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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.

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