Genes for alkaline/neutral invertase in rice: alkaline/neutral invertases are located in plant mitochondria and also in plastids
Two cDNA clones (OsNIN1 and OsNIN3) encoding an alkaline/neutral invertase localized in organelles were identified from rice. The deduced amino acid sequences of these cDNA clones showed high homology to other plant alkaline/neutral invertases. Semi-quantitative reverse transcription polymerase chain reaction revealed that the expression of OsNIN1 was constitutive and independent of organ difference, although its expression level was low. Analyses using five types of web software for the prediction of protein localization in the cell, Predotar, PSORT, Mitoprot, TargetP, and ChloroP, strongly supported the possibility that OsNIN1 is transported into the mitochondria and that OsNIN3 is transported into plastids. Transient expression of fusion proteins combining the amino terminal region of these two proteins with sGFP demonstrated that N-OsNIN1::GFP and N-OsNIN3::GFP fusion proteins were transported into the mitochondria and plastids, respectively. We expressed the OsNIN1 protein in vitro and revealed that the translated protein had an invertase activity. These results clearly indicate that some of alkaline/neutral invertases are located in plant organelles, mitochondria and plastids, and that they might have a novel physiological function in plant organelles.
KeywordsAlkaline/Neutral invertase Mitochondria Oryza Plastids Rice
Cetyl trimethyl ammonium bromide
Green fluorescent protein
Reverse transcription polymerase chain reaction
We thank Drs. Yasuo Niwa and Hirokazu Kobayashi (University of Shizuoka, Shizuoka, Japan) for the generous gift of the enhanced GFP expression vector. We express our appreciation to Dr. Hiroyuki Kawahigashi (National Institute of Agrobiological Sciences, Tsukuba, Japan) for providing us with the RNA samples for quantitative RT-PCR. We also thank Drs. Midori Yoshida, Akira Kawakami (National Agricultural Research Center for Hokkaido Region, Sapporo, Japan), and Dr. Hanae Kaku (Meiji University, Kawasaki, Japan) for their valuable suggestions. This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science and Culture of Japan (No. 17580012).
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