Reactive Oxygen Species and Alternative Respiration ... - Springer Link

J Plant Growth Regul (2013) 32:83–91 DOI 10.1007/s00344-012-9278-4

Reactive Oxygen Species and Alternative Respiration in the Developing Flowers of Two Subtropical Woody Plants Nan Liu • Zhifang Lin

Received: 5 February 2012 / Accepted: 23 April 2012 / Published online: 17 June 2012 Springer Science+Business Media, LLC 2012

Abstract Petals, sepals, pistils, and stamens from flowers of two landscape tree species (Elaeocarpus hainanensis Oliv and Michelia alba DC) were analyzed to determine the changes in levels of reactive oxygen species (ROS), lipoxygenase (LOX) activity, and alternative pathway respiration throughout flower development. Histochemical and quantitative analyses revealed that ROS levels differed among tissues and stages of flower development. ROS levels were high in all flower tissues early in development and then declined to low levels late in development. ROS levels were highest in the stamen. In contrast, LOX activity, heat energy evolution, and the percentage of total respiration represented by alternative respiration in M. alba petals increased with flower development. The results suggest that ROS are involved in the growth of various flower tissues at early developmental stages and not only located on the tip site of growing tissues. The reduction in ROS generation later in development is probably due to the significant elevation in alternative respiration. The increase in LOX activity and heat energy evolution might contribute to the formation and release of some aromatic compounds and to flower opening. LOX, however, might not mediate ROS generation in the petal during development. Keywords Alternative respiration pathway Landscape plant Lipoxygenase Ontogenesis Reproduction

N. Liu Z. Lin (&) Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, 723 Xingke Road, Tianhe District, Guangzhou 510650, Guangdong, China e-mail: [email protected]

Introduction Reactive oxygen species (ROS) are primarily known as toxic by-products of various redox processes in plants. In recent years, however, researchers have recognized that ROS have both harmful and beneficial functions. There is increasing evidence that by acting as secondary messengers in signal transduction pathways and as cell-wall-loosening agents, ROS could control many key metabolic processes, including growth, development, overall metabolism, and stress responses (Schopfer and others 2001; Schopher and Liszkay 2006; Kovaćˇik and others 2009, 2010). Numerous reports have documented that ROS mediate seed germination and seedling and leaf growth (Rodrı´guez and others 2002; Kranner and others 2010), hypocotyl and coleoptile growth (Frahry and Schopfer 2001; Pereyra and others 2010), root and root hair growth (Dunand and Penel 2007), pollen tube growth (Potocky and others 2007), and seed dormancy (Oracz and others 2007). Until now, most of these investigations have concentrated on how ROS affect the growth and development of vegetative organs of model species, and much less attention has been paid to the role of ROS in the development of reproductive organs. McInnis and others (2006) found that flowers from 20 species of angiosperms accumulated ROS/H2O2 in their stigmas, particularly in the stigmatic papillae. The high amount of ROS/H2O2 in the stigmatic papillae of Senecio squalidus and Arabidopsis thaliana was then reduced as pollen grains adhered. Because nitric oxide (NO) can reduce the amount of ROS/H2O2 in stigmas, McInnis and others (2006) suggested that stigmatic ROS/H2O2 might be involved in the signaling network of the pollen–stigma interaction and in the defense against attack by pathogens. Hiscock and others (2007) demonstrated that accumulation of ROS/H2O2 in stigmas is probably a general feature of

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angiosperms and they proposed a potential new signaling role for ROS/H2O2 and NO in pollen–stigma recognition processes. Zafra and others (2010) found that ROS accumulation on the surfaces of olive stigmas and anthers was stage- and tissue-specific. When the stigma became receptive to pollen late in the development of olive flowers, an intense interaction between the pollen and pistil occurred, during which ROS were downregulated in stigmas and NO was actively produced in pollen grains and tubes. These results concern mainly H2O2 levels and the signaling role of ROS and NO in the interaction between stigma and pollen at the late developmental stage of flowers. There is still limited information about the role of ROS in different flower tissues during all stages of flower development. In nonphotosynthetic tissues, ROS may originate from the mitochondrial respiratory chain or through the action of enzymes such as NADPH oxidases and peroxidases (Bailly 2004). Lipoxygenase (LOX) also can generate O–• 2 via oxidation of unsaturated fatty acid and pyridine nucleotides (Roy and others 1994). The alternative respiration has been proposed to lower mitochondrial ROS formation (Maxwell and others 1999). This article describes changes in ROS levels in developing flowers of Elaeocarpus hainanensis Oliv and Michelia alba DC. These trees are important urban landscape and garden species in South China and other tropical Asian areas. In addition, the flower of M. alba is an important source of oils used in perfumes. The purpose of our study is to answer two questions: (1) How do levels of ROS (•OH, O–• 2 , and H2O2) change with the growth and development of various flower tissues? (2) How do LOX activity and the fraction of alternative respiration in petals change during the late stages of flower development? Answering these questions will provide initial insight into how ROS might affect the development of plant reproductive organs.

Materials and Methods Plant Materials Trees of E. hainanensis and M. alba were grown on the campus of the South China Botanical Garden, Guangzhou, China. Flowers were sampled in May and June 2010 and classified into developmental stages as described below. Flower development for E. hainanensis was divided into seven stages, from small green buds to fully opened (Fig. 1a). The lengths of green buds at stage 1 through stage 5 were 10, 14, 18, 21, and 25 mm, respectively; at stage 6, the sepals were completely dehiscent; and at stage 7, the flower was just fully opened.

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J Plant Growth Regul (2013) 32:83–91

Fig. 1 Changing patterns of superoxide radical (O–• 2 ) generation in different flower tissues of E. hainanensis at seven developmental stages as indicated by staining with NBT. a Seven stages of E. hainanensis flower development. b O–• 2 levels in the petal during –• flower development. c Reduction of O–• 2 level in the petal by the O2 –• scavenger tiron. d Enhancement of O2 level in the petal in the presence of NADH. e O–• 2 level in the sepal during flower develop–• ment. f O–• 2 level in the pistil during flower development. g O2 level in the stamen during flower development

Flower development for M. alba was divided into five stages (Fig. 3a): stage 1 (green bud 12 mm long), stage 2 (green bud 18 mm long), stage 3 (green bud 23 mm long), stage 4 (white bud 32 mm long; petals were slightly dehiscent), and stage 5 (recently opened flower with white petals separated). Thermogenesis Measurement Heat produced by flowers was measured as an indicator of activity in the alternative respiration pathway (Ordentlich and others 1991) and temperature increase. Because heat evolution in an intact flower sample would provide a more accurate assessment in vivo than the detached tissue sample, in this study temperature was measured based on the flower;


this is a revision of the method used by other studies. For the temperature measurement, the flowers of M. alba were collected at three late developmental stages: the green calyx stage (25 mm long 9 6 mm wide), the white calyx and slightly dehiscent stage (29 mm long 9 7 mm wide), and the recently opened flower stage (34 long 9 8 mm wide). The flowers were immediately put in a sealed container (150 ml) with a thermograph inserted and the container was kept at room temperature (28.6–28.9 C). The air temperature inside the container was measured at 0 h and after 1 and 2 h. Each container had eight flowers (both green calyx and white calyx stages) or three flowers (opened flower stage). Heat energy evolution in joules per flower was calculated using a heat formula (Katz and others 2006; Physics Tutorials 2012, www.PhysicsTutorials.org): Q ¼ m Cp DT where Q is heat energy (in joules), m is air mass (mg) in a 150 ml container, 4T is the change in temperature (C), and Cp is specific heat capacity (in J/g C), which for air is 1.01.

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Histochemical Localization of ROS Five flowers at seven developmental stages for E. hainanensis and at five developmental stages for M. alba were separated into petals, sepals, pistils, and stamens. The localization of the superoxide radical (O–• 2 ) and H2O2 in the tissues was determined using the stain NBT (nitroblue tetrazolium) for O–• 2 and DAB (dimethyl azobenzene) for H2O2 as described previously (Lin and others 2009, 2011). Specific staining for O–• 2 by NBT was confirmed by addition of 20 mM tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid), which is an O–• 2 scavenger, and specific staining for H2O2 by DAB was confirmed by addition of 10 mM ascorbic acid (AsA), which is a H2O2 scavenger. After the stained tissues were photographed, the tissues stained with NBT were retained for future quantitative determination of O–• 2 . ROS Quantification ROS were quantified in the petals, pistils, and anthers of M. alba flowers at five developmental stages. Flower tissues were separated immediately and stored in liquid nitrogen before use.

Respiration Measurement H2O2 determination Five outermost petals sampled from five flowers of M. alba at five developmental stages were cut into small pieces. This petal sample was first kept in phosphate buffer (pH 7.0) for 30 min to eliminate wound respiration. Oxygen uptake was then polarographically determined using a Clark-type oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH, USA) in a reaction chamber containing 3 ml of phosphate buffer at 25 C in the dark with or without KCN (1 mM). The use of KCN, which is a cytochrome pathway inhibitor (Kumar and Sinha 1994), enabled the measurement of respiration flux through the alternative pathway. The measurements were repeated three times. The fraction of total respiration flux through the alternative pathway was calculated as the percentage of alternative pathway to total respiration.

LOX Activity Determination Petals of M. alba flowers were homogenized in chilled phosphate buffer (pH 7.0). After centrifugation of the homogenate at 10,0009g for 10 min at 4 C, LOX activity in the supernatant was assayed mainly as described by Fischer and others (1999). The 3 ml reaction mixture contained 2 mM linoleic acid, 0.1 % Tween-20, 0.2 M phosphate buffer (pH 6.5), and 0.2 ml of the petal extract. The oxygen consumption rate was measured with an oxygen electrode as described for measurement of respiration.

A 120–150 mg quantity of each petal, pistil, and anther sample per stage was homogenized in chilled phosphate buffer (50 mM, pH 7.0). The extract obtained by centrifugation at 10,0009g for 10 min at 4 C was used to quantify both H2O2 and the hydroxyl radical (•OH). H2O2 production was determined fluorometrically following the fluorescence emission enhancement of homovanillic acid (a fluorescence probe) in the presence of horseradish peroxidase (POD) according to the methods reported by Romero-Puertas and others (2004). The 3 ml reaction mixtures contained 5 mM homovanillic acid, 50 mM Hepes buffer (pH 7.6), 200 ll of sample extract, and 50 units of POD. The reaction was started by adding POD, and the fluorescence intensity was detected with a fluorescence spectrophotometer (LS-55, PerkinElmer, Waltham, MA, USA) using excitation of 315 nm and emission of 440 nm. The relative level of H2O2 generation was expressed as Em440nm/g FW. O–• 2 determination For the quantification of O–• 2 , the NBT-stained sample with blue formazan precipitate was ground in a 2 M KOHDMSO (1:5 v/v) solution to obtain soluble formazan. The O–• 2 generation level was then read in the supernatant with a spectrophotometer (Lambda 650, PerkinElmer) at 630 nm (Kilinc and others 2009) and expressed as A630nm/g FW.

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OH determination

The tissue extract used for •OH quantification was the same extract used for H2O2 quantification. In the extracts from different parts of the flower, •OH generation was detected using terephthalic acid (TPA) as a dosimeter (Liu and others 2010). The product of the reaction between TPA and • OH (TPA–OH) becomes fluorescent, so the fluorescent signal was monitored with a fluorescence spectrophotometer (LS-55, PerkinElmer) at 450-nm emission by 326-nm excitation. •OH generation was expressed as relative fluorescence intensity (Em450nm/g FW).

Statistical Analyses Each assay was carried out using five flowers (for respiration, LOX activity, and ROS localization) or eight flowers (for ROS quantification) with three replicate measurements. Results are presented as mean ± SD. Data were subjected to one-way ANOVAs, and Tukey’s test was applied for multiple comparisons.


the anther in the early stages and was less evident in the late stages (Fig. 1g). Staining for H2O2 was similar to that for O–• 2 with respect to tissue location and the developmental stage of the E. hainanensis flower (Fig. 2). The dark-brown color of the DAB–H2O2 reaction product was evident in almost all parts of the petals, sepals, and anthers at stages 1–5 but was less evident at later stages (Fig. 2a, c, e). In the pistil, however, H2O2 was localized in the basal part of the style and the top of the ovary (Fig. 2d). H2O2 was not detected in the ovary or even in the pistil at stage 7, but H2O2 was abundant below the ovary at all the developmental stages (Fig. 2d). H2O2 was not detected in petals and stamens (Fig. 2b, f) when AsA (a scavenger of H2O2) was added, which confirmed that the staining detected H2O2 in the flower tissues in situ. ROS Localization and Quantification in Flower Tissues of M. alba During Differential Developmental Stages Histochemical localization of O–• 2 and H2O2 in the petals, pistils, and stamens at five developmental stages of M. alba

Results ROS Localization in E. hainanensis Flower Tissues at Different Developmental Stages As indicated by staining, the O–• 2 generation level was substantial in all tissues at the early stages of E. hainanensis flower development (Fig. 1b). The insoluble blue formazan, produced by the binding of NBT to O–• 2 , was evident in the petals at stages 1–3, and especially at early stage 1. The staining intensity declined as flower development continued and appeared only at the base of the petal at the middle or late developmental stages (Fig. 1b). The blue formazan staining of the petals was abolished by the addition of the O–• 2 scavenger tiron (see stages 3 and 4 in Fig. 1c) and enhanced by the addition of NADH (Fig. 1d), indicating that the blue color resulted from the –• staining of O–• 2 in the petals. Similar trends in O2 generation were observed in the tissues of sepals, pistils, and stamens during flower development. O–• 2 levels were high in stages 1–4 for sepals (Fig. 1e), stages 1–5 for pistils (Fig. 1f), and stages 1–4 for stamens (Fig. 1g). O–• 2 was present throughout the sepal tissue at stage 1, was absent from sepals from late stage 5 to stage 7, and was present mainly in the tips and bases of sepals at other stages (Fig. 1e). The ovary of the pistil had strong staining by blue formazan in stages 1–5, but only the ovary base was stained at later stages (Fig. 1f). In the stamen, O–• 2 was localized mainly in the anther; it was present throughout

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Fig. 2 Changing patterns of H2O2 generation in different flower tissues of E. hainanensis at seven developmental stages as indicated by staining with DAB. a H2O2 generation level in the petal during flower development. b The same as panel a but in the presence of AsA, a H2O2 scavenger. H2O2 generation levels in the sepal (c), pistil (d), and stamen (e) during flower development. f The same as panel e, but staining intensity was reduced in the presence of AsA


flowers was conducted with NBT or DAB staining as described for E. hainanensis. Stamens, pistils, and petals at stage 1 had an intense blue coloration corresponding to the generation of O–• 2 (Fig. 3b). Staining became less intense in the petal and pistil as development continued. In the pistil, staining was evident mainly in the ovary and stigma. In the stamen, however, staining was not evident at stage 3 but was evident again at stages 4 and 5. The intensity of H2O2–DAB staining also depended on the tissue and stage of M. alba (Fig. 3c). H2O2 was evident throughout the small petal at stage 1 but occurred in only small spots on the larger petals at stages 2–5. Staining of the pistil for H2O2 was intense at stages 1–3 but became less intense with further development and was evident only in the stigma at the last stage. H2O2 staining in the anther was intense at stages 1 and 2 and at stage 5 but was faint at stage 4. ROS generation level in different tissues and at different stages of M. alba flower development was also determined by quantitative analysis of tissue extracts (Fig. 4). The fluorescent probe indicated that H2O2 in petals was most abundant at stage 1 and then declined as development continued (Fig. 4a). The stamen contained more H2O2 than the petal or pistil; its average fluorescent signal intensity across all five stages was 2.15 times and 1.92 times greater than that of the petal and pistil, respectively. The higher fluorescence in the stamen at stage 5 was consistent with the higher staining at stage 5 (Fig. 3c). In the pistil,

Fig. 3 O–• 2 and H2O2 generation in the petal, pistil, and stamen of M. alba flowers at five developmental stages as indicated by histochemical staining. a Five developmental stages of M. alba flowers. b O–• 2 generation (visualized by staining with NBT) in the stamen (left), pistil (middle), and petal (right). c H2O2 generation (visualized by staining with DAB) in the stamen (left), pistil (middle), and petal (right)

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however, fluorescence was highest at stages 4 and 5 and lowest at stage 1, which was inconsistent with the staining pattern in Fig. 3c. Figure 4b shows the results of the spectrophotometric determination of O–• 2 in M. alba petals, pistils, and stamens. In petals, the gradual reduction in O–• 2 with development was consistent with the staining data (Fig. 3b) and with the H2O2 data (Fig. 4a). Likewise, O–• 2 generation in the pistil was high at the early stages and then declined with further development (Fig. 4b). Like H2O2, O–• 2 was more abundant in the stamen than in the petal or pistil; the O–• 2 level in the stamen (averaged across five developmental stages) was 8.03 and 1.56 times higher than in the petal and pistil, respectively. The generation of •OH in the tissues of the M. alba flower at different developmental stages was detected with fluorescence intensity (Fig. 4c). •OH was more abundant in the stamen than in the pistil or petal. The changes in •OH levels in the pistil and stamen with flower development resembled the changes in H2O2 levels in the same tissues

• Fig. 4 Quantification of H2O2 (a), O–• 2 (b), and OH (c) in the petals (left), pistils (middle), and stamens (right) of M. alba flowers at five developmental stages. For each tissue type, values with different letters are significantly different (P \ 0.05)

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Respiration and Heat Evolution in the Petals of M. alba Flower During Different Developmental Stages Total respiration (Vt) and the fraction of total respiration attributed to the alternative respiration pathway (Valt) were assayed in the petals of M. alba flowers at five developmental stages (Table 1). Calculated on a fresh-weight basis in petals, Vt ranged from 77.48 to 186.19 lmol O2 g-1 FW h-1; Vt increased from stages 1 to 3 and then declined. In the presence of KCN, an inhibitor of the cytochrome respiration pathway (Cyt), the rate of respiration was significantly inhibited. Valt ranged from 31 to 48 % at stages 1–3 but increased to 73–78 % at stages 4–5. These results indicated that in the early stages, petals were actively respiring to provide energy for petal growth and that most of the respiration was through the Cyt pathway. At later stages, when the flower was about to open, Valt increased and became the dominant pathway. Because heat is a major product of Valt, heat produced by whole flowers at stages 3–5 was measured. Heat production during a 2-h period increased from stages 3 to 5 (Table 2), which was consistent with the higher percentage of Valt respiration at the same stages (Table 1). LOX Activity in the Petals of M. alba Flowers During Different Developmental Stages Lipoxygenase (LOX, EC.1.13.11.12) is a ubiquitous enzyme that catalyzes the dioxygenation of polyunsaturated fatty acids with a cis, cis-1,4-pentadiene structure to produce hydroperoxy fatty acids, that is, LOX can contribute to the production of O2-. Here, LOX activity in petals of M. alba flower was assayed at various developmental stages. LOX activity was low at stages 1–3, Table 1 Total respiration (Vt), alternative respiration (Valt), and the percentage of total respiration represented by alternative respiration in petals of M. alba flowers at five developmental stages Vt (lmol O2 g-1 FW h-1)

Valt (lmol O2 g-1 FW h-1)

Valt (%)

1

146.46 ± 14.14b

45.22 ± 9.12d

30.88 ± 6.23c

2

176.20 ± 18.59a

84.56 ± 15.84a

47.99 ± 0.98b

3

186.19 ± 15.20a

66.32 ± 4.92b

35.62 ± 2.64c

4

100.08 ± 10.82c

72.71 ± 6.24a

72.65 ± 1.76a

5

77.48 ± 14.67d

60.41 ± 4.80c

77.98 ± 7.56a

Stage

Values in a column followed by different letters are significantly different (P \ 0.05)

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Table 2 Temperature of air surrounding flowers (3 or 8 flowers were kept in a sealed, 150 ml container) and heat energy evolution by whole flowers of M. alba at stages 3–5 during a 2 h period Stage (No. flowers tested)

Temperature (C)

Heat energy released (mJ/flower)

0h

1h

2h

3 (8)

28.9

29.3

29.4

4 (8)

28.8

29.8

30.0

9.00

5 (3)

28.6

28.9

29.3

14.10

3.75

increased greatly at stage 4, and then declined to a low level (Fig. 5). Because LOX activity was high at stage 4, and stage 4 is the stage just before flower opening, LOX activity might be involved with petal expansion.

Discussion Although ROS are the by-products of metabolism in aerobic organisms, they also contribute to the regulation of plant growth and development (Gapper and Dolan 2006). In the present study, histochemical and quantitative analyses suggest that ROS are found in flower tissues and accompany flower development, which might involve the regulation of flower growth and development of both tree species, especially in the early developmental stages. The high levels of O–• 2 and H2O2 detected in the petals, sepals, pistils, and stamens of green flower buds indicated that these reproductive tissues were metabolically active during maturation. Previous studies showed high levels of O–• 2 and H2O2 in the vegetative tissue of plants undergoing rapid growth (Carol and Dolan 2006; Gapper and Dolan 2006). ROS generation in vegetative tissues was located mainly in the active growing tip site. However, ROS generation could occur in all parts of the flower and covered the whole surface of flower tissues in our study. ROS generation in LOX activity n mol O2 mg-1 protein min-1

(Fig. 4c vs. a). •OH was most abundant at stage 4 in the pistil and at stages 3 and 5 in the stamen. In petals, on the other hand, •OH was most abundant at stage 1 and decreased as development continued.

1000

a 800

600

400

200

0

c

1

d 2

c

3

b

4

5

Developmental stage

Fig. 5 LOX activity in petals of M. alba flowers at five developmental stages. Values with different letters are significantly different (P \ 0.05)


flowers differed within different tissues and development • stages. In M. alba petals, ROS (O–• 2 , H2O2, and OH) declined gradually as the flower developed from the small green bud stage to the fully opened stage. We infer that the high levels of ROS in the early developing stages of the petal might participate in expansion of petal cells. In pistils and stamens, however, ROS generation fluctuated and the amount detected increased or decreased irregularly during flower development. To be specific, in pistils, H2O2 and • OH levels were highest at stage 4 and lowest at stage 1, whereas the O–• 2 level was highest at stage 2 and lowest at stage 5. Interestingly, ROS levels in the stamen had two peaks during flower development, with H2O2 and •OH peaks at stages 3 and 5, but with the O–• 2 peak at stages 1 and 5. The reason for this ROS generation pattern is unclear but we infer that the rise of ROS in the pistil and stamen at the late stages might possibly be related to the development of pollen grains and the pollen–stigma interaction. As revealed before, NADH is the electron donor of both important ROS-generating enzymes, NAD(P)H oxidase and peroxidase (when acting in the NADH oxidation mode) (Foreman and others 2003; Passardi and others 2006). Therefore, the enhancement of O–• 2 –NBT staining intensity (O–• production) by adding NADH 2 revealed that O–• 2 generation in flower organs may be partially due to the activation of NAD(P)H oxidase/ peroxidase in cell membranes. The changing kinetics of three types of ROS in the petals during five developmental stages of M. alba flowers showed maximum levels of •OH and H2O2 in the pistil at stages 4 and 5, as well as high levels of O–• 2 and H2O2 at stage 4 in the stamen, indicating –• that O2 and H2O2 are the precursors of •OH generation in flower tissues. The activity of the alternative respiration pathway depends on developmental stage, tissue type, and physiological status (Ordentlich and others 1991). In lotus flowers, the alternative respiration pathway accounted for 55–75 % of the respiratory flux in thermogenic tissue and 43 % of total respiration in nonthermogenic tissue (Watling and others 2006). Moreover, by stabilizing the redox state of the mitochondrial ubiquinone pool and allowing the continued operation of the citric acid cycle, alternative respiration is thought to reduce mitochondrial ROS formation (Puris and Shewfelt 1993; Maxwell and others 1999). Suppression of alternate oxidase activity or inhibition of the cytochrome pathway resulted in an increase of ROS formation (Maxwell and others 1999). In the current study, alternative respiration represented a lower percentage of respiration and a higher ROS generation in petals at stage 1, as well as a higher percentage of respiration and a lower ROS generation in petals at stage 5. It is indicated that alternative respiration may be involved in the regulation of ROS generation in petals. We conclude that ROS

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reduction in petals at late developmental stages could be partially due to an increase in the flux of the alternative respiration pathway. Siegelman and others (1958) reported a marked rise in total respiration during the expansion of rose petals, whereas a respiratory drop followed the complete opening of camellia flowers (Bonner and Honda 1950). These results indicate that the respiration pattern in petals during flower development can differ among species. In our experiment, the 50 % drop in total respiration in petals at full expansion was in agreement with the results from camellia flowers (Bonner and Honda 1950). The alternative respiration pathway dissipates most of the chemical energy in its respiratory substrates as heat, and heat evolution is an indicator of increased activity in the alternative pathway (Ordentlich and others 1991). Kumar and Sinha (1994) found a very high correlation between temperature and alternative respiration activity in the top leaves of nonirrigated sorghum and pearl millet. Watling and others (2006) confirmed that the alternative pathway produced heat in sacred lotus flowers. In our study, during development stages 3–5 of M. alba flowers, the increased evolution of heat energy in petals along with the increase of alternative respiration also indicated that alternative respiration elevated flower temperature. Thermogenesis is thought to provide the optimum temperature for floral development or pollen-tube growth and for volatilization of compounds that attract pollinator insects (Seymour and Schulze-Motel 1996; Lampecht and others 2002). Compared to the thermogenic tissue (in which the temperature can increase more than 10 C), the increase in temperature of M. alba flowers at the opening stage is small but probably sufficient to benefit the opening of petals and the release of odors. Previous research has demonstrated that LOX activity changes significantly during different growth phases of olive callus cultures (Williams and others 2000). The highest LOX activity in the common bean was found in rapidly growing tissues; LOX mRNA was abundant in the growing region of hypocotyls and almost absent in the mature region (Porta and others 1999). High LOX activity has been observed in fully opened rose flowers (Sood and others 2006) and in the leaf chloroplasts of flowering Arabidopsis plants (Ban˜uelos and others 2008). In cut carnation flowers, LOX activity increased from day 0 to day 12 (Zhang and others 2007), and this increment appeared before the increasing electrolyte leakage from the membrane, whereas the fluidity of the microsome membrane in fully expanded flowers increased by 28 % over young flowers (Fobel and Lynch 1987). The pattern of LOX activity in the petals of M. alba flowers changed with development stages, which showed that LOX activity was low at stages 1–3 and then increased substantially at later stages. This changing pattern of LOX activity was similar

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to that described for rose flowers and cut carnation flowers but different from that described for the common bean. One of the functions of LOX is thought to be that it mediates the production of ROS (Lynch and Thomson 1984; Chamulitrat and others 1991; Siedow 1991; Roy and others 1994; Cho and others 2011). In the current study, however, the high LOX activity in the late stages of M. alba flower development was not accompanied by an increase of ROS in petals. As LOXs are widely distributed in plant organs and function in the regulation of plant growth and development, the formation of flavor and aroma, changes in membrane permeability, and so on (Feussner and Wasternack 2002; Sood and others 2006; Kumar and others 2008), we speculate that the high LOX activity in M. alba flowers might function in the formation of some aromatic molecules and in an increase in membrane permeability for the release of such molecules.

Conclusion • Overall, ROS (O–• 2 , H2O2, and OH) generation is detected in any sites of flower tissues (petals, sepals, pistils, and stamens) of the tested landscape tree species and the amount differs with tissue type and developmental stage. It is concluded that high ROS levels in the flower tissues at early developmental stages may contribute to cell expansion associated with flower growth. Meanwhile, low ROS levels and higher LOX activity in petals at late flower development stages may contribute to the formation of aromatic compounds for attracting pollinators. Moreover, the decreased ROS levels associated with the increase in alternative respiration at late flower development stages are accompanied by an increase in heat energy evolution (thermogenesis), which would contribute to petal opening. The present study provides a starting point for the investigation of ROS generation, LOX activity, and alternative respiration in various flower components during flower development. More extensive analyses are required to clarify how changes in ROS levels occur in flowers and how these changes affect flower tissue development.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31070305). The authors are grateful to the English editing work by Bruce Jaffee.

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