水稻强弱势籽粒灌浆差异的分子生态学机制研究
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摘要
水稻尤其是大穗型的水稻，存在着强弱势籽粒灌浆差异的现象，弱势籽粒灌浆差限制了水稻高产潜力的发挥，如何提高弱势籽粒的灌浆饱满度及其千粒重是当前水稻高产研究的热点问题。强弱势籽粒灌浆差异的形成是个复杂的分子生态学过程，涉及多个基因的表达调控。虽然前人已就此问题在分子水平上开展了大量的研究，但是关于强弱势籽粒之间的蛋白表达及蛋白磷酸化修饰的差异未见报道。本研究运用双向电泳技术及Pro-Q磷酸化原位检测技术，选用大穗型常规稻“金恢809”作为研究对象，动态比较分析了强弱势籽粒灌浆之间的分子生理差异。主要研究结果如下：
1、通过形态学及干物质动态分析发现水稻“金恢809”存在显著的强弱势籽粒灌浆差异现象。强势籽粒在花后5-20天，干物质重增长迅速，而弱势籽粒花后存在着一个滞育期，直到花后15天才启动灌浆，其籽粒的大小和干物质重都比强势籽粒低。在滞育期内弱势籽粒可溶性糖含量较高，说明碳水化合物的供应不是限制弱势籽粒灌浆启动的主要原因。通过考察不同种植季节以及不同栽培调控措施下水稻强弱势籽粒干物质动态变化，发现强势籽粒灌浆稳定，而弱势籽粒灌浆易受环境所调控。合理的水肥调控仅能够一定程度促进弱势籽粒的灌浆，但无法促使弱势籽粒灌浆启动的提前。
2、为了明确限制弱势籽粒灌浆启动的分子机制，构建了弱势籽粒滞育期及灌浆前期的蛋白表达及磷酸化蛋白表达图谱，在弱势籽粒灌浆启动过程中，共鉴定到86个蛋白点和38个磷酸化蛋白点出现显著的表达变化，其中即出现蛋白表达变化的又出现磷酸化修饰变化的蛋白点有6个。经过MALDI-TOF/TOF-MS及LC-MS/MS的分析，蛋白点全部成功得到鉴定。通过蛋白丰度及功能的分析发现，萌发素蛋白的上调表达及α-微管蛋白的磷酸化修饰能够打破弱势籽粒胚乳细胞发育的停滞，导致弱势籽粒胚乳膨大，形成库容，为下一步淀粉累积做准备，而蔗糖合酶、UDP葡糖焦酸酶、ADP葡萄糖焦磷酸化酶、颗粒结合型淀粉合成酶、丙酮酸磷酸二激酶的上调表达，以及丙酮酸磷酸二激酶,ADP葡萄糖焦磷酸化酶，磷酸甘油酸酯变位酶和14-3-3蛋白的磷酸化修饰导致了淀粉合成行为的启动。与此同时，弱势籽粒从滞育期到灌浆期，磷酸戊糖途径减弱，糖酵解途径增强，产生更多的能量及中间产物供弱势籽粒胚乳细胞的增殖及淀粉的合成。
3、为了明确强弱势籽粒灌浆差异形成的分子机制，构建了强弱势籽粒灌浆启动后对应的不同灌浆时期的蛋白表达及磷酸化蛋白表达图谱，共鉴定到156个蛋白点和54个磷酸化蛋白点在强弱势籽粒之间出现表达变化，其中即出现蛋白表达变化的又出现磷酸化修饰变化的蛋白点数有16个。经过MALDI-TOF/TOF-MS及LC-MS/MS质谱分析，蛋白点全部成功得到鉴定。研究发现，灌浆的前期，弱势籽粒中可溶性糖含量高于强势籽粒，淀粉合成关键蛋白的低丰度表达，限制了淀粉的合成；灌浆的中期，弱势籽粒中蔗糖及淀粉相关蛋白的低丰度表达是导致其淀粉合成效率低的主要原因；而到了灌浆的后期，碳水化合物供应的不足又成为限制其淀粉合成的主要原因。磷酸化分析发现，葡糖磷酸变位酶，UDP葡糖焦酸酶，ADPG焦磷酸化酶，颗粒结合型淀粉合成酶、磷酸化酶及丙酮酸磷酸二激酶在强弱势籽粒之间存在着磷酸化修饰的差异，暗示着在磷酸化修饰层面调控强弱势籽粒淀粉合成关键酶活性差异的可能性，同时也发现这些淀粉合成关键酶会通过磷酸化修饰并在14-3-3的调控下，形成淀粉酶复合体。此外，在弱势籽粒灌浆过程中糖酵解、三羧酸循环及乙醇代谢途径相关蛋白的表达量底，导致其碳流通量低，产生供胚乳细胞分化及淀粉合成的能量及前体相对强势籽粒较少，限制了弱势籽粒库容的形成及淀粉的合成，而肌动蛋白，肿瘤翻译调控蛋白及微管蛋白又限制了弱势籽粒胚乳细胞的分化。
4、依据比较蛋白组学结果，筛选了7个限制弱势籽粒灌浆启动及9个导致弱势籽粒灌浆不充分的关键蛋白，蛋白丰度分析发现这些蛋白在强弱势籽粒之间存在着时空表达差异，其中14-3-3蛋白被认为在强弱势籽粒灌浆差异形成的过程中具有重要调控作用。进一步通过亲和层析技术探讨了籽粒中14-3-3蛋白的互作网络，共鉴定到24个互作蛋白，发现淀粉合成过程的ADPG焦磷酸化酶、淀粉分支酶、支链淀粉酶，转化酶及丙酮酸磷酸二激酶与14-3-3蛋白存在着紧密的互作关系。 LC-MS/MS/MS分析发现籽粒中14-3-3蛋白存在着丝氨酸和苏氨酸的磷酸化位点，据此认为14-3-3在强弱势籽粒之间的蛋白及磷酸化修饰差异是导致强弱势籽粒淀粉合成差异及对环境产生不同响应机制的一个关键信号调控因子。
综上所述，本研究发现水稻“金恢809”存在着显著的强弱势籽粒灌浆差异现象，进一步运用比较蛋白组学策略分析了强弱势籽粒灌浆过程中基因的时空表达变化差异，揭示了弱势籽粒灌浆启动及强弱势籽粒灌浆差异形成的蛋白作用及磷酸化信号调控机制，筛选出关键的信号调控因子14-3-3蛋白。最后，依据本研究的结果，提出了改善弱势籽粒灌浆的分子遗传改良及分子生态调控措施。
The asynchronous filling of superior and inferior spikelets is a commonphenomenon in rice (especially the extra-heavy panicles cultivars) and the poorgrain-filling in inferior spikelets limited the high yield potential of rice. Currently,improvement of the grain-filling rate and grain weight in inferior spikelets is one ofthe major tasks to enhance rice yield. The formation of asynchronous filling ofsuperior and inferior spikelets is a complicated ecopisiological process, whichinvolved in multiple gene expression. Although many researches focus on themolecular mechanism of the asynchronous filling, few studies were carried out at theprotein and phosphoprotein level. In order to further understand the molecularphysiology of asynchronous filling of superior and inferior spikelets, we analyzed thechanges of protein expression and phosphorylation in the superior and inferiorspikelets during the grain filling of rice “jinhui809” with the proteomic approaches,including the technology of two-dimensional electrophoresis (2-DE) and Pro-QDiamond phosphoprotein gel stain.
The morphological analyses showed that rice “jinhui809” has an obviousphenomenon of asynchronous filling of superior and inferior spikelets. The size andgrain weight of superior spikelets increased rapidly at5–20day after flowering (DAF),while that of inferior spikelets hardly increased until15DAF, which indicated thedevelopment of inferior spikelets was stagnant at0-15DAF. The size and grain weighof superior spikelets were better than those of inferior spikelets. In the stagnant stage,the inferior spikelets accumulated high concentrations of soluble carbohydrate，which indicated that the carbohydrate supply didn’t limit the starting of grain-fillingin inferior spikelets. The analyses of grain weigh of superior and inferior spikeletsunder different cultivation measures showed that the grain-filling of inferior spikeletsis more sensitive to the environmental factors than that of superior spikelets. Thesuitable water and fertilizer management was only favorable for the grain-filling ofinferior spikelets in some degree, but could not promote the starting of grain-filling ofinferior spikelets. The results obtained from the present study suggested that the genetic diversity between the superior and inferior spikelets was the major reason forthe poor grain-filling of inferior spikelets.
To better understand the molecular mechanisms of the starting of inferiorspikelets filling, we took two-dimensional gel-based proteomic andphosphoproteomic approaches to profile the proteins with abundance andphosphorylation state changes in inferior spikelets at stagnant stage and earlygrain-filling stage. A total of86protein spots and38phosphoprotein spots showeddifferential abundance changes by more than1.5-fold, and all of them weresuccessfully identified by the MALDI-TOF/TOF MS and LC-MS/MS. Only6protein spots showed significant changes in both abundance and phosphorylation. Theresults showed that the up-regulated expression of Germin-like protein (GLP) and thephosphorylation change of Alpha-tubulin may favorable to break out the stagnant ofinferior spikelets and promote the cell enlargement, thus leading to the sink capacityformation. The up-regulated expression of Sucrose synthase (SUSase), UDP-glucosepyrophosphorylase(UDPase), ADP glucose pyrophosphorylase (AGPase), Granule-boundstarch synthase(GBSS), Pyruvate orthophosphate dikinase (PPDK) and the phosphorylationchange of UDPase, AGPase, Phosphoglucomutase (PGM),14-3-3protein may help tostart the starch synthesis. At the same time, the Embden-Meyerhof-Parnas pathway (EMP)activity was increased and the Pentose Phosphate Pathway (PPP) activity was decreased,and thereby, more energy and building blocks for cell enlargement and starchsynthesis in the inferior spikelets could be available.
To gain a better understanding of the molecular mechanisms of the poorgrain-filling of inferior spikelets, comparative proteomic and phosphoproteomicanalysis between the superior and inferior spikelets at different grain-filling stagewere also carried out. A total of156protein spots and54phosphoprotein spotsshowed differential abundance changes between superior and inferior spikelets bymore than1.5-fold, and all of them were successfully identified by the MALDI-TOF/TOF MS and LC-MS/MS. Only16protein spots showed significant changes inboth abundance and phosphorylation. Based on our results, the reasons for the poorgrain-filling of inferior spikelets can be documented as follows. Firstly, in the early grain-filling stage, the lower abundance of starch synthesis related protein in inferiorspikelets compare to the superior spikelets limited the starch accumulated. Secondly,in the middle grain-filling stage, the down-regulate expression of sugar conversionand starch synthesis related protein resulting in slow grain-filling of inferior spikelets.Finally, in the late grain-filling stage, the assimilate supply is the main factor thatleads to poor grain-filling of inferior spikelets. Significant phosphorylation changesbetween superior and inferior spikelets were also found for6proteins involved instarch synthesis, such as PGM, UDPase, AGPase, GBSS, PPDK, and phosphorylase,suggesting that the activity of key enzyme of starch synthesis in inferior spikelets canbe modificated by the phosphorylation. The results also showed that thephosphorylation of the key enzyme of starch synthesis was necessary for theformation of a starch synthesizing protein complex (SSPC) and14-3-3protein mayacts as a scaffold protein holding enzymes of starch synthesis together in aphosphorylation-dependant SSPC. In addition, down-regulated expression of theenzyme proteins related to EMP, Tricarboxylicacidcycle (TCA) and alcohol decreased thecarbon flux in inferior spikelets during the grain-filling stage, thus leading less energyand building blocks for cell enlargement and starch synthesis in the inferior spikeletscompared to the superior spikelets. The author also found that low protein expressionof Translationally controlled tumor protein (TCTP）and tubulin, phosphorylation change ofalpha-tubulin, and early embryogenesis protein caused the low cell division rate in inferiorspikelets.
According to the results of comparative proteomic analysis,7key proteinsrelated to the starting of the inferior spikelets and9key proteins related to the poorgrain-filling of inferior spikelets were selected. The key proteins in the superior andinferior spikelets exhibited different temporal and spatial expression patterns, whichcaused significant differences in grain-filling at different spikelet positions on thepanicle. Among all of the key proteins,14-3-3protein is considered to be an importantregulatory protein for the poor grain-filling of inferior spikeletss. Then, the14-3-3protein was over-expressed, immobilized and used to affinity purify14-3-3bindingproteins from rice spikelets. These proteins were fractionated by PAGE and identified by LC-MS/MS. In total,2414-3-3binding proteins were identified. These proteinsfell into5functional categories. The largest category was for carbohydratemetabolism, including enzymes for starch synthesis and modification (e.g. AGPase,Starch branching enzyme, Pullulanase, Invertase and PPDK). In the present study, twophosphorylation sites at the serine and threonine residue of14-3-3protein wereidentified by the LC-MS/MS/MS. This work illustrates the starch synthesis in which14-3-3prtoein may be involved, and augments the different expression andphosphorylation changes in superior and inferior spikelets, which demonstrated thekey roles of14-3-3protein in asynchronous filling of superior and inferior spikelets.
In conclusion, the present field experiments showed that rice “jinhui809” hasobvious asynchronous filling of superior and inferior spikelets. The comparativeproteomic and phosphoproteome analyses further revealed the changes of proteinexpression and phosphorylation in the stagnant stage of inferior spikelets and thegrain-filling stage of inferior spikelets compared to the superior spikelets. Thefindings explained the asynchronous filling properties of superior and inferiorspikelets which were attributed to differential expression of proteome level intemporal and space. In addition, one of the important regulatory protein called as14-3-3protein was selected and its function was studied by the affinitychromatography technology. Finally, according to the present results, the authorproposed two effective ways for molecular genetic manipulations and molecularecological regulations to enhance the grain-filling of inferior spikelets.
1、通过形态学及干物质动态分析发现水稻“金恢809”存在显著的强弱势籽粒灌浆差异现象。强势籽粒在花后5-20天，干物质重增长迅速，而弱势籽粒花后存在着一个滞育期，直到花后15天才启动灌浆，其籽粒的大小和干物质重都比强势籽粒低。在滞育期内弱势籽粒可溶性糖含量较高，说明碳水化合物的供应不是限制弱势籽粒灌浆启动的主要原因。通过考察不同种植季节以及不同栽培调控措施下水稻强弱势籽粒干物质动态变化，发现强势籽粒灌浆稳定，而弱势籽粒灌浆易受环境所调控。合理的水肥调控仅能够一定程度促进弱势籽粒的灌浆，但无法促使弱势籽粒灌浆启动的提前。
2、为了明确限制弱势籽粒灌浆启动的分子机制，构建了弱势籽粒滞育期及灌浆前期的蛋白表达及磷酸化蛋白表达图谱，在弱势籽粒灌浆启动过程中，共鉴定到86个蛋白点和38个磷酸化蛋白点出现显著的表达变化，其中即出现蛋白表达变化的又出现磷酸化修饰变化的蛋白点有6个。经过MALDI-TOF/TOF-MS及LC-MS/MS的分析，蛋白点全部成功得到鉴定。通过蛋白丰度及功能的分析发现，萌发素蛋白的上调表达及α-微管蛋白的磷酸化修饰能够打破弱势籽粒胚乳细胞发育的停滞，导致弱势籽粒胚乳膨大，形成库容，为下一步淀粉累积做准备，而蔗糖合酶、UDP葡糖焦酸酶、ADP葡萄糖焦磷酸化酶、颗粒结合型淀粉合成酶、丙酮酸磷酸二激酶的上调表达，以及丙酮酸磷酸二激酶,ADP葡萄糖焦磷酸化酶，磷酸甘油酸酯变位酶和14-3-3蛋白的磷酸化修饰导致了淀粉合成行为的启动。与此同时，弱势籽粒从滞育期到灌浆期，磷酸戊糖途径减弱，糖酵解途径增强，产生更多的能量及中间产物供弱势籽粒胚乳细胞的增殖及淀粉的合成。
3、为了明确强弱势籽粒灌浆差异形成的分子机制，构建了强弱势籽粒灌浆启动后对应的不同灌浆时期的蛋白表达及磷酸化蛋白表达图谱，共鉴定到156个蛋白点和54个磷酸化蛋白点在强弱势籽粒之间出现表达变化，其中即出现蛋白表达变化的又出现磷酸化修饰变化的蛋白点数有16个。经过MALDI-TOF/TOF-MS及LC-MS/MS质谱分析，蛋白点全部成功得到鉴定。研究发现，灌浆的前期，弱势籽粒中可溶性糖含量高于强势籽粒，淀粉合成关键蛋白的低丰度表达，限制了淀粉的合成；灌浆的中期，弱势籽粒中蔗糖及淀粉相关蛋白的低丰度表达是导致其淀粉合成效率低的主要原因；而到了灌浆的后期，碳水化合物供应的不足又成为限制其淀粉合成的主要原因。磷酸化分析发现，葡糖磷酸变位酶，UDP葡糖焦酸酶，ADPG焦磷酸化酶，颗粒结合型淀粉合成酶、磷酸化酶及丙酮酸磷酸二激酶在强弱势籽粒之间存在着磷酸化修饰的差异，暗示着在磷酸化修饰层面调控强弱势籽粒淀粉合成关键酶活性差异的可能性，同时也发现这些淀粉合成关键酶会通过磷酸化修饰并在14-3-3的调控下，形成淀粉酶复合体。此外，在弱势籽粒灌浆过程中糖酵解、三羧酸循环及乙醇代谢途径相关蛋白的表达量底，导致其碳流通量低，产生供胚乳细胞分化及淀粉合成的能量及前体相对强势籽粒较少，限制了弱势籽粒库容的形成及淀粉的合成，而肌动蛋白，肿瘤翻译调控蛋白及微管蛋白又限制了弱势籽粒胚乳细胞的分化。
4、依据比较蛋白组学结果，筛选了7个限制弱势籽粒灌浆启动及9个导致弱势籽粒灌浆不充分的关键蛋白，蛋白丰度分析发现这些蛋白在强弱势籽粒之间存在着时空表达差异，其中14-3-3蛋白被认为在强弱势籽粒灌浆差异形成的过程中具有重要调控作用。进一步通过亲和层析技术探讨了籽粒中14-3-3蛋白的互作网络，共鉴定到24个互作蛋白，发现淀粉合成过程的ADPG焦磷酸化酶、淀粉分支酶、支链淀粉酶，转化酶及丙酮酸磷酸二激酶与14-3-3蛋白存在着紧密的互作关系。 LC-MS/MS/MS分析发现籽粒中14-3-3蛋白存在着丝氨酸和苏氨酸的磷酸化位点，据此认为14-3-3在强弱势籽粒之间的蛋白及磷酸化修饰差异是导致强弱势籽粒淀粉合成差异及对环境产生不同响应机制的一个关键信号调控因子。
综上所述，本研究发现水稻“金恢809”存在着显著的强弱势籽粒灌浆差异现象，进一步运用比较蛋白组学策略分析了强弱势籽粒灌浆过程中基因的时空表达变化差异，揭示了弱势籽粒灌浆启动及强弱势籽粒灌浆差异形成的蛋白作用及磷酸化信号调控机制，筛选出关键的信号调控因子14-3-3蛋白。最后，依据本研究的结果，提出了改善弱势籽粒灌浆的分子遗传改良及分子生态调控措施。
The asynchronous filling of superior and inferior spikelets is a commonphenomenon in rice (especially the extra-heavy panicles cultivars) and the poorgrain-filling in inferior spikelets limited the high yield potential of rice. Currently,improvement of the grain-filling rate and grain weight in inferior spikelets is one ofthe major tasks to enhance rice yield. The formation of asynchronous filling ofsuperior and inferior spikelets is a complicated ecopisiological process, whichinvolved in multiple gene expression. Although many researches focus on themolecular mechanism of the asynchronous filling, few studies were carried out at theprotein and phosphoprotein level. In order to further understand the molecularphysiology of asynchronous filling of superior and inferior spikelets, we analyzed thechanges of protein expression and phosphorylation in the superior and inferiorspikelets during the grain filling of rice “jinhui809” with the proteomic approaches,including the technology of two-dimensional electrophoresis (2-DE) and Pro-QDiamond phosphoprotein gel stain.
The morphological analyses showed that rice “jinhui809” has an obviousphenomenon of asynchronous filling of superior and inferior spikelets. The size andgrain weight of superior spikelets increased rapidly at5–20day after flowering (DAF),while that of inferior spikelets hardly increased until15DAF, which indicated thedevelopment of inferior spikelets was stagnant at0-15DAF. The size and grain weighof superior spikelets were better than those of inferior spikelets. In the stagnant stage,the inferior spikelets accumulated high concentrations of soluble carbohydrate，which indicated that the carbohydrate supply didn’t limit the starting of grain-fillingin inferior spikelets. The analyses of grain weigh of superior and inferior spikeletsunder different cultivation measures showed that the grain-filling of inferior spikeletsis more sensitive to the environmental factors than that of superior spikelets. Thesuitable water and fertilizer management was only favorable for the grain-filling ofinferior spikelets in some degree, but could not promote the starting of grain-filling ofinferior spikelets. The results obtained from the present study suggested that the genetic diversity between the superior and inferior spikelets was the major reason forthe poor grain-filling of inferior spikelets.
To better understand the molecular mechanisms of the starting of inferiorspikelets filling, we took two-dimensional gel-based proteomic andphosphoproteomic approaches to profile the proteins with abundance andphosphorylation state changes in inferior spikelets at stagnant stage and earlygrain-filling stage. A total of86protein spots and38phosphoprotein spots showeddifferential abundance changes by more than1.5-fold, and all of them weresuccessfully identified by the MALDI-TOF/TOF MS and LC-MS/MS. Only6protein spots showed significant changes in both abundance and phosphorylation. Theresults showed that the up-regulated expression of Germin-like protein (GLP) and thephosphorylation change of Alpha-tubulin may favorable to break out the stagnant ofinferior spikelets and promote the cell enlargement, thus leading to the sink capacityformation. The up-regulated expression of Sucrose synthase (SUSase), UDP-glucosepyrophosphorylase(UDPase), ADP glucose pyrophosphorylase (AGPase), Granule-boundstarch synthase(GBSS), Pyruvate orthophosphate dikinase (PPDK) and the phosphorylationchange of UDPase, AGPase, Phosphoglucomutase (PGM),14-3-3protein may help tostart the starch synthesis. At the same time, the Embden-Meyerhof-Parnas pathway (EMP)activity was increased and the Pentose Phosphate Pathway (PPP) activity was decreased,and thereby, more energy and building blocks for cell enlargement and starchsynthesis in the inferior spikelets could be available.
To gain a better understanding of the molecular mechanisms of the poorgrain-filling of inferior spikelets, comparative proteomic and phosphoproteomicanalysis between the superior and inferior spikelets at different grain-filling stagewere also carried out. A total of156protein spots and54phosphoprotein spotsshowed differential abundance changes between superior and inferior spikelets bymore than1.5-fold, and all of them were successfully identified by the MALDI-TOF/TOF MS and LC-MS/MS. Only16protein spots showed significant changes inboth abundance and phosphorylation. Based on our results, the reasons for the poorgrain-filling of inferior spikelets can be documented as follows. Firstly, in the early grain-filling stage, the lower abundance of starch synthesis related protein in inferiorspikelets compare to the superior spikelets limited the starch accumulated. Secondly,in the middle grain-filling stage, the down-regulate expression of sugar conversionand starch synthesis related protein resulting in slow grain-filling of inferior spikelets.Finally, in the late grain-filling stage, the assimilate supply is the main factor thatleads to poor grain-filling of inferior spikelets. Significant phosphorylation changesbetween superior and inferior spikelets were also found for6proteins involved instarch synthesis, such as PGM, UDPase, AGPase, GBSS, PPDK, and phosphorylase,suggesting that the activity of key enzyme of starch synthesis in inferior spikelets canbe modificated by the phosphorylation. The results also showed that thephosphorylation of the key enzyme of starch synthesis was necessary for theformation of a starch synthesizing protein complex (SSPC) and14-3-3protein mayacts as a scaffold protein holding enzymes of starch synthesis together in aphosphorylation-dependant SSPC. In addition, down-regulated expression of theenzyme proteins related to EMP, Tricarboxylicacidcycle (TCA) and alcohol decreased thecarbon flux in inferior spikelets during the grain-filling stage, thus leading less energyand building blocks for cell enlargement and starch synthesis in the inferior spikeletscompared to the superior spikelets. The author also found that low protein expressionof Translationally controlled tumor protein (TCTP）and tubulin, phosphorylation change ofalpha-tubulin, and early embryogenesis protein caused the low cell division rate in inferiorspikelets.
According to the results of comparative proteomic analysis,7key proteinsrelated to the starting of the inferior spikelets and9key proteins related to the poorgrain-filling of inferior spikelets were selected. The key proteins in the superior andinferior spikelets exhibited different temporal and spatial expression patterns, whichcaused significant differences in grain-filling at different spikelet positions on thepanicle. Among all of the key proteins,14-3-3protein is considered to be an importantregulatory protein for the poor grain-filling of inferior spikeletss. Then, the14-3-3protein was over-expressed, immobilized and used to affinity purify14-3-3bindingproteins from rice spikelets. These proteins were fractionated by PAGE and identified by LC-MS/MS. In total,2414-3-3binding proteins were identified. These proteinsfell into5functional categories. The largest category was for carbohydratemetabolism, including enzymes for starch synthesis and modification (e.g. AGPase,Starch branching enzyme, Pullulanase, Invertase and PPDK). In the present study, twophosphorylation sites at the serine and threonine residue of14-3-3protein wereidentified by the LC-MS/MS/MS. This work illustrates the starch synthesis in which14-3-3prtoein may be involved, and augments the different expression andphosphorylation changes in superior and inferior spikelets, which demonstrated thekey roles of14-3-3protein in asynchronous filling of superior and inferior spikelets.
In conclusion, the present field experiments showed that rice “jinhui809” hasobvious asynchronous filling of superior and inferior spikelets. The comparativeproteomic and phosphoproteome analyses further revealed the changes of proteinexpression and phosphorylation in the stagnant stage of inferior spikelets and thegrain-filling stage of inferior spikelets compared to the superior spikelets. Thefindings explained the asynchronous filling properties of superior and inferiorspikelets which were attributed to differential expression of proteome level intemporal and space. In addition, one of the important regulatory protein called as14-3-3protein was selected and its function was studied by the affinitychromatography technology. Finally, according to the present results, the authorproposed two effective ways for molecular genetic manipulations and molecularecological regulations to enhance the grain-filling of inferior spikelets.
引文
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