Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

Abstract

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth Xowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and Xow cytometry for DAPI Xuorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic Xuoroprobe DiOC 6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA Xuorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no eVect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.

Gene expression in opening and senescing petals of morning glory (Ipomoea nil) flowers

“…An increase in the number of DNA masses with decreased fluorescence started at about t = 12 and was still found between t = 24 and 30 h (Yamada et al 2006). In the present experiments, the DAPI fluorescence of 5,000 DNA masses was determined at t = 30 h. At this time the total amount of DNA in the untreated control has become degraded to about half, and the number of DNA masses has considerably increased (Yamada et al 2006). ActD inhibited the measured DAPI staining parameter, if the onset of treatment was t = 0 or 2 h. If ActD treatment started later, the effect gradually decreased.…”

“…These parameters include: an increase in the activity of a variety of degradative enzymes, a decrease in lipid, protein, and nucleic acid levels, a larger number of DNA masses with decreased 4,6-diamino-2-phenylindole (DAPI) staining, showing less DNA per DNA mass (nucleus), an increase in the number of DNA masses, rupture of the tonoplast, and degradation of the cell walls (Winkenbach 1970;Yamada et al 2006) Effect of actinomycin D We tested the effect of ActD on petal opening and petal inrolling. ActD was supplied continuously.…”

“…4. A color change started from t = 3 h and was clear by t = 5 h. Inward rolling started about t = 9 h and went on until at least t = 22 h. Using agarose gels, DNA degradation was observed from t = 9 to 30 h. Apparent nuclear fragmentation probably started at about t = 0 h but was clearly shown between t = 12 and 18 h. It persisted at least until t = 24 h (Yamada et al 2006). Expression of SAGs was related to visible senescence symptoms and some PCD parameters.…”

“…The expression of these two genes was induced by drought stress (Koizumi et al 1993), but it is not clear if this occurred independently of water-stress induced senescence. (Yamada et al 2006) In29 encoded a putative leucine-rich repeat transmembrane receptor protein kinase. Since other protein kinases are involved in signal transduction, it is tempting to speculate that the present protein has a similar function.…”

“…This species and the closely related I. tricolor have been used in several previous studies on petal senescence (Matile and Winkenbach 1971;Kende and Baumgartner 1974;Baumgartner et al 1975;Yamada et al 2006). We previously determined the time line of various visible symptoms of programmed cell death (PCD; here taken to be synonymous to senescence) and processes such as DNA degradation, chromatin condensation, and nuclear fragmentation, during petal PCD in I. nil (Yamada et al 2006). The purpose of the present paper was to relate the expression of identified SAGs to the visible senescence symptoms and to some of the known changes at the cellular level.…”