The Function Of Spermidine In Chilling Stress And Signal Transduction In Tomato Seedlings

Chilling stress is a major biotic stresses factors; it can influence plants growth and development. As a warm-season vegetable, tomato (Lycopersicon esculentum Mill.) is sensitive to low temperature. Polyamines (PAs) are low-molecular weight aliphatic amines, mainly including putrescine (Put), spermidine (Spd), and spermine (Spm), which exist in all plant cells. It has been suggested that PAs could involve in several fundamental processes in plants. However, the mechanism of PAs on chilling tolerance and signal transduction in tomato are still not very clear. In this study, chilling-sensitive tomato cultivar (’Moneymaker’) was used to investigate the effect of exogenous Spd on tomato seedlings under chilling stress, and we analyzed if nitric oxide (NO) involve in Spd-induced chilling tolerance. The interactional mechanism between PAs and ascisic acid (ABA) under chilling stress was also investigated. This study analyzed the relation among mitogen-activated protein kinase (MAPK), NO, and hydrogen peroxide (H2O2) in antioxidant defense reaction regulated by PAs. In order to clarify the physiological mechanism of PAs in enhancing chilling tolerance, and explore the function of PAs in signal transduction pathway. The main results are as follows:1. Application exogenous Spd on Tomato seedlings cv. Moneymaker (chilling-sensitive) under chilling stress (15/6℃). In this study, exogenous Spd could alleviate the damage induced by chilling stress. Applying with Spd significantly increased the maximum fluorescence ratio (Fv/Fm), solule sugars, and proline contents, as well as decreased malondialdehyde (MDA) and reactive oxygen species (ROS) production in tomato leaves during chilling stress. Exogenous Spd caused much greater increase in free and conjugated endogenous Spd and Spm levels; enhanced the activities of arginine decaroxylase (ADC), ornithine decaroxylase (ODC), diamine oxidase (DAO), and polyamine oxidase (PAO) activities. In addition, during chilling stress, treatment with Spd improved the activities and transcript levels of antioxidant enzymes. Application of Spd remarkale increased ASA and GSH contents, ASA/DHA and GSH/GSSG. These results suggested that Spd enhance chilling stress tolerance in tomato plants, through increasing compatile solute content, modulating PAs metaolism and ROS scavenging system.2. Using cv. Moneymaker, application of exogenous Spd and different inhibitors under chilling stress (4℃) showed exogenous Spd enhanced chilling tolerance of tomato seedlings, through inducing NO generation. Application of Spd significantly increased NO production, pre-treatment with MGBG (an inhibitor of Spd synthesis) and PTIO (a scavenger of NO) greatly reduced the effect of Spd. L-NAME (a specific inhibitor of nitric oxide) and Tungstate (an inhibitor of nitrite reductase) also caused NO accumulation decreased in tomato seedlings under chilling stress. Therefore, in present study, Spd induced NO production through NOS-like and NR pathways in tomato leaves under chilling stress. During stress condition, the content of H2O2 was oviously detected by exogenous Spd, DPI (an inhibitor of NADPH) and DMTU (OH and H2O2 scavenger) treatment significantly reduced the content of H2O2 induced by Spd. However, spraying with PTIO, L-NAME, and Tungstate had little effect on Spd-induced H2O2 production. It indicated that H2O2 may act upstream of NO in Spd-induced stress response signal pathways.3. Compared to the control, chilling stress significantly reduced Fv/Fm and increased electrolyte leakage, exogenous Spd caused Fv/Fm increased and electrolyte leakage reduced. Treatment with L-NAME, Tungstate, and PTIO inhibited the effect of Spd. Therefore, NO played an important role in Spd-induced tolerance against chilling stress. Application of Spd to chilling stressed plant enhanced antioxidant enzymes activities and corresponding genes expressions, NO scavenger (PTIO) and inhibitor (L-NAME, Tungstate) treatment resulted in decreased activities and expressions of antioxidant enzymes and genes. The results supported that NO is implicated in the regulation of antioxidant ability by Spd, whereby improving chilling tolerance of tomato seedlings.4. Application exogenous Put and D-Arg (a synthesis inhabitor of Put) on tomato seedlings (cv. Moneymaker) under chilling stress (4℃) suggested the endogenous ABA levels were significantly increased under chilling stress condition. Exogenous Put led to ABA contents increased relative to control, pre-treatment with D-arginine (D-Arg, a synthesis inhabitor of Put) reduced ABA levels under chilling stress, while adding Put to plants, ABA contents were remarkale lower than Put application alone. Meanwhile, chilling treatment up-regulated 9-cis-Epoxycarotenoid dioxygenase (NCED1), exogenous Put increased NCED1 gene expression, Applying with D-Arg down-regulated NCED1 transcript levels induced by Put. Compared to the control, D-Arg treatment greatly increased electrolyte leakage of tomato seedling; the effect was alleviated by ABA treatment. These results indicated that ABA is involved in Put-induced chilling stress response in cv. Moneymaker.5. In order to further examine the relationship between ABA and Put. ABA-deficient mutant tomato (Sit) and wild-type (RR) tomato cultivar were used in this study, treatment at 4℃ for 24h. The results suggested that chilling stress treatment caused endogenous contents of Put, Spd, and Spm increased in wild-type (RR) tomato during chilling stress, Put content in mutant tomato was increased, Spd and Spm contents were lower than control. It indicated that chilling treatment result in Put and ABA accumulation in both mutant and wild-type tomato seedling, and Put content was higher in wild-type tomato seedlings. Exogenous application of ABA or Put had no obvious effect on endogenous ABA and Put levels under normal temperature; exogenous ABA caused endogenous ABA contents increased in the two tomato cultivars; ABA treatment decreased endogenous Put content in wild-type tomato leaves, and had little influence in Put content of mutant tomato during chilling stress,. In addition, application of Put treatment could increase endogenous ABA and Put contents in the two tomato cultivars. There is little change in electrolyte leakage in the two tomato cultivars under normal temperature. Treatment with Put or ABA obviously reduced the effect of chilling stress condition. For the mutant tomato seedlings, exogenous ABA inhibited the increased electrolyte leakage, while Put treatment had no this effect. These results revealed that ABA induced chilling tolerance is independent of Put in the two cultivars. Put could enhance chilling tolerance of wild-type tomato via increasing ABA content, however, the ABA levels induced by exogenous Put in mutant tomato leaves were unable to resist the chilling stress.6. Under chilling stress, exogenous Spd greatly increased MAP 1,2,3,4,6, and 16 transcript levels, MAPK inhibitors (PD98059 and U0126) alleviated the change induced by Spd. On the other hand, application of Spd could increase Fv/Fm, elevate antioxidant enzymes activities, and up-regulated corresponding genes expressions. However, the effect was locked by MAPK inhibitors. The result shown that MAPK play a vital role in Spd-induced antioxidant defense reaction.7, Applying with SNP (NO donor) or H2O2 resulted in increased antioxidant enzymes activities and relevant transcript levels. Antioxidant enzymes and corresponding genes expressions were lower in tomato seedlings with MAPK inhibitors than SNP or H2O2 treatment alone. Moreover, MAPK inhibitors treatment significantly decreased H2O2 content, but no ovious change was obvious in NO production. These results suggested there is a linear relation between MAPK and NO; the cross-talk was also existed in MAPK and H2O2.