Relationship between petal abscission and programmed cell death in Prunus yedoensis and Delphinium belladonna

Abstract

Depending on the species, the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid. Wilting at the end of petal life is due to programmed cell death (PCD). It is not known whether the abscission of turgid petals is preceded by PCD. We studied some parameters that indicate PCD: chromatin condensation, a decrease in nuclear diameter, DNA fragmentation, and DNA content per nucleus, using Prunus yedoensis and Delphinium belladonna which both show abscission of turgid petals at the end of floral life. No DNA degradation, no chromatin condensation, and no change in nuclear volume was observed in P. yedoensis petals, prior to abscission. In abscising D. belladonna petals, in contrast, considerable DNA degradation was found, chromatin was condensed and the nuclear volume considerably reduced. Following abscission, the nuclear area in both species drastically increased, and the chromatin became unevenly distributed. Similar chromatin changes were observed after dehydration (24 h at 60 degrees C) of petals severed at the time of flower opening, and in dehydrated petals of Ipomoea nil and Petunia hybrida, severed at the time of flower opening. In these flowers the petal life span is terminated by wilting rather than abscission. It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators, whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission. Dehydration of the petal cells, after abscission, was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration. This nuclear morphology has apparently not been described previously, for any organism.

Fig. 1

Petal abscission and petal dehydration in P. yedoensis, indicated by a P (a) and D.belladonna, indicated D (b). Cut branches of (P. yedoensis) and potted plants (D.belladonna) stood in a growth chamber (24°C, about 70% RH). The time to abscission and to dehydration symptoms (in days from full flower opening) is shown in the left lower corner of the pictures (n = 6). Stages P1 and D1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated. All pictures have the same scale (bars = 10 mm)

Fig. 2

Agarose gel analysis of total DNA isolated from the petals of P. yedoensis, annotated as P (a) and D. belladonna, annotated as D (b). 3 μg of total DNA was extracted from the petals and electrophoresed in a 3% agarose gel. Lanes P1–P4 and SD1–SD4 refer to DNA isolated from petals at stages P1–P4 and D1–D4 as shown in Fig. 1. Stages P1 and D1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated. Pixel ratios below the lanes are means of two repeat experiments

Fig. 3

Morphology of DAPI-stained nuclei in the petals of P. yedoensis, indicated by a P (a) and D.belladonna, indicated D (b). Two representative nuclei (-1 and -2) are shown at each stage of development. Nuclei were isolated from the petals and observed using a fluorescence microscope under U-excitation. Bars: 5 μm (a) or 15 μm (b). Stages as in Fig 1. P1 and D1 1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated

Fig. 4

Fluorescence of DAPI-stained nuclei. Histograms were obtained by flow cytometry of 5,000 nuclei isolated from the petals of P. yedoensis (indicated by a P) as shown in a, and petals of D.belladonna (D) shown in b. Petals were severed from cut twigs placed in water (Prunus) or potted plants (Delphinium), which stood both in a growth chamber (24°C, about 70% RH). Stages as in Fig. 1. P1 and D1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated

Fig. 5

Effect of ethylene on petal abscission and DNA fluorescence of nuclei in the petals of P. yedoensis and D.belladonna. Time to petal abscission and petal dehydration symptoms (a) and flow cytometric determination of 5,000 DAPI-stained nuclei (b) of ethylene-treated branches of P. yedoensis and potted plants of D.belladonna. Ethylene treatment is annotated by E. Compare stages of development with untreated controls shown in Fig. 1, and with fluorescence data of controls in Fig.4. The left lower corner of the pictures series a show the times to abscission (EP2 and ED2) and the time to dehydration symptoms (both expressed in days from full flower opening; n = 6). Stages P1 and D1: full flower opening; EP2 and ED2, petal abscission; EP3 and ED3, onset of petal dehydration; EP4 and ED4, petals desiccated. All pictures have the same scale (bars = 10 mm)

Fig. 6

Effect of dehydration on DNA degradation, DNA fluorescence of nuclei, and nuclear morphology in the petals of P. yedoensis (P) and D.belladonna (D). Dehydration in the two species is indicated as DP and DD, respectively. a Flower morphology before (P1 and D1) and after (DP and DD) dehydration at 60°C for 24 h. Photographs show small branches with fully opened flowers and desiccated flowers of P. yedoensis (P1 and DP) and the same for individual flowers of D.belladonna (D1 and DD). All pictures have the same scale (bars = 10 mm). b Agarose gel analysis of total DNA isolated from the petals. c Flow cytometric determination of nuclei isolated from the petals. d Morphology of nuclei in the petals. Two representative nuclei (-1 and -2) are shown for each species, after the dehydration treatment (DP and DD). Bars = 5 μm (P1 and DP) or 15 μm (D1 and DD)

Fig. 7

Effect of dehydration on fresh weight, DNA degradation, and DNA fluorescence of nuclei in the petals of I. nil and P. hybrida. Potted plants were grown in a greenhouse and then placed in a climate-controlled chamber (24°C, about 70% RH). Petals were detached from flowers that had just opened, and were placed at 60°C for 24 h to dehydrate. In a the time until about half of each petal had wilted (in days from full flower opening; n = 6) is shown in the left lower corner of the pictures. a Morphology of the flowers, at opening (left) and at petal wilting (right). All pictures have the same scale (bars = 10 mm). b Agarose gel analysis of total DNA isolated from the petals. c, d Flow cytometric determination of 5,000 DAPI-stained nuclei isolated from the petals of I. nil (c) and P. hybrida (d). Ip, Ipomoea; Pe, Petunia, D, dehydration

Fig. 8

Effect of dehydration on nuclear morphology in the petals of I. nil and P. hybrida. Nuclei were isolated from the petals of I. nil (a) and P. hybrida (b) after the indicated treatments, then observed using a fluorescence microscope under U-excitation. For comparison, the nuclei are shown during PCD of petals that remained attached to the plant (for data of Ipomoea and Petunia see also Yamada et al. 2006a, b). Stage S1: full flower opening; S2: onset of petal wilting; S3: full petal wilting; and S4: petals desiccated but still attached to the flower. Bars = 10 (μm