EYFP fusions to HD-Zip IV transcription factors enhance their stability and lead to phenotypic changes in Arabidopsis

Green fluorescent protein (GFP) and its derivatives are extensively used for labeling cells, monitoring gene expression and/or tracking the localization or interactions of proteins. Previous reports of detrimental effects of fluorescent protein (FP) expression include cytotoxicity and interference with fusion protein function or localization. Only a few studies have documented the fluorescent tag-specific effects in plants. Here, we show that placing an enhanced yellow FP (EYFP) tag on the amino-terminus of GLABRA2 (GL2) and PROTODERMAL FACTOR2 (PDF2), two developmentally important HD-Zip IV transcription factors from Arabidopsis, enhances their protein stability. Additionally, expression of EYFP:GL2 not only rescued the gl2 null mutant but also resulted in the abnormal development of abaxially curled leaves associated with EYFP-tag induced GL2 overexpression. Our study raises concerns on the use of FPs regarding their effects on the native properties of target proteins as well as biological consequences of fusion protein expression on morphology.