Activity

Gen and starch synthase activities, leaves have been extracted with 100 mM MOPS, pH 7.two, 1 mM EDTA, 1 mM DTT, ten (v/v) glycerol (300 mg leaf ml?). Extracts (32-ll) had been loaded onto nondenaturing polyacrylamide gels containing 0.3 (w/v) glycogen. Just after incubation for 16 h at 20 in 100 mM HEPES-NaOH, pH 7.5, two mM DTT, 10 (v/v) glycerol, 0.five mM EDTA, 0.five M Na-citrate, two mM ADPglucose, starch and glycogen synthase activities were revealed by iodine staining. Immunoblotting An antiserum was raised commercially in rabbits employing the synthetic peptide DIGHDDGKNLDNIT (present in SS4 but not other starch synthase isoforms). Antibodies have been affinity-purified working with this peptide. Leaf tissue was powdered in liquid nitrogen then extracted in Laemmli sample buffer containing SDS and 1 (v/v) protease inhibitor cocktail (Sigma, http://www.sigmaaldrich. com). Electrophoresis and immunoblotting were as described by Barratt et al. (2001).(c)(d)Fig. 1 Light micrographs of sections of Arabidopsis leaves stained with toluidine blue. (a) Mature leaf, wild-type plant. (b) Mature leaf, ss4 plant. (c) Immature leaf, wild-type plant. (d) Immature leaf, ss4 plant (see also Fig. 4a). (a ) Arrows indicate starch granules. Bars, ten lm.ResultsImmature leaves of ss4 plants contain pretty much no starch We extended the characterisation of ss4 mutants, utilizing the ss4-1 mutant allele (Roldn et al., 2007) along with a further T-DNA insera tion line, ss4-3, which lacks SS4 protein (Fig. S1a). Examination of whole rosettes and mature leaves largely confirmed earlier reports of your ss4 phenotype (Roldn et al., a 2007). Compared with wild-type plants, ss4 plants had slightly decreased soluble starch synthase activities, 40 less chlorophyll (Table S2), less degradation of starch for the duration of the evening (measured as end-of-day minus end-of-night starch contents) and higher end-of-night starch contents (Fig. S1b), and slower growth prices under both lengthy and short photoperiods (Fig. S1c). As reported previously (Roldn et al., 2007), mature ss4 leaves a appeared to possess only one large, rounded starch granule per chloroplast (Figs 1a,b, S1d,e). Quantification of granule numbers (using Technique two from Crumpton-Taylor et al., 2012) revealed that mature, nonflowering rosettes of wild-type and ss4-3 plants had five.54 ?0.28 and 0.87 ?0.14 granules per chloroplast, respectively (imply ?SE from eight chloroplast preparations in both circumstances). We identified a different situation in immature leaves. Whereas immature leaves of wild-type plants have more starch granules per chloroplast than mature leaves (Crumpton-Taylor et al., 2012), no starch granules have been visible by light or Orexin A custom synthesis electron microscopy in immature leaves of ss4 plants (Fig. 1c,d). Consistent withNew Phytologist (2013) 200: 1064?075 http://www.newphytologist.comthis observation, the youngest leaves of ss4 rosettes didn’t stain with iodine in the end of your day (Fig. 2a), and quantitative measurements revealed very low starch contents and small diel starch turnover (Fig. 2c,d). Starch c.