pH-sensitivity of YFP provides an intracellular indicator of programmed cell death

Abstract

BackgroundProgrammed cell death (PCD) is an essential process for the life cycle of all multicellular organisms. In higher plants however, relatively little is known about the cascade of genes and signalling molecules responsible for the initiation and execution of PCD. To aid with the discovery and analysis of plant PCD regulators, we have designed a novel cell death assay based on low cytosolic pH as a marker of PCD.ResultsThe acidification that occurs in the cytosol during plant PCD was monitored by way of the extinction of YFP fluorescence at low pH. This fluorescence was recovered experimentally when bringing the intracellular pH back to 7, demonstrating that there was no protein degradation of YFP. Because it uses YFP, the assay is none-destructive, does not interfere with the PCD process and allows time-lapse studies to be carried out. In addition, changes of sub-cellular localisation can be visualised during PCD using the protein of interest fused to RFP. Coupled to a transient expression system, this pH-based assay can be used to functionally analyse genes involved in PCD, using point mutations or co-expressing PCD regulators. Transfecting mBAX and AtBI-1in onion epidermal cells showed that the pH shift is downstream of PCD suppression by AtBI-1. In addition, this method can be used to score PCD in tissues of stably transformed transgenic lines. As proof of principle, we show the example of YFP extinction during xylogenesis in Arabidopsis. This demonstrates that the assay is applicable to PCD studies in a variety of tissues.ConclusionsThe observation that YFP fluorescence is lost during the plant PCD process provides a new tool to study the genetic regulation and cell biology of the process. In addition, plant cell biologists should make a note of this effect of PCD on YFP fluorescence to avoid misinterpretation of their data and to select a pH insensitive reporter if appropriate. This method represents an efficient and streamlined tool expected to bring insights on the process leading to the pH shift occurring during PCD.

I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

“…~5% to ~60% for sfGFP, or ~9% to ~67% for mCitrine). However, it was never restored back to 100% which is in line with previous experiments using GFP‐S65T and EYFP, presumably because protein conformation changes induced at pH < 5 require longer recovery times (Kneen, Farinas, Li, & Verkman, ; Young et al., ). Notably, the fluorescence of two proteins could not be restored even to 50% of the initial values: mEYFP (~38%) and pH‐tdGFP (~45%).…”

“…Yellow proteins are notorious for their sensitivity to low pH and their relatively high p K a (Shaner et al., ; Young, Wightman, Blanvillain, Purcel, & Gallois, ). In this range, we selected mCitrine (p K a mCitrine = 5.7) which has a p K a significantly lower than that of its parent protein mEYFP (p K a mEYFP = 6.9), included as pH‐sensitive control (Figure c) (Griesbeck, Baird, Campbell, Zacharias, & Tsien, ; Kremers, Goedhart, van Munster, & Gadella, ; Miyawaki et al., ; Ormö et al., ; Rizzo, Springer, Granada, & Piston, ).…”

Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca 2+ ] cyt Patterns in Arabidopsis

“…Wild-type Aequorea victoria GFP and several of its derivates are sensitive to changes in pH (Kneen et al, 1998; Young et al, 2010). Also R-GECO1, derived from circular permutated mApple (Shaner et al, 2008), is pH sensitive in vitro (Zhao et al, 2011).…”

Microautophagy involves programmed cell semi‐death of sieve elements in developing caryopsis of Triticum aestivum L.

“…Cytosolic pH is also reduced in transgenic onion epidermis during PCD (Matsuyama et al, 2000;Nilsson et al, 2006;Young et al, 2010). Cytosolic pH is also reduced in transgenic onion epidermis during PCD (Matsuyama et al, 2000;Nilsson et al, 2006;Young et al, 2010).…”