| 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. |
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