Non‐invasive quantitative detection and applications of non‐toxic, S65T‐type green fluorescent protein in living plants

Abstract

Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.

Mutations in the FIE and MEA Genes That Encode Interacting Polycomb Proteins Cause Parent-of-Origin Effects on Seed Development by Distinct Mechanisms

“…The GREEN FLUORESCENT PROTEIN (GFP) gene used in the construction of reporter transgenes is the synthetic variant S65T (sGFP[S65T]) and lacks any subcellular localization sequences (Heim et al, 1995;Chiu et al, 1996;Niwa et al, 1999). The complete coding sequence of sGFP(S65T) and a polyadenylation signal from the nopaline synthase gene (nos3Ј) were excised together from a donor plasmid (CaMV35S-sGFP[S65T]-nos3Ј) by use of BamHI and EcoRI…”

Visualization of Nuclear Localization of Transcription Factors with Cyan and Green Fluorescent Proteins in the Red Alga Porphyra yezoensis

“…First, molecular cloning and characterization of cDNAs encoding transcription factors were carried out. Secondly, we successfully showed 5 expression of humanized fluorescent proteins ZsGFP and ZsYFP, whose codon usage is similar to that of genes from P. yezoensis, and that of plant-adapted fluorescent protein sGFP(S65T), which provides up to 100-fold brighter fluorescence signals than the original GFP from jellyfish Aequorea victoria in plants (Niwa et al 1999). Thirdly, we showed successful application of the above transient gene expression system for visualization of the nuclear localization of transcription factors from P. yezoensis by fusion with the above fluorescent proteins.…”

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase

“…A sequence encoding GFP was incorporated into the construct to monitor the accumulation of the transgene products in vivo by CLSM [26]. The sequence for the CAO-GFP fusion protein was placed downstream from the Cauliflower mosaic virus 35S promoter for constitutive overexpression of the transgene.…”