| Grain filling directly determines yield and quality in rice.Nevertheless,key factors affecting grain filling remain poorly understood.Pyruvate kinase(PK)is a key rate-limiting enzyme in glycolysis pathway.Its catalytic substrate(PEP)and product(pyruvate)are important precursors for intracellular biosynthesis and metabolism.Pyruvate kinase genes play crucial roles in diverse physiological processes during plant growth,such as regulating seed development,storage substances synthesis,sugar metabolism in leaf and the transport of assimilates in stem.Therefore,it is important to further explore the function of rice PK genes to improve the regulation network of grain filling.In this tudy,we identified a mutant with defective grain filling,named ospk3.Further studies found that a putative PKc gene OsPK3 was responsible for the ospk3 phenotype.Therefore,we systematically studied the expression pattern,subcellular localization,interaction with other subunits of OsPK3 and so on.We obtained the main results as below:1.The agronomic traits of ospk3 mutant were comprehensively investigated.The pleiotropic defects of ospk3 included dwarfism,short panicle length and a reduced seed-setting rate,moreover,the proportion of unfilled grain that failed to produce integrated endosperm was significantly increased,and ospk3 mutant also exhibited a markedly slower grain filling rate that led to decreased grian thickness and grain weight.Rice quality analysis showed that ospk3 had severely chalky grains with opaque and floury white mature endosperm.Cytological analysis showed that ospk3 endosperm revealed small,round and loose starch grains,and the number of starch grain was reduced compared to wild type.2.The location of the T-DNA was identified in ospk3 mutant.A T-DNA was inserted at the nucleotide position 1843 bp downstream of the ATG start codon of Os04g58110(OsPK3)in the ospk3 and caused significant reduction of OsPK3 m RNA transcript levels in the leaf and endosperm compared to wild type.Moreover,we obtained knockout mutants of OsPK3 in the Dongjin background,and concluded that the T-DNA insertion in OsPK3(Os04g58110)was responsible for the ospk3 phenotype.3.QPCR analysis showed that OsPK3 was constitutively expressed but had high expression levels in the leaf and developing caryopsis.GUS activity was mainly detected in the leaf,stem and developing caryopsis.GUS histochemical analysis revealed OsPK3 had specific expression signals in tissues involved in sucrose transport and unloading.GUS activity was detected in mesophyll cells and phloem companion cells in the leaf and in phloem companion cells in the vascular bundles of internodes.The expression signal was detected at the parenchyma cell bridge(PCB)of enlarged vascular bundles(EVBs)in the nodes.In the caryopsis at 9 DAF,GUS signal was observed in the nucellar projection,nucellar epidermis,aleurone layer cells and ovular vascular trace.Overall,the expression pattern of OsPK3 suggest that it plays vital roles in leaf,stem and caryopsis in rice.On the other hand,subcellular localization showed that OsPK3 is associated with the mitochondrial surface and hardly exist in the cytosol.4.The loss of OsPK3 function led to reduced PK activity and pyruvate content in rice.5.During the grain-filling stage,compared to wild type,the sucrose and total temporary storage starch content were noticeably increased in the mutant leaves,however,the sucrose content was decreased in stems,and the soluble sugar(sucrose,glucose and fructose),starch and amylose content were markedly reduced in the mutant endosperm.QPCR results showed that the expression levels of several genes(OsSUT1,OsSUT4,OsSWEET11,OsSWEET15 and GIF1)involved in sucrose delivery and unloading were up-regulated except that OsSUT5 was down-regulated in mutant caryopses at 15 DAF.Several starch synthesis-associated genes were obviously up-regulated in the mutant endosperm.Taken together,these results indicated that OsPK3 is involved in starch synthesis,compound granule formation and grain filling by regulating sucrose translocation from leaf to caryopsis in rice.6.The interaction between OsPK3 and other cytosolic pyruvate kinase isozymes was analyzed by Bi FC.OsPK3 interacted with the OsPK1 and OsPK4 PKc subunits,but it did not bind itself or interact with the three remaining PKc isozymes(Os03g20880,Os01g16960 and Os11g10980).Co-immunoprecipitation assays verified the interaction of OsPK3 with both OsPK1 and OsPK4,but no interaction between OsPK1 and OsPK4.Native-PAGE gels showed that OsPK3 physically interacts and forms heterodimers with OsPK1 and OsPK4 in vitro.7.Both OsPK1 and OsPK4 were widely expressed in multiple tissues but nevertheless had distinct expression characteristics.OsPK1 was highly expressed in the leaf and developing caryopsis,which was highly similar to that of OsPK3;however,OsPK4 was mainly highly expressed in the developing caryopsis.Subcellular localization indicated that OsPK1 and OsPK4 exist in both the cytosol and mitochondria and were recruited to the mitochondria by OsPK3.8.The agronomic traits of ospk1 and ospk4 were investigated.Similar to ospk3,the ospk1 mutants had pleiotropic defects including dwarfism,reduced grain weight and increased grain chalkiness.In contrast,ospk4 mutants mainly had severely chalky grains.ospk3;ospk1 and ospk3;ospk4 both double mutants were similar to the single ospk3 mutant.Taken together,OsPK3 recruits its counterpart isozymes OsPK1 and OsPK4 to the mitochondrial surface to form different PK heterodimers.Throughout rice growth,OsPK1 assists OsPK3 to regulate sucrose translocation from leaf to caryopsis.In contrast,the OsPK3-OsPK4 heterodimer may be specifically involved in accelerating sucrose unloading and transport in the developing caryopsis to meet starch synthesis,compound granule formation and grain filling. |
