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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

Sequence and phylogenetic analysis of AtRS4 and AtRS5. (A) Schematically shows the 783 amino acid long sequence of AtRS5 (A5g40390) from Arabidopsis thaliana. (B) Shows a section of a sequence alignment performed with Clustal Omega (Sievers et al., 2011) of RafS and StaS amino acid sequences, which revealed very high amino acid identity and similarity, except for a 80 amino acid long sequence block insertion, which is only present in the StaS. (C) Schematically shows the 876 amino acid long sequence of AtRS4 (At4g01970) from A. thaliana. RafS amino acid sequence from A. thaliana (AtRafS, gi/332195171), Oryza sativa subsp japonica (OsRafS, gi/115471135), Cucumis sativus (CsRafS, gi/124057819), Pisum sativum (PsRafS, gi/18181865) and Glycine max (GmRafS, gi/187610414). StaS amino acid sequence from A. thaliana (AtStaS, gi/332656706), Vigna angularis (VaStaS, gi/6634701), P. sativum (PsStaS, gi/13992585), Cucumis melo (CmStaS, gi/659101177) and Alonsoa meridionalis (AmStaS, gi/21038869).
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Figure 2: Sequence and phylogenetic analysis of AtRS4 and AtRS5. (A) Schematically shows the 783 amino acid long sequence of AtRS5 (A5g40390) from Arabidopsis thaliana. (B) Shows a section of a sequence alignment performed with Clustal Omega (Sievers et al., 2011) of RafS and StaS amino acid sequences, which revealed very high amino acid identity and similarity, except for a 80 amino acid long sequence block insertion, which is only present in the StaS. (C) Schematically shows the 876 amino acid long sequence of AtRS4 (At4g01970) from A. thaliana. RafS amino acid sequence from A. thaliana (AtRafS, gi/332195171), Oryza sativa subsp japonica (OsRafS, gi/115471135), Cucumis sativus (CsRafS, gi/124057819), Pisum sativum (PsRafS, gi/18181865) and Glycine max (GmRafS, gi/187610414). StaS amino acid sequence from A. thaliana (AtStaS, gi/332656706), Vigna angularis (VaStaS, gi/6634701), P. sativum (PsStaS, gi/13992585), Cucumis melo (CmStaS, gi/659101177) and Alonsoa meridionalis (AmStaS, gi/21038869).

Mentions: A database search using AtRS4 sequence from A. thaliana clearly revealed homologous sequences in Vigna angularis, Cucumis melo, Pisum sativum and Alonsoa meridionalis, which are all coding for StaSs, as well as homologous sequences in P. sativum, C. sativus, Glycine max, and Oryza sativa subsp japonica, which are all coding for RafSs (Figure 2B). Both AtRS4 (Figure 2C) as well as AtRS5 (Figure 2A) amino acid sequence contain a typical motif of AmyAc_family superfamily (α-amylase catalytic domain) for α-amylase family, the largest family of glycosidehydrolases. These enzymes catalyze the transformation of α-1,4- and α-1,6-glucosidic linkages. Furthermore, the AtRS5 (Figure 2A) amino acid sequence displays a typical motif of Raffinose_syn (RafS), whereas AtRS4 only displays a 400 amino acid long C-terminal typical motif sequence (Figure 2C), which represents several RafSs (InterPro: IPR017853), also known as seed imbibition proteins (SIP), from the glycoside hydrolase family 36°C (InterPro: IPR008811).


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

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

Sequence and phylogenetic analysis of AtRS4 and AtRS5. (A) Schematically shows the 783 amino acid long sequence of AtRS5 (A5g40390) from Arabidopsis thaliana. (B) Shows a section of a sequence alignment performed with Clustal Omega (Sievers et al., 2011) of RafS and StaS amino acid sequences, which revealed very high amino acid identity and similarity, except for a 80 amino acid long sequence block insertion, which is only present in the StaS. (C) Schematically shows the 876 amino acid long sequence of AtRS4 (At4g01970) from A. thaliana. RafS amino acid sequence from A. thaliana (AtRafS, gi/332195171), Oryza sativa subsp japonica (OsRafS, gi/115471135), Cucumis sativus (CsRafS, gi/124057819), Pisum sativum (PsRafS, gi/18181865) and Glycine max (GmRafS, gi/187610414). StaS amino acid sequence from A. thaliana (AtStaS, gi/332656706), Vigna angularis (VaStaS, gi/6634701), P. sativum (PsStaS, gi/13992585), Cucumis melo (CmStaS, gi/659101177) and Alonsoa meridionalis (AmStaS, gi/21038869).
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Sequence and phylogenetic analysis of AtRS4 and AtRS5. (A) Schematically shows the 783 amino acid long sequence of AtRS5 (A5g40390) from Arabidopsis thaliana. (B) Shows a section of a sequence alignment performed with Clustal Omega (Sievers et al., 2011) of RafS and StaS amino acid sequences, which revealed very high amino acid identity and similarity, except for a 80 amino acid long sequence block insertion, which is only present in the StaS. (C) Schematically shows the 876 amino acid long sequence of AtRS4 (At4g01970) from A. thaliana. RafS amino acid sequence from A. thaliana (AtRafS, gi/332195171), Oryza sativa subsp japonica (OsRafS, gi/115471135), Cucumis sativus (CsRafS, gi/124057819), Pisum sativum (PsRafS, gi/18181865) and Glycine max (GmRafS, gi/187610414). StaS amino acid sequence from A. thaliana (AtStaS, gi/332656706), Vigna angularis (VaStaS, gi/6634701), P. sativum (PsStaS, gi/13992585), Cucumis melo (CmStaS, gi/659101177) and Alonsoa meridionalis (AmStaS, gi/21038869).
Mentions: A database search using AtRS4 sequence from A. thaliana clearly revealed homologous sequences in Vigna angularis, Cucumis melo, Pisum sativum and Alonsoa meridionalis, which are all coding for StaSs, as well as homologous sequences in P. sativum, C. sativus, Glycine max, and Oryza sativa subsp japonica, which are all coding for RafSs (Figure 2B). Both AtRS4 (Figure 2C) as well as AtRS5 (Figure 2A) amino acid sequence contain a typical motif of AmyAc_family superfamily (α-amylase catalytic domain) for α-amylase family, the largest family of glycosidehydrolases. These enzymes catalyze the transformation of α-1,4- and α-1,6-glucosidic linkages. Furthermore, the AtRS5 (Figure 2A) amino acid sequence displays a typical motif of Raffinose_syn (RafS), whereas AtRS4 only displays a 400 amino acid long C-terminal typical motif sequence (Figure 2C), which represents several RafSs (InterPro: IPR017853), also known as seed imbibition proteins (SIP), from the glycoside hydrolase family 36°C (InterPro: IPR008811).

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