@misc{oai:ir.soken.ac.jp:00004089,
 author = {高原, 正裕 and タカハラ, マサヒロ and TAKAHARA, Masahiro},
 month = {2016-02-26, 2016-02-17},
 note = {The interaction of legumes with N2-fixing bacteria collectively called rhizobia, results in the root nodule development. The number of nodules is tightly restricted through the negative feedback regulation by hosts. The fact that the HAR1-mediated control of nodule number needs the HAR1 expression in the shoots exhibits a long distance communication between the shoot and the root. This long-distance regulation of nodulation is termed auto regulation of nodulation (AON). However, the large part of the mechanism remains to be elucidated. A previous study has shown that too much love-1 (tml-1, formerly tml), a hypernodulating mutant in Lotus japonicus, has a defect in the negative feedback regulation and that TML functions in the roots downstream of HAR1. To better understand the mechanism by which legume plants control the number of nodules, the author performed molecular biological and genetic analyses using tml mutant alleles.
Firstly, the author examined the genetic interaction between TML and PLENTY, another root factor that regulates the number of nodules. The tml-1 plenty double mutant showed an increased number of nodules compared to those of the respective single mutants, indicating that TML and PLENTY function in different genetic pathways and that if TML and the other genes act in different genetic pathways, the double mutant indeed illustrates the additional effect on the nodule number. In contrast, the tml-1 har1-7 double mutant did not show an additive effect on nodulation. Taken together, he concluded that TML and HAR1 function in the same genetic pathway.
Secondly, the genetic interaction between TML and CLE-RS1/RS2 (the genes encoding the putative root-derived signals transporting to the shoot) was investigated. In the tml mutant background, the roots overexpressing either CLE-RS1 or CLE-RS2 developed as many nodules as the control GUS expressing roots. This result indicates that CLE-RS1/RS2 suppresses nodulation in a TML-dependent manner. Therefore, he concluded that TML functions in nodule development downstream of CLE-RS1/RS2.
Thirdly, the author investigated the genetic interaction between TML and Snf2 (the gain-of-function mutant of the cytokinin receptor LHK1) to assess the role of TML in AON. The tml-1 Snf2 double mutant spontaneously developed many small nodules similar to those generally observed in the tml mutant upon rhizobial infection, whereas the Snf2 single mutant developed spontaneous nodules similar to those produced in the wild type upon infection, indicating that TML inhibits the nodule organogenesis induced by the LHK1-mediated cytokinin signaling.
Taken together, he concluded that TML acts at the final stage of AON downstream of CLE-RS1/RS2 and HAR1 to negatively regulate the nodule number by inhibiting the organogenesis induced by the cytokinin signaling.
In an attempt to localize the TML gene, inverse PCR was performed and the deleted regions in the large deletion alleles tml-1, tml-2 and tml-3 were determined. In addition, the fine mapping was performed using the EMS allele tml-4 (formerly rdh1). Together with the results, the TML gene locus was delimited to a region of approximately 117 kb. Of the 21 genes predicted in the region, the whole genome resequencing of tml-4 using a next generation sequencer revealed only one non-synonymous single nucleotide alteration in the gene corresponding to the EST sequence (GenBank accession number AK339024), which results in a premature stop codon. The number of nodules developed on the candidate gene-silenced roots increased approximately 7.5-fold compared to those on the control roots, indicating that the gene corresponding to AK339024 is indeed responsible for the tml hypernodulating phenotype.
The sequence analysis revealed that TML encodes a Kelch repeat-containing F-box protein with three types of conserved domains: the F-box domain, the Kelch-repeat domain and two nuclear localization signals (NLSs). The F-box domain is a conserved domain in the component of E3 ubiquitin ligase that is involved in the proteasome-mediated protein degradation. The fluorescence of TML-sGFP was observed in the nucleus in the transgenic hairy roots in the wild-type plants. Taken together, TML might function by degrading its target protein in the nucleus. A phylogenetic analysis revealed that the TML-related Kelch repeat-containing F-box proteins were widely conserved in embryophytes. It also showed that at least one ortholog of TML in each legume plant exists in the TML clade, which supported the possibility that there is the conserved function of TML among legume species.
The qPCR analysis showed that TML is constitutively expressed in the roots and nodules and the expression was not detected in shoots from the plants with nodules nor shoots from the plants without infection, suggesting that TML is a root-specific gene. In addition, the author and his collaborator demonstrated that TML is constitutively expressed in the root tip region, including the meristematic region and elongation zone, which might explain why the tml mutant develops an excessive number of infection threads. Moreover, the expression pattern of ProTML-GUS was detected in root nodule primordia after several cell divisions of the cortical cells but not soon after the initial cell division, suggesting that TML inhibits the nodule development after the initiation of cortical cell division. This hypothesis is consistent with the appearance of arrested root nodule primordia in roots overexpressing either CLE-RS1 or CLE-RS2 genes. 
In this thesis, the author clarified the acting point of TML during AON through the characterization of the tml mutant. Genetic and molecular analyses indicated that TML and PLENTY act in different genetic pathways and TML acts downstream of LjCLE-RS1/2 and HAR1 to suppress the nodulation signaling downstream of the cytokinin receptor LHK1/CRE1. He also revealed that the TML gene encodes a Kelch repeat-containing F-box protein with two NLSs and potentially functions in proteasome-mediated degradation of its target protein. In conclusion, the author identified the F-box protein TML as a key factor in maintaining proper nodulation at the final stage during AON., 総研大甲第1609号},
 title = {Molecular genetic analyses and molecular cloning of TOO MUCH LOVE; the root regulatory gene in the long-distance regulation of the root-nodule symbiosis in Lotus japonicus.},
 year = {}
}