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Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana.

Gangl R, Behmüller R, Tenhaken R - Front Plant Sci (2015)

Bottom Line: Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product.Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds.We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Physiology, Department of Cell Biology, University of Salzburg Salzburg, Austria.

ABSTRACT
Stachyose is among the raffinose family oligosaccharides (RFOs) one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, RFOs are indigestible for humans and can contribute to diverse abdominal disorders. In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the RFO physiology in A. thaliana. In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970) as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 μM) as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress.

No MeSH data available.


Related in: MedlinePlus

Enzyme kinetics of recombinant AtRS4. Enzyme activity of recombinant AtRS4 was measured with varying concentrations of substrates (50 to 1,200 μM) under standard conditions for 60 min with HPAEC-PAD enzyme assays. Values are averages of three independently performed assays (±SD). (A) Substrate saturation by Michaelis–Menten curve for Raf is shown. Km with 259.2 ± 21.15 μM and Vmax 4,722 ± 132.3 pkat mg-1 protein was calculated. (B) Substrate saturation by Michaelis–Menten curve for Gol is shown. Km with 1,170 ± 246.8 μM and Vmax with 8,911 ± 1,105 pkat mg-1 protein was calculated. (C) Substrate saturation by Michaelis–Menten curve for Sta measuring Raf is shown. Km with 1,059 ± 269,3 μM and Vmax 1,610 ± 233,3 pkat mg-1 protein was calculated. (D) Substrate saturation by Michaelis–Menten curve for Sta measuring Gal is shown. Km with 2,832 ± 1,043 μM and Vmax 2,944 ± 820.4 pkat mg-1 protein was calculated. (E) Substrate saturation by Michaelis–Menten curve for Gol measuring Gal is shown. Km with 548.6 ± 152 μM and Vmax 1,653 ± 205.3 pkat mg-1 protein was calculated.
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Figure 8: Enzyme kinetics of recombinant AtRS4. Enzyme activity of recombinant AtRS4 was measured with varying concentrations of substrates (50 to 1,200 μM) under standard conditions for 60 min with HPAEC-PAD enzyme assays. Values are averages of three independently performed assays (±SD). (A) Substrate saturation by Michaelis–Menten curve for Raf is shown. Km with 259.2 ± 21.15 μM and Vmax 4,722 ± 132.3 pkat mg-1 protein was calculated. (B) Substrate saturation by Michaelis–Menten curve for Gol is shown. Km with 1,170 ± 246.8 μM and Vmax with 8,911 ± 1,105 pkat mg-1 protein was calculated. (C) Substrate saturation by Michaelis–Menten curve for Sta measuring Raf is shown. Km with 1,059 ± 269,3 μM and Vmax 1,610 ± 233,3 pkat mg-1 protein was calculated. (D) Substrate saturation by Michaelis–Menten curve for Sta measuring Gal is shown. Km with 2,832 ± 1,043 μM and Vmax 2,944 ± 820.4 pkat mg-1 protein was calculated. (E) Substrate saturation by Michaelis–Menten curve for Gol measuring Gal is shown. Km with 548.6 ± 152 μM and Vmax 1,653 ± 205.3 pkat mg-1 protein was calculated.

Mentions: Kinetic analysis of the StaS enzyme activity of recombinant AtRS4 was performed for the substrates Raf and Gol. The enzyme kinetic of recombinant AtRS4 for Raf (Figure 8A) showed a hyperbolic curve from which a Km value for Raf of 259.2 ± 21.15 μM and a Vmax value of 4,722 ± 132.3 pkat mg-1 protein was calculated using curve regression analysis with SigmaPlot 13.0 software. Substrate saturation curves of recombinant AtRS4 for Gol (Figure 8B) followed a hyperbolic curve according to Michaelis–Menten kinetic. The Km value of recombinant AtRS4 for Gol in a substrate range from 50 to 1,200 μM was calculated as 1,170 ± 246.8 μM and a Vmax value of 8,911 ± 1,105 pkat mg-1 protein.


Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana.

Gangl R, Behmüller R, Tenhaken R - Front Plant Sci (2015)

Enzyme kinetics of recombinant AtRS4. Enzyme activity of recombinant AtRS4 was measured with varying concentrations of substrates (50 to 1,200 μM) under standard conditions for 60 min with HPAEC-PAD enzyme assays. Values are averages of three independently performed assays (±SD). (A) Substrate saturation by Michaelis–Menten curve for Raf is shown. Km with 259.2 ± 21.15 μM and Vmax 4,722 ± 132.3 pkat mg-1 protein was calculated. (B) Substrate saturation by Michaelis–Menten curve for Gol is shown. Km with 1,170 ± 246.8 μM and Vmax with 8,911 ± 1,105 pkat mg-1 protein was calculated. (C) Substrate saturation by Michaelis–Menten curve for Sta measuring Raf is shown. Km with 1,059 ± 269,3 μM and Vmax 1,610 ± 233,3 pkat mg-1 protein was calculated. (D) Substrate saturation by Michaelis–Menten curve for Sta measuring Gal is shown. Km with 2,832 ± 1,043 μM and Vmax 2,944 ± 820.4 pkat mg-1 protein was calculated. (E) Substrate saturation by Michaelis–Menten curve for Gol measuring Gal is shown. Km with 548.6 ± 152 μM and Vmax 1,653 ± 205.3 pkat mg-1 protein was calculated.
© Copyright Policy
Related In: Results  -  Collection

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Figure 8: Enzyme kinetics of recombinant AtRS4. Enzyme activity of recombinant AtRS4 was measured with varying concentrations of substrates (50 to 1,200 μM) under standard conditions for 60 min with HPAEC-PAD enzyme assays. Values are averages of three independently performed assays (±SD). (A) Substrate saturation by Michaelis–Menten curve for Raf is shown. Km with 259.2 ± 21.15 μM and Vmax 4,722 ± 132.3 pkat mg-1 protein was calculated. (B) Substrate saturation by Michaelis–Menten curve for Gol is shown. Km with 1,170 ± 246.8 μM and Vmax with 8,911 ± 1,105 pkat mg-1 protein was calculated. (C) Substrate saturation by Michaelis–Menten curve for Sta measuring Raf is shown. Km with 1,059 ± 269,3 μM and Vmax 1,610 ± 233,3 pkat mg-1 protein was calculated. (D) Substrate saturation by Michaelis–Menten curve for Sta measuring Gal is shown. Km with 2,832 ± 1,043 μM and Vmax 2,944 ± 820.4 pkat mg-1 protein was calculated. (E) Substrate saturation by Michaelis–Menten curve for Gol measuring Gal is shown. Km with 548.6 ± 152 μM and Vmax 1,653 ± 205.3 pkat mg-1 protein was calculated.
Mentions: Kinetic analysis of the StaS enzyme activity of recombinant AtRS4 was performed for the substrates Raf and Gol. The enzyme kinetic of recombinant AtRS4 for Raf (Figure 8A) showed a hyperbolic curve from which a Km value for Raf of 259.2 ± 21.15 μM and a Vmax value of 4,722 ± 132.3 pkat mg-1 protein was calculated using curve regression analysis with SigmaPlot 13.0 software. Substrate saturation curves of recombinant AtRS4 for Gol (Figure 8B) followed a hyperbolic curve according to Michaelis–Menten kinetic. The Km value of recombinant AtRS4 for Gol in a substrate range from 50 to 1,200 μM was calculated as 1,170 ± 246.8 μM and a Vmax value of 8,911 ± 1,105 pkat mg-1 protein.

Bottom Line: Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product.Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds.We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Physiology, Department of Cell Biology, University of Salzburg Salzburg, Austria.

ABSTRACT
Stachyose is among the raffinose family oligosaccharides (RFOs) one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, RFOs are indigestible for humans and can contribute to diverse abdominal disorders. In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the RFO physiology in A. thaliana. In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970) as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 μM) as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress.

No MeSH data available.


Related in: MedlinePlus