Involvement of alpha-amylase I-1 in starch degradation in rice chloroplasts.
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.
Asatsuma
Satoru
S
Laboratories of Plant and Microbial Genome Control, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181 Japan.
Sawada
Chihoko
C
Itoh
Kimiko
K
Okito
Mitsutoshi
M
Kitajima
Aya
A
Mitsui
Toshiaki
T
D002736:Chloroplasts / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D012275:Oryza / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D030821:Plants, Genetically Modified; D018087:Plastids / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D012333:RNA, Messenger; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013213:Starch; D000516:alpha-Amylases