Regulation of nitric oxide by phytoglobins in Lotus japonicus is involved in mycorrhizal symbiosis with Rhizophagus irregularis.

Various reactive molecular species are generated in plant-microbe interactions, and these species participate in defense and symbiotic responses. Leguminous plants successfully establish symbiosis by maintaining an appropriate level of nitric oxide (NO), which is generated in the roots and nodules during root nodule symbiosis. Phytoglobin (plant hemoglobin) controls NO levels in plants. In this study, we investigated mycorrhizal symbiosis, which occurs in more than 80% of land plants, between Rhizophagus irregularis and Lotus japonicus to clarify the involvement of phytoglobin-mediated NO regulation. The mycorrhizae of L. japonicus exhibited higher NO levels in the presence of R. irregularis than in its absence, especially at the infection site. LjGlb1-1, a phytoglobin that regulates NO level in L. japonicus, was upregulated during symbiosis with R. irregularis. In transformed hairy roots carrying the ProLjGlb1-1:GUS construct, LjGlb1-1 expression was observed at the R. irregularis infection site. We further examined the symbiotic phenotypes of L. japonicus lines with high and low LjGlb1-1 expression with R. irregularis. During mycorrhizal symbiosis, the high LjGlb1-1 expression line exhibited better growth than the wild-type, whereas the low expression line exhibited poor growth. In addition, the expression of LjPT4, a phosphate transporter specific to mycorrhizal symbiosis, was higher in the high LjGlb1-1 expression line, whereas that of the tubulin gene of R. irregularis was lower in the low LjGlb1-1 expression line than in the wild-type. These results confirm that NO regulation by LjGlb1-1 is involved in mycorrhizal symbiosis in L. japonicus, as it is reportedly in nitrogen-fixing symbiosis.

Reactive Sulfur Species Produced by Cystathionine γ-lyase Function in the Establishment of Mesorhizobium loti-Lotus japonicus Symbiosis.

Reactive sulfur species (RSS) are present in root nodules; however, their role in symbiosis and the mechanisms underlying their production remain unclear. We herein investigated whether RSS produced by the cystathionine γ-lyase (CSE) of microsymbionts are involved in root nodule symbiosis. A cse mutant of Mesorhizobium loti exhibited the decreased production of hydrogen sulfide and other RSS. Although the CSE mutation of M. loti did not affect the early stages of symbiosis, i.e., infection and nodulation, with Lotus japonicus, it reduced the nitrogenase activity of nodules and induced their early senescence. Additionally, changes in the production of sulfur compounds and an increase in reactive oxygen species (ROS) were observed in the infected cells of nodules induced by the cse mutants. The effects of CSE inhibitors in the L. japonicus rhizosphere on symbiosis with M. loti were also investigated. All three CSE inhibitors suppressed infection and nodulation by M. loti concomitant with decreased RSS levels and increased ROS and nitric oxide levels. Therefore, RSS derived from the CSE activity of both the microsymbiont and host plant are required for symbiosis, but function at different stages of symbiosis, possibly with crosstalk with other reactive mole-cular species.

Nitric Oxide Detoxification by Mesorhizobium loti Affects Root Nodule Symbiosis with Lotus japonicus.

Root nodule symbiosis between legumes and rhizobia involves nitric oxide (NO) regulation by both the host plant and symbiotic rhizobia. However, the mechanisms by which the rhizobial control of NO affects root nodule symbiosis in Lotus japonicus are unknown. Therefore, we herein investigated the effects of enhanced NO removal by Mesorhizobium loti on symbiosis with L. japonicus. The hmp gene, which in Sinorhizobium meliloti encodes a flavohemoglobin involved in NO detoxification, was introduced into M. loti to generate a transconjugant with enhanced NO removal. The symbiotic phenotype of the transconjugant with L. japonicus was examined. The transconjugant showed delayed infection and higher nitrogenase activity in mature nodules than the wild type, whereas nodule senescence was normal. This result is in contrast to previous findings showing that enhanced NO removal in L. japonicus by class 1 phytoglobin affected nodule senescence. To evaluate differences in NO detoxification between M. loti and L. japonicus, NO localization in nodules was investigated. The enhanced expression of class 1 phytoglobin in L. japonicus reduced the amount of NO not only in infected cells, but also in vascular bundles, whereas that of hmp in M. loti reduced the amount of NO in infected cells only. This difference suggests that NO detoxification by M. loti exerts different effects in symbiosis than that by L. japonicus.

Elevated Nitrogen Priming Induced Oxinitro-Responses and Water Deficit Tolerance in Rice.

Under water deficit conditions, the essential macronutrient nitrogen becomes limited as a result of reduced dissolved nitrogen and root nitrogen uptake. An elevated nitrogen level might be able to mitigate these effects, integrated with the idea of using nitric oxide as abiotic stress tolerant inducers. In this study, we evaluated the potential of using elevated nitrogen priming prior to water shortage to mitigate plant stress through nitric oxide accumulation. We grew rice plants in 300 mg L-1 nitrogen for 10 weeks, then we primed plants with four different nitrogen concentrations: 100, 300 (control), 500 and 1000 mg L-1 nitrogen prior to inducing water deficit conditions. Plants primed with 500 mg L-1 nitrogen possessed a higher photosynthetic rate, relative water content, electrolyte leakage and lipid peroxidation under water deficit conditions, compared to control plants. The induction of water deficit tolerance was supported with the activation of antioxidant defense system, induced by the accumulation of nitric oxide in leaves and roots of rice plants. We originally demonstrated the accumulation of nitric oxide in leaves of rice plants. The elevated nitrogen priming can be used to enhance water deficit tolerance in irrigated paddy fields, instead of nitric oxide donors.

Mechanisms of Rice Endophytic Bradyrhizobial Cell Differentiation and Its Role in Nitrogen Fixation.

Bradyrhizobium sp. strain SUTN9-2 is a symbiotic and endophytic diazotrophic bacterium found in legume and rice plants and has the potential to promote growth. The present results revealed that SUTN9-2 underwent cell enlargement, increased its DNA content, and efficiently performed nitrogen fixation in response to rice extract. Some factors in rice extract induced the expression of cell cycle and nitrogen fixation genes. According to differentially expressed genes (DEGs) from the transcriptomic analysis, SUTN9-2 was affected by rice extract and the deletion of the bclA gene. The up-regulated DEGs encoding a class of oxidoreductases, which act with oxygen atoms and may have a role in controlling oxygen at an appropriate level for nitrogenase activity, followed by GroESL chaperonins are required for the function of nitrogenase. These results indicate that following its exposure to rice extract, nitrogen fixation by SUTN9-2 is induced by the collective effects of GroESL and oxidoreductases. The expression of the sensitivity to antimicrobial peptides transporter (sapDF) was also up-regulated, resulting in cell differentiation, even when bclA (sapDF) was mutated. This result implies similarities in the production of defensin-like antimicrobial peptides (DEFs) by rice and nodule-specific cysteine-rich (NCR) peptides in legume plants, which affect bacterial cell differentiation.

Reactive Sulfur Species Interact with Other Signal Molecules in Root Nodule Symbiosis in Lotus japonicus.

Reactive sulfur species (RSS) function as strong antioxidants and are involved in various biological responses in animals and bacteria. Few studies; however, have examined RSS in plants. In the present study, we clarified that RSS are involved in root nodule symbiosis in the model legume Lotus japonicus. Polysulfides, a type of RSS, were detected in the roots by using a sulfane sulfur-specific fluorescent probe, SSP4. Supplying the sulfane sulfur donor Na2S3 to the roots increased the amounts of both polysulfides and hydrogen sulfide (H2S) in the roots and simultaneously decreased the amounts of nitric oxide (NO) and reactive oxygen species (ROS). RSS were also detected in infection threads in the root hairs and in infected cells of nodules. Supplying the sulfane sulfur donor significantly increased the numbers of infection threads and nodules. When nodules were immersed in the sulfane sulfur donor, their nitrogenase activity was significantly reduced, without significant changes in the amounts of NO, ROS, and H2S. These results suggest that polysulfides interact with signal molecules such as NO, ROS, and H2S in root nodule symbiosis in L. japonicus. SSP4 and Na2S3 are useful tools for study of RSS in plants.

Ectopic or Over-Expression of Class 1 Phytoglobin Genes Confers Flooding Tolerance to the Root Nodules of Lotus japonicus by Scavenging Nitric Oxide.

Flooding limits biomass production in agriculture. Leguminous plants, important agricultural crops, use atmospheric dinitrogen gas as nitrogen nutrition by symbiotic nitrogen fixation with rhizobia, but this root-nodule symbiosis is sometimes broken down by flooding of the root system. In this study, we analyzed the effect of flooding on the symbiotic system of transgenic Lotus japonicus lines which overexpressed class 1 phytoglobin (Glb1) of L. japonicus (LjGlb1-1) or ectopically expressed that of Alnus firma (AfGlb1). In the roots of wild-type plants, flooding increased nitric oxide (NO) level and expression of senescence-related genes and decreased nitrogenase activity; in the roots of transgenic lines, these effects were absent or less pronounced. The decrease of chlorophyll content in leaves and the increase of reactive oxygen species (ROS) in roots and leaves caused by flooding were also suppressed in these lines. These results suggest that increased levels of Glb1 help maintain nodule symbiosis under flooding by scavenging NO and controlling ROS.

Antimicrobial Activities of Cysteine-rich Peptides Specific to Bacteriocytes of the Pea Aphid Acyrthosiphon pisum.

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.

Stably Transformed Lotus japonicus Plants Overexpressing Phytoglobin LjGlb1-1 Show Decreased Nitric Oxide Levels in Roots and Nodules as Well as Delayed Nodule Senescence.

The class 1 phytoglobin, LjGlb1-1, is expressed in various tissues of the model legume Lotus japonicus, where it may play multiple functions by interacting with nitric oxide (NO). One of such functions is the onset of a proper symbiosis with Mesorhizobium loti resulting in the formation of actively N2-fixing nodules. Stable overexpression lines (Ox1 and Ox2) of LjGlb1-1 were generated and phenotyped. Both Ox lines showed reduced NO levels in roots and enhanced nitrogenase activity in mature and senescent nodules relative to the wild-type (WT). Physiological and cytological observations indicated that overexpression of LjGlb1-1 delayed nodule senescence. The application to WT nodules of the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) or the phytohormones abscisic acid (ABA) and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) repressed nitrogenase activity, induced the expression of three senescence-associated genes and caused cytological changes evidencing nodule senescence. These effects were almost completely reverted by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Our results reveal that overexpression of LjGlb1-1 improves the activity of mature nodules and delays nodule senescence in the L.japonicus-M.loti symbiosis. These beneficial effects are probably mediated by the participation of LjGlb1-1 in controlling the concentration of NO that may be produced downstream in the phytohormone signaling pathway in nodules.

Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis.

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels.