Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression

Abstract

Summary An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β‐glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)‐mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin‐mediated induction of chloramphenicol acetyl‐transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin‐inducible PS‐IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.

A molecular pathway for CO2 response in Arabidopsis guard cells

“…The indicated pairs of expression constructs 46 were co-transformed into Arabidopsis mesophyll protoplasts by PEG-mediated transfection 54 . Protoplast images were taken by confocal microscopy (Zeiss 5-LIVE) operated with LSM Image Browser software.…”

“…Arabidopsis plants grow under 13 h light/11 h dark for 3-4 weeks; well expanded leaves are used to isolated protoplasts. Isolation and transient protoplast transformation was performed as described previously 54 . Briefly, leaves were sliced on a paper and immediately transferred into enzyme solution, the digestion was continued for about 3 h with gentle shaking in the dark.…”

“…After polyethylene glycol (PEG)-mediated transfection, protoplasts were incubated at 25°C in the dark for 24 h. Subcellular localization. The expression constructs 35S::RHC1, 35S::SLAC1-GFP or 35S::GFP were transformed into Arabidopsis protoplasts by PEG-mediated transfection 54 . Protoplasts were stained with 2 mM FM4-64 dye for 5 min to stain the plasma membrane.…”

Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

“…Arabidopsis protoplasts were isolated and transfected using a modified polyethylene glycol method as described previously (Abel & Theologis, 1994). Typically, 1610 6 -2610 6 protoplasts were transfected with 30-50 mg pBI221-GFP or pBI221-S9-GFP and then incubated at 22 uC for 16-24 h in the dark.…”

SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

“…Plant protoplasts can be cell-or tissue-specific populations of single cells used to study a broad spectrum of processes from physiology to gene function/regulation (Abel and Theologis, 1994;Chiu et al, 1996;Davey et al, 2005;Thorpe, 2007;Yoo et al, 2007). Freshly isolated protoplasts retain cell and transcriptome identity, differentiated state (without dedifferentiation), and original biochemical and regulatory activity (Cocking, 1972;Sheen, 2001;Birnbaum et al, 2003;Yoo et al, 2007;Faraco et al, 2011).…”