New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems

Abstract

Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein (YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co‐cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co‐transformation with the peroxisome marker, CFP‐PTS1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP‐PTS1, allowing piggy‐back import into peroxisomes. Indeed, if co‐expressed with monomeric Cerulean‐PTS1 (mCer‐PTS1), the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization‐based piggy‐back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co‐expressed with CFP‐PTS1 but not with mCer‐PTS1. By contrast, piggy‐back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.

Peroxisomes: versatile organelles with diverse roles in plants

“…In transient expression systems using Arabidopsis seedlings and tobacco leaves, certain fluorophore combinations that weakly heterodimerize can lead to false positives as a result of the so-called piggy-back mechanism of peroxisomal protein import (Falter et al, 2019). Caution should be exercised when selecting fluorophores.…”

“…Caution should be exercised when selecting fluorophores. In transient expression systems using Arabidopsis seedlings and tobacco leaves, certain fluorophore combinations that weakly heterodimerize can lead to false positives as a result of the so-called piggy-back mechanism of peroxisomal protein import (Falter et al, 2019). However, this phenomenon has not been observed in Arabidopsis protoplasts, onion epidermal cells or stable transgenic plants.…”

Evolutionary Maintenance of the PTS2 Protein Import Pathway in the Stramenopile Alga Nannochloropsis

“…The latter elements were reamplified from an episomal CRISPR/Cas9 vector (Addgene accession number: #101009, Poliner et al, 2018; Supplementary Table 3) and subcloned by ApaI and NotI. The Venus reporter gene was replaced by the blue fluorescent reporter gene mCerulean extended by a C-terminal PTS1 (Falter et al, 2019) using the given primer pairs ( Supplementary Table 3) and the restriction enzymes SgsI and SacI.…”

“…Onion (Allium cepa L.) epidermal cells were transformed biolistically as described (Falter et al, 2019), followed by microscopic analyses 1-7 days post transformation (dpt). Arabidopsis protoplasts were transiently transformed as described (Yoo et al, 2007) and analyzed 1-2 dpt.…”

Plant peroxisomes: Small organelles with versatility and complexity

“…However, when certain fluorescent proteins are co‐expressed, such as yellow fluorescent protein (YFP)‐tagged candidate together with cyan fluorescent protein (CFP)‐PTS1, heterodimerization can occur between these two proteins thus causing false positives as a result of the so‐called piggy‐back import. After a systematic investigation, Falter et al (2019) provided a guideline for the usage of fluorophores and transient plant expression systems for reliable validation of novel plant peroxisomal proteins.…”