VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

Abstract

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn 2 þ concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.

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

“…Proteolytic activity of recombinant mcII-Pa and P. abies cell extracts (16) were assayed in 150-l reaction mixtures containing 0.3 g of recombinant protein or 22.5 g of total protein, respectively, and 50 M individual substrate in the optimized assay buffer: 100 mM Hepes, pH 7.0͞50 mM CaCl 2 ͞0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate͞5 mM DTT (Fig. 7, which is published as supporting information on the PNAS web site).…”

“…7, which is published as supporting information on the PNAS web site). First-order kinetics of substrate cleavage was measured as described (16). For each independent experiment, at least three assays were performed, each assay containing triplicate reactions.…”

“…Previously, we have reported that activation of protease(s) cleaving the caspase substrate Val-Glu-Ile-Asp (VEIDase activity) is essential for PCD and embryogenesis in the gymnosperm Norway spruce (Picea abies) (16). Silencing of P. abies metacaspase gene mcII-Pa inhibited VEIDase activity, suppressed PCD in the embryos, and blocked suspensor differentiation (15).…”

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

“…The SI-induced caspase-3-like/DEVDase exhibits maximum substrate cleavage in vitro at pH 5, with peak activity 5 h after SI induction in vivo (Bosch and Franklin-Tong, 2007). The low pH optimum for this caspase-3-like/DEVDase activity is unusual, because most of the cytosolic plant caspase-like activities identified to date have optimal activity close to normal physiological pH (approximate pH, 6.5-7.0; Korthout et al, 2000;Bozhkov et al, 2004;Coffeen and Wolpert, 2004). Because the SI-induced cytosolic-located DEVDase requires a low pH for activity, this suggested that, during SI, the pollen tube cytosol undergoes dramatic acidification.…”

“…There is considerable biochemical evidence for the involvement of caspase-like activities in plant PCD (van Doorn and Woltering, 2005). For example, the vacuolar processing enzyme has YVADase (caspase-1-like) activity (Hatsugai et al, 2004;Rojo et al, 2004;Hara-Nishimura et al, 2005), DEVDase (caspase-3-like) and YVADases are associated with PCD in several plant systems (del Pozo and Lam, 1998;Korthout et al, 2000;Danon et al, 2004), and VEIDase (caspase-6-like) is the main caspase-like activity involved in embryonic pattern formation (Bozhkov et al, 2004). However, because plants have no caspase gene homologs (Sanmartín et al, 2005), the nature of their caspase-like enzymes is the subject of considerable debate.…”