Petal blackening and lack of bud opening in cut lotus flowers (Nelumbo nucifera): Role of adverse water relations

Postharvest Biology and Technology 79 (2013) 32–38

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Petal blackening and lack of bud opening in cut lotus flowers (Nelumbo nucifera): Role of adverse water relations Wachiraya Imsabai a,b , Preeyapon Leethiti a,b , Petcharat Netlak a,b , Wouter G. van Doorn c,∗ a b c

Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA

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Article history: Received 28 September 2012 Received in revised form 17 December 2012 Accepted 20 December 2012 Keywords: Nelumbo nucifera Blackening Flower opening Latex Laticifers Petals Water relations

a b s t r a c t Lotus flowers (Nelumbo nucifera Gaertn.) are commercially sold as closed buds. When placed in water the buds fail to open and the outer petals show rapid blackening. We investigated whether this is due to adverse water relations. Placing a plastic bag over the flower head delayed petal blackening, indicating that it was induced by early water stress. This treatment did not result in bud opening. A rapid occlusion of the stem xylem was found. Four possible causes of this occlusion were investigated:air uptake into the xylem, microorganisms in the vase solution, a plant-induced effect, and exuded latex. Preventing the uptake of air into the stem ends did not affect water uptake. Inclusion in the vase water of antibacterial compounds, or antioxidants that inhibit the plant-induced xylem blockage in other species, similarly did not alleviate the xylem occlusion. Cut stems exuded copious latex, close to the opened xylem conduits. Latex exudation was prevented by cutting under water, allow the latex to flow out, and cut again in air, within 1 cm from the previous cut. This treatment did not promote water uptake of the cut stems. A pulse treatment with citric acid also reduced latex flow, but also did not prevent the decrease in water uptake. Treatment with ethephon or GA3 delayed the xylem occlusion, which suggests that it is induced by the plant itself. Only GA3 delayed petal blackening. None of these treatments promoted flower opening. It is concluded that adverse water relations are a cause of early petal blackening in cut lotus, but is not a cause of the lack of bud opening. The adverse water relations are apparently due to a plant-induced xylem occlusion which is different from those studied thus far in other species. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Lotus plants (Nelumbo nucifera spp. nucifera) are mainly found in Asia. The leaves and the flower stalk emerge from a tuberous rhizome that grows in the sediment of a body of water, usually 0.5–1.0 m deep. The flowers are single and develop at the apex of an erect floral stalk (peduncle). The peduncle has become lignified by the time of bud opening (Mosely and Uhl, 1985; Hayes et al., 2000). In Thailand one of the two main commercial cultivars has several green outer petals and many white inner petals. It is locally called cv. Saddabutra, and is probably identical to cv. Album Plenum. If left uncut, the full-grown floral bud opens in the morning and closes at night, for a few consecutive days. In uncut flowers the time between bud opening and the end of floral life-span is usually about 4–5 days. In intact plants the end of floral life is determined by petal abscission. Some signs of petal blackening can sometimes be observed by the time of petal abscission.

∗ Corresponding author. E-mail address: [email protected] (W.G. van Doorn). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.12.017

Floral buds are harvested just before they would open (mature bud stage) and are transported dry and subsequently placed in the temple by the religious devotee. When the cut stems are placed in water the buds do not open and the outer petals show early blackening. We investigated whether opening could be promoted and whether the petal blackening symptoms could be delayed. Imsabai et al. (2010) reported that treatment with 1-methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, reduced the rate of bud ethylene production and delayed the onset of petal blackening by about 2 days. 1-MCP did not promote the opening of the floral bud. These data suggested that ethylene is a cause of petal blackening but not of the lack of bud opening. Additionally, Imsabai and van Doorn (2013) reported that cytokinins and gibberellic acid (GA3 ) delayed petal blackening but had no effect on bud opening. Here, we tested the hypotheses that the lack of bud opening and the early petal blackening in lotus flowers are due to adverse water relations. Many species of cut flowers, even with their stems placed in vase water, rapidly exhibit serious water stress. This is due to a xylem occlusion in the stem. Depending on the species, the xylem occlusion can be due to factors such as bacteria in the vase water, a plant-induced effect, and the presence of air bubbles in the xylem

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conduits (van Doorn, 1997, 2012). Furthermore, the stems of lotus flowers exude copious latex after cutting. The latex flows into the vase solutions and might be taken up with the vase water and then block the xylem. We tested if lotus flowers showed such a xylem occlusion, and if so, whether it caused early petal blackening and lack of flower opening. It was observed that petal blackening was delayed by decreasing water stress and that a hitherto not described type of xylem occlusion induced this water stress. 2. Materials and methods 2.1. Plants Lotus flower buds (Nelumbo nucifera Gaertn., cv. Saddhabutra, likely the same as cv. Album Plenum) were harvested in the morning. The buds were picked at their normal commercial stage, i.e. with the floral buds still fully closed but about to open. Workers walked in the water of the lotus pond, which stood about 1 m deep, or collected stems by boat. Stems were broken under water, close to their junction with the rhizome. Directly after harvest, the stems were held dry or were placed in purified water (tap water, after passing through reverse osmosis equipment). Stem length at harvest varied from 40 to 60 cm. Stems were brought to the laboratory within about 1 h of harvest. In the laboratory the stems were recut in air, to a length of 25 cm, and then placed individually in glass vials or glass graduated cylinders, containing purified water. The flower stalks were held in a temperature-controlled room at 25 ◦ C, 65–75% RH, and natural light supplemented with TL light (light from about 7 a.m. to 7 p.m., with total photon flux density about 15 ␮mol m−2 s−1 ). In some tests, the flower stalks with buds were brought to the laboratory as described, and were then held dry at 20 ◦ C for 24 h, after which the stems were recut in air to a length of 25 cm, removing about 30 cm. This was compared with the same treatment without the period of dry storage. 2.2. Petal blackening and flower opening During vase life, petal blackening was assessed visually, at the end of the morning. The length of vase life was defined as the period until half of the visible petals showed black patches. Flower opening was determined visually. The flower was defined to open if the petals left an opening at their tips. 2.3. Chemicals Treatment with citric acid has been described as a means to stop latex flow from cut stems (van Doorn, 1997). Citric acid (Sigma–Aldrich) at 150 mg L−1 was used as a pulse treatment, just before vase life, or was continuously included in the vase water during vase life. The following antibacterial chemicals were included in the vase water at the onset of vase life and were not renewed: hydroxyquinoline sulphate (HQS; from Sigma–Aldrich, at 100, 200, 300, or 400 mg L−1 ), sodium dichloroisocyanuric acid (DICA; from BDH, at 10, 20, 30, 40, or 50 mg L−1 ), or silver nitrate (Sigma–Aldrich, at 25, 50, 75, or 100 mg L−1 ). In other tests, some chemicals were used that are known to prevent cutting-induced xylem occlusion in the stems of cut flowers of other species (van Doorn and Cruz, 2000; Vaslier and van Doorn, 2003; Loubaud and van Doorn, 2004). Several of these chemicals were included in the vase water at the onset of vase life, thus were tested as a continuous treatment. These chemicals were n-propylgallate (1, 2, and 5 mM), phloroglucinol (0.1 and 0.5 mM), n-nitrophenol (1 mM), p-nitrophenol (4-nitrophenol, 1, 2, and 5 mM), and phenylhydrazine (0.1 and 0.5 mM). Tropolone was tested continuously at 0.25 and 0.50 mM. 4-Hexylresorcinol,

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included in purified water at 2 and 10 mM, was given as a 5 h pulse treatment, prior to placement of the stems in purified water. In other tests it was applied continuously at 4, 40 and 400 ␮M. These chemicals were obtained from the same sources as mentioned by van Doorn and Vaslier (2002). S-carvone, the main etherical oil from caraway seed, was tested continuously at 32 and 64 ␮M. It was obtained from Sigma–Aldrich. Gibberellic acid (GA3 ; PhytoTechnology Laboratories, Shawnee Mission, KS, USA) was applied at 0.12 mM. The cytokinins benzyladenine (BA; Sigma–Aldrich) and thidiazuron (TDZ; PhytoTechnology Laboratories, Shawnee Mission, KS, USA) were tested at 0.1 mM and at 2.5 ␮M, respectively. Ethephon (ProCrop Co. Ltd., Thailand), which releases ethylene, was applied at 200 mg L−1 . This treatment reduced vase solution pH to 2.9. Controls for pH 2.9 were prepared using 1 M HCl, or HEPES buffer (Sigma–Aldrich), whereby the pH was adjusted with 1 M HCl. HEPES buffers were also applied at pH 3, 4, 5, 6, and 7. These chemicals were included in the vase solution at the onset of vase life and were not replenished. Finally, the surfactants Tween-20 and Agral-LN were tested at 0.01, 0.1 and 1.0 mL L−1 . 2.4. Water uptake, transpiration, water balance, and fresh weight The rate of water uptake was measured by placement of stems in graduated cylinders and daily measuring of the solution level. The rate of transpiration was assessed by weighing. The water balance (the difference between the rate of water uptake and transpiration) was calculated from the uptake and transpiration data. Flower fresh weight (FW) was determined by weighing. Results were expressed as the percentage of initial FW. 2.5. Covering with plastic bags Polypropylene (PP) bags, 12.7 cm × 15.2 cm, were used to cover the flower heads. The open end of the bag was wrapped around the stem but was not tied to the stem. Four or eight small holes (diameter about 0.55 mm) were made by using a needle. 2.6. Stem anatomy and latex flow Stems were cut and the exudation of latex was observed, using a binocular microscope magnifying 10 times. Stem anatomy was studied by cutting thin slices by hand and observation under a microscope. Latex flow was determined by removal of the exuded latex and weighing. 2.7. Preventing latex flow and air uptake When stems were cut at 1 cm or more from the original cut surface, new latex was observed to flow out. However, when re-cutting the stems in air at 0.5 cm or less from the initial cut, no new latex flowed out. This difference was used to test whether exudation of latex is a cause of early petal blackening. The stems were again cut in air, removing 0.5 cm (no new latex flow), and stems were immediate placed in water. This was compared with recutting the stems at 2 cm from the original cut surface (resulting in latex flow into the water) and immediate placement in water. Another method to reduce latex flow involved placement of freshly cut stems in an aqueous citric acid solution at 150 mg L−1 (pH about 3.3). We prevented any air from entering the stem end by recutting the harvested stems under water, gently shaking the ends for 3 min to allow the exuded latex to flow off. The stem end was then placed in a cup, under water. Stems were brought to a bucket of water while inside this cup. The stems were transported to the laboratory in this bucket with water. In the laboratory a graduated cylinder was held under water in the bucket and the stem was placed (under

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water) into this cylinder, which then served as the vial in which the flower stood. This treatment was compared with one which was identical, except that in the laboratory the stems were taken out of the bucket and placed on the laboratory table of 1 h (at 25 ◦ C, and about 60% RH). Alternatively, the stems were harvested normally, i.e. they were taken out of the pond water and held dry for about 15–20 min before placement in water. When in the laboratory, these stems were again taken out of water but immediately placed in the vase.

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All experiments used 8–10 flowers (replications) per treatment. Where possible, the means between treatments were compared after calculating LSD. All experiments were repeated at least once at a later date, with very similar results.

Blackening was initially observed at the tips of the green petals that enclose the unopened buds. When placed in vase water for 1 or 2 days, almost all the green visible petals that enclose the bud had black spots, while the remainder of the petal surface had become dark gray. The length of vase life, defined as the time until half of the visible petals showed black patches, was 3–5 days. In most experiments the rate of water uptake was relatively high during the first day of vase life, but rapidly dropped to low levels. A typical example is given in Fig. 1. On days 0–1 the rate of transpiration was much lower than the rate of water uptake, indicating that this high initial water uptake is compensating water loss during the previous period. The rate of transpiration was about the same throughout vase life (Fig. 1). The water balance (transpiration minus water uptake) usually became negative before day 2 of vase life (Fig. 1). The fresh weight (FW) of the buds did not decrease on days 0–1 of vase life (Fig. 1), suggesting absence of severe water stress during this period. The FW started to decrease from day 1 of vase life,

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Time (d) Fig. 2. Effect of placing a plastic bag over the floral bud on cut stems of the sacred lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). (A) Rate of water uptake, (B) rate of transpiration, and (C) water balance. Data refer to controls (), floral buds covered with plastic (polypropylene) bags PP bag with no holes (), or covered with bags with 8 holes having a diameter of about 0.55 mm each (). Data on day 1 in the graph refer to days 0–1, etc. Data are means (n = 10) ± SD.

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coinciding with the time the water balance had became negative. FW often reached about 80% of initial by day 5 (Fig. 1), but in other experiments the FW showed a smaller decrease. 3.2. Placing a plastic bag over the floral buds Placement of a polypropylene (PP) bag over the bud reduced both the rate of transpiration and the rate of water uptake (Fig. 2). However, this treatment had no effect on the water balance (Fig. 2). The presence of the polypropylene bag delayed petal blackening (Table 1).

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Time (d) Fig. 1. Typical water relations of cut floral buds of lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). Data refer to stems with a large apical floral bud that were placed in vase water shortly after harvest. Symbols are the rate of water uptake (), the rate of transpiration (), water balance (), and fresh weight (䊉). Data on day 0 in the graph refer to 2–3 h of the experiment, data on day 1 to 3–24 h, data on day 2 to days 1–2, etc. Data are means (n = 10) ± SD.

3.3. Antibacterial compounds and surfactants Three antibacterial compounds (HQC, DICA, and silver nitrate) were tested at a range of concentrations (see Section 2). These treatments had no effect on the rate of water uptake or the rate of transpiration. The treatments also did not affect the time to petal blackening (data not shown).

W. Imsabai et al. / Postharvest Biology and Technology 79 (2013) 32–38 Table 1 Effect of a plastic cover over the flower bud on petal blackening in the sacred lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). The bags were not tightly closed at the peduncle subtending the floral bud. Additionally, holes were made with a needle, diameter 0.55 mm. Blackening is expressed as the time until black spots are found on half of the visible petals. PP = polypropylene. Blackening (%) on day 4a

Time to 50% petals black (d)a

Not covered with a plastic (PP) bag PP bag, no holes PP bag, 8 holes

82.0a 47.0b 43.9b

3.8b 4.5a 4.4a

a Data are means of 10 replicate stems. Data with different letters, in each column, are significantly different (P ≤ 0.05).

Two surfactants (Tween-20 and Agral-LN) were continuously tested at three concentrations (0.01, 0.1 and 1.0 mL L−1 ). These treatments did not promote the rate of water uptake, and had no effect on the time to petal blackening (data not shown). 3.4. Chemicals that prevented a cutting-induced xylem occlusion in other cut flowers

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Time (d) In previous experiments with chrysanthemum flowers, antioxidative chemicals, and some inhibitors of catechol oxidase and peroxidase, prevented a cutting-induced reaction that led to xylem blockage. Some of these compounds (n-propylgallate, phloroglucinol, p-nitrophenol, phenylhydrazine, 4-hexylresorcinol and tropolone), used at concentrations used previously for continuous treatments (see Section 2), were tested. These treatments did not change the rates of water uptake and transpiration, or the time to petal blackening (data not shown). Additionally, 4hexylresorcinol, used as a 5 h pulse treatment at concentrations that were effective in other species (see Section 2), did not affect water uptake or petal blackening (data not shown). Carvone, tested continuously at 32 and 64 ␮M, also had no effect on the rate of water uptake or on petal blackening (data not shown). 3.5. Ethephon, gibberellic acid and cytokinins Ethephon was included in the vase solution at 200 mg L−1 . It resulted in an increase in water uptake but had no effect on transpiration (Table 2). Ethephon lowered the pH of the vase to 2.9. The effects of ethephon were apparently not due to this low pH, as controls for the low pH (HCl in water at pH 2.9, or a HEPES buffer at pH 3.0) did not affect water uptake or transpiration (Table 2). HEPES applied in the vase solution at a pH varying from 3 to 7 also showed no effect of lowering the pH on water uptake and transpiration (data not shown). Ethephon treatment induced early petal blackening (Table 2), whereas the other treatments did not affect blackening (Table 2 and data not shown).

Fig. 3. Effect of the inclusion of GA3 at 0.12 mM in the vase solution on the water uptake of cut floral buds of the sacred lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). The stems held in water () or in GA3 solution (䊉). Data on day 1 in the graph refer to days 0–1. Data are means (n = 10).

Experiments with GA3 , included in the vase water at 0.12 mM, were carried out seven times. This treatment invariably delayed the decrease in the rate of water uptake (Fig. 3) and invariably had no effect on the rate of transpiration (Table 2). In five of these experiments the treatment delayed the time to petal blackening by 0.6–1.5 days, whereas in two experiments it had no effect. These two experiments were carried out in the hot and rainy season, whereas all other experiments were in the dry and cooler season (Imsabai and van Doorn, 2013). Inclusion of 0.1 mM of the cytokinin BA in the vase water had no effect on the rate of transpiration or the rate of water uptake (Table 3). Yet, this treatment delayed the time to blackening by about 1.0 day (Table 2; Imsabai and van Doorn, 2013). TDZ at 2.5 ␮M also did not affect the rate of water uptake or the rate of transpiration (Table 2). TDZ delayed the time to petal blackening, usually by 1.5–2.0 days (Table 2; Imsabai and van Doorn, 2013). 3.6. Stem anatomy, laticifers, latex flow from cut stems and its prevention Thin transverse sections of the floral stalks (peduncles), observed using light microscopy, showed 6–7 large cavities, which

Table 2 Effects of hormone treatments on water uptake, transpiration, and petal blackening in cut flower buds of the sacred lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). Water uptake and transpiration rates refer to day 3 of vase life. Blackening is expressed as the time to the presence of black spots in half of the visible petals. Treatment Exp. 1 Control (deionised water) Ethephon 200 mg L−1 , pH 2.9 HCl in water, pH 2.9 HEPES buffer, pH 3.0 Exp. 2 Control (deionised water) GA3 , 0.12 mM Benzyladenine, 0.1 mM Thidiazuron, 2.5 ␮M a b

Water uptake rate (mL flower−1 d−1 )a

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Time to 50% petals black (d)a

3.9b 8.0a 4.1b 4.0b

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4.3b 5.6a 5.6a 6.3a

Data are means of 10 replicate stems. Data with different letters, in each column and experiment, are significantly different (P ≤ 0.05). See Fig. 3.

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Table 3 Effect of the uptake of air into the cut stem end on petal blackening in cut flower buds of the sacred lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). In one treatment no air entered the cut ends of the stems, as the stems were continuously held under water, in another air was allowed to enter for 1 h. Additionally, in a noair treatment the stems were recut under water twice, in order to prevent latex from flowing out (see also Table 3 This recutting occurred daily, starting from day 1. Blackening is expressed as the percentage of tepals that show black spots on day 4 and as the time until black spots are found on half of the visible petals. Treatments

Blackening (%) on day 4a

Time to 50% petals black (d)a

Control, stem ends exposed to air for 1 h Stem ends not exposed to air Stem ends not exposed to air, stems recut under water to prevent latex flow

34.2a

5.5a

34.9a 21.3a

5.4a 6.0a

a Data are means of 10 replicate stems. Data with different letters, in each column, are significantly different (P ≤ 0.05).

are separated by strands of parenchyma tissue. This gives the impression of a wheel having 6–7 ‘spokes’. A few vascular bundles are found in the tissue that form the ‘spokes’ whilst many vascular bundles are found in the central tissue (the ‘axis’ of the wheel) and in the large outer ring of tissue. Small cavities are sometimes found at the end of the spokes and in the middle of the central tissue. Upon cutting the stem, white latex flows out of laticifers, which are all in very close proximity to the phloem side of the vascular bundles. When the stems are cut, therefore, the exuded latex can readily flow into the xylem vessels and tracheids. Latex that flows into the vase water can also become taken up into the xylem, together with the uptake of vase water. Upon cutting of the stem in air and leaving it, exuded latex first shows as small drops of latex which then covers the whole cut surface, within 30 s of cutting. The amount of exuded latex depended on the distance from the flower bud. Cutting at 40 cm from the bud produced less than 0.5 mL of latex whilst cutting at 20 cm from the flower head resulted in about 1.5 mL. The latex showed no staining with ruthenium red, indicating that it does not contain high levels of polysaccharides, but stained with Folin–Ciocalteu reagents, which suggest that it contains phenols. The latex did not dissolve in water. If the stems are placed in water immediately after cutting, latex became suspended in the water, as fluffy white threads. When cut under water, the latex flow into the water ceased within about 3 min. When stems were cut again in air, after 3 min, the flow of latex depended on the position along the stem where this second cut was made. If cut at 1 cm or more from the original cut surface, new latex was observed to flow out. However, when re-cutting the stems in air at 0.2–0.5 cm from the initial cut, no new latex flowed out. We used this in an attempt to test whether latex exudation is a cause of xylem occlusion and early petal blackening. The stems were again cut in air, removing about 0.5 cm (no new latex flow), and stems were immediate placed in water. This was compared with recutting the stems at 2 cm from the original cut surface (resulting in latex flow into the water) and immediate placement in water. No difference was found in water uptake or transpiration. Also no difference petal blackening was observed (data not shown). When stems of 40 cm length were cut and placed in an aqueous citric acid solution at 150 mg L−1 (pH about 3.3), latex flow was considerably reduced but not completely prevented (controls exuded on average 0.5 mL of latex, n = 5; the treated stems 0.1 mL, n = 5). Citric acid treatments (150 mg L−1 ) were given as a 1, 2, 3, 6, 9 or 12 h pulse, or continuously. These treatments had no effect on the rate of water uptake and the rate of transpiration. It did also not affect petal blackening (results not shown).

3.7. Effect of air uptake by the stems We prevented any air from entering the stem as described in Section 2. The treatment without aspired air was compared to one in which the stems were treated such that no air could enter, but in the laboratory these stems were taken out of the water and placed dry on the laboratory table of 1 h (at 25 ◦ C, and about 60% RH), after which the stems were individually placed in water. There was no difference in water uptake between these two treatments (Fig. 4A). The two treatments showed the same rate of petal blackening (Table 3). In another type of experiment the absence of air in the stem was combined with re-cutting the stem twice under water, once at 4 cm from the previous cut surface, whereby the latex was allowed to flow out by gently shaking the stem end for 3 min in water, which was followed by a second recutting at 0.5 cm from the last cut surface. This procedure started on day 0.5 of vase life and was repeated on days 1, 2, 3, 4 and 5. The double recutting prevented the visible outflow of latex. Controls were treated as the controls in all other experiments (i.e. the stem ends absorbed air when taken out of the pond water, and possibly more when taken out of water and placement in the vase), or their stem ends were treated as described above (treated such that no air could enter, then placed in air for 1 h and subsequently placed in water). None of the treatments promoted water uptake (Fig. 4B). These treatments also had no effect on the time to petal blackening (data not shown). 3.8. Effects of the treatments on flower opening None of the treatments resulted in opening of the flower buds. 4. Discussion Adverse water relations seem at least one of the causes of early petal blackening in cut lotus flowers. This follows from the experiment in which a plastic bag was placed over the bud. The presence of the bag considerably reduced the rate of transpiration and the rate of water uptake and delayed petal blackening. The bag was open at the peduncle to allow some exchange of gas with the outside air, in order to prevent accumulation of ethylene or carbon dioxide inside the bags. The data therefore suggest that the main effect of the plastic bag was to reduce transpiration. In the controls the water balance (water uptake minus transpiration) became negative already by about day 1. Also the rate of water uptake drastically decreased from days 0 to 1. This shows rapid development of an occlusion in the xylem, which inhibits water uptake. The presence of the bag did not change the time to the negative water balance, thus did not prevent the xylem occlusion. We tested several possible causes of the xylem occlusion in lotus. The inclusion of three antibacterial compounds in the vase solution of lotus flowers, at various concentrations, had no effect. Additionally, in lotus the water uptake was low already by day 1, a time when the number of bacteria in water in other cut flowers is generally still too low to cause a xylem occlusion (van Doorn, 1997). In some flowers a wounding-induced xylem occlusion in the stem ends has been found (Vaslier and van Doorn, 2003; Loubaud and van Doorn, 2004; He et al., 2006; C¸elikel et al., 2011). This occlusion was prevented by antioxidant treatments and by inhibitors of peroxidase or polyphenol oxidase (PPO). The catechol oxidase activity of PPO seemed partially responsible for the occlusion in these flowers (van Doorn and Cruz, 2000; van Doorn and Vaslier, 2002). However, treatment of lotus stems with antioxidant compounds was without effect. The tested phenylhydrazine is an inhibitor of both peroxidases and polyphenoloxidases, whilst

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Time (d) Fig. 4. Effect of preventing air uptake into the cut stem, and that of preventing latex from flowing out of the stems, on the rate of water uptake of cut floral buds of the sacred lotus (Nelumbo nucifera spp. nucifera var. Saddabutra). (A) Stems were treated such that either no air could enter the xylem at the cut surface (), or stems were exposed to air for 1 h at 20 ◦ C (). (B) No air could enter the xylem at the cut surface and the stems were recut under water such that no latex was flowing out of the stems. Data are controls that were cut as in all other experiments, thus the stem end was taken out of the pond, and these stems were not recut under water during vase life (䊉), or stems that were cut at harvest under water and were continuously held under water, thus did not aspire air into xylem conduits opened at the cut surface, whereby the stems were not recut during vase life to prevent latex blockage (). The open symbols refer to treatments in which the stems had not aspired air into xylem conduits opened at the cut surface (as the stem ends were always held under water). These stems were recut under water on days 0.5, 1, 2, 3, 4 and 5. Recutting occurred twice, once at 4 cm from the previous cut, after which the stem ends were gently shaken in water to release the latex, and then again at 0.5 cm from the previous cut, which did not result in latex flow. This treatment was given to stems indicated by (). Other stems were recut twice under flowing tap water (at the same time points) in an attempt to further improve the release of latex after cutting (ⵦ). Data on day 0.5 in the graph refer to days 0–0.5, data on day 1 to days 0.05–1.0, data on day 2 to days 1–2, etc. Data are means (n = 10) ± SD.

p-nitrophenol and 4-hexylresorcinol are inhibitors of polyphenoloxidases. Tropolone is a specific inhibitor of polyphenol oxidase (van Doorn and Vaslier, 2002). These inhibitors were tested at a range of concentrations, but had no effect. This indicated that the xylem occlusion in lotus stems is different from the one found in, for example, cut chrysanthemum flowers. S-carvone, a monoterpene from seeds such as caraway and dill, is an antimicrobial compound that also inhibits phenylalanine

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ammonia lyase (PAL) activity. It slightly extended the vase life of Hakea francisiana flowers (Williamson et al., 2002), Grevillea inflorescences (He et al., 2006a) and Baeckia frutescens flowers (Damunupola et al., 2010), but had no effect in some other species (C¸elikel et al., 2011). It also had no effect in lotus. The inclusion of surfactants in the vase solution often promotes water uptake in cut flowers. Surfactants seem to bypass a bacterial occlusion in the xylem, and also help refill the xylem conduits that have become filled with air (van Doorn, 1997). However, two surfactants tested did not promote water uptake in cut lotus flowers, and had no effect on blackening. The stems of lotus flowers exude latex upon cutting, which might be a possible cause of the xylem occlusion. Vascular bundles were present throughout cross-sections of the stems, at very regular positions with regard to the air lacunae and the stem exterior. Laticifers were present very close to the vascular bundle, next to the phloem. These data confirm those of Esau and Konsakai (1975) and Schneider and Carlquist (1996). Latex flowing out upon cutting rapidly covered the xylem conduits that had been opened by cutting, and entered these xylem conduits. When the stems are placed in vase water, copious latex becomes suspended in the water. This latex can be taken up together with the vase water. Latex might thus be a cause of the xylem occlusion. Two methods were used to reduce or prevent (at least visibly) latex flow. One was recutting at a short distance (0.5 cm) from the original cut. No new closed laticifers were apparently opened by this recutting, but it is very likely that the recutting opened some xylem conduits that were previously closed. The effect was compared with recutting at a larger distance from the original cut (2 cm), which again produced copious latex. The second method to reduce latex flow was a treatment with citric acid. Neither of these methods prevented the early drop in the rate of water uptake and the early onset of a negative water balance. These data might be interpreted in a few ways: (a) latex can bypass the pits in the pore membranes between xylem conduits, hence the conduits opened by the second cutting already contained latex and did therefore not become available for water transport, (b) the methods used to reduce the second latex flux were not fully effective, thus did not prevent latex from blocking the xylem, and (c) there is a xylem blockage other than due to latex. The water uptake data might be interpreted against latex being a main cause of the xylem blockage. If latex were the cause we would expect a very low rate of water uptake from the very first day of vase life. In contrast, we found that the rate of water uptake was still relatively high by 2–3 h after cutting (Fig. 1) and by 12 h after cutting (Fig. 4B), and only rapidly declined thereafter. The uptake of air is a cause of xylem occlusion in a few flowers, and combined with other causes of the xylem occlusion the uptake of air was found to add to the occlusion (van Doorn, 1997, 2012). However, preventing any air from entering the cut lotus stems did not promote water uptake. This suggests that the occlusion is mainly due to factors other than air uptake. Interestingly, the xylem occlusion in lotus flowers was inhibited both by ethephon and by GA3 . These chemicals have not been reported to inhibit a xylem occlusion in other cut flowers. The effect of ethephon was found not to be due to its low pH, so seems due to ethylene. This is remarkable, as in cut lilac flowers the opposite has been demonstrated: a xylem occlusion was induced by ethylene (van Doorn et al., 1991). Ethephon hastened petal blackening in lotus, which confirmed our previous report that this blackening was hastened by endogenous ethylene (Imsabai et al., 2010). In contrast to ethephon, GA3 resulted in a delay of visible petal blackening (Imsabai and van Doorn, 2013). GA3 prevented the decrease in the rate of water uptake in all seven experiments carried out, thus it delayed the xylem occlusion. The finding that ethephon and GA3 delayed the occlusion suggests that it is not due to bacteria, air, or latex, but to a plant-induced phenomenon.

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It was also reported previously that BA or thidiazuron (TDZ) delayed lotus petal blackening (Imsabai and van Doorn, 2013). We now found that this was not accompanied by higher water uptake rates. These data indicate that petal blackening, which is at least in part due to early adverse water relations, can be delayed with and without improvement of the water relations. Browning reactions in plants, such as those responsible for the color change of cut apples left in air, are often mediated by catechol oxidase (Koval et al., 2006; Mayer, 2006). Petal blackening might therefore be directly affected by inhibitors of catechol oxidase, apart from effects on water uptake. However, tropolone, a specific inhibitor of the enzyme, and other less specific inhibitors, did not delay petal blackening in lotus, suggesting that catechol oxidase might not be involved. As none of the treatments promoted the opening of the closed buds, it seems that the problem of lack of bud opening is not related to adverse water relations, or at least is not due to adverse water relations alone. It is concluded that the very early petal blackening in cut lotus flowers, compared with flowers left attached to the plant, is at least partially due to the rapid development of a xylem occlusion in the stem xylem. This occlusion is different from any other species tested thus far, as it was not alleviated by preventing air uptake, by treatment with antimicrobial compounds, after treatment with compounds that prevent a wounding-induced xylem occlusion in some other cut flowers, or after treatment with surfactants. The lotus stem xylem occlusion was inhibited by ethephon and by GA3 . These compounds are not known to prevent a xylem occlusion in any other species. The xylem occlusion in lotus flowers therefore seems plant-induced, but it is quite different from the ones that are known to date. It is not known how this occlusion is induced. As in some other types of plant-induced xylem occlusion it might be due to cutting of the stem, hence to wounding. The data suggest that a type of xylem occlusion is to be added to the one induced by microorganisms, and to the two types of occlusion that are known to be induced by the plant itself. One of these is regulated by ethylene, the other is not but is apparently under the control of catechol oxidase and peroxidase. Aspiration of air can add to these three types of occlusions, and in some rare chrysanthemum cultivars is adequate to cause an occlusion (van Doorn, 2012). Additionally, it has been suggested that in some species a xylem occlusion can be due to mucilage exuding from the cut stem surface (van Doorn, 1997). The present occlusion, therefore, is the fourth known type.

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