Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in cervical carcinoma

HLA class I loss is a significant mechanism of immune evasion by cervical carcinoma, interfering with the development of immunotherapies and cancer vaccines. We report the systematic investigation of HLA class I and antigen processing machinery component expression and association with clinical outcome. A tissue microarray containing carcinoma lesions from 109 cervical carcinoma patients was stained for HLA class I heavy chains, β2-microglobulin, LMP2, LMP7, LMP10, TAP1, TAP2, ERAP1, tapasin, calreticulin, calnexin and ERp57. A novel staining evaluation method was used to ensure optimal accuracy and reliability of expression data, which were correlated with known clinicopathological parameters. Partial HLA class I loss was significantly associated with decreased 5-years overall survival (61% vs. 83% for normal expression; P < 0.05) and was associated with decreased 5-years disease-free survival (DFS) (65% vs. 82% for normal expression; P = 0.05). All APM components except LMP10, calnexin and calreticulin were down-regulated in a substantial number of cases and, except ERAP1, correlated significantly with HLA class I down-regulation. LMP7, TAP1 and ERAP1 loss was significantly associated with decreased overall and (except LMP7) DFS (P < 0.05 and 0.005, respectively). ERAP1 down-regulation was an independent predictor for worse overall and DFS in multivariate analysis (HR 3.08; P < 0.05 and HR 2.84; P < 0.05, respectively). HLA class I and APM component down-regulation occur frequently in cervical carcinoma, while peptide repertoire alterations due to ERAP1 loss are a major contributing factor to tumour progression and mortality.
Introduction
Tumour cells utilize several mechanisms to escape immune-mediated recognition and destruction [1]. Loss of surface-expressed human leukocyte antigen (HLA) class I molecules is particularly important, as this enables tumour cells to evade recognition and lysis by cytotoxic T-lymphocytes [2–5]. These molecules consist of a glycoprotein heavy chain (encoded by genes within the HLA regions of chromosome 6p) and a β2-microglobulin (β2-m) light chain (encoded on chromosome 15q) [6, 7].
Defects in HLA class I expression are caused by multiple mechanisms, including loss of heterozygosity at chromosome 6p; β2-m or HLA class I mutations; and defective expression and/or function of components of the antigen processing machinery (APM) [8–10]. The APM is the combination of cellular processes responsible for the presentation of endogenous peptides by HLA class I molecules. These peptides are generated by specialized immunoproteasomes (composed of LMP2, LMP7 and LMP10 subunits); and transported from the cytoplasm to the endoplasmic reticulum by TAP (transporter associated with antigen presentation) which is composed of TAP1 and TAP2 subunits. Transported peptides undergo length-specific trimming by ERAP1 (endoplasmic reticulum aminopeptidase associated with antigen presentation 1) and are loaded onto empty HLA class I molecules (with involvement of tapasin and the chaperone molecules calnexin, calreticulin and ERp57) [11–14].
Although various in vitro studies have demonstrated partial or total APM component loss in human malignancies, the extent to which this occurs in vivo is largely unknown. Two recent reports describe down-regulation of several APM components in head and neck squamous cell carcinoma and astrocytoma, and demonstrate an association of these defects with prognosis [15, 16]. As HLA class I-restricted processing and presentation of endogenous peptides is crucial to the generation of a virus-specific cytotoxic T-lymphocyte-mediated reaction, APM defects are assumed to contribute significantly to virus-mediated carcinogenesis. Several studies have confirmed interference in various APM processes by viral peptides [17]. However, the extent to which defective expression of APM components contributes to HPV-induced cervical carcinogenesis remains to be established. One study has assessed the expression of various APM components, although only at the mRNA level [18]. Other studies of APM component expression in cervical carcinoma were largely limited to TAP1 and TAP2 and encompassed relatively small sample sizes. Moreover, the expression data yielded by these studies are conflicting, with TAP1 loss being reported in 0–50% of cases [18–20]. Though TAP1 and HLA class I down-regulation has occasionally been shown to be associated with progressive malignant transformation [21–24], a comprehensive study of the association of APM defects with prognostic parameters has yet to be performed.
Since information regarding defective APM component expression in cervical cancer can contribute to the development of T cell-based immunotherapies, we have investigated the expression of all known APM components in a large number of cervical cancer specimens using a tissue microarray (TMA)-based approach [25, 26]. Additionally, we demonstrate for the first time a negative association of APM defects with prognosis and other clinicopathological variables.
Materials and methods
Clinical characteristics and tissue samples
From 109 patients with cervical carcinoma who underwent radical hysterectomy with bilateral pelvic lymphadenectomy (by the same surgical team) between 1985 and 1999, formalin-fixed, paraffin-embedded tissue blocks were retrieved from the archives of the Department of Pathology, Leiden University Medical Centre, The Netherlands. Twenty-six adenocarcinomas and 83 squamous carcinomas were obtained. These cases were not consecutive, but chosen on the basis of availability of sufficient material. The use of clinical material was approved by the institutional review board according to the guidelines of the Dutch Federation of Medical Research Associations.
All patients were inhabitants of The Netherlands and had not received pre-operative radiotherapy or chemotherapy. Mean age was 48.5 years, the youngest patient being 24 years and the oldest 87 years at the time of surgery. Follow-up of these patients until 2005 provided information concerning recurrence rate and performance state.
Tissue microarray construction
The archival slides for all the cases were reviewed; a slide containing representative tumour was selected, and an area of tumour was encircled on the slide. The corresponding tissue blocks were recovered from the archives and the selected area on the slide was circled on the block for tissue microarray construction. Using a manual tissue microarrayer (Beecher Instruments, Silver Spring, MD, USA), the area of interest in the donor block was cored with a 0.6-mm diameter needle and transferred to a recipient paraffin block. The microarray was constructed with a threefold redundancy (three spots for each patient) to increase accuracy. The finalized arrays were then cut into 3 μm-thick sections and mounted on glass slides using an adhesive tape-transfer system (Instrumedics Inc., Hackensack, NJ, USA) with ultraviolet cross-linking.
Antibodies
The mouse monoclonal antibody (mAb) HCA2 which recognizes a determinant expressed on β2-m-free HLA-A (excluding HLA-A24), HLA-B7301 and HLA-G heavy chains [27, 28], the mAb HC10 which recognizes a determinant on all β2-m-free HLA-B and HLA-C heavy chains and on β2-m-free HLA-A10, -A28, -A29, -A30, -A31, -A32 and -A33 heavy chains [27, 29], the LMP2-specific mAb SY1 [30], the LMP7-specific mAb HB-2 [30], the LMP10-specific mAb TO-7 [30], the TAP1-specific mAb NOB-1 [31], the TAP2-specific mAb NOB-2 [31], the calnexin-specific mAb TO-5 [32], the calreticulin-specific mAb TO-11 [32], the Erp57-specific mAb TO-2 [32] and the tapasin-specific mAb TO-3 [32] were developed and characterized as described.
The rabbit polyclonal anti-β2-m-antibody A-139 was purchased from DAKO, Copenhagen, Denmark. The rabbit polyclonal anti-ERAP1 antibody has been described previously and was kindly provided by Dr. M. Tsujimoto, RIKEN, Wako, Saitama, Japan [33–35].
Immunohistochemical staining
The slides were dried for 16 h at 56°C before being dewaxed with standard xylene and rehydrated through graded alcohols into water. Standard immunohistochemical staining was performed as described previously [36]. Stromal cells, including infiltrating leukocytes, were used as internal positive controls. Negative control sections of each specimen were processed with omission of the primary antibodies.
Evaluation of TMA immunostaining
Two researchers (A.M. and E.J.) evaluated all patient cores, and scores were averaged to produce a single evaluation with the scorers being blind to the source of the samples. When slides were scored differently, which occurred rarely (<5%), a consensus was reached by simultaneous evaluation. Staining was scored semi-quantitatively by the quality control system proposed by Ruiter et al. [37]. The intensity and percentage of positive cells in the tumour and the stromal cells surrounding the tumour were determined. The intensity of staining was scored as 0, 1, 2, or 3 indicating absent, weak, clear, or strong expression, respectively. The percentage of positive cells was scored as 0 for 0%; 1 for 1–5%; 2 for 5–25%; 3 for 25–50%; 4 for 50–75% and 5 for 75–100%. The sum of both scores was used to identify three categories of expression: normal expression (total score 7–8), partial loss (3–6) and total loss (0–2). Immunohistochemical staining demonstrated strong positive expression of all examined markers in stromal tissue and tumour-infiltrating inflammatory cells, thereby providing an internal positive control.
Statistical analysis
The Chi-square test or, where appropriate, Fisher’s exact test was used for evaluation of associations between expression and clinicopathological parameters. All statistical analyses were performed with the SPSS Version 12 software package. Five-year survival rates were calculated and survival rates were compared according to the Kaplan–Meier method using the log rank test, while multivariate analysis of survival was performed according to Cox proportional hazard models. Overall survival (OS) was defined as survival till death due to cervical carcinoma (the two patients that dyed from other causes were excluded), i.e. disease-specific survival, while disease-free survival (DFS) was defined as time to disease recurrence, metastasis, or disease-specific death. All tests were two-sided and the significance level was set to 5%.
Results
Clinical features
Of the total group of 109 patients, 52 were diagnosed as FIGO stage IB1, 30 as IB2, 21 as FIGO IIa and 2 as FIGO IIb. For four patients no FIGO staging was available. Fifty-five patients (50%) received post-operative radiotherapy due to narrow tumour-free margins of excision, lymph node metastases, vaso-invasive growth, parametrial infiltration, depth of infiltration exceeding 15 mm and/or tumour size exceeding 4 cm. Twenty-seven patients had lymph node metastases. Fifty-one patients (58%) were HPV16-positive, 21 (24%) were HPV18-positive, while the remaining patients were positive for HPV31, HPV33, HPV45, HPV52 or HPV68. At the end of follow-up, 80 patients were alive, 2 suffered from recurrent disease, 3 had a metastasis, 22 had died of the disease and 2 had died of causes unrelated to the primary disease. The median follow-up time was 45 months.
HLA class I and APM component expression
Representative staining patterns for the HLA class I heavy chains and various APM components are shown in Figs. 1 and 2. Expression of the HLA class I heavy chains and β2-m is summarized in Table 1. Of the 109 cases, eight exhibited total β2-m loss; correspondingly, these cases were also negative for HLA heavy chain cell surface expression. As HLA class I expression, and therefore tumour immunogenicity, is not affected by the APM in a β2-m-negative background these eight cases were excluded from further analysis of APM effects. Furthermore, as HLA class I expression is regulated by the APM in a β2-m-independent manner, the overall HLA class I expression score was based on expression of the heavy chains. If either or all the heavy chains were expressed normally, overall HLA class I expression was considered to be normal. Total loss of all heavy chains was counted as total HLA class I loss, and all other cases were counted as partial loss.
Fig. 1Immunohistochemical analysis. Staining patterns of a–c HLA-A, d–f HLA-B/-C, g–i LMP7 and j–l TAP1 showing a, d, g, j normal expression, b, e, h, k partial loss of expression and c, f, i, l total loss of expressionFig. 2Immunohistochemical staining patterns of ERAP1. a Normal expression. b Partial loss of expressionTable 1Expression of HLA class I heavy chains, β2-microglobulin and overall HLA class IHLA-AHLA-B/-Cβ2-microglobulinHLA class ITotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionOverall (n = 109)36 (33)22 (20)51 (47)24 (22)29 (27)56 (51)8 (7)28 (26)73 (67)21 (19)24 (22)63 (59)Tumour type AC (n = 26)6 (23)5 (19)15 (58)4 (15)8 (31)14 (54)1 (4)7 (27)18 (69)4 (15)5 (19)17 (66) SCC (n = 83)30 (36)17 (21)36 (43)20 (24)21 (25)42 (51)7 (8)21 (25)55 (67)17 (21)19 (23)46 (56)Data shown as n (%)
As shown in Table 2, expression of all APM components except LMP10, calnexin, and calreticulin was lost in some of the cases. This down-regulation was significantly associated with HLA class I down-regulation for all components, except ERAP1. Moreover, defects in overall TAP expression (defined as partial or total loss of either or both of the TAP subunits) were significantly associated with HLA class I down-regulation (P < 0.001). Down-regulation of any APM component was significantly associated with HLA class I down-regulation (P < 0.001).
Table 2Overall and histological type-specific expression of APM componentsOverall (n = 101)AC (n = 25)SCC (n = 76)Total lossPartial lossNormal expressionPTotal lossPartial lossNormal expressionPTotal lossPartial lossNormal expressionPLMP214 (14)30 (30)57 (56)0.0145 (20)11 (44)9 (36)0.7729 (12)19 (25)48 (63)0.001LMP73 (3)16 (16)82 (81)<0.0012 (8)9 (36)14 (56)0.1001 (1)7 (9)68 (90)<0.001LMP1000101 (100)TAP14 (4)19 (19)78 (77)<0.0010 (0)4 (16)21 (84)0.2304 (5)15 (20)57 (75)0.001TAP211 (11)26 (26)64 (63)<0.0014 (16)6 (24)15 (60)0.0057 (9)20 (26)49 (65)<0.001ERAP1014 (15)80 (85)0.3140 (0)2 (9)20 (91)0.582012 (16)60 (84)0.307Tapasin1 (1)24 (24)76 (75)<0.0010 (0)10 (40)15 (60)0.2841 (1)14 (18)61 (81)<0.001Calnexin00101 (100)ERp5712 (12)40 (40)49 (48)0.0171 (4)6 (24)18 (72)0.61511 (15)34 (45)31 (40)0.002Calreticulin00101 (100)Data shown as n (%). P values are shown for association with overall HLA class I down-regulation
Association with histopathological parameters
Depth of invasion exceeding 15 mm and vaso-invasive growth are unfavourable prognostic indicators. Of the cases examined, 51 (45%) demonstrated depth of invasion greater than 15 mm and 38 (33%) demonstrated vaso-invasive growth. Total loss of overall HLA class I expression and of any APM component was found to be significantly associated with depth of invasion exceeding 15 mm (P = 0.034 and 0.045, respectively), while no significant association was found for defective expression of any marker and vaso-invasion.
None of the markers was found to be significantly associated with FIGO stage, corresponding to the lack of a significant association with histopathological parameters. No association of any of the markers with specific HPV status of the patients was detected. Interestingly, partial LMP2 and/or LMP7 loss was significantly associated with an absence of detectable lymph node metastases (P = 0.023 and 0.021, respectively).
Association with overall and disease-free survival
As shown in Table 3 and Figs. 3 and 4, partial loss of overall HLA class I expression was significantly associated with decreased OS, and exhibited a trend towards a significant association with DFS, whereas total loss was not significantly associated with survival. Moreover, partial HLA-A loss (evaluated by HCA2 reactivity) was significantly associated with decreased OS and DFS, whereas total loss was not significantly associated with survival. Partial, but not total HLA-B/-C loss (assessed by HC10 reactivity) showed a trend towards significant association with decreased survival.
Table 3HLA class I and APM component expression and 5-years survival ratesOverall survivalDisease-free survivalNormal expressionPartial lossTotal lossNormal expressionPartial lossTotal lossHLA-A42 (83)12 (55)29 (81)42 (83)13 (60)28 (77)HLA-B/-C45 (81)19 (64)18 (75)46 (82)19 (67)18 (73)HLA class I52 (83)15 (61)16 (77)52 (82)16 (65)15 (75)LMP724 (74)17 (87)1 (33)62 (76)13 (80)2 (50)TAP161 (78)8 (70)1 (33)63 (81)13 (67)1 (25)ERAP166 (82)5 (38)–65 (81)7 (50)–Data shown as n (%)Fig. 3Kaplan–Meier curves for overall survival. Patients with normal expression, partial loss, and total loss of a HLA-A, b HLA-B/-C, c overall HLA class I, d LMP7, e TAP1 and f ERAP1 expression. P values are shown for comparison with normal expressionFig. 4Kaplan–Meier curves for disease-free survival. Patients with normal expression, partial loss, and total loss of a HLA-A, b HLA-B/-C, c overall HLA class I, d LMP7, e TAP1 and f ERAP1 expression. P values are shown for comparison with normal expression
Decreased OS and DFS was significantly associated with total TAP1 loss, but not with normal expression and partial loss. A similar pattern was observed for partial LMP7 loss, though this was not significantly associated with DFS. ERAP1 loss was significantly associated with decreased OS and DFS as compared to normal ERAP1 expression.
Multivariate analysis
As expected, well known prognostic parameters such as depth of infiltration exceeding 15 mm, presence of vaso-invasive growth, and presence of lymph node metastases were significantly associated with a shorter survival, while FIGO-stages of IB2 or more showed a significant association with shorter DFS (Table 4). HPV type was not significantly associated with survival (data not shown). Subsequently, multivariate analysis was performed on overall HLA class I, TAP1, LMP7 (for OS only) and ERAP1 expression, combined with the aforementioned prognostic factors. ERAP1 down-regulation was demonstrated to be an independent prognostic parameter for shorter OS (hazard ratio (HR) 3.08; 95% CI 1.07–8.90; P = 0.037) and shorter DFS (HR 2.84; 95% CI 1.01–8.03; P = 0.049), as was presence of lymph node metastases (HR 7.02; 95% CI 2.55–19.32; P < 0.001 for OS and HR 6.64; 95% CI 2.53–17.44; P < 0.001 for DFS). Total TAP1 loss was also an independent predictor for shorter DFS (HR 6.72; 95% CI 1.29–35.06; P = 0.024).
Table 4Histopathological parameters and 5-years survival ratesOverall survivalDisease-free survival%P%PDepth of infiltration <15 mm (n = 59)8890 ≥15 mm (n = 51)60<0.00157<0.001Vaso-invasion Absent (n = 68)8185 Present (n = 38)590.031540.002Lymph nodes Negative (n = 80)8686 Positive (n = 27)43<0.00139<0.001Histopathological class SCC (n = 84)7576 AC (n = 26)770.924740.662FIGO stage ≤IB1 (n = 52)8291 ≥IB2 (n = 53)690.060570.002
Discussion
In the present study we have investigated HLA class I and APM component expression in cervical carcinoma patients and have evaluated the relationship of these factors with survival and other clinicopathological parameters.
Our results show that HLA class I down-regulation occurs frequently (∼40% of cases) in cervical carcinoma, corresponding to previous reports [19, 21]. Synchronous partial or total loss of various APM components was found in a substantial number of cases and was demonstrated to be significantly associated with HLA class I down-regulation. We observed partial HLA class I loss to be significantly associated with decreased OS. Of the APM components, TAP1, LMP7 and ERAP1 down-regulation exhibited significant associations with decreased survival. ERAP1 down-regulation was shown to be an independent, significant predictor of shorter survival.
Though defective APM component expression is hypothesized to be an important mechanism of HLA class I down-regulation and therefore immune evasion by tumour cells, information regarding APM down-regulation in carcinomas is limited. This is partly due to the scarcity of reliable antibodies for most APM components other than the TAP subunits. Furthermore, most previous studies of APM expression in cervical cancer were limited by small sample sizes. Lastly, the use of various scoring methods for evaluation of immunohistochemical staining has rendered proper interpretation of findings difficult, leading to conflicting data, e.g. regarding the frequency of TAP1 loss [9, 18–20].
In this study, a panel of recently reported monoclonal antibodies was used to investigate expression of all known APM components in a large set of cervical carcinoma lesions using a TMA-based approach [30–33]. Staining was evaluated according to a previously reported quality control system [37], which is more stringent in defining total and partial expression loss than other evaluation methods, which consider ≤25% positive cells as total loss. The system used here defines total loss as complete or near-complete negativity and is therefore more reliable. Moreover, by excluding β2-m-negative cases from the analysis, all outcome parameters can be reliably correlated to expression levels of the various APM components, as any influence of defective APM expression will only be detectable in a β2-m-positive setting.
As an APM component, ERAP1 has been shown to be an important determinant of the repertoire of HLA class I-presented peptides [14, 34, 38–41]. However, to date no systematic investigation of its potential role in HLA class I down-regulation in tumours has been performed. To our knowledge, this is the first description of ERAP1 down-regulation in a human carcinoma. Additionally, we have identified ERAP1 loss as an independent predictor of shorter survival, in the absence of associated HLA class I down-regulation. A possible explanation for this observation is the role of ERAP1 in shaping the antigen repertoire: ERAP1 down-regulation may lead to the preferential loading and presentation of non-tumour-associated peptides, thereby yielding a less immunogenic phenotype and facilitating tumour growth and progression. This hypothesis is supported by previous reports on the disrupted nature of presented peptide repertoire in ERAP1-deficient mice [41, 42]. Also, the effect of down-regulation of several APM components on the presentation of specific HPV 16 E6-derived epitopes has been firmly established [43–45].
Although we found that total TAP1 loss was an independent predictor for shorter DFS, this finding should be interpreted with caution as only four patients exhibited this loss. The decreased significance of ERAP1 as an independent predictor for shorter DFS can be explained by the interdependency of ERAP1-mediated peptide trimming and TAP-mediated peptide transport.
An interesting finding in this study is the observed association of LMP2 and LMP7 down-regulation with an absence of lymph node metastasis; this finding is seemingly corroborated by reports that certain immunogenic peptides in melanoma and renal-cell carcinoma are not generated by immunoproteasomes and that presentation of these peptides is reduced upon up-regulation of the immunoproteasomal subunits [46]. Though altered proteasomal cleavage of endogenous proteins can lead to an alternative, less immunogenic antigen repertoire, an adequate mechanism for the association of LMP down-regulation with an absence of lymph node metastases remains to be postulated.
Interestingly, we have recently observed that genetic variation in various APM components is significantly associated with increased cervical carcinoma risk. Although the underlying mechanisms of APM component loss are largely unknown, the occurrence of single nucleotide polymorphisms in the various APM genes could form a genetic basis predisposing to either mutations, or regulatory down-regulation [47, 48].
Partial HLA class I loss was observed to be significantly associated with decreased OS; correspondingly, reports in colon cancer have shown partial, but not total HLA class I loss to be associated with worse OS [49]. This finding can be explained by the ability of low levels of HLA class I molecules to provide tumour cells with an escape not only from cytotoxic T lymphocyte-mediated, but also from natural killer (NK) cell-mediated cytotoxicity [2]. This hypothesis is further supported by the association of decreased survival with total TAP1 loss, which has been shown previously to lead to partial HLA class I loss [2, 50–52]. In the present study, an association of loss or down-regulation of expression of the various APM molecules with aberrant HLA expression was observed. Moreover, the partial HLA loss observed is most likely an underestimation of the extent of HLA alterations present as immunohistochemical staining on paraffin sections does not reveal loss of specific HLA alleles [8]. However, there are also other aberrations known to lead to HLA class I alterations and it is plausible to hypothesize that all these mechanisms can coexist in the same tumour sample.
In conclusion, we have shown here that defective expression of HLA class I and of various APM components occurs frequently in cervical carcinoma. Partial HLA class I loss and total TAP1 loss are associated with decreased survival, while down-regulation of immunoproteasomal subunits is associated with decreased risk of lymphogenic metastasis. Moreover, we report the first description of ERAP1 down-regulation in a human carcinoma, and have found that this is an independent prognostic parameter for decreased survival. A complete understanding of the mechanisms and relevance of HLA class I and APM component down-regulation and immune evasion may contribute to the rational design of tumour vaccines and T-cell-based immunotherapies.

antigen processing machinery
cervical carcinoma
erap1


Reproducibility of onset and recovery oxygen uptake kinetics in moderately impaired patients with chronic heart failure

Oxygen (O2) kinetics reflect the ability to adapt to or recover from exercise that is indicative of daily life. In patients with chronic heart failure (CHF), parameters of O2 kinetics have shown to be useful for clinical purposes like grading of functional impairment and assessment of prognosis. This study compared the goodness of fit and reproducibility of previously described methods to assess O2 kinetics in these patients. Nineteen CHF patients, New York Heart Association class II–III, performed two constant-load tests on a cycle ergometer at 50% of the maximum workload. Time constants of O2 onset- and recovery kinetics (τ) were calculated by mono-exponential modeling with four different sampling intervals (5 and 10 s, 5 and 8 breaths). The goodness of fit was expressed as the coefficient of determination (R2). Onset kinetics were also evaluated by the mean response time (MRT). Considering O2 onset kinetics, τ showed a significant inverse correlation with peak- (R = −0.88, using 10 s sampling intervals). The limits of agreement of both τ and MRT, however, were not clinically acceptable. O2 recovery kinetics yielded better reproducibility and goodness of fit. Using the most optimal sampling interval (5 breaths), a change of at least 13 s in τ is needed to exceed normal test-to-test variations. In conclusion, O2 recovery kinetics are more reproducible for clinical purposes than O2 onset kinetics in moderately impaired patients with CHF. It should be recognized that this observation cannot be assumed to be generalizable to more severely impaired CHF patients.
Introduction
Oxygen (O2) kinetics describe the rate change of oxygen uptake
during onset or recovery of exercise and reflect changes in cardiac output and tissue oxygen extraction. Compared to healthy individuals, patients with chronic heart failure (CHF) have slower O2 onset and recovery kinetics, resulting in early fatigue and slow recovery after exertion due to a greater reliance on anaerobic metabolism (Wasserman et al. 1996; Koike et al. 1995). Although peak
is widely accepted as a reliable indicator of maximal aerobic capacity in CHF patients (Weber et al. 1982; Janicki et al. 1990), O2 kinetics provide additional objective information on the ability to adapt to and recover from exercise that is indicative of daily life (Riley et al. 1994; Koike et al. 1995). Furthermore, O2 kinetics are potentially useful for risk stratification of CHF patients (Brunner-La Rocca et al. 1999; Schalcher et al. 2003) and for measuring the effects of exercise training, which has already been demonstrated in healthy individuals (Carter et al. 2000) and patients with chronic obstructive pulmonary disease (COPD) (Puente-Maestu et al. 2000).
In order to use O2 kinetics for these clinical purposes it is necessary to know more about the applicability and reproducibility of these exercise parameters in this specific patient group. Until now there has been no uniformity in the assessment of O2 kinetics in patients with CHF (Arena et al. 2001). In addition, the reproducibility of O2 kinetics at submaximal exercise in CHF patients has not been studied extensively. Two studies that assessed O2 onset kinetics by different modeling techniques, suggest an acceptable reproducibility of nonlinear regression and an algebraic method (Belardinelli et al. 1998; Sietsema et al. 1994). In both studies, however, intra-class correlations and limits of agreement were not mentioned.
The purpose of this study is to evaluate the goodness of fit and reproducibility of previously described clinically applicable methods to characterize O2 onset and recovery kinetics in moderately impaired patients with CHF. Furthermore, we aim to define interventional changes that are required to distinguish from the normal test-to-test variations.
Methods
Subjects
Nineteen patients (15 men, 4 women) with stable CHF (New York Heart Association class II–III and echocardiographical ejection fraction ≤40%) attributed to idiopathic dilated cardiomyopathy (n = 4) or ischemic heart disease due to myocardial infarction (n = 15) were selected at the cardiology outdoor clinic of the Máxima Medical Centre (Veldhoven, The Netherlands). Fifteen patients were in NYHA functional class II and four in class III. Subject characteristics are listed in Table 1. Patients with recent myocardial infarction (<3 months), angina pectoris at rest, atrial fibrillation, or atrial flutter were not included. All patients performed a pulmonary function test using a spirometer (Masterlab, Jaeger, Würzburg, Germany) including measurement of forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) during a maximal forced expiratory effort. Patients with chronic airways obstruction, defined as FEV1/FVC < 60% were excluded.
Table 1Characteristics of included patients with CHF (N = 19)VariablesMean ± SDRangeAge (years)62 ± 843–78Height (cm)172 ± 8155–184Weight (kg)85 ± 1054–97Body mass index (kg/m2)29 ± 422–37Fat mass (%)a30 ± 720–43Time since diagnosis (months)20 ± 246–96Left ventricular ejection fraction (%)33 ± 719–40a Fat mass was assessed by skinfold measurements (biceps, triceps, subscapular, and suprailiacal) according to standard procedures (WHO 1995)
Fifteen patients used beta-blockers and angiotensin-converting enzyme inhibitors, three patients used an angiotensin-converting enzyme inhibitor only, and one patient used a beta-blocker only. Sixteen patients used diuretics. The average duration that patients were using beta-blockers was 34 ± 33 months (range 7–112 months) and 32 ± 29 months for ACE inhibitors (range 7–118 months). Patients who did not use beta-blockers were not different from the other patients with respect to age, peak-
or left ventricular ejection fraction.
The research protocol was approved by the local Research Ethics Committee of the Máxima Medical Centre, and all patients provided written informed consent.
Exercise testing
Subjects performed a symptom-limited, incremental exercise test, and on a separate day (at least 3 days later), a constant-load test at 50% of the maximum workload achieved at the first test. This test was repeated at the same time on another day within 2 weeks (mean difference between tests 6.7 ± 3.9 days). All subjects took their medication at the usual time and were instructed not to perform any extra physical activity on testing days. During the testing period all patients were on sinus rhythm. Furthermore, none of the patients reported changes in symptoms, functional status or medication use. Therefore, they could be considered to be in a stable physical condition during the study period.
All exercise tests were performed in an upright seated position on an electromagnetically braked cycle ergometer (Corival, Lode, Groningen, The Netherlands). Measurements of
and respired fractions of
and
were obtained breath by breath (Oxycon α, Jaeger, Germany). Volumes and gas analysers were calibrated before each test.
The incremental exercise test was performed using an individualized ramp protocol with a total test duration of 8–12 min (Working Group on Cardiac Rehabilitation & Excercise Physiology and Working Group on Heart Failure of the European Society of Cardiology 2001). During the tests all patients were instructed to maintain a pedaling frequency of 70 per minute. A 12-lead electrocardiogram was registered continuously, and blood pressure was measured every 2 min (Korotkoff). The test was ended when the patient was not able to maintain the required pedaling frequency. Maximal workload was defined as the final registered workload, peak-
as the average
of the last 30 s of the test. Predicted value of peak-
was calculated with use of the Wasserman equation, normalizing peak-
for age, gender, weight and height (Hansen et al. 1984).
The ventilatory threshold was determined by two independent observers using the V-slope method as described by Beaver et al. (1986). The constant-load tests included 2 min of rest, 2 min of unloaded pedaling, 6 min at 50% of the maximum workload and 5 min of rest.
Data analysis
Mono-exponential model
Time constants (τ) were calculated by fitting the
data of the constant-load tests to a first-order (mono-exponential) model using the non-linear least squares method (Whipp and Wasserman 1972). Calculations were performed with breath-by-breath data averaged into four different sampling intervals that were used in previous studies: 5 s (5 s) (Matsumoto et al. 1999), 10 s (10 s) (Arena et al. 2002), 5 breaths (5b) (Koike et al. 1995) and 8 breaths (8b) (Pavia et al. 1999). The following formulas were used: A=B=Td=time delayτ=time constant (s)Baseline-
was defined as the average
of the last minute of the unloaded-cycling stage and steady-state-
as the average
of the last minute of exercise. The time delay (Td) is a parameter allowed to vary in order to optimize the fit, representing the time between onset of exercise and the start of the mono-exponential increase of
One of the determinants of this time delay is the lag time between the computer signal to deliver the work rate and the actual response of the ergometer, which amounted to 2.2 ± 0.6 s in this study. Occasional errant breaths (e.g., due to coughing, swallowing or talking) were deleted from the data set when
exceeded three standard deviations of the mean, defined as the average of two following and two preceding sampling intervals (Lamarra et al. 1987). In total, about 1% of the breaths had to be deleted.
Algebraic method
O2 onset kinetics were also evaluated by an algebraic method calculating mean response time (MRT) (Sietsema et al. 1994), using the following formulas:MRTO2 deficitThe expected amount of O2-uptake
was calculated by multiplying the O2-amplitude
with exercise duration (6 min). Oxygen deficit was calculated by subtracting the summed
above the baseline-value
from the expected
Statistical analysis
All data (presented as mean ± SD) were analyzed using a statistical software program (SPSS 11.0). The ‘goodness of fit’ for mono-exponential modeling was evaluated by the coefficient of determination (R2). The fitting procedure was considered acceptable when R2 ≥ 0.85, as previously described by de Groote et al. (1996). Differences between calculation methods were evaluated by one-way ANOVA with repeated measures and Bonferroni post hoc analyses. In order to assess differences between kinetic parameters of the two tests the paired Student’s t test was used. Linear regression was used to define correlations between variables. Agreement between the kinetic parameters was assessed by intra-class correlation coefficients, limits of agreement (mean difference ±1.96 × SD) (Bland and Altman 1986) and coefficients of variation (SD of difference as a percentage of the mean value). Probability values <0.05 were considered statistically significant.
Results
Symptom-limited exercise tests
All subjects completed the exercise tests. The maximum workload was 109 ± 32 W, peak-
was 20.0 ± 4.0 ml min−1 kg−1 (73 ± 9% of predicted peak-
) and the maximal respiratory exchange ratio was 1.13 ± 0.13. The ventilatory threshold could not be determined in three patients (16%) because of excessive ventilatory oscillations. In the remaining 16 patients the independent observers agreed on the determination of the ventilatory threshold (mean
:16.4 ± 3.2 ml min−1 kg−1, 60 ± 11% of predicted peak-
).
Constant-load exercise tests
The mean value of
during the second minute of unloaded pedaling was 655 ± 78 ml min−1 (30 ± 5% of predicted peak-
), and the steady-state value at 50% of the maximal work load was 1,185 ± 228 ml min−1 (53 ± 7% of predicted peak-
). Figure 1 shows changes in
during a constant-load test in a representative subject.
Fig. 1-response to steady-state exercise at 50% of the maximal workload (50 W) in a representative subject. The solid line represents 10 s averages of
The curved dashed line is the computer-derived representation of the best fit of the mono-exponential model to the
-response. The first dashed vertical line indicates onset of exercise and the second vertical line the end of exercise
In 16 subjects, in whom the ventilatory threshold could be determined reliably, steady-state-
was below the ventilatory threshold. None of the other three subjects demonstrated a significant rise of
defined as in increase from the third to the sixth minute of exercise of more than two times the SD of the mean
in the fourth minute. This indicates that these three patients also exercised below the ventilatory threshold (Whipp 1994).
Comparison of calculation methods
Concerning the onset phase, there were no significant differences between mono-exponential modeling and the algebraic method (differences between τ and MRT −6.6–1 s, SD 11–17 s). In the recovery phase, the use of different sampling intervals yielded significantly different time constants (P < 0.001). Paired comparisons showed that in two cases the time constants were not significantly different (5 s vs. 5 breaths, P = 0.14 and 10 s vs. 8 breaths, P = 0.31).
When comparing the applicability of the mono-exponential model to the
-response during onset and recovery of exercise, the results indicate a better ‘goodness of fit’ during recovery (P < 0.001) (Table 2). Parameters of O2 onset kinetics showed significant correlations with peak-
when calculating τ with sampling intervals of 10 s, 5 breaths and 8 breaths (Table 3). However, mainly due to the limited number of patients, the differences between these correlation coefficients were not statistically significant. During recovery of exercise τ was only correlated with peak-
when 5 breath sampling intervals were used.
Table 2O2 Kinetics using an algebraic method and a mono-exponential model with different sampling intervals (n = 19)Kinetic parametersMean ± SD (s)Goodness of fit (R2) Mean ± SDUseful measurements (%)aO2 Onset kinetics MRT71 ± 19–– τ-5 s67 ± 200.69 ± 0.1526 τ-10 s74 ± 22 0.81 ± 0.1242 τ-5b75 ± 300.87 ± 0.0879 τ-8b74 ± 220.91 ± 0.0784O2 Recovery kinetics τ-5 s60 ± 130.89 ± 0.0684 τ-10 s66 ± 140.94 ± 0.04100 τ-5b61 ± 130.96 ± 0.03100 τ-8b64 ± 130.98 ± 0.02100Average duration of 5 breath sampling interval 15.9 ± 3.6 s at baseline, 13.0 ± 3.0 s at steady-state. Average duration of 8 breath sampling interval 25.0 ± 5.2 s at baseline, 20.9 ± 4.7 s at steady stateMRT Mean response time, τ time constant calculated with a mono-exponential model, 5s 5 s, 10 s 10 s, 5b 5 breaths, 8b 8 breathsa The assessment of τ was considered useful when R2 ≥ 0.85Table 3Correlation coefficients between averaged kinetic parameters and peak
Kinetic parametersCorrelation with peak
95% Confidence intervalO2 Onset kineticsMRT−0.33−0.680.15τ−5 s–τ-10 s−0.88*−0.99−0.24τ-5b−0.67*−0.90−0.16τ-8b−0.57 *−0.83−0.10O2 Recovery kineticsτ-5 s−0.43−0.760.08τ-10 s−0.45−0.750.01τ-5b−0.47*−0.76−0.02τ-8b−0.39−0.720.08Only data with R2 > 0.85 when using a mono-exponential model were included; differences between correlation coefficients were not statistically significant* P < 0.05MRT Mean response time, τ = time constant calculated with a mono-exponential model, 5s 5 s, 10s 10 s, 5b 5 breaths, 8b 8 breaths
Reproducibility
In the two constant-load tests there were no statistically significant differences between
during unloaded pedaling (655 ± 78 vs. 633 ± 98 ml min−1),
at steady-state exercise (1,185 ± 228 vs. 1,202 ± 235 ml min−1), and respiratory exchange ratios at steady-state exercise (0.94 ± 0.04 vs. 0.94 ± 0.06). In addition, there were no statistically significant differences between the kinetic parameters of both tests. Considering limits of agreement, coefficients of variation and intra-class correlation coefficients, recovery kinetics show better reproducibility than onset kinetics (Table 4).
Table 4Comparison of kinetic parameters in two consecutive constant-load testsKinetic parameterNDifference mean ± SDLimits of agreementCoefficient of variation (%)Intraclass correlation coefficientO2 Onset kinetics
(ml min−1 kg−1)190.1 ± 1.2−2.32.58.50.97 MRT (s)192.8 ± 12.9−232918.40.86τ−5 s (s)0––––τ-10 s (s)69.3 ± 11.8−143317.10.79τ-5b (s)125.9 ± 19.8−344628.40.63τ-8b (s)165.6 ± 16.7−283923.00.77O2 Recovery kineticsτ-5 s (s)16−0.4 ± 7.2−151412.20.91τ-10 s (s)19−0.9 ± 10.7−222116.30.86τ-5 b (s)19−0.9 ± 6.4−141210.40.94τ-8 b (s)19−1.4 ± 8.6−191613.40.91Only data with R2 > 0.85 when using a mono-exponential model were included Oxygen uptake at steady-state exercise, MRT mean response time; τ time constant calculated with a mono-exponential model, 5 s 5 s; 10 s 10 s; 5b 5 breaths, 8b 8 breaths
Figure 2 shows Bland Altman plots of the kinetic parameters during onset and recovery of exercise with the highest intra-class correlation coefficients.
Fig. 2Bland Altman plots showing the difference of O2 kinetics between two constant-load tests during onset (upper graph) and recovery of exercise (lower graph), using an algebraic method and mono-exponential modeling with 5 breath sampling intervals, respectively. The solid lines represent the mean difference between the two tests, the dashed lines indicate the 95% confidence intervals of the difference. MRT mean response time. τ -Rec = time constant of
during recovery with 5-breath sampling intervals
Discussion
The principal finding of this study is that, using the applied exercise protocol, O2 recovery kinetics were more reproducible than O2 onset kinetics in moderately impaired patients with CHF. It should be recognized, however, that this observation cannot be assumed to be generalizable to more severely impaired CHF patients.
O2 onset kinetics
Because of the lack of standardized protocols, previous authors used both time constants (τ, non-linear regression) (Belardinelli et al. 1998; Koike et al. 1995; Matsumoto et al. 1999) and mean response times (MRT, algebraic method) (Sietsema et al. 1994) to assess O2 onset kinetics in CHF patients. Considering non-linear regression, the use of 10 s sampling intervals yielded the best results in terms of reproducibility in this study. However, the goodness of fit, which was comparable with the study of Arena et al. (2002) (0.81 vs. 0.78, respectively), was insufficient in 58% of the patients. Moreover, the wide limits of agreement restrict its use for clinical applications. While the calculation of τ with larger sampling intervals resulted in a better goodness of fit, their reproducibility was even lower. To our knowledge, only one study previously addressed reproducibility of O2 onset kinetics using mono-exponential modeling in CHF patients. In that study five patients with a peak-
that was comparable to our study performed three constant-load tests at a workload of 50 W, starting from unloaded cycling. Although the low mean difference between the tests (2 s) suggests good reproducibility, the actual variability between the tests was not mentioned, making it impossible to compare these results to our study (Belardinelli et al. 1998). In spite of the fact that almost all of the onset procedures showed a significant correlation with peak-
(Table 3), differences between these correlations were not statistically significant. Therefore, one could not suggest a preferential use of one of the procedures.
Although showing a slightly higher intra-class correlation coefficient (Table 4), the wide limits of agreement of the MRT also indicate low reproducibility of this parameter. In addition, this parameter did not correlate significantly with peak
Sietsema et al. (1994) previously addressed reproducibility of the MRT in 18 CHF patients, reporting small mean differences between two tests. Again these results cannot be compared to our study, because intra-class correlations and limits of agreement were not mentioned.
When comparing reproducibility of O2 onset kinetics in CHF patients with healthy subjects (Kilding et al. 2005) and patients with COPD (Puente-Maestu et al. 2001) we found a lower reproducibility of τ (coefficients of variation 17.1, 6.2 and 8.7%, respectively). There are several physiological and methodological factors that may explain this discrepancy.
One significant physiological factor that may cause the large variability of O2 onset kinetics in CHF patients is the influence of typical ventilatory oscillations, especially when small sampling intervals are used (Francis et al. 2002). Although we did not quantify the effect of oscillations, we did observe ventilatory oscillations more clearly in patients with low coefficients of determination. Furthermore, it is postulated that ventilatory oscillations in patients with CHF increase in the transition from rest to exercise (Kremser et al. 1987) and diminish with increasing exercise (Francis et al. 2002). This suggests that these oscillations have a greater influence on O2 onset kinetics than O2 recovery kinetics. Based on our study, variations in τ during onset of at least 24 s are needed to exceed the limits of the 95% confidence interval, whereas a change of only 13 s in τ during recovery is sufficient to exceed the normal test-to-test variations. This latter variability is also observed during the on-transient response in patients with COPD (Puente-Maestu et al. 2001), which supports our view that the relatively large variability of the on-kinetic parameters was mainly caused by ventilatory oscillations.
Another explanation for the limited reproducibility of O2 onset kinetics in CHF patients is their low exercise capacity, which reduces the amplitude of O2 uptake, and consequently the reliability of the determination of the time constant (Lamarra et al. 1987). Furthermore, the period between the tests (6.7 ± 3.9 days) could be a long enough period of time to induce changes in the cardio circulatory condition of the patients. However, all patients were in a stable clinical or functional condition during the study period.
Considering methodological factors, the fact that all patients started the constant-load test with unloaded pedaling in stead of rest resulted in a relatively low
-amplitude. This could be a major factor contributing to the relatively low reproducibility of the on-kinetic parameters found in this study. The reasons for the authors, however, to apply this exercise protocol were threefold. First, starting exercise from unloaded pedaling results in a reduction of the duration of the early rapid increase of oxygen uptake, representing an initial increase of pulmonary blood flow (cardiodynamic phase). Since in this phase the change of
is functionally distinct from the subsequent mono-exponential increase of
(phase II), reducing the relative contribution of this phase might result in a better fit of the mono-exponential model to the data. Second, starting exercise from unloaded cycling might reduce ventilatory oscillations at the onset of exercise because of a reduction in variation of pedaling frequency and upper limb muscle activity. Third, in daily life exercise will be frequently started from an active state. Given the results of this study, however, the authors feel that additional research is required to re-assess the reproducibility of O2 onset kinetics using an exercise protocol starting from rest in stead of unloaded cycling. Starting exercise from rest might result in a better reproducibility of O2 onset kinetics than observed in this study, which may in particular be of importance for more severely impaired CHF patients than used in this study.
Another factor that may have influenced reproducibility of O2 onset kinetics is the choice of the workload (i.e. 50% of the maximum workload). The approach to maximize
-amplitude by relating the workload to the ventilatory threshold (e.g. 90% of ventilatory threshold) was not used in this study, because it was postulated that such a fixed threshold cannot be determined reliably in a substantial number of CHF patients (Meyer et al. 1996). When looking at the patients of this study in whom determination of this threshold was successful,
at steady state amounted to 86% of
at the ventilatory threshold, with none of the patients exercising above this threshold. This suggests that the applied constant-load exercise protocol was adequate, because the exercise intensity remained below the ventilatory threshold.
O2 recovery kinetics
O2 recovery kinetics were more reproducible in moderately impaired patients with CHF than O2 onset kinetics. This difference in reproducibility is at least partly explained by the larger
-amplitude in recovery due to the fact that subjects were not cycling in the recovery phase. From the results of this study it is difficult to conclude to what extent this difference between onset and recovery kinetics is caused by a smaller influence of the cardiodynamic phase during recovery or a more stable breathing pattern during recovery causing less ventilatory oscillations. Nevertheless, these observations are in line with previous studies in healthy individuals, which also reported a better reproducibility of O2 recovery kinetics than O2 onset kinetics (Kilding et al. 2005; Ozyener et al. 2001).
In terms of reproducibility, the most optimal method to characterize O2 recovery kinetics was mono-exponential modeling with sampling intervals of 5 breaths (intra-class correlation 0.94). Using this method a change of at least 13 s in τ is needed to exceed the normal test-to-test variations. In addition, this method yielded an excellent goodness of fit (R2 0.96 ± 0.03). To our knowledge, other data on the reproducibility of the recovery kinetics after submaximal exercise in CHF patients are not available. Cohen-Solal et al. (1995) studied reproducibility of O2 recovery kinetics after maximal exercise in 10 patients with CHF (NYHA II-III) using a mono-exponential model with sampling intervals of 7 breaths. They found a coefficient of variation comparable to our study with 5 breath sampling intervals (12.3 vs. 10.4%, respectively).
Conclusion
This study shows that the reproducibility of O2 onset kinetics assessed by mono-exponential modeling is too low to warrant their use for measuring effects of therapeutic interventions in moderately impaired patients with CHF. This might be mainly attributable to physiological factors like ventilatory oscillations and the applied exercise protocol. Future studies should address the effect of different exercise protocols.
The determination of time constants of O2 recovery kinetics has been shown to be feasible and reproducible when using mono-exponential modeling with 5 breath sampling intervals. Since this variable represents recovery from exercise that is indicative of daily life, it is potentially useful for clinical purposes like grading of functional impairment in patients with CHF and measuring effects of therapeutic interventions.

oxygen uptake kinetics
time constant
mean response time
exercise testing
cardiac disease


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

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 Delphiniumbelladonna 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°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.
Introduction
Floral life span depends on the fate of the petals. In several species the end of floral life span is due to petal withering or wilting. The petals in most of these species desiccate, and finally abscise or fall due to growth of the fruit. Only in a few species the desiccated petals remain attached to the fruit. In another large group of species the end of floral life span is determined by abscission of petals that are mostly or fully turgid.
Wilting in petals, at the end of their lifespan, is due to a series of highly regulated processes that lead to cell death, thus is a form of programmed cell death (PCD; van Doorn et al. 2003). Prior to visible PCD, many macromolecules in the petal cells are degraded to molecules that are suitable for phloem transport. These mobile molecules are transported to other parts of the plant (Winkenbach et al. 1970a, b). Typical for PCD in wilting petals is the disappearance of most of the organelles and accumulation of degrading enzymes in the vacuole. This is followed by rupture of the vacuolar membrane, whereby several hydrolases are released. Tonoplast rupture in numerous cells results in their collapse, which is visible as wilting (Winkenbach 1970a, b; van Doorn et al. 2003). PCD in several plant parts (Gunawardena et al. 2001; Wojciechowska and Olszewska 2003), including petals, is also usually accompanied by DNA degradation (van der Kop et al. 2003; Wagstaff et al. 2003; Yamada et al. 2006a, b) and chromatin condensation (Yamada et al. 2006a, b).
It is not known if the abscission of turgid petals is preceded by cellular changes that are indicative of PCD. We therefore investigated the time line of some of these changes, in Prunus yedoensis and Delphiniumbelladonna. In both species the petals abscise without any visible sign of turgor loss. We used DNA degradation (measured both on agarose gels and by using flow cytometry), chromatin condensation, and a decrease in nuclear diameter as parameters that indicate PCD. According to our hypothesis these parameters would not detectably change, prior to petal abscission.
Materials and methods
Plant material
Plants were grown in the garden of National Institute of Floricultural Science of Japan (P. yedoensis cv. Someiyoshino) or the greenhouse of the institute (D. belladonna cv. Bellamosum, Ipomoea nil cv. Violet, and Petunia hybrida cv. Double Duo Pink). Just before the onset of flower opening, cut branches (P. yedoensis) or potted plants (the other species) were transferred into a growth chamber at 24 ± 1°C, about 70% relative humidity (RH), and 12 h of daily illumination from cool-white fluorescent lights (100 μmol m−2 s−1). The time to petal abscission and petal desiccation was observed daily, on plant material in the growth chamber. The timing of these symptoms was noted in six flowers from two plants. All determinations were made using petals that were collected from plant material in the chamber.
DNA degradation
DNA breakdown was detected by agarose gel analysis. Total DNA was extracted according to Yamada et al. (2003). The amount of DNA was estimated by molecular absorption spectrophotometry. Equal amounts of total DNA (3 μg) were immediately electrophoresed in a gel containing 3% agarose and stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR, USA). The gel pattern was photographed, using an electronic UV transilluminator system (model FAS-III mini + DS-30; Toyobo, Tokyo, Japan). The DNA degradation index was calculated according to Yamada et al. (2006b).
Two biological replications, each containing several petals, were used for each stage of development. Depending on the species, two to several petals were used in each sample.
Flow cytometry
The petal was chopped in nuclear extraction buffer, part of the kit (High Resolution kit) for plant DNA analysis (Partec, Münster, Germany), a reagent set provided by the manufacturer of the flow cytometer used (Ploidy Analyzer, Partec). The extraction buffer in this kit was a low pH solution containing Triton X-100. The extract was filtered through 50 μm nylon mesh. The medium with the isolated DNA masses (nuclei) was collected, the buffer with the fluorescent DNA stain 4, 6-diamidino-2-phenylindole (DAPI) from a standard reagent set (Partec) was added, and the solution was vortex mixed. The DNA content of the isolated nuclei was analysed using the flow cytometer. The flow cytometer apparatus can be set to count all nuclei in a sample, and can be set to analyse a fixed number of nuclei. We here set it to detect 5,000 nuclei. DNA levels were obtained in a total of 5,000 nuclei. It should be noted that the large histogram peak was adjusted to 10 on the fluorescence scale. This means that nuclear condensation was not taken into account in the flow cytometry data.
Two biological replications, each containing several petals, were used for each stage of development. Depending on the species, three to several petals were used in each sample. In each species, the number of petals was the same at all stages of development.
Nuclear morphology; chromatin condensation
Nuclei were isolated from petals in the same way as for flow cytometry. Isolated nuclei were stained with DAPI, which stains DNA. The nuclei were observed under a fluorescence microscope (model PROVIS AX70, Olympus, Tokyo, Japan) using U-excitation (330–385 nm). Digital photography of nuclei was obtained for each stain using low light cool CCD camera (model DP30BW, Olympus).
Dehydration and ethylene treatment
Flowers were excised when the petals were fully open. Cut flowers were placed in air in the growth chamber (24°C, about 70% RH) or in a desiccator (at 60°C). For ethylene treatments, stems of cut flowers were placed in sterile distilled water, in a closed 70 l clear plastic box containing 2 μl/l ethylene. Petal abscission was checked at hourly intervals and abscised petals were collected immediately. The petals were then used instantly for the measurement of the various parameters mentioned.
Statistics
The number of replications is mentioned in the text and/or in the figure legends. Statistical analysis used one way analysis of variance, at P < 0.05. All experiments were repeated at least once.
Results
Symptoms of petal life span cessation
P. yedoensis flowers have five pale pink petals. After flower opening the petals remained attached to the flower for about 4 days, and then fell without visible wilting (Fig. 1a, P2). During the first day following abscission, petals that were held at 24°C and 70% RH changed colour from pale pink to darker pink, and subsequently became infiltrated with liquid (results not shown). By the end of the first day following abscission, the petals started to shrivel at their margins (Fig. 1a, P3). By the end of the second day after abscission the petals had much more decreased in size (Fig. 1a, P4). The stages 2–4 (pictures P2–P4) in Fig. 1a use the same scale thus show the decrease in petal size.
Fig. 1Petal 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)
Flowers of Delphinium species contain five petals one of which has a large spur. The petals of D. belladonna fell about 7 days after flower opening (Fig. 1b, D2). They did not show any visible wilting symptom by the time of abscission. Two days after abscission the petals had slightly shrivelled (Fig. 1b, D3) but by day-3 after abscission the shrivelling had become very clear (Fig. 1b, D4). The stages 2–4 (pictures D2–D4) in Fig. 1b have the same scale.
DNA degradation
Figure 2 shows the petal DNA agarose gels of one of two repeat experiments. The pixel ratios under the lanes are the means of two experiments. In both species, the agarose gels showed no DNA fragments at the time of flower opening (Fig. 2, lanes P1 and D1). By the time of abscission, the petals of P. yedoensis did not show a statistically significant (P < 0.05) increase in DNA fragmentation, compared with the time of floral opening (Fig. 2, lane P2). In contrast, the petals of D. belladonna showed a considerable increase in DNA fragmentation by the time of abscission (Fig. 2b, lane D2, pixel intensity had increased from 4.3 to 17.4). Following petal abscission the degree of DNA fragmentation, as observed on agarose gels, increased in P. yedoensis (Fig. 2a, compare lane P3 with lane P2) and further increased in D. belladonna (Fig. 2b, compare lane D3 with lane D2). By the time of petal desiccation (P4 and D4) the amount of fragmented DNA had become considerably lower than at the previous stage investigated, when the abscised petal showed visible wilting and dehydration (Fig. 2).
Fig. 2Agarose 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
Nuclear morphology
In P. yedoensis petals the nuclear morphology had not clearly changed by the time of petal abscission (Fig. 3a). This is in contrast with D. belladonna where visual inspection showed that the nucleus was much smaller by the time of abscission, compared with the size observed at flower opening. The small nuclei of D. belladonna showed brighter DAPI fluorescence than the nuclei at flower opening. This indicates a high concentration of DNA per unit volume, thus chromatin condensation (Fig. 3b, D2). Nuclear morphology was studied using light microscopy, giving a two-dimensional impression only. For this reason nuclear size will be referred to as nuclear area.
Fig. 3Morphology 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
After abscission, during the period of dehydration, the nuclei of both species showed an increase in area (Fig. 3) In P. yedoensis the nuclear area at stage P4 seemed even considerably larger than that at floral opening (Fig. 3a). In D. belladonna the nuclear area was apparently similar at D4 compared to the stage of floral opening (D1), but it was ostensibly much larger compared with the area at the stage of petal abscission (P2; Fig. 3b). In both species the nuclear periphery was no longer smooth at stage P4 or D4 (Fig. 3). In both species investigated, numerous nuclei showed the morphology shown in Fig. 3, stages P4 and D4. The area of the remaining nuclei did not increase as much, whilst a few nuclei had not changed in nuclear area with respect to controls at flower opening.
Flow cytometry of nuclei at various stages prior to and after abscission
Flow cytometry of nuclei stained for DNA (with DAPI) shows the distribution of nuclei in fluorescence classes. The X-axis in Fig. 4 is divided in numerous classes of increasing fluorescence, and the Y-axis shows the number of nuclei per fluorescence class. Flow cytometry of 5.000 petal nuclei at the time of P. yedoensis flower opening showed a peak of nuclei at phase G0/G1 (which was set at 101 on the arbitrary logarithmic scale of the X-axis), and no mitotic nuclei (G2/M), which have a fluorescence about two times that of normal G0/G1 nuclei. By the time of petal abscission, no change had occurred in the DNA fluorescence histogram (Fig. 4a, P2). Two days after abscission, the height of the G0/G1 peak had diminished but the peak had not shifted. This is shown by mixing nuclei from day-0 to -6 (Fig. 4a, P1 + P4). The data suggest that a small amount of DNA had become degraded by day-2 after abscission.
Fig. 4Fluorescence 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
By the time of petal abscission in D. belladonna, flow cytometry revealed a decrease in the height of the G0/G1 phase nuclei, and several nuclei with a fluorescence less than that of G0/G1 phase nuclei (Fig. 4b, D2). During the 3 days following abscission the G0/G1 peak height further decreased and the number of nuclei with fluorescence lower than G0/G1 further increased. No clear shift in the G0/G1 peak was observed: a mix of nuclei at day-0 and -10 did not result in a bimodal curve (Fig. 4b, D1 + D4). The results indicate extensive DNA degradation, before, during, and after abscission.
Effects of ethylene
Ethylene treatment greatly hastened the time to petal abscission, in both species (Fig. 5a, compared with Fig. 1a). In both species, the rate of petal dehydration after abscission was not much affected by the ethylene treatment (Fig. 5a) compared with the control without ethylene (Fig. 1a). For example, 3 days after petal fall in D. belladonna the petals had extensively shrivelled, independent of the time of petal fall (day-7 in controls and day-2 in ethylene-treated flowers).
Fig. 5Effect 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)
In both species, flow cytometry revealed no effect of ethylene treatment on DNA fluorescence at the time of petal fall, compared to the fluorescence at the time of flower opening (Fig. 5b). After petal fall, a clear shift to lower DNA fluorescence of the G0/G1 peak of fluorescence was observed in P. yedoensis (Fig. 5b, upper sequence), which was not found in controls without ethylene treatment (Fig. 4, upper sequence). A small shift to lower DNA fluorescence (Fig. 5b, lower sequence), also not found in controls (Fig. 4 lower sequence), was also observed after ethylene treatment of D. belladonna. Superimposition of the stages P1 and EP4 (control and ethylene-treated stage 4 of P. yedoensis) showed two peaks of DNA fluorescence (Fig. 5b, upper sequence). A similar tendency was observed by superimposing the stages D1 and ED3 (Fig. 5b, lower sequence).
Dehydration of petals cut at flower opening
Flowers were harvested just after opening and subjected to a dehydration treatment at 60°C for 24 h. The treatment resulted in a petal FW of about 20% of that of controls. Preliminary experiments showed that such a treatment resulted in a decrease of FW, and in changes of other parameters such as DNA degradation and nuclear morphology, that were very similar to dehydration on the laboratory bench (about 20°C and 70% RH) for about 96 h.
Treatment of P. yedoensis flowers at 60°C for 24 h (Fig. 6a, DP) resulted in a slight increase in petal DNA degradation, as shown on agarose gel (Fig. 6b, DP). In contrast, this treatment induced a large increase in DNA degradation in D. belladonna petals (Fig. 6b, DD).
Fig. 6Effect 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)
The dehydration treatment also had a small effect on DNA fluorescence of nuclei from P. yedoensis petals (Fig. 6c). Superimposition of the nuclei prior to and after treatment showed no shift to lower DNA fluorescence (Fig. 6c, P1 + DP). In D. belladonna, the dehydration treatment resulted in lower G0/G1 DNA fluorescence, and many nuclei with a lower than G0/G1 fluorescence (Fig. 6c). Superposition of the treatment and the control showed no shift of the G0/G1 peak of DNA fluorescence (Fig. 6c, D1 + DD).
Following the dehydration treatment, the nuclei of P. yedoensis had apparently somewhat increased in area, but no clear increase in nuclear area was discernable in D. belladonna (Fig. 6d). The chromatin distribution in the nuclei, after the dehydration treatment (Fig. 6d) was similar to that after dehydration due to abscission (Fig. 3). Visual inspection indicated that about 80% of all nuclei had the morphology shown in Fig. 6c, d. The area of the remaining nuclei had not as much increased.
Dehydration of petals in species that show petal wilting (Ipomoea and Petunia)
Similar dehydration treatments (60°C for 24 h) were carried out with I. nil and Petunia hybrida, species in which the petals wilt by the end of flower life span. Wilting is shown as inrolling in I. nil, which occurs at about 12 h after flower opening, in plants held at 24°C (Fig. 7a). Wilting is observed after 7 days at 24°C in flowers attached to P. hybrida plants (Fig. 7a).
Fig. 7Effect 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
Flowers were detached from potted plants, just after full opening. Fig. 7b shows considerable DNA fragmentation on agarose gels, after the dehydration treatment (60°C for 24 h), in both species tested. Flow cytometry of Ipomoea petals held at 60°C for 24 h showed a large decrease in DNA fluorescence, compared to controls of Ipomoea (Ip) before desiccation (Fig. 7c, compare DIp with Ip, whereby D means dehydrated). Fig. 7d shows that DNA degradation was also found in Petunia (Pe). Superimposition of the fluorescence in controls and the treated samples showed two peaks (Fig. 7c, d).
The morphology of DAPI-stained nuclei of I. nil and P. hybrida, after petal desiccation at 60°C for 24 h, is shown in Fig. 8. Compared to nuclei of petals in flowers that had just fully opened (two panels upper left), relatively large parts not stained with DAPI were observed (two panels upper right). In I. nil the nuclear area had not become clearly increased, compared to those in petals that had not been desiccated (Fig. 8a), but inflated nuclei were observed in P. hybrida (Fig. 8b). Visual inspection indicated that about 80% of all nuclei showed the morphology shown in Fig. 8b. The area of the remaining nuclei had not as much increased.
Fig. 8Effect 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
Both in I. nil (Fig. 8a, S1–S4) and in P. hybrida (Fig. 8b, S1–S4) the nuclear morphology after the dehydration treatment was quite different from the one observed during normal PCD. During PCD of petals that remained attached to the plant the area of the nuclei became increasingly smaller, whereas the brightness of DNA fluorescence remained high at least until the nuclei had become quite small.
Discussion
In reports on animal cells the suggestion that PCD occurs is usually backed up by showing changes such as phosphatidylserine externalization to the outer leaflet of the plasma membrane, caspase activation, and DNA degradation. Only the addition of a number of such parameters seems indicative of PCD. In the present study we took massive DNA degradation (measured in two ways), chromatin condensation, and a decrease in nuclear diameter as indicators of PCD. In other studies on petal PCD, it was shown that these parameters were each correlated with other indicators of PCD and also correlated with visible PCD symptoms such as petal wilting (Yamada et al. 2003, 2006a, b). We therefore suggest that these parameters, which separately represent only circumstantial evidence for PCD, are, when occurring together, adequate to indicate PCD.
We hypothesized that PCD parameters will not show in petal cells, prior to the abscission of turgid petals. The results of D. belladonna petals contradict the hypothesis, while those of P. yedoensis are in agreement with it. The data suggest that the degree of PCD, prior to abscission of turgid petals, depends on the species. The present data indicate that abscission of fully turgid petals may coincide with an advanced stage of PCD in many petal cells. The absence of visible wilting in abscising petals, therefore, does not seem to show absence of PCD. This is so because the wilting that is often associated with PCD is quite a late stage in the PCD process.
In petals the timing of PCD and that of abscission may be regulated independently. Independent regulation would explain the large variation of their relative timing, among the Angiosperms. In many species PCD comes much earlier than abscission (species showing petal wilting at the end of floral life), whereas in other species abscission comes first. In most species in which floral life span is ended by petal wilting, the petals eventually abscise, even when most of the petal surface has become desiccated. In these species the petal base, where abscission takes place, remains alive. Only in a few species fully desiccated petals are torn-off by the growing fruit, or (in still other species) remain attached to the fruit (van Doorn and Stead 1997; van Doorn 2001).
The agarose gel data (Fig. 2) showed substantial DNA degradation prior to and during abscission in D. belladonna and less DNA degradation prior to and during petal fall in P. yedoensis. Flow cytometry data (Fig. 4) confirmed the agarose gel data. DNA degradation seems a relatively late PCD process. Mass protein degradation, a late process, may serve as a yardstick. In Ipomoea petals, bulk degradation of proteins and DNA occurred at about the same time (Winkenbach 1970a, b). In Iris petals, 40% of total DNA had become degraded (van der Kop et al. 2003) by the time of the onset of mass protein degradation (Pak and van Doorn 2005). In Alstroemeria petals DNA breakdown (Wagstaff et al. 2003) started slightly before the decrease in protein content (Wagstaff et al. 2002).
When using agarose gels to show DNA breakdown during PCD, a ladder of DNA fragments is often observed, at least in association with apoptosis in animal cells. DNA laddering on gels is the result of oligonucleosomal cleavage, resulting in DNA sequences of multiples of about 180 bp. In contrast to apoptosis in animal cells, the DNA breakdown pattern during plant PCD is quite variable and depends on the system. For example, during petal PCD clear DNA laddering without a background of DNA fragments of various lengths is rarely found. Most reports show DNA laddering against a background of fragments of various lengths (Orzáez and Granell 1997; Wagstaff et al. 2003; Yamada et al. 2003) or only fragments of various lengths (van der Kop et al. 2003). The present results fall in the last category.
Nuclear morphology at the time of petal abscission also suggested advanced PCD in the petal cells of D. belladonna and little PCD in those of P. yedoensis. The nuclei of D. belladonna were much smaller and showed bright DAPI fluorescence, indicating chromatin condensation. No chromatin condensation was observed in P. yedoensis petals, but here it might possibly have occurred between the stages P2 and P3 and therefore may have been missed. We found that most but not all nuclei underwent the changes shown at the stages 2–4 of Fig. 3. The difference might relate to differences in PCD progress, within the petal. It is known that cells associated with the vascular bundle undergo PCD at a later stage than the other petal cells. In some species the mesophyll cells also show earlier PCD than the epidermis cells (van Doorn et al. 2003).
Little is known about the physiology of petal cells, after abscission. The cells of abscised petals, as long as they remain alive, will be subject to a considerable decrease in water potential. Using young detached corollas (petals) of Ipomoea flowers, we observed that FW was about 65% of initial after 24 h on the laboratory bench (at about 20°C and 70% RH) and 50% of initial after 48 h (unpublished data). This indicated that some physiological processes would occur at least during day-1 and -2, but that these processes would be influenced by lower water potential.
Both agarose gel (Fig. 2, P3 and D3) and flow cytometry analysis (Fig. 4) showed DNA degradation after the petals had abscised. The data indicate that the PCD process, once started, goes on even under conditions of increasing water stress. However, by the time of total desiccation of the petals, the amount of degraded DNA on gels was smaller than at the time of visible petal wilting of the abscised petals (Fig. 2, P4 and D4). This indicated that the DNA breakdown became halted by that time. This could be due to the fact that there was no more DNA, or might relate to the very low water potential in the tissue.
After petal abscission, the petal nuclei unexpectedly increased in size. In both species the chromatin became unevenly distributed. The chromatin became also much less condensed, as indicated by decreasing DAPI brightness. The increase in nuclear size and uneven and reduced DAPI brightness were also observed after dehydration of young D. belladonna and P. yedoensis petals. In these experiments flowers were cut from the plant just after floral opening and then subjected to a desiccation treatment (24 h at 60°C). This treatment, apart from inducing dehydration, also involved an increase in temperature. The effects did not seem to be due to high temperature though, as preliminary experiments had shown that 72–96 h desiccation at room temperature had the same effects as 24 h treatment at 60°C. This indicates that the effect was due to dehydration rather than high temperature, and that it was not only taking place in cells that were relatively old, but also in much younger cells.
I. nil and P. hybrida flowers both exhibit petal wilting rather than abscission at the end of floral life span. An increase in nuclear volume and a decrease in DAPI brightness were also found in the nuclei, if the flowers of these species were severed when just fully open and then subjection to a 24 h 60°C desiccation treatment. Preliminary experiments with these species had also shown that 72–96 h desiccation at room temperature had the same effects as 24 h treatment at 60°C. These results indicate that the particular nuclear morphology observed after dehydration in P. yedoensis and D. belladonna is also found in the petals of other species, which show petal wilting rather than petal abscission at the end of flower life. The particular nuclear morphology, therefore, seemed related to the low water potential as a result of the dehydration treatment. After the dehydration treatment in I. nil or P. hybrida the nuclear morphology was quite different from the one observed during wilting as the final phase of PCD. At the time of the normal senescence-related petal wilting in Ipomoea (Yamada et al. 2006b) and Petunia (Yamada et al. 2006a) flowers, the nuclei had become much smaller than at previous phases of development, and had a rather bright DAPI fluorescence. The desiccation treatment, therefore, induced changes quite different from those observed during PCD: a large nucleus with faint DAPI fluorescence rather than a small one with bright fluorescence.
It is concluded that the tested parameters indicated advanced stages of PCD prior to petal abscission in D. belladonna whereas PCD was much less obvious by the time of petal abscission in P. yedoensis. Dehydration of the petal cells, after abscission, was associated with a remarkable change in nuclear morphology and chromatin distribution. Similar changes in the nucleus were found after dehydration of younger petal cells, and after dehydration of the petal cells in two other species. This nuclear morphology has apparently not been described previously.

abscission
programmed cell death
wilting
dna degradation
flow cytometry
petal senescence