A Transient Gene Expression Protocol for Subcellular Protein Localization and Protein Secretion Analyses in Rice

Wang, Yiming; Wu, Jingni; Kim, Pil Joo; Kim, Sun Tae; Kang, Kyu Young

doi:10.1038/protex.2013.064

Cited by 4 publications

(2 citation statements)

References 30 publications

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“…Transient expression in rice sheath cells was conducted according to Wang et al (2013). Plasmids pOS493 and pAT485 were introduced into leaf sheath cells of 7-wk-old rice using a Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA, USA).…”

Section: Transient Expression Assaysmentioning

confidence: 99%

Subcellular localization of rice acyl‐CoA‐binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes

et al. 2014

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Summary Acyl‐CoA‐binding proteins (ACBPs) show conservation at the acyl‐CoA‐binding (ACB) domain which facilitates binding to acyl‐CoA esters. In Arabidopsis thaliana, six ACBPs participate in development and stress responses. Rice (Oryza sativa) also contains six genes encoding ACBPs. We investigated differences in subcellular localization between monocot rice and eudicot A. thaliana ACBPs. The subcellular localization of the six OsACBPs was achieved via transient expression of green fluorescence protein (GFP) fusions in tobacco (Nicotiana tabacum) epidermal cells, and stable transformation of A. thaliana. As plant ACBPs had not been reported in the peroxisomes, OsACBP6::GFP localization was confirmed by transient expression in rice sheath cells. The function of OsACBP6 was investigated by overexpressing 35S::OsACBP6 in the peroxisomal abc transporter1 (pxa1) mutant defective in peroxisomal fatty acid β‐oxidation. As predicted, OsACBP1::GFP and OsACBP2::GFP were localized to the cytosol, and OsACBP4::GFP and OsACBP5::GFP to the endoplasmic reticulum (ER). However, OsACBP3::GFP displayed subcellular multi‐localization while OsACBP6::GFP was localized to the peroxisomes. 35S::OsACBP6‐OE/pxa1 lines showed recovery in indole‐3‐butyric acid (IBA) peroxisomal β‐oxidation, wound‐induced VEGETATIVE STORAGE PROTEIN1 (VSP1) expression and jasmonic acid (JA) accumulation. These findings indicate a role for OsACBP6 in peroxisomal β‐oxidation, and suggest that rice ACBPs are involved in lipid degradation in addition to lipid biosynthesis.

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Section: Transient Expression Assaysmentioning

confidence: 99%

Subcellular localization of rice acyl‐CoA‐binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes

et al. 2014

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“…Leaf sheaths from young rice seedlings at the 4–6 leaf stage were sectioned with a double folded razor blade, exactly as described in Wang et al (2013). With a scalpel blade, the greenish regions were removed from the sections.…”

Section: Methodsmentioning

confidence: 99%

Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants

Singh

Siddiqui

et al. 2016

J Plant Res

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Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement.

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Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants

et al. 2016

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Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

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A Transient Gene Expression Protocol for Subcellular Protein Localization and Protein Secretion Analyses in Rice

Cited by 4 publications

References 30 publications

Subcellular localization of rice acyl‐CoA‐binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes

Subcellular localization of rice acyl‐CoA‐binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes

Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants

Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants

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