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The identification of novel loci required for appropriate nodule development in Medicago truncatula.

Domonkos A, Horvath B, Marsh JF, Halasz G, Ayaydin F, Oldroyd GE, Kalo P - BMC Plant Biol. (2013)

Bottom Line: Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci.Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agricultural Biotechnology Center, Gödöllő 2100, Hungary. kalo@abc.hu.

ABSTRACT

Background: The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.

Results: Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

Conclusions: The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

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The gross phenotypes of nodulation defective mutants. The nodulation phenotype of the Fix- mutant dnf5-2 as compared to wild-type M. truncatula 4 weeks postinoculation (wpi) with S. meliloti 1021. Ineffective mutants, such as dnf5-2 display retarded growth and show other symptoms of nitrogen deficiency, such as leaf yellowing (A). Wild-type plants developed pink cylindrical nodules (B). Spherical or slightly elongated white nodules were found on ineffective mutant roots. (C), dnf5-2; (D), 13U mutant. Scale bars: 2 cm in A, 1 mm in B to D.
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Figure 1: The gross phenotypes of nodulation defective mutants. The nodulation phenotype of the Fix- mutant dnf5-2 as compared to wild-type M. truncatula 4 weeks postinoculation (wpi) with S. meliloti 1021. Ineffective mutants, such as dnf5-2 display retarded growth and show other symptoms of nitrogen deficiency, such as leaf yellowing (A). Wild-type plants developed pink cylindrical nodules (B). Spherical or slightly elongated white nodules were found on ineffective mutant roots. (C), dnf5-2; (D), 13U mutant. Scale bars: 2 cm in A, 1 mm in B to D.

Mentions: The Fix- mutants were defined by symptoms of nitrogen starvation under symbiotic conditions and the development of small white nodules (Figure 1). Supplementation with combined nitrogen restored normal growth habits in eight of the Fix- mutants, indicating specific defects in the ability to establish appropriate conditions for nitrogen fixation. This was further validated using acetylene reduction that measures the activity of bacterial nitrogenase [28]. After 18 days post inoculation with S. meliloti wild-type nodules displayed high levels of acetylene, but no acetylene reduction was detected on any of the 8 backcrossed Fix- mutants, except one mutant, dnf8 (see below), in which a very low rate of nitrogen fixation was observed (Figure 2).


The identification of novel loci required for appropriate nodule development in Medicago truncatula.

Domonkos A, Horvath B, Marsh JF, Halasz G, Ayaydin F, Oldroyd GE, Kalo P - BMC Plant Biol. (2013)

The gross phenotypes of nodulation defective mutants. The nodulation phenotype of the Fix- mutant dnf5-2 as compared to wild-type M. truncatula 4 weeks postinoculation (wpi) with S. meliloti 1021. Ineffective mutants, such as dnf5-2 display retarded growth and show other symptoms of nitrogen deficiency, such as leaf yellowing (A). Wild-type plants developed pink cylindrical nodules (B). Spherical or slightly elongated white nodules were found on ineffective mutant roots. (C), dnf5-2; (D), 13U mutant. Scale bars: 2 cm in A, 1 mm in B to D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852326&req=5

Figure 1: The gross phenotypes of nodulation defective mutants. The nodulation phenotype of the Fix- mutant dnf5-2 as compared to wild-type M. truncatula 4 weeks postinoculation (wpi) with S. meliloti 1021. Ineffective mutants, such as dnf5-2 display retarded growth and show other symptoms of nitrogen deficiency, such as leaf yellowing (A). Wild-type plants developed pink cylindrical nodules (B). Spherical or slightly elongated white nodules were found on ineffective mutant roots. (C), dnf5-2; (D), 13U mutant. Scale bars: 2 cm in A, 1 mm in B to D.
Mentions: The Fix- mutants were defined by symptoms of nitrogen starvation under symbiotic conditions and the development of small white nodules (Figure 1). Supplementation with combined nitrogen restored normal growth habits in eight of the Fix- mutants, indicating specific defects in the ability to establish appropriate conditions for nitrogen fixation. This was further validated using acetylene reduction that measures the activity of bacterial nitrogenase [28]. After 18 days post inoculation with S. meliloti wild-type nodules displayed high levels of acetylene, but no acetylene reduction was detected on any of the 8 backcrossed Fix- mutants, except one mutant, dnf8 (see below), in which a very low rate of nitrogen fixation was observed (Figure 2).

Bottom Line: Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci.Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agricultural Biotechnology Center, Gödöllő 2100, Hungary. kalo@abc.hu.

ABSTRACT

Background: The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.

Results: Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

Conclusions: The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

Show MeSH
Related in: MedlinePlus