Physiological And Biochemical Characteristics Of Salt Tolerance In Grafted Cucumber Using Salt Tolerant Rootstock

In recent years, protected cultivation of cucumber has expanded rapidly in China. However, due to the continuous cultivation of vegetables and inappropriate fertilizer management measures during the cultivating process, salt accumulation in greenhouse soil has become a severe problem in cucumber protected cultivation. The growth of cucumber is unfavorably influenced by the secondary salinization of soil, resulting in a serious reduction of quality and yield of cucumber. It is showed that graft can improve vegetable tolerance to environmental stresses, become an effective approach overcoming salt stress. Cucumber (Cucumis sativus L.) is one of the important vegetables in protected culture, researchs on cucumber grafted technique, identification and the plant's response to disease and pest attack have been carrying on, but few reports on physiological and biochemical mechanisms of grafted cucumber in salt stress have been showed. We use hydroponically-grown grafted and own-root cucumber ('Shintosa Supreme' pumpkin and 'Xintaimici' as rootstock and scion respectively) plants as trial material, comparisons of physiological and biochemical changes were made between grafted and own-root cucumber plants under 100 mM NaCl stress. There were 4 treatments (own-root, own-root +NaCl, grafted, grafted +NaCl).1. In the aspect of characteristics of ion absorption and distribution, Inhibition of biomass production in grafted plants was significantly lower than that of own-root plants. The contents of Na⁺ and Cl^- in both grafted (G2) and own-root plants (O2) were increased significantly after exposing to NaCl stress. The contents of Na⁺ in different organs except root of G2 were significantly lower than those of O2. The contents of Cl^- in old leaves, petiole and root of G2 were significantly lower than those of O2. The main organ of Na⁺ accumulation in G2 was root, but the main organ of Cl^- accumulation was stem. The contents of K⁺, Ca²⁺, Mg²⁺ in young leaves and roots of G2 were significantly higher than those of O2. G2 had significantly higher ratios of K⁺/Na⁺, Ca²⁺/Na⁺, Mg²⁺/Na⁺ than those of O2. The above results demonstrated that adaptation to salt stress in grafted plants was caused by factors including salt accumulation in root and stem, stronger K⁺, Ca²⁺, Mg²⁺ selective absorption and transport abilities, therefore, the grafted plants had a better compartmentalization of ionic distribution in different organs and showed a stronger salt tolerance.2. The following results were obtained. Net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ contentration (Ci) and chlorophyll content of grafted plants were significantly higher than those of own-root plants. Photosynthesis was inhibited under NaCl stress, but the performance of the grafted plants was superior to that of own-root plants. The contents of free proline, soluble sugar and soluble protein in the leaves of grafted plants were significantly higher than those of own-root plants. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate-specific peroxidase (APX) in leaves of grafted plants were significantly higher than those of own-root plants. O₂·^- producing rate and malondiadehyde (MDA) content in leaves of grafted plants were significantly lower than those of own-root plants. The peroxidation of membrane lipid of grafted plants was lower than that of own-root plants. Based on these results, it could be concluded that the resistance to NaCl stress of grafted cucumber plants was stronger than that of own-root plants.3. Polyamines are low-molecular-weight biologically active compounds, play important roles in plant growth and development, configuration formation, response to environmental stresses. Contents of free putrescence (Put) in leaves of grafted plants (G2) were significantly higher than those of own-root plants (O2) except on the second day of stress. Free spermidine (Spd) and free spermine (Spm) contents of G2 were significantly higher than those of O2 during the whole course of stress. Total free polyamine content of G2 peaked on the fourth day of stress. Free Put/PAs value of G2 was significantly lower than that of O2 except on the fourth day of stress, but (Spd+Spm)/Put value was significantly higher than that of O2 during the whole course of stress. The contents of conjugated and bound Put, Spd, Spm in leaves of G2 were significantly higher than those of O2 during the whole course of stress. Either total conjugated or total bound polyamine content of G2 peaked on the sixth day of stress. Changing trends of both conjugated Put/PAs value and conjugated (Spd+Spm)/Put value were similar to those of free polyamine. Bound Put/PAs value of G2 was significantly lower than that of O2 except on the sixth day of stress, but bound (Spd+Spm)/Put value was significantly higher than that of O2 during the whole course of stress. The above results demonstrated that grafted cucumber plants possessed the characteristics of stronger salt tolerance.4. The results showed that contents of free putrescence (Put), spermidine (Spd) and spermine (Spm) in roots of grafted plants (G2) were significantly higher than those of own-root plants (O2) during the whole course of stress. Total free polyamine content of G2 peaked on the fourth day of stress. Free Put/PAs value of G2 was significantly lower than that of O2 except on the second day of stress, but (Spd+Spm)/Put value was significantly higher than that of O2 except on the second day of stress. The contents of conjugated and bound Put, Spd and Spm in roots of G2 were significantly higher than those of O2 during the whole course of stress. Total conjugated polyamine content of G2 peaked on the sixth day of stress, but total bound polyamine peaked on the eighth day of stress. Conjugated Put/PAs value was significantly lower than that of O2 during the whole course of stress, but conjugated (Spd+Spm)/Put value was significantly higher. Bound Put/PAs value of G2 was significantly lower than that of O2 except on the second day of stress, but bound (Spd+Spm)/Put value was significantly higher than that of O2 during the whole course of stress.5. The following results were obtained. Changes of hormone in plants was correlated to salt tolerance, ABA,GA and IAA were very important to enhance salt tolerance, high ABA, IAA content and ABA/GA ratio were very important to resist salt stess. With the stress time, ABA and IAA contents of grafted plants (G2) were increased during the whole course of stress, but those of own-root plants (O2) were increased at the beginning and then declined. The accumulation of ABA contents were faster and the value was higher in grafted plants, rapidly perceiving salt stress, which induced a series of relative genes expression by signal transduction pathways, regulated plant response to environmental stress. IAA contents and ABA/GA ratio in leaves, roots and exudates of G2 were significantly higher than those of O2 during the whole course of stress, but contents of GA were significantly lower than those of O2. The above results demonstrated that high ABA, IAA content and ABA/GA ratio might be involved in stronger salt tolerance of grafted cucumber plants.6. Comparison of the differences in protein components by two-dimensional electrophoresis analysis in leaves was made between own-root and grafted cucumber plants under NaCl stress. The gels were scanned by the densitometer VersaDoc 3000, which generated the digitalized images. The computer analyses including automatic detection and quantification of protein spots, as well as alignment, matching between the different 2-DE gels, were carried out by using PDQuest software. The results showed that 16 specific proteins were identified to be related to salt stress in leaves of grafted cucumber after 2 days of NaCl stress. Their molecular weights (Mw) is 110.6 kD, 89.7 kD, 76.5 kD, 56.4 kD, 49.4kD, 39.6kD, 36.2kD, 35.8kD, 34.7kD, 34.4kD, 34.3 kD, 33.4kD, 22.7 kD, 19.8 kD, 15.9 kD and 12.9 kD respectively. 5 proteins were selected and identified using MALDI-TOF-MS, but only one of them had been reported. By searching SwissProt 45.2 protein database, the map was matched with O-methyltransferase (with accession number P47917, theoretical Mw 39.58 kD, and theoretical PI 5.45). O-methyltransferase was reported to be related to salt tolerance. In addition, the synthesis of other proteins in the leaves of grafted plants was up-regulated compared with that of the own-root plants. These results indicated that specific proteins were synthesized and the synthesis of other proteins was up-regulated in the leaves of grafted plants under NaCl stress, hence increased the salt tolerance of grafted plants.