NIBB led group clarifies the role of "cytokinin" in legume and rhizobia symbiosis

Legumes can fix atmospheric nitrogen and grow in low-nutrient environments through symbiosis with rhizobia. However, if root nodules are unevenly distributed, the plant is unable to get sufficient water and other nutrients from the ground, and growth will be adversely affected. Therefore, the positions of symbiont rhizobia are precisely controlled, but the regulatory mechanism remained unclear.

Research on Lotus corniculatus, a model legume species, was conducted by Associate Professor Takashi Soyano, Professor Masayoshi Kawaguchi, and Specially Appointed Assistant Professor Nao Okuma (currently at RIKEN) of the Division of Symbiotic Systems at the National Institute for Basic Biology; Professor Keiji Nakajima and Assistant Professor Tatsuaki Goh of the Graduate School of Science and Technology at Nara Institute of Science and Technology; Associate Professor Masaaki Watahiki of Hokkaido University; Professor Naoya Takeda and Assistant Professor Akira Akamatsu (currently at RIKEN) of Kwansei Gakuin University; Senior Visiting Researcher Hitoshi Sakakibara, Specialist Technician Mikiko Kojima, and Technical Staff I Yumiko Takebayashi of the RIKEN Center for Sustainable Resource Science; and Professor Emeritus Norio Suganuma of Aichi University of Education. The research team has shown that the response of roots to rhizobia is accompanied by oscillating gene functioning with constant intervals, and that this periodicity regulates the distribution patterns of root nodules by determining the size of root zones infected by rhizobia. Furthermore, they found that the plant hormone cytokinin is necessary to maintain the periodicity of gene expression. Their results were published in Science.

Rhizobia primarily infects a narrow area (susceptible region) near the root tip of legumes. The susceptible region is continually formed as the root grows because the root apical meristem located even closer to the root tip produces new cells that make up the root. If rhizobial infection always occurs at similar frequencies in the susceptible region, the infected areas should be evenly distributed in all parts of the root, but the infected areas are spaced at regular intervals. The same is true for the expression of a host gene (NSP1 gene) that is rapidly upregulated in response to rhizobia.

The research team observed the spatiotemporal expression pattern of the NSP1 gene using live luminescence imaging, which can detect gene expression with high sensitivity and temporal resolution. They found that gene expression increased and decreased in a cycle of about 6 hours in the susceptible region after rhizobial inoculation and that new NSP1-expression regions were formed intermittently and repeatedly on the apical side of the expression regions formed in the previous cycle as the roots grew. Most of the root nodules formed in the regions where the oscillating NSP1 expression was strong, suggesting that expression of this gene is closely related to the nodulation process. Furthermore, NSP1 and many other pre-existing genes important for root nodule symbiosis showed oscillatory expression patterns, indicating for the first time that the early response to rhizobia is associated with periodic gene expression.

Many of the periodically expressed genes were related to the biosynthesis, metabolism, and signaling of the plant hormone "cytokinin." The exploration of the sites where the response was activated using the TCSn reporter, a marker of cytokinin response, revealed that the activated and non-activated sites alternate along the root growth axis. The TCSn activity detected through live luminescence imaging showed that the response to cytokinin after rhizobial inoculation also occurred in a 6-hour cycle. The periodicity was also observed in changes in cytokinin accumulation. The expression patterns of genes responsible for biosynthesis and inactivation of cytokinin were also temporally aligned with the cytokinin responses.

To investigate the relationship between the cytokinin response and the periodicity of gene expression, Lotus corniculatus was genetically modified to express a loss-of-function mutant of the cytokinin receptor LHK1 (lhk1) or an activated form of LHK1 (gofLHK1). In the gofLHK1 strain with activated responses, NSP1 expression at the time of rhizobial inoculation was reduced, and the periodicity of expression was also disrupted. This expression pattern was similar to that observed when rhizobia were inoculated on roots directly treated with cytokinin, suggesting that the expression of symbiosis-related genes is suppressed when the cytokinin response is activated.

In contrast, the cytokinin response was reduced in the lhk1 mutant, incapable of cytokinin reception. The interval lengths of the periodic NSP1 expression and the NSP1- expression area were increased while the NSP1 expression level was maintained. These results indicate that the cytokinin response is required to maintain the periodicity of NSP1 expression and control the size of the NSP1-expression region. Furthermore, the densely infected region was expanded in the lhk1 loss-of-function mutant compared to the normal strain. In another mutant, in which the number of infection sites increases but the expression cycle of the NSP1 gene does not change, the size of the region was comparable to that of the normal strain.

Meanwhile, the gofLHK1 strain, of which periodicity is disrupted, showed a decrease in the number of infection sites and an increase in the overall size variability of the infected regions with a slight increase in size. The distribution of root nodules formed just below the infection site in the epidermis also showed changes consistent with the distribution of these sites.

These results indicate that the normal cytokinin reception maintains the periodicity of expression of symbiosis-related genes, and disruption of this pathway causes changes in the size of the expression regions of these genes and affects the distribution of infection sites and root nodules. Root nodule symbiosis is found in the orders Rosales, Cucurbitales, and Fagales in addition to the order Fabales. It is thought that the common ancestor of these orders acquired root nodule symbiosis. However, there are virtually no reports on cytokinin action in plants other than legumes, suggesting the possibility that the cytokinin pathway was independently incorporated by legumes as an important regulatory component of root nodule symbiosis. The findings of this study have also raised many questions that need to be addressed in future studies, such as the molecular mechanism by which the periodicity is formed and the mechanism by which the periodic responses establish the infection regions.

Soyano said, "Many collaborators helped us with various things in this study, such as measurement and imaging, and we could see spatial changes, which led to new discoveries. The high temporal resolution gene expression data that we obtained will help advance the elucidation of the gene networks of the regulatory pathways."

Journal Information
Publication: Science
Title: Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule developmen
DOI: 10.1126/science.adk5589