Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis

Abstract

The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development. INTRODUCTIONPlant protoplasts without cell walls offer a versatile cell-based experimental system. Macromolecules such as DNA, RNA and proteins can be delivered into protoplasts using various methods, e.g., PEG-calcium fusion, electroporation and microinjection. Signal transduction and metabolic pathways as well as transcription and translation machineries can be transiently manipulated to investigate cell-autonomous regulation and responses. The method is especially powerful in functional genomics analysis when combined with rich genetic resources, such as insertion libraries, available in the model plant Arabidopsis thaliana. We describe here a protocol for a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) and provide troubleshooting advice and detailed information on useful reporter constructs. Applications, advantages and limitations of the TEAMP assay are also discussed. More general information about plant protoplasts can be found in a review 1 and a live performance of the TEAMP procedure can be seen at the Sheen lab website (http://genetics. mgh.harvard.edu/sheenweb/protocols_reg.html).

PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length

“…For FRET in A. thaliana cells, protoplasts from adult leaves of plants grown under 12‐h light/12‐h dark were isolated by previously described methods (Yoo et al , 2007; Wu et al , 2009). In these experiments, 9 μg of plasmids carrying 35S:: PRR:CFP or 35S:: CO:YFP was used for transfection to ~100 000 protoplasts.…”

Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism

“…For detection by fluorescence microscopy, the red fluorescent protein (RFP)-and green fluorescent protein (GFP)-coding sequences were fused in-frame to the 3′ ends of the IDD14α and IDD14β sequences, respectively, and the fusions were subcloned into the p326-RFP vector for RFP fusion and the pB7FWG2 vector for GFP fusion. The expression constructs were transformed into Arabidopsis protoplasts by polyethylene glycol-calcium transfection 26 . The subcellular distribution of the IDD14 proteins was visualized by differential interference contrast (DIC) microscopy and fluorescence microscopy.…”

“…BiFC assays were carried out by co-transfection of the IDD14α-nYFP and IDD14β-cYFP vectors or vice versa into the Arabidopsis mesophyll protoplasts as described previously 26 . Sixteen hours after transfection, reconstitution of yellow fluorescent protein (YFP) fluorescence was observed using a confocal microscope with the following YFP filter set up: excitation 515 nm, 458/514 dichroic and emission 560-615 BP filter.…”

The wheat NB‐LRR gene TaRCR1 is required for host defence response to the necrotrophic fungal pathogen Rhizoctonia cerealis

“…The resulting p35S:GFP‐TaRCR1 and p35S:GFP constructs were separately introduced into white onion epidermal cells and wheat protoplasts following Yoo et al . (2007) and Zhang et al . (2007).…”