Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles

Abstract

Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed cysteine endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 “ex vivo”. The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 μm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 μm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-013-0157-6) contains supplementary material, which is available to authorized users.

Expression analysis of KDEL-CysEPs programmed cell death markers during reproduction in Arabidopsis

“…1i, m, n, o). Similar to previous studies in roots (Hierl et al 2014), the signal of AtCEP1 reflected localization of the proenzyme instead of the mature enzyme, as AtCEP1 localized to network structures likely representing subdomains of the ER and also to punctuated ricinosome-like structures (Fig. 1j, l), which likely represent stored vesicles.…”

“…The Arabidopsis thaliana genome encodes three KDELCysEPs named as AtCEP1 (At5g50260), AtCEP2 (At3g48340) and AtCEP3 (At3g48350). Tissue expression data from previous studies showed broad expression of all AtCEPs in vegetative and reproductive tissues as well as during biotic interactions in Arabidopsis (Helm et al 2008;Hierl et al 2014). However, although the tissue-specific expression pattern indicated that all three AtCEPs were present in tissues undergoing PCD, there is only experimental evidence for AtCEP1 involved in tapetum PCD during pollen development but no functional report for the other two AtCEPs (Zhang et al 2014).…”

“…The reporter lines P CEP1 ::pre-pro-3xHA-EGFP-AtCEP1-KDEL (AtCEP1-EGFP) and P CEP2 ::pre-pro-3xHA-mCherry-KDEL (AtCEP2-mCherry) both in Arabidopsis Columbia-0 background were generated as described in Höwing et al (2014) and Hierl et al (2014), respectively. The two functional reporter lines localize to the ER and ER-derived compartments.…”

“…KDEL-CysEPs exhibit a characteristic and unusual broad substrate specificity. In addition to the papain-type preference for neutral amino acids with large aliphatic and nonpolar or aromatic side chains in the P2 position of the cleavage site, they accept proline in the P1 and P1 0 position (Than et al 2004) and are able to digest hydroxyproline-rich glycoproteins such as extensins that form the basic scaffold of the cell wall (Cannon et al 2008;Helm et al 2008;Hierl et al 2014). KDEL-CysEPs seem to have a dual set of substrates: they digest cytoplasmic components in cells of dying tissues for recycling to the surviving parts of the plant or in cells of germinating seedlings for mobilization of storage proteins.…”

A conserved core of PCD indicator genes discriminates developmentally and environmentally induced programmed cell death in plants

“…Additionally, pCEP1 ..H2A-GFP conveys a strong expression in epidermal cells in the root hair zone, though these are not known to undergo cell death (data not shown). Interestingly, the close CEP1 homolog CEP2 is highly expressed in LRC cells in the root transition zone (Hierl et al, 2014) and might take over CEP1 functions here. In the developing seed, pCEP1 is active in the embryonic suspensor during early embryo development (data not shown) and is present in later stages both in the seed coat and the differentiating endosperm (Supplemental Fig.…”