Insights into Plant Programmed Cell Death Induced by Heavy Metals-Discovering a Terra Incognita

Abstract

Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.

Keywords: abiotic stress; cell culture; gene expression; heavy metal stress; necrosis; plant PCD vs. animal PCD; programmed cell death.

Figure 1

Cells and tissues of Viola tricolor cultured in vitro. Suspended cells (a), microcallus (b) and callus (c,d). (a)—viable cells after fluorescein diacetate staining. (b)—TUNEL-positive nuclei emitting light yellow-green fluorescence (arrowheads) in cells treated with Pb for 72 h. (c,d)—xylem vessels with lignified cell wall thickening among other callus cells as a result of dPCD (stars). Note the variation in size and shape of callus cells. (c,d)—Sections of calli stained with toluidine blue. Based on Sychta et al. [18], new photos were obtained from the private collection of K. Sychta.

Figure 2

Summary of subcellular heavy metal transport in plant cells. Metal transporter proteins participating in heavy metal transport into and out of cellular organelles are presented. ABCC—ATP-binding cassette subfamily C protein; As—arsenic; CAX—Ca²⁺ exchanger; COPT—copper transporter; CCH—copper chaperone; Cu—copper; GS—glutathione conjugates; GSH—glutathione; HMA—heavy metal ATPase; Me—heavy metal; MTP—metal tolerance proteins; NIP—nodulin 26-like intrinsic protein; NRAMP—natural resistance-associated macrophage protein; PS—phytochelatin conjugates; PCS—phytochelatin synthase; Pi-transporter—phosphorus transporter; VTL—vacuolar-iron transporter-like; ZIF—zinc induced facilitator; ZIP—zinc regulated transporters; YSL—yellow stripe like (metal-nicotianamine transporter) (based on [97,98,99]).

Figure 3

Pb deposition in the vacuoles (a) and cell walls (b) of V. tricolor cells treated with 2000 µM Pb for 72 h visible in transmission electron microscopy. Based on Sychta et al. [100], new photos were obtained from the private collection of K. Sychta.

Figure 4

Vacuolization of V. tricolor cells visible in transmission electron microscopy. (a)—control cells. (b)—cells treated with 2000 µM Zn for 72 h. (c)—cells treated with 2000 µM Pb for 72 h. v—vacuole. Based on Sychta et al. [100], new photos were obtained from the private collection of K. Sychta.