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White (end of night). Soluble glucan: dark grey (finish of day) and light grey (end of evening). Values are means of measurements on at the least 5 rosettes. Error bars, ?SE.Fig. 3 Effects of your ss4 mutation within a sex1 background. (a) Presence of starch in rosettes of sex1, and two independently chosen ss4sex1 double mutant Title Loaded From File Arabidopsis lines. Decolourised rosettes were stained with iodine solution. (b) Transmission electron micrographs of (left) a cell inside a mature sex1 leaf and (correct) element of a cell inside a mature ss4sex1 leaf. The arrows indicate starch granules. Note that sex1 starch granules are flattened, whereas ss4sex1 granules are rounded and of really variable sizes. Bars, four lm.Some profiles of chloroplasts in sections of mature ss4sex1 leaves also had no starch granules and others contained few, rounded granules of variable size, whereas sex1 chloroplasts in mature leaves had been packed with flattened granules (Fig. 3). Hence, introduction of a mutation that drastically reduces starch degradation in a wild-type background had only minor effects on starch granule formation in ss4 leaves. Leaves of ss4 mutants accumulate incredibly high concentrations of ADPglucose The experiments described above show that neither starch granules nor soluble glucans accumulate in immature ss4 leaves. This observation suggests that the actions of starch synthase isoforms aside from SS4 might be dependent on SS4. To test this concept, we measured the effect of your loss of SS4 on concentrations with the starch synthase substrate ADPglucose. Concentrations were 50?00 times higher in ss4-1 and ss4-3 rosettes than in wild-type rosettes, and were elevated in both mature and immature leaves (Tables 1, S3). This was a highly precise effect. Evaluation of mutants lacking any of the other 3 isoforms of soluble starchNew Phytologist (2013) 200: 1064?075 http://www.newphytologist.comthis possibility, we introduced into the ss4 background a mutation that blocks starch degradation in wild-type plants. The sex1 mutation affects glucan, water dikinase (GWD), a starch-phosphorylating enzyme that renders the surface from the starch granule accessible to starch degrading enzymes at night (Stitt Zeeman, 2012). Mutants lacking GWD possess a strongly reduced price of starch degradation at night, and accumulate really high concentrations of starch in all leaves (Zeeman ap Rees, 1999; Yu et al., 2001). The ss4sex1 double mutants grew far more slowly than ss4 mutants, at about the identical price as sex1 mutants (Fig. S3). Mature leaves had high starch contents at the end of each the day and also the evening. Having said that, immature leaves contained extremely tiny starch and most chloroplasts appeared to contain no granules (Figs three, S3).?2013 The Authors New Phytologist ?2013 New Phytologist Trust1068 ResearchTable 1 Metabolite contents of Arabidopsis rosettes lacking starch synthase isoforms Amount (nmol g? FW) Metabolite ADPglucose UDPglucose Trehalose 6-P Glucose 6-P Glucose 1-P Fructose 6-P Fructose 1,6-bisphosphate Glycerol 3-P 3-Phos.