Acta Physiologiae Plantarum (2020) 42:48 https://doi.org/10.1007/s11738-020-03044-w

ORIGINAL ARTICLE

Genotypic and phenotypic diferences in fresh weight partitioning of cut rose stems: implications for water loss

Dimitrios Fanourakis1 · Dimitris Bouranis2 · Georgios Tsaniklidis3 · Abdolhossein Rezaei Nejad4 · Carl‑Otto Ottosen5 · Ernst J. Woltering6,7

Received: 20 July 2019 / Revised: 27 November 2019 / Accepted: 18 March 2020 © Franciszek Górski Institute of Physiology, Polish Academy of Sciences, Kraków 2020

Abstract In vase life studies, cut fower fresh weight is often recorded, but mass distribution is not. Here, we addressed the variation in mass distribution among the diferent cut fower organs, and assessed its role in water relations. In the frst part of the study, excised , fower, and stem were exposed to desiccation. Water loss (per fresh mass) of both fower and stem was low, relatively constant over time and comparable between the three studied cultivars. Instead, water loss (per fresh mass) of leaves was initially much higher, and decreased upon desiccation due to stomatal closure. Leaves had the greatest contribu- tion to cut fower water loss, while this contribution was diferent among the tested cultivars. Similar fndings were obtained following evaluation of the contribution of leaves, stem, and fower to cut fower transpirational water loss under conditions where water supply was not limiting. A strong correlation between the weight loss in the desiccation experiment and the length of vase life was found. Low evaporative demand during vase life evaluation increased vase life, and alleviated vase life diferences between cultivars. Instead, high evaporative demand during evaluation shortened vase life, and increased the noted diferences in vase life between cultivars. In the second part of the study, fresh weight partitioning was assessed within and among cut rose cultivars. Among eight cultivars, same weight fowering stems may have over 11% diference in leaf weight. In conclusion, cultivar diferences in transpirational water loss between cut fowers of the same weight may be attributed to variations in both stomatal characteristics and mass partitioning to the leaves.

Keywords · Mass allocation · Vase life

Abbreviations Introduction RH Relative air humidity VPD Vapor pressure defcit Long keeping quality is a very important factor determining consumers’ satisfaction and thus choice (Fanourakis et al. 2015b; Onozaki 2018). A basic requirement for a long keeping quality is a positive water balance (i.e., water uptake > water Communicated by P. Wojtaszek. loss; Fanourakis et al. 2013b; In et al. 2016). A yet unexplored strategy to improve cut fower longevity could be to select gen- Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s1173​8-020-03044​-w) contains otypes with a reduced rate of water loss (Giday et al. 2013b; supplementary material, which is available to authorized users.

* Dimitrios Fanourakis 4 Department of Horticultural Sciences, Faculty of Agriculture, [email protected] Lorestan University, P.O. Box 465, Khorramabad, Iran 5 Department of Food Science, Aarhus University, 1 Giannakakis SA, Export Fruits and Vegetables, Tympaki, Kirstinebjergvej 10, 5792 Årslev, Denmark Greece 6 Wageningen Food & Biobased Research, Bornse Weilanden 2 Plant Physiology and Morphology Laboratory, Crop Science 9, 6708 WG Wageningen, The Netherlands Department, Agricultural University of Athens, Athens, Greece 7 Horticulture & Product Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, 3 Institute of Olive Tree, Subtropical and Viticulture, The Netherlands Hellenic Agricultural Organization ‘Demeter’ (NAGREF), P.O. Box 2228, 71003 Heraklio, Greece

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Carvalho et al. 2015). Until now, this discussion mostly cent- in a multispan plastic greenhouse. Two harvests were con- ers around a better closure of the stomatal pores on the leaf ducted (6 and 20 July, 2015), supplying material for the two surface (Fanourakis et al. 2016b; Woltering and Paillart 2018). respective experiments. In the frst harvest, three cultivars However, cut fower water loss is not one-dimensional. The were tested (Bordeaux, Lenny, and Testarossa). These three mechanism of leaf stomatal closure is crucial (Fanourakis et al. cultivars were selected based on preliminary measurements, 2019a, b) albeit not a unique parameter that infuences the cut showing similar fresh mass partitioning to the leaves. In the fower water loss trait, while other attributes such as biomass second experiment, fve more cultivars (Avalanche, Gladi- allocation to the leaves should also be taken into consideration ator, Jumilia, Sorbet avalanche, and Talea) were included, (Giday et al. 2014). A research gap currently exists on how thus making eight ones in total. Twenty days prior to each variable biomass allocation to the leaves is among as well as harvest (corresponding to the growth period), climate within cut rose cultivars, which evidently afects the relation- parameters were automatically recorded. Preceding the frst ship between leaf and cut fower water loss. harvest, mean air temperature was 22.3 ± 0.4 °C, and prior to Leaf water loss is often employed to make assumptions the second harvest it was 23.7 ± 0.7 °C. Relative air humidity regarding the whole cut fower transpiration (Woltering and (RH) averaged 53 ± 6% in either period, resulting in vapor Paillart 2018; Fanourakis et al. 2019b). However, leaf water pressure defcits (VPDs) of 1.27 ± 0.16 kPa (experiment 1) loss responses may not always correlate to the whole cut fower and 1.38 ± 0.18 kPa (experiment 2). Based on the proxim- transpiration, especially under several specifc storage scenar- ity of the two harvest dates and the comparable evaporative ios that are nevertheless not uncommon during the postharvest demand during the two growth periods, it is safe to attribute life of the fowers. For instance, although most gas exchange the noted phenotypic diferences to the genotype (thus limit- indeed occurs through leaf stomata when they are open (Car- ing its interaction with the growth environment). penter and Rasmussen 1974; Mayak et al. 1974), transpira- In either experiment, harvested shoots had a length of tion rate through the stem and the fower bud may potentially approximately 0.7 m and a fower bud with a cylindrical become increasingly important for cut fower water loss when shape and pointed tip. Replicate shoots were collected from stomata are closed (e.g., during the dark period of vase life, or diferent plants. Cut fowers were collected in the morning upon water defcit). Further research is thus required to quan- (08:00–10:00 h), and immediately placed in buckets with tify the impact of leaf transpiration on total cut fower water aqueous sodium hypochlorite solution (1%, v/v). The stems loss under diferent stomatal closure states. Moreover, stem were transferred to the laboratory in these buckets at the and fower bud transpiration data have not been previously day of harvest and using refrigerated transport (2 °C). Upon recorded in diferent rose cultivars, and therefore, their poten- arrival, the leaves on the lower 0.15 m of the shoot were tial contribution to the cultivar diferences in cut fower water stripped. Cut fowers were stored overnight in buckets with loss remains elusive. Such divergences that directly afect the water-containing sodium hypochlorite (1%, v/v), at 2 °C and cut fower water balance during the postharvest life can be darkness. critical for the quality and marketability of the cut fowers. To prevent bacterial growth, which would cause vascular The aims of the current study are: (1) to quantify the con- blockage leading to low water uptake, sodium hypochlorite tribution of the stem, the leaves and the fower bud to total cut was added in the water, where the cut fowers were placed fower water loss under diferent stomatal closure states (i.e., throughout handling and evaluation (Fanourakis et al. under both ample water supply, and upon water defcit), and 2016b). (2) to evaluate variation in mass distribution among and within At the end of each experiment, leaf area (using ImageJ; cultivars. The examination of the results obtained by these two Koubouris et al. 2018), number of leaves, stem and pedi- objectives combined are expected to provide not only a better cel length, top (below the pedicel) and bottom (above insight into cultivar diferences in transpirational water loss, the cut point) stem diameter together with pedicel diam- which in many cases underlie variation in vase life (Spinarova eter (assessed midway its length) were evaluated in all cut and Hendriks 2007; Fanourakis et al. 2012, 2016b), but also fowers. to facilitate the scaling of water loss data from organ level to processes occurring to whole cut fower level. Contribution of stem, leaves, and fower bud to cut fower water loss upon water defcit (experiment 1)

Materials and methods Cultivar diferences in the contribution of each individ- ual part involved in cut fower transpirational water loss Plant material and growth conditions were investigated in the course of desiccation. The fully hydrated cut fowers were taken from the refrigerated stor- Cut roses were obtained from a commercial grower age (2 °C and darkness) and placed into the test room, (Polioudakis G., Rethymno, Greece). Plants were grown where they were kept for 2 h prior to measurements. This

1 3 Acta Physiologiae Plantarum (2020) 42:48 Page 3 of 10 48 period served the dual aim of inducing stomatal open- Vase life following defoliation and at 10, 50, ing and bringing cut fowers to test room temperature and 95% RH (experiment 1) (Fanourakis et al. 2016b). Subsequently, cut fowers were separated into stem, leaves, and peduncle plus fower bud. It was investigated whether the noted variation in the con- These parts were then placed on empty vases, and the rate tribution of each individual part to cut fower water loss of water loss over time was gravimetrically recorded for impacts keeping quality, as well as whether this effect 6 h (± 0.01 g; MXX-412; Denver Instruments, Bohemia, depends on conditions that afect transpiration. The night NY, USA). Test room conditions were air temperature of before the experiment, the cut fowers were kept in the dark 25.0 ± 0.2 °C, RH equal to 50 ± 3%, and light intensity refrigerated storage (2 °C) for 12 h, to ensure maximal of 50 μmol m−2 s−1 provided by fuorescent lamps (T5 hydration (Fanourakis et al. 2012, 2016b). Next day, vase fuorescent lamp; GE lighting, Cleveland, OH, USA). As life was determined on cut fowers that were placed in the an indication of air velocity, the rate of evaporation from vase (one fower per fask). The vases contained 250 mL two glass beakers was recorded in the test room during sodium hypochlorite solution (1%, v/v), employed to inhibit measurements (± 0.0001 g; Mettler AE 200, Giessen, bacterial growth. The fasks were then moved to three cli- Germany). The evaporation rate of distilled water was mate-controlled rooms, set at diferent levels of RH [i.e., −2 −1 0.74 ± 0.01 mmol ­H2O ­m s , which indicated adequate 10% (dry), 50% (moderate), or 95% (humid)]. At 50% RH, air circulation. The employed environmental conditions an additional set of cut fowers was placed, which previ- are typical for dehydration experiments (Giday et al. ously underwent defoliation. The remaining environmental 2013a; Carvalho et al. 2016; Fanourakis et al. 2019a, b). variables were identical among the three test rooms. Air Diferent cut fower parts were always assessed simultane- temperature was set to 25 °C, resulting in VPDs of 2.85, ously. Three cultivars (Bordeaux, Lenny, and Testarossa) 1.27, and 0.16 kPa, respectively. Light level was set at were evaluated (n = 14). 15 μmol m−2 s−1 photosynthetic photon fux density (deter- mined by LI-250A, LI-COR, Lincoln, NE, USA) for 12 h per day, and was supplied by fuorescent tubes (TLD 58 W/84, Contribution of stem, leaves, and fower bud to cut Philips, Eindhoven, The Netherlands). The height of the vase fower water loss under continuous water supply solution column was held constant at 0.07 ± 0.01 m over the (experiment 1) evaluation period to avoid hydrostatic pressure diferences between fowers with diferent transpiration rates (Fanoura- Cultivar diferences in the contribution of each individual kis et al. 2012, 2016b). Flower quality was determined daily, part involved in cut fower transpirational water loss were while end of fower life was based on the occurrence of at also evaluated under vase life evaluation conditions, thus least one of the following criteria: (i) bending of the pedicel unhindered water supply. Cut fowers were kept in dark (i.e., fower angle becomes larger than 90° from the verti- refrigerated storage (2 °C) for 12 h before experimentation. cal position); (ii) abscission of more than two petals; (iii) The following three treatments were applied: (a) intact cut visible wilting of the fower (i.e., loss of petal turgor); and fowers, (b) defoliated cut fowers (i.e., leaves were excised), (iv) more than 50% of the number of leaves had abscised, and (c) peduncle plus fower (i.e., stem and leaves were turned yellow, or had desiccated (VBN 2005). To reduce excised). The treatments were placed in a vase (one treat- subjectivity, three observers independently evaluated the end ment per fask) containing 250 mL sodium hypochlorite of vase life (without being aware of treatment assignment), solution (1%, v/v). The top of each vase was covered with and decision was based on majority vote. In this study, no Paraflm to prevent direct evaporation from the vase solu- Botrytis cinerea infections were observed. Twelve fowers tion. The fasks were moved to a climate control room with per treatment were assessed in three cultivars (Bordeaux, the following environmental conditions: 50% RH, 25 °C Lenny, and Testarossa). air temperature (i.e., 1.27 kPa VPD), and 15 μmol m−2 s−1 photosynthetic photon fux density (determined by LI-250A, Contribution of stem, peduncle, leaves, LI-COR, Lincoln, NE, USA) for 12 h per day provided by and fower bud to the total cut fower mass fuorescent tubes (TLD 58 W/84, Philips, Eindhoven, The within and among cultivars (experiment 2) Netherlands). The water loss over a 4 day period was meas- ured (± 0.01 g; MXX-412; Denver Instruments, Bohemia, To evaluate cultivar diferences in fresh mass allocation, NY, USA), and this was expressed per initial fresh weight. stem, pedicel, leaf, and fower bud masses of each cut fower Treatments were compared based on the average transpira- were recorded (± 0.01 g; MXX-412; Denver Instruments, tion rate over the 4 day period. Diferent treatments were Bohemia, NY, USA). For each fower bud, the weights of assessed simultaneously. Three cultivars (Bordeaux, Lenny, sepals, petals, and the remaining structure (i.e., androecium, and Testarossa) were evaluated (n = 12). gynoecium, and the receptacle) were measured. For each

1 3 48 Page 4 of 10 Acta Physiologiae Plantarum (2020) 42:48 leaf, the weights of petiole and leafet lamina were assessed. Replicate stems were selected to have similar (total) weight 2,4 per cultivar (n = 20). These measurements were conducted ter loss on all eight cultivars under study. )

-1 1,6 wa To evaluate within cultivar diferences in fresh mass allo- h er cation, stem, pedicel, leaf and fower bud masses of each (g cut fower were assessed. Replicate stems were selected 0,8 to belong to diferent (total) weight categories per cultivar Cut flow (n = 20). These measurements were conducted on three cul- 0,0 tivars (Bordeaux, Lenny, and Testarossa). 0246 Time (h) Statistical design and analysis

Fig. 1 Cut fower transpirational water loss as a function of dehydra- Data analysis was performed using the R software (version tion time in three cut rose cultivars [Lenny (flled circle), Testarossa 2.14.2; www.r-proje​ct.org). Treatment efects were tested (blue flled square), and Bordeaux (red flled diamond); Experiment at 5% probability level and mean separation was carried out 1; n = 14]. Both the water loss (data in Fig. 2) and fresh mass frac- t test tion of each component were considered for the computation. Before using least signifcant diferences based on Tukey’s the onset of desiccation, cut fowers were well hydrated, while start- (P ≤ 0.05). ing weight was similar among cultivars (Suppl. Table S1). Several morphological parameters of the employed cut fowers are provided in Suppl. Table S1 Results desiccation, since leaf transpiration decreased, whereas Contribution of stem, leaves, and fower bud to cut water loss through the other two components remained fower transpirational water loss following water nearly stable. During the sixth hour of desiccation, the severity or under ample water supply (experiment water loss through the leaves was still more prominent as 1) compared to the other two cut fower components alone or combined in cvs. Testarossa and Bordeaux (65.0 and 70.8% Before the onset of desiccation, cut fower starting weight of total, respectively; Fig. 3). Instead, at the sixth hour of was similar among cultivars (Suppl. Table S1). In all three desiccation, water loss by the leaves was 42.3% of the total cultivars under study, cut fower transpiration decreased over cut fower water loss for cv. Lenny (Fig. 3). Thus, at the end desiccation time (Fig. 1). Cv. Lenny underwent the lowest of desiccation, stem and fower bud combined contributed water loss, as compared to the other two cultivars, through- more to cut fower water loss, as compared to leaves, in cv. out desiccation (Fig. 1). Cv. Bordeaux lost more water, as Lenny. compared to cv. Testarossa, during the frst hour of desic- The water loss (per initial weight) was also recorded cation (Fig. 1). under vase life evaluation conditions, and thus under con- The water loss (per initial weight) of each of the three tinuous water supply (Fig. 4; for percentages, see Fig. 5). cut fower components was separately recorded (Fig. 2). In These data also confrm that leaves not only account for most all tested cultivars, the leaf weight loss decreased over time of the cut fower water loss under conditions where water is (Fig. 2a, c, e), indicating stomatal closure. Instead, stem not a limiting factor, but also give rise to cultivar diferences. and fower bud weight loss was almost constant over time (Fig. 2b, d, f). Leaf weight loss was much higher as com- Vase life following defoliation and at 10, 50, pared to stem and fower bud weight loss in all the cultivars and 95% RH (experiment 1) (Fig. 2a, c, e). Strong cultivar diferences were observed in leaf weight loss (Fig. 2a, c, e), whereas diferences were Vase life was assessed under conditions that afect cut fower small when comparing stem and fower bud weight loss transpiration. These conditions included diferent evapora- (Fig. 2b, d, f). The highest leaf weight loss was noted in cv. tive demands (by means of diferent RH levels; Fig. 6), as Borbeaux, while the lowest was observed in cv. Lenny. well as defoliation (Fig. 7). Cv. Lenny not only had the The contribution of each cut fower component to total longest vase life at control (50%) RH, but also underwent cut fower water loss was also expressed as a percentage the smallest vase life decrease when evaluation took place (Fig. 3). During the frst hour of desiccation, the water loss at 10% RH (Fig. 6). Cv. Bordeaux had similar vase life as through the leaves was 70.8 to 88.5% of the total cut fower Testarossa at control (50%) RH, though underwent a larger water loss (cvs. Lenny and Bordeaux, respectively; Fig. 3). vase life decrease at 10% evaluation RH (Fig. 6). At condi- However, this fraction decreased in the course of cut fower tions where cut fower water loss was minimized (i.e., 95%

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0,24 A Testarossa B Testarossa 0,02 ) 0,16 ) -1 -1 h h -1 -1 g g ter loss ter loss 0,01 (g (g Wa Wa 0,08

0,00 0,00 0246 0246 0,24 Χρόνος (ώρες) Χρόνος (ώρες) C Lenny D Lenny 0,02

) 0,16 ) -1 -1 h h loss loss -1 -1 g g ter ter 0,01 (g (g Wa Wa 0,08

0,00 0,00 0246 0246 0,24 Χρόνος (ώρες) Χρόνος (ώρες) E Bordeaux F Bordeaux 0,02 ) 0,16 ) -1 -1 h h loss loss -1 -1 g g ter ter 0,01 (g (g Wa Wa 0,08

0,00 0,00 0246 0246 Time (h) Time (h)

Fig. 2 Transpirational water loss, per initial weight, of leaves (flled using a diferent scale. Error bars indicate SEM. When the SE bars circle), stem (blue flled square), and peduncle plus fower bud (red are not visible, the SE is smaller than the symbol. Several morpho- flled diamond) as a function of dehydration time in three cut rose logical parameters of the employed cut fowers are provided in Suppl. cultivars (Experiment 1; n = 14). The fgures b, d, and f (on the Table S1 right) represent the same data as the fgures a, c, and e (on the left), evaluation RH or after defoliation), all three cultivars had number did not signifcantly difer among the studied cul- similar vase life (Figs. 6, 7). tivars (n = 5; data not shown). Flower bud fresh weight was mostly allocated (72.5–86.2%) to the petals (Suppl. Fig. Allocation of cut fower mass to stem, peduncle, S3A), while the least weight was allocated (3.7–5.7%) to leaves, or fower bud within and among cultivars sepals (Suppl. Fig. S3B). Petal number was poorly correlated (experiment 2) with both fower bud weight (R2 = 0.44; Suppl. Fig. S2B) and its percentage allocated to petals (R2 = 0.12; Suppl. Fig. In most cultivars, the stem had the greatest contribution to S2C). Within the leaf, lamina acquired the largest portion of the total fresh mass (32.3–42.3%; Fig. 8c), followed by the fresh mass (85.1–90.4%), as compared to the petiole (Suppl. fower bud (27.9–36.7%; Fig. 8b), the leaves (20.5–31.3%; Fig. S4). Fig. 8a), and the peduncle (3.1–6.6%; Fig. 8d). Among cul- Within a given cultivar, as cut fower weight changes, the tivars, diferences in fresh mass allocation were most promi- weight of each component is afected. This does not hold nent in leaf weight (i.e., up to 10.8% diference; Fig. 8). Leaf for the percentage of fresh weight acquired by each com- weight was highly correlated with leaf area among cultivars ponent. In all three cultivars under study, as cut fower total (R2 = 0.91; Suppl. Fig. S1). weight increases, the relative fresh mass allocation to the Large cultivar diferences in the number of petals were stem increases, whereas the relative fresh mass allocation recorded (28.9–50.9; Suppl. Fig. S2A), whereas sepal to the fower bud decreases (Fig. 9). Instead, relative fresh

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60 -1 0,02 g

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e lo r Wa (% 30 te 0,01

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li li li In In Χρόνος (ώρες) In er er er fo fo 90 fo ow ow B Lenny ow De De De Fl Fl Fl BordeauxTestarossaLenny Treatment 60 Cultivar total) ter loss of Fig. 4 Transpirational water loss, per initial weight, of intact cut fow- Wa (% 30 ers, defoliated cut fowers, and peduncle plus fower over a 4 day period under vase life evaluation conditions in three cut rose cultivars (Experiment 1; n = 14). Cut fowers were well hydrated at the onset 0 of the experiment, while starting intact cut fower weight was similar 0246among cultivars (Suppl. Table S1). Several morphological parameters Χρόνος (ώρες) of the employed cut fowers are provided in Suppl. Table S1. Means 90 C Bordeaux followed by diferent letters indicate signifcant diferences according to the Tukey’s t test. P value is provided in Suppl. Table S2 ) 60 90 ter loss of total

Wa (% 30

total) 60 of 0 0246 Time (h) loss (% ter 30 Wa Fig. 3 Transpirational water loss, as a percentage of total cut fower water loss (data in Fig. 1), of leaves (flled circle), stem (blue flled square), and peduncle plus fower bud (red flled diamond) as a func- 0 tion of dehydration time in three cut rose cultivars (Experiment 1; Leaves Stem Flower Leaves Stem Flower Leaves Stem Flower n = 14). Both the water loss (data in Fig. 2) and fresh mass fraction of bud bud bud each component were considered for the computation. Several mor- Bordeaux Testarossa Lenny phological parameters of the employed cut fowers are provided in Treatment Suppl. Table S1 Cultivar

Fig. 5 Transpirational water loss, as a percentage of total cut fower mass allocation to the leaves remains nearly constant, as cut water loss (data in Fig. 4), of leaves, stem, and peduncle plus fower fower weight changes (Fig. 9). bud over a four day period under vase life evaluation conditions in three cut rose cultivars (Experiment 1; n = 14). Both the water loss (data in Fig. 4) and fresh mass fraction of each component were con- sidered for the computation. Several morphological parameters of the employed cut fowers are provided in Suppl. Table S1 Discussion

This study examines the reasons which underlie cultivar Cut fower water loss varied depending on the culti- diferences in transpirational water loss, together with the var (Figs. 1, 4). These cultivar diferences in water loss potential role of those diferences on the vase life under vari- were observed under ample water supply (Fig. 4) and ous postharvest conditions. at the onset of desiccation (Fig. 1), whereas these were

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9 This study for the frst time also shows that under ample water supply (Fig. 5) or when water defcit is still mild (e.g.,

) at the onset of desiccation; Fig. 3), more water (≥ 64% of 6 the total) is lost though the leaves as compared to stem and fower bud combined. However, under severe water defcit se life (d (e.g., at the end of desiccation), leaves retained the highest

Va 3 fraction of total cut fower water loss, as compared to stem and fower bud combined, in cvs. Bordeaux and Testarossa, 0 whereas in cv. Lenny, the opposite was true (Fig. 3). This 0255075100 indicates that water loss through both the stem and fower Evaluation RH (%) bud becomes increasingly important under water-defcit conditions, and may eventually exceed the leaf water loss Fig. 6 Vase life as a function of relative air humidity (RH) during depending on the cultivar. evaluation in three cut rose cultivars [Lenny (flled circle), Testarossa Why more water is lost through the leaves? The ratio of (blue flled square), and Bordeaux (red flled diamond); Experiment (transpiring) area per fresh mass in leaves is much higher 1]. Values are the means of twelve cut fowers ± SEM. When the SE bars are not visible, the SE is smaller than the symbol. Several mor- as compared to both stem and fower bud, which at least phological parameters of the employed cut fowers are provided in partly contributes to the higher transpirational water loss of Suppl. Table S1. Statistics are provided in Suppl. Table S3 leaves. More importantly, the declining pattern of leaf water loss indicates stomatal closure, whereas the nearly stable transpiration of stem and fower bud hints to the absence of (functional) stomata (Fig. 2). This is in agreement with the 10 c c c early work reporting the absence of stomata in both the stem b and petals of cut roses (Carpenter and Rasmussen 1974). 8 Therefore, the large diference in water loss between leaves a a and other organs is also related to the presence of stomata. ) 6 (d Tissues bearing stomata have been accounted to contribute

life to most of the cut fower water loss in other ornamental spe-

ase cies (Huang et al. 2018).

V 4 Exposing detached cut fower organs to desiccation also indicates that cultivar diferences in cut fower water loss are 2 predominantly caused by the leaves (Fig. 2). Similar fndings were also obtained under conditions where water supply was 0 not limiting (Fig. 4). Since cultivar diferences in leaf cuticu- ControlDefoliatedControl Defoliated ControlDefoliated lar water loss were found to be rather small (Fanourakis et al. Bordeaux Testarossa Lenny Treatment 2013a, 2019b), the noted genetic variation in leaf water loss Cultivar ought to be related to stomatal characteristics. Therefore, genetic variation in stomatal aperture in the absence of stress Fig. 7 Vase life of intact or defoliated cut fowers in three cut rose (Fig. 4), in combination with stomatal response to a closing cultivars (Experiment 1). Relative air humidity during evaluation was stimulus, such as desiccation assessed here (Fig. 2) or dark- 50%. Values are the means of 12 cut fowers ± SEM. Several morpho- logical parameters of the employed cut fowers are provided in Suppl. ness taking place during the dark phase of vase life, explains Table S1. Means followed by diferent letters indicate signifcant dif- cultivar diferences in cut rose water loss. ferences according to the Tukey’s t test. P value is provided in Suppl. Under control conditions (50% RH), the cultivar with the Table S3 lowest leaf weight loss (Lenny) exhibited the longest vase life, as compared to the other two cultivars (Bordeaux, and Testarossa) where no signifcant diference was found in minimized as cut fower was progressively exposed to vase life (Figs. 2, 6). Previous studies have also highlighted dehydrating conditions (Fig. 1). By separating leaves, that a better stomatal closing ability is related to a longer stem, and fower bud, it was found that the majority of vase life (Mayak et al. 1974; Spinarova and Hendriks 2007; water loss occurred through the leaves, as compared to Fanourakis et al. 2016a; Woltering and Paillart 2018). We the other two components alone (Figs. 3, 5). Thus, as here further show that under high evaporative demand con- expected, leaves are the main driver of cut fower water ditions (10% RH), the leaf weight loss was inversely related loss, as compared to either stem or fower bud (see also to vase life (Figs. 2, 6). Cv. Lenny had longer vase life than Carpenter and Rasmussen 1974; Mayak et al. 1974). Testarossa, which in turn had longer vase life than Bordeaux

1 3 48 Page 8 of 10 Acta Physiologiae Plantarum (2020) 42:48 l l) a t 45 Α ta 45 Β o g t to f e

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Cultivar Cultivar

Fig. 8 Fresh weight distribution over the four individual parts of employed cut fowers are provided in Suppl. Table S4. Means fol- the cut fower in eight cut rose cultivars (Experiment 2; n = 20). lowed by diferent letters indicate signifcant diferences according to Error bars indicate SEM. Several morphological parameters of the the Tukey’s t test. P values are provided in Suppl. Table S5

(Figs. 2, 6). Instead, under low evaporative demand condi- Notably, the fresh weight allocation to the leaves was found tions (95% RH) or following defoliation, vase life was inde- to be reasonably constant within a given cultivar (Fig. 9). pendent of leaf weight loss (Figs. 2, 6 and 7). This suggests These data indicate that the water loss diferences between that under favorable postharvest conditions of low evapora- same weight cut fowers belonging to diferent cultivars are tive demand, the impact of leaf weight loss on vase life is not only related to stomatal traits (Figs. 2, 4), but also to reduced. Instead, the vase life-promoting efect of low leaf diferences in leaf weight (Fig. 8a). This analysis focused on weight loss is amplifed under conditions that promote tran- the cultivar efects on fresh mass partitioning to the leaves. spiration (i.e., high temperature, low RH, high light inten- However, it must be kept in mind that growth environment sity, or increased air velocity). The entirety of these results can also have a major impact on mass partitioning to the indicates that the efect of leaf weight loss (an indication of leaves (reviewed by Poorter et al. 2012). stomatal closing ability; Fanourakis et al. 2015a) on vase life Overlooking or ignoring the importance of leaf mass on is strongly associated with the postharvest scenario under cut fower water loss will not only hinder a full answer to study, with this efect being increasingly important as cut one’s research question, but will also make it difcult to fower water loss is stimulated. refect the obtained fndings to the ones of other studies. On Considering the importance of a long vase life to the hor- top of this, the relative impact of variation in leaf mass on ticultural sector (Fanourakis et al. 2015b; Onozaki 2018), the vase life will difer excessively depending on the experimen- selection of cultivars with a better ability to control water tal setup (Figs. 6, 7), thus infuencing the relative diferences loss is essential in real-world situations, where the supply between treatments. We conclude that it is important for chain often includes a wide range of conditions (Fanourakis researchers to minimize such efects in vase life studies by et al. 2013b; In et al. 2016; Pouri et al. 2017). This selection considering genotypic and/ or environmental efects on mass of cultivars with stomata responsive to closing cues (e.g., partitioning to the leaves. desiccation or darkening) currently appears to be realistic, given the existence of a large genetic variation regarding the stomatal closing ability under water unavailability (Fan- Conclusion ourakis et al. 2013a; Giday et al. 2013a, b, 2015; Carvalho et al. 2015). Assessments were conducted under conditions of water def- Given that the main origin of cut fower water loss is cit, as well as under unhindered water supply. It was found located in the leaves (Figs. 2, 4), it was also here noted that that water is mainly transpired through the leaves in cut cut roses of the same weight sampled from diferent cultivars roses, with stem and fower bud being of lower importance. may have up to 10.8% diference in leaf weight (Fig. 8a). Therefore, cultivar diferences in cut fower transpirational

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6 A Testarossa B 45

4 of total) of total)

30 (% (% 2 15 Weight Weight

0 0 20 35 50 65 20 35 50 65 Βάροςτριαντάφυλλου (gr) 6 Βάροςτριαντάφυλλου (gr) C Lenny D 45

4 of total) of total)

30

ht (% 2

15 ight (% Weig We

0 0 20 35 50 65 20 35 50 65 Βάροςτριαντάφυλλου (gr) Βάροςτριαντάφυλλου (gr) E Bordeaux 6 F 45

4 of total) of total)

30 ht (% ht (% 15 2 Weig Weig

0 0 20 35 50 65 20 35 50 65 Cut flower weight (g) Cut flower weight (g)

Fig. 9 Fresh weight distribution in the four individual parts of the cut n = 20). The fgures b, d, and f (on the right) represent the same fower [stem (flled circle), fower bud (red flled diamond), leaves data as the fgures a, c, and e (on the left), using a diferent scale. (purple flled triangle), and peduncle (green flled square)] as a func- Error bars indicate SEM. When the SE bars are not visible, the SE is tion of cut fower weight in three cut rose cultivars (Experiment 2; smaller than the symbol water loss mainly originate in the leaves. Cultivars with a and wrote the manuscript. GT and ARN assisted the data better ability to control water loss have a longer vase life analysis and interpretation. DB, C-OO, and EJW partici- under conditions that stimulate transpiration. Instead, the pated in data interpretation and supervised the study. All ability to control water loss is not related to vase life under authors contributed in writing, editing, proof-reading, and conditions that impede transpiration. It is consequently approving the fnal manuscript. beyond doubt that cultivar development will beneft from incorporating the low leaf weight loss trait. Cultivar diferences in cut fower water loss are not only Acknowledgements This work was funded by the Project “Research & Technology Development Innovation Projects”-AgroETAK, MIS related to stomatal characteristics, but also to mass allo- 453350, in the framework of the Operational Program ‘Human cation to the leaves, a cultivar-dependent variable. There- Resources Development’, through a post-doctoral grant to DF. The fore, considering mass allocation to the leaves will greatly Project is co-funded by the European Social Fund through the National enhance the quality of phenotypic data in vase life studies, Strategic Reference Framework (Research Funding Program 2007– 2013), and is coordinated by the Hellenic Agricultural Organization– and thus will not only simplify addressing one’s research DEMETER. The valuable comments of four anonymous reviewers question, but will also facilitate comparisons across experi- are greatly acknowledged. The authors also wish to thank Dr. Roland ments and laboratories. Pieruschka for critically reviewing the manuscript.

Author contribution statement DF performed the experi- mental work, carried out the data analysis and interpretation,

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