Plant material and abiotic stress treatments
The RS5 mutants (rs 5–1 and 5–2) in the Col-0 background were obtained from the SALK collection (SALK_049583 and _085989, respectively). The mutants carry a T-DNA insert in the second intron of At5g40390. Homozygous mutants were identified by PCR using two different primer pairs: the At5g40390 wild-type allele was amplified with the primers rs 5–1 (fwd 5′ CTCTTCTTGAAGGCTCCTTCC, rev 5′ ATGACATCAACTTTAACGCCG) and rs 5–2 (fwd 5′ATGGAACTCAGCACAAGGATG, rev 5′TTATTGAAATCCTCACACC). The mutant allele was amplified with the SALK Lb1.3 primer (5′TTTTGCCGATTTCGGAAC) and either the rs 5–1 or 5–2 reverse primer, respectively. Wild-type plants used in this study represented a pool (3) of individual plants which genotyped as wild-type in the screen described above.
Following seed stratification (48 h, 4°C), Arabidopsis plants were propagated in soil (Einheitserde, type ED73, Gebr. Patzer GmbH & Co. KG, Schopfheim, Germany) in a controlled environment chamber (8 h light, 30 μmol photons m-2 s-1, 22°C, 16 h dark, 60% RH). Five-week-old plants were used for abiotic stress treatments as follows. Experiments were conducted twice, using 4 pools of 6 plants (24 plants) per experiment.
(i) cold stress: plants were transferred into an acclimation chamber with identical settings to those listed above but with a constant temperature of 4°C, for a period of 14 d; (ii) high salinity was imposed by irrigating soil with 150 ml of NaCl solution (25 mM, after 24 h 50 mM and 24 h thereafter 100 mM); (iii) water deficit: soil was passed through a sieve (0.5 x 0.5 cm mesh size) to remove large detritus and 60 g was weighed into pots prior to seed sowing. The pots were sub-irrigated twice weekly until plants reached the desired growth stage (5-week-old). Water deficit was subsequently imposed by withholding water until leaves showed the first visible signs of leaf wilting, typically occurring between 7 and 9 d under our growth conditions, (iv) oxidative stress was imposed as previously described , with minor modifications. Leaves were liberally sprayed with 25 μM MV (paraquat) followed by a 3 h exposure to high light-intensity (700 μmol photons m-2 s-1); (v) heat shock was imposed by transferring plants to 30°C for 6 h.
RNA isolation and semiquantitative PCR (sqPCR)
Total RNA was extracted from leaves using the RNeasy mini kit (Qiagen, Hombrechtikon, Switzerland), following the manufacturer’s instructions. The cDNA template for sqPCR were obtained by reverse transcription of 1 μg total RNA with an oligo (dT15) primer and the M-MLV reverse transcriptase (Promega AG, Dübendorf, Switzerland) following the manufacturer’s instructions. The sqPCR was carried out in 50 μl containing 5 μl cDNA, 0.5 mM of each dNTP, and 0.5 μmol of each primer, 1X PCR buffer and 1.25 U GoTaq DNA polymerase (Promega), at a primer annealing temperature of 56°C for 24 cycles. The number of cycles chosen for the sqPCR was determined to occur in the linear range of the constitutively expressed ACTIN2 gene (ACT2, At3g18780). The following primer pairs were used to amplify a 1Kb fragment of the corresponding cDNAs ACTIN2: ACT2
5′TTAGAAACATTTTCTGTGAACGAT; RS5: RS5
5′ ATGGCTTCGCCGTGTTTGACC and RS5
Heterologous expression of At5g40390 in E. coli
Total RNA was isolated from the leaves of cold-stressed (4°C, 14 d) Col-0 Arabidopsis plants using the Plant RNeasy kit (Qiagen AG, Hombrechtikon, Switzerland). The cDNA template for high fidelity PCR of the At5g40390 open reading frame (ORF) was obtained by reverse transcribing 1 μg total RNA with an oligo (dT15) primer and M-MLV (H–) reverse transcriptase (Promega AG, Dübendorf, Switzerland) following the manufacturer’s protocol. The high fidelity PCR was conducted with 2 μl first strand cDNA using the Expand High Fidelity PCR kit (Roche) following the manufacturer’s instructions. The primer pair amplified the entire 2.48 kB ORF of At5g40390 (RS5
5′ ATGGCTTCGCCGTGTTTGACC and RS5
5′ CTAAAACAAATACTGAATAGAAGACAAACC). The resultant amplicon was cloned into the pGEMT-Easy vector (Promega) and subcloned into the pPROEx HTc vector (Invitrogen) using the NotI restriction endonuclease. This construct (RS5::pPROExHTc) was transformed via standard heat shock procedure, into E. coli (BL21 Codon Plus, Stratagene).
Induction of recombinant RS5 expression and crude extract preparations were conducted as previously described . Aliquots (25 μl) of crude extracts were assayed for RS activity in a 50 μl final volume containing 25 μl assay buffer (50 mM HEPES-KOH, pH 7.5, 100 mM Suc, 10 mM Gol). Assays were incubated for 1 h at 30°C and subsequently desalted and analysed by HPLC-PAD as previously described [4, 13, 22, 23]. Control reactions represented crude extracts from cell cultures transformed with the empty pPROExHTc vector and processed as outlined above.
To confirm that heterologous RS5 actually produced Raf, fractions of enzyme assay reactions were collected after HPLC separation and digested with a fungal (Aspergillus niger) acid α-Gal, as previously described . Further, to confirm that RS5 did not display an alkaline α-Gal activity, crude extracts were incubated in the presence of 50 mM Raf .
Enzyme extractions, GolS and RS activity assays
Freshly harvested Arabidopsis leaf material (200 mg) was ground in 400 μl of chilled extraction buffer [50 mM HEPES/KOH, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 20 mM dithiothreitol (DTT), 0.1% (v/v) Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride (PMSF), 50 mM Na-ascorbate, 2% (w/v) polyvinylpyrrolidone (PVP)]. Samples were centrifuged at 12,000 x g (5 min, 4°C). A 200 μl aliquot of supernatant was desalted by gel filtration at 1,400 x g (2 min, 4°C) through 5 ml Sephadex G-25 columns (fine, final bed volume of 3 ml). Columns were pre-equilibrated with assay buffer (50 mM HEPES/KOH, pH 7.5, 2 mM MnCl2, 10 mM DTT). Pre-equilibration was performed twice with 2 ml of assay buffer. Aliquots (20 μl) of desalted extract were assayed for GolS activity in a final volume of 40 μl containing 20 μl assay buffer (50 mM HEPES-KOH, pH 7.5, 100 mM Ino, 10 mM UDP-Gal) at 30°C for 20 min. Similarly, RS activity was assayed at 30°C for 60 min with assay buffer (50 mM HEPES-KOH, pH 7.5, 100 mM Suc, 10 mM Gol). To determine the degree of Raf contamination of Suc (substrate impurity), assay buffer was incubated with 20 μl dH20 and processed as described above. Samples were desalted and analysed by HPLC-PAD as previously described [4, 22, 23]. Enzyme activities were expressed as a measure of dry weight.
WSCs were extracted using an ethanol series, as previously described [4, 13, 22, 23], with minor modifications. Ground, freeze-dried Arabidopsis leaf material (100 mg) from 5-week-old soil-grown plants was flash-frozen in liquid N2 and macerated, by hand, using plastic pestle in a 1.5 ml Eppendorf tube. WSCs were extracted twice (per step) in a three-step sequential process, using 1 ml of 80% (v/v) EtOH, 50% (v/v) EtOH, and dH2O, respectively. Extractions were conducted at 80°C for 10 min and the tubes centrifuged at 15,000 x g (5 min, 4°C). Samples were desalted and analysed by HPLC-PAD as previously described [4, 13, 22, 23].
Desalted WSC extracts and enzyme assay reactions were analysed and quantified by HPLC-PAD as previously described [4, 13, 22, 23]. Briefly, a Ca2+/Na+-moderated ion partitioning carbohydrate column (Benson BC100, BC200 columns, 7.8 × 300 mm; Benson Polymeric, Reno, Nevada, USA) was used to separate carbohydrates. Quantification was done using the Chromeleon v6.4 software package (Dionex) against a series of 5 nmol of standard sugars, the concentration of which corresponded to the linear response range of both chromatographic systems.