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OPEN
received: 04 December 2014 accepted: 14 April 2015 Published: 08 June 2015
Nodulation Characterization and Proteomic Profiling of Bradyrhizobium liaoningense CCBAU05525 in Response to Water-Soluble Humic Materials Tong Guo Gao1,2,*, Yuan Yuan Xu1,*, Feng Jiang1, Bao Zhen Li1, Jin Shui Yang1, En Tao Wang3 & Hong Li Yuan1 The lignite biodegradation procedure to produce water-soluble humic materials (WSHM) with a Penicillium stain was established by previous studies in our laboratory. This study researched the effects of WSHM on the growth of Bradyrhizobium liaoningense CCBAU05525 and its nodulation on soybean. Results showed that WSHM enhanced the cell density of CCBAU05525 in culture, and increased the nodule number, nodule fresh weight and nitrogenase activity of the inoculated soybean plants. Then the chemical compounds of WSHM were analyzed and flavonoid analogues were identified in WSHM through tetramethyl ammonium hydroxide (TMAH)-py-GC/MS analysis. Protein expression profiles and nod gene expression of CCBAU05525 in response to WSHM or genistein were compared to illustrate the working mechanism of WSHM. The differently expressed proteins in response to WSHM were involved in nitrogen and carbon metabolism, nucleic acid metabolism, signaling, energy production and some transmembrane transports. WSHM was found more effective than genistein in inducing the nod gene expression. These results demonstrated that WSHM stimulated cell metabolism and nutrient transport, which resulted in increased cell density of CCBAU05525 and prepared the bacteria for better bacteroid development. Furthermore, WSHM had similar but superior functions to flavone in inducing nod gene and nitrogen fixation related proteins expression in CCBAU05525.
Soybean is an important legume that forms symbiotic root nodules with Bradyrhizobium liaoningense, bacteria that reduce atmospheric nitrogen to ammonium, which is a form of nitrogen available for plant uptake. To initiate this symbiosis process, flavonoids secreted by soybean would activate the transcription of nod genes of rhizobia, which encode lipochitooligosaccharide signal molecules, also termed Nod factors. After being recognized by the epidermal cells of the host, these Nod factors induce root hair curling and cell division, and result in root nodule formation. Rhizobia entrapped in the curled root hair could enter the roots through infection threads1. Agricultural systems could acquire approximately 40 million tonnes of nitrogen each year through symbiotic nitrogen fixation between legumes and rhizobia2. Symbiotic nitrogen fixation is a gift from the nature which is beneficial to increasing crop yields and nutrient-use efficiency meanwhile reducing 1
State Key Laboratory of AgroBiotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China. 2College of Life Science, Agricultural University of Hebei, Baoding 071001, P. R. China. 3Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F. 11340, Mexico. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.L.Y. (email: [email protected]) Scientific Reports | 5:10836 | DOI: 10.1038/srep10836
1
www.nature.com/scientificreports/ WSHM concentration (mg L−1) Nodulation Number of nodules Fresh weight of nodules (g) Nitrogenase activity (nmol C2H4 h−1)
0 (control) 36.0± 1.00b 0.436± 0.0610b 22.0± 2.93c
300 43.0± 4.35a 0.547± 0.0330ab 28.6± 1.30a
500 47.0± 3.00a
1000 38.0± 5.00b
0.593± 0.113a
0.554± 0.0950ab
25.3± 0.320b
26.9± 0.810ab
Table 1. Effects of water-soluble humic material (WSHM) treatments on the nodulation of soybean in greenhousea. aValues are given as mean ± standard deviation of triplicate samples for nodule analysis; values with different alphabets in the same line are significantly (P
OPEN
received: 04 December 2014 accepted: 14 April 2015 Published: 08 June 2015
Nodulation Characterization and Proteomic Profiling of Bradyrhizobium liaoningense CCBAU05525 in Response to Water-Soluble Humic Materials Tong Guo Gao1,2,*, Yuan Yuan Xu1,*, Feng Jiang1, Bao Zhen Li1, Jin Shui Yang1, En Tao Wang3 & Hong Li Yuan1 The lignite biodegradation procedure to produce water-soluble humic materials (WSHM) with a Penicillium stain was established by previous studies in our laboratory. This study researched the effects of WSHM on the growth of Bradyrhizobium liaoningense CCBAU05525 and its nodulation on soybean. Results showed that WSHM enhanced the cell density of CCBAU05525 in culture, and increased the nodule number, nodule fresh weight and nitrogenase activity of the inoculated soybean plants. Then the chemical compounds of WSHM were analyzed and flavonoid analogues were identified in WSHM through tetramethyl ammonium hydroxide (TMAH)-py-GC/MS analysis. Protein expression profiles and nod gene expression of CCBAU05525 in response to WSHM or genistein were compared to illustrate the working mechanism of WSHM. The differently expressed proteins in response to WSHM were involved in nitrogen and carbon metabolism, nucleic acid metabolism, signaling, energy production and some transmembrane transports. WSHM was found more effective than genistein in inducing the nod gene expression. These results demonstrated that WSHM stimulated cell metabolism and nutrient transport, which resulted in increased cell density of CCBAU05525 and prepared the bacteria for better bacteroid development. Furthermore, WSHM had similar but superior functions to flavone in inducing nod gene and nitrogen fixation related proteins expression in CCBAU05525.
Soybean is an important legume that forms symbiotic root nodules with Bradyrhizobium liaoningense, bacteria that reduce atmospheric nitrogen to ammonium, which is a form of nitrogen available for plant uptake. To initiate this symbiosis process, flavonoids secreted by soybean would activate the transcription of nod genes of rhizobia, which encode lipochitooligosaccharide signal molecules, also termed Nod factors. After being recognized by the epidermal cells of the host, these Nod factors induce root hair curling and cell division, and result in root nodule formation. Rhizobia entrapped in the curled root hair could enter the roots through infection threads1. Agricultural systems could acquire approximately 40 million tonnes of nitrogen each year through symbiotic nitrogen fixation between legumes and rhizobia2. Symbiotic nitrogen fixation is a gift from the nature which is beneficial to increasing crop yields and nutrient-use efficiency meanwhile reducing 1
State Key Laboratory of AgroBiotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China. 2College of Life Science, Agricultural University of Hebei, Baoding 071001, P. R. China. 3Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F. 11340, Mexico. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.L.Y. (email: [email protected]) Scientific Reports | 5:10836 | DOI: 10.1038/srep10836
1
www.nature.com/scientificreports/ WSHM concentration (mg L−1) Nodulation Number of nodules Fresh weight of nodules (g) Nitrogenase activity (nmol C2H4 h−1)
0 (control) 36.0± 1.00b 0.436± 0.0610b 22.0± 2.93c
300 43.0± 4.35a 0.547± 0.0330ab 28.6± 1.30a
500 47.0± 3.00a
1000 38.0± 5.00b
0.593± 0.113a
0.554± 0.0950ab
25.3± 0.320b
26.9± 0.810ab
Table 1. Effects of water-soluble humic material (WSHM) treatments on the nodulation of soybean in greenhousea. aValues are given as mean ± standard deviation of triplicate samples for nodule analysis; values with different alphabets in the same line are significantly (P
