Kozue Nitta 2, Akiko A. Yasumoto 3, and Tetsukazu Yahara

Transcription

1 American Journal of Botany 97(2): V ARIATION OF FLOWER OPENING AND CLOSING TIMES IN F1 AND F2 HYBRIDS OF DAYLILY ( HEMEROCALLIS FULVA ; HEMEROCALLIDACEAE) AND NIGHTLILY ( H. CITRINA ) 1 Kozue Nitta 2, Akiko A. Yasumoto 3, and Tetsukazu Yahara Department of Biology, Faculty of Sciences, Kyushu University Hakozaki, Higashi-Ku, Fukuoka, , Japan In flowering plants, pollination success is strongly dependent on the timing of when flowers start to bloom and when they start to close. To elucidate the genetic mechanism influencing the timing of flower opening and closing, we obtained F1 and F2 hybrids of Hemerocallis fulva (a diurnally blooming species, pollinated by swallowtail butterflies) and H. citrina (a nocturnally blooming species, pollinated by nocturnal hawkmoths) and observed their flowering behavior from blooming to closing with the use of digital cameras. For flower opening times, F1 hybrids were highly variable, and F2 hybrids showed a bimodal distribution of flower opening times with peaks in both the morning and evening. The ratio of morning flowering and evening flowering among F2 hybrids did not deviate from 1 : 1. For the start to close time, both F1 and F2 hybrids were similar in showing the major peak in the evening. The ratio of evening closing and morning closing among F2 hybrids did not deviate from 3 : 1. These results suggest that the time of flower opening and the start of closing are regulated by different major genes. Key words: circadian rhythms; daylily; flower opening time; flower closing time; Hemerocallis ; F2 hybrids; Hemerocallidaceae; reproductive isolation; sphingophyly. In flowering plants, the pollination success of flowers is strongly dependent on when flowers start to bloom and when they start to close ( Miyake and Yahara, 1999 ). Each species is therefore expected to have a specific flowering schedule that is adapted to a particular pollination environment. For example, while most flowering plants bloom diurnally to attract the more numerous day-active pollinators, some flowers open in the evening and close in the morning as an adaptation to pollination by nocturnal pollinators, especially hawkmoths ( Miyake and Yahara, 1998 ). Despite the apparently obvious adaptive significance, few studies have been made on the genetic mechanism of flowering schedules. This paucity is in high contrast to our relatively rich knowledge about other floral traits such as flower color (as noted in Koes et al., 2005 ) or flower fragrance (as in van Schie et al., 2006). Previous studies, although limited, have contributed to the development of hypotheses accounting for the control of flower opening and closing. In 2003, van Doorn and van Meeteren first published a comprehensive review on flower opening and closing concluding that the processes were under the complex control of endogenous and exogenous factors. According to this study, flower opening is a process associated with cell elongation, and the timing of opening is usually regulated by many factors such as temperature, the quality and quantity of light, and the duration of light and darkness. Flower closure may be a 1 Manuscript received 1 January 2009; revision accepted 20 November The authors are grateful to E. Kasuya and colleagues of the ecology laboratory of Kyushu University for discussion and comments that greatly improved this manuscript. They also thank C. Wood for correcting the English and providing valuable comments on this manuscript. This research was partially supported by Grant-in-Aid for Scientific Research (B) (No ) and Grant-in-Aid for JSPS Fellows (No ) from Japan Society for the Promotion of Science. 2 Author for correspondence ( knittscb@kyushu-u.org) 3 Present address: Center for Ecological Research, Kyoto University; Institute of Plant Biology, University of Zurich doi: /ajb process related to senescence, and its timing is again affected by many environmental factors. On the other hand, the two processes are often coordinated by endogenous rhythms, ensuring the repeated opening and closing of flowers. Little is known, however, about the molecular mechanisms behind such coordination. van Doorn and van Meeteren (2003) suggested the use of Arabidopsis mutants related to the circadian clock for the further study of flower opening and closing. The circadian oscillation in Arabidopsis is based on feedback loops through which clock proteins activate and repress transcription of their own genes and thus generate their daily rhythm ( Young and Kay, 2001 ; Eriksson and Millar, 2003 ; Hayama and Coupland, 2004 ; McClung, 2006 ). Those transcription factors negatively regulate the expression of downstream genes by binding to motifs in their promoter regions. Thus, a single mutation to a regulatory element may largely change the expression timing of a particular gene ( Harmer and Kay, 2005 ). Specific flower opening genes or flower closing genes could be examples of such genes. The daylily ( H. fulva ) and nightlily ( H. citrina ) provide an extraordinary opportunity for the genetic study of flowering schedules as hybrids of these two species remain highly fertile despite the fact that the two species have contrasting phenotypes of flower opening and closing times. Hemerocallis fulva starts to flower early in the morning and starts to close in the evening, while H. citrina starts to flower in the evening and starts to close in the morning. In both species, the longevity of flowers is only half a day, varying from 11 to 15 h. Hasegawa et al. (2006) observed the timings of flower opening and closing in a natural hybrid population. It was reported that the natural hybrid population showed a bimodal distribution of flower opening time, while most F1 hybrids exhibited diurnal flowering. Their finding suggested that few major genes regulate whether flowers open in the morning or in the evening. In this study, we observed segregations for the timings of flower opening and closing in F2 hybrids of H. citrina and H. fulva. We show evidence suggesting that the timing of flower opening and the timing of closing are largely determined by

2 262 American Journal of Botany [Vol. 97 major genes. On the basis of this finding and also on our understanding about plant circadian rhythms, we propose a two-gene model that can explain the observed patterns of segregation for flower opening and closing. MATERIALS AND METHODS Species and collection Plants of Hemerocallis fulva L. var. aurantiaca (Baker) M. Hotta and H. citrina var. vespertina (H. Hara) M. Hotta were used for the experiments. The biology of these species is reviewed in Matsuoka and Hotta (1966), Hotta et al. (1985), Hasegawa et al. (2006), and Yasumoto and Yahara (2006). Hemerocallis fulva plants were collected at Haifuku (33 14 N, E) and H. citrina were collected at Tsutsumi (33 15 N, E) on Hirado Island of Nagasaki Prefecture, Japan in Plants were maintained in pots placed in the outdoor nursery of Department of Biology, Faculty of Sciences, Kyushu University. Crossing experiments From July to August 2001, a plant of H. fulva was crossed with pollen of a plant of H. citrina. These parental individuals were chosen because they had flower traits typical of each species; the H. fulva plant has a deep reddish color, and the H. citrina plant has a strong scent. We obtained 14 seeds of a full-sib family from a single fruit that was sown in the autumn of And 11 F1 hybrids started to flower in late June of All 11 F1 plants were randomly crossed to produce F2 hybrids in 2003 and In 2003, 26 flowers of five genets of F1 hybrids were crossed; those five genets acted both as female and male parents. We obtained 199 seeds of F2 hybrids from 13 fruits in In 2004, 278 flowers of 11 genets of F1 hybrids, including the five genets above, were crossed; 11 genets acted as female parents and 10 genets acted as male parents. We generated 1153 seeds of F2 hybrids from 119 fruits in In total, we generated 1352 seeds of F2 hybrids, all of which were sown in autumn of 2003 and From those seeds, 445 seedlings germinated, 413 surviving until late June 2006 when the flowering of the 116 plants was observed. All plants were kept in pots. They were placed outdoors until flowering season. All crossing experiments were made by hand pollination in the greenhouse of Kyushu University. We did not use any special measures to induce flowering. These plants flowered at the same time of year that they flower in natural populations. Measurement of traits From late June to August 2006, we observed flowers in the greenhouse of Kyushu University. Digital cameras (Optio W10, PEN- TAX, Tokyo, Japan) were used to record images of a flower at 15-min intervals. A flower bud that was expected to flower soon was selected and observed with a digital camera until the flower closed. We defined six stages of the flowering process as follows ( Fig.1 ), and the time of each stage was recorded to the nearest 15-min increment: (1) Flower opening time; the time when the point of a bud starts opening. (2) Inner petal opening time; the time when internal petals open and stamens appear among opening petals. (3) Maximal opening time; the time when petals are fully opened and cease any further opening movement. (4) Start to close time; the time when petals start to close. (5) Internal petal closing time; the time when internal petals closed. (6) Flower closing time; the time when petals cease any further closing movement. We defined the flower opening time as stage 1 as described above because it is less variable than Inner petal opening time (stage 2) and may better represent the internal physiological process of flowering initiation. We observed three plants of H. citrina propagated by separating single genet, three plants of H. fulva propagated by separating single genet, 22 plants of F1 hybrids propagated by separating nine genets (the number of replicates per genet; 6, 4, 3, 2, 2, 2, 1, 1, and 1), and 116 plants of F2 hybrids all of which were grown from seeds. The genets of H. fulva and H. citrina observed were the same genets used in crosses. For each plant, we observed three flowers and recorded times of stages 1 6. The median of three flower records was used as a representative value for each plant of F1 and F2 hybrids that showed high variability within an individual. Means and medians of three flower records were highly correlated across individuals (Pearson s product-moment correlation, r = 0.94, P < 0.01 for flower opening time, and r = 0.91, P < 0.01 for start to close time). For the two parent species, F1 hybrids and F2 hybrids, variation in time of flower opening and closing was illustrated using the following two methods; the times of stages 1 and 4 were plotted on a circle diagram of 24 h ( Fig. 2 ) using the software R (version 2.4.1, R Development Core Team, org/), and histograms were also drawn for each stage ( Fig. 5 ). We tested for deviations from 1 : 1 and 3 : 1 ratios by χ 2 test using the software R (version 2.4.1). RESULTS In H. fulva, flower opening time varied from 0245 to 0415 hours with a peak at 0345 hours. In contrast, the flower opening time of H. citrina varied from 1630 to 1830 hours with a peak at 1830 hours. In the F1 hybrids, flower opening time was extremely variable; 14 of the 22 total plants started flowering from 0145 to 0745 hours with a low peak at 0345 hours, six plants opened in the daytime (from 0930 to 1415 hours), and two plants opened at night (from 1615 to 1715 hours) ( Fig. 2 ). Remarkably, F1 hybrids showed high variability of flower opening time even within an individual; for example, three flowers of the same plant opened at 0700, 1315, and 1830 hours. Difference of flowering time over 6 h was observed in nine plants (six genets). In F2 hybrids, within-individual variability was lower; flower opening time varied over 6 h within an individual for 30 plants (genets) of the 116 total. On the other hand, F2 hybrids had a wider range of median time of flower opening than did F1 hybrids; some of them started to flower from 1900 to 0000 hours. The overall pattern of flower opening time showed an apparent bimodal distribution with peaks in the morning and evening ( Fig. 2 ); 14 plants (12.1%) opened from 0245 to 0415 hours as in H. fulva, and 18 plants (15.5%) opened from 1630 to 1830 hours as in H. citrina. Thus, we classified the phenotypes of flower opening time to two categories, morning flowering (flower opening time from 0000 to 1200 hours) and evening flowering (flower opening time from 1200 to 2400 hours). Fifty-three plants were morning flowering, and 63 were evening flowering. The ratio of these numbers did not deviate from 1 : 1 ( χ 2 = 0.86, df = 1, p = 0.35). In H. fulva, start to close time varied from 1800 to 2030 hours with a peak at 2030 hours. On the other hand, H. citrina started to close its flowers from 0015 to 0415 hours with a peak at 0015 hours. In the F1 hybrids, 19 (86%) of the 22 total plants started to close their flowers from 1600 to 2115 hours with a peak at 1900 and 2000 hours, and three plants (14%) started to close their flowers in the morning (from 0045 to 0445 hours) ( Fig. 2 ). Within-individual variability was observed also for start to close time; start to close time varied over 6 h within an individual for seven plants (five genets) of the F1. In F2 hybrids, within-individual variability was lower, and a difference over 6 h was observed for 35 plants (genets) of the 116 total. On the other hand, F2 hybrids had a wider range of start to close time than F1 hybrids. The overall pattern of variation was unimodal; flowers of 56 plants (48.3%) started to close from 1800 to 2030 hours as in H. fulva, and only 15 plants (12.9%) had flowers start to close from 0015 to 0415 as in H. citrina ( Fig. 2 ). Eighty-seven plants were evening closing (start to close time from 1200 to 2400), and 29 were morning closing (start to close time from 0000 to 1200 hours). The ratio of these numbers did not deviate from 3 : 1 ( χ 2 = 0, df = 1, p = 1). The distributions of flower opening and start to close times in F1 and F2 significantly deviated from a normal distribution ( P < 0.05, Kolmogorov Smirnov test). To quantify how much of the variation in flower opening and closing times of F1 plants is partitioned among genets, among clonal replicates within genets, and among flowers within clonal replicates, we carried out hierarchical ANOVA, using a linear mixed-effects model fit by restricted maximum likelihood (REML). For flower opening time, variance component partitioned among clonal replicates within genets was effectively zero, the among-genets component

3 February 2010] Nitta et al. Variation in flower opening and closing time in daylily 263 Fig. 1. Six stages of flowering. (1) Flower opening time. (2) Inner petal opening time. (3) Maximal opening time. (4) Start to close time. (5) Internal petal closing time. (6) Flower closing time. Scale bar = 8 cm. These images are the same flower of one F2 hybrid of Hemerocallis fulva and H. citrina. was 49.6%, and the within-plants component (residual) was 50.4%. For flower closing time, the among-clonal replicates within the genets component was effectively zero, among genets was 32.9%, and within plants was 67.1%. The relationship between flower opening time and start to close time (stage 1 and stage 4) in F2 is shown in Fig. 3. Most plants that started to flower in the morning started to close in the evening. On the other hand, plants that started to flower in the evening had a large variation in closing time; 28 of the 63 total started to close flowers in the morning, while the remaining 35 started to close in the evening of next day. The variation of flowering duration (from stage 1 to stage 4) among F2 hybrids is shown in Fig. 4 in which plants are ordered by flower opening time. F2 plants that started to flower in the morning (from 0000 to 1200 hours) had flowering duration from 9.25 to 21 h (mean = 15.16, SD = 2.73, variance = 7.47), while the other plants that started to flower in the afternoon or evening (1200 to 2400 hours) had a higher variability of flower duration from 5 to 31.5 h (mean = 17.62, SD = 7.24, variance = 52.44). We also used an F test to compare two variances ( F 52,62 = 0.14, P < 0.01). To examine how bimodal distribution of flower opening time is shifted to unimodal distribution of start to close time, histograms of inner petal opening time, maximum opening time, and start to close time are shown in Fig. 5 in which F2 plants are divided to two classes for flower opening time, and hours. In F2 plants that started to flower from , inner petal opening time of 52 (44.8%) plants ranged also from hours (designated by dark gray in Fig. 5 ). In F2 plants that started to flower from hours, inner petal opening time of 53 (45.7%) plants ranged also from hours (shown in light gray), but 10 (8.6%) of them showed delayed inner petal opening time from hours (shown in black). F2 plants of the dark gray class (both flower opening time and inner petal opening time from ) showed a unimodal distribution of the maximal flowering stage (stage (3) in Fig. 5 ) with a sharp peak from These started to close flowers from hours, with a sharp peak from hours. F2 plants of the light gray class (both flower opening time and inner petal opening time from hours) had markedly

4 264 American Journal of Botany [Vol. 97 Fig. 2. Circle diagrams of 24 hours showing variations of flower opening time (stage 1; A D) and start to close time (stage 4; E H) in the two parent species (Hf, Hemerocallis fulva ; Hc, H. citrina ), F1 hybrids and F2 hybrids. Each dot represents a measured value of each flower for two parent species; the length of the mark indicates frequency. For F1 and F2 hybrids, the dot is the median of three measurements for each plant. different flowering behavior. Among the 53 total, 35 (66%) plants shifted to stage (3) from hours, indicating that the larger proportion of plants that started to flower in the afternoon finished flower opening by midnight. On the other hand, the remaining 18 (34%) plants shifted to stage 3 from 0000 to 1100 hours, indicating a delayed finalization of flower opening. The former group of F2 plants started to close flowers in the next morning or evening, and the latter group of F2 plants started to close flowers in the evening of the same day. As a result, the distribution of start to close time showed a sharp peak in the evening ( hours) and had another lower peak in the morning ( hours). F2 plants of the black class (flower opening time from 1200 to 2400 hours and inner petal opening time from 0000 to 1200) shifted to stage 3 within the morning and started to close flowers until 2100 hours; then shifted to stage 5 until 2300 hours. However, in this case, flower closing time was often delayed until the next morning. Based on the observations described, a scheme of flowering behavior is shown in Fig. 6. Distribution of flower opening times was bimodal ( Fig. 2 ), and F2 plants started to flower in the evening ( Fig. 6, upper N = 63) or in the morning ( Fig. 6, lower N = 52). Many F2 plants that started to flower in the evening soon shifted to stages 2 and 3, and most of them started to close early in the morning ( N = 28). However, others failed to close flowers in the morning and continued to flower during the daytime. In addition, some F2 plants that started to flower in the evening delayed in shifting to stage 3 until the next morning and continued to flower during the daytime. Those flowers ( N = 35) that extended blooming until next day started to close in the evening; thus, the flower longevity was prolonged from half a day to 1 day. It is notable that the start to close time of 1-day flowers had a sharp peak from 1900 to 2000 hours, irrespective of their flowering behavior at stages 2 and 3. This suggests that the start to close time was synchronized. For F2 plants that started to flower in the morning ( Fig. 6, lower, N = 52), 47 plants soon shifted to stages 2 and 3, and the remaining five delayed in shifting to stage 3 until the afternoon. However, all started to close around 1900 hours. Irrespective of flowering behavior from stage 1 to stage 4, timing of shifting from stage 5 to stage 6 was highly variable. Fig. 3. The relationship between flower opening time and start of closing time in F2 hybrids. Each dot shows the median of three measurements for each F2 plant. F2 plants are classified into four groups based on a combination of (1) flower opening time and (2) inner petal opening time as follows. Open circle: (1) hours, (2) hours; cross: (1) hours, (2) hours; open triangle: (1) hours, (2) hours; and solid square: (1) hours, (2) hours.

5 February 2010] Nitta et al. Variation in flower opening and closing time in daylily 265 Fig. 4. Variation in flowering duration among the F2 hybrids. The length of the line shows the flowering duration of each plant defined as time from stage 1 to stage 4. Lines are ordered by flower opening time. The horizontal axis shows time measured from midnight (0000 hours). DISCUSSION As far as we know, this is the first genetic study of the timing of flower opening and closing within a day. A number of studies have focused on the genetic basis of flowering time within a year, primarily dealing with the transition from vegetative growth to flowering (such as Simpson and Dean, 2002 ; Imaizumi and Kay, 2006 ; Kobayashi and Weigel, 2007 ). However, in contrast, little has been revealed regarding how the timing of flower opening and the timing of closing are regulated within a single day. The distributions of flower opening and start to close times in F1 and F2 hybrids deviated significantly a normal distribution. Thus, it is unlikely that flower opening and starting to close times are regulated only by polygenes. In this study, F1 hybrids had highly variable flower opening times, with no evident peaks either in the morning or in the evening ( Fig. 2 ). In contrast, for F2 hybrids, flower opening times had a clear bimodal distribution ( Fig. 2 ), the two modes corresponding to the flower opening times of H. fulva (in the morning) and H. citrina (in the evening). The ratio of morning flowering and evening flowering in F2 hybrids did not deviate from 1 : 1, the segregation ratio expected for a single-gene locus having two codominant alleles. These results suggest that the flower opening time is regulated primarily by a major gene, and the morning phase and evening phase alleles are codominant. Most of the F1 hybrids started to close their flowers in the evening ( Fig. 2 ). The distribution of start to close time of F2 hybrids had a major peak at the evening, from 1800 to 2030 hours as in H. fulva ( Fig. 2 ). The ratio of evening closing and morning closing in F2 hybrids did not deviate from 3 : 1, the segregation ratio expected for a single-gene locus having dominant and recessive alleles. These results suggest that start to close time is regulated primarily by a major gene, and flower closure in the evening is a dominant trait. Recent advances in our understanding about plant circadian rhythms may help us to interpret the observed dichotomy between the mechanisms of the timing of flower opening and flower closing. In Arabidopsis thaliana, the transcription factors CCA1 and LHY negatively regulate the expression of TOC1 by binding to a motif in the TOC1 promoter known as the evening element (EE) ( Harmer et al., 2000 ; Alabadi et al., 2001, 2002 ; Mizoguchi et al., 2002 ). Recently, Harmer and Kay (2005) showed that the evening element (EE) itself is necessary and sufficient to confer evening-phase rhythms on a reporter gene. They also identified a new motif termed the morning element (ME), that confers morning-phase rhythms. Interestingly, ME activity was shown to be masked or modified by EE, and simple changes in the sequences of the EE resulted in an almost 180 change in the phase of the reporter gene expression. We suggest that the gene determining flower opening time of Hemerocallis fulva may have a motif in the promoter region to which a morning-phase oscillator such as CCA1 or LHY binds, and H. citrina may have a motif to which an evening-phase oscillator like TOC1 binds. Hereafter, we will refer to the hypothetical motifs as a morningphase oscillator-binding motif (MM) and an evening-phase oscillator-binding motif (EM). In F1 and F2 hybrids, flower opening time varied widely even within an individual, and some flowers opened in the daytime while others opened in the evening in the same individuals. This apparently puzzling variability of flower opening time within an individual can be explained by the described model because the promoter region of the flower

6 266 American Journal of Botany [Vol. 97 Fig. 6. Scheme of flower opening and closings time for F2 hybrids. The horizontal axis is time of day. The numbers represent the following flowering stages: (1) Flower opening time, (2) inner petal opening time, (3) maximal opening time, (4) start to close time, (5) internal petal closing time, (6) flower closing time. The arrow indicates the time durations. The numbers of N indicate how many of the F2 hybrids followed each flowering trajectory. Fig. 5. Histograms of flowering time drawn for each stage (from stage 2 to 6) and divided into two groups for stage 1: F2 plants that started to flower (left) from 0000 to 1200 hours ( N = 53) and (right) from 1200 to 2400 hours ( N = 63). The horizontal axis is each flowering time, and the vertical axis is frequency. In the histograms, the four classes based on the combination of stage 1 and 2 are shown by different levels of shading. Dark gray: (1) hours, (2) hours; white: (1) hours, (2) hours; black: (1) hours, (2) hours; and light gray: (1) hours, (2) hours. opening gene is heterozygous for MM and EM in F1 hybrids, and thus both oscillators (a CCA1 like morning-phase oscillator and a TOC1 like evening-phase oscillator) are expected to bind to heterozygous motifs and promote the expression of the gene determining flower opening time. Under this heterozygous condition, F1 plants are expected to be arrhythmic, and the time of flower opening may depend on other endogenous and exogenous regulatory factors such as size of flower bud and temperature. Flowers of F2 plants started to close in the evening irrespective of the flower opening time. Most F2 plants that started to flower in the morning started to close in the evening ( Fig. 3 ). Among F2 plants that started to flower in the evening, 28 started to close flowers in the morning, but the remaining 35 extended blooming until the next day and started to close in the evening (see also Figs. 5, 6 ). These facts suggest that closure in the evening is a dominant trait. Our observation suggested that a major gene is the primary determiner of when flowers start to close. This gene also appears to be controlled by circadian rhythms, but its regulation must differ from that of the flower opening gene because closure in the evening is a dominant phenotype. We suggest that the gene determining the time of flower closure in H. citrina has a morning-phase oscillator-binding motif (MM), the gene in H. fulva has an evening-phase oscillatorbinding motif (EM), and EM is dominant to MM. This dominance model explains well why flowers that have a non-uniform start to bloom times begin to close in the evening, mostly from 1800 to 2030 hours, in a clear and uniform peak period ( Fig. 6 ), and why some flowers starting to bloom in the evening proceeded to start to close the next morning. The trends shown in Figs. 3 and 5, however, do not seem entirely consistent with independent genetic control of flower opening and closing. Instead, phenotypic association remains between flower opening and closing. While 35 F2 plants of the 63 total that started to flower in the evening extended their flowering until the next evening, almost none of the F2 plants flowers that flowered in the morning extended flowering until the next morning. This phenotypic association suggests that the phenotypic effects of the flower opening gene and the flower closing gene may be mediated by common regulatory systems.

7 February 2010] Nitta et al. Variation in flower opening and closing time in daylily 267 After the start of closure, the duration required for full closure of a flower was highly variable ( Figs. 5, 6 ). The process of flower closure after its start may thus be regulated by many genes. Regarding this area of closing activity, the petals of daylily flowers have attracted some recent attention from plant physiologists because this species provides an unusual opportunity for organ senescence studies in that the floral longevity is as short as 1 day, much shorter than leaf longevity ( Bieleski and Reid, 1992 ; Lay-Yee et al., 1992 ; Valpuesta et al., 1995 ; Panavas et al., 1999, 2000 ). These studies revealed that the senescence of daylily petals is a complex process associated with many simultaneously proceeding events such as loss of differential membrane permeability, lipid peroxidation, increase of H 2 O 2, and increased activity of proteinases and nucleases ( Panavas et al., 1999, 2000 ), suggesting that genetic control of the senescence of daylily petals may also be complex. According to our model, flowers of Hemerocallis are compelled to close within 24 h under the regulation of the start to close gene. This hypothetical gene might be a multifunctional transcription factor, located in the upstream of signal transduction pathways regulating physiological changes between day and night, and its mutation might have pleiotropic, deleterious effects in various processes because no variant having floral longevity of more than one day has been discovered during the long history of daylily breeding. In many plants including Hemerocallis, cdnas whose amounts increase during floral senescence have been isolated. However, those genes are induced after the process of senescence is triggered and thus are unlikely candidates for the start to close gene. In this study, we show evidence supporting the idea that the sequence from flower opening to closure is regulated primarily by a gene that determines flower opening time and another gene that determines start to close time. However, our observations also showed that both flower opening time and flower closing time are highly variable traits, among-genets variance was 49.6% for flower opening time and 32.9% for flower closing time. Although observed ratios of phenotypic segregation did not deviate from expectations of a single-gene locus model, the high variability of phenotypes suggests phenotypic expression is not exclusively determined by two major genes but also likely includes the involvement of some others. Further genetic studies such as mapping of quantitative trait loci (QTLs) for times of flower opening and closing using Hemerocallis will continue to greatly improve our understanding of the mechanisms of flower opening and closing. LITERATURE CITED Alabadi, D., T. Oyama, M. J. Yanovsky, F. G. Harmon, P. Mas, and S. A. Kay Reciprocal regulation between TOC1 and LHY/CCA1 within the Aradidopsis circadian clock. Science 293 : Alabadi, D., M. J. Yanovsky, P. Mas, S. L. Harmer, and S. A. Kay Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Aradidopsis. Current Biology 12 : Bieleski, R. L., and M. S. Reid Physiological changes accompanying senescence in the ephemeral daylily flower. Plant Physiology 98 : Eriksson, M. E., and A. J. Millar The circadian clock: A plant s best friend in a spinning world. Plant Physiology 132 : Harmer, S. L., J. B. Hogenesch, M. Straume, H.-S. Chang, B. Han, T. Zhu, X. Wang, J. A. Kreps, and S. A. 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