【作者】 孙学成；

【导师】 胡承孝；

【作者基本信息】 华中农业大学 ， 植物营养学， 2006， 博士

【摘要】 钼是植物必需的微量元素之一，钼在植物体内的生理功能主要通过含钼酶来实现。本文在总结国内外植物钼营养和抗寒机理研究进展的基础上，通过土壤培养和营养液培养试验，以冬小麦钼高效品种和钼低效品种为材料，应用生理生化研究法并结合荧光定量PCR技术、双向电泳和质谱分析技术，深入研究了钼提高冬小麦抗寒力的生理基础，初步分析了钼提高冬小麦抗寒力的分子机制。主要研究结果如下：1．研究了在低温胁迫下施钼对冬小麦叶片半致死温度(LT₅₀)、电解质渗透率和丙二醛（MDA）含量的影响。结果表明：随着低温胁迫的延长，2冬小麦品种（97003和97014）施钼处理叶片半致死温度(LT₅₀)均呈一直下降趋势，缺钼处理半致死温度(LT₅₀)先下降后有所回升；低温胁迫前后2个冬小麦品种施钼处理叶片半致死温度(LT₅₀)均显著低于缺钼处理，低温胁迫后，2个冬小麦品种缺钼处理电解质渗透率和丙二醛（MDA）含量均显著高于施钼处理。在低温胁迫进程中，钼低效品种缺钼处理半致死温度(LT₅₀)、电解质渗透率、丙二醛（MDA）含量的上升幅度高于钼高效品种，说明低温胁迫下冬小麦钼低效品种对缺钼更敏感。2．采用营养液培养的方法，研究了在低温胁迫下施钼对冬小麦根及叶片含钼酶活性的影响。低温胁迫前后施钼均显著提高2个冬小麦品种根及叶片中NRA_max、NRA_act活性和NRA活化状态；低温胁迫前后施钼显著提高了2个冬小麦品种根及叶片中AO和XDH活性，低温胁迫时间越长，缺钼处理AO和XDH活性下降幅度越大。低温胁迫下冬小麦含钼酶活性存在基因型差异，钼低效品种施钼处理含钼酶活性上升幅度更大。3．采用营养液培养的方法，研究了在低温胁迫下施钼对冬小麦根及叶片内源激素的影响。结果表明，低温胁迫前后施钼均显著提高2个冬小麦品种根及叶片中ABA和IAA含量，低温胁迫前期缺钼与施钼处理根及叶片中GA₃和Z含量差异不显著，低温胁迫后期施钼显著降低了冬小麦根及叶片中GA₃含量，显著提高了冬小麦根及叶片中Z含量。低温胁迫下缺钼冬小麦根及叶片ABA／GA比值显著下降破坏了的冬小麦激素稳态平衡。钼低效品种施钼处理叶片及根中ABA、IAA、Z含量和ABA／GA比值的上升幅度，GA₃含量下降幅度均远高于钼高效品种，说明缺钼对冬小麦内源激素及其稳态平衡的影响存在基因型差异。4．研究了在低温胁迫下施钼对冬小麦叶片抗氧化酶活性影响。结果表明，低温处理2、4和6d时施钼均显著提高了超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）和抗坏血酸过氧化物酶（APX）活性，显著降低了2个冬小麦品种叶片中超氧阴离子产生速率；施钼后，随着低温胁迫时间的延长，2个冬小麦品种叶片中4种抗氧化酶活性均先升高而后呈下降或突降趋势，说明施钼冬小麦能通过正常的低温锻炼，有利于植株在经受更长时间低温胁迫时维持较高的抗寒力；钼对冬小麦钼高、低效品种叶片中抗氧化酶活性的影响存在基因型差异，与钼高效品种相比，钼低效品种缺钼处理叶片SOD、CAT、POD和APX等抗氧化酶活性下降幅度更大，活性氧自由基积累速率更大。5．低温胁迫下钼对冬小麦光合作用气体交换参数的影响。结果表明，低温胁迫下施钼显著提高2个品系叶片的净光合速率（P_n）和气孔限制值（L_s），显著降低叶片气孔导度（G_s）、胞间CO₂浓度（C_i）和蒸腾速率（T_r）。随着低温处理时间的延长P_n下降，施钼处理C_i降低、L_s升高，说明其P_n下降主要由气孔限制因素引起；不施钼处理低温胁迫前期（0-4d）C_i下降、L_s升高，而后期（4-6d）C_i升高、L_s下降，说明不施钼处理P_n下降在低温胁迫前期可能主要由气孔限制因素引起，而低温胁迫后期可能主要由非气孔因素引起。钼对冬小麦叶片光合参数的影响存在基因型差异，低温处理前后钼低效品系施钼处理叶片P_n上升幅度和T_r下降幅度均显著高于钼高效品系。6．研究了低温胁迫下施钼对冬小麦钼高效品系97003和钼低效品系97014叶片光合作用光响应曲线和CO₂响应曲线参数的影响。结果表明，低温胁迫下施施钼显著提高了2个冬小麦品种叶片最大净光合速率(A_max)、光饱和点（LSP），表观量子效率（AQY）、CO₂饱和点（CSP）、表观羧化效率（CE）、Rubisco最大羧化效率(V_cmax)和最大电子传递速率(J_max)，显著降低冬小麦叶片光补偿点（LCP）、光下呼吸速率(R_day)和CO₂补偿点（CCP），这可能是施钼提高冬小麦叶片净光合速率的原因。7．研究了低温胁迫下施钼对冬小麦叶片光合色素、细胞色素和内囊体膜蛋白质复合体含量的影响。结果表明，低温胁迫下冬小麦叶片施钼提高了冬小麦叶片叶绿素总量、叶绿素a和叶绿素b含量，缺钼导致chla／b比值逐渐升高，说明低温胁迫下缺钼冬小麦叶绿素a向叶绿素b转化受阻；施钼显著提高冬小麦叶片的类胡萝卜素含量，从而降低光抑制，有利于活性氧自由基的清除。低温胁迫前后施钼均显著提高了冬小麦叶片中的细胞色素f、细胞色素b563、光系统Ⅰ和细胞色素b6／f复合体含量，低温胁迫后期施钼显著提高了冬小麦叶片中细胞色素b559和光系统Ⅱ的含量，提高对光能的吸收和电子传递能力。低温胁迫下PSI复合体和细胞色素b6／f复合体对缺钼的反应更为敏感，推测低温下缺钼首先导致光系统Ⅰ和细胞色素b6／f复合体的损伤，进而才导致光系统Ⅱ的损伤。8．研究了在低温胁迫下施钼对冬小麦叶片ABA含量、ABA依赖型抗寒基因、CBF／DREB转录因子基因和ABA非依赖型抗寒基因表达的影响。结果表明：随着低温胁迫的延长，2个冬小麦品种叶片ABA依赖型抗寒基因、CBF／DREB转录因子基因、ABA非依赖型基因的mRNA表达量均呈先快速上升后下降的趋势。低温胁迫前后2个冬小麦品种施钼处理ABA含量均显著增加，低温胁迫3h时施钼处理ABA依赖型抗寒基因（Wrab15、Wrab17、Wrab18和Wrabl9）mRNA表达量开始显著增加，推测钼可以通过醛氧化酶（AO）→ABA→bZIP→ABRE→COR基因表达这一途径来调控冬小麦的抗寒力。低温胁迫前缺钼与施钼处理CBF／DREB转录因子基因（TaCBF和Wcbf2-1）和ABA非依赖型基因（WCS120、WCS19、Wcor14和Wcor15）表达量差异均不显著，低温处理3h时，施钼冬小麦CBF／DREB转录因子基因（TaCBF和Wcbf2-1）表达量丌始显著增加，低温处理6h时，施钼处理ABA非依赖型基因（WCS120、WCS19、Wcor14和Wcor15）表达量开始显著增加，推测钼还能通过低温信号→CBF／DREB转录因子→CRT／DRE元件→COR基因表达这一途径来调控冬小麦的抗寒力的形成。9．通过双向电泳和质谱技术相结合的方法，研究了低温胁迫下钼对冬小麦叶片蛋白质表达的影响，并应用荧光定量PCR技术对编码差异蛋白质点的基因的mRNA表达量进行了动态分析。结果表明，低温胁迫前，施钼后叶片中的产生特异蛋白质点有2个；缺钼与施钼处理中表达量差异达1.5倍以上的蛋白质点15个；低温胁迫后，施钼处理叶片中的产生特异蛋白质点有3个，缺钼与施钼处理中表达量差异达1.5倍以上的蛋白质点13个。对12个蛋白质点的质谱鉴定结果表明，有5个蛋白质点（spot814、spot1296、spot1297、spot1255和spot1157）的功能与光合作用光反应过程有关，4个蛋白质点（spot918、spot548、spot1386和spot648）的功能与光合作用暗反应过程有关，1个蛋白质点（spot991）的功能与叶绿体基因的转录过程有关，1个蛋白质点（spot1305）功能与蛋白质翻译过程有关，1个蛋白质点（spot1058）在数据库中未找到匹配结果，这说明说明钼营养通过蛋白质表达调控冬小麦光合作用、叶绿体基因转录及蛋白质翻译等生理生化过程，但钼对光合作用的影响更为突出。10．结合含钼酶、激素、抗氧化系统、光合特性及内囊体膜组成的分析结果，提出了钼提高冬小麦抗寒力和增强光合作用的生理及分子机制。该机制以含钼酶活性的变化为基础，以植物钼营养对基因表达与蛋白质翻译的调控为核心，明确了叶绿体是缺钼条件下各种生化反应与代谢过程发生改变的关键部位，低温胁迫是各种基因表达及生理过程发生剧烈变化的诱发因素。更多还原

【Abstract】 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.更多还原