Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis

Abstract

Programmed cell death (PCD) is indispensable for eukaryotic development. In animals, PCD is executed by the caspase family of cysteine proteases. Plants do not have close homologues of caspases but possess a phylogenetically distant family of cysteine proteases named metacaspases. The cellular function of metacaspases in PCD is unknown. Here we show that during plant embryogenesis, metacaspase mcII-Pa translocates from the cytoplasm to nuclei in terminally differentiated cells that are destined for elimination, where it colocalizes with the nuclear pore complex and chromatin, causing nuclear envelope disassembly and DNA fragmentation. The cell-death function of mcII-Pa relies on its cysteine-dependent arginine-specific proteolytic activity. Accordingly, mutation of catalytic cysteine abrogates the proteolytic activity of mcII-Pa and blocks nuclear degradation. These results establish metacaspase as an executioner of PCD during embryo patterning and provide a functional link between PCD and embryogenesis in plants. Although mcII-Pa and metazoan caspases have different substrate specificity, they serve a common function during development, demonstrating the evolutionary parallelism of PCD pathways in plants and animals.

Fig. 1.

McII-Pa is a processive cysteine protease with arginine-specific enzymatic activity. (A) McII-Pa zymogen consists of p20 caspase-like subunit with catalytic Cys-139, a linker region, and p10 caspase-like subunit. Autoprocessing of mcII-Pa in E. coli occurs by autocleavage after Arg-188 and Lys-269 between the domains. (B) Substrate specificity of recombinant mcII-Pa tested on five different substrates containing Arg, Lys, or Asp at the P1 position. (C) Western blotting demonstrating that WT recombinant mcII-Pa (lane WT), but not catalytic Cys-139 mutant (lane C139A), undergoes autoprocessing. The graph shows that Cys-139 mutant is proteolytically inactive.

Fig. 2.

Cell-death function of mcII-Pa relies on its proteolytic activity. (A) Western blotting of WT (WT) and mcII-Pa silenced lines probed with anti-mcII-Pa shows reduction of mcII-Pa level in the silenced line. TUNEL of WT and silenced lines shows suppression of PCD in the silenced lines (Top). The EGRase proteolytic activity is reduced in the silenced lines (Bottom). Data are representative of three silenced clones. (B) A developmentally arrested cell line unable to form the embryos exhibits a reduced level of TUNEL (Top), lack of mcII-Pa processing (Western blot), and a reduced level of EGRase activity (Bottom). Error bars indicate SEM.

Fig. 3.

McII-Pa colocalizes with nuclei containing fragmented DNA in the embryo-suspensor cells. (A) Epifluorescence microscopy of DAPI, TUNEL, and anti-mcII-Pa labeling in the whole embryo. A representative example of one of ≈500 embryos analyzed. EM, embryonal mass. (B) Confocal microscopy of embryonal mass cells (Top), and suspensor cells with morphologically intact (Middle), and degraded (Bottom) nuclear envelope. A representative example of 1 of the 100 embryos analyzed. [Bars, 100 (A) and 10 μm(B).]

Fig. 4.

McII-Pa translocates from the cytoplasm into the nucleus during PCD in the embryo suspensor. Phase-contrast image of the representative embryo (Left; Bar, 100 μm) analyzed by immunogold electron microscopy with antibody to mcII-Pa (Center). Upper Center corresponds to the embryonal mass cells. Lower Center corresponds to the cells from the middle zone of the suspensor. Left Center shows cell ultrastructure (Bars, 5 μm), whereas Right Center shows immunogold labeling of the cytoplasm and nucleus, respectively (Bars, 200 nm). Gold particles are associated with the condensed chromatin in the suspensor nucleus. Graph (Right) shows density of the gold particles in the cytoplasm and nuclei of the embryonal mass and upper and middle zones of the suspensor. Error bars indicate SEM. n, nucleus; v, vacuole; asterisk indicates autophagosome.

Fig. 5.

Immunogold labeling of mcII-Pa at the sites of nuclear pore complex disassembly in the embryo-suspensor cells. (Left) Lobed nucleus typical for the early stages of nuclear degradation. Asterisk denotes the area shown enlarged (Right). The gold particles marked by arrows are associated with nuclear pore complex disassembly. c, cytoplasm; n, nucleus. [Bars, 5 μm (Left) and 100 nm (Right).]

Fig. 6.

McII-Pa induces nuclear degradation in the cell-free system. (A) Frequency of TUNEL-positive nuclei before and after treatment with cell extract, recombinant WT (WT) mcII-Pa in the absence or presence of H-EGR-cmk, and catalytically inactive mutant mcII-Pa (C139A), as indicated below each bar. (B) Representative examples of normal nuclei and three stages of nuclear degradation after staining with DAPI and TUNEL. (C) Frequency of nuclei at each stage of degradation in the cell-free system experiments conducted as described in A. Error bars indicate SEM.