IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ... IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Crosstalk between GmSAT1 and yeast unveils a novel family of eukaryotic ammonium transport proteins Authors Danielle Mazurkiewicz 1 , Patrick Loughlin 2 , David Chiasson 1 , Mamoru Okamoto 3 , David A. Day 4 , Stephen D. Tyerman 1 & Brent N. Kaiser 1 Poster Board Number 7 1 School of Agriculture, Food and Wine. The University of Adelaide, 2 School of Biological Sciences. The University of Sydney, 3 Australian Centre For Plant Functional Genomics. The University of Adelaide, 4 Flinders University GmSAT1 is a soybean basic helix-loop-helix (bHLH) transcription factor localised to the peribacteroid membrane of nitrogen fixing nodules 1 . Overexpression of GmSAT1 restores the growth of a yeast ammonium (NH4 + ) transport mutant 26972c on low (1 mM) NH4 + concentrations (Kaiser et al., 1998). GmSAT1 overexpression in yeast also increases the uptake of methylammonium (MA), a toxic NH4 + analogue. The objective of this study was to identify and characterise the NH4 + /MA transporter(s) regulated by GmSAT1 in yeast. We have evaluated the transcriptional activity of GmSAT1 in 26972c cells grown for 12 hours in media containing 1mM NH4 + , using microarray (Affymetrix Yeast Genome 2.0 Array) and quantitative RT-PCR. We have characterised one of the genes, ScAMF1, found to be upregulated by GmSAT1. ScAMF1 (Saccharomyces cerevisiae Ammonium Facilitator 1), previously uncharacterised, shows sequence similarity to predicted drug:H + antiporters of the DHA2 family of major facilitator proteins. Overexpression of ScAMF1 in 26972c and a second NH4 + transport mutant yeast strain, 31019b, enhanced 14 C-MA uptake and established a related toxicity phenotype when grown at elevated MA (0.1M) concentrations. The loss of ScAMF1 activity in 26972c:Δamf1 and 31019b:Δamf1 mutants eliminated the ability of GmSAT1 to accumulate 14 C-MA and instate the sensitivity phenotype. The MA associated phenotypes were also lost when the bHLH region of GmSAT1 was mutated. Mutagenesis of the bHLH region of GmSAT1 was shown to abolish the expression of ScAMF1. Stopped-flow spectroscopy demonstrated the ability of ScAMF1 to transport NH4 + and MA into yeast spheroplasts. Parallel experiments with a soybean homolog (GmAMF1) also showed enhanced accumulation of 14 C-MA and transport of NH4 + and MA into spheroplasts. The functional similarity of ScAMF1 homologues identified in Arabidopsis and Medicago truncatula are currently being investigated. Kaiser, B.N., Finnegan, P.M., Tyerman, S.D., Whitehead, L.F., Bergersen, F.J., Day, D.A. and Udvardi, M.K. 1998. Characterisation of an ammonium transport protein from the peribacteroid membrane of soybean nodules. Science 281: 1202-6. 122 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Titles Co-inoculation of rhizobia with highly induced acc deaminase bacteria could alleviate the stress of legumes growing under stress conditions Authors Panlada Tittabutr, Pongdaj Piromyou, Nantakorn Boonkerd & Neung Teaumroong Poster Board Number 8 School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand Since it was previously found that silver thiosulfate (STS) could lower the level of ethylene production in mungbean and resulted in increasing plant growth under both normal and various stress conditions. It is interesting to investigate whether highly 1-amino-cyclopropane-1-carboxylate (ACC) deaminase activity producing bacteria promote the growth of mungbean under various stress conditions. ACC deaminase producing bacteria were isolated from mungbean root growing in the field, and three isolates ACC1, ACC2, and ACC3, which are highly homology to Enterobacter sp. ZJUPD4, Enterobacter sp. M.D.E.NA4-3, and Chryseobacterium sp. KR200, respectively were obtained with high ACC deaminase activity. The experiments of co-inoculation with mungbean bradyrhizobia PRC008 were tested under each condition of high temperature-, water-, salt-stress, and under normal condition. The results showed that ethylene was highly produced by plant growing under all stress conditions. The amount of ethylene production in plant directly influenced mungbean growth and nodulation efficiency under stress conditions. However, co-inoculation of PRC008 with high ACC deaminase activity producing bacteria showed the better plant growth and nodulation than those inoculated with PRC008 alone. Co-inoculation with ACC3 obviously alleviated the stress of legumes growing under water- and high temperature-stress conditions, which coincided with high inducton of ACC deaminase activity in the cell cultured under stress conditions even ACC3 produced lower ACC deaminase activity when compared with ACC1 and ACC2 under normal condition. These results revealed the role and relationship of ethylene and the ACC deaminase producing bacteria on the growth of leguminous plant under stress conditions. This information will be useful for further development of high efficient bacterial inoculant using in agriculture under global warming situation. 123 2011
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17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Crosstalk between GmSAT1 and yeast unveils a novel family of eukaryotic ammonium<br />
transport proteins<br />
Authors Danielle Mazurkiewicz 1 , Patrick Loughlin 2 , David Chiasson 1 , Mamoru Okamoto 3 , David A.<br />
Day 4 , Stephen D. Tyerman 1 & Brent N. Kaiser 1<br />
Poster Board Number 7<br />
1 School of Agriculture, Food and Wine. The University of Adelaide,<br />
2 School of Biological Sciences. The University of Sydney,<br />
3 Australian Centre For Plant Functional Genomics. The University of Adelaide,<br />
4 Flinders University<br />
GmSAT1 is a soybean basic helix-loop-helix (bHLH) transcription factor localised to the peribacteroid membrane<br />
of nitrogen fixing nodules 1 . Overexpression of GmSAT1 restores the growth of a yeast ammonium (NH4 + )<br />
transport mutant 26972c on low (1 mM) NH4 + concentrations (Kaiser et al., 1998). GmSAT1 overexpression in<br />
yeast also increases the uptake of methylammonium (MA), a toxic NH4 + analogue. The objective of this study<br />
was to identify and characterise the NH4 + /MA transporter(s) regulated by GmSAT1 in yeast. We have evaluated<br />
the transcriptional activity of GmSAT1 in 26972c cells grown for 12 hours in media containing 1mM NH4 + , using<br />
microarray (Affymetrix Yeast Genome 2.0 Array) and quantitative RT-PCR. We have characterised one of the<br />
genes, ScAMF1, found to be upregulated by GmSAT1. ScAMF1 (Saccharomyces cerevisiae Ammonium<br />
Facilitator 1), previously uncharacterised, shows sequence similarity to predicted drug:H + antiporters of the DHA2<br />
family of major facilitator proteins. Overexpression of ScAMF1 in 26972c and a second NH4 + transport mutant<br />
yeast strain, 31019b, enhanced 14 C-MA uptake and established a related toxicity phenotype when grown at<br />
elevated MA (0.1M) concentrations. The loss of ScAMF1 activity in 26972c:Δamf1 and 31019b:Δamf1 mutants<br />
eliminated the ability of GmSAT1 to accumulate 14 C-MA and instate the sensitivity phenotype. The MA<br />
associated phenotypes were also lost when the bHLH region of GmSAT1 was mutated. Mutagenesis of the<br />
bHLH region of GmSAT1 was shown to abolish the expression of ScAMF1. Stopped-flow spectroscopy<br />
demonstrated the ability of ScAMF1 to transport NH4 + and MA into yeast spheroplasts. Parallel experiments with<br />
a soybean homolog (GmAMF1) also showed enhanced accumulation of 14 C-MA and transport of NH4 + and MA<br />
into spheroplasts. The functional similarity of ScAMF1 homologues identified in Arabidopsis and Medicago<br />
truncatula are currently being investigated.<br />
Kaiser, B.N., Finnegan, P.M., Tyerman, S.D., Whitehead, L.F., Bergersen, F.J., Day, D.A. and Udvardi, M.K.<br />
1998. Characterisation of an ammonium transport protein from the peribacteroid membrane of soybean nodules.<br />
Science 281: 1202-6.<br />
122<br />
2011
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