Study On Physiological Basis And Molecular Mechanism Of Cold Resistance Enhanced By Molybdenum Application In Winter Wheat

Molybdenum is an essential micronutrient for plants. Mo itself seems to gain biological function until it is complexed by Mo-containing enzymes. Based on the reviews of plant molybdenum nutrition and cold resistant mechanism, solution culture and soil culture experiments were conducted to investigate the physiological basis and molecular mechanism of cold resistance enhanced by molybdenum application in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014) through the biochemical methods combined with Real-time PCR, 2-DE and MS. The main results were as following.1. The effects of molybdenum on lethal dose-50 temperature(LT₅₀), electrolyte leakage and malondialdehyde(MDA) in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014) were investigated under low temperature stress. The results showed that the lethal dose-50 temperature(LT₅₀) in the leaves of Mo-fertilized treatment in both winter wheat cultivars decreased gradually with the prolongation of low temperature stress, whereas LT₅₀ in Mo-deficient treatment decreased first and then increased slightly in both winter wheat cultivars. LT₅₀ in Mo-fertilized treatment decreased significantly compared with Mo-deficient treatment in both winter wheat cultivars before and after low temperature stress. The electrolyte leakage and malondialdehyde(MDA) contents in Mo-deficient treatment increased significantly compared with Mo-fertilized treatment in both winter wheat cultivars during the low temperature stress. The increase rates of LT₅₀, electrolyte leakage and MDA in Mo-deficient treatment in Mo-inefficient cultivar 97014 were higher than those in Mo-efficient cultivar 97003 under low temperature stress, which indicated that Mo-inefficient cultivar 97014 in response to Mo deficiency was more sensitive than Mo-efficient cultivar 97003.2. Liquid nutrient culture experiments were conducted to investigate the effects of molybdenum on Mo-enzymes in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014) under low temperature stress. The results showed that the maximum NR activities(NRA_max), actual NRA activities(NRA_act) and NRA activation state(%) increased in the roots and leaves of Mo-treated in both winter wheat cultivars under low temperature stress. The activities of aldehyde oxidase(AO) and xanthine dehydrogenase(XDH) increased in the roots and leaves of Mo-treated in both winter wheat cultivars under low temperature stress. The decreased rates in Mo-deficient winter wheat became greater with the prolongation of low temperature stress. Genotypic differences in response to the effects of molybdenum on the Mo-enzymes activities exist between Mo-efficient line 97003 and Mo-inefficient line 97014. The increase rates of Mo-enzymes in 97014 were significantly higher than those of 97003 in Mo-fertilized treatment.3. Liquid nutrient culture experiments were conducted to investigate the effects of molybdenum on endogenesis hormone in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014) under low temperature stress. The results showed that the contents of abscisic acid(ABA) and indole-3-acetic acid(IAA) increased in the roots and leaves of Mo-treated winter wheat under low temperature stress. The contents of gibberellin(GA₃) increased significantly, and zeatin(Z) decreased significantly in the roots and leaves of Mo treated winter wheat in the later stage of low temperature stress, whereas no significant difference in the contents of GA₃ and Z was detected between Mo-deficient and Mo treated winter wheat in the earlier stage of low temperature stress. The hormone homeostasis in winter wheat was damaged by the drastic decrease of the ABA/GA in Mo deficient winter wheat under low temperature stress. The increase rates of ABA, IAA and Z contents, the ABA/GA ratio and the decrease rates of GA₃ contents in Mo inefficient cv. 97014 were higher than those in Mo efficient cv. 97003, which indicated that genotypic difference in response to molybdenum on hormone and its homeostasis in Mo efficient cv. 97003 and Mo inefficient cv. 97014.4. The effects of molybdenum on antioxidative enzymes in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014) were investigated under low temperature stress. The Results showed that the activities of superoxide dismutase(SOD), catalase(CAT), peroxidase(POD) and ascorbate peroxidase(APX) increased and the production rates of free radicals decreased in Mo-treated winter wheat under low temperature stress. In Mo-treated winter wheat, with the prolongation of low temperature stress, the activities of the four antioxidative enzymes increased at first 2 or 4 days of low temperature stress, and then decreased or dropped drastically. The tendency in the activities of these antioxidative enzymes indicated that a valid cold acclimation experienced in the Mo-treated winter wheat and that molybdenum helped to maintain the higher level of cold resistance under the following durative low temperature stress. Genotypic differences in response of activities of molybdenum on antioxidative enzymes were found in Mo efficient cv. 97003 and Mo inefficient cv. 97014. The decrease rates of the SOD, CAT, POD and APX activities and the accumulative rate of free radicals for-Mo treatment in Mo inefficient cv. 97014 were higher than those in Mo efficient cv. 97003, which maybe one of the reasons why the cold injury happened frequently in Mo inefficient cultivar for the Mo deficient treatment. Molybdenum regulates reactive oxygen species(ROS) metabolism to affect the cold resistance of winter wheat.5. Effects of molybdenum(Mo) on several photosynthetic parameters in winter wheat were investigated under low temperature stress in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014). The results indicated that the net photosynthetic rate(P_n) and stomatal limitation(L_s) increased significantly(P<0.05), while stomatal conductance(G_s), intercellular CO₂ concentration(C_i) and transpiration rate(T_r) decreased significantly(P<0.05) in Mo-fertilized treatment of both wheat lines under low temperature stress. From 0 to 6 days of low temperature stress in Mo-fertilized treatment, with the decline of P_n, C, decreased and L_s increased, indicating that stomatal limitation was the main factor to P_n decline. In Mo-deficient treatment, with the decline of P_n, C_i decreased and L_s increased at earlier stage of low temperature stress(0 to 4 days), whereas C_i increased and L_s decreased at later stage(4 to 6 days), implying that the probable main factors to P_n decline were stomatal limitation at earlier stage of low temperature stress, and non-stomatal limitation at later stage. Genotypic differences in response to the effects of molybdenum on the photosynthetic parameters exist between Mo-efficient cv. 97003 and Mo-inefficient cv. 97014. The changing rates of P_n and T_r of 97014 were significantly higher than those of 97003 in Mo-fertilized treatment.6. Effects of molybdenum(Mo) on photosynthetic light reaction curve and CO₂ reaction curve in winter wheat were investigated under low temperature stress in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014). The results indicated that the Maximum Net Photosynthetic rate(A_max), Light-Saturation Point(LSP), Apparent Quantum Yield(AQY), CO₂ Saturation Point(CSP), Carboxylation Efficiency(CE), Maximum Carboxylation Velocity of Rubisco(V_cmax) and Maximum Potential Rate of Electron Transport(J_max) increased significantly(P<0.05), while Light-Compensation Point(LCP), Day Respiration Rate(R_day) and CO₂ Compensation Point(CCP) decreased significantly(P<0.05) in Mo-fertilized treatment of both wheat lines under low temperature stress.7. Effects of molybdenum(Mo) on photosynthetic pigment, cytochrome, and protein complex of thylakoids in winter wheat were investigated under low temperature stress in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014). The results indicated that the total chlorophyll, chlorophyll a, and chlorophyll b increased significantly(P<0.05) in Mo-fertilized treatment, while the chlorophyll a/chlorophyll b decreased significantly(P<0.05) in Mo-deficient treatment in both wheat lines under low temperature stress, implying that the transformation from the chlorophyll a to chlorophyll b was inhibited. The increase of the carotenoid contents in Mo-fertilized treatment decreased the photoinhibition and improved scavenging of reactive oxygen species in winter wheat. The contents of cytochrome f, cytochrome b563, cytochrome b559, PSI and cytochrome b6/f complex increased significantly(P<0.05) in Mo-fertilized treatment from 0 to 6 days of low temperature stress, while the contents of cytochrome b559 and PSII increased significantly(P<0.05) at later stage(4 to 6 days) of low temperature stress, implying that the capacity of light absorption and electron transfer were improved in Mo-fertilized treatment. The response to Mo deficiency in PSI and cytochrome b6/f complex was more sensitive under low temperature stress, implying that PSI and cytochrome b6/f complex were damaged firstly and then PSII was damaged in Mo deficient winter wheat.8. The effects of molybdenum on ABA concentration, expression of ABA-dependent COR(cold-regulated) genes, CBF/DREB transcription factors and ABA-independent COR genes in winter wheat(Mo efficient cv. 97003 and Mo inefficient cv. 97014) were investigated under low temperature stress. The results showed that expression of ABA-dependent COR genes, CBF/DREB transcription factors and ABA-independent COR genes increased first and then decreased slightly in the leaves of Mo-deficient and Mo-fertilized treatment in both winter wheat cultivars with the prolongation of low temperature stress. The concentration of ABA in Mo-fertilized treatment in both winter wheat cultivars increased before and after the low temperature stress. Expression of ABA-dependent COR genes(Wrab15, Wrab17, Wrab18 and Wrab19) in Mo-fertilized winter wheat were significantly up-regulated from 3 h of low temperature stress. It suggested that molybdenum regulated the cold resistance of winter wheat from the ABA-dependent signal pathway(AO→ABA→bZIP→ABRE→COR genes). No significant difference existed between Mo-deficient and Mo-fertilized treatment in the expression of CBF/DREB transcription factor genes(TaCBF and Wcbf2-1) and ABA-independent COR genes(WCS120, WCS19, Wcor14 and Wcor15) before low temperature stress. Expression of CBF/DREB transcription factor genes(TaCBF and Wcbf2-1) in Mo-fertilized winter wheat was significantly up-regulated from 3 h of low temperature stress, and expression of ABA-independent COR genes(WCS120, WCS19, Wcor14 and Wcor15) in Mo-fertilized winter wheat was significantly up-regulated from 6 h of low temperature stress. It suggested that molybdenum also regulated the cold resistance of winter wheat from the ABA-independent signal pathway(low temperature signaling→transcription factors→CRT/DRE motif→COR genes).9. The effects of molybdenum on protein express in winter wheat under low temperature stress were investigated using 2-DE combined with MS analysis and Expression of mRNA for genes encoded the differential proteins above was analyized by real-time PCR. In total, Fifteen proteins with quantitatively differential expression(>1.5-fold increase or decrease, Student's t-test, p<0.01) between Mo-deficient and Mo-fertilized winter wheat and two specific proteins in Mo-fertilized winter wheat were found before low temperature stress. Similarly, thirteen proteins with quantitatively differential expression(>1.5-fold increase or decrease, Student's t-test, p<0.01) between Mo-deficient and Mo-fertilized winter wheat and three specific proteins to Mo-deficient and Mo-fertilized treatment were found after low temperature stress. Twelve proteins interested were identified by mass spectrometry(MS or MS-MS). Among them five protein spots(spot814, spot1296, spot1297, spot1255 and spot1157) were closely related to light reactions of photosynthesis, four protein spots(spot918, spot548, spotl386 and spot648) closely related to dark reactions of photosynthesis, one spot(spot991) related to the transcription of chloroplast gene, one spot(spot 1305) related to the translation of proteins and one spot(spot 1058) not detected in protein database. The results showed Mo regulated the physiological processes such as photosynthesis, transcription of chloroplast gene and translation of proteins through the protein express, and focused on the regulation of photosynthesis.10. Combined with the results of Mo-enzymes, hormones, antioxidative enzymes, photosynthetic parameters and composition of thylakoids, molecular mechanism on how cold resistance and photosynthesis enhanced by molybdenum was put forward. Base on the changes of Mo-enzyme activities, the mechanism focus on the process of gene express and protein translation regulated by molybdenum, and testified that the chloroplast was the key subcellular structures where significant differences in biochemical reactions and metabolism progress existed between Mo-fertilized and Mo-deficient winter wheat. Low temperature stress was the induction factors for Mo-deficient symptom.