【作者】 张毅；

【导师】 邹志荣；

【作者基本信息】 西北农林科技大学 ， 设施园艺工程， 2013， 博士

【摘要】 盐碱胁迫是造成作物减产的主要非生物胁迫之一，关于提高作物耐盐碱性的研究已引起人们的广泛关注。多胺（PAs）是生物在其生命活动过程中产生的一类次生代谢物，在植物中具有调节基础代谢、影响生长发育、控制形态建成、延缓衰老等功能，主要包括腐胺（Put）、亚精胺（Spd）和精胺（Spm），其中Spd与植物抗逆性的关系最为密切。研究表明，外源Spd处理能够在一定程度上缓解盐胁迫的抑制效应，甚至能提高作物在高盐胁迫下的生产潜力。然而，目前关于外源Spd对植物抗盐性的影响机理研究多集中在NaCl上，而对碱性盐的研究报道极少。番茄（Solanum lycopersicum）是一种世界范围内广泛栽培和消费的蔬菜作物，也是植物遗传学及茄科作物研究中的模式植物。栽培番茄属于中度盐敏感型作物，盐碱逆境极易给其生产造成严重损失。本文以耐性不同的两个番茄品种（盐碱耐性较强的‘金棚朝冠’和盐碱耐性较弱的‘中杂9号’）为试材，采用水培法，通过模拟盐碱生态条件（NaCl:Na₂SO4: NaHCO₃: Na₂CO₃=1:9:9:1），结合外源施用0.25mmol·L^-1Spd（叶面喷施或浸种），研究Spd对盐碱胁迫下番茄幼苗生长、光合荧光特性、主要矿质元素含量、抗氧化保护系统、渗透调节系统、氮代谢和PAs代谢的影响，探讨外源Spd在提高番茄耐盐碱性中的生理调节功能。同时，对盐碱和外源Spd处理下盐碱敏感型品种‘中杂9号’的叶片差异蛋白进行了质谱分析和功能鉴定，以期初步揭示外源Spd对番茄响应盐碱胁迫的蛋白质表达调控基础。主要结果如下：1.盐碱胁迫下，两品种番茄幼苗生长受抑，叶片净光合速率、气孔导度、蒸腾速率、叶绿素含量、PSII光化学量子效率、光合电子传递速率、光化学淬灭系数、全P含量及叶片/根系全N、全K、Ca²⁺、Mg²⁺含量均显著降低，叶片非光化学淬灭系数及叶片/根系Na⁺含量均显著升高，且‘中杂9号’受胁迫的影响程度较大；在处理第5d，盐碱逆境对‘中杂9号’的光合限制以非气孔因素为主，而对‘金棚朝冠’的则以气孔因素为主。外源喷施Spd能够显著改善盐碱胁迫下番茄幼苗地上部的生长状况，提高叶绿素含量，增强PSII反应中心的光化学活性，有效减轻盐碱逆境对番茄叶片光合电子传递的抑制及其对光合作用的气孔限制和非气孔限制，并有助于维持番茄植株主要矿质营养元素间的相对平衡，且对耐盐碱性相对较弱的‘中杂9号’盐碱毒害的缓解效果更为明显。2.盐碱胁迫下，两品种番茄幼苗的游离氨基酸总量、NH₄⁺含量及谷氨酸脱氢酶（GDH）活性增加，而NO₃^-含量及硝酸还原酶、亚硝酸还原酶、谷氨酰胺合成酶（GS）、谷氨酸合酶（GOGAT）、谷草转氨酶、谷丙转氨酶活性均有所降低，同时，盐碱逆境的这种效应在外源Spd作用下有所减缓，尤其对‘中杂9号’的效果更明显。表明，外源喷施Spd可通过加强GDH、GS/GOGAT和转氨三大途径的协同作用以促进盐碱条件下过量氨的同化，使细胞内的NH₄⁺及其代谢酶维持在适度平衡状态，进而减轻氨毒害作用并有效缓解盐碱胁迫引起的氮代谢紊乱，从而促进植株生长。3.盐碱胁迫下，两品种番茄幼苗的超氧阴离子产生速率、过氧化氢含量增加，较高的活性氧（ROS）水平导致膜脂过氧化程度加剧，丙二醛含量明显升高；同时盐碱处理在一定程度上诱导了超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）的活性及脯氨酸、可溶性糖的积累。外源喷施Spd处理一方面通过提高盐碱逆境下细胞内SOD、POD、CAT的活性来降低ROS水平，从而减轻对膜脂的过氧化损伤；另一方面通过诱导盐碱胁迫植株体内渗透调节物质的进一步合成或积累来提高细胞的吸水能力，进而增强番茄幼苗的盐碱耐性。4.盐碱胁迫下，两番茄品种的根系不同种类、不同形态PAs含量均显著增加。与‘中杂9号’相比，‘金棚朝冠’的根系游离态和束缚态Spd、结合态Spd和Spm含量，及其S-腺苷蛋氨酸脱羧酶（SAMDC）和二胺氧化酶（DAO）活性在盐碱胁迫下的增幅较大。盐碱条件下，外源Spd浸种处理抑制了两品种的根系游离态Put积累，显著促进游离态Spd和Spm、结合态Spd及束缚态Put含量的增加。可见，盐碱胁迫下Spd浸种处理促进了游离态Put向游离态Spd和Spm的转化，以及结合态和束缚态PAs的积累。盐碱胁迫下，Spd浸种处理提高了‘中杂9号’根系鸟氨酸脱羧酶（ODC）、SAMDC和DAO活性，降低了其精氨酸脱羧酶（ADC）和PAs氧化酶（PAO）活性；同时，外源Spd降低了‘金棚朝冠’根系ADC和ODC活性，但提高了其SAMDC和DAO活性。这表明，外源Spd参与了盐碱胁迫下番茄幼苗体内PAs代谢的调节，同时，Spd浸种处理对耐性不同品种的效应存在着一定的差异。5.对盐碱敏感型番茄品种‘中杂9号’叶片的双向电泳分析结果显示，盐碱+Spd、单纯Spd喷施处理、对照、单纯盐碱处理植株的叶片蛋白点数分别为1172、1243、1084和954个。选取其中82个差异蛋白点进行质谱分析和数据检索，其中72个蛋白点得到了有效的搜库结果，这些蛋白主要参与了胁迫防御反应、氨基酸代谢、光合作用、光呼吸、能量转换、信号转导、蛋白折叠和转录后调控等生理过程。进一步表明，盐碱和外源Spd处理下植株可通过诱导多种代谢途径的协调作用来适应外界环境的变化。更多还原

【Abstract】 Saline-alkaline stress is one of the major abiotic stresses which causes crop failures, sothe research for improving the saline-alkaline tolerance of crops has been attracted greatattention. As a kind of secondary metabolites in organisms, polyamines （PAs） in plants havemulti-functions, such as regulating basic metabolisms, influencing growth and development,adjusting morphogenesis and slowing the aging process. Putrescine （Put）, spermidine （Spd）and spermine （Spm） are common PAs, and Spd is the most closely associated PA with stresstolerance in plants. Under salt stress conditions, exogenous Spd application has beensuccessfully used for alleviating the growth inhibitory effects, and even enhancing theproductive potential of crops exposed to high-salinity stress. However, the current researchabout the influence mechanisms of exogenous Spd on plant resistance, mostly focus on theneutral NaCl solution, while there is nearly no related report on the alkaline salt tolerance.Tomato （Solanum lycopersicum） is one of the widely cultivated and consumed crop inthe world, and it is also a good model for the plant genetics and solanaceous crops research.Cultivated tomatoes are moderately sensitive to salt stress, and salinity-alkalinity seriouslyreduces its production. In the present study, the effects of exogenous0.25mmol·L^-1Spd（foliar spraying or seed soaking） on the plant growth, photosynthetic fluorescencecharacteristics, main mineral elements contents, antioxidant system, osmotic adjustmentsystem, nitrogen metabolism and PAs metabolism were investigated, in two cultivars oftomato seedlings （‘Jinpengchaoguan’ and ‘Zhongza No.9’, the former being more tolerant tosaline-alkaline stress than the latter）, grown under salinity-alkalinity stress condition （NaCl:Na₂SO4: NaHCO₃: Na₂CO₃=1:9:9:1）, to explore the physiological functions of exogenous Spdon improving the saline-alkaline tolerance of tomato. Meanwhile, the differentially expressedproteins in leaves of ‘Zhongza No.9’ tomato seedlings under each treatment were identifiedand classified by MALDI-TOF/MS, in order to preliminarily reveal the protein foundationsinduced by Spd under saline-alkaline stress. Main research results were as follows:1. Under salinity-alkalinity stress, the seedling growth of the two tomato cultivars wasinhibited. The net photosynthetic rate, stomatal conductance, transpiration rate, chlorophyllcontents, photochemical quantum efficiency of PSII, photosynthetic electron transport rate, photochemical quenching coefficient and total P contents in leaves, as well as the total N,total K, Ca²⁺, Mg²⁺contents in leaves and roots, were all significantly reduced undersaline-alkaline stress. However, the leaf non-photochemical quenching coefficient and the Na⁺contents in leaves and roots were all significantly increased. Besides, there was greaterinfluence on ‘Zhongza No.9’. At the fifth day of treatment, the main limiting factor ofphotosynthesis in ‘Zhongza No.9’ tomato seedling was non-stomatal factor undersaline-alkaline stress, while it was stomatal factor for‘Jinpengchaoguan’. Under stressconditions, exogenous foliar spraying Spd could significantly improve the shoots growth, theleaf chlorophyll contents and PSII photochemical activities of both tomato cultivars.Moreover, the inhibition of leaf photosynthetic electron transportation, as well as the stomataland non-stomatal limitation, caused by salinity-alkalinity stress, could be effectively reducedby exogenous Spd, meanwhile, the main mineral elements contents were maintained in aproper balance, and the alleviative effect of Spd treatment on saline-alkaline damages wasmore obvious in ‘Zhongza No.9’ cultivar.2. Exogenous Spd may help reduce salinity-alkalinity stress-induced increases in freeamino acids, ammonium （NH₄⁺） contents, and glutamate dehydrogenase （GDH） activities;depress stress-induced decreases in nitrate contents, and nitrate reductase, nitrite reductase,glutamine synthetase （GS）, glutamate synthase （GOGAT）, glutamate oxaloacetatetransaminase （GOT）, and glutamate pyruvate transaminase （GPT） activities, especially for‘Zhongza No.9’. The results showed that exogenous Spd promotes the assimilation of excesstoxic NH₄⁺, by coordinating and strengthening the synergistic action of GDH, GS/GOGAT,and transamination pathways, all during saline-alkaline stress. Subsequently, NH₄⁺and itsrelated enzymes （GDH, GS, GOGAT, GOT and GPT）, in vivo, are maintained in a proper andbalanced state, to enable mitigation of stress-resulted nitrogen metabolic disturbances, andeventually promote plant growth.3. Under saline-alkaline stress, the Oˉ2.production rate and H2O2contents in leaves androots of both tomato cultivars increased at different degrees, and the higher levels of reactiveoxygen species （ROS） aggravated the membrane lipid peroxidation, which facilitated theproduction of malondialdehyde. Moreover, the activities of superoxide dismutase （SOD）,peroxidase （POD） and catalase （CAT）, as well as the accumulation of proline and solublesugar, could be induced by salinity-alkalinity stress to some extent. Exogenous Spd, on onehand, may help to alleviate the membrane lipid peroxidative injury, by further increasing thestress-induced SOD, POD and CAT activities to reduce the excessive ROS level. On the otherhand, Spd treatment can enhance the saline-alkaline tolerance of tomato seedlings, throughinducing the further biosynthesis and accumulation of osmotic adjustment materials. 4. PAs content, in any form, increased significantly during salinity-alkalinity stress. Theactivities of S-adenosylmethionine decarboxylase （SAMDC） and diamine oxidase （DAO）,contents of free Spd, soluble conjugated Spd and Spm, and insoluble bound Spd in roots wereincreased to a greater extent in ‘Jinpengchaoguan’ tomato seedlings exposed to saline-alkalinestress. Exogenous Spd markedly suppressed the accumulation of free Put, but promoted anincrease in free Spd and Spm contents, as well as soluble conjugated Spd and insoluble boundPut in both cultivars. From these data, we deduced that exogenous Spd promotes theconversion of free Put into free Spd and Spm, and soluble conjugated forms and insolublebound forms PAs under salinity-alkalinity stress. Furthermore, under saline-alkaline stress,exogenous Spd enhanced the activities of ornithine decarboxylase （ODC）, SAMDC and DAO,and reduced the activities of arginine decarboxylase （ADC） and polyamine oxidase （PAO） in‘Zhongza No.9’ roots. In addition, exogenous Spd reduced the activities of ADC and ODC,and increased the activities of DAO and SAMDC in ‘Jinpengchaoguan’ roots undersaline-alkaline stress. These results suggest that Spd treatment can regulate the metabolicstatus of PAs caused by saline-alkaline stress, and the effects of exogenous Spd on differentcultivars with differential salinity-alkalinity tolerance exist certain differences.5. Two dimensional gel electrophoresis analysis results showed that the leaf protein spotsin control, single Spd, single salinity-alkalinity, and salinity-alkalinity plus Spd treated plantswere1084,1243,954and1172, respectively. Eighty-two differentially expressed proteinspots were identified by MALDI-TOF/MS and related databases, in which72protein spotsgot valid results. The identified proteins were associated with the regulation of photosynthesis,photo respiration, energy pathway, signal transduction, amino acid metabolism, proteinfolding, post-transcriptional regulation, and so on. This provides further evidence thatsaline-alkaline stress and exogenous Spd treatment could induce the synergy of numerousmetabolic processes in plants to adapt to the changing environment.更多还原