METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

Abstract

We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs) that undergo programmed cell death (PCD) and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9) was reduced using RNAi (MC9-RNAi). Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2) was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells.

Keywords: Arabidopsis thaliana; Autophagy; Intercellular signalling; Metacaspase; Programmed cell death; Tracheary element.

Fig. 1.

MC9 is involved in TE differentiation in cell suspensions. (A) cLSM micrographs of proMC9::MC9:GFP cell cultures stained with propidium iodide (PI, cell wall stain, magenta) five days after induction. Asterisks indicate TEs and arrowheads indicate non-TEs. Scale bars=10 µm. (B) Proportions of living and dead TEs (top) and MC9 transcript abundance measured by qPCR (bottom) during TE differentiation (n=3). (C) 3D projection of cLSM micrographs showing the middle part of PI-stained wild-type TEs to exemplify autolysed (top) and remnant-containing (bottom) TEs ten days after induction of TE differentiation. Note that in addition to staining cell walls, PI also stains protoplast remnants after cell death. Scale bars=20 µm. (D) Proportion of TEs (among all TEs) containing a remnant in wild-type and two MC9-RNAi lines ten days after induction of TE differentiation (n=3). (E) Proportion of dead TEs (among all TEs) during TE differentiation in wild-type and two MC9-RNAi lines (n=3). Error bars show mean±s.d.; *P<0.05.

Fig. 2.

Downregulation of MC9 influences intercellular signalling during TE differentiation. (A) Fluorescence micrographs of wild-type (left) and two MC9-RNAi cell lines (middle and right) stained with FDA (viability, green) and PI (cell walls, red) 10 days after induction. Asterisks indicate TEs. Scale bars=50 µm. (B) Proportions of living TEs (among all TEs) and non-TEs (among all non-TEs) during TE differentiation in wild-type and two MC9-RNAi lines. R2 indicates the square Pearson's correlation coefficient between the both aforementioned variables (n=3). (C) Proportion of living non-TEs (among all non-TEs) during TE differentiation in wild-type and two MC9-RNAi lines and in a 1:1 mix of wild-type and MC9-RNAi 2 cells. Cell viability in the non-induced conditions after ten days demonstrates that ectopic cell death occurs in the MC9-RNAi cells only upon induction of TE differentiation (n=3). (D) Proportions of living non-TEs (among all non-TEs) in wild-type and MC9-RNAi 2 lines ten days after being induced to differentiate with normal or suboptimal hormone levels (resulting in ∼50% reduction of TE differentiation) (n=3). (E) Proportion of TEs (among all TEs) containing a remnant in wild-type, two MC9-RNAi lines and in a 1:1 mix of wild-type and MC9-RNAi 2 cells ten days after induction of TE differentiation (n=3). Error bars show mean±s.d.; *P<0.05 in C; means that do not share any letter are significantly different (P<0.05) in D,E.

Fig. 3.

MC9 regulates autophagy specifically in TEs. (A) cLSM micrographs of differentiating cells stained with FDA (viability, green) for viability and with lysotracker (magenta) for acidic bodies in the vacuole. Asterisks indicate living TEs and arrowheads indicate examples of lysotracker-stained vacuolar bodies. Scale bars=20 µm. (B) DIC micrographs (top) and cLSM micrographs (bottom) of wild-type and MC9-RNAi 1 cells treated with Concanamycin A and stained with FDA (viability, green) and PI (cell walls, magenta) five days after induction. Asterisks indicate living TEs and arrowheads indicate example of vacuolar bodies in the displayed focal plane. Scale bars=20 µm. (C) Abundance of vacuolar bodies in TEs (top) and non-TEs (bottom) of Concanamycin A-treated wild-type and MC9-RNAi 1 lines, five days after induction of TE differentiation (n≥3). Error bars show mean±s.d.; *P<0.05.

Fig. 4.

MC9:GFP is present in the vacuole of cells undergoing PCD. (A) 3D projections of cLSM micrographs of a PI-stained (cell walls, magenta) proMC9::MC9:GFP (green) seedling's root treated or not with Concanamycin A and without or with wortmannin. Asterisks indicate TEs. Scale bars=20 µm. (B) Blow up of the GFP signal in single focal planes from (A) used for vacuolar GFP-punctate quantification (C). Vacuoles are indicated by white arrowheads. Scale bars=5 µm. (C) Quantification of vacuolar MC9:GFP punctates in TEs of proMC9::MC9:GFP seedling's root treated or not with Concanamycin A and without or with wortmannin (n=3). Error bars show mean±s.d.; *P<0.05. (D) 3D projection of cLSM micrographs of the lateral root cap of a PI-stained proMC9::MC9:GFP seedling treated with Concanamycin A. Scale bar=20 µm. (E) Blow up of two cells from (D). Scale bar=5 µm.

Fig. 5.

Autophagy in TEs controls ectopic cell death and TE autolysis downstream of MC9. (A) Abundance of vacuolar bodies in TEs (left) and non-TEs (right) of Concanamycin A-treated cell cultures five days after induction of TE differentiation in wild-type (n=11 for TEs and n=13 for non-TEs), MC9-RNAi 1 (n=15 for TEs and n=11 for non-TEs) and MC9-RNAi proIRX1::ATG2-RNAi line 1 (n=12 for TEs and n=13 for non-TEs), 2 (n=7 for TEs and n=8 for non-TEs) and 3 (n=9 for TEs and n=11 for non-TEs), as well as in wild-type (n=6 for TEs and n=6 for non-TEs) and MC9-RNAi 1 (n=8 for TEs and n=7 for non-TEs) simultaneously treated with the autophagy inhibitor wortmannin. (B) Proportions of living non-TEs (among all non-TEs; left) and of TEs with remnants (among all TEs; right) in wild-type, MC9-RNAi 1 and three MC9-RNAi proIRX1::ATG2-RNAi lines ten days after induction of TE differentiation (n=3). Error bars show mean±s.d. Means that do not share any letter are significantly different (P<0.05).