Downregulation of a chloroplast‐targeted β‐amylase leads to a starch‐excess phenotype in leaves

Abstract

SummaryA functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation. One clone isolated had a sequence very similar to a recently described chloroplast-targeted b-amylase of Arabidopsis. Expression of the gene in E. coli showed that the protein product was a functional b-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the b-amylase was imported and processed into pea chloroplasts. To study the function of the protein in transitory starch degradation, transgenic potato plants were generated where its activity was reduced using antisense techniques. Analysis of plants reduced in the presence of this b-amylase isoform showed that their leaves had a starch-excess phenotype, indicating a defect in starch degradation. In addition, it was shown that the antisense plants degraded only 8±30% of their total starch, in comparison with 50% in the wild type, over the dark period. This is the ®rst time that a physiological role for a b-amylase in plants has been demonstrated.

Glucan, water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubilization

“…Arabidopsis mutants with a diminished expression of BAM3 possess elevated starch levels (Kaplan and Guy, 2005), and these results have been confirmed using a null mutant of BAM3 (Fulton et al, 2008). Similar results have been reported for Solanum tuberosum L. (Scheidig et al, 2002). The in vitro breakdown of native starch particles, as catalyzed by BAM3, is stimulated by the simultaneous phosphorylation of starch by GWD (Edner et al, 2007).…”

Repression of a Novel Isoform of Disproportionating Enzyme (stDPE2) in Potato Leads to Inhibition of Starch Degradation in Leaves But Not Tubers Stored at Low Temperature

“…No significant difference was found in starch phosphorylase activity between leaves of the transgenic and control plants; however, the activities of all the other enzymes were significantly (P # 0.05) increased in the transgenic lines in comparison with the control. It is not unusual for the activities of enzymes involved in starch degradation to increase in plants demonstrating a starch-excess phenotype (Zeeman et al, 1998;Critchley et al, 2001;Scheidig et al, 2002), and these data demonstrate that the decreased starch degradation found in the transgenic plants of this study is probably not due to some pleiotropic effect on another starch degradative enzyme.…”

“…The TSM90 strain demonstrates a negative phenotype when grown on MacConkey agar supplemented with maltose, unless expressing a protein that gives it the ability to metabolize maltose (Ehrmann and Vogel, 1998). TSM90 cells were transformed with a plasmid cDNA library constructed using RNA isolated from potato leaves during the light-to-dark transition (Scheidig et al, 2002), and plasmid DNA was isolated from colonies demonstrating a positive phenotype. Digestion of the resulting plasmids with restriction enzymes indicated that they could be separated into two different classes, and DNA sequence information was obtained from the inserts of a representative of both.…”

“…The amounts of no other malto-oligosaccharide were consistently altered in the transgenic lines. Maltose must be produced in plastids during starch breakdown, as it is the only product of b-amylase activity, and a b-amylase isoform has recently been demonstrated to be involved in starch degradation in potato leaves (Scheidig et al, 2002). It has always been presumed that maltose would be degraded by a-glucosidase isoforms; however, in the only study in which the activity of an a-glucosidase was repressed in plants, it was demonstrated that it was most likely involved in processing glycoproteins (Taylor et al, 2000).…”

“…a-amylase and b-amylase were determined by monitoring the degradation of malto-oligosaccharides linked to a p-nitrophenyl group by a glucosidic bond at the reducing end, using assay kits from Megazyme (Wicklow, Ireland) according to the method of Scheidig et al (2002). Fifty microliters of plant extract were mixed with 225 mL of buffer containing 100 mM MES-KOH, pH 6.2, 1 mM EDTA, and 0.1% (v/v) b-mercaptoethanol.…”