Downregulating a Type I Metacaspase in Petunia Accelerates Flower Senescence

in Journal of the American Society for Horticultural Science

Metacaspases are cysteine proteases from plants, fungi, and protozoans that have structural similarity to metazoan caspases. They play a critical role in programmed cell death (PCD) induced by developmental cues and environmental signals. In this study, a type I metacaspase (PhMC1) was identified and characterized from Petunia ×hybrida ‘Mitchell Diploid’ (MD) (petunia). The recombinant PhMC1 had activity against the metacaspase substrate Boc-GRR-AMC (GRR). Activity was highest at pH 7–9 and was dependent on the active site C237. Quantitative polymerase chain reaction (qPCR) showed that PhMC1 transcripts increased at a later stage of petal development, when corollas were visibly senescent in both pollinated and unpollinated flowers. Gene expression patterns were similar to that of the senescence-related gene PhCP10, a homolog of Arabidopsis thaliana (arabidopsis) AtSAG12. PhMC1 transcripts were upregulated in the petals by ethylene treatment. This ethylene regulation did not require protein synthesis, indicating that PhMC1 is a primary ethylene response gene. Metacaspase-like activity against Boc-GRR-AMC increased in protein extracts from senescing petals. RNAi was used to knock down the expression of PhMC1. Transgenic PhMC1 petunias had no abnormal, vegetative growth phenotypes under normal greenhouse conditions, but flower senescence was accelerated by an average of 2 days.

Contributor Notes

This research was funded by an OARDC SEED Grant, The Ohio State University D.C. Kiplinger Endowment, and the Floriculture and Nursery Research Initiative. Salaries and research support were provided in part by State and Federal funds appropriated to the OARDC, The Ohio State University. Journal Article Number HCS 17-08.

Current address: Division of Plant and Soil Science, West Virginia University, Morgantown, WV 26506.

Corresponding author. E-mail: jones.1968@osu.edu.

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Article Figures

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    Characterization of substrate specificity and pH optima for Petunia ×hybrida metacaspase1 (PhMC1). Relative enzymatic activity in each sample is presented as the percentage of the activity compared with the sample with the highest activity. All data are the means of three replicates ±se (n = 3). The experiment was performed twice with similar results. Different letters represent statistically significant difference between the means (P ≤ 0.05). (A) The activity of the recombinant PhMC1 against the fluorogenic metacaspase substrate Boc-GRR-AMC (GRR) and the fluorogenic caspase substrate Ac-DEVD-AMC (DEVD) was determined at various pH. (B) The effect of cysteine protease inhibitor (iodoacetamide) and arginal protease inhibitor (leupeptin) on the activity of recombinant PhMC1 using GRR as substrate was evaluated at pH 8.0. All GRRase activity of the recombinant metacaspases in the presence of inhibitors is expressed as a percentage of the relative activity without inhibitors. (C) A recombinant metacaspase mutant, PhMC1C237A, was created with the catalytic cysteine converted to alanine. The activity of this mutant was determined using the substrate GRR at pH 8.0.

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    Gene expression was analyzed in petals of unpollinated Petunia ×hybrida ‘Mitchell Diploid’ flowers. (A) Photos show flowers from anthesis (0 d) through senescence and corolla wilting (7 d). Numbers indicate the days after anthesis. Transcript levels of (B) PhMC1 and (C) PhCP10 (mean ± sd, n = 3) in petals during flower development. Relative mRNA abundance compared with two reference genes (PhACTIN and PhRPS13) was determined by reverse transcription quantitative polymerase chain reaction. Different letters represent statistically significant difference between the means (P ≤ 0.05).

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    Gene expression was analyzed in petals of pollinated Petunia ×hybrida ‘Mitchell Diploid’ flowers. (A) Photos show flowers at 0, 12, 24, and 48 h after pollination. Transcript levels of (B) PhMC1 and (C) PhCP10 (mean ± sd, n = 3) in petals from pollinated flowers. Relative mRNA abundance compared with two reference genes (PhACTIN and PhRPS13) was determined by reverse transcription quantitative polymerase chain reaction. Different letters represent statistically significant difference between the means (P ≤ 0.05).

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    Gene expression was analyzed in Petunia ×hybrida ‘Mitchell Diploid’ petals from flowers treated with ethylene following pretreatment with cycloheximide or water. Detached flowers were treated with 0, 0.1, 1.0, or 10.0 µL·L−1 ethylene for 4 h after pretreating with either 0 (water) or 50 µm cycloheximide for 3 h (n = 6). Transcript levels of (A) PhMC1 and (B) PhCP10 (mean ± sd, n = 3) in petals. Relative mRNA abundance compared with two reference genes (PhACTIN and PhRPS13) was determined by reverse transcription quantitative polymerase chain reaction. Different letters represent statistically significant difference between the means (P ≤ 0.05).

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    Metacaspase-like activity was quantified in Petunia ×hybrida ‘Mitchell Diploid’ petals. Activity against the fluorogenic metacaspase substrate Boc-GRR-AMC was analyzed in total petal protein extracts from three flower developmental stages (anthesis, nonnsenescing, and senescing). Fluorescence units are reported and bars represent the mean ± sd (n = 3). Different letters represent statistically significant difference between the means (P ≤ 0.05).

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    Corolla senescence was evaluated in Petunia ×hybrida PhMC1 RNAi transgenic lines. The time to flower senescence and PhMC1 gene expression were evaluated in T2 plants from three independent transgenic lines (F05-53, F05-6, and F05-65) compared with ‘Mitchell Diploid’ (MD, wild type). (A) Flower longevity was determined as the time from flower opening (anthesis) until corolla wilting in unpollinated, naturally senescing flowers (n = 15). (B) Relative gene expression of PhMC1 in corollas at 48 h after pollination (n = 3) was determined by reverse transcription quantitative polymerase chain reaction. Transcript levels are expressed relative to PhACTIN and PhRPS13. Bars represent mean ± sd, and different letters represent statistically significant difference between the means (P ≤ 0.05).

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    Multiple alignment of predicated metacaspase amino acid sequences from Petunia ×hybrida PhMC1 (GenBank JQ314004) and Arabidopsis thaliana (Arabidopsis AtMC1–9; AtMC1, AY322525; AtMC2, AY322526; AtMC3, AY322527; AtMC 4, AY322528; AtMC5, AY322532; AtMC6, AY322533; AtMC7, AY322529; AtMC8, AY322530; AtMC9, AY322531. Cysteine residues in the zinc finger motif of the prodomain of type I metacaspases are indicated in yellow and by an open triangle (∆). The putative histadine and cysteine catalytic residues are designated in dark blue and by a closed diamond (♦). The second cysteine involved in activity and autolysis is green and indicated by a closed square (▪). The lysine or arginine involved in the predicted autocatalytic cleavage site is pink and indicated by a closed circle (•). Sequence alignment was performed using Clustal Omega (1.2.4) multiple sequence alignment software (Larkin et al., 2007). Identical amino acids are indicated with as asterisk. Double dots indicate conserved substitutions and single dots indicate semi-conserved substitutions.

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