【作者】 李耕；

【导师】 董树亭； 刘鹏；

【作者基本信息】 山东农业大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 本试验于2010-2012年在山东农业大学黄淮海区域玉米技术创新中心和作物生物学国家重点实验室进行。采用两个保绿差异明显的玉米自交系为材料（保绿型：齐319（Q319）、非保绿型：黄早四（HZ4））。采取单株盆栽种植模式。氮素处理分为缺氮（N0）和正常施氮（N40）两种，P、K肥按照大田标准正常施入，正常施氮处理苗期基肥50%+大口期追肥50%。利用叶绿素荧光诱导动力学和820nm光吸收曲线系统研究了不同保绿型玉米花后衰老过程中叶片光合作用衰退的原因及氮素调控机理。同时利用双向电泳技术，分析衰老过程中叶片差异蛋白质组学的变化规律及氮素调控关键位点。主要结果如下：1不同保绿型玉米花后叶片光合特性的变化差异衰老中前期叶片净光合速率与光系统性能间存在极显著的相关性，该阶段光系统性能是限制叶片净光合速率强弱的主要原因，但两者之间的相关性在衰老中后期呈减弱趋势，同时叶片净光合速率与羧化反应系统性能间的相关性呈现增强趋势，因此在叶片衰老中后期，羧化系统活性成为限制叶片净光合速率强弱的主要因素。两品种相比，保绿型玉米Q319叶片光系统性能在衰老中前期显著高于非保绿型玉米HZ4，高性能维持期也显著长于HZ4；同时，羧化反应性能衰老中后期下降显著低于HZ4，对减缓净光合速率后期下降起到了稳定作用。2氮素对玉米花后叶片光合特性的影响施氮条件下两品种叶片光系统性能与净光合速率间的相关性关系向衰老后期延续。羧化反应关键酶活性与净光合速率之间呈显著相关性的时期向衰老前期提前，同时向衰老后期延续。氮素对光系统性能和羧化反应系统性能同时提升的作用是维持同化力形成与消耗平衡的关键。品种间相比，HZ4叶片光系统性能和羧化反应关键酶活性在施氮条件下较缺氮处理随略有提高，但不足以改善叶片衰老过程中两系统的整体变化趋势，因此衰老中前期光系统性能下降明显，中后期羧化反应系统性能快速降低，同化力形成与消耗之间存在着不同步现象，客观表现为叶片衰老中前期“稳定态”和“缓降态”维持时间较短，衰老中后期“速降态”过早出现。3不同保绿型玉米花后叶片差异蛋白质组变化两品种叶片衰老过程中差异蛋白主要分布在叶绿体、细胞间质和细胞膜内。按主要功能分类在整个衰老过程占有比例最高的隶属于碳代谢、光合作用、能量代谢和ATP互变4个领域。其中，占比例最大的两个功能组分别是碳代谢和光合作用功能组，两组的比例在衰老中期和后期显著降低，其中保绿型玉米Q319两组随衰老的降幅显著高于HZ4。两品种差异蛋白中权重蛋白分布也主要集中于光合作用组和碳代谢组。HZ4权重蛋白活性随衰老呈下调趋势，蛋白变化范畴集中于羧化反应酶系，且前期无活性上调现象，而Q319权重蛋白活性则随衰老呈先升后降趋势，蛋白变化范畴广，在羧化反应酶系、光反应酶系及抗氧化酶系中均有表达，尤其是前期光反应相关酶系活性的上调显著。4缺氮对玉米花后叶片差异蛋白质组的影响缺氮条件下，保绿型玉米Q319叶片在衰老前期差异蛋白质表达量以上调为主，上调蛋白数量显著多于施氮处理，但表达量显著小于施氮处理。该时期表达量上调的差异蛋白主要功能集中于超氧化物清除酶系、光合作用酶系以及碳代谢和能量代谢酶系，其中具有光合作用功能和碳代谢功能的上调蛋白在衰老前期占有比例显著多于其他功能。缺氮处理叶片在衰老中期和后期，具有超氧化物清除、光合作用及碳代谢功能的权重差异蛋白表达数量显著增加，同时表达量下调幅度增大，导致叶片衰老中后期活性氧清除性能下降，光合作用性能降低，同时伴随碳代谢能力下降。5不同保绿型花后叶片光/羧相关蛋白质组变化差异两品种叶片衰老过程中，隶属于PSI电子传递链末端具有同化力形成功能的蛋白质组活性下降速度显著快于定位于PSII电子传递链前段具有光能/电能转化的蛋白质组活性，导致用于羧化反应的同化力数量下降，与本试验光合生理所阐述的光系统在衰老过程中性能的强弱主要受控于PSI性能的高低结果一致。品种间相比，非保绿型玉米HZ4同化力形成能力下降在衰老过程中明显快于保绿型玉米Q319，与两品种衰老过程中光系统差异结果一致。衰老中前期Q319叶片羧化系统同化力消耗能力显著高于HZ4，利于维持同化力形成与消耗之间的平衡，较好的解释了Q319叶片衰老中前期存在着明显的光合高值持续期，而HZ4则衰老快速下降的分子生物学原因。6缺氮对玉米花后叶片光/羧相关蛋白质组的影响缺氮条件下，叶片衰老过程中光系统中下调数量最多和下调量最大的差异蛋白主要集中于光合电子传递链末端PSI处，多以具有氧化还原功能的铁硫蛋白和ATP合成蛋白为主，且光系统差异蛋白数量占光/羧总数量的比例显著增加，其中隶属于PSI具有同化力形成功能的蛋白活性下降更为明显。同时，缺氮条件下叶片羧化系统相关差异蛋白的表达量在衰老前期上调幅度显著低于施氮处理，衰老中后期羧化系统相关差异蛋白表达量下调趋势显著高于施氮处理。总体上，氮素显著的提高了叶片衰老前期的羧化系统关键酶的活性，使之消耗同化力的能力增强，同时叶片衰老前期光系统同化力形成能力在施氮条件下显著上升，因此，施氮条件下叶片中前期光系统提供的大量同化力能够被羧化系统充分消耗，从而促进了该阶段叶片光合高值持续时间。更多还原

【Abstract】 In this research we compared the discrepancy of physiology and biochemistry druingplant senescence in two different stay green maize varieties (Stay green maize variety: Q319;Non stay green maize variety: HZ4), as well as the regulation of two level N-fertilizertreatment by using of chlorophyll a fluorescence transient and light absorbance at820nm, andanalysed the changes of differential proteomics in leaf of two different stay green maizevarietise during senescence and the critical control point under N-fertilizer applied treatment.The experiment was carried out in the Yellow, Huaihe and Haihe River Basin MaizeTechnological Innovation Center and the States Key Laboratory of Crop Biology of ShandongAgricultral University from2010to2012. The experiments take the potted plant growingmethods, providing non-fertilization (N0) and normal fertilization treatment (N40), and the P,K-fertilizer applied as normal treatment. In normal N-fertilizer treatment, the total N-fertilizerwas applied by base fertilizer50%at seeding stage and topdressing50%at anthesis stage.The main results as follow:1Discrepancy of photosynthetic characteristics during leaf senescence in two differentstay green maize varietiesAt early and middle senescence stage, the significant correlation was observed betweennet photosynthetic rate (Pn) and photosystem performance, which indicating Pn was restrictedthe photosystem performance at this period. The correlation between Pn and photosystemperformance tended to be weaker from middle to later senescence stage, nevertheless thecorrelation between Pn and carboxylation system performance tended to be stronger as aresult of which the carboxylation system performance restricted Pn at middle and latersenescence stage. Compared to non stay green cultivar HZ4, relatively higher photosystemperformance and longer duration were observed at early and middle senescence stage instay-green cultivar Q319. Meanwhile, relatively lower reduction of carboxylation systemperformance in Q319postponed the reduction of Pn at late senescence stage. 2Effect of the N fertilizer on leaf photosynthetic characteristics after anthesisThe significant correlation between the photosystem performance and net photosyntheticrate (Pn) in leaf of the two varieties last to the later senescence stage under N-fertilizerapplied, and the significant correlation between activity of the key carboxylation enzymes andPn the revealed in the early period of senescence and last to the later senescence stage. Theimproved carboxylation and photosystem performance by N-fertilizer applying played a keyrole to maintain the balance between forming and consuming of assimilatory power.Compared with the N-fertilizer lacking treatment, although the perfomance of photosystemand carboxylation of HZ4were both increased under N-fertilizer applied, but the increasedmagnitude did not ameliorate the changes tendency of two system performance in the processof senescence. Therefore, a decreasing tendency of the photosystem performance wasobserved at the early stage of senescence, and the performance of carboxylation declinedrapidly at the later stage, which resulted in the forming and consuming of assimilatory powerout-sync, and showed a short maintaining time for the “stable state” and “slowly declinestate” at the early stage of senescence, but the “rapidly decline state” was presented early atthe later stage of senescence.3Changes of differentail proteomics during leaf senescence in two different stay greenmaize varietiesThe differential proteomics of leaf in two varieties was mainly distributed in thechloroplast, interstitial cell and cell membrane. According to the classification of the mainfunction, the differential proteomics during leaf senescence can be distributed in4areas:carbon metabolism, photosynthesis, energy metabolism and ATP interconversion. The largestproportions among these areas were accounted for two functional groups: carbon metabolismand photosynthesis, which decreased significantly in the aging metaphase and anaphase. Intwo different stay green maize varieties, the declined rate of spots number in Q319of twomain groups was significantly higher than HZ4during leaf senescence. The change of weightprotein during senescence showed that, the activity of weight protein in HZ4isdown-regulated with aging, which was mainly concentrated in carboxylation enzymes. Butthe activity of weight protein in Q319was increased first and then decreased with aging, andthe category of these weight protein changes in a wide range, including carboxylationenzymes, photosystem enzymes and antioxidant enzymes. Importantly, the photosystem-related enzyme of Q319was increased significantly at early senescence stage. 4Effects on leaf differential proteomics after anthesis under nitrogen deficiencyUnder nitrogen deficiency condition, the expression of leaf differential protein in earlyaging of Q319was up-regulated, the number of which was significantly more than N-fertilizerapplied condition, but, the expression quantity was markedly less than N-fertilizer appliedcondition. During early aging period, the function of up-regulated proteins were mainlydistributed in superoxide scavenging, photosynthesis, carbon metabolism and energymetabolism system. Specially, the up-regulated protein with the function of photosynthesisand carbon metabolism group in early aging period was significantly more than other groups.Compared with early senescence stage, the expression number of weight protein with thefunction of superoxide scavenging, photosynthesis and carbon metabolism were significantlyincreased at later senescence stage, but the expression quantity was markedly decreased. As aconsequence, the performance of activated oxygen scavenging, photosynthesis and carbonmetabolism ability of leaf were significantly decreased in later aging period.5Changes of photosystem and carboxylation-related differentail proteomics during leafsenescence in two different stay green maize varietiesIn the process of leaf senescence, the quantity of assimilation power provide tocarboxylation was signifcantly decreased, that was caused by the activity of the proteins withassimilatory power synthetic function belong to the end of electron transport chain (ETC) ofPSI declining heavier than that of proteins belong to ETC front with function of translateinglight energy to electronic power. This conclusion was accordant with the photosyntheticphysiology results that showed the photosystem performance was manily controlled by PSIactivity in the process of aging. Varieties compared showed that the capability of assimilationpower forming decreased faster during leaf senescence in HZ4than Q319, this conclusionwas accordant with the photosystem performance results that changed during the process ofsenescence. The details showed, in the early-middle aging period, the dissipation capacity ofassimilation power by carboxylation system was greater higher in Q319than HZ4, it waspropitious to maintain the balance between assimilation power forming and consumption, andexplained the reason why the high Pn could be maintained at the early senescence stage inQ319, but not exsit in HZ4.6Effects on photosystem and carboxylation-related proteomics after anthesis undernitrogen deficiencyUnder N-fertilizer deficiency condition, the most down-regulated protein was iron-sulfurprotein and ATP synthetase with the redox capability, and both the expression number andquantity in which mainly centralized in end of ETC of PSI during the process of leafsenescence, and the amount of differential protein in photosystem was significantly more than that in the carboxylation system. In carboxylation system, the expression quantity ofup-regulated differential protein was less under N-fertilizer condition than N-fertilizerapplied in early aging period, but this tendency was turned at middle-last senescence stage. Ingeneral, the activity of key enzyme in carboxylation was markedly increased underN-fertilizer applied in the early aging period. Meanwhile, the capability of assimilation powerconsuming and forming was greater improved too. As a consequence, the performance ofactivated oxygen scavenging, photosynthesis and carbon metabolism ability of leaf weresignificantly decreased in later aging period, under N-fetilizer applied condition, thephotosystem provided large amounts of assimilation power at early senescence stage, andwhich can be consumed by assimilation process in carboxylation system timely. Thereby, theleaf photosynthetic duration of high value was promoted in the early-middle aging period.更多还原