Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy

• Published in: Plant cell, tissue and organ culture Vol. 104; no. 2; pp. 199 - 207
• Main Authors: Xu, Shi-Xiao, Cai, Xiao-Dong, Tan, Bin, Guo, Wen-Wu
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.02.2011; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Actin; Agrobacterium; Agrobacterium tumefaciens; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Callus; Cell fusion; Cell lines; Cell walls; Citrus fruits; Citrus sinensis; Confocal microscopy; Cytoplasm; Cytoskeleton; Endoplasmic reticulum; Epidermis; Fluorescence; Fruits; Fundamental and applied biological sciences. Psychology; Fusion protein; Green fluorescent protein; Guard cells; Leaves; Life Sciences; Localization; Mitochondria; Original Paper; Parenchyma; Peptides; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; Plant Sciences; Plant tissues; Proteins; Protoplasts; Scanning microscopy; Signal peptides; Transgenic plants; Citrus; Agrobacterium tumefaciens; Co-localization; Confocal laser scanning microscope; Green fluorescent protein; Vegetals; Targeting; Citrus sinensis; Rutaceae; Confocal microscopy; Orange; Target; Dicotyledones; Angiospermae; Laser; Bacteria; Spermatophyta; Rhizobiaceae; Localization; Genetically modified organism; Transgenesis; Comparative study
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-010-9819-0

The green fluorescent protein (GFP) has become an ideal visual marker to monitor and quantify the expression of the transgene. It can be targeted to specific subcellular locations, including the endoplasmic reticulum, mitochondria, actin cytoskeleton and nuclei through the addition of signal peptides. Our previous work has resulted in transgenic citrus plants expressing cytoplasmic targeted GFP (Cy-GFP) or endoplasmic reticulum targeted GFP (Er-GFP) gene. To evaluate the localization of three different subcellular targeted GFP, i.e., Cy-GFP, Er-GFP and mitochondria targeted GFP (Mt-GFP) in citrus tissues and to utilize cell lines containing Mt-GFP for basic research in cell fusion, the plasmid pBI-mgfp4-coxIV encoding the Mt-GFP gene was successfully transferred into embryogenic callus of Valencia sweet orange (Citrus sinensis (L.) Osbeck) via Agrobacterium tumefaciens-mediated transformation. Furthermore, we compared the specific expression of these three different subcellular localized GFP constructs in cells of different mature leaf tissues (upper epidermis, palisade parenchyma, spongy parenchyma and lower epidermis) by a confocal laser scanning microscope (CLSM). Cytoplasmic-localized GFP expression was observed throughout the cytoplasm but appeared to accumulate within the nucleoplasm. The Er-GFP occurred within a layer very close to the cell wall. In addition, a stable fluorescence on the ER network throughout the guard cells was detected. Interestingly, the Mt-GFP specifically expressed in the guard cells to particles of about 1-2 μm within the cytoplasm in this case. To verify that the fluorescent particles observable in the guard cells are indeed mitochondria, we co-localize the Mt-GFP fusion protein with a mitochondrial-specific dye in citrus protoplasts. These results demonstrate that the subcellular distribution of the three subcellular targeted GFP is very distinct in citrus leaf cells and the cell lines containing Mt-GFP gene can be further used in citrus basic cell fusion research.