Plant caspase-like proteases in plant programmed cell death

Abstract

Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, we will cover the recent results on the existence of plant caspase-like proteases and introduce major technologies used in detecting the activation of caspase-like proteases during plant PCD.

Introduction

Programmed cell death (PCD) is a fundamental genetically-controlled process that is responsible for removal of unwanted and potentially dangerous cells.¹ It was reported that PCD was crucially involved in various aspects of plant life cycle,¹ including the biotic defenses such as hypersensitive response (HR) to plant pathogens attack,² the death response to abiotic stresses such as ultraviolet-C (UV-C) exposure or heat shock treatment,^3,4 and a number of development processes including embryo formation, degeneration of the aleurone layer during monocot seed germination, differentiation of tracheary elements in water-conducting xylem tissues, formation of root aerenchyma and epidermal trichomes, anther tapetum degeneration, floral organ abscission, pollen self-incompatibility, remodeling of some types of leaf shape, and leaf senescence.^5,6 Moreover, many dying plant cells undergo biochemical and morphological changes similar to those in apoptotic mammalian cells, including DNA fragmentation (laddering) and chromatin condensation.⁷

In animal cells, a central core execution switch for apoptosis, which is a defined type of PCD in the animal kingdom, is the activation of caspases (Cysteine-containing Aspartate-specific proteases), which is triggered by the release of cytochrome c (Cty C) from the mitochondrial intermembrane space and mediates the cleavage of a variety of apoptotic proteins, ultimately leading to cell demise.⁸ Similarly, accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although there are many types of plant cell death that do not depend upon caspase-like proteases and do not share aspects of apoptosis.⁹ Here, an overview of the characteristics and the role of caspase-like proteases in plant PCD will be presented, and major methods for detecting the caspase-like protease activity will be comparatively introduced.

Plant caspase-like proteases

There are up to eight distinct caspase-like activities in plants until now, including YVADase,^10,11 DEVDase,^3,10,12 VEIDase,¹³ IETDase,^13,14 VKMDase,¹⁴ LEHDase,¹⁵ TATDase and LEVDase.¹³ Most of these activities have been detected multiple times in various species and in various tissues or cell types. For example, it has been demonstrated that vacuolar processing enzyme (VPE), which has YVADase activity and can cleave caspase-1 substrate (YVAD), is required for tobacco mosaic virus (TMV)-induced cell death in Nicotiana benthamiana.¹⁶ The plant VPE was initially be characterized as active against the substrates ESEN and AAN, and as being involved in various protein maturation processes in seed or leaf.¹⁷ Subsequent research in Arabidopsis has shown that VPE is also required in two more cell death systems: fumonisin-induced cell death and developmental cell death in seed integuments.^11,18 Interestingly, a serine protease with caspase-like activity was also purified and characterized to be associated with victorin-induced PCD in oats (Avena sativa).¹⁹

Recent evidence suggests that some metacaspases were involved in the regulation of plant PCD. Metacaspases, with a greater diversity of caspase-related proteases in sequence and structure, are present in other eukaryotes including yeast, fungi and plants.²⁰ However, several reports suggest that plant metacaspases are unable to cleave caspase substrates and their enzymatic activity is not inhibited by caspase inhibitors.²¹ Although metacaspases do not have caspase-like activities, some evidence have shown that metacaspases have an important role in PCD. For, example, metacaspase-8, a member of the metacaspase gene family, is strongly upregulated by UV-C, H₂O₂ and methyl viologen, and metacaspase-8 KO lines showed a reduced cell death triggered by UV-C or H₂O₂ in protoplasts.²¹

Methods Used to Detect the Activation of Caspase-Like Proteases

Because of the absence of true caspase genes in plants, how to detect caspase-like proteases activity in plant PCD? During the past decade yeas, synthetic fluorogenic substrates and synthetic peptide inhibitors to caspases have been widely used to study caspase-like activity and its functional involvement in plant PCD induced by biotic or abiotc stimuli. Based on the use of the synthetic tetrapeptide fluorogenic substrate to caspase-1 (Ac-YVAD-AMC), caspase-like activity has been demonstrated in extracts from TMV-infected tobacco leaves, and this caspase-like activity could be inhibited with caspase-1 (Ac-YVADCMK) and caspase-3 (Ac-DEVD-CHO) inhibitors but not by caspase unrelated protease inhibitors.²² Likewise, it has also been demonstrated that cytosolic extracts from barley (Hordeum vulgare) embryonic suspension cells exhibited both caspase1-like and caspase-3-like activity by the assay using fluorogenic substrates to caspase-1 (Ac-YVAD-AMC) and caspase-3 (Ac-DEVD-AMC).²³

Recently, employing caspase-1 inhibitors (Ac-YVAD-CHO, biotin-X VAD-fmk, and biotin-YVAD-chloromethylketone) and a caspase-1 substrate (Ac-YVAD-MCA), a vacuole-localized protease called VPE has been identified as a protease that exhibits mammalian caspase-1 activity and is essential for TMV-induced HR in tobacco leaves and fumonisin B1 (FB1)-induced cell death in Arabidopsis plants, although it is structurally unrelated to caspases.^11,16 However, the evidence for the existence of caspase-like proteases in plants is still indirect and largely based on in vitro assays using synthetic caspase-specific inhibitors or fluorogenic substrates, which tell us little about the characteristics of the activation of capase-3-lile proteases at the single cell level.

Recently, the assays performed in incompatible pollen using commercially available fluorescent substrates demonstrated that the caspase-3-like proteases are activated remarkably rapidly during self-incompatibility induced PCD in Papaver pollen. Also in this study, the temporal and spatial activation of caspase-like enzymes has been demonstrated in living cells.¹³ It is possible to detect DEVD activity and to follow activation of caspase-like proteases in vivo using fluorescent caspase substrates and synthetic caspase inhibitors;^11,13,16 however, this tells us little about the characteristics of the activation of caspase-like proteases in specific tissues. Therefore, it is intriguing to develop new strategies for real-time monitoring of the key events of PCD in specific tissues or cells.²⁴

During the past decade, fluorescence resonance energy transfer (FRET) has been proved as a powerful technique to monitor compartmentation and sub-cellular targeting as well as to visualize protein-protein interactions and proteases activity in living cells.²⁵ FRET is a quantum-mechanical process by which the excitation energy is transferred from a donor fluorochrome to a neighboring acceptor fluorochrome. FRET will occur when: (1) the absorption spectrum of the acceptor chromophore overlaps with the fluorescence emission spectrum of the donor, and (2) the proximity of the two chromophores is less than 10 nm.²⁶ During the process of staurosporine-induced apoptosis of COS-7 cells, FRET technique has been successfully employed to reveal the timescale of the activation of capase-3 at the single cell level by measuring the extent of FRET within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e., CFPDEVD-YFP).²⁷ Likewise, FRET has been successfully used to detect caspase 1, caspase 7, caspase 8 and caspase 9 activation during apoptosis in animal cell induced by different stimulis.²⁸

Excitingly, a FRET probe to monitor caspase-3-like proteases activation in single living plant cells in real time has been constructed in Xing's laboratory. Moreover, the activation of caspase-3-like was first detected in vivo by transfecting the Arabidopsis protoplasts with the constructed recombinant caspase substrate.²⁴ This recombinant plasmid is composed of enhanced cyan fluorescent protein (ECFP) as the FRET donor and enhanced yellow fluorescent protein (EYFP) as the acceptor, linked by a peptide containing the sequence DEVD, which have been defined as an optimal cleavage sequence for animal caspase-3. Careful optimization of transfection efficiency, it is clearly demonstrated that the caspase-3-like activity in single living plant cells could be detected in real time during the process of UV-C-induced plant PCD based on the FRET analysis. This method can not only reveal the timescale of the activation of capase-3 at the single cell level but also allow study of the activation of caspase-3-like protease and the key events of PCD in specific tissues or cells using tissue-specific or cell type-specific promoters and transgenic plants.²⁴

Conclusion and Perspective

During the past years, using caspase substrates and caspase inhibitors have brought significant progress in our understanding of the role of caspase like protease in plant PCD. The involvement of caspase-like proteases in the control of cell death activation in plants has been shown in various forms of plant PCD. Some progress has been made towards the identification of the protease involved. However, these evidences about the existence of caspaselike proteases were mainly indirectly, and these methods used to detect the activation of caspase-like proteases tell us little about the characteristics of the activation of capase-lile proteases. Interestingly, in Xing's laboratory, FRET technology has successfully been shown to detect the caspase-3-like protease activity in plant PCD, and this method can not only reveal the timescale of the activation of capase-3 at the single cell level but also allow study of the activation of caspase-3-like protease and the key events of PCD in specific tissues or cells using tissue-specific or cell type-specific promoters and transgenic plants.²⁴

Abbreviations

Cty C

cytochrome c

FRET

fluorescence resonance energy transfer

PCD

programmed cell death

UV-C

ultraviolet-C

VPE

vacuolar processing enzyme

Addendum to: Zhang LR, Xu QX, Xing D, Gao CJ, Xiong HW. Real-time detection of caspase-3-like proteases activation in vivo using fluorescence resonance energy transfer during plant programmed cell death induced by ultraviolet-C overexposure. Plant Physiol. 2009 doi: 10.1104/pp.108.125625. In press.