Differential responses of the sunn4 and rdn1-1 super-nodulation mutants of Medicago truncatula to elevated atmospheric CO2

Background and Aims Nitrogen fixation in legumes requires tight control of carbon and nitrogen balance. Thus, legumes control nodule numbers via an autoregulation mechanism. ‘Autoregulation of nodulation’ mutants super-nodulate and are thought to be carbon-limited due to the high carbon-sink strength of excessive nodules. This study aimed to examine the effect of increasing carbon supply on the performance of super-nodulation mutants. Methods We compared the responses of Medicago truncatula super-nodulation mutants (sunn-4 and rdn1-1) and wild type to five CO2 levels (300-850 μmol mol -1). Nodule formation and N2 fixation were assessed in soil-grown plants at 18 and 42 days after sowing. Key results Shoot and root biomass, nodule number and biomass, nitrogenase activity and fixed-N per plant of all genotypes increased with increasing CO2 concentration and reached the maximum around 700 μmol mol -1. While the sunn-4 mutant showed strong growth-retardation compared to wild-type plants, elevated CO2 increased shoot biomass and total N content of rdn1-1 mutant up to two-fold. This was accompanied by a four-fold increase in nitrogen fixation capacity in the rdn1-1 mutant. Conclusions These results suggest that the super-nodulation phenotype per se did not limit growth. The additional nitrogen fixation capacity of the rdn1-1 mutant may enhance the benefit of elevated CO2 on plant growth and N2 fixation.

Shoot Extracts from Two Low Nodulation Mutants Significantly Reduce Nodule Number in Pea

E107 and E132 are pea mutants that nodulate poorly. Because they have a shoot-controlled nodulation phenotype, we asked if their mutated genes were implicated in the autoregulation of nodulation (AON), a mechanism which consists of two systemic circuits, the positive CEP/CRA2 and the negative CLE/SUNN, coordinated via NIN and miR2111. We further characterized the mutants’ phenotype by studying nodule distribution and nodulation efficiency. E107 was similar to wild-type (WT) in its nodule distribution, but E132 had an extended nodulation zone with nodules forming distally on its lateral roots. Moreover, we tested whether their shoots produced a compound inhibitory to nodulation. We made ethyl-acetate extracts of roots and shoots of both mutants and WT, which we applied to rhizobia-inoculated WT seedlings and to pure rhizobial cultures. Whereas free-living bacteria were unaffected by any of the extracts, WT treated with shoot extracts from either inoculated mutant had fewer nodules than that of control. E107 and E132 shoot extracts led to a 50% and a 35% reduction in nodule number, respectively. We propose that E107 and E132 belong to a new sub-class of AON mutants, i.e., hypo-nodulators, and that their respective gene products are acting in the AON descending branch, upstream of TML signaling.

New Nodulation Mutants Responsible for Infection Thread Development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

A model for the development of the rhizobial and arbuscular mycorrhizal symbioses in legumes and its use to understand the roles of ethylene in the establishment of these two symbioses

We propose a model depicting the development of nodulation and arbuscular mycorrhizae. Both processes are dissected into many steps, using Pisum sativum L. nodulation mutants as a guideline. For nodulation, we distinguish two main developmental programs, one epidermal and one cortical. Whereas Nod factors alone affect the cortical program, bacteria are required to trigger the epidermal events. We propose that the two programs of the rhizobial symbiosis evolved separately and that, over time, they came to function together. The distinction between these two programs does not exist for arbuscular mycorrhizae development despite events occurring in both root tissues. Mutations that affect both symbioses are restricted to the epidermal program. We propose here sites of action and potential roles for ethylene during the formation of the two symbioses with a specific hypothesis for nodule organogenesis. Assuming the epidermis does not make ethylene, the microsymbionts probably first encounter a regulatory level of ethylene at the epidermis – outermost cortical cell layer interface. Depending on the hormone concentrations there, infection will either progress or be blocked. In the former case, ethylene affects the cortex cytoskeleton, allowing reorganization that facilitates infection; in the latter case, ethylene acts on several enzymes that interfere with infection thread growth, causing it to abort. Throughout this review, the difficulty of generalizing the roles of ethylene is emphasized and numerous examples are given to demonstrate the diversity that exists in plants.Key words: AM, epidermis, evolution, pea, rhizobia, sym mutant.

Nodule-Expressed Cyp15a Cysteine Protease Genes Map to Syntenic Genome Regions in Pisum and Medicago spp.

PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules. To further the understanding of nodular PsCyp15a expression, a region 5′ to the coding sequence of the gene was cloned. Varying lengths of 5′ untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into “hairy roots” of Vicia hirsuta. In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization. An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M. truncatula was also shown to express strongly in nodules. With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.