based on crossed layers of cellulose microfibrils, but the involvement of microtubules in such wall lamellation is unclear. Recently, using a long-term movie system in which Arabidopsis seedlings were grown in a biochamber, the tracks along which cortical microtubules move were shown to undergo slow rotary movements over the outer surface of hypocotyl epidermal cells. Because microtubules are known to guide cellulose synthases over the short term, we hypothesised that this previously unsuspected microtubule rotation could, over the longer term, help explain the cross-ply structure of the outer epidermal wall. Here, we test that hypothesis using Arabidopsis plants expressing the cellulose synthase GFP-CESA3 and show that cellulose synthase trajectories do rotate over several hours. Neither microtubule-stabilising taxol nor microtubule-depolymerising oryzalin affected the linear rate of GFP-CESA3 movement, but both stopped the rotation of cellulose synthase tracks. Transmission electron microscopy revealed that drug-induced suppression of rotation alters the lamellation pattern, resulting in a thick monotonous wall layer. We conclude that microtubule rotation, rather than any hypothetical mechanism for wall self-assembly, has an essential role in developing cross-ply wall texture.

SummaryMutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln : Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N 2 was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N2 on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

SUMMARYThe glucose-6-phosphate/phosphate antiporter GPT1 is a major route of entry of carbon into nonphotosynthetic plastids. To discover its importance in oilseeds, we used a seed-specific promoter to generate lines of Arabidopsis thaliana with reduced levels of GPT1 in developing embryos. Strong reductions resulted in seed abortion at the end of the globular stage of embryo development, when proplastids in normal embryos differentiate and acquire chlorophyll. Seed abortion was partly dependent on the light level during silique development. Embryos in seeds destined for abortion failed to undergo normal morphogenesis and were 'raspberry-like' in appearance. They had ultrastructural and biochemical defects including proliferation of peroxisomes and starch granules, and altered expression of genes involved in starch turnover and the oxidative pentose phosphate pathway. We propose that GPT1 is necessary for early embryo development because it catalyses import into plastids of glucose-6-phosphate as the substrate for NADPH generation via the oxidative pentose phosphate pathway. We suggest that low NADPH levels during plastid differentiation and chlorophyll synthesis may result in generation of reactive oxygen species and triggering of embryo cell death.