Seeds of barley (Hordeum vulgare L. cv. Clarine) were sown in 2 l pots (25 seeds per pot) containing perlite; these were placed in a growth room with 350 µmol m−2 s−1 photon flux density (fluorescent plus incandescent) in a 16-h photoperiod, 22°C day/16°C night temperatures and 70% RH. Plants received water and a nutrient solution (Morcuende et al., 2004). In a second experiment, carried out during winter, the pots were moved into an unheated glasshouse when three leaves had emerged. The glasshouse had maximum/minimum temperatures of 17.8/1.8°C for 10 d, followed by 10.0/−0.4°C for 12 d. Irradiance was >500 µmol m−2 s−1 in the middle of the day. Plants were left in the glasshouse for 24 d, until the fifth leaf was fully expanded.
Treatment of excised leaves
The youngest fully expanded leaf in a shoot was cut with a sharp scalpel and the cut end placed immediately in water for 30 min, and then in 5-cm-high Petri dishes with the cut end dipping in water or the test solutions through slots in the covers, as described (Morcuende et al., 2004). In a first experiment, leaves developed in the growth room were incubated in water or 5 mm K2HPO4/KH2PO4 (1.7 : 1 w/w); in the second experiment cold-treated leaves were incubated in water or 0.5, 2 or 5 mm phosphate. The incubations were carried out for 24 h under continuous light or darkness under the growth room conditions described above. Treatments were arranged at random in four blocks in the first experiment and three in the second, each consisting of four Petri dishes (two leaves per dish) per treatment. At the end of the incubation period leaves were cut above the dish cover, rapidly transferred in situ to liquid N and stored at −80°C until analysed.
Analysis of compounds and enzyme activities
The pool of metabolically accessible phosphate was determined by feeding leaves (two additional leaves per treatment and block) with 200 mm glucose in water or phosphate (at the concentrations used during incubations) for 30 min following the 24-h incubations, and analysing the increases in glucose-6 phosphate (G6P) and fructose-6 phosphate (F6P) (Strand et al., 1999), as described below.
The content of fructans, other carbohydrates and amino acids, and fructosyltransferase activity were determined in subsamples of frozen leaves as described by Morcuende et al. (2004), and hexose-phosphates as described by Pérez et al. (2001). For extraction of total proteins, frozen leaf material (100 mg f. wt) was ground to a fine powder using a pestle and mortar precooled with liquid N and homogenized with 1 ml ice-cold extraction buffer containing 50 mm Tricine buffer (pH 8), 75 mm sucrose, 10 mm NaCl, 5 mm MgCl2, 1 mm ethylenediaminetetraacetic acid (EDTA), 5 mmɛ-aminocaproic acid, 2 mm benzamidine, 0.14% (v/v) β-mercaptoethanol, and 1 mm phenylmethylsulfonylfluoride (PMSF). An aliquot of the homogenate was used to precipitate proteins with one volume of cold acetone containing 0.07% (v/v) β-mercaptoethanol and 20% (w/v) trichloroacetic acid in 100% acetone. After incubation of the mixture at −20°C for 2 h, the extract was centrifuged at 20 000g for 15 min at 4°C to pellet the precipitated proteins, and the supernatant was removed. The pellet was washed three times with ice-cold 100% acetone with 0.07% (v/v) β-mercaptoethanol until it was completely white. The rest of the acetone from the pellet was removed by heating in a drying chamber at 40°C for 30 min. Proteins were solubilized with 1 ml 50 mm Tris–HCl buffer pH 8 containing 100 mm sucrose, 3.5% (w/v) SDS, 1 mm EDTA and 0.07% (v/v) β-mercaptoethanol, by incubation at room temperature, shaking for 20 min, and further incubation at 70°C for another 20 min. After cooling to room temperature, samples were centrifuged at 20 000g for 15 min and the supernatant was decanted. Total protein content in the supernatant was determined spectrophotometrically at 750 nm using the method of Lowry et al. (1951) with slight modifications (Peterson, 1977), using bovine serum albumin as a standard.
For cytosolic FBPase assays, frozen leaf subsamples were ground to a fine powder using a mortar and pestle precooled with liquid N, and extracted in ice-cold 50 mm N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)–KOH (pH 7.5) buffer containing 12 mm MgCl2, 1 mm EDTA, 1 mm ethylene glycol-bis-(β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 1 mm benzamidine, 1 mmɛ-aminocaproic acid, 1 mm dithiothrenitol (DTT), 0.1% Triton X-100, 1 mm PMSF and 1% polyvinylpolypyrrolidone (PVPP). A spectrophotometric method coupled to NADP reduction was used for the FBPase assays (Pérez et al., 2001). SPS was assayed by measuring either the sucrose plus sucrose-6-phosphate or the UDP produced from F6P and UDP-glucose. Replicate analyses were performed with high substrate and effector concentrations to measure both activated and inactivated forms of SPS (Va activity, Trevanion et al., 2004), or with low concentrations of these in the presence of the inhibitor phosphate to measure only the activated form (Vb activity, Trevanion et al., 2004); SPS activation was estimated as the Vb : Va ratio. First, the widely used procedure described for spinach (Huber et al., 1989) was followed. However, very low-Va SPS was obtained in leaves incubated with phosphate. This was overcome using the method of Trevanion et al. (2004), optimized for wheat, in which enzyme activity involves greater changes in affinity for UDP-glucose and reduced sensitivity to inhibition by phosphate than in spinach. However, the desalting step with Sephadex G25 (Amersham Biosciences Europe GmbH, Barcelona, Spain) failed sufficiently to remove the sugars, probably fructans, giving high blanks and variability in the anthrone test. Thus numerous assays were required to obtain reliable results. The alternative assay of UDP was unsuitable because of low recovery of UDP, probably because of high UDP phosphatase activity (Trevanion et al., 2004). An alternative method was used in which leaves were extracted in ice-cold 50 mm Hepes–KOH (pH 7.5) buffer containing 10 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm benzamidine, 5 mmɛ-aminocaproic acid, 5 mm DTT, 10 µm leupeptin, 0.5% BSA, 0.1% Triton X-100, 1 mm PMSF and 2% PVPP. After centrifugation at 17 000g at 5°C for 5 min, the undesalted supernatants were made 40% polyethylene glycol (PEG)-6000, allowed to stand on ice for 40 min, then centrifuged at 17 000g at 5°C for 5 min. The precipitate was then resuspended in the extraction buffer without PVPP and assayed with the concentrations of substrate, effectors and phosphate described by Trevanion et al. (2004). The PEG precipitation step substantially decreased the amount of interfering sugars so that reliable results could be obtained. The SPS assay was validated using tests of linearity of enzyme activity with respect to time and amount of leaf extract.
The anovas for a randomized block design experiment were performed with the genstat 6.2 statistical package. From these analyses the least significant differences (P < 0.05) among treatments were derived, as shown in the figures.