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.

Figure 1

RS activity in crude extracts from E. coli transformed with RS5:pPROExHTc (lower chromatogram). Crude extracts were incubated with 100 mM Suc and 10 mM Gol at pH 7.5 for 1 h. The empty vector control, pPROEx HTc, did not show any RS activity (upper chromatogram). Raf, raffinose (7.2 min); Suc, sucrose (8.8 min); Gol, galactinol (11.2 min); Ino, myo-inositol (13.9 min). The Raf biosynthetic reaction yields Ino consequent to the transfer of a galactose moiety from Gol to Suc.

Figure 2

HPLC-PAD chromatograms confirming the identity of Raf (Raf^R) produced by recombinant RS5. A, Fractions representing 40 nmol of a commercial Raf standard (Raf^St) were collected after separation by HPLC, hydrolysed with a fungal (Aspergillus niger) acid α-Gal and rechromatographed. B, Fractions representing Raf^R were collected from RS enzyme activity assays after separation by HPLC, hydrolysed as above and rechromatographed. Raf, raffinose (8.2 min); Suc, sucrose (9.2 min); Gal, galactose (12.4 min).

Figure 3

Water-soluble carbohydrates in seeds of Arabidopsis wild-type (Col-0) and rs 5–1 and 5–2 mutant plants. Gol, galactinol; Suc, sucrose; Raf, raffinose; Sta, stachyose. Values are the means ± SE of three independent replicates. Statistical significance of a two-tailed t-test is represented by stars (Gol, **p < 0.002, ***and p < 0.0002; Raf, **p < 0.003 and 0.002 for rs 5–1 and 5–2, respectively).

Figure 4

Analyses of 4°C cold stressed-leaves from wild-type (Col-0) and RS5 mutants. A, Semi-quantitative PCR of RS5 transcript abundance in the leaves of Col-0, rs 5–1 and 5–2 mutant plants stressed for 24 h. The ACTIN2 gene (ACT2, At3g18780) was used as a constitutively expressed control. B, GolS activity in the leaves of Col-0, rs 5–1 and 5–2 plants stressed for 14 d. C, RS activity in the leaves of Col-0, rs 5–1 and 5–2 plants stressed for 14 d.