【作者】 杨卫兵；

【导师】 王振林；

【作者基本信息】 山东农业大学 ， 作物栽培学与耕作学， 2014， 博士

【摘要】 小麦籽粒灌浆期是小麦籽粒形成的一个重要阶段。灌浆期间，小麦茎、叶等器官光合作用产生的糖类和转化的蛋白质通过同化作用贮存在小麦种子内。灌浆期干旱，不利于籽粒灌浆，导致减产。因此，研究小麦籽粒灌浆对水分亏缺的响应机制及调节途径，对提高小麦粒重有着重要的意义。研究表明，乙烯和多胺在作物的生长发育中发挥重要作用。然而，水分亏缺条件下，（1）小麦籽粒中多胺、1-氨基环丙烷-1-羧酸（ACC）浓度变化及其与小麦籽粒灌浆特性和旗叶光合特性的关系，外源调节物质调节籽粒灌浆的生理生化机制等尚不明确。（2）小麦籽粒腹部韧皮部的发育和结构及筛管伴胞（SE-CCs）复合体质膜ATPase活性，籽粒中蔗糖运输蛋白基因TaSUT1的表达与籽粒光合物质储存的关系等仍需进一步研究。明确以上问题对于丰富水分亏缺条件下的小麦栽培理论具有重要意义，并为外源化学物质的应用提供理论依据与技术支持。1粒重对水分亏缺响应的品种和粒位效应及原因分析粒重对水分亏缺的响应依品种类型及籽粒粒位而异。水分亏缺条件下，水浇地类型品种济麦22强势粒和弱势粒的粒重分别降低7.38%和23.5%，旱地类型品种山农16强势粒和弱势粒的粒重分别降低13.8%和2.2%。未分粒位，则济麦22粒重降低16.2%，而山农16粒重降低7.6%。利用Richards模型分析产生品种和粒位效应的原因，结果表明，水分亏缺条件下，济麦22强势粒和弱势粒的活跃灌浆期分别降低10%和6.1%，山农16强势粒和弱势粒的活跃灌浆期分别降低6.7%和6.7%；济麦22强势粒和弱势粒的平均灌浆速率分别降低15.8%和9.9%，山农16强势粒和弱势粒的平均灌浆速率分别降低0.5%和12.3%。总而言之，活跃灌浆持续期及平均灌浆速率的变化幅度可以解释水分亏缺条件造成粒重降低的品种效应，但是无法对水分亏缺造成粒重降低的粒位效应做出解释。喷施外源亚精胺（Spd）或胺基乙氧基乙烯甘氨酸（AVG）均显著提高籽粒在水分亏缺条件下的粒重，而喷施外源丙脒腙（MGBG）或乙烯利则造成相反的影响。水分亏缺条件下，综合分析了籽粒TaSUT1表达量和籽粒蔗糖含量随灌浆持续期的变化趋势，表明：灌浆前期，蔗糖运输能力较弱可能是限制灌浆的重要因素；灌浆中期，淀粉合成相关酶活性低是限制灌浆的重要因素；灌浆后期，淀粉合成相关酶活性低和蔗糖运输能力低共同成为限制籽粒灌浆的重要因素。2小麦籽粒多胺和乙烯合成对水分亏缺响应的品种和粒位效应及其与籽粒灌浆的关系水分亏缺显著提高籽粒中ACC浓度，进而提高籽粒的乙烯释放速率。水分亏缺显著提高籽粒中腐胺（Put）的含量。Put和乙烯合成对水分亏缺的响应依品种类型和籽粒粒位而异，以花后21天为例，水分亏缺条件下，济麦22强势粒和弱势粒中ACC浓度分别上升90%和164%，山农16强势粒和弱势粒中ACC浓度分别上升65%和13.2%；济麦22强势粒和弱势粒中Put浓度分别上升1.04%和7.9%，山农16强势粒和弱势粒中Put浓度分别上升34.4%和10.3%；济麦22强势粒和弱势粒中Spd浓度下降幅度差异不显著，Spd浓度分别下降33.7%和43.5%，山农16强势粒和弱势粒中Spd浓度分别下降15.1%和8.9%。在基因表达水平上分析表明，水分亏缺显著提高籽粒ACC和Put合成相关酶基因（ACS、ADC1、ADC2、ODC、AGM）的表达量，而降低亚精氨合成相关酶基因（Spd1、Spd2、SAMDC）表达量。相关分析表明，籽粒灌浆速率与Spd、Spm浓度及Spd/ACC、Spm/ACC的值存在显著正相关关系，与Put浓度在一定范围内存在正相关关系，与乙烯释放速率及ACC浓度存在显著负相关关系。另有相关分析表明，ACC和Put浓度与SuSase和AGPase存在显著负相关关系，而Spd及Spd/ACC的值分别与SuSase和AGPase存在显著正相关关系。因此，可以得出多胺和乙烯释放是通过影响淀粉合成相关酶活性进而影响籽粒灌浆速率的结论。研究还表明，小麦籽粒淀粉粒的体积和表面积分布呈双峰曲线，而数量分布呈单峰曲线。水分亏缺导致淀粉积累量的降低，同时，导致B-型淀粉颗粒的体积和表面积百分数下降，而提高A-型淀粉颗粒体积和表面积百分数。喷施外源Spd或AVG均可显著提高水分亏缺条件下的B-型淀粉颗粒的体积和表面积百分数。相关分析表明，乙烯和Spd在B-型淀粉颗粒形成的过程中呈现相互拮抗的关系，因此，水分亏缺条件下，提高Spd的含量、降低ACC浓度及乙烯释放速率有利于B-型淀粉颗粒的形成。3小麦籽粒腹部韧皮部SE-CCs复合体超微结构特征、其质膜ATPase酶活性对水分亏缺的响应及其与籽粒灌浆的关系针对济麦22弱势粒粒重对水分亏缺的响应较强势粒敏感的特征，本试验研究了水分亏缺条件下的济麦22籽粒腹部韧皮部SE-CCs复合体超微结构特征、其质膜ATPase酶活性及其与籽粒灌浆的关系，结果表明，正常供水条件下，花后15～20天的强势粒中，韧皮部SEs趋于成熟，呈现出清晰的细胞结构，且CCs有突出的细胞核位于细胞质的中央。随着水分亏缺处理时间的延长，ICs开始出现，且在花后15或20天，强势粒和弱势粒中CCs较少。花后25天，在正常供水条件下的强势粒中只观察有少量CCs。正常供水条件下的强势粒、水分亏缺条件下的强势粒和弱势粒中几乎观察不到CCs。花后25天，水分亏缺处理下的弱势粒韧皮部PPC出现质壁分离及细胞核浓缩现象，且SEs出现膜降解的现象。正常供水条件下，SE-CCs复合体、ICs和PPC细胞质膜及ICs和PPC细胞之间的胞间连丝呈现较强的ATPase活性（铅颗粒呈连续的带状分布），而水分亏缺条件下，SEs和ICs质膜及ICs与ICs之间的胞间连丝呈现较弱的ATPase活性（铅颗粒呈间断的点状分布），且CCs和PPC细胞质膜未发现有ATPase酶存在。以上结果表明，水分亏缺条件下，ICs较早的出现以弥补SEs纵向运输能力的不足，灌浆后期，弱势粒的韧皮部SE-CCs复合体的运输蔗糖的能力减弱，进而影响籽粒灌浆。4小麦旗叶乙烯合成对水分亏缺的响应及其与旗叶光合特性的关系针对济麦22粒重对水分亏缺的响应较山农16敏感的特征，本试验系统分析了济麦22旗叶光合特性对水分亏缺的响应及其与旗叶乙烯释放的关系。远红外成像分析表明，水分亏缺显著提高济麦22旗叶的温度，可能与水分亏缺条件下旗叶的蒸腾速率降低有关，而喷施外源Spd或AVG均可显著降低旗叶的温度。水分亏缺显著降低灌浆前期济麦22旗叶的胞间CO2浓度（Ci）、气孔导度（gs）及灌浆中后期的电子传递速率（ETR）和光系统II实际光化学效率（ΦPSII）,从而降低旗叶的光合速率，而喷施外源Spd或AVG均可显著提高旗叶的光合速率。研究还表明，灌浆前期气孔因素是导致水分亏缺条件下旗叶光合速率降低的主要原因。随着灌浆时间的持续，除了气孔因素之外，非气孔因素也成为造成旗叶光合速率降低的重要因素。水分亏缺显著提高旗叶ACC浓度和乙烯释放速率，喷施外源Spd或AVG均可降低水分亏缺条件下的乙烯释放速率。相关分析表明，旗叶ACC浓度及乙烯释放速率与PN、ΦPSII及ETR存在显著负相关关系，而ACC浓度与非光化学猝灭（NPQ）呈显著正相关关系。因此，水分胁迫条件下光合速率的降低与乙烯释放速率的增加密切相关。更多还原

【Abstract】 The stage of grain filling plays an important role in the grain formation. During grain filling,the sugars and proteins produced by leaves or stems of wheat are stored in the seed by theassimilaton function. Water deficit during grain filling had a negative effect on grain fillingand wheat production, and it indicated that the response mechanism of grain filling to waterdeficit and regulation pathway play important roles in improving grain weight. It proved thatethylene and polyamines play key roles in the responses of crops growth to abiotic stress.However, the relationship of polyamines and ethylene biosynthesis to grain filling and flagleaves photosynthetic characteristics as well as the regulatory mechanism of exogenouschemicals were unclear. The relationships of the ultrastructure of the abdominal phloemtissues characters, the ATPase activity on abdominal phloem tissues and the TaSUT1geneexpression to photoassimilate storage of grains under water deficit need to be furtherinvestigation. It can rich the theory of wheat cultivation under water deficit conditions, andsupply theoretical basis and technical support for the application of exogenous substances.The main results were as follows.1Grain weight of different variety and grain-bit in response to water deficitThe responses of grain weight to water deficit differed with the varieties and grain types.SD reduced the weight of superior and inferior grains, by6.2and23.54%in JM22,13.8and2.2%in SN16, respectively. SD reduced the total grain weight of SN16and JM22by7.6and16.2%respectively. The analysis by using the Richards model indicated that SD reduced the active grain filling stage of superior and inferior grains, by10%and6.1%in JM22,6.7%and6.7%in SN16, respectively. It also suggested that SD educed the average grain-filling rate ofsuperior and inferior grains, by15.8%and9.9%in JM22,0.5%and12.3%in SN16,respectively. Application of exogenous Spd or AVG significantly increased the grain weightunder water deficit, while MGBG or ethephon showed the opposite effects. Changes ofTaSUT1expression levels and sucrose content with the grain filing showed that sucrosetransport capacity may be a major factor limiting grain filling at initial and later grain fillingstage, while the key enzymes involved in the sucrose-to-starch conversation becomes a majorfactor limiting grain filling at middle filling stage.2The responses of polyamines and ethylene biosynthesis to water deficit in wheat and itsrelations to grain fillingHigher Spd and Spm concentration and Put concentration, EER and ACC concentrationwere found in superior grains than those in inferior ones. Opposite to the variations of Spdand Spm concentration, ACC, Put concentration and EER were significantly increased underSD. The percentage variations of PAs and ACC differed with cultivars and grain types. ACCconcentration of superior and inferior grains under SD increased significantly at21DPA, by90%and164%in JM22,65%and13.2%in SN16, respectively. The equivalent value of Putconcentration was1.04%and7.9%in JM22,34.4%and10.3%in SN16. Spd concentrationof superior grains showed a higher decrease than that of inferior ones in both cultivars, whileSpm exhibited an opposite trend between both grain types. These percentage variations werehighly consistent with the differed responses of weight of both grain types to SD in JM22andSN16. SD apparently increased the ACC and Put concentration as well their biosynthesisrelated genes expression, while degraded the Spd biosynthesis. Grain filling rate wasnegatively correlated with EER and ACC concentration, while positively correlated with Spdand Spm concentration as well as the ratio of Spd or Spm to ACC. Exogenous Spd oraminoethoxyvinylglycine obviously reduced ACC concentration and EER and increased Spdand Spm concentration, while exogenous ethephon or methylglyoxal-bis showed the oppositeeffects. SuSase, AGPase and SSSase negatively correlated with ACC and Put concentrations,but positively correlated with Spd concentration and Spd/ACC, indicated that ACC and Spdinteract in mediating the influence of SD on filling. The volume and surface area distribution of starch granules showed a bimodal curve, while the number distribution exhibited aunimodal curve. SD caused a marked drop in grain weight, grain number and starch content,also led to a significant reduction in the proportion (both by volume and by surface area) ofB-type starch granules (<10μm), with an increase in those of A-type starch granules (>10μm). Application of Spd or AVG increased the proportion (both by volume and by surfacearea) of B-type starch granules under SD. Correlation analysis suggested that ethylene andSpd showed an antagonism relation in the formation of B-type granules. These resultssuggested that it would be good for the formation of B-type starch granules to have thephysiological traits of higher Spd and lower ACC concentration and ethylene emission underSD.3Abdominal phloem tissues characters of wheat caryopsis in the responses of grainfilling to water deficitMost of the SEs of the phloem are associated with CCs and have walls contiguous withthose of the PPC. The superior grains under WW at15or20DPA, of which SEs of abdominalphloem were structurally mature, and exhibited a clear lumen and plasma membrane, and CCshad prominent nuclei distributed in dense cytoplasm. At15and20DPA, ICs were found toreside predominantly in SE-CCs complexes. Whereas CCs were hardly observed in inferiorgrains under WW, superior grains under SD and inferior grains under SD. As a consequence,ICs played the functions of long-distance assimilate transport to compensate the decreasedassimilate transport functions of SEs with the development of grain and the extension of waterdeficit. A few CCs was showed in superior grains under WW, while it was hardly observed ininferior ones under WW, superior and inferior grains under SD at25DPA. It is noteworthythat the PPC in inferior grains under SD showed the phenomenon of plasmolysis and nuclearchromatin condensation, and the SEs exhibited the characters of membrane degradation atlater filling stage, suggested that the characters of PPC and SEs in inferior grains was moresensitive to SD than the superior ones. Under WW, ATPase activity on plasma membrane ofSE-CCs complexes and ICs and PPC visualized by particles of lead phosphate precipitate wasconsiderably increased when compared with the control. The band of lead phosphateprecipitate on the plasma membrane was intensified, widened and became continuous asconsequence of the precipitate increase. Also the plasmodesm between ICs and PPC showed a high density of lead phosphate precipitate. Under SD, the plasma membrane of SEs and ICsand the plasmodesm between ICs and ICs showed a lower density of lead phosphateprecipitate. Unlike SEs and ICs, ATPase activity was hardly observed on the plasmamembrane of CCs and PPC. These observations suggested that apoplastic transport based onthe driving of H+-ATPase on plasma membrane was easily affected by SD condition, and thenlead to a low photosynthetic substance accumulation in inferior grains.4The responses of ethylene biosynthesis to water deficit in flag leaves and its relations tophotosynthesis and photochemical efficiencyThermal images indicated that SD obviously increased the temperature of flag leaves,mainly due to the decrease in E under SD. The marked increases in both EER and ACCconcentration were observed under SD, which can be effectively reversed by exogenous Spdor AVG. Exogenous Spd or AVG to some extent decreased the temperature of flag leaves. Thestrongly decreased of PNand gsas well as the photo-damage of PSII under SD at14and21DPA were also observed. Ciwas reduced at7DPA, but slightly increased at14and21DPAunder SD, indicating that the decreased PNat7DPA might result from stomatal limitations,while its decrease at14and21DPA might be attributed to nonstomatal limitations.Correlation analysis suggested that EER and ACC showed negative relations tophotosynthesis and photochemical efficiency. Data obtained suggested that the effects of SDwere predominantly mediated by the increase in EER and ACC concentration, which greatlydecreased the leaves photosynthesis and photochemical efficiency. Application of Spd or AVGevidently decreased the EER and ACC concentration, and thus exhibited a positive influenceon the leaves photosynthesis and photochemical efficiency under SD.更多还原