A 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 beta-amylase of Arabidopsis. Expression of the gene in E. coli showed that the protein product was a functional beta-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the beta-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 beta-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 first time that a physiological role for a beta-amylase in plants has been demonstrated.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2002.01317.x

Downregulation of a chloroplast-targeted beta-amylase leads to a starch-excess phenotype in leaves.

A quantitative assessment of the importance of barley seed α-amylase, β-amylase, debranching enzyme, and α-glucosidase in starch degradation

This review describes recent progress in discovering the pathway of starch breakdown in leaves. The synthesis of starch from photo-assimilated carbon is one of the major biochemical fluxes in plants. Despite this, the pathway through which this starch is remobilized has not been defined. Numerous enzymes that could participate in starch breakdown are present in leaves, but until recently, the relative importance of each had not been determined. Through studies using model species such as Arabidopsis and potato, significant progress has now been made in determining the roles of known enzymes, and in the discovery of novel proteins necessary for breakdown. These data allow a tentative pathway for starch breakdown to be mapped out, involving hydrolysis primarily to maltose and subsequent maltose export to the cytosol. This provides a framework for complete discovery of the pathway and for the analysis of its regulation. Contents Summary 247 I. Introduction 247 II. Structure of the starch granule 248 III. Initial attack on the granule and the role of glucan, water dikinase 249 IV. Debranching of branched glucans 250 V. The metabolism of linear glucans 251 VI. Export of starch catabolites 254 VII. Metabolism of glucose and maltose 255 VIII. The emerging pathway of starch breakdown and its regulation 256 Acknowledgements 258 References 258.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.2004.01101.x

The breakdown of starch in leaves

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants Expression of the ct-Bmy cDNA in E coli confirmed that the encoded protein possesses beta-amylase activity CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase.

β-Amylase of sweet potato (Ipomoea batatas L.), which constitutes about 5% of the total soluble protein of the tuberous root, is absent or is present in only small amounts in organs other than the tuberous roots of the normal, field-grown plants. However, when leaf-petiole cuttings from such plants were supplied with a solution that contained sucrose, the accumulation of β-amylase was induced in both leaf and petiole portions of the explants. The sucrose-induced accumulation of β-amylase in leaf-petiole cuttings occurred concomitant with the accumulation of starch and of sporamin, the most abundant storage protein of the tuberous root. The accumulation of β-amylase, of sporamin and of starch in the petioles showed similar dependence on the concentration of sucrose, and a 6% solution of sucrose gave the highest levels of induction when assayed after 7 days of treatment. The induction of mRNAs for β-amylase and sporamin in the petiole could be detected after 6 hours of treatment with sucrose, and the accumulation of β-amylase and sporamin polypeptides, as well as that of starch, continued for a further 3 weeks. In addition to sucrose, glucose or fructose, but not mannitol or sorbitol, also induced the accumulation of β-amylase and sporamin, suggesting that metabolic effects of sucrose are important in the mechanism of this induction. Treatment of leaf-petiole cuttings with water under continuous light, but not in darkness, also caused the accumulation of small amounts of these components in the petioles, probably as a result of the endogenous supply of sucrose by photosynthesis. These results suggest that the expression of the gene for β-amylase is under metabolic control which is coupled with the expression of sink function of cells in the sweet potato.

Sucrose-Induced Accumulation of β-Amylase Occurs Concomitant with the Accumulation of Starch and Sporamin in Leaf-Petiole Cuttings of Sweet Potato

Differences in starch metabolism during seed development and germination of two soybean [Glycine max (L.) Merrill] genotypes with normal seed β-amylase activity [‘Williams’ (Sp1b and ‘Altona’ (Sp1b)] and two soybean genotypes with undetectable seed β-amylase activity [‘Chestnut’ (Sp1au) and ‘Altona’ (Sp1)] were investigated. Starch and soluble sugar profiles were essentially the same during seed development and germination. Total amylase activity of Williams and Altona (Sp1b) peaked just prior to seed maturity and then dropped off slowly; whereas, the total amylase activity of Chestnut and Altona (sp1) was very low throughout seed development and germination. The differences in amylase activity between Altona (Sp 1 b) and Altona (sp 1) was also seen in leaves. α-Amylase activity was similar in the four genotypes when β-amylase was inhibited with Hg2+ but was higher in the two genotypes with normal β-amylase activity when β-amylase was inhibited with heat plus Ca2+. Low levels of starch phosphorylase activity were detected throughout seed development and germination, and the activity was similar in three of the genotypes and higher in Altona (sp 1).



The protein, oil and oligosaccharide contents of mature seeds of the four genotypes were similar. Altona (sp 1 b) and (sp 1), which appear to be near isogenic lines, were not different in any morphological character or yield.



Altona (Sp 1 b) showed greater hydrolysis of soybean seed starch than Altona (sp 1), but the evidence indicates that the mutation resulting in greatly reduced or missing β-amylase activity has no effect on starch metabolism of developing and germinating soybean seeds.

T. Hymowitz

Papers

Role of β‐amylase in starch metabolism during soybean seed development and germination