Involvement of α-Amylase I-1 in Starch Degradation in Rice Chloroplasts

Abstract

To determine the role of alpha-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of alpha-amylase I-1. In the lines with suppressed expression of alpha-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing alpha-amylase I-1. In immunocytochemical analysis with specific anti-alpha-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of alpha-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of alpha-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that alpha-amylase I-1 targets the chloroplasts through the endoplasmic reticulum-Golgi system and plays a significant role in the starch degradation in rice leaves.

Rice PlastidialN-Glycosylated Nucleotide Pyrophosphatase/Phosphodiesterase Is Transported from the ER-Golgi to the Chloroplast through the Secretory Pathway

“…Although consistent with recent reports showing the occurrence of N-glycosylated proteins in plastids (Asatsuma et al, 2005;Villarejo et al, 2005), the finding of NPP1 in the interior of a subcellular compartment unrelated to the ER-Golgi system was quite surprising and totally unpredictable in the context of the chloroplast genomics and proteomics (Miras et al, 2002;Leister, 2003). A mechanism of protein traffic from the ER-Golgi to the chloroplast must be invoked that is distinct from the wellestablished pathway of protein traffic between the cytosol and plastids (Heins et al, 1998).…”

Double Knockout Mutants of Arabidopsis Grown under Normal Conditions Reveal that the Plastidial Phosphorylase Isozyme Participates in Transitory Starch Metabolism

“…Single knockout mutants lacking functional AMY3 do not exhibit any starch-related phenotype and perform nocturnal starch degradation similar to the wild type, and therefore, in Arabidopsis (as opposed to rice [Oryza sativa]), the endoamylase was thought not to be essential for the mobilization of transitory starch (Asatsuma et al, 2005;Yu et al, 2005). By contrast, Arabidopsis triple knockout mutants deficient in two plastidial debranching enzymes plus AMY3 accumulate more starch than the double mutants lacking the two debranching enzymes, and starch degradation is largely blocked.…”

Effect of time of day for harvest and postharvest treatments on the sugar metabolism of broccoli (Brassica oleracea var. italica)

“…In photosynthetic tissues, starch accumulates transiently in chloroplasts during daylight hours by action of photosynthesis. However, for extended periods of photosynthesis, storage takes place in the amyloplasts in reserve tissues (Asatsuma et al 2005). The transient starch is degraded releasing maltose and glucose units, which are then exported to the cytosol to be used as source of energy for synthesis of sucrose.…”

“…Sugars derived from photosynthesis also are transported via phloem to target organs, where can be stored as starch reserves. Although α-amylase has an important role in the degradation of starch in the endosperm of cereals (Asatsuma et al 2005) it does not appear to have an essential role in arabidopsis leaves (Yu et al 2005). The α-amylase is required for the initiation of the degradation of the starch during the germination of grains, but is not required for normal degradation of transient starch in leaves.…”