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Activities of nucleases in senescing daylily petals
Plant Physiol. Biochem. 38 (2000) 837−843 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S098194280001192X/FLA
Activities of nucleases in senescing daylily petals Tadas Panavasa,b§, Rebbecca LeVangiec, John Mistlera, Philip D. Reidc, Bernard Rubinsteina,b* a
Department of Biology, University of Massachusetts, Amherst, MA 01003-5810, USA
b
Plant Biology Graduate Program, University of Massachusetts, Amherst, MA 01003-5810, USA
c
Department of Biological Sciences, Smith College, Northampton, MA 01063, USA
Received 3 January 2000; accepted 17 July 2000 Abstract – The activity of nucleases during organ death was investigated using daylily petals (Hemerocallis hybrid cv. Stella d’Oro), in which the processes associated with senescence are rapid and clearly ordered. The number of nuclei with fragmented DNA as well as activities of various nucleases increase before certain other events that are related to senescence. Furthermore, DNA breakage and activities of nucleases occur earlier when senescence is accelerated by abscisic acid and occur later when senescence is retarded by cycloheximide. These results suggest that the activities of nucleases contribute to the senescence of daylily petals. Therefore, studying the regulation of nuclease gene expression may be useful for understanding components of the signal transduction system that leads to the death of these organs. © 2000 Éditions scientifiques et médicales Elsevier SAS abscisic acid / daylily flower senescence / DNA breakage / Hemerocallis / nuclease ABA, abscisic acid / CHI, cycloheximide / DS, double stranded / PCD, programmed cell death / SS, single stranded / TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
1. INTRODUCTION The program leading to cell death (PCD) in plants, like that in animals [10], involves a series of overlapping and often interacting processes, some of which may only be incidental to the death that eventually occurs [14, 19, 20, 29]. One approach for understanding more thoroughly the mechanisms that are closely involved with PCD of plant organs is to characterize one pathway that appears to be a factor leading to cell death. A decrease in extractable RNA and DNA [6, 16, 29], the degradation of nuclear DNA (e.g. [2, 8, 17, 21, 35, 37]) and an upregulation of enzymes that degrade nucleic acids (e.g. [7, 15, 16, 30]) suggest that the appearance of various nuclease activities may be an important component of PCD. * Correspondence and reprints: fax +1 413 545 3243. E-mail address: [email protected] (Bernard Rubinstein). § Present address: Department of Plant Pathology, University of Kentucky, S-305, Agr. Sci. Bldg.-N, Lexington, KY 40546, USA.
A requirement for determining a role for any particular mechanism related to organ PCD is the selection of an amenable plant system in which the time course of senescence can be well-defined. The senescence of petals, especially those from the ephemeral flowers of daylily, is one such system. Although genetic manipulation is difficult, the petals senesce rapidly and synchronously, and the biochemical and molecular processes investigated so far occur in a fixed order that can be easily related to morphological changes associated with senescence [4, 13, 25]. In this paper, we are asking whether nuclease activities represent an important and easily detectable component of the program for cell death in daylily petals. We report that breakage of nuclear DNA increases even before the flowers open, and that upregulation of various nuclease activities occurs at about the same time. Furthermore, the fragmentation of nuclear DNA and the activities of nucleases occur prematurely after addition of abscisic acid (ABA), which stimulates senescence, and are delayed in the presence of cycloheximide (CHI), which retards senescence.
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2. RESULTS 2.1. Evidence for breakage of nuclear DNA A role for nuclease activity in PCD is indicated by the TUNEL assay, in which fragmented nuclear DNA can be observed as brown, compared to blue-stained nuclei. At 30 h before the flower opens (–30 h), an average of about 10 % of the petal nuclei stain positively for breaks in DNA (figure 1A). This value increases to about 20 % by –6 to +6 h and then increases sharply at +18 h. The trends are similar regardless of whether the abaxial or adaxial epidermis, or mesophyll cells are examined. Because of possible artifacts due to the TUNEL assay after tissues are embedded in paraffin [33], we compared the data from figure 1A with those obtained from cryo-sectioned material. The results are nearly identical. Average values of the three tissues for TUNEL-positive nuclei at –6 h in the cryo-sectioned material is 16 ± 5 %, compared to 20 ± 7 % for paraffinembedded sections. At +18 h, the cryo-sectioned material has 40 ± 8 % TUNEL-positive nuclei and 44 ± 14 % for paraffin-embedded. This represents an increase from –6 to +18 h of 2.5 and 2.2 times for cryosectioned and paraffin-embedded tissue, respectively. Furthermore, cryo-sectioned, like paraffin-embedded, material shows no clear differences between the cells of different petal tissues. We also studied the effects of a stimulator of daylily PCD, abscisic (ABA), and a PCD inhibitor, cycloheximide (CHI), on the numbers of TUNEL-positive nuclei. The data in figure 1B indicate that after ABA is applied to the petals from –30 to –6 h, the number of TUNEL-positive nuclei is twice the value of controls at –6 h; in fact, the values for ABA-treated petals approach those seen in the controls 24 h later (at +18 h). There appears to be no significant difference between the two epidermides and the mesophyll cells. The addition of CHI at –6 h (figure 1C) results in only a small increase in TUNEL-positive nuclei by +18 h compared to controls at –6 h (figure 1B), and clearly below the levels seen in the +18-h controls. There are also no significant differences between the cells of various tissues after CHI applications (figure 1C). One effect of increased nuclease activity in daylily petals could be the degradation of nuclear DNA into oligonucleosomal fragments. There is a smear at the top of the ethidium bromide gel (figure 2B) and in the Southern blot (figure 2C) for DNA extracted at +24 h, but no laddering was detected either before or after flower opening. However, DNA laddering is clearly present in wheat seed extracts using identical proce-
Figure 1. Numbers of nuclei that stain for fragmented DNA using the TUNEL procedure in three different daylily petal tissues. A, Time course of TUNEL-positive nuclei from 30 h before flower opening (–30 h) to 28 h after opening (+28 h); B, effect of 100 µM abscisic acid (ABA) applied from –30 to –6 h on the number of TUNELpositive nuclei at –6 h (black bars) compared to untreated controls at –6 h (open bars); C, effect of 20 µg·mL–1 cycloheximide (CHI) applied from –6 to +18 h on the number of TUNEL-positive nuclei at +18 h (gray bars) compared to untreated controls at +18 h (open bars). Data represent the mean of TUNEL-positive nuclei as a percent of the total nuclei counted (approx. 300 to 400) for each tissue type ± SE. AD, Adaxial epidermis; AB, abaxial epidermis; M, mesophyll. The differences between ABA- or cycloheximide-treated and control petals are significant with P ≤ 0.05.
dures (figure 2A), indicating that our methods are incapable of detecting oligonucleosomal fragments in daylily petals.
2.2. Activities of nucleases during petal senescence The onset of nuclease activities can be visualized on activity gels (figure 3). Clear bands representing zones
T. Panavas et al. / Plant. Physiol. Biochem. 38 (2000) 837–843
Figure 2. Electrophoretic separation of DNA from senescent tissues. A, Ethidium bromide staining of molecular mass standards in bp (MM) and wheat endosperm DNA approximately 30 d after flowering (WE); B, ethidium bromide staining of daylily petal DNA prepared at 0 h (0), +12 h (+12), and +24 h (+24) after flower opening; C, Southern blot analysis of daylily genomic DNA using a probe obtained by random primer labeling. The blot was made from the gel shown in B. The daylily results shown are typical of three independent analyses.
of DNA-digesting activity are seen 6 h before the flowers open; the existing bands increase in intensity and new bands appear at +18 h (figure 3A). Similar effects occur when RNA is the substrate (figure 3B).
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An arrow points to a band on the RNA gel that is not seen at the same position on the DNA gel, suggesting that RNA-specific nuclease activity appears with kinetics similar to the other bands. For both the DNase and RNase activity gels, incubation of the petals in ABA for 24 h (from –30 to –6 h) to stimulate senescence, induces a very similar pattern of nuclease activity, but at least 18 h earlier than the untreated controls (figure 3). Incubation in CHI for 24 h (from –6 to +18 h) to retard senescence, almost completely eliminates the presence of both DNase and RNase activities (figure 3). The activity of single stranded (SS) and double stranded (DS) nucleases is plotted over time in figure 4A. The SS activity is undetectable 30 h before flower opening, but the activity is evident at –12 h and rises rapidly thereafter. DS activity is detectable throughout the time period assayed, but the increase, while appearing significant from –30 to –6 h and from –6 to +18 h, is much less than that observed for SS activity. Addition of ABA at –30 h stimulates SS nuclease activity so that by 24 h after treatment (i.e. at –6 h) this activity is about twice the control value and almost the same as the activity of the controls 24 h after flower opening (figure 4B). DS activity shows the same trends, but the incremental changes are less than SS nucleases. When CHI is added at 6 h before flower opening, SS activity 24 h later (i.e. at +18 h) is 40 % less than the corresponding +18-h control, but is 40 % greater than the activity of –6-h controls, which was the activity of the nucleases when the CHI was added (figure 4C). The DS activity in the presence of CHI (figure 4C) is qualitatively similar to those for SS activity. The nuclease activities after CHI additions,
Figure 3. Nuclease activities of daylily petal proteins on polyacrylamide gels containing single stranded DNA (A) or RNA (B). Control lanes have homogenates extracted from petals 30 or 6 h before, or 18 h after, flower opening. Lanes labeled ABA have homogenates from –6 h petals treated from –30 to –6 h with 100 µM abscisic acid. Lanes labeled CHI have homogenates from +18 h petals treated from –6 to +18 h with 20 µg·mL–1 cycloheximide. Clear areas represent enzyme activities. Arrow points to bands on the RNA-containing gel that have no bands at a similar location on the SS DNAcontaining gel. The gel shown is typical of three independent experiments.
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Figure 4. Activities of single stranded (SS) and double stranded (DS) nucleases in homogenates of daylily petals. A, Time course of SS and DS activities from 30 h before flower opening (–30 h) to 18 h after opening (+18 h); B, effect of 100 µM abscisic acid (ABA) applied from –30 to –6 h on SS and DS nuclease activities at –6 h (black bars) compared to controls at –6 h (open bars); C, effect of 20 µg·mL–1 cycloheximide (CHI) applied from –6 to +18 h on SS and DS nuclease activities at +18 h (black bars) compared to controls at +18 h (gray bars). Values are absorbance at A260 after subtracting the blank. Data are the means ± SE for three independent experiments, each with replicate treatments. The differences between ABA- or cycloheximide-treated and control petals are significant with P ≤ 0.05.
however, were not high enough to be detected on activity gels (figure 3).
3. DISCUSSION Because of the importance of undegraded DNA and RNA for normal cellular functions, we are analyzing the role of various nucleases on PCD of daylily petals,
a relatively facile model system for the study of organ senescence. Using the TUNEL detection method, nuclease-induced breakage of nuclear DNA has been correlated with the death of cells in many different plant systems, e.g. conditions related to biotic stress [12, 17, 28, 33], tracheary element formation [8], embryo development [9], barley aleurone during germination [35], as well as floral organs such as anthers [34], and the hormone-induced senescence of carpels, and petals of pea [21, 22]. Here, we present quantitative data that the fragmentation of nuclear DNA in an entire organ, the petals of daylily, is increasing 6 h before the flowers even open (figure 1A). Furthermore, the number of stained nuclei increases when senescence is accelerated by ABA (figure 1B) and decreases when senescence is delayed by CHI (figure 1C). The degradation of nuclear DNA and its presumptive deleterious effect on cell integrity appears to occur synchronously for most of the cells of the daylily petal. Cells associated with vascular tissues, however, remain functional for many hours after other petal cells have died [3]. The changes of the TUNEL assay parallel the changes of nuclease activity. The possibility that the increased nuclease activity indicated by the TUNEL assay is a result of increased nicking of DNA during the fixation process cannot be excluded. Along with TUNEL staining, PCD in animals as well as in many plant systems is accompanied by a laddering of DNA that results as nucleases cleave nuclear DNA into multiples of nucleosomal-sized fragments (e.g. [11, 21, 22, 37]). Interestingly, laddering is not obvious in extracts from daylily petals (figure 2A, B), although the TUNEL assay clearly indicates that breaks in the DNA are occurring (figure 1A). The sensitivity of the TUNEL technique is greater than that of electrophoresis [27], however, so breaks in the DNA may not be detectable. It is also possible that only large DNA fragments are formed that are subsequently hydrolyzed by exonucleases. This would lead to an increase in free ends that are labeled by the TUNEL reaction, but would not provide an oligonucleosomal ladder. The smear at the top of the gel at 24 h (figure 2C) may be an indication of this sort of digestion. Nucleosomal fragments are not always seen during animal cell apoptosis [38], nor are they evident during the hypersensitive response [17] or during DNA degradation in barley aleurone [2]. Furthermore, in anther cells where TUNEL labeling is clearly evident, laddering on ethidium bromide gels is not very pronounced [35]. The program for cell death in daylily consists of
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many events found in other senescing systems, but production of nucleosome-sized fragments is not one that is easily detectable by our methods. DNase and RNase activity gels (figure 3A, B) indicate that new enzymes appear at least 6 h before flowers open and the number of different nuclease enzymes increases when flowers senesce. The activity gels were run to demonstrate the appearance of new enzymes and were normalized for the same amount of protein. We are aware that the protein content per petal decreases after flower opening and the quantification of the activity gel would not reflect the change of total nuclease activity per cell. For that reason the quantitative assay for total nuclease activity was normalized per petal. No cell divisions were observed in sections after –30 h. Therefore the changes in nuclease activity per petal should reflect the changes in activity on a per cell basis. SS and DS DNase activities (figure 4A) indicate that total nuclease activity increases at least 6 h before the flowers open, which is approximately when TUNEL labeling increases (figure 1A). Activity gels were also used to demonstrate an upregulation of RNase activities in dark-treated [5, 36] as well as in senescing and salt-stressed barley leaves [18]. Furthermore, cytosolic nuclease activity degrades nuclear DNA in vitro when mannitol-treated Arabidopsis roots and maize cell cultures are used as model systems for senescence [30]. A further relationship between the onset of senescence and nuclease activities in daylily petals is suggested by the fact that increases in nuclease activities are inhibited by CHI (figures 3A, B, 4C) which retards senescence. This result also suggests that the nucleases are newly translated during petal senescence or that a newly synthesized protein regulator is an important part of the increase of activity. What is more, nuclease activities occur prematurely, and with the same pattern, on activity gels after a 24-h treatment with ABA, which hastens daylily senescence (figures 3A, B, 4C). A cDNA for a leaf nuclease is upregulated by ABA in tomato [15] and in barley leaves [18]. Because nuclease activities and their effects on DNA breakage and RNA levels increase 18 h before the visible senescence of cells, which is 12 h before the loss of differential membrane permeability [4, 23] and somewhat earlier than the large increases in proteinase activities [31], we suggest that a close relationship exists between heightened nuclease activity and senescence leading to death of daylily petals. This suggestion is strengthened by a stimulation of the above parameters by ABA and a retardation by CHI.
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Thus, it may be worthwhile to study more fully the putative S1-type nuclease (DSA6) whose cDNA has been cloned from daylily [24]. A S1-type nuclease has also been correlated with PCD in aleurone cells and during tracheary element formation [1]. The message for DSA6 begins to increase as early as flower opening, and its mRNA is upregulated 40-fold by ABA. Furthermore, expression of DSA6 is petal specific, and there appears to be only a single copy of the gene. The expression of DSA6 may be controlled in a manner similar to that of nucleases with other specificities that are upregulated at the same time. An understanding of the regulation of this nuclease gene may help identify some of the components of the signal transduction scheme that result in the death of whole organs.
4. METHODS 4.1. Plant culture Hemerocallis hybrid (cv. Stella d’Oro) plants were grown in a growth chamber as described earlier so that the flowers opened synchronously at 21:00 hours, referred to hereafter as 0 h [25]. Only the inner whorl of three petals was used for analysis. For treatment of ABA (100 µM) or (CHI) (20 µg·mL–1), individual petals were placed in 1 mL appropriate solutions in 5-mL vials, using distilled water as the control. The ABA was applied for 24 h from –30 to –6 h. CHI was also applied for 24 h, but from –6 to +18 h.
4.2. Analysis of nuclear DNA fragmentation 4.2.1. TUNEL assay The middle 1/3 portion of the petals was infused with 4 % (w/v) paraformaldehyde (pH 6.8) for 1 h, then washed and dehydrated in graded concentrations of butanol prior to embedding in paraffin. Sections of 10 µm were mounted on poly-L-lysine-coated slides, deparaffinized with xylene and rehydrated with a graded series of ethanol concentrations. The sections were then digested with 20 µg·mL–1 proteinase K for 15 min. For cryo-sectioning, the middle 1/3 of petals were fixed in 4 % paraformaldehyde in PBS buffer (pH 7.2) for 1 h, rinsed twice with PBS and then incubated overnight in 30 % sucrose in PBS (pH 7.2). The samples were then quick frozen in Tissue-Tek OCT embedding compound and stored at –80 °C for at least 24 h. Sections of 10 µm were prepared in a cryostat maintained at –20 °C. The sections were post
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fixed with 4 % (w/v) paraformaldehyde for 10 min then treated with a graded series of ethanol concentrations. The TUNEL assay was performed using the protocol of the Oncor Apoptag kit (Oncor Co., Gaithersburg, MD, USA). Endogenous peroxidases were quenched with 2 % (v/v) H2O2 (for cryo-sections, the H2O2 concentration was diluted to 0.002 % (v/v) to avoid cellular damage), and digoxigenin-dUTP containing terminal deoxynucleotidyl transferase (TdT) was applied to the surface of the slides for 1 h. Following several washes, anti-digoxigenin-peroxidaseconjugated antibody was applied to the sections. The specific binding of this antibody provides the enzyme needed to generate a brown color from the chromogenic substrate, 0.05 % (w/v) diaminobenzidine, which also contained 0.02 % (v/v) H2O2. After 10 min, the slides were washed and counterstained with 0.5 % (w/v) methyl green, which stained nuclei with unfragmented DNA as well as the cell walls a blue-green color. Approximately, 300 to 400 nuclei were counted in the abaxial (lower) and adaxial (upper) epidermides as well as in the mesophyll tissue for each timepoint or treatment. Many more sections were counted from mature tissues because of fewer cells per section. Consecutive sections and damaged sections were not counted. Data are presented as the number of TUNELpositive (brown) nuclei as a percent of the total counted ± SE.
4.2.2. Analysis of size of DNA Genomic DNA was extracted and precipitated according to the methods of Young and Gallie [37]. The redissolved DNA was separated on agarose gels (1.8 % w/v) and stained with ethidium bromide. Five micrograms of DNA was used for 0 and +12 h time points and 3 µg for +24 h time point. After visualization of the DNA on the gel, it was blotted to a nitrocellulose membrane and probed with radioactive DNA fragments prepared with random hexamers.
4.3. DNase and RNase activity gels SDS-PAGE was performed as described previously [26, 32] using a 16 % (w/v) resolving gel that contained either 3 mg·mL–1 yeast RNA or salmon sperm DNA that had been made single stranded by boiling for 10 min. Petal extracts in sample buffer containing 5 % (w/v) SDS and 5 µg protein was loaded onto each lane. Following electrophoresis, the SDS was removed by washing twice in Tris-HCl (pH 7.0) containing 2.5 % (v/v) Triton X-100 for 30 min, and the slabs were incubated in 0.1 M imidazole at 37 °C for 1 h.
Gels were stained with 0.1 % toluidine blue and destained in distilled water.
4.4. Single stranded and double stranded DNase assays Homogenates were prepared and activities measured by the methods of Wood et al. [36]. Single stranded (SS) DNA was prepared from native double stranded (DS) salmon sperm DNA by boiling and shearing the sample. The reaction mixture consisted of 140 µL SS or DS DNA, 70 µL 1 mg·mL–1 BSA, 70 µL 1 M Tris-HCl (pH 7.4), and 300 µL extraction buffer. The reaction was initiated by addition of 115 µL petal extract and incubated for 15 min at 34 °C. The reference tube contained the same constituents, except that the DNA was added after the incubation period. Following incubation, 3.4 % (v/v) perchloric acid was added to terminate the reaction, and the absorbance was read at A260 after centrifugation.
Acknowledgments. This work was partially supported by grants to T.P. from the American Hemerocallis Society and from the R.I. Davis Fund of the Biology Department, University of Massachusetts. P.D.R. received grants from the Blakeslee Fund and the Wilens Fund of Smith College. B.R. was awarded a Faculty Research Grant from the University of Massachusetts. We would like to thank Dr Pamela Cascone for her help and Mr Ronald Beckwith for growing the daylilies. REFERENCES [1] Aoyagi S., Sugiyana M., Fukada H., BENI and ZENI cDNAs encoding S1-type DNases that are associated with programmed cell death in plants, FEBS Lett. 429 (1998) 134–138. [2] Bethke P.C., Lousdale J.E., Fath A., Jones R.L., Hormonally regulated programmed cell death in barley aleurone cells, Plant Cell 11 (1999) 1033–1045. [3] Bieleski R.L., Onset of phloem export from senescent petals of daylily, Plant Physiol. 109 (1995) 557–565. [4] Bieleski R.L., Reid M.S., Physiological changes accompanying senescence in the ephemeral daylily flower, Plant Physiol. 98 (1992) 1042–1049. [5] Blank A., McKeon T.A.S., Expression of three RNase activities during natural and dark-induced senescence of wheat leaves, Plant Physiol. 97 (1991) 1409–1413. [6] Brady C.J., Nucleic acid and protein synthesis, in: Nooden L.D., Leopold A.C. (Eds.), Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 147–179.
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Activities of nucleases in senescing daylily petals Tadas Panavasa,b§, Rebbecca LeVangiec, John Mistlera, Philip D. Reidc, Bernard Rubinsteina,b* a
Department of Biology, University of Massachusetts, Amherst, MA 01003-5810, USA
b
Plant Biology Graduate Program, University of Massachusetts, Amherst, MA 01003-5810, USA
c
Department of Biological Sciences, Smith College, Northampton, MA 01063, USA
Received 3 January 2000; accepted 17 July 2000 Abstract – The activity of nucleases during organ death was investigated using daylily petals (Hemerocallis hybrid cv. Stella d’Oro), in which the processes associated with senescence are rapid and clearly ordered. The number of nuclei with fragmented DNA as well as activities of various nucleases increase before certain other events that are related to senescence. Furthermore, DNA breakage and activities of nucleases occur earlier when senescence is accelerated by abscisic acid and occur later when senescence is retarded by cycloheximide. These results suggest that the activities of nucleases contribute to the senescence of daylily petals. Therefore, studying the regulation of nuclease gene expression may be useful for understanding components of the signal transduction system that leads to the death of these organs. © 2000 Éditions scientifiques et médicales Elsevier SAS abscisic acid / daylily flower senescence / DNA breakage / Hemerocallis / nuclease ABA, abscisic acid / CHI, cycloheximide / DS, double stranded / PCD, programmed cell death / SS, single stranded / TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
1. INTRODUCTION The program leading to cell death (PCD) in plants, like that in animals [10], involves a series of overlapping and often interacting processes, some of which may only be incidental to the death that eventually occurs [14, 19, 20, 29]. One approach for understanding more thoroughly the mechanisms that are closely involved with PCD of plant organs is to characterize one pathway that appears to be a factor leading to cell death. A decrease in extractable RNA and DNA [6, 16, 29], the degradation of nuclear DNA (e.g. [2, 8, 17, 21, 35, 37]) and an upregulation of enzymes that degrade nucleic acids (e.g. [7, 15, 16, 30]) suggest that the appearance of various nuclease activities may be an important component of PCD. * Correspondence and reprints: fax +1 413 545 3243. E-mail address: [email protected] (Bernard Rubinstein). § Present address: Department of Plant Pathology, University of Kentucky, S-305, Agr. Sci. Bldg.-N, Lexington, KY 40546, USA.
A requirement for determining a role for any particular mechanism related to organ PCD is the selection of an amenable plant system in which the time course of senescence can be well-defined. The senescence of petals, especially those from the ephemeral flowers of daylily, is one such system. Although genetic manipulation is difficult, the petals senesce rapidly and synchronously, and the biochemical and molecular processes investigated so far occur in a fixed order that can be easily related to morphological changes associated with senescence [4, 13, 25]. In this paper, we are asking whether nuclease activities represent an important and easily detectable component of the program for cell death in daylily petals. We report that breakage of nuclear DNA increases even before the flowers open, and that upregulation of various nuclease activities occurs at about the same time. Furthermore, the fragmentation of nuclear DNA and the activities of nucleases occur prematurely after addition of abscisic acid (ABA), which stimulates senescence, and are delayed in the presence of cycloheximide (CHI), which retards senescence.
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2. RESULTS 2.1. Evidence for breakage of nuclear DNA A role for nuclease activity in PCD is indicated by the TUNEL assay, in which fragmented nuclear DNA can be observed as brown, compared to blue-stained nuclei. At 30 h before the flower opens (–30 h), an average of about 10 % of the petal nuclei stain positively for breaks in DNA (figure 1A). This value increases to about 20 % by –6 to +6 h and then increases sharply at +18 h. The trends are similar regardless of whether the abaxial or adaxial epidermis, or mesophyll cells are examined. Because of possible artifacts due to the TUNEL assay after tissues are embedded in paraffin [33], we compared the data from figure 1A with those obtained from cryo-sectioned material. The results are nearly identical. Average values of the three tissues for TUNEL-positive nuclei at –6 h in the cryo-sectioned material is 16 ± 5 %, compared to 20 ± 7 % for paraffinembedded sections. At +18 h, the cryo-sectioned material has 40 ± 8 % TUNEL-positive nuclei and 44 ± 14 % for paraffin-embedded. This represents an increase from –6 to +18 h of 2.5 and 2.2 times for cryosectioned and paraffin-embedded tissue, respectively. Furthermore, cryo-sectioned, like paraffin-embedded, material shows no clear differences between the cells of different petal tissues. We also studied the effects of a stimulator of daylily PCD, abscisic (ABA), and a PCD inhibitor, cycloheximide (CHI), on the numbers of TUNEL-positive nuclei. The data in figure 1B indicate that after ABA is applied to the petals from –30 to –6 h, the number of TUNEL-positive nuclei is twice the value of controls at –6 h; in fact, the values for ABA-treated petals approach those seen in the controls 24 h later (at +18 h). There appears to be no significant difference between the two epidermides and the mesophyll cells. The addition of CHI at –6 h (figure 1C) results in only a small increase in TUNEL-positive nuclei by +18 h compared to controls at –6 h (figure 1B), and clearly below the levels seen in the +18-h controls. There are also no significant differences between the cells of various tissues after CHI applications (figure 1C). One effect of increased nuclease activity in daylily petals could be the degradation of nuclear DNA into oligonucleosomal fragments. There is a smear at the top of the ethidium bromide gel (figure 2B) and in the Southern blot (figure 2C) for DNA extracted at +24 h, but no laddering was detected either before or after flower opening. However, DNA laddering is clearly present in wheat seed extracts using identical proce-
Figure 1. Numbers of nuclei that stain for fragmented DNA using the TUNEL procedure in three different daylily petal tissues. A, Time course of TUNEL-positive nuclei from 30 h before flower opening (–30 h) to 28 h after opening (+28 h); B, effect of 100 µM abscisic acid (ABA) applied from –30 to –6 h on the number of TUNELpositive nuclei at –6 h (black bars) compared to untreated controls at –6 h (open bars); C, effect of 20 µg·mL–1 cycloheximide (CHI) applied from –6 to +18 h on the number of TUNEL-positive nuclei at +18 h (gray bars) compared to untreated controls at +18 h (open bars). Data represent the mean of TUNEL-positive nuclei as a percent of the total nuclei counted (approx. 300 to 400) for each tissue type ± SE. AD, Adaxial epidermis; AB, abaxial epidermis; M, mesophyll. The differences between ABA- or cycloheximide-treated and control petals are significant with P ≤ 0.05.
dures (figure 2A), indicating that our methods are incapable of detecting oligonucleosomal fragments in daylily petals.
2.2. Activities of nucleases during petal senescence The onset of nuclease activities can be visualized on activity gels (figure 3). Clear bands representing zones
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Figure 2. Electrophoretic separation of DNA from senescent tissues. A, Ethidium bromide staining of molecular mass standards in bp (MM) and wheat endosperm DNA approximately 30 d after flowering (WE); B, ethidium bromide staining of daylily petal DNA prepared at 0 h (0), +12 h (+12), and +24 h (+24) after flower opening; C, Southern blot analysis of daylily genomic DNA using a probe obtained by random primer labeling. The blot was made from the gel shown in B. The daylily results shown are typical of three independent analyses.
of DNA-digesting activity are seen 6 h before the flowers open; the existing bands increase in intensity and new bands appear at +18 h (figure 3A). Similar effects occur when RNA is the substrate (figure 3B).
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An arrow points to a band on the RNA gel that is not seen at the same position on the DNA gel, suggesting that RNA-specific nuclease activity appears with kinetics similar to the other bands. For both the DNase and RNase activity gels, incubation of the petals in ABA for 24 h (from –30 to –6 h) to stimulate senescence, induces a very similar pattern of nuclease activity, but at least 18 h earlier than the untreated controls (figure 3). Incubation in CHI for 24 h (from –6 to +18 h) to retard senescence, almost completely eliminates the presence of both DNase and RNase activities (figure 3). The activity of single stranded (SS) and double stranded (DS) nucleases is plotted over time in figure 4A. The SS activity is undetectable 30 h before flower opening, but the activity is evident at –12 h and rises rapidly thereafter. DS activity is detectable throughout the time period assayed, but the increase, while appearing significant from –30 to –6 h and from –6 to +18 h, is much less than that observed for SS activity. Addition of ABA at –30 h stimulates SS nuclease activity so that by 24 h after treatment (i.e. at –6 h) this activity is about twice the control value and almost the same as the activity of the controls 24 h after flower opening (figure 4B). DS activity shows the same trends, but the incremental changes are less than SS nucleases. When CHI is added at 6 h before flower opening, SS activity 24 h later (i.e. at +18 h) is 40 % less than the corresponding +18-h control, but is 40 % greater than the activity of –6-h controls, which was the activity of the nucleases when the CHI was added (figure 4C). The DS activity in the presence of CHI (figure 4C) is qualitatively similar to those for SS activity. The nuclease activities after CHI additions,
Figure 3. Nuclease activities of daylily petal proteins on polyacrylamide gels containing single stranded DNA (A) or RNA (B). Control lanes have homogenates extracted from petals 30 or 6 h before, or 18 h after, flower opening. Lanes labeled ABA have homogenates from –6 h petals treated from –30 to –6 h with 100 µM abscisic acid. Lanes labeled CHI have homogenates from +18 h petals treated from –6 to +18 h with 20 µg·mL–1 cycloheximide. Clear areas represent enzyme activities. Arrow points to bands on the RNA-containing gel that have no bands at a similar location on the SS DNAcontaining gel. The gel shown is typical of three independent experiments.
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Figure 4. Activities of single stranded (SS) and double stranded (DS) nucleases in homogenates of daylily petals. A, Time course of SS and DS activities from 30 h before flower opening (–30 h) to 18 h after opening (+18 h); B, effect of 100 µM abscisic acid (ABA) applied from –30 to –6 h on SS and DS nuclease activities at –6 h (black bars) compared to controls at –6 h (open bars); C, effect of 20 µg·mL–1 cycloheximide (CHI) applied from –6 to +18 h on SS and DS nuclease activities at +18 h (black bars) compared to controls at +18 h (gray bars). Values are absorbance at A260 after subtracting the blank. Data are the means ± SE for three independent experiments, each with replicate treatments. The differences between ABA- or cycloheximide-treated and control petals are significant with P ≤ 0.05.
however, were not high enough to be detected on activity gels (figure 3).
3. DISCUSSION Because of the importance of undegraded DNA and RNA for normal cellular functions, we are analyzing the role of various nucleases on PCD of daylily petals,
a relatively facile model system for the study of organ senescence. Using the TUNEL detection method, nuclease-induced breakage of nuclear DNA has been correlated with the death of cells in many different plant systems, e.g. conditions related to biotic stress [12, 17, 28, 33], tracheary element formation [8], embryo development [9], barley aleurone during germination [35], as well as floral organs such as anthers [34], and the hormone-induced senescence of carpels, and petals of pea [21, 22]. Here, we present quantitative data that the fragmentation of nuclear DNA in an entire organ, the petals of daylily, is increasing 6 h before the flowers even open (figure 1A). Furthermore, the number of stained nuclei increases when senescence is accelerated by ABA (figure 1B) and decreases when senescence is delayed by CHI (figure 1C). The degradation of nuclear DNA and its presumptive deleterious effect on cell integrity appears to occur synchronously for most of the cells of the daylily petal. Cells associated with vascular tissues, however, remain functional for many hours after other petal cells have died [3]. The changes of the TUNEL assay parallel the changes of nuclease activity. The possibility that the increased nuclease activity indicated by the TUNEL assay is a result of increased nicking of DNA during the fixation process cannot be excluded. Along with TUNEL staining, PCD in animals as well as in many plant systems is accompanied by a laddering of DNA that results as nucleases cleave nuclear DNA into multiples of nucleosomal-sized fragments (e.g. [11, 21, 22, 37]). Interestingly, laddering is not obvious in extracts from daylily petals (figure 2A, B), although the TUNEL assay clearly indicates that breaks in the DNA are occurring (figure 1A). The sensitivity of the TUNEL technique is greater than that of electrophoresis [27], however, so breaks in the DNA may not be detectable. It is also possible that only large DNA fragments are formed that are subsequently hydrolyzed by exonucleases. This would lead to an increase in free ends that are labeled by the TUNEL reaction, but would not provide an oligonucleosomal ladder. The smear at the top of the gel at 24 h (figure 2C) may be an indication of this sort of digestion. Nucleosomal fragments are not always seen during animal cell apoptosis [38], nor are they evident during the hypersensitive response [17] or during DNA degradation in barley aleurone [2]. Furthermore, in anther cells where TUNEL labeling is clearly evident, laddering on ethidium bromide gels is not very pronounced [35]. The program for cell death in daylily consists of
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many events found in other senescing systems, but production of nucleosome-sized fragments is not one that is easily detectable by our methods. DNase and RNase activity gels (figure 3A, B) indicate that new enzymes appear at least 6 h before flowers open and the number of different nuclease enzymes increases when flowers senesce. The activity gels were run to demonstrate the appearance of new enzymes and were normalized for the same amount of protein. We are aware that the protein content per petal decreases after flower opening and the quantification of the activity gel would not reflect the change of total nuclease activity per cell. For that reason the quantitative assay for total nuclease activity was normalized per petal. No cell divisions were observed in sections after –30 h. Therefore the changes in nuclease activity per petal should reflect the changes in activity on a per cell basis. SS and DS DNase activities (figure 4A) indicate that total nuclease activity increases at least 6 h before the flowers open, which is approximately when TUNEL labeling increases (figure 1A). Activity gels were also used to demonstrate an upregulation of RNase activities in dark-treated [5, 36] as well as in senescing and salt-stressed barley leaves [18]. Furthermore, cytosolic nuclease activity degrades nuclear DNA in vitro when mannitol-treated Arabidopsis roots and maize cell cultures are used as model systems for senescence [30]. A further relationship between the onset of senescence and nuclease activities in daylily petals is suggested by the fact that increases in nuclease activities are inhibited by CHI (figures 3A, B, 4C) which retards senescence. This result also suggests that the nucleases are newly translated during petal senescence or that a newly synthesized protein regulator is an important part of the increase of activity. What is more, nuclease activities occur prematurely, and with the same pattern, on activity gels after a 24-h treatment with ABA, which hastens daylily senescence (figures 3A, B, 4C). A cDNA for a leaf nuclease is upregulated by ABA in tomato [15] and in barley leaves [18]. Because nuclease activities and their effects on DNA breakage and RNA levels increase 18 h before the visible senescence of cells, which is 12 h before the loss of differential membrane permeability [4, 23] and somewhat earlier than the large increases in proteinase activities [31], we suggest that a close relationship exists between heightened nuclease activity and senescence leading to death of daylily petals. This suggestion is strengthened by a stimulation of the above parameters by ABA and a retardation by CHI.
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Thus, it may be worthwhile to study more fully the putative S1-type nuclease (DSA6) whose cDNA has been cloned from daylily [24]. A S1-type nuclease has also been correlated with PCD in aleurone cells and during tracheary element formation [1]. The message for DSA6 begins to increase as early as flower opening, and its mRNA is upregulated 40-fold by ABA. Furthermore, expression of DSA6 is petal specific, and there appears to be only a single copy of the gene. The expression of DSA6 may be controlled in a manner similar to that of nucleases with other specificities that are upregulated at the same time. An understanding of the regulation of this nuclease gene may help identify some of the components of the signal transduction scheme that result in the death of whole organs.
4. METHODS 4.1. Plant culture Hemerocallis hybrid (cv. Stella d’Oro) plants were grown in a growth chamber as described earlier so that the flowers opened synchronously at 21:00 hours, referred to hereafter as 0 h [25]. Only the inner whorl of three petals was used for analysis. For treatment of ABA (100 µM) or (CHI) (20 µg·mL–1), individual petals were placed in 1 mL appropriate solutions in 5-mL vials, using distilled water as the control. The ABA was applied for 24 h from –30 to –6 h. CHI was also applied for 24 h, but from –6 to +18 h.
4.2. Analysis of nuclear DNA fragmentation 4.2.1. TUNEL assay The middle 1/3 portion of the petals was infused with 4 % (w/v) paraformaldehyde (pH 6.8) for 1 h, then washed and dehydrated in graded concentrations of butanol prior to embedding in paraffin. Sections of 10 µm were mounted on poly-L-lysine-coated slides, deparaffinized with xylene and rehydrated with a graded series of ethanol concentrations. The sections were then digested with 20 µg·mL–1 proteinase K for 15 min. For cryo-sectioning, the middle 1/3 of petals were fixed in 4 % paraformaldehyde in PBS buffer (pH 7.2) for 1 h, rinsed twice with PBS and then incubated overnight in 30 % sucrose in PBS (pH 7.2). The samples were then quick frozen in Tissue-Tek OCT embedding compound and stored at –80 °C for at least 24 h. Sections of 10 µm were prepared in a cryostat maintained at –20 °C. The sections were post
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fixed with 4 % (w/v) paraformaldehyde for 10 min then treated with a graded series of ethanol concentrations. The TUNEL assay was performed using the protocol of the Oncor Apoptag kit (Oncor Co., Gaithersburg, MD, USA). Endogenous peroxidases were quenched with 2 % (v/v) H2O2 (for cryo-sections, the H2O2 concentration was diluted to 0.002 % (v/v) to avoid cellular damage), and digoxigenin-dUTP containing terminal deoxynucleotidyl transferase (TdT) was applied to the surface of the slides for 1 h. Following several washes, anti-digoxigenin-peroxidaseconjugated antibody was applied to the sections. The specific binding of this antibody provides the enzyme needed to generate a brown color from the chromogenic substrate, 0.05 % (w/v) diaminobenzidine, which also contained 0.02 % (v/v) H2O2. After 10 min, the slides were washed and counterstained with 0.5 % (w/v) methyl green, which stained nuclei with unfragmented DNA as well as the cell walls a blue-green color. Approximately, 300 to 400 nuclei were counted in the abaxial (lower) and adaxial (upper) epidermides as well as in the mesophyll tissue for each timepoint or treatment. Many more sections were counted from mature tissues because of fewer cells per section. Consecutive sections and damaged sections were not counted. Data are presented as the number of TUNELpositive (brown) nuclei as a percent of the total counted ± SE.
4.2.2. Analysis of size of DNA Genomic DNA was extracted and precipitated according to the methods of Young and Gallie [37]. The redissolved DNA was separated on agarose gels (1.8 % w/v) and stained with ethidium bromide. Five micrograms of DNA was used for 0 and +12 h time points and 3 µg for +24 h time point. After visualization of the DNA on the gel, it was blotted to a nitrocellulose membrane and probed with radioactive DNA fragments prepared with random hexamers.
4.3. DNase and RNase activity gels SDS-PAGE was performed as described previously [26, 32] using a 16 % (w/v) resolving gel that contained either 3 mg·mL–1 yeast RNA or salmon sperm DNA that had been made single stranded by boiling for 10 min. Petal extracts in sample buffer containing 5 % (w/v) SDS and 5 µg protein was loaded onto each lane. Following electrophoresis, the SDS was removed by washing twice in Tris-HCl (pH 7.0) containing 2.5 % (v/v) Triton X-100 for 30 min, and the slabs were incubated in 0.1 M imidazole at 37 °C for 1 h.
Gels were stained with 0.1 % toluidine blue and destained in distilled water.
4.4. Single stranded and double stranded DNase assays Homogenates were prepared and activities measured by the methods of Wood et al. [36]. Single stranded (SS) DNA was prepared from native double stranded (DS) salmon sperm DNA by boiling and shearing the sample. The reaction mixture consisted of 140 µL SS or DS DNA, 70 µL 1 mg·mL–1 BSA, 70 µL 1 M Tris-HCl (pH 7.4), and 300 µL extraction buffer. The reaction was initiated by addition of 115 µL petal extract and incubated for 15 min at 34 °C. The reference tube contained the same constituents, except that the DNA was added after the incubation period. Following incubation, 3.4 % (v/v) perchloric acid was added to terminate the reaction, and the absorbance was read at A260 after centrifugation.
Acknowledgments. This work was partially supported by grants to T.P. from the American Hemerocallis Society and from the R.I. Davis Fund of the Biology Department, University of Massachusetts. P.D.R. received grants from the Blakeslee Fund and the Wilens Fund of Smith College. B.R. was awarded a Faculty Research Grant from the University of Massachusetts. We would like to thank Dr Pamela Cascone for her help and Mr Ronald Beckwith for growing the daylilies. REFERENCES [1] Aoyagi S., Sugiyana M., Fukada H., BENI and ZENI cDNAs encoding S1-type DNases that are associated with programmed cell death in plants, FEBS Lett. 429 (1998) 134–138. [2] Bethke P.C., Lousdale J.E., Fath A., Jones R.L., Hormonally regulated programmed cell death in barley aleurone cells, Plant Cell 11 (1999) 1033–1045. [3] Bieleski R.L., Onset of phloem export from senescent petals of daylily, Plant Physiol. 109 (1995) 557–565. [4] Bieleski R.L., Reid M.S., Physiological changes accompanying senescence in the ephemeral daylily flower, Plant Physiol. 98 (1992) 1042–1049. [5] Blank A., McKeon T.A.S., Expression of three RNase activities during natural and dark-induced senescence of wheat leaves, Plant Physiol. 97 (1991) 1409–1413. [6] Brady C.J., Nucleic acid and protein synthesis, in: Nooden L.D., Leopold A.C. (Eds.), Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 147–179.
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