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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

Kinetic of WT and ΔAtRS4 seed germination. WT and ΔAtRS4 mutant seeds for germination experiment were sowed on 0.5% plant agar and grew under control growth conditions. Germination was defined as the time between sowing and protrusion of the radicle. Values are averages of three independently performed experiments (±SD).
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Figure 11: Kinetic of WT and ΔAtRS4 seed germination. WT and ΔAtRS4 mutant seeds for germination experiment were sowed on 0.5% plant agar and grew under control growth conditions. Germination was defined as the time between sowing and protrusion of the radicle. Values are averages of three independently performed experiments (±SD).

Mentions: To test whether ΔAtRS4 mutant seeds show any differences in the kinetic of imbibition and subsequent germination, we put WT and ΔAtRS4 seeds either on 0.5 × MS agar plates or on 0.5% plant agar plates to let them germinate under standard growth conditions and observed the time period the seeds need to germinate. On 0.5 × MS agar plates, almost all ΔAtRS4 mutant seeds started to germinate after 2 days, while WT seeds did not seem to germinate as fast as ΔAtRS4 mutant seeds, whereas after 3 days, no difference could be observed (Supplementary Figure S6). On 0.5 × MS agar plates supplemented with 100 mM and 150 mM NaCl kinetic of germination showed no differences, while after 20 days on plates supplemented with 200 mM NaCl 66% of WT seeds and in contrast 90% of ΔAtRS4 seeds germinated and died subsequently (Supplementary Figure S7). To avoid dormancy breaking signals like nitrate in plate media, we performed a seed germination experiment on 0.5% plant agar (Supplementary Figure S8) and observed germination of 75% of ΔAtRS4 seeds compared to 16% of WT seeds 2 days after sowing, while 82% of WT seeds germinated 3 days after sowing (Figure 11). To test whether segregating seeds from a heterozygous ΔAtRS4 mutant plant show a genotype-phenotype correlation on the kinetic of germination, we put those seeds on 0.5% plant agar plates and separated each day 5 just germinated seeds for genotyping. We did not find a correlation between the kinetic of germination and ΔAtRS4 genotype.


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

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

Kinetic of WT and ΔAtRS4 seed germination. WT and ΔAtRS4 mutant seeds for germination experiment were sowed on 0.5% plant agar and grew under control growth conditions. Germination was defined as the time between sowing and protrusion of the radicle. Values are averages of three independently performed experiments (±SD).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587089&req=5

Figure 11: Kinetic of WT and ΔAtRS4 seed germination. WT and ΔAtRS4 mutant seeds for germination experiment were sowed on 0.5% plant agar and grew under control growth conditions. Germination was defined as the time between sowing and protrusion of the radicle. Values are averages of three independently performed experiments (±SD).
Mentions: To test whether ΔAtRS4 mutant seeds show any differences in the kinetic of imbibition and subsequent germination, we put WT and ΔAtRS4 seeds either on 0.5 × MS agar plates or on 0.5% plant agar plates to let them germinate under standard growth conditions and observed the time period the seeds need to germinate. On 0.5 × MS agar plates, almost all ΔAtRS4 mutant seeds started to germinate after 2 days, while WT seeds did not seem to germinate as fast as ΔAtRS4 mutant seeds, whereas after 3 days, no difference could be observed (Supplementary Figure S6). On 0.5 × MS agar plates supplemented with 100 mM and 150 mM NaCl kinetic of germination showed no differences, while after 20 days on plates supplemented with 200 mM NaCl 66% of WT seeds and in contrast 90% of ΔAtRS4 seeds germinated and died subsequently (Supplementary Figure S7). To avoid dormancy breaking signals like nitrate in plate media, we performed a seed germination experiment on 0.5% plant agar (Supplementary Figure S8) and observed germination of 75% of ΔAtRS4 seeds compared to 16% of WT seeds 2 days after sowing, while 82% of WT seeds germinated 3 days after sowing (Figure 11). To test whether segregating seeds from a heterozygous ΔAtRS4 mutant plant show a genotype-phenotype correlation on the kinetic of germination, we put those seeds on 0.5% plant agar plates and separated each day 5 just germinated seeds for genotyping. We did not find a correlation between the kinetic of germination and ΔAtRS4 genotype.

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