Crop Science and Horticulture Burst of reactive oxygen species (ROS) in pedicel mediated fruit abscission after carbohydrate supply was cut off in longan (Dimocarpus longan) Ziqin Yang, Xiumei Zhong, Yan Fan, Huicong Wang, Jianguo LI and Xuming Huang Journal Name: Frontiers in Plant Science ISSN: 1664-462X Article type: Original Research Article Received on: 17 Feb 2015 Accepted on: 06 May 2015 Provisional PDF published on: 06 May 2015 Frontiers website link: www.frontiersin.org Citation: Yang Z, Zhong X, Fan Y, Wang H, Li J and Huang X(2015) Burst of reactive oxygen species (ROS) in pedicel mediated fruit abscission after carbohydrate supply was cut off in longan (Dimocarpus longan). Front. Plant Sci. 6:360. doi:10.3389/fpls.2015.00360 Copyright statement: © 2015 Yang, Zhong, Fan, Wang, Li and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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Fully formatted PDF and full text (HTML) versions will be made available soon. 1 Burst of reactive oxygen species (ROS) in pedicel mediated 2 fruit abscission after carbohydrate supply was cut off in 3 longan (Dimocarpus longan) 4 Zi-Qin Yang1,2, Xiu-Mei Zhong1,3, Yan Fan3, Hui-Cong Wang1, Jian-Guo Li1, 5 Xu-Ming Huang1 6 7 1 Physiological Laboratory for South China Fruits, College of Horticulture, South 8 China Agricultural University, Guangzhou 510642, China 9 2 Tropical Crops Genetic Resource Institute, Chinese Academy of Tropical 10 Agricultural Science, Danzhou 571737, China 11 3 Dongguan Agricultural Research Center, Dongguan, Guangdong, China 12 13 14 15 Correspondence 16 Dr. Xu-Ming Huang 17 Physiological Lab for South China Fruits, College of Horticulture, South China 18 Agricultural University, Guangzhou 510642, China 19 [email protected] 20 21 22 The first two authors contributed equally to the work 23 24 Running title: Carbohydrate stress, H2O2 and abscission 25 26 1 27 Abstract 28 Cutting off carbohydrate supply to longan (Dimocarpus longan Lour.) fruit by 29 girdling and defoliation or by detachment induced 100% abscission within a few days. 30 We used these treatments to study the involvement of reactive oxygen species (ROS) 31 in fruit abscission. Girdling plus defoliation decreased sugar concentrations in the 32 fruit and pedicel and depleted starch grains in the chloroplasts in the cells of 33 abscission zone. Prior to the occurrence of intensive fruit abscission, there was a burst 34 in ROS in the pedicel, which peaked at 1 day after treatment (DAT), when H2O2 in 35 the abscission zone was found to be chiefly located along the plasma membrane (PM). 36 H2O2 was found exclusively in the cell walls 2 DAT, almost disappeared 3 DAT, and 37 reappeared in the mitochondria and cell walls 4 DAT. Signs of cell death such as 38 cytoplasm breakdown were apparent from 3 DAT. The burst of ROS coincided with a 39 sharp increase in the activity of PM-bound NADPH oxidase in the pedicel. At the 40 same time, activities of antioxidant enzymes including superoxide dismutase (SOD), 41 catalase and peroxidase (POD) were all increased by the treatment and maintained 42 higher than those in the control. Accompanying the reduction in H2O2 abundance, 43 there was a sharp decrease in PM-bound NADPH oxidase activity after 1 DAT in the -1 44 treated fruit. H2O2 scavenger dimethylthiourea (DMTU, 1 g L ) significantly inhibited 45 fruit abscission in detached fruit clusters and suppressed the increase in cellulase 46 activity in the abscission zone. These results suggest that fruit abscission induced by 47 carbohydrate stress is mediated by ROS. Roles of ROS in regulating fruit abscission 48 were discussed in relation to its subcellular distribution. 49 50 Key words: fruit abscission, carbohydrate stress, reactive oxygen species, plasma 51 membrane-bound NADPH oxidase, cellulase, longan. 52 53 2 54 Introduction 55 Carbohydrates serve as the “hard currency” in plants, representing the costs for 56 various biological functions including growth, maintenance and defense. Fruit are net 57 importers of carbohydrates from the tree reserves or leaf photosynthesis (Mehouachi 58 et al. 2000; Hieke et al. 2002; Iglesias et al. 2003). Fruit trees generally produce more 59 fruitlets than they can support to harvest, and fruit abscission is a normal 60 physiological event during fruit development due to a self-regulatory mechanism to 61 reduce fruit load (Bangerth 2000). However, under adverse conditions, such as 62 shading (Yuan and Huang 1988, Zhou et al. 2008, Li et al., 2013), high temperatures 63 (Atkinson et al. 2001; Gazit and Degani 2002), or abrupt temperature fluctuations 64 (Yang et al. 2010), this mechanism may cause excessive fruit abscission. According to 65 Lakso et al. (2006), environmental factors affect fruit abscission based on the 66 carbohydrate supply–demand balance, and higher carbohydrate availability reduces 67 sensitivities to abscission-inducing stresses or fruit-thinning chemicals. Mehouachi et 68 al. (1995) suggested the existence of a threshold carbohydrate concentration in citrus 69 below which fruit shedding was intensified. 70 71 There is limited evidence on how a shortage of carbohydrates initiates the activity 72 of the abscission zone leading to fruit shedding. Gomez-Cadenas et al. (2000) found 73 that defoliation increased the concentration of the ethylene precursor 74 1-aminocyclopropane-1-carboxylic acid (ACC) and abscisic acid (ABA). They 75 suggested that these hormones participate in the self-regulatory mechanism that 76 adjusts fruit load depending on the availability of carbohydrates. Iglesias et al. (2006) 77 observed high ethylene evolution in the fruit and the pedicel and intensive fruit 78 abscission after the fruit stalk was girdled. While the hormone ethylene is a 79 well-known hormone that triggers fruit abscission (Taylor and Whitelaw, 2001), there 80 is much less information about roles of reactive oxygen species (ROS) in abscission 81 regulation. 82 83 ROS is involved in the responses of plants to stresses and is generated by a 84 number of mechanisms including NADPH oxidation catalyzed by plasma membrane 85 (PM)-bound NADPH oxidase (Cheeseman 2007; Tripathy and Oelmüller, 2012). 86 Reports about the roles ROS in fruit abscission have been inconsistent. Lai et al. 87 (2001) found that H2O2 reduced wax apple abscission under low temperatures, while 3 88 Ueda et al. (1991) reported that H2O2 stimulated abscission of bean petioles under 89 high light levels independent of ethylene. Sakamoto et al. (2008) showed that H2O2 90 was involved in salt-induced abscission of pepper petioles and that it acted 91 downstream of ethylene in signaling abscission. Botton et al. (2011) suggested that an 92 increase in sugar concentration in the cortex of apple fruit serves as an initial 93 senescing signal that induced H2O2 and ethylene production, causing abortion of seed, 94 reduction in IAA export and fruit drop. 95 96 Longan (Dimocarpus longan Lour.) is a tropical fruit tree that generally sets 97 heavily and requires thinning to produce large fruit, although natural fruit abscission 98 occurs during fruit development. The fruit are borne in multi-fruit panicles. Unlike 99 apple, whose fruit abscission is highly predictable with obvious dominant central fruit 100 and weak side fruit, longan fruit within a panicle are similar in size and vigor and it is 101 difficult to predict which and when fruit will abscise. In this study, longan fruit 102 clusters were starved for carbohydrates by girdling and defoliating, or detaching the 103 fruit clusters. These treatments induced 100% fruit abscission within a few days, 104 providing a convenient experimental system to study signals involved in fruit 105 abscission. Using this system, we examined the occurrence and roles of ROS in 106 regulating abscission under carbohydrate stress. 107 108 Materials and methods 109 Materials and treatments 110 The study was carried out during the mid stage of fruit development (50-60 days after 111 anthesis), after the early wave of fruit drop had ended and before the rapid aril (flesh) 112 growth initiated. The on-tree experiments involved girdling and defoliation treatments, 113 which were performed on 12 to 14-year-old ‘Chuliang’ trees at the South China 114 Agricultural University or Dongguan Agricultural Research Center. The off-tree 115 experiments used detached fruit clusters harvested from these trees. 116 117 Effectiveness of girdling plus defoliation in inducing fruit abscission was 118 examined. Twenty bearing shoots from different positions of the canopy, each with 119 more than 20 leaves and one terminal fruit cluster bearing 40-50 fruit were selected 120 from a tree. They were randomly allocated to four treatment groups, each with 5 121 replicates consisting of 5 bearing shoots as the experimental plots: no girdling or 4 122 defoliation (control); or girdled at a width of 5 mm at around 60 cm from the fruit 123 cluster base and defoliated to leave 0, 5 or 10 top leaves above the girdle. The number 124 of fruit on each panicle was counted every day until 5 days after treatment, when all 125 fruit in the “0 leaf” group had been shed. Since girdling plus complete defoliation 126 induced 100% fruit drop, we adopted this treatment for the other on-tree experiments 127 in later seasons. Twenty bearing shoots with a similar sized terminal panicle were 128 selected from different positions of five trees (n=5, one tree as one experimental block) 129 and girdled and defoliated 50 days after anthesis as mentioned above, and 20 130 untreated panicles with similar fruit load from each tree used as controls.