- Top of page
- Materials and Methods
- Supporting Information
In plants, genetic programmes for cellular suicide are triggered in response to various environmental biotic and abiotic stresses (Danon et al., 2004; Greenberg & Yao, 2004; Lam, 2004). The term programmed cell death (PCD) in plants includes various forms of cell death composed of a number of orderly processes mediated by intracellular signaling molecules, regardless of the triggers or the hallmarks it exhibits (Zhang & Xing, 2008; Li & Xing, 2011). Heat shock (HS), one of the important environmental stresses, could trigger PCD and several apoptosis-like characters upon exposure to HS have been described in plant cells including DNA ladder, fragmentation of the nucleus and the release of cytochrome C (Vacca et al., 2006; Zuppini et al., 2006). In our previous paper, we investigated the reactive oxygen species (ROS) production, changes of mitochondrial function and morphology, as well as the protective roles of HsfA2 (Zhang et al., 2009a). However, the mechanistic analysis of plant response to HS stress, especially the signaling pathway leading to the execution of PCD, is still lacking in many studies.
Previous work has reported the vacuole-localized cysteine proteases called vacuolar processing enzymes (VPEs), which were originally discovered in the maturation of seed storage proteins (Hara-Nishimura et al., 1991). VPEs are endopeptidases with a substrate-specificity towards asparagine residues. They are synthesized as inactive larger pro-protein precursors, from which the C-terminal and N-terminal propeptides are sequentially removed self-catalytically to produce the active mature forms at acidic condition (pH 5.5) (Kuroyanagi et al., 2002). Studies have provided evidence that VPEs are involved in virus-induced hypersensitive cell death in tobacco and exhibit caspase-1-like activity (Hatsugai et al., 2004; Kuroyanagi et al., 2005). Although the two enzymes VPEs and caspase 1 share several structural properties, there is limited sequence identity between them. Full-genome analysis indicates that there are no caspase-encoding genes in Arabidopsis genome; furthermore, the subcellular localizations of the two proteases are different: VPEs are localized in the vacuoles, unlike animal caspases which are localized in the cytosol (Hatsugai et al., 2006).
The Arabidopsis genome has four VPE genes: αVPE, βVPE, γVPE and δVPE, which can be separated into two subfamilies: vegetative-type VPEs and seed-type VPEs. αVPE and γVPE are expressed in vegetative organs, whereas βVPE and δVPE are expressed in seeds (Kinoshita et al., 1999; Gruis et al., 2002, 2004). The βVPE is essential for the proper processing of storage proteins (Shimada et al., 2003), and δVPE specifically expressed in the seed coat is associated with cell death (Nakagami et al., 2005). By contrast, the vegetative αVPE and γVPE are upregulated during wounding, senescence and pathogen infection, and may play vital roles in various types of cell death in plants (Kinoshita et al., 1999; Yamada et al., 2004). Recently, VPEs have been identified as plant-specific caspases and a VPE-mediated vacuolar system has been considered as a cellular suicide strategy in plant development and cell death programmes (Hatsugai et al., 2004, 2006). Previous research into plant VPEs has mostly focused on plant senescence, terminal differentiation and pathogen-induced hypersensitive cell death. By contrast, the molecular mechanisms underlying the roles of VPEs in response to abiotic stresses are poorly understood.
Alteration to the phosphorylation state of proteins plays a central role in cellular signal transduction. Mitogen-activated protein kinase (MAPK) cascades are conserved pathways by which extracellular stimuli can be transduced into intracellular responses in all eukaryotic cells (Widmann et al., 1999; Davis, 2000; Kyriakis & Avruch, 2001; Tena et al., 2001; Zhang & Klessig, 2001; Asai et al., 2002; Nakagami et al., 2005). Each MAPK cascade minimally consists of three kinases: MAPKKK, MAPKK and MAPK. In the Arabidopsis genome, 20 MAPKs, 10 MAPKKs and 60 MAPKKKs have been identified (Asai et al., 2002; Ichimura et al., 2002). It is well documented that MAPK plays key roles in the regulation of innate immunity and adverse stress responses (Ichimura et al., 2002; Xing et al., 2008). Under HS treatment, MAPK activity has also been detected (Chen et al., 2008), but it remains to establish a specific MAPK cascade which mediates the response to HS stress. A decade ago, MKK1 was first identified as a member of the group of phosphorelay signaling pathway that controls MAPK activation (Morris et al., 1997). Recent investigations have verified a MAPK cascade, extending from MEKK1 through MKK1/2 to MPK4/6 in response to abscissic acid and environmental stresses including cold and high salinity (Teige et al., 2004; Xing et al., 2008). MPK6, as a well-characterized terminal of MAPK cascade in Arabidopsis, can be activated by various environmental stresses and participate in the regulation of several functional proteins including catalase, nitrate reductase, ethylene response factor 104 and so on (Morris et al., 1997; Teige et al., 2004; Xing et al., 2008, 2009; Bethke et al., 2009; Wang et al., 2010).
ROS and calcium (Ca2+) are believed to be the key signaling molecules in plant cells (Fluhr & Bowler, 2000; Romeis et al., 2001), and previous reports have implicated both of them in the activation of MAPK cascades under various stimuli (Xing et al., 2008; Wang et al., 2010). Calmodulin (CaM), an ubiquitous second messenger, acts as the crucial sensor protein in plant signal transduction. In Arabidopsis, CaM has several isoforms and different isoforms can interact with their particular targets upon the different exogenous stimuli. For example, CaM is believed to be necessary for the cellular signaling transduction in Arabidopsis response to cold and heat stresses (Gong et al., 1997; Tahtiharju et al., 1997). Among the isoforms of CaM, CaM3 is considered as a key component of Arabidopsis HS signaling pathway, and the Ca2+-CaM3 cascade participates in the activation of downstream functional proteins under HS treatment (Gong et al., 1997; Xuan et al., 2010).
In this paper, the possible molecular mechanisms underlying the process of VPE-mediated PCD under HS treatment were investigated. Our result indicated that γVPE, among the four Arabidopsis VPEs, was upregulated by HS treatment and played an important role in HS-induced Arabidopsis PCD. Moreover, a MPK6-modulated signaling cascade was demonstrated to be responsible for the activation of γVPE under HS stress.
- Top of page
- Materials and Methods
- Supporting Information
This study is an attempt to understand the possible molecular mechanisms underlying the cellular process of VPE-mediated Arabidopsis PCD induced by HS treatment. The results shown in this work provide evidence for the cellular signaling cascade of the activation and function of VPEs in Arabidopsis response to HS stress.
High temperature is one of the limiting factors for plants growth (Zuppini et al., 2006; Zhang et al., 2009a), and recent studies have reported that HS challenge can cause several apoptosis-like characters and trigger PCD in plant cells (Vacca et al., 2006; Zuppini et al., 2006; Zhang et al., 2009a). In both animal and plant PCD, the caspase-like activation is believed to be the key and final step, and the activation of caspase-3-like protease, which is considered as the major executioner of animal PCD, has been detected under UV-C and aluminum stresses (Zhang et al., 2009b; Li & Xing, 2011). The present study observed the caspase-3-like activation in HS-induced Arabidopsis death, indicating the vital role of caspase-3-like protease in the Arabidopsis HS response (Fig. 4).
The vacuole may be involved in the process of plant development and stress response (Hatsugai et al., 2004), and ultrastructural analysis has shown that the vacuole is the organelle site most sensitive to HS treatment (Jones, 2001). In plants, a vacuole-localized cysteine protease called VPE has been identified and experimental evidence has demonstrated that VPEs exhibit caspase-1-like activity and participate in development, senescence, hypersensitive cell death and hormone signaling (Kinoshita et al., 1999; Hatsugai et al., 2004, 2006; Kuroyanagi et al., 2005). Recent work has further reported that a cellular suicide strategy mediated by the vacuole and VPEs can regulate the development and cell death programmes in plants (Hatsugai et al., 2004). In our work, it was found that HS caused γVPE activation and vacuolar disruption (Figs 2, 3), suggesting the possible involvement of γVPE-mediated vacuolar system in HS-induced Arabidopsis PCD.
In animal apoptosis, there are several kinds of caspases with different functions. Compared with caspase 3, which acts as the executioner of PCD to induce DNA fragmentation and chromatin condensation, several other caspases (such as caspase 8 and caspase 1) can function as mediators activating downstream signaling cascades of PCD (Fan et al., 2005). Our data showed that γVPE might exhibit caspase-1-like activity (Fig. S4) and promote vacuolar disruption and caspase-3-like activation in Arabidopsis PCD induced by HS (Figs 1, 3, 4, S3). The vacuole contains many hydrolases and proteases which can lyse proteins and cellular compartments to regulate the process of plant senescence and death (Hatsugai et al., 2004; Muntz, 2007); notably, in the vacuole-mediated PCD signaling pathway, VPEs function as the key molecules through processing these hydrolases and proteases and disrupting the vacuole (Kuroyanagi et al., 2002; Hatsugai et al., 2004). Hence, it was supposed that γVPE or other vacuolar enzymes released into the cytosol from the vacuole through γVPE-mediated vacuolar disruption, functioned in the activation of a downstream caspase-like pathway in HS-induced Arabidopsis PCD.
In the vacuole-mediated PCD pathway, the upstream signaling cascades of VPE activation are important components but remain unclear. MAPK cascades can be activated by various stimuli and play central roles in the process whereby extracellular stimuli are transduced into intracellular responses (Widmann et al., 1999; Davis, 2000; Kyriakis & Avruch, 2001; Tena et al., 2001; Zhang & Klessig, 2001; Asai et al., 2002; Nakagami et al., 2005). Besides their protective roles, MAPK cascades also can function as negative regulators in plant stress response. For example, MKK7 is shown to negatively regulate polar auxin transport and a mutation in MKK9 results in the enhanced stress tolerance and alleviated senescence in plants (Dai et al., 2006; Alzwiya & Morris, 2007; Zhou et al., 2009). Furthermore, Li et al. (2007) report that a 56-KD MAPK protein mediates self-incompatibility-induced PCD by activating caspase-like activity. Our experiment demonstrated that MAPK cascades also participated in the activation of γVPE in HS-induced PCD (Fig. 5). Among various kinds of MAPK proteins, MPK6 is a well-established signaling protein in Arabidopsis, which can be activated by various stimuli, including low temperature, wounding, heavy metals, drought, oxidative stress and plant hormones (Morris et al., 1997; Teige et al., 2004; Xing et al., 2008, 2009; Wang et al., 2010). In HS-treated Arabidopsis, the activation of MPK6 was proved to be responsible for the upregulation of γVPE activity and the subsequent execution of PCD (Figs 6–8).
ROS and Ca2+, as important signal messengers in plant cells, can function in the upstream activation of MAPK cascade under various stimuli (Fluhr & Bowler, 2000; Romeis et al., 2001; Xing et al., 2008; Wang et al., 2010). Under HS stress, ROS production and cytoplasmic calcium concentration ([Ca2+]cyt) increase are early events (Gong et al., 1998; Zhang et al., 2009a), and the Ca2+-CaM3 cascade can regulate plant HS response through activating downstream signal transduction (Gong et al., 1997; Xuan et al., 2010). Our data presented a cellular signaling cascade, composed of ROS production, [Ca2+]cyt increase and the upregulation of CaM3 transcript level, which functioned in the upstream activation of MPK6 in response to HS (Figs S5–S9, S11, S12; Methods S4, S5; Notes S3, S4, S6, S7).
In conclusion, our data show that MPK6-mediated activation of Arabidopsis γVPE modulated HS-induced Arabidopsis PCD. According to our experimental results, a potential cascade of cellular events during HS-induced PCD occurred (Fig. 9): HS treatment caused ROS production and increase of [Ca2+]cyt, and the Ca2+-CaM3 cascade, in turn, activated MPK6 protein; then the activated MPK6 protein upregulated the transcript level of γVPE, resulting in the accumulation of inactive VPE precursors in the vacuole which can self-process into activated VPEs; subsequently, activated VPEs processed vacuolar hydrolases and proteases and disrupted the vacuole to promote the caspase-3-like activation. These results suggested a possible molecular mechanism underlying the process of HS-induced PCD, and provided a new insight into the cellular signaling cascade of plant VPEs.
Figure 9. Proposed working model for MPK6-dependent activation of γVPE in heat shock-induced programmed cell death (PCD). VPE, vacuolar processing enzyme.
Download figure to PowerPoint
- Top of page
- Materials and Methods
- Supporting Information
Fig. S1 Difference in Vacuolar processing enzyme (VPE) activity of detached leaves of WT, vpe and γvpe at 6 h recovery period after heat shock (HS) treatment.
Fig. S2 Identification of mpk3-1, mpk4-1, mpk6-2, mpk6-3, MPK6-OE and cam3 insertion mutants using semi-quantitative RT-PCR analysis.
Fig. S3 Effects of Vacuolar processing enzyme and caspase-1 inhibitors on the HS-induced vacuolar rupture and caspase-3-like activation in Arabidopsis.
Fig. S4 HS-induced activation of caspase-1-like activity in detached Arabidopsis leaves.
Fig. S5 Imaging of ROS production and cytoplasmic Ca2+ content in HS-treated Arabidopsis wild type protoplasts.
Fig. S6 Estimation of HS-induced changes of ROS and Ca2+ content by flow cytometry analysis using fluorescence probes H2DCFDA and Fluo-3 respectively.
Fig. S7 Kinetics of changes in ROS and Ca2+ content by flow cytometry analysis after HS treatment.
Fig. S8 Change of CaM3 transcript level in detached Arabidopsis leaves under HS stress.
Fig. S9 Roles of Ca2+-CaM3 in HS-induced activation of MPK6.
Fig. S10 Effect of HS treatment on the growth of Arabidopsis roots.
Fig. S11 Kinetics of changes in ROS level by flow cytometry analysis in mpk6-2 and mpk6-3 protoplasts after HS treatment.
Fig. S12 Effect of PD98059 on HS-induced MPK6 activation in detached Arabidopsis leaves.
Table S1 Quantitative analysis of vacuolar state in wild-type protoplasts after HS treatment
Table S2 Primers for several genes
Table S3 The ATG numbers for the cited genes
Methods S1 Chemical reagents.
Methods S2 Identification of Arabidopsis mutants using semi-quantitative RT-PCR.
Methods S3 Detection of caspase-1-like activity.
Methods S4 Confocal microscopy observation.
Methods S5 Flow cytometry analysis.
Methods S6 Phenotypic analysis of root growth.
Methods S7 Treatment with VPE and caspase-1 inhibitors.
Notes S1 Identification of T-DNA insertion mutants.
Notes S2 Effects of VPE and caspase-1 inhibitors on the HS-induced vacuolar rupture and PCD in Arabidopsis.
Notes S3 ROS production and cytoplasmic calcium concentration ([Ca2+]cyt) increase in HS-treated protoplasts.
Notes S4 Ca2+-CaM3 cascade functions upstream of MPK6 activation under HS treatment.
Notes S5 Effect of HS treatment on the growth of Arabidopsis roots.
Notes S6 Flow cytometry analysis of the ROS production in mpk6-2 and mpk6-3.
Notes S7 Effect of PD98059 on the HS-induced MPK6 activation.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.