Involvement of [alpha]-Amylase I-1 in Starch Degradation in Rice Chloroplasts
2005
Asatsuma, Satoru | Sawada, Chihoko | Itoh, Kimiko | Okito, Mitsutoshi | Kitajima, Aya | Mitsui, Toshiaki
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.
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