Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula.

The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.

A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation.

N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 µM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.

Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti.

We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.

Defective long-distance auxin transport regulation in the Medicago truncatula super numeric nodules mutant.

Long-distance auxin transport was examined in Medicago truncatula and in its supernodulating mutant sunn (super numeric nodules) to investigate the regulation of auxin transport during autoregulation of nodulation (AON). A method was developed to monitor the transport of auxin from the shoot to the root in whole seedlings. Subsequently, the transport was monitored after inoculation of roots with the nodulating symbiont Sinorhizobium meliloti. The sunn mutant showed an increased amount of auxin transported from the shoot to the root compared to the wild type. The auxin transport capacity of excised root segments was similar in wild type and sunn, suggesting that the difference in long-distance auxin transfer between them is due to loading in the shoot. After inoculation, wild-type seedlings showed decreased auxin loading from the shoot to the root; however, the sunn mutant failed to reduce the amount of auxin loaded. The time of reduced auxin loading correlated with the onset of AON. Quantification of endogenous auxin levels at the site of nodule initiation showed that sunn contained three times more auxin than wild type. Inoculation of sunn failed to reduce the level of auxin within 24 h, as was observed in the wild type. We propose a model for the role of auxin during AON of indeterminate legumes: 1) high levels of endogenous auxin are correlated with increased numbers of nodules, 2) inoculation of roots reduces auxin loading from the shoot to the root, and 3) subsequent reduction of auxin levels in the root inhibits further nodule initiation.

Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus.

This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus. Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose. The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum. The carbon balance in the medium with glucose and xylose was virtually 100%. The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate. Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate. The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L x h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9-11 mmol/[L x h]). The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate.