Dataset Information

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390).

ABSTRACT:

Background

The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves.

Results

By heterologously expressing At5g40390 in E.coli, we demonstrate that crude extracts synthesise Raf in vitro, contrary to empty vector controls. Using two independent loss-of-function mutants for At5g40390 (rs 5-1 and 5-2), we confirm that this RS is indeed responsible for Raf accumulation during low temperature-acclimation (4°C), as previously reported. Surprisingly, leaves of mutant plants also fail to accumulate any Raf under diverse abiotic stresses including water-deficit, high salinity, heat shock, and methyl viologen-induced oxidative stress. Correlated to the lack of Raf under these abiotic stress conditions, both mutant plants lack the typical stress-induced RafS activity increase observed in the leaves of wild-type plants.

Conclusions

Collectively our findings point to a single abiotic stress-induced RS isoform (RS5, At5g40390) being responsible for Raf biosynthesis in Arabidopsis leaves. However, they do not support a single RS hypothesis since the seeds of both mutant plants still contained Raf, albeit at 0.5-fold lower concentration than seeds from wild-type plants, suggesting the existence of at least one other seed-specific RS. These results also unambiguously discount the existence of six stress-inducible RS isoforms suggested by recent reports.

SUBMITTER: Egert A

PROVIDER: S-EPMC3878221 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Publications

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390).

Egert Aurélie A Keller Felix F Peters Shaun S

BMC plant biology 20131220

<h4>Background</h4>The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Ind ...[more]

PMID: 24354450

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Project description:Soybean [Glycine max (L.) Merr.] is an important world economic crop. It is rich in oil, protein, and starch, and soluble carbohydrates in soybean seeds are also important for human and livestock consumption. The predominant soluble carbohydrate in soybean seed is composed of sucrose and raffinose family oligosaccharides (RFOs). Among these carbohydrates, only sucrose can be digested by humans and monogastric animals and is beneficial for metabolizable energy, while RFOs are anti-nutritional factors in diets, usually leading to flatulence and indigestion, ultimately reducing energy efficiency. Hence, breeding efforts to remove RFOs from soybean seeds can increase metabolizable energy and improve nutritional quality. The objective of this research is to use the multiplex Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing system to induce the knockout of soybean raffinose synthase (RS) genes RS2 and RS3 simultaneously to reduce RFOs in mature seeds. First, we constructed five types of multiplex gene editing systems and compared their editing efficiency in soybean hairy roots. We confirmed that the two-component transcriptional unit (TCTU) and single transcriptional unit (STU) systems with transfer RNA (tRNA) as the cleavage site performed better than other systems. The average editing efficiency at the four targets with TCTU-tRNA and STU-tRNA was 50.5% and 46.7%, respectively. Then, we designed four single-guide RNA (sgRNA) targets to induce mutations at RS2 and RS3 by using the TCTU-tRNA system. After the soybean transformation, we obtained several RS2 and RS3 mutation plants, and a subset of alleles was successfully transferred to the progeny. We identified null single and double mutants at the T2 generation and analyzed the seed carbohydrate content of their progeny. The RS2 and RS3 double mutants and the RS2 single mutant exhibited dramatically reduced levels of raffinose and stachyose in mature seeds. Further analysis of the growth and development of these mutants showed that there were no penalties on these phenotypes. Our results indicate that knocking out RS genes by multiplex CRISPR/Cas9-mediated gene editing is an efficient way to reduce RFOs in soybean. This research demonstrates the potential of using elite soybean cultivars to improve the soybean meal trait by multiplex CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated gene editing.

Dataset Information

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390).

Background

Results

Conclusions

Publications

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390).

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