ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 4, с. 506-513


УДК 581.1



© 2015 H. Y. Yang, C. H. Zhang, W. L. Wu, W. L. Li, Y. L. Wei, S. S. Dong

Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, P.R. China

Received October 14, 2014

In order to study the potential antioxidant defense mechanisms, the blackberry cultivar "Ningzhi 1", a new flo-ricane-fruiting hybridberry (Rubus sp.), was subjected to 20-day drought stress by withholding irrigation, followed by rewatering for 5 days, then the leaf water content (LWC), membrane electrolyte leakage, contents of photosynthetic pigments, protein, soluble sugar, hydrogen peroxide (H2O2), and malondialdehyde (MDA), activities of superoxide dismutase (SOD) and peroxidase (POD), and the levels of antioxidants such as ascorbate (AsA) and reduced glutathione (GSH) in leaves were investigated. The results showed that LWC was greatly decreased during the 20-day drought treatment period. After rewatering, water content restored. Drought stress induced significant accumulation of photosynthetic pigments, protein, soluble sugar, H2O2, and MDA as well as an increase in membrane electrolyte leakage, which were all decreased after rewatering. The activities of SOD and POD were elevated under drought stress, which were still at higher levels compared with control after rewa-tering. The contents of AsA and GSH ascended first and were then followed by a decline during the whole drought period, after rewatering, the contents increased and remained at a higher level than that of controls. The plants showed a rapid and almost complete recovery after rewatering, and the physiological alterations could represent a set of adaptive mechanisms employed by "Ningzhi 1" to cope with drought stress. It was suggested that increased drought tolerance of "Ningzhi 1" was due to higher antioxidant enzymes, reduced lipid peroxidation, better accumulation of osmolytes, and maintenance of tissue water content.

Keywords: Rubus sp. — blackberry — drought stress — oxidative stress — antioxidant system

DOI: 10.7868/S0015330315040181


Drought stress is one of the most frequent environmental stresses which affect plant growth and commonly constitute serious threats to agriculture [1]. Thus, improving our understanding of the plant response can greatly enhance our ability to increase plant performance under such conditions. In plant, the first response is stomatal closure to prevent tran-spirational water loss during drought stress. Stomatal closure limits CO2 absorption and results in the over-reduction of components within the electron transport chain leading to the generation of oxidative stress [2]. Oxidative stress is characterized by the overproduction of highly reactive oxygen species (ROS), such as superoxide anion (O2- ), hydrogen peroxide (H2O2), hy-

1 This text was submitted by the authors in English.

Abbreviations: AsA — ascorbate; Chl — chlorophyll; Car — caro-tenoids; GSH — reduced glutathione; H2O2 — hydrogen peroxide; LWC — leaf water content; MDA — malondialdehyde; NBT — nitro blue tetrazolium; POD — peroxidase; ROS — reactive oxygen species; SOD — superoxide dismutase.

Corresponding author: H. Y. Yang. Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing 210014, P.R. China; e-mail addresses: lwlcnbg@mail.cnbg.net, haiyanyang_025@126.com

droxyl radical (OH '), and singlet oxygen [1—3]. These ROS can cause membrane lipid peroxidation, protein degradation, enzyme inactivation, and DNA modification, resulting in cell damage and eventually cell death. To protect against oxidative stress, a highly efffi-cient antioxidant defense system is present in plant cells, which include the enzymes superoxide dismu-tase (SOD), peroxidase (POD), and catalase (CAT) and low molecular weight antioxidants, such as glu-tathione (GSH), ascorbate (AsA), and carotenoids [1—4]. The scavenging of superoxide anion is achieved through SOD, which catalyses the dismutation of superoxide to H2O2. The H2O2 is then further scavenged by CAT and POD into H2O and O2 [1]. Besides, the soluble antioxidants AsA and GSH act directly as reducing agents or indirectly as co-substrates of different enzymatic reactions [3]. Carotenoids also prevent photodamage mainly by acting as physical quenchers of electronically excited molecules, besides functioning as photoreceptors [5].

Blackberry belonging to the Rubus genus and subgenus (formerly Eubatus) of Rosaceae family, is generally considered to be broadly adapted to a wide range of climates and soils [6], which can be grown through-

out much of the temperate regions in the world [7]. Beside, blackberries are a good source of natural antioxidants. In addition to vitamins and minerals, extracts of blackberries are also rich in phenolic compounds such as anthocyanins, flavonols, chlorogenic acid, and procyanidins, which have high biological activities and may provide benefits to human health [8]. Recently, blackberry is widely cultivated commercially in Oregon, Mexico, Serbia, and China owing to the increasing international demand [7], its importance as a crop has dramatically increased in the past 10 years.

In China, blackberry is grown under irrigated conditions. However, water for irrigation is not always available at the time and amount needed by the plant. Moreover, dry years are occurring more frequently probably due to climate change, and the probability of water deficit for blackberry is increasing, especially in the summer months [6]. Prolonged water deficits result in negative impacts on plant growth and fruit production. Hence, it is essential to elucidate the mechanisms of this plant associated with water deficit tolerance. Up to date, very few reports are available on physiological adaptation of blackberry to drought stress [9, 10]. In this study, "Ningzhi 1" is a new flori-cane-fruiting hybridberry (Rubus sp.) released by the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences in 2010 [11, 12], which was selected from an induced primocane mutation of hybridberry "Boysenberry". It can grow in hilly areas of Jiangsu, Zhejiang, Anhui, Shandong provinces in China [11]. However, to the best of our knowledge, no work has so far been carried out to study drought stress induced metabolic changes therein.

In this investigation, we researched the physiological responses of "Ningzhi 1" to water deficit/drought stress, with reference to: (1) changes in leaf water content and the membrane electrolyte leakage; (2) changes in the contents of photosynthetic pigments, protein, soluble sugar, H2O2, and MDA; (3) changes in the activities of superoxide dismutase (SOD) and peroxidase (POD); (4) changes in the levels of antioxidants such as reduced ascorbate (AsA) and reduced glutathione (GSH). As a fruit tree plant, blackberry had excellent adaptability on multiple abiotic stresses including drying climate. This study will be helpful in elucidating the mechanism of drought tolerance in blackberry plants.


Plant material. This experiment was carried out with the blackberry cultivar "Ningzhi 1", a new flori-cane-fruiting hybridberry (Rubus sp.), from June to July 2014 in the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China. The one-year-old seedlings of "Ningzhi 1", which were obtained by layering, we used in the tests. Plants were grown in pots (26 cm in diameter x 24 cm in depth) containing a mixture of loamy garden soil and peat soil (3 : 2, v/v) and maintained in a greenhouse

with a mean temperature of 26 ± 3°C, relative humidity of 86 ± 20%, and natural light/dark cycle. All pots initially weighed the same (6.0 kg) to facilitate calculations. After applying standard irrigation (to 75% field capacity) for five months in the greenhouse, morphologically uniform seedlings were selected for drought stress experiments.

Well-watered plants were used as controls. Control plants were watered every other day to field capacity. Plants undergoing drought stress treatment were subjected to 20-day drought stress by withholding irrigation, and then plants were rewatered to field capacity and followed by a recovery for 5 days. The experiment was carried out using a completely randomized split-plot design with six replicates, giving a total of six plants per treatment. Samples were harvested at the 0, 5, 10, 15, and 20th day during drought stress, and also post-drought after 5-day rewatering. Fully expanded leaves at the fourth or fifth position from the apex of the shoot were sampled.

Assay of leaf water content. For the measurement of leaf water content (LWC) fully developed leaves were harvested and weighed immediately to determine the fresh weight (FW) and were then oven dried at 60°C till constant weight for dry weight (DW) [13]. The LWC was determined as: LWC (%) = [(FW - DW)/FW] x 100.

Assay of membrane permeability. Membrane permeability, which reflects membrane damage, was measured by an electrical conductivity method described by Yan et al. [14]. Six fresh leaf discs (diameter = 5 mm) from a recently fully expanded leaf were used to assess the electrolyte leakage percentage. Samples were washed three times with deionized water to remove surface-adhered electrolytes. Leaf discs were placed in closed vials containing 10 mL deionized water under a vacuum (via a vacuum pump) for 10 min and then surged for 1 h. Electrical conductivity of the solution (Lj) was determined at 25°C. Samples were then incubated in boiling water for 10 min and the final electrical conductivity (L0) was obtained after equilibration at 25°C. Electrolyte leakage percentage was defined by the formula:

Electrolyte leakage (%) = (Lt/L0) x 100.

Assay of photosynthetic pigment. Chlorophylls and carotenoids (~0.2 g fresh samples) were extracted with 80% acetone a

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