Apoptotic-like cell death marks the early stages of gypsophila (Gypsophila paniculata) petal senescence

Abstract

Flower senescence is accompanied by the rapid death of large numbers of cells. In situ DNA degradation was studied in gypsophila (Gypsophila paniculata) petals during flower opening and senescence, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). In flowers that had just opened, TUNEL staining was exclusively observed in nuclei in the vicinity of (developing) vessels, presumably reflecting xylem vessel differentiation. In fully open flowers, TUNEL positive nuclei appear in all the cells well before the increase in ethylene production and visible signs of senescence. Occurrence of TUNEL positive nuclei was stimulated by ethylene but not prevented by silver thiosulphate. The results show that gypsophila petal senescence is a form of PCD with features of animal apoptosis and suggest that nuclear DNA degradation is an early regulatory event rather than a result of massive cell death in the final stage of senescence.

Introduction

Petal senescence is a highly regulated process that marks the final stage of a flower's lifespan. In general, senescence is viewed as a programmed series of events, leading to the degradation and remobilisation of proteins, lipids, and nucleic acids and, ultimately leading to cell death. Petal senescence complies with this general view as extensive breakdown of proteins, lipids and nucleic acids, as well as mobilisation of the released nutrients from senescing petals to other parts of the same flower (e.g. the developing ovary) have been observed (reviewed in Rubinstein, 2000).

Senescence of flower petals (and other plant organs) also conforms with the general definition of programmed cell death (PCD). The term PCD is used for cell death that is part of the normal life of organisms. It refers to any process by which cells are eliminated as part of a developmental or adaptive event in the life cycle of organisms. PCD is found throughout animal and plant kingdoms (Greenberg, 1996, Raff, 1998).

Most cases of PCD described in animals have the appearance of apoptosis, which is characterised by specific morphological and biochemical features such as cell shrinkage, condensation and fragmentation of the nucleus, internucleosomal DNA cleavage (DNA laddering), and phagocytosis (Hengartner, 2000). It is still a matter of debate if, or to what extend the mechanisms of PCD and, more specifically, apoptosis are conserved across kingdoms. Morphological and biochemical similarities have been found between animal cells undergoing apoptosis and dying plant cells (reviewed in Hoeberichts and Woltering, 2003) and, although only few homologs of animal apoptotic genes have been identified in plants so far, it has been suggested that a certain degree of functional conservation between cell death pathways in animals and plants exists (Lam et al., 2001, Woltering et al., 2002, Hoeberichts and Woltering, 2003).

The involvement of apoptotic-like cell death during flower petal senescence has been studied by other authors. Internucleosomal DNA fragmentation, a widely used marker for (apoptotic-like) PCD, has been detected during petal senescence in pea, petunia, freesia, alstroemeria, and gladiolus (Orzáez and Granell, 1997, Xu and Hanson, 2000, Yamada et al., 2001, Yamada et al., 2003, Wagstaff et al., 2003). In all of these cases, this phenomenon was observed during the advanced stages of senescence and it is hard to decide if the breakdown of nuclear DNA is a programmed event or a result of decompartmentalisation following cell collapse. However, microscopic analysis in Sandersonia aurantiaca, iris, and alstroemeria, have revealed that mesophyll cells in petal or tepal tissue may degrade before senescence becomes visible to the naked eye, whilst epidermal cells appear to remain intact (Bailly et al., 2001, O’Donoghue et al., 2002, Van der Kop et al., 2003, Wagstaff et al., 2003). This indicates that cell death occurs early in petal senescence.

Gypsophila flowers are small structures containing 20–30 individual flower petals of 2–3 mm in length and about five cell layers thick making them most suitable to study whole mount in situ DNA degradation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Gypsophila belongs to the caryophyllaceae subfamily that also includes carnation (Dianthus caryophyllus). Gypsophila flowers are highly sensitive to ethylene and display a transient increase in ethylene production at the onset of senescence (Woltering and Van Doorn, 1988, Van Doorn and Reid, 1992).

Here, we show that nuclear DNA degradation in gypsophila petals occurs well before the increase in ethylene production and the associated visible signs of senescence, indicating the involvement of apoptotic-like cell death in flower petal senescence.