Analysis of ENOD40 expression in alb1, a symbiotic mutant of Lotus japonicus that forms empty nodules with incompletely developed nodule vascular bundles

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical β-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root’s stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

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Structural and Expression Analysis of Uricase mRNA from Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.10.1156 ◽

2000 ◽

Vol 13 (10) ◽

pp. 1156-1160 ◽

Cited By ~ 8

Author(s):

Ken-ichi Takane ◽

Shigeyuki Tajima ◽

Hiroshi Kouchi

Keyword(s):

Phylogenetic Analysis ◽

Expression Analysis ◽

Lotus Japonicus ◽

Vascular Bundles ◽

Model Legume

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.

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Lotus japonicus Gene Ljsbp Is Highly Conserved Among Plants and Animals and Encodes a Homologue to the Mammalian Selenium-Binding Proteins

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.4.313 ◽

2002 ◽

Vol 15 (4) ◽

pp. 313-322 ◽

Cited By ~ 25

Author(s):

Emmanouil Flemetakis ◽

Adamantia Agalou ◽

Nektarios Kavroulakis ◽

Maria Dimou ◽

Anna Martsikovskaya ◽

...

Keyword(s):

Polyclonal Antibodies ◽

Lotus Japonicus ◽

Root Hairs ◽

Physiological Role ◽

Cdna Clones ◽

Vascular Bundles ◽

Root Tips ◽

Root Epidermis ◽

Infected Cells ◽

Low Levels

We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L. japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L. japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a poly-peptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both.

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A Dicarboxylate Transporter, LjALMT4, Mainly Expressed in Nodules of Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-16-0071-r ◽

2016 ◽

Vol 29 (7) ◽

pp. 584-592 ◽

Cited By ~ 10

Author(s):

Kojiro Takanashi ◽

Takayuki Sasaki ◽

Tomohiro Kan ◽

Yuka Saida ◽

Akifumi Sugiyama ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus ◽

Specific Gene ◽

Vascular Bundles ◽

Transport Activity ◽

Organ Specific ◽

Infected Cells ◽

Photosynthetic Products ◽

Strong Candidate

Legume plants can establish symbiosis with soil bacteria called rhizobia to obtain nitrogen as a nutrient directly from atmospheric N2 via symbiotic nitrogen fixation. Legumes and rhizobia form nodules, symbiotic organs in which fixed-nitrogen and photosynthetic products are exchanged between rhizobia and plant cells. The photosynthetic products supplied to rhizobia are thought to be dicarboxylates but little is known about the movement of dicarboxylates in the nodules. In terms of dicarboxylate transporters, an aluminum-activated malate transporter (ALMT) family is a strong candidate responsible for the membrane transport of carboxylates in nodules. Among the seven ALMT genes in the Lotus japonicus genome, only one, LjALMT4, shows a high expression in the nodules. LjALMT4 showed transport activity in a Xenopus oocyte system, with LjALMT4 mediating the efflux of dicarboxylates including malate, succinate, and fumarate, but not tricarboxylates such as citrate. LjALMT4 also mediated the influx of several inorganic anions. Organ-specific gene expression analysis showed LjALMT4 mRNA mainly in the parenchyma cells of nodule vascular bundles. These results suggest that LjALMT4 may not be involved in the direct supply of dicarboxylates to rhizobia in infected cells but is responsible for supplying malate as well as several anions necessary for symbiotic nitrogen fixation, via nodule vasculatures.

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cDNA Macroarray Analysis of Gene Expression in Ineffective Nodules Induced on the Lotus japonicus sen1 Mutant

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.11.1223 ◽

2004 ◽

Vol 17 (11) ◽

pp. 1223-1233 ◽

Cited By ~ 20

Author(s):

Norio Suganuma ◽

Atsuko Yamamoto ◽

Ai Itou ◽

Tsuneo Hakoyama ◽

Mari Banba ◽

...

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Nitrogenase Activity ◽

Cysteine Proteinase ◽

Lotus Japonicus ◽

Vascular Bundles ◽

Cdna Macroarray ◽

Plant Genes ◽

Macroarray Analysis ◽

Mutant Forms

The Lotus japonicus sen1 mutant forms ineffective nodules in which development is arrested at the stage of bacterial differentiation into nitrogen-fixing bacteroids. Here, we used cDNA macroarray systems to compare gene expression in ineffective nodules induced on the sen1 mutant with gene expression in wild-type nodules, in order to identify the host plant genes that are involved in nitrogen fixation. Macroarray analysis coupled with Northern blot analysis revealed that the expression of 18 genes was significantly enhanced in ineffective sen1 nodules, whereas the expression of 30 genes was repressed. Many of the enhanced genes encoded hydrolase enzymes, such as cysteine proteinase and asparaginase, that might function in the early senescence of sen1 nodules. By contrast, the repressed genes encoded nodulins, enzymes that are involved in carbon and nitrogen metabolism, membrane transporters, enzymes involved in phytohormone metabolism and secondary metabolism, and regulatory proteins. These proteins might have a role in the establishment of nitrogen fixation. In addition, we discovered two novel genes that encoded glutamate-rich proteins and were localized in the vascular bundles of the nodules. The expression of these genes was repressed in the ineffective nodules, which had lower levels of nitrogenase activity.

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Promoters of Orthologous Glycine max and Lotus japonicus Nodulation Autoregulation Genes Interchangeably Drive Phloem-Specific Expression in Transgenic Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-7-0769 ◽

2007 ◽

Vol 20 (7) ◽

pp. 769-780 ◽

Cited By ~ 59

Author(s):

Sureeporn Nontachaiyapoom ◽

Paul T. Scott ◽

Artem E. Men ◽

Mark Kinkema ◽

Peer M. Schenk ◽

...

Keyword(s):

Glycine Max ◽

Hairy Roots ◽

Lotus Japonicus ◽

Transcript Level ◽

Regulatory Elements ◽

Vascular Bundles ◽

Specific Expression ◽

Translation Start ◽

Young Leaves ◽

Rapid Amplification

The nodule autoregulation receptor kinase (GmNARK) of soybean (Glycine max) is essential for the systemic auto-regulation of nodulation. Based on quantitative reverse-transcriptase polymerase chain reaction, GmNARK is expressed to varying levels throughout the plant; the transcript was detected at high levels in mature leaves and roots but to a lesser extent in young leaves, shoot tips, and nodules. The transcript level was not significantly affected by Bradyrhizobium japonicum during the first week following inoculation. In addition, the activities of the promoters of GmNARK and Lotus japonicus HAR1, driving a β-glucuronidase (GUSPlus) reporter gene, were examined in stably transformed L. japonicus and transgenic hairy roots of soybean. Histochemical GUS activity in L. japonicus plants carrying either a 1.7-kb GmNARKpr∷GUS or 2.0-kb LjHAR1pr∷GUS construct was clearly localized to living cells within vascular bundles, especially phloem cells in leaves, stems, roots, and nodules. Phloem-specific expression also was detected in soybean hairy roots carrying these constructs. Our study suggests that regulatory elements required for the transcription of these orthologous genes are conserved. Moreover, rapid amplification of 5′ cDNA ends (5′ rapid amplification of cDNA ends) revealed two major transcripts of GmNARK potentially originating from two TATA boxes. Further analysis of the GmNARK promoter has confirmed that these two TATA boxes are functional. Deletion analysis also located a region controlling phloem-specific expression to a DNA sequence between 908 bp and 1.7 kb upstream of the translation start site of GmNARK.

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