2024 Volume 93 Issue 3 Pages 216-223
In the most popular fragrant rose cultivar in Japan, ‘Yves Piaget’, the petal edges are frequently malformed, curving toward the adaxial side. These malformed petals prevent normal flowering and weaken the flower fragrance, which significantly decreases the quality of this cultivar and increases financial losses of cut flowers. We refer to such malformed flowers as ‘incurved flowers’. It has been reported that jasmonic acid (JA) affects petal growth. Therefore, we attempted to control the number of incurved flowers by applying exogenous JA, methyl jasmonate (MeJA), during flower development before harvest. Two types of spray treatment were applied to the flower buds before flower opening; (1) 100 μM MeJA or (2) deionized water as a control. The 100 μM MeJA spray treatment before harvest reduced the incurved flower rate, with fewer incurved petals, and resulted in a significantly larger maximum flower diameter and longer stamen length. In addition, the 100 μM MeJA spray treatment before harvest tended to increase the number of days from the commercial harvest stage to full bloom and also significantly increased the maximum flower diameter of fully-bloomed flowers. We also analyzed the endogenous phytohormone content in the petals of normal and incurved flowers at each flower developmental stage. The results showed that at the beginning of the flower opening stage the petals of incurved flowers had higher indoleacetic acid (IAA) content and lower JA/jasmonoyl isoleucine (JA-Ile) content than those of normal flowers. In particular, the JA and JA-Ile contents in incurved petals were approximately one quarter of those in normal flowers. These results suggest that IAA, JA, and JA-Ile may be involved in the development of incurved flowers.
In Japan, approximately 189 million roses were produced in the fiscal year 2022, making them the third most produced cut flowers in Japan after chrysanthemums and carnations (Ministry of Agriculture, Forestry, and Fisheries, 2023). However, the import of inexpensive cut roses has been increasing recently, which means that domestic cut roses will require improved quality to compete with the imported cut roses. One of the most important qualities of domestic roses is freshness and it is an important factor in the growing demand for fragrant cut roses.
In Japan, the most popular fragrant cut rose cultivar is ‘Yves Piaget’. It has gorgeous peony-like double flowers comprising around fifty pink petals, but it often produces malformed flowers with petal edges that curve toward the adaxial side (Fig. 1). These malformed petals prevent normal flowering and reduce the flower fragrance, which significantly decreases the quality of this cultivar. This type of malformed flower is called an “incurved flower” (Kaneeda et al., 2019).
Flower developmental stages and morphological differences between normal and incurved flowers. (A): Stage 0, (B): Stage 1, (C): Stage 2, (D): Stage 3, (E) and (F): Normal flower at stage 4, (G) and (H): Incurved flower at stage 4. Arrows indicate incurved petals.
The process of flower bud development and subsequent flower opening is associated with petal growth during which petal cells divide and expand (Yamada et al., 2009b). In rose flowers, the petal growth during flower opening is mainly due to cell expansion (Yamada et al., 2009b), which requires the accumulation of osmolytes and an influx of water (Yamada et al., 2009a). Previous studies have reported that the levels of soluble carbohydrates and activity of invertase change during the growth of rose petals (Ito et al., 2007; Yamada et al., 2007). Large amounts of soluble carbohydrates accumulate in rose petals during flower opening (Ichimura et al., 2003), especially in petal cell vacuoles (Yamada et al., 2009b). Cell wall acid invertase converts sucrose into hexoses after the sucrose has been translocated from the phloem to the apoplast (Roitsch and Gonzalez-Garcia, 2004), and this enables petals to take up more sucrose from the phloem. Vacuolar invertase also plays an important role in biological functions associated with sucrose metabolism. It is thought that this hydrolysis of sucrose supplies the hexoses necessary for cell growth and development (Tang et al., 1999; Tymowska and Kreis, 1998). Therefore, it is thought that the hexose supply requiring water absorption into petal cells is inhibited in incurved flowers resulting in petal growth suppression (Kaneeda et al., 2019).
Phytohormones, such as auxins and gibberellins, are key factors for controlling cell division and expansion, but their precise functions in flower development are unclear because of their pleiotropic roles and complex signaling interactions (Weiss et al., 2005). The lipid-derived phytohormone, jasmonic acid (JA), including free-acid conjugates, regulates a wide range of biological processes, such as biotic or abiotic plant stress responses, and developmental processes like root growth, seed germination, anther development, and senescence (Browse, 2005; Devoto and Turner, 2003). However, little is known about the role played by JA in petal development (Brioudes et al., 2009). Horibe et al. (2013) reported that the postharvest addition of α-naphthalene acetic acid (NAA), a kind of synthetic auxin, and methyl jasmonate (MeJA), a derivative of JA, affected invertase activity in cut rose flowers. Also, Ochiai et al. (2013) reported that the promotion of early flower opening by MeJA treatment was due to loosening of petal cell walls due to accelerated expression of expansin and xyloglucan endotransglycosylase/hydrolase. Additionally, it has been reported that, whereas treatment of cut rose flowers with MeJA slows down flower opening and extends the vase life of the flowers (Horibe and Makita, 2019), treatment with NAA promotes flower opening and petal growth (Horibe and Makita, 2021). Furthermore, Singh et al. (2022) reported that JA treatment delayed flower opening, as well as petal abscission, by more than 24 h in the fragrant rose Rosa × bourboniana.
In our previous study, 500 μM MeJA pulse-uptake treatment after harvest improved cut flower quality by reducing the number of incurved malformed petals and extending the number of days to full bloom in ‘Yves Piaget’ (Kaneeda et al., 2023a). However, this post-harvest treatment was not sufficient to suppress emergence of incurved flowers. In addition, previous studies have shown that exogenous application of JA to non-blooming flower buds restored flower phenotype in Arabidopsis (Brioudes et al., 2009), Chinese cabbage (Peng et al., 2019), and the double-flowered lily cultivar ‘Doubleen’ (Fukasawa et al., 2023). Therefore, we considered that the MeJA treatment on the flower buds before harvest would be more effective to reduce the numbers of incurved flowers; this may also help to reduce losses caused by incurved flowers.
In this study, we attempted to reduce the number of incurved flowers of the cut rose cultivar ‘Yves Piaget’ by MeJA spray treatment before harvest, and investigated whether such treatment would affect cut flower quality. In addition, we compared the contents of endogenous acidic phytohormones at each flower developmental stage in normal and incurved flowers.
Rosa × hybrida ‘Yves Piaget’ plants were grown in a greenhouse at Meiji University from 2018 to 2022, in 30 cm diameter plastic pots containing 8 L of culture soil (loamy soil). The cultivation method was based on the “arching” (shoot-bending) technique of Shimomura et al. (2003). The average temperature in the greenhouse was 27.0 ± 0.1°C, and relative humidity was 43.7 ± 0.1%. A two-liquid mixing unit (IM-N6-001-0412B; ES-Water Net, Japan) equipped with an irrigation controller (Watermaster; ES-Water Net) was used for combined irrigation and fertilization. The irrigation was performed using a drip-type tentacle. The culture medium used for irrigation was pH 5.8 and contained a mixture of solution A (Sumitomo Chemical, Japan) for solution cultivation (N:P:K = 7:0:3) diluted 928 times and solution B (Sumitomo Chemical) for solution cultivation (N:P:K = 1:3:7) diluted 309 times.
Spray treatment and morphological surveyTwo spray treatments were conducted in 2020: (1) 100 μM MeJA (MeJA 100), and (2) deionized water as a control. The 100 μM MeJA concentration was used based on the results of a preliminary study that showed a lower number of incurved petals with 100 μM MeJA spray treatment compared with 50 μM or 200 μM MeJA spray (data not shown). The MeJA (Wako Pure Chemical Industries Ltd., Japan) was dissolved in a small amount of 70% ethanol and diluted to 100 μM with distilled water. The same amount of 70% ethanol was mixed with the deionized water as control. The spray treatment started when flower buds on the basal shoots reached a diameter of 6 to 10 mm and the sepals were broken, enabling colored petals to start to appear (Stage 0; Fig. 1). The buds were then sprayed each day until 10 outer petals had opened (Stage 4; Fig. 1), at which point the flowers were harvested. For the daily spray treatment, each flower bud was evenly wetted with 3 mL spray each morning between 10:00–12:00. To prevent splashes on other parts of the flower stem, the flower buds were covered with a paper cone during each spray treatment. Flower stems were harvested between 10:00–12:00 from April to July 2020. A morphological survey was then conducted to measure the following parameters of the harvested flowers: proportion of incurved flowers (%), number of incurved petals, flower diameter (mm), petal fresh weight (g), stamen length (mm), stamen fresh weight (g), pistil length (mm), ovary diameter (mm), ovary fresh weight (g), and calyx fresh weight (g). These items make up the morphology that characterizes incurved flowers. In addition, jasmonic acid is known to be essential for stamen and petal development in Arabidopsis (Tabata et al., 2010). In this experiment, 15 potted plants were used per treatment, resulting in a total of 34 stems in the 100 μM MeJA spray treatment and 27 stems in the control.
Quality evaluation of cut flowersRosa × hybrida ‘Yves Piaget’ plants were grown in a greenhouse at Meiji University as described. Two spray treatments in 2022 were conducted the same as in the 2020 experiment: (1) MeJA 100, or (2) deionized water as a control. In this experiment, cut flowers were obtained at the commercial harvest stage (Stage 2; Fig. 1) in May 2022. Flower stems of cut flowers were soaked in water after harvest and transported to the laboratory. Promptly after transport, flowers were cut at their stem bases in deionized water to remove any air embolism and cavitation then kept in a refrigerator at 4 ± 2°C with 90 ± 10% relative humidity in the dark for 2 h before the start of the experiment. The flower stem bases were placed in deionized water with 0.005% (w/v) KathonTM CG (an antibacterial agent; Rohm and Hass company, PA, USA). The flowers were kept in a plant growth incubator at 24 ± 2°C with 60 ± 10% relative humidity, and 16 h light photoperiod (PPFD: 20–40 μmol·m−2·s−1). The days from harvest to full bloom and the vase life were determined as described by Kaneeda et al. (2023a). The number of incurved petals and maximum flower diameter (mm) were measured when flowers were in full bloom. We used six stems for the 100 μM MeJA spray treatment and three stems for the control.
Measurement of endogenous acidic phytohormones during flower developmentPetals from flower buds of ‘Yves Piaget’ grown in a glasshouse at Meiji University in 2021 were used for the measurement of endogenous acidic phytohormones. Whole petals were collected from flowers at each flower developmental stage (Stage 1–4; Fig. 1) from April to July 2021. Three flower stems of normal and incurved flowers at the following stages were collected (Fig. 1). Stage 1 means immature flower buds with sepals expanded at 90°. There is no morphological distinction between normal and incurved flowers at this stage. Therefore, three randomly selected flower buds were used. Stage 2 (commercial harvest stage) means mature flower buds with sepals expanded to 180°, just before petals develop. At this stage, differences in shoot length and ovary diameter enable normal flowers to be distinguished from incurved flowers (data not shown). Stage 3 means flower opening stage with the outer five petals expanded. At this stage, the incurved petals are clearly visible. Stage 4 completes flower development with 10 outer petals expanded.
The fresh weight of each petal in each developmental stage was measured after it was collected, and then the petals were frozen in liquid nitrogen and stored at −80°C until analysis. The petals were analyzed for the following endogenous acidic phytohormones; gibberellic acids (GA1 and GA4), indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), jasmonoyl isoleucine (JA-Ile), and salicylic acid (SA). Extraction, purification, and quantification of acidic phytohormones were performed as described previously (Kanno et al., 2016). Petal samples were extracted with 20 mL of 80% (v/v) acetonitrile containing 1% (v/v) acetic acid, and the extracts were purified first with Oasis HLB (Waters, USA) and then with an Oasis WAX column (Waters). Acidic phytohormones including GA1, GA4, ABA, IAA, JA, and JA-Ile were eluted with 80% (v/v) acetonitrile containing 1% (v/v) acetic acid. SA was eluted with 80% (v/v) acetonitrile containing 1%(v/v) formic acid. An LC-MS/MS system consisting of a quadrupole/time-of-flight tandem mass spectrometer (Triple TOF 5600; Shimadzu, Japan), and a Nexera HPLC system (Shimadzu) was used in the analysis. Phytohormone content was calculated on a dry weight basis. Seven replications were conducted with three flower stems per replication.
Statistical analysisA Student’s t-test was used to make comparisons between the two treatment groups. Values are means of each replication ± SE. The significance level was 5% or 1%.
There was no difference between treatments in the number of days for complete flower development (about 10 days) from the day the treatments began when flower buds reached a diameter of 6 to 10 mm on the basal shoots (stage 0) until complete flower development when 10 outer petals opened (stage 4). When the flowers were harvested at stage 4, the rate of incurved flowers in the 100 μM MeJA spray treatment (23.53%) was much lower than that of the control (77.78%) (Table 1). The diameter of the 100 μM MeJA spray-treated flowers was significantly larger (96.14 mm) than that of the control flowers (84.60 mm), and the stamen length was also significantly longer (8.82 mm vs 8.67 mm). However, there were no significant differences between the treatments for stamen weight, pistil length, ovary fresh weight, or calyx fresh weight. In Table 1, the standard errors are large relative to the number of replicates. This variation is due to inclusion of both incurved flowers and normal flowers in these results.
Effect of 100 μM MeJA (MeJA 100) spray treatment before harvest on the morphology of floral organs.
For the flowers harvested at the commercial harvest stage (stage 2), there was a similar vase life of about six days for both treatments (Table 2). The 100 μM MeJA spray treatment increased the number of days from harvest to full bloom, although this difference was not statistically significant. However, there was a big difference in the numbers of incurved petals, with seven incurved petals in the fully blooming control flowers compared with no incurved petals in the flowers of the 100 μM MeJA spray treatment. Also, at full blooming, the diameter of the 100 μM MeJA spray-treated flowers was significantly larger (101.36 mm) than that of the control flowers (79.20 mm).
Effect of 100 μM MeJA (MeJA 100) spray treatment before harvest on cut flower quality.
No GA1 or GA4 were detected in the flower buds at any stage (data not shown). There were no significant differences between the normal and incurved flowers in the contents of abscisic acid or salicylic acid at any stage (Fig. 2B, E). There was a higher content of IAA in the incurved flowers at stage 2 (Fig. 2A), but no significant difference was observed at stage 3 or 4. The JA content decreased from stage 1 to 2 in both normal and incurved flowers (Fig. 2C), with a much more rapid decrease in the incurved flowers than in the normal flowers. By stage 3, the JA content in the normal flowers had also fallen to very low levels, and thereafter there was no significant difference at stage 3 or 4 between the normal and incurved flowers. Similarly, the JA-Ile content decreased from stage 1 to stage 2 in both normal and incurved flowers (Fig. 2D), again with a much more rapid decrease in the incurved flowers. The JA-Ile content in the normal flowers had also fallen to very low levels by stage 3; thereafter, there was no significant difference in JA-Ile content at stage 3 or 4 between the normal and incurved flowers.
Endogenous acidic phytohormone content of petals at different flower developmental stages. Error bars indicate standard errors (n = 7). (A): indole-3-acetic acid (IAA), (B): abscisic acid (ABA), (C): jasmonic acid (JA), (D): jasmonoyle isoleucine (JA-Ile), (E): salicylic acid (SA). Stage 1 shows the average of all flowers because it was too early to distinguish between normal and incurved flowers. Different lowercase letters indicate a significant difference at P < 0.05; NS indicates no significant difference (Student’s t-test for comparisons between treatments).
The 100 μM MeJA spray treatment on flower buds of ‘Yves Piaget’ during flower development before harvest was effective in inhibiting the number or incurved flowers and improving flower quality. The 100 μM MeJA spray treatment before harvest reduced the rate of incurved flowers and numbers of incurved petals, and resulted in a larger flower diameter and stamen length (Table 1). These results are in common with a previous report showing that JA is required for elongation of petals and stamens in Arabidopsis (Tabata et al., 2010). Furthermore, in conjunction with the results of a previous study, 500 μM MeJA pulse-uptake treatment on cut flowers of ‘Yves Piaget’ suppressed the number of incurved petals (Kaneeda et al., 2023a). These results strongly suggest that MeJA is effective in suppressing incurved petals of this cultivar. The current study suggests that 100 μM MeJA spray treatment before harvest may be better at suppressing incurved flowers and petals than 500 μM MeJA pulse-uptake treatment after harvest. While there was no difference in flowering days (10 days) or vase life (6 days) between the 100 μM MeJA spray treatment and the control, the number of days from commercial harvest to full bloom tended to be a day longer for the 100 μM MeJA spray-treated flowers (Table 2). The increase in days to full bloom with 100 μM MeJA spray treatment before harvest suggests that MeJA slowed down the flowering process after harvest (Horibe et al., 2013). Therefore, the 100 μM MeJA spray treatment before harvest could also improve the quality of cut roses because their value lies in the blooming process from bud to full bloom (Horibe and Makita, 2019). Previous studies found that MeJA treatment on cut rose flowers could extend vase life (Horibe and Makita, 2019, 2021), which differs from the results in the current study. In addition, the 500 μM MeJA pulse-uptake treatment on cut rose flowers did not prevent petal bluing, similar to our previous study (Kaneeda et al., 2023a). The failure to increase vase life in the current study may have been due to insufficient amounts of soluble sugars, energy or osmolytes. Therefore, in future studies, it will be necessary to experiment with adding sugars to a vase solution after harvest to further extend vase life. Furthermore, it will be necessary to investigate a more effective MeJA concentration. Incurved petals did not emerge in cut flowers with 100 μM MeJA spray treatment when harvested at stage 2, but they were only observed in the control after harvest (Table 2). This result suggests that the 100 μM MeJA spray treatment before harvest may have suppressed the occurrence of incurved petals in the treated flowers after harvest. In commercial cultivation, flowers are harvested as mature buds (stage 2), so they are often shipped and distributed without identification of incurved flowers. Therefore, it is possible that MeJA treatment before harvest may contribute to suppressing potential incurved flowers. In rose flowers, petal growth during flower opening is mainly due to cell expansion (Yamada et al., 2009b), which requires the accumulation of osmolytes and an influx of water (Yamada et al., 2009a). It is thought that sugar accumulation in petal cells reduces petal water potential, thus promoting water influx for cell expansion, which leads to flower opening (Ho and Nichols, 1977). In our previous study, the petals of incurved flowers had a smaller water potential and smaller change in turgor pressure, making it more difficult for water to flow into these petals (Kaneeda et al., 2023b). Other studies have reported changes in the levels of soluble carbohydrates and the invertase activities during the growth of rose petals (Ito et al., 2007; Yamada et al., 2007). In general, high invertase activity promotes the conversion of sucrose to hexose, which causes a reduction in petal water potential promoting water influx, resulting in petal growth (Horibe et al., 2013). In the same study, it was also reported that MeJA could maintain higher levels of invertase activity in cut rose flowers. These results suggest that 100 μM MeJA spray treatment of flower buds may increase vacuolar invertase activity and promote petal growth. In addition, cell wall-loosening proteins (Dai et al., 2012; Takahashi et al., 2007; Yamada et al., 2009c) and aquaporins (Chen et al., 2013) are also involved in the growth of rose petals. Therefore, these proteins will also need to be investigated.
In the current study, we found significant differences in the contents of the endogenous acidic phytohormones IAA, JA, and JA-Ile between normal and incurved flowers at stage 2, which is the beginning of flower opening and the commercial harvest stage (Fig. 2). This suggests that development of incurved flowers emerges at the beginning of the flower developmental stage. Previous studies of various malformed rose flowers have consistently reported that the occurrence of incurved flowers is induced during a very short period of flower development, and caused by factors such as high temperature (Hubbard, 1953), low temperature (Halevy and Zieslin, 1969; Ma et al., 2015), drought (Chimonidou-Pavlidou, 2004), and other stresses. Bullhead flowers, a type of malformed rose flower, often occur when roses are grown under unsuitable temperature conditions such as low or high temperature. It has been shown that endogenous cytokinin activity increased in bullhead flowers, while the activity of endogenous gibberellin-like substances decreased (Zieslin et al., 1979). Therefore, it is likely that some stress at the onset of flowering will also cause changes in endogenous phytohormones in incurved flowers. In the current study, the IAA content of incurved flowers was significantly higher than that of normal flowers at stage 2 (Fig. 2C). In a previous study (Aloni et al., 2006), it was reported that high concentrations of IAA in the anthers of Arabidopsis during flower bud development delayed the growth of adjacent floral organs such as petals and nectaries and synchronized their growth with stamen development. Therefore, the high IAA concentration of the incurved flowers at stage 2 in this study may also have been a factor limiting petal growth. The JA and JA-Ile contents of the incurved flowers were significantly lower than those of the normal flowers at stage 2, with only about 25% of the normal flower content (Fig. 2C, D). A previous study on Arabidopsis thaliana (Brioudes et al., 2009) showed that loss of function of the opr3 gene, which is involved in the synthesis of JA, lead to changes in petal size, cell expansion and vein patterns, but that exogenous JA restored the OPR3 petal phenotype. These results suggest that signals initiated by JA during petal development are involved in the regulation of cell expansion and vein patterning in the later stages of petal growth (Brioudes et al., 2009). In a petal degeneration mutant (pdm) of Chinese cabbage (Brassica campestris ssp. pekinensis), mutation of Bra040093 was found to lead to reduced JA levels compared with the wild-type ‘FT’, thereby influencing petal development (Peng et al., 2019). Furthermore, in the double-flowered lily cultivar ‘Doubleen’, in which the JA biosynthesis site was lost, low concentrations of JA in the tepals strongly suppressed elongation, but the tepals expanded when external MeJA was supplied (Fukasawa et al., 2023). Therefore, these results suggest that low concentrations of JA in petals are involved in the formation of incurved flowers, but that this phenomenon may be suppressed by exogenous jasmonic acids.
In conclusion, 100 μM MeJA spray treatment of flower buds may inhibit the number of incurved flowers and increase the maximum flower diameter. Furthermore, the results of the current study suggest that 100 μM MeJA spray treatment of flower buds before harvest more strongly suppresses the emergence of incurved petals than the 500 μM MeJA pulse-uptake treatment of cut flowers after harvest that was investigated in our previous study (Kaneeda et al., 2023a). This is probably because the significantly lower endogenous JA content in the incurved flowers at the beginning of flower opening compared with that in the normal flowers could be compensated for by the 100 μM MeJA spray treatment, which could suppress the number of incurved petals. In future studies, we will investigate whether there is any relationship between the reduction of incurved petals and endophytic phytohormones. Also, related to this, it is necessary to clarify which factors in the petals change IAA, JA, and JA-Ile during cultivation and cause incurved flowers.
The authors wish to deeply thank Professor Yoshiya Seto for his kind advice, and Professor Iain McTaggart for English proof reading.
