Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response

Tree Physiology 30, 914–922 doi:10.1093/treephys/tpq030 Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response JIE SHI,1 XING-ZHENG FU,1 TING PENG,1 XIAO-SAN HUANG,1 QI-JUN FAN1 and JI-HONG LIU1,2 1 National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China 2 Corresponding author () Received November 24, 2009; accepted March 16, 2010; published online May 12, 2010 Summary Polyamines, small aliphatic polycations, have been suggested to play key roles in a number of biological processes. In this paper, attempts were made to investigate the possibility of improving dehydration tolerance of citrus in vitro plants by exogenous application of spermine (Spm). ‘Red Tangerine’ (Citrus reticulata Blanco) in vitro plants pretreated with 1 mM Spm exhibited less wilted phenotype and lower water loss and electrolyte leakage than the control under dehydration. Spm-pretreated plants contained higher endogenous polyamine content during the course of the experiment relative to the control, particularly at the end of dehydration, coupled with higher expression levels of ADC and SPMS. Histochemical staining showed that the Spm-pretreated leaves were stained to a lower extent than those without Spm pretreatment, implying generation of less reactive oxygen species (ROS). On the contrary, activities of peroxidase (POD) and superoxide dismutase (SOD) in the Spmpretreated samples were higher than the control at a given time point or during the whole experiment, suggesting that Spm exerted a positive effect on antioxidant systems. In addition, significantly smaller stomatal aperture size was observed in Spm-pretreated epidermal peels, which showed that stomatal closure was promoted by polyamines. All of these data suggest that Spm pretreatment causes accumulation of higher endogenous polyamines and accordingly leads to more effective ROS scavenging (less tissue damage) and stimulated stomatal closure (lower water loss) upon dehydration, which may function collectively to enhance dehydration tolerance. Keywords: antioxidant enzymes, Citrus reticulata, dehydration tolerance, polyamine, reactive oxygen species, stomatal response. Introduction The citrus industry worldwide is always threatened by adverse environmental stresses, among which drought is one of the most devastating factors. Drought retards plant growth, reduces fruit size and yield and promotes leaf abscission, leading to irreversible damage in some cases (García-Sánchez et al. 2007). Accordingly, it is important to develop appropriate strategies that can be taken to tackle the drought stress. Although selection and breeding of drought-tolerant cultivars have been suggested to be an effective solution to this issue, progress in drought-tolerance-oriented breeding has been fairly slow in Citrus due to several reproductive barriers, such as polyembryony, long juvenility, high heterozygosity and male/female sterility. Therefore, it is still a favorable way to take other measures in order to minimize drought-derived stress damage. Drought results in water deficit and loss of cell turgor. In addition, it evokes overproduction of highly reactive oxygen species (ROS) like superoxide (O2−) and hydrogen peroxide (H2O2), leading to oxidative stress. It has been well documented that plants have developed an array of mechanisms to cope with these abnormal physiological disorders. Accumulating evidence has been acquired to show that under stressful conditions plants may undergo physiological, biochemical, cellular and molecular alterations (YamaguchiShinozaki and Shinozaki 2005). One approach for the plants to respond and adapt to adverse milieus is the accumulation of compatible solutes, also known as osmoprotectants, for osmotic adjustment and maintenance of cell turgor. Moreover, the plants evolve an endogenous defensive mechanism to protect cellular and subcellular components against oxidative stress, in which ROS is primarily removed by enzymatic or non-enzymatic antioxidant systems (Arbona and GómezCadenas 2008). These two mechanisms might work independently or in synergy to mitigate stress-induced cell death and consequently enhance stress tolerance. It is thus conceivable that a given compound that can function both as osmoprotectant and ROS scavenger will serve as a robust effector to counteract the drought stress. In this regard, polyamines can be regarded as a satisfactory candidate to meet the two requirements. Polyamines, primarily spermidine (Spd), spermine (Spm) and their diamine precursor, putrescine (Put), are low- © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: DEHYDRATION TOLERANCE OF CITRUS IN VITRO PLANTS molecular-weight aliphatic polycations that are ubiquitously present in almost all living organisms. Being positively charged at physiological pH, they can interact with various cellular macromolecules like nucleic acids, protein and membrane phospholipids and regulate relevant processes (MartinTanguy 2001). These properties provide the basis for the involvement of polyamines in a wide spectrum of physiological processes, including cell division, embryogenesis, morphogenesis, growth and development (Evans and Malmberg 1989, Liu et al. 2006a). Moreover, intensive work has revealed that polyamines play important roles in stress response, although the definitive modes of action remain a matter of speculation (Liu et al. 2007, Kusano et al. 2008). So far, a growing body of research has revealed accumulation of polyamines in many plants upon exposure to a variety of stresses, including salinity, chilling, drought, ozone and pathogen invasion (Liu et al. 2007, Kusano et al. 2008, references therein). Based on these phenomena, it has been proposed to enhance stress tolerance through augmenting endogenous cellular polyamine content via either genetic transformation or exogenous replenishment of polyamines. For example, transformation of an arginine decarboxylase gene Datura stramonium in rice led to remarkable drought tolerance (Capell et al. 2004). Interestingly, over-expression of Cucurbita ficifolia spermidine synthase (SPDS) gene in Arabidopsis thaliana and apple SPDS gene (MdSPDS1) in European pear confers tolerance to multiple abiotic stresses (Kasukabe et al. 2004, Wen et al. 2008, 2009). Exogenously applied polyamines have also been shown to effectively alleviate stress-derived injury caused by acid rain (Velikova et al. 2000), ozone (Navakoudis et al. 2003), heavy metals (Zhao and Yang 2008), chilling (Shen et al. 2000), salinity (Iqbal and Ashraf 2005, Liu et al. 2006b) and water stress (Kubiś 2008, Farooq et al. 2009, Yiu et al. 2009). The above-mentioned illustration suggests that modulation of cellular polyamine content can be regarded as a convenient and effective strategy to en (...truncated)