Introduction

Rose (Rosa hybrida L.) is one of the most economically important flowers and has a significant role in the cut flower industry. In Korea, cut roses were harvest at the stage of maturity (onset of outer petal reflex) and then transported long distances for exportation. However, during harvest and transportation, water deficit stress is one of the main factors influencing on flower quality such as incomplete flower opening, flower wilting, bending of peduncles, and petal abscission (Zieslin 1989). The shorter vase life and a decrease in postharvest quality after harvest and shipping cause a sharp decline in the market share of Korean-grown roses (Reid 2005). It has been shown that the fresh weight and vase life of cut flowers were harvested and exported in dry conditions are often lower and shorter than in wet harvest and shipping (Yang et al. 2014). Therefore, it is important to understand the effects of harvest methods on the water relations and postharvest longevity of cut roses to improve future postharvest techniques.

The water relations of cut roses are dependent on their physiological and morphological characteristics that regulate water uptake and water loss such as stomatal function, stomatal density, and leaf surface area (Fanourakis et al. 2013a;van Doorn 2012). These characteristics are established by the interactions of genotype with growth environmental factors such as temperature, photon flux density (PFD), and relative humidity (RH) in the greenhouse during cultivation (Fanourakis et al. 2012;Fanourakis et al. 2013b;In and Lim 2018;Torre and Fjeld 2001). Water balance is an important factor determining postharvest quality and longevity of cut roses. It is determined by the balance between water uptake and transpiration. When the volume of water uptake is lower than the amount of transpiration, water stress will occur and cut flowers wilting rapidly (van Doorn 2012). The accumulation of bacteria in the basal of the cut stem ends may be a major cause water stress due to a low rate of water uptake, resulting in an early vase life termination (van Doorn et al. 1995;Woltering 1987). It has been indicated that tap water used for cut flower handling during and after harvest acts as a source of bacteria (van Doorn and de Witte 1997). However, previous studies have also shown that bacterial population in cut rose stems was increased or excluded during dry storage (van Doorn and de Witte 1991) or after exposure to air (van Doorn et al. 1990).

Flower wilting and/or incomplete flower opening in response to water stress is related to petal cell expansion, which requires water entrance across biological membranes. Aquaporins, the primary channels of water transport across biological membranes, play an important role in the regulation of plant cell expansion. Plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) are two of four subfamilies of aquaporins in plants (Maurel et al. 2015;Zardoya 2005). TIPs mediate water exchange between the cytosolic and vacuolar compartments and therefore are thought to play a central role in the regulation of cell turgor in plants (Tyerman et al. 2002). When plants undergo water deficit stress, TIPs expression is decreased in most cases, while in some cases, water deficit stress was found to increase the expression of TIPs (Alexandersson et al. 2005;Lopez et al. 2004;). PIPs can be divided into two major subgroups, PIP1 and PIP2. PIP2 usually has a higher channel activity than PIP1 (Fetter et al. 2004;Johansson et al. 2000). Rh-PIP2;1 and Rh-TIP, rose aquaporin genes, were isolated in Rosa hybrida ‘Samantha’ and expressed highly in petals among floral tissues. The expression of Rh-TIP and Rh-PIP2;1 in rose flowers is related to petal cell expansion and response to water deficit stress and ethylene (Ma et al. 2008;Xue et al. 2009). Therefore, it is important to identify the effects of air-exposure time on the expression of rose aquaporin genes and their relationship to flower opening of cut rose flowers in this study.

In the present study, we used the standard roses ‘Dolcetto’ (Rosa hybrida L.) as the plant materials. After harvest, cut roses were exposed to air for 0, 1, 2, and 3 h and then placed in tap water for recovery. We determined the effect of air-exposure time on postharvest longevity, morphological and physiological changes, bacterial contamination, water relations, and gene expression levels of cut roses ‘Dolcetto’. The transcript levels of rose aquaporin genes, Rh-PIP2;1 and Rh-TIP, were monitored in rose petals to investigate how water stress after air-exposure affected the expression of these genes and flower opening. The results from our study may provide evidence for suggestions for improving postharvest technologies in the cut rose flower industry.

Materials and methods

Plant materials and air-exposure treatment

The flower stems of Rosa hybrida L. ‘Dolcetto’ were harvested at stage 2, loose pointed bud of cylindrical shape in accordance to scale of VBN, 2014, from a commercial grower in Jangsu, Korea. The rose flowers were grown in the greenhouse on rockwool slabs using the “arching” method and were drip-irrigated with a nutrient solution containing NH₄NO₃, Ca(NO₃)₂.4H₂O, KH₂PO₄, K₂SO₄, MgSO₄.7H₂O, and small amounts of other compounds.

The rose stems were randomly harvested on March 25 and April 24 in 2019. The stem length was 60 - 65 cm, stems were then quickly recut in the air to a length of 50 cm. Immediately after re-cutting, the cut flower stems were exposed to air for 0 (E-0), 1 (E-1), 2 (E-2), and 3 h (E-3). E-0 treatment was used as a control in this study. For air-exposure treatment, the cut flower stems were exposed to air by placing them on the bench, about 15 cm apart, inside the greenhouse at 22 ± 2°C, and 77 ± 2% RH. After 0, 1, 2, and 3 h of air-exposure treatment, the cut rose flowers were placed in tap water to recovery and transported by car to the laboratory within 3 h.

Assessment of vase life, water relations, and physiological characteristics of cut roses

At the laboratory, the lowermost leaves of cut flowers were removed, leaving three upper leaves and then placed in a glass vase containing distilled water to a height of 8 cm. Among the thirty cut flowers of each treatment, 24 were selected for vase life assessment. The remaining 6 cut roses were used for RNA extraction, soluble solids content (SSC; %), and leaf stomatal size measurement. All cut flowers were then maintained in growth chambers (VS-91G09M-1300-0, Vision Scientific Co., LTD, Daejeon-si, Korea) at 23 ± 2°C, relative humidity 40 - 50%, and 12 h of light with a flux density of 20 μmol·m^-2·s^-1. In this study, cut roses with uniform size, development stage, color, and free of Botrytis cinerea were selected for all experiments.

The longevity of cut rose was determined as days of vase life from the time flowers were placed into the vases (day 0) to the end of vase life. Cut rose flowers were considered to be at the end of their vase life when the flower showed at least one of the following senescence symptoms: wilting of flower (the flower is visibly limp), leaf or petal abscission (≥ 50% leaf or petal drop), and bending of pedicel (bent-neck; when the flower is at an angle greater than 90°) (VBN 2014).

The day after air-exposure treatment, leaf stomatal characteristics (densities and sizes) were measured in dark in growth chambers (after 12 h in dark condition) and the light in growth chambers (after 1 h of exposure to light at 20 μmol·m^-2·s^-1). The stomata on the abaxial surface of the leaves were taken by Suzuki’s Universal Micro-Printing method. Impressions of the leaf surfaces were photographed with a digital camera (PL-A662, PixeLink, Ontario, Canada). The length and width (except the guard cells) and the number of stomata were analyzed from the images using Image J software (Version 1.49p, NIH, Bethesda, MD, USA).

Changes in water uptake and fresh weight of cut flowers were measured daily at 9:30 to determine the effect of air-exposure time on postharvest quality of cut roses. The water balance of cut flowers was determined by deducting daily evaporation loss from a vase and transpiration from daily water uptake.

To determine the effect of air-exposure time on the microbial contamination at cut rose stem ends, the total bacterial population was assessed at the base of the cut stem ends on day 1 and day 3 of the vase life. The bacterial counts were performed using swab samples taken from the basal 2-cm of the cut stem ends. Each sample was diluted in 10 mL NaCl buffer (3M Pipette Swab; 3M Health Care, St. Paul, MN, USA), and 1 mL of each diluted sample was poured into an aerobic count plate (Petrifilm 6400; 3M Health Care, St. Paul, MN, USA). After two days incubation at 37 ± 2°C, the bacterial count was assessed by counting the number of colonies formed on the plates.

The flower diameter was determined by measuring the maximum diameter and the diameter perpendicular to it (Fanourakis et al. 2012) using digital calipers. The SSC of cut flowers was determined in the uppermost leaves. Leaf tissues (100 mg) were placed in an Eppendof containing 500 μL distilled water, then ground using a TissueLyser (TissueLyser II; Quiagen, Hilden, Germany). The SSC (%) was measured by a portable refractometer (PR-104, Atago, Tokyo, Japan).

The effect of air-exposure time on the water stress status of cut roses was evaluated by measuring the chlorophyll fluorescence parameters on day 3 of the vase life using an Imaging Fluorometer (FluorCam 700MF, Photon Systems Instruments, Drásov, Czech Republic). For leaf chlorophyll fluorescence measurement, minimal fluorescence (F₀) was detected in 20 min dark-adapted leaves and maximal fluorescence (F_m) was also detected in the same leaves in full light-adapted conditions. Maximal variable fluorescence in dark-adapted states (Fᵥ = F_m − F₀), and the maximal PSII quantum yield (F_v/F_m) were automatically calculated from the measured parameters. The F_v/F_m ratios in leaves represent the water stress status of cut roses (Maxwell and Johnson 2000).

RNA extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)

Rose petals were detached from cut flowers after 3 days of air-exposure treatment. Total RNA from petals (200 mg) was isolated using the RibospinTM Plant kit (Gene All Biotechnology Co., LTD, Seoul, Korea). The concentration of RNA was determined at 260 nm/280 nm using a NanoDrop spectrophotometer (NanoDrop One, Thermo Fisher Scientific 5225 Verona Rd. Madison, WI, USA).

First-strand of cDNA was synthesized from the purified total RNA (1 μg) using an oligo(dT)₁₅ primer (INTRON Biotechnology, Inc., Seongnam, Korea). The reverse reaction was performed in a SimpliAmp Thermal Cycler machine (AB Amplied Biosystems, Singapore) for an hour at 42°C and followed by 5 min at 70°C to terminate the reaction using Power cDNA Synthesis Kit (INTRON Biotechnology, Inc., Seongnam, Korea).

Transcript levels of the two rose aquaporin genes in the rose petals of the cut flowers were measured using the StepOnePlusTM real-time PCR system (Applied Biosystems, CA, USA). The primer sequences using in this study were shown in Table 1. A fragment of the Rosa hybrida actin (Rh-ACT1) was used as an internal control to confirm the amount of the RNA. The qRT-PCR reactions were performed following the fast thermal cycles: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. The threshold cycle (Ct) value was automatically detected for each reaction by the qRT-PCR system with default parameters. The final Ct value was the mean of three independent biological replicates and the coefficient of variance for each gene was also calculated. The relative level of the gene expression was calculated as the absolute integrated absorbency normalized to the relative actin. In all the experiments, six samples per treatment were collected, and each analysis was performed with three replications.

Experiment design and data analysis

The air-exposure experiments were performed two times on March 25 and April 24 in 2019. The vase life experiments followed a completely randomized block design with 48 replicates for each experiment. The measurement of chlorophyll fluorescence was performed with 24 biological replicates. Data were presented as means ± standard errors (SE). One-way analyses of variance (ANOVA) were conducted for the treatment comparison. When significant effects were detected, post-hoc pairwise comparison of group means was executed with Duncan’s multiple range test, with a significance level of p ˂ 0.05. Statistical analyses were performed using SPSS version 16.0 (IBM, Somers, NY, USA).

Result

Effect of air-exposure time on the vase life and senescence symptoms of cut roses

The vase life and senescence symptoms of cut flowers varied considerably after exposure to air. The longest vase life was observed in E-0 treatment (13.3 d), followed by E-1 (12.5 d), E-2 (10 d), E-3 (9.1 d) (Fig. 1A). Exposure to air for 2 and 3 h significantly decreased the postharvest longevity of cut rose flowers by 3.3 and 4.2 d, comparing to control flowers (E-0) (Fig. 1A).

In this study, cut roses ‘Dolcetto’ showed wilting of leaves and flowers or bent neck after exposure to air for 2 and 3 h (data not shown). The main senescence symptom that terminated the vase life of cut flowers was petal wilting (80%) and petal abscission (20%) (Fig. 1B). Although air-exposure treatment primarily accelerated the bent neck of cut flowers, it also stimulated leaf wilting in cut roses (Fig. 1B).

Effect of air-exposure time on flower opening, SSC, and bacterial contamination

Flower diameter was daily measured to determine the effect of air-exposure time on flower opening. Overall, exposure to air for 2 and 3 h significantly decreased flower diameter of cut flowers (Fig. 2A). Interestingly, the variation in SSC (%) of leaves among treatments did not show a similar pattern as that of the flower diameter of cut roses (Fig. 2B), indicating that leaf SSC may not be related to flower opening and air-exposure treatment also does not influence on the SSC of cut rose flowers.

The bacterial population at the basal of the stem ends were found to increase after exposure to air. E-3 treatment showed the highest number of colonies after 1 and 3 days of air-exposure treatment (Fig. 3). The bacterial proliferation at the cut stem ends on day 1 and 3 was lowest in E-0 treatment (Fig. 3). In this study, E-1 and E-2 treatments also increased the bacterial population at the basal of the cut stem ends on day 3 (Fig. 3).

Effect of air-exposure time on fresh weight, water uptake, and water balance of cut roses

When flower stems were exposed to air for 3 h, fresh weight of cut flowers was lowest during vase life (data not shown). Exposure to air for 2 and 3 h resulted in a decrease of time that cut flowers maintained their initial fresh weight by 1.5 and 2 days, respectively, compared with control flowers (E-0) (Fig. 4A). Whereas the fresh weight of cut flowers was recovered when placed in tap water after 1 h of air-exposure treatment (Fig. 4A).

Water balance of cut roses was determined by the difference between water loss from leaves and water uptake by flower stem. Similarly to changes in initial fresh weight, control flowers (E-0) showed the longer the number of days that cut flowers retained a positive water balance than air-exposure flowers (Fig. 4B). Cut roses were exposed to air for 3 h (E-3) exhibited the shortest time maintaining the positive water balance (Fig. 4B).

When cut roses ‘Dolcetto’ were exposed to air for 3 h (E-3), the rate of water uptake was lowest during vase periods. E-2 treatment also decreased the water uptake rate of cut flowers (Fig. 4C). Overall, these results revealed that exposure to air for 2 and 3 h increased water stress of cut rose flowers.

Effect of air-exposure time on stomatal characteristics, transpiration, and chlorophyll fluorescence

The stomatal sizes under both light (at 20 μmol·m^-2·s^-1) and dark condition of the control flowers (E-0) were smaller than other treatments (Fig. 5A). Exposure to air for 2 and 3 h significantly increased the size of stomata under both light and dark conditions (Fig. 5A). The results indicate that cut flowers were exposed to air for 2 and 3 h have less functional stomata, which are less able to close during the transition to the dark condition. Consequently, the transpiration rate on day 1 of cut roses in E-2 and E-3 treatments was significantly higher than that of other treatments (Fig. 5B). The flower stems in E-0 treatment showed the lowest transpiration rate on day 1. Exposure to air for 1 h slightly increased the transpiration rate of cut flowers on day 1 (Fig. 5B).

The leaf chlorophyll fluorescence ratios (F_v/F_m) were measured to assess the effect of air-exposure time on water stress status of cut flowers. In this study, F_v/F_m ratios in leaves of cut flowers on day 3 progressively declined with increasing air-exposure time (Fig. 5C). F_v/F_m ratios of cut roses in E-0 treatment were significantly higher than that of other treatments (Fig. 5C). The cut flowers were exposed to air for 3 h had the lowest leaf F_v/F_m ratio on day 3 (Fig. 5C). Exposure to air for 2 h also decreased F_v/F_m ratio in leaves of cut roses (Fig. 5C).

Effect of air-exposure time on the expression of Rh-PIP2;1 and Rh-TIP in rose petals

Expression of two rose aquaporin genes, Rh-PIP2;1 and Rh-TIP, were detected in rose petals using qRT-PCR analysis after 3 days of air-exposure treatment. The transcript levels of Rh-PIP2;1 and Rh-TIP were highest in the petals of the control flowers (E-0) (Fig. 6). The expression of Rh-PIP2;1 and Rh-TIP was found to respond to water stress after exposure to air. E-2 and E-3 treatments significantly suppressed the transcript levels of Rh-PIP2;1 and Rh-TIP in the rose petals on day 3 (Fig. 6). When cut roses were re-watered with tap water after 1 h of air-exposure treatment, they quickly recovered from the leaf and petal wilting symptoms. Water recovery was able to restore the transcript levels of Rh-PIP2;1 and Rh-TIP in rose petals of E-1 treated flowers, which reached a similar level of expression with control flowers (Fig. 6).

Discussion

The postharvest quality and vase life of cut roses, one of a major floriculture crop in the world, are often unsatisfactory due to water stress under unfavorable harvest and storage conditions. After harvest or during vase period, premature signs of water deficit stress occur, such as wilting of both the leaves and the flowers, poor flower opening, and bending of the pedicel of the flowers (Doi et al. 2000;Zieslin 1978, 1989). These senescence symptoms are due to incompetency to take up a sufficient amount of water from the vase solution, which in turn is due to an occlusion in the basal of the cut rose stem ends (van Doorn and Reid 1995). In this study, we examined the air-exposure time in cut roses ‘Dolcetto’ to develop the effective harvest method for a high quality of cut rose flowers. The rose flowers were placed in the tap water directly after harvest (E-0), thereby avoiding the effects of aspired, or were exposed to air for 1, 2, and 3 h prior placement in tap water to recovery, thereby avoiding the effects of bacterial growth. We evaluated the effect of air-exposure time on the vase life, flower opening, physiological characteristics, bacterial contamination, water stress, water relations, and gene expression of cut rose flowers during vase periods.

The present study revealed that the vase life of cut roses ‘Dolcetto’ was shortest in E-3 treatment. The results from our experiments showed that the reduction in vase life of cut flowers in E-3 treatment was related to the deteriorations in water relations such as larger stomatal size under the light and dark, higher transpiration and the bacterial population at the cut stem ends. These results are supported by previous works that indicated that leaf transpiration is responsible for most water loss after harvest and is the major cause of short postharvest vase life in cut rose flowers (In and Lim 2018;van Doorn 2012). After harvest, the high water loss of cut flowers and proliferation of bacteria at the basal of stem or in vase solution caused a failure in the recovery of water balance and led to early bent neck and wilting of flowers and leaves, resulting in a decrease vase life of cut rose flowers, consistent with previous studies (Doi et al. 2000;van Doorn 2012). In this work, the bacterial population at the stem ends of cut flowers was increased upon the air-exposure time and was significantly highest on day 1 and day 3 in cut flowers after exposed to air 3 h. This is consistent with previous observations showing that bacterial and vascular occlusion numbers in cut rose stems were increased during dry storage (van Doorn et al. 1989; van Doorn and de Witte 1991). This caused a blockage in cut rose stems and decrease in water uptake rate of cut flowers. In addition, exposure to air with a long time resulted in a reduction in the maximum penetration depth and decrease in water uptake rate of cut roses (van Doorn and Reid 1995).

The present study showed that E-0 treatment exhibited a superior postharvest quality of cut rose flowers as the longest vase life and good water relations. Previous work indicated that tap water used for cut flower handling during and after harvest acts as a source of bacteria. The bacterial population was found to increase in the vase solution after 3 or 4 d of vase life (van Doorn and de Witte 1997). Our results revealed that the cut rose were placed in the tap water immediately after harvest avoiding the effect of the bacteria contamination only on day 1. However, the bacterial proliferation at the basal of the stems was increased on day 3 in E-0 treated flowers. Thus, the use of an antibacterial solution for non-exposed harvest is necessary to improve water uptake of cut flowers.

The result from our experiments revealed that the vase life of cut roses exposed to air for 2 and 3 h rapidly decreased due to an early failure of water relations. Chlorophyll fluorescence (CF) ratios (F_v/F_m) reflect the photosynthetic efficiency and is often used for assessing stress conditions of plant tissues. An optimal CF ratio in many plant species ranges from 0.79 to 0.84, the decrease in CF ratio in leaves indicates plant stress such as water stress (Maxwell and Johnson 2000). In this work, the decline of F_v/F_m ratios in leaves of cut roses were related to the increase in air-exposure time. The E-0 treatment effectively inhibited water stress and the reduction in F_v/F_m ratios in leaves of cut roses. Exposure to air for a longer time increased water stress, accelerated the early wilting of leaves or flowers and bent neck, and consequently decreased F_v/F_m ratios in leaves and vase life of cut flowers.

Notably, in this study, the cut flowers in E-0 treatment exhibited the largest flower diameter on day 3 of the vase period. Exposure to air 2 and 3 h significantly reduced flower opening of cut flowers. However, the variation in the soluble solids content in leaves was not related to the flower diameter increase as well as the postharvest longevity of cut roses among treatments. These results support the idea that the soluble solids content did not influence on the vase life of cut roses (In and Lim 2018;In et al. 2007;Marissen and Benninga 2001) and air-exposure time may not influence on flower diameter through congenial sucrose contents. Previous experiments showed that Rh-PIP2;1 and Rh-TIP were mainly expressed in the petals of cut roses and had an important role in flower opening through the regulation of petal expansion (Ma et al. 2008;Xue et al. 2009). In addition, the transcript levels of Rh-PIP2;1 and Rh-TIP were abundant in rose petals during rapid flower opening (Ma et al. 2008;Xue et al. 2009). PIPs and TIPs, as the water channel proteins, play a regulatory role in plant growth and development and confer resistance against environmental stresses. In Arabidopsis, the expression of TIPs was decreased during water deficit stress and could be restored to the original level after water recovery (Alexandersson et al. 2005). The similar expression patterns for TIPs were found in other plants such as Nicotiana glauca, sunflowers, and in rice (Kirch et al. 2000;Sarda et al. 1997;Smart et al. 2001). In the present study, we found that the transcript levels of Rh-PIP2;1 and Rh-TIP were repressed in the rose petals by exposure to air for 2 and 3 h. Whereas the expression of Rh-PIP2;1 and Rh-TIP was highest in the petals of control flowers. Exposure to air caused an increase in water stress, resulting in significantly decreased the expression of Rh-PIP2;1 and Rh-TIP in rose petals. This reduced petal water content and inhibited petal expansion and consequently decreased flower opening in cut roses, consistent with previous observations (Ma et al. 2008;Xue et al. 2009). On the other hand, placing cut roses into tap water immediately after harvest decreased water stress, thus increased the transcript levels of Rh-PIP2;1 and Rh-TIP and maintained water content in petals and cell turgor, consequently increased flower opening of cut roses.

Conclusion

The present study revealed that E-0 treatment exhibited higher postharvest quality and longer vase life of cut roses ‘Dolcetto’. The higher postharvest quality of cut flowers in E-0 treatment was related to the good water relations such as maintenance positive water balance, smaller stomatal size in the dark condition, and lower transpiration. E-0 treatment significantly inhibited the bacterial growth at the basal of the stem ends on day 1, enhanced water uptake, and retained fresh weight and leaf chlorophyll fluorescence of cut flowers during vase period. E-0 also effectively improved flower opening of cut roses through the reduction in water stress and increase in the transcript levels of Rh-PIP2;1 and Rh-TIP in rose petals. Based on these results, the non-exposed harvest combination with an antibacterial agent is recommended for harvest stage to improve the quality of cut rose flowers.