1. Introduction
The
Orchidaceae family includes groups of ethylene-sensitive genera, which show a great variable sensitivity to ethylene among the species and cultivars [
1,
2,
3,
4]. The previous study of Woltering and Van Doorn (1988) [
4] reported that the orchid flowers of
Cattleya,
Paphiopedilum,
Dendrobium and
Phalaenopsis are highly sensitive to ethylene.
Cymbidium cut flowers were sensitive to exogenous ethylene, manifested in color fading and wilting of sepal tips, induction of anthocyanin formation in female reproductive parts, and floret abscission [
5,
6]. Cut flowers of
Dendrobium orchid cv. “Karen” responded to endogenous ethylene by abscission of young floral buds and fastening the opening of mature buds [
7].
Recently we reported that exposure of cut flowers of
Vanda orchids cv. “Patchara Delight”, “Pure Wax” and “Sansai Blue” to exogenous ethylene (10 µL·L
−1) for 24 h significantly reduced their vase life by 50% [
8]). A further study by our group suggested that the ethylene-induced rapid bleaching in petals of
Vanda “Sansai Blue” cut flowers is independent of the flower senescence process, and is an outcome of anthocyanin degradation, partially mediated by increase in peroxidase activity [
9].
During development and senescence, ethylene biosynthesis in plants is under metabolic regulation. S-adenosyl methionine (SAM) is converted to 1-aminocyclopropene-1-carboxylic acid (ACC) by ACC synthase (ACS) enzyme, which is the first rate-limiting step in the ethylene biosynthesis pathway. Subsequently, ACC is converted to ethylene by ACC oxidase (ACO) enzyme [
10,
11,
12]. Cut flowers can be categorized as either climacteric or nonclimacteric according to whether or not, respectively, ethylene plays a pivotal role in coordinating their senescence [
13,
14,
15,
16]. Climacteric flowers may also have an increase in respiration during senescence. The ethylene climacteric peak in cut flowers appears to be under autocatalytic regulation, since exposure to ethylene induces endogenous ethylene biosynthesis by turning on genes and enzymes responsible for this process, which coincides with the ethylene climacteric rise [
13,
14,
15,
16].
Various ethylene inhibitors were developed and commercially used to overcome the deleterious effects of ethylene and prolong the vase life of many ornamentals including orchids [
17]. Thus, an inhibitor of ethylene biosynthesis, aminooxyacetic acid (AOA) [
18], and two inhibitors of ethylene perception, silver thiosulfate (STS) [
19], and 1-methylcyclopropene (1-MCP) [
20,
21] are commonly used with ornamentals. Therefore, it is possible that these inhibitors could be effective in
Mokara hybrid orchids.
Mokara orchids are a special group of artificially created tri-genetic hybrids of
Vanda ×
Arachnis ×
Ascocentrum [
22]. The
Mokara orchid flowers are tropical and exotic flowers, with several freckled and broad starfish-shaped florets on the stem, and therefore the inflorescences are commonly used as cut flowers. The information about the postharvest physiology of
Mokara orchid cut flowers is extremely limited [
23]. The vase life is often shortened due to floret senescence and problems in water uptake due to xylem plugging by the presence of bacteria and fungi [
24,
25]. Addition of leaf extracts with antifungal activity from
Jatropha curcas,
Psidium guajava, and
Andrographis paniculata to the vase solution, reduced microbial populations, improved water uptake, and extended the inflorescence longevity comparing to 8-hydroxyquinoline citrate (8-HQC) control [
24,
25].
To the best of our knowledge, there is no report regarding ethylene involvement in the senescence of Mokara orchid cut flowers. Here, we report for the first time on a negative correlation between ethylene production rates and vase life of five Mokara hybrids, which widely vary in their vase life longevity. To investigate further the effects of ethylene on the flowers’ quality, we challenged two Mokara hybrids with exogenous ethylene, and analyzed their responses. In addition, we examined the effects of inhibitors of ethylene biosynthesis or perception on inflorescences quality and vase life, as well as on activities of ethylene biosynthesis enzymes, which represent autocatalytic effects. The results suggest that ethylene is involved in the regulation of the Mokara orchid flower senescence, and pretreatment with ethylene inhibitors can improve their vase life longevity. Accordingly, the rate of endogenous ethylene biosynthesis can be used as a criterion for selection of Mokara hybrids for cut flowers with long vase life.
2. Materials and Methods
2.1. Plant Material and Treatments
Inflorescences of five hybrids of
Mokara (
Arantha ×
Ascocentrum ×
Vanda) orchids were obtained from Thai Orchid Co. Ltd., Bangkok, Thailand. The appearance of the florets of the five hybrids, “Moo-deang”, “Jao-pra-ya”, “Duang-porn”, “Nora-pink”, and “Dao-lai”, is presented in
Figure 1. The
Mokara inflorescences were harvested at a commercial maturity stage (7–9 open florets and 4–5 buds), were selected for uniformity and lack of defects, packed dry, and transported to King Mongkut’s University of Technology Thonburi (KMUTT), Bangkhuntien Campus. Within 1 h after arrival to the laboratory, inflorescence stems were recut (in water) to a 30-cm length. The inflorescences were held in vases with distilled water in the observation room maintained at 21 + 2 °C, 70–80% RH, and cool-white fluorescent light for a 12-h photoperiod.
Application of (2-chloroethyl)-phosphonic acid (ethephon): Inflorescences of “Moo-deang” and “Dao-lai” hybrids were pulsed with either distilled water (control) or with 10 µL·L−1 ethephon for 24 h in the observation room.
Application of inhibitors of ethylene biosynthesis or perception: Inflorescences of “Moo-deang” and “Dao-lai” hybrids were pulsed with either 0.5 mM AOA or 0.05 mM STS for 24 h in the observation room. For application of 1-MCP, the inflorescences were exposed to 200 nL·L−1 1-MCP (0.14% w:w 1-MCP; Floralife, Inc., Walterboro, SC, USA) in 43-L glass chambers for 6 h in the observation room. Control flowers were exposed to air under the same conditions.
Following treatments, all treated inflorescences were transferred to vases with distilled water and were held in the observation room throughout the experimental period. Samples of 45 inflorescences per treatment were used for all the experiments.
2.2. Measurement of Fresh Weight and Water Uptake
Fresh weight (FW) changes of Mokara orchid inflorescences were calculated daily and expressed as % FW relative to the initial FW on day 0 (g g−1 initial FW day−1). Changes of water uptake (mL day−1) were recorded daily from the volume losses of the vase solutions during the vase life evaluation period.
2.3. Measurement of Bud Opening, Floret Senescence, and Vase Life Duration
Open buds and senescent florets were monitored every two days, and their numbers were presented as percent of open buds and senescent florets in each inflorescence. Vase life was defined according to the number of days until 30% senescent florets were observed. The percentage of senescent florets in each inflorescence was calculated according to the sum of florets with petal enrolling and abscised florets.
2.4. Measurement of Ethylene Production and Respiration Rates
Ethylene production rate of whole inflorescences was monitored by enclosing three inflorescences in an airtight polyethylene container and allowing ethylene to accumulate for 1 h at 21 + 2 °C. Three replicates were analyzed per each time point. Gas samples were collected with an 1-mL syringe and analyzed for ethylene in a gas chromatograph (GC8A, Shimadzu, Kyoto, Japan), equipped with a 2-m stainless steel column packed with Unibeads C 80/100 mesh, and a flame ionized detector. Nitrogen was used as the carrier gas, and the temperatures of the column, injector and detector were 220, 100 and 100 °C, respectively. Ethylene production rate was expressed as µL C2H4 kg−1FW h−1.
Respiration rate of the inflorescences was monitored by recording the carbon dioxide concentration, which was analyzed in a gas chromatograph (GC2014, Shimadzu, Kyoto, Japan), equipped with a 1.8-m stainless steel column packed with WG100 KA1144 and a flame ionized detector. Helium was used as the carrier gas, and the temperature of the column, injector and detector was 50 °C. Respiration rate was expressed as mg CO2 kg−1FW h−1.
2.5. Evaluation of ACC Content and Activities of ACS and ACO Enzymes
ACO was extracted and assayed according to the method described by Kato and Hyodo [
26]. The tissues of two lower florets per inflorescence were pooled, immediately frozen in liquid nitrogen, and stored at −70 °C until used. Samples of 3-g florets were homogenized in 2 mL of extraction buffer consisting of 0.1 M Tris-HCl, pH 7.2, 5 mM dithiothreitol (DTT), 30 mM Na-ascorbate, and 10% glycerol (
v/
w) at 2 °C. The homogenate was centrifuged at 14,000×
g for 20 min at 4 °C, and the supernatant was used for assays of the ACC content and ACO enzyme activity.
The assay tubes were held in an ice bath and after 3 min a gas sample was withdrawn for measurement of ethylene concentration in a gas chromatograph (GC8A, Shimadzu, Kyoto, Japan). The efficiency of ACC conversion to ethylene in each sample was determined by adding a known amount of ACC (at least three times the anticipated ACC content of the tissue sample), as an internal standard to each replicate assay tube. The identity of ACC in the Mokara orchid tissue was verified by cochromatography with authentic ACC on a Whatman No. 3 paper developed with butanol-acetic acid-water (4:1:5,
v/
v), as described by Lizada and Yang [
27].
ACO activity in the supernatant was assayed in a reaction medium consisting of 0.1 M Tris-HCl buffer, pH 7.2, 30% glycerol, 1 mM ACC, 10 mM sodium ascorbate, 50 mM FeSO4 and 10 mM NaHCO3 in a total volume of 1 mL. The test solution was placed in 6-mL sealed test tubes, which were incubated at 37 °C and gently shacked for 30 min. A 1-mL sample of the headspace gas was then withdrawn for ethylene determination in a gas chromatograph (GC-8A, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. ACO activity was determined as the amount of ethylene produced from ACC during the reaction period, and expressed as nL C2H4 mg protein−1 h−1.
For the analysis of ACS activity, a 5-g sample of floret tissue was rinsed twice with distilled water, blotted with a paper towel, and ground in 100 mM
N-2-hydroxyethylpiperazine propane sulfonic acid (EPPS) buffer (pH 8.5) containing 4 mM DTT and 0.5 mM pyridoxal phosphate (1:2,
w/
v), using mortar and pestle. The homogenate was centrifuged at 12,000×
g for 20 min. The supernatant extract was placed in a dialysis bag, soaked in dialysis buffer containing 2 mM EPPS buffer, 0.1 mM DTT and 0.2 µM pyridoxal phosphate (1:10,
v/
v), and dialyzed for 24 h. All steps were carried out on ice or at 4 °C. The ACS activity was assayed as described by Hoffman and Yang [
28], by incubating 0.4-mL extract in a 6-mL tube with 50 µL of 0.5 mM SAM and 90 µL of distilled water at 30 °C for 2 h. The ACC produced was determined as described above. ACS activity was expressed as nmol ACC mg protein
−1 h
−1.
2.6. Experimental Design and Statistics Analysis
The experiments were conducted in a completely randomized design (CRD). The quality parameters (FW, water uptake, percent of open buds and senescent florets) were analyzed using 8–10 replicate inflorescences per treatment, and the physiological and enzymatic parameters (rates of respiration and ethylene production, ACC content, and activities of ACS and ACO enzymes) were analyzed using three replicate samples. The data were analyzed using ANOVA, and differences among means were compared by Duncan’s New Multiple Rang Test (DMRT), using SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA). The coefficient of determination between vase life and ethylene production rates was analyzed by linear regression model in Excel.
4. Discussion
The vase life of cut orchid flowers is often terminated by floret wilting and withering, accompanied with improper bud opening, which wither and abscise due to a failure in water relations. The floret senescence symptoms are usually expressed in petal enrolling, wilting and abscission [
29,
30]. The information about the postharvest performance and physiology of
Mokara orchid cut flowers is extremely limited [
23,
24,
25]. Therefore, studying various
Mokara hybrids (
Figure 1), which differ in their vase life longevity, can serve as a research tool for developing effective postharvest treatments. The results of the present research show that the vase life of five hybrids of
Mokara orchids held in distilled water varied from 7.6 to 11.5 days after harvest (
Table 1). The termination of the inflorescences’ vase life was defined when 30% of their florets were senesced. The main senescence symptoms, manifested in petal enrolling, wilting and floret abscission (
Figure 3E), are known to be regulated by ethylene, and are followed by water deficit symptoms [
3,
4,
29,
30,
31,
32,
33]. Thus, the ethylene-induced petal enrolling followed by wilting in florets of
Phalaenopsis,
Doritaenopsis,
Dendrobium and
Cymbidium orchid cut flowers, were accompanied by water loss from cells of the upper layer of the petals, leading to their upward folding [
31]. Therefore, the question was whether the variation in vase life duration of the five
Mokara hybrids stems from the differences in their water relations or in ethylene production parameters.
The results of the present study showed significant differences among the five
Mokara hybrids, in their water relations parameters (
Figure 2A–D). Lower rates of water uptake and higher rates of transpiration resulted in the lowest ratio of water uptake/transpiration, and in the fastest decrease in the relative FW during vase life of the ‘Jao-pra-ya’ hybrid, as compared to the ‘Dao-lai’ hybrid. These patterns of water relations may apparently lead to the significant differences in the vase life longevity of these two hybrids, varying between 7.6 and 11.5 days, respectively (
Table 1). However, the ‘Duang-porn’ hybrid, which also showed a fast decrease in its relative FW, had a long vase life of 11.5 days. In addition, all the hybrids, including ‘Jao-pra-ya’, did not show water deficit symptoms, such as inflorescences wilting and apex bending, or reduced bud opening. Actually, the two hybrids with the shortest vase life longevity, ‘Jao-pra-ya’ and ‘Moo-deang’ (
Table 1), showed the highest percent of bud opening during vase life, and their buds opened in the fastest rate compared to the other hybrids (
Figure 2E). The process of bud opening usually requires active water uptake and high turgor, as it results in increased floret FW [
34]. Therefore, to our opinion, the differences in the vase life longevity observed among the five
Mokara hybrids are not exclusively due to the differences in their water relations parameters. Additionally, the slight and sustained decline of the water uptake rates, observed from day 4 to the end of vase life in all hybrids (
Figure 2B), further suggests that there was no significant blockage in the vascular system of the inflorescences, although the vase solution contained only distilled water without addition of antimicrobial compounds. This conclusion is in contrast to a previous study, performed with
‘Mokara Red’ orchid flowers, which suggested that blockage in the vascular system, resulting from a rapid growth of bacteria in the vase solution, is the cause of its short vase life [
24,
25]. However, to avoid possible contamination during vase life, it is suggested to use preservatives also for
Mokara hybrids.
It was previously reported that ethylene can affect the water status in some cut orchid flowers. For example, ethephon treatment decreased water uptake and enhanced the decrease in flower FW of
Vanda orchid flowers cv. ‘Sansai Blue’ [
35], while the ethylene action inhibitors STS or 1-MCP delayed their FW loss or negated the ethephon effects, respectively [
35,
36]. Similarly, 1-MCP treatment delayed the decrease in FW of two
Dendrobium cultivars ‘Red Sonia’ and ‘Burana Jade’ [
37,
38]. Accordingly, the possibility that the differences in the water relations parameters observed among the
Mokara hybrids during vase life, such as decreased FW and reduced ratios of water uptake to transpiration, can be ascribed in part to the differences in their endogenous ethylene production rates.
Orchid flowers are classified as climacteric flowers in which senescence is accompanied by a climactic increase in rates of respiration and ethylene production [
29,
30,
39]. Ethylene climacteric peak in cut flowers appears to be under autocatalytic regulation, since exposure to exogenous ethylene induces endogenous ethylene biosynthesis and higher respiration rates [
13,
14,
15,
16]. The results of the present study show that ethylene production rates in
Mokara orchid hybrids had different patterns during vase life. While the patterns of ethylene production rates showed climacteric peaks that varied in timing and magnitude among the hybrids (
Figure 2G), their respiration rates did not change much during vase life after the sharp decrease from day 0 to day 2 (
Figure 2H). These results suggest that the
Mokara orchid hybrids could be categorized by the combined differences in their respiration and ethylene production rates, according to the following three patterns: (
1) low respiration and ethylene production rates (‘Doa-lai’); (
2) moderate respiration and ethylene production rates (‘Duang-porn’ and ‘Nora-pink’); (
3) high respiration and ethylene production rates (‘Moo-deang’ and ‘Jao-pra-ya’). Thus, the differences in respiration rates, as well as ethylene production patterns and rates, which are probably genetically derived, can serve as markers for developing other
Mokara cultivars.
The increase in ethylene production rates in the
Mokara hybrids closely coincided with their senescence symptoms that terminated their vase life longevity. Indeed, ethylene production rates were negatively correlated with the vase life longevity of the five hybrids, which were statistically significant on day 4 (
Table 1). These results suggest that the differences in vase life longevity among the five
Mokara hybrids are due to the differences in their ethylene production rates, especially at the day of their peak production, which regulate their flower development manifested in bud opening and floret senescence.
It was previously suggested that the variation in the postharvest life among flower species and cultivars can be partly ascribed to differences in their endogenous ethylene biosynthesis, as well as to differences in their sensitivity to endogenous and exogenous ethylene [
3,
4,
40]. The responses to ethylene vary widely according to the species [
18], although they are often consistent within either families or subfamilies [
29]. This sensitivity to exogenous ethylene plays a vital role in their quality and lasting characteristics [
41]. Variation in ethylene sensitivity may be related to differences in the concentration and affinity of the ethylene receptors and/or the activity of downstream components in the signal transduction pathway, which activates gene transcription and translation [
42]. It was previously reported that
Orchidaceae flowers are highly sensitive to ethylene, even after exposure to very low concentrations of ethylene [
4], and this sensitivity is generally manifested in flower bud drops or abscission [
43]. Ethylene also hastened senescence of petals in flowers of the
Orchidaceae family, e.g.,
Cymbidium and
Dendrobium, that initially stay attached to the flower [
44].
In the present study, we exposed two
Mokara hybrids, ‘Moo-deang’ and ‘Dao-lai’, to exogenous ethylene using ethephon treatment. These hybrids were chosen based on the differences in their vase life duration, and the symptoms causing their vase life termination. The ‘Moo-deang’ hybrid showed a fast floret abscission after petal enrolling started, which ended in vase life of 8 days, and ‘Dao-lai’ florets showed petals enrolling and delayed abscission after 11.5 days of vase life (
Figure 2H;
Table 1). Both hybrids showed high sensitivity to the same ethephon treatment, which significantly enhanced the typical senescence symptoms of each hybrid (
Figure 3C,D), and reduced their vase life longevity very significantly (
Table 2). In addition, the sensitivity to ethephon was manifested in enhanced bud opening (
Figure 3A,B), and acceleration of various parameters of ethylene autocatalysis (
Figure 4). Ethylene was reported to promote or inhibit flower opening, depending on the species and the cultivar [
34,
45]. In the five
Mokara hybrids examined in the present study, ethylene promoted bud opening, as buds opened faster (
Figure 2E) in the hybrids that produced higher ethylene (
Figure 2G). Additionally, in the two selected Mokara hybrids, bud opening and florets senescence were enhanced by ethephon treatment (
Figure 3), and were slowed down by ethylene inhibitors (
Figure 5). It seems therefore, that in
Mokara orchids, ethylene regulates the flower developmental processes, manifested in bud opening, floret senescence and abscission. The ethylene autocatalysis in response to ethephon treatment was more pronounced in the ‘Dao-lai’ hybrid than in the ‘Moo-deang’ hybrid, as all its ethylene biosynthesis parameters were significantly increased by ethephon during vase life (
Figure 4). Unlike this, in the ‘Moo-deang’ hybrid, the increases in ethylene production rates and in ACC content in response to ethephon were minor and were observed only on day 4. A similar pattern of response to exogenous ethylene as that obtained in the
Mokara ‘Moo-deang’ hybrid was reported in
Cattleya Aliliances orchids, showing reduced vase life, increase in ACO activity and no effect on ACS activity and ACC content [
46]. In general, it seems that the increases in ACS and mainly in ACO activities are very sensitive to ethylene in these two
Mokara hybrids.
To avoid detrimental effects of endogenous ethylene on cut flower quality we treated the two hybrids with inhibitors of ethylene biosynthesis and perception. It was our interest to find whether inhibitors of ethylene can inhibit senescence of
Mokara orchid cut flowers and increase their vase life longevity. The effects of three ethylene inhibitors, AOA, STS, and 1-MCP, were examined on various quality parameters of the two
Mokara hybrids, ‘Moo-deang’ with a short vase life, and ‘Dao-lai’ with long lasting vase life. Our results revealed that all the ethylene inhibitors effectively and significantly extended the vase life duration of both hybrids (
Table 3). These effects could be ascribed to the delaying effects of the inhibitors on floret senescence (
Figure 5C,D), as well as to their effects in reducing the endogenous ethylene production rates and ACO activities in both hybrids (
Figure 6A–D). However, there were several differences in the responses of the two hybrids to the ethylene inhibitors, which probably resulted from the differences in their original vase life longevity. Thus, the ethylene inhibitors significantly decreased the percentage of bud opening but kept them open for a longer time in the ‘Moo-deang’ hybrid (
Figure 5A), while they had almost no effect on the bud opening in the ‘Dao-lai’ hybrid (
Figure 5B). Similarly, the ethylene inhibitors significantly decreased ACS activity and ACC content only in the ‘Moo-deang’ hybrid (
Figure 6E,G), probably because these parameters were already very low in the control inflorescences of the ‘Dao-lai’ hybrid (
Figure 6F,H). These differences indicate that the longer vase life of the ‘Dao-lai’ hybrid as compared to that of the ‘Moo-deang’ hybrid, could be due to its lower sensitivity to ethylene and its lower ethylene production rates. Taken together, these ethylene inhibitors can be further used as effective postharvest treatments for preserving the quality of
Mokara hybrid cut flowers.