HighlightMissense and null Lotus japonicus mutants allowed us to demonstrate that hemoglobin LjGlb1-1 is required for infection thread elongation and nodule formation, probably by regulating nitric oxide production in the roots.
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 N 2 -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-D,L-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.
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.
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.
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.
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