Nodulation regulation: Legumes convert atmospheric nitrogen into biologically useful ammonium with the help of symbiotic bacteria housed in root nodules. Much of nodule development is controlled by the transcription factor NODULE INCEPTION (NIN). Feng et al . show that NIN is proteolytically processed to release a fragment that regulates the later stages of nodulation when the nodules acquire nitrogen-fixing capability. In related work, Jiang et al . identified members of the NIN-like protein (NLP) transcription factor family as being regulators of leghemoglobin expression acting through an unusual promotor motif shared across legumes. —PJH

The specific partnership between legumes and rhizobia relies on a chemical dialogue. Plant flavonoids activate the bacterial transcription factor NodD, which triggers production of Nod factors that are recognized by the plant. Structural studies of the Pisum sativum (pea) symbiont Rhizobium leguminosarum NodD revealed two pockets that are essential for its activation by flavonoids. Comparative studies with NodD1 of Sinorhizobium medicae , the symbiont of Medicago truncatula , revealed that this specificity is determined by the shape of the pocket and by specific amino acids. A chimeric NodD containing the flavonoid recognition residues from S. medicae NodD1 in the R. leguminosarum NodD backbone was sufficient to complement nitrogen fixation in M. truncatula by an S. medicae nodD1 mutant, confirming the critical role of flavonoid recognition in host range.

The Agrobacterium rhizogenes hairy root transformation system is widely used in symbiotic studies of model legumes. It typically relies on fluorescent reporters, such as DsRed, for identification of transgenic roots. The MtLAP1 transcription factor has been utilized as a reporter system in Medicago truncatula based on production of anthocyanin pigment. Here, we describe a version of this reporter driven by a root-cap specific promoter for direct observation of anthocyanin accumulation in root tips, which allows the identification of transgenic hairy roots by the naked eye. Results from our analysis suggest that the reporter had no significant effects on nodulation of M. truncatula. This approach, by virtue of its strong and specific expression in root cap cells, greatly reduces false positives and false negatives, and its use of an easily scored visible pigment should allow greater versatility and efficiency in root biology studies.

The NIN-like protein 2 (NLP2) transcription factor is highly expressed in the infected cells of the N2 fixation zone of mature nodules in Medicago truncatula. In these cells, NLP2 directly activates the expression of leghemoglobin genes and is required for the full expression of nitrite reductase (NiR). Histological examination of nodules formed under different nitrate regimes revealed that the infected cells of nlp2 display abnormal vacuole morphology under high nitrate (5 mm KNO3). This phenotype is associated with starch accumulation, higher expression of starch biosynthesis genes, and increased levels of nitrite and nitric oxide. A transcriptomic comparison of nlp2 and wild-type nodules across nitrate concentrations revealed that under high nitrate, nlp2 shows lower expression of genes important for hypoxia adaptation, such as alcohol dehydrogenase and pyruvate decarboxylase (PDC), and increased expression of S-nitrosoglutathione reductase (GSNOR), which encodes a key enzyme for nitric oxide homeostasis. These changes in gene expression were associated with lower nodule ATP levels and a higher NAD+/NADH ratio, suggesting that energy metabolism is specifically compromised in nlp2 nodules under high nitrate. Transgenic expression of NiR in the nodules of nlp2-1 mutants grown under high nitrate rescued the vacuole phenotype and restored the expression of GSNOR and PDC to normal levels. Overall, our data indicate a role for NLP2 in the regulation of NiR in N2-fixing cells, suggesting a role for nitrate reduction in nodule energy homeostasis.