| Sucrose,as the main product of photosynthesis and the main form of long-distance transport of assimilation,is synthesized in the source organs and transported through the phloem to the sink organs,requiring the participation of sucrose transporter?SUT?.The flow of assimilation between"source"and"sink"is determined by both"source strength"and"sink strength".The distribution of assimilation between different sink tissues determines the de-velopment of various organs and the formation of final yield factors,and the driving force generated by the transport of sucrose can also promote the transportation of other photosyn-thetic products in the phloem.At present,studies have reported that SUT plays an important role in various physiological processes such as sucrose absorbed and excreted by the phloem,sucrose supply in the sink tissue,sucrose storage,regulation of sucrose transport,and transport of other small molecules.In addition,previous studies have confirmed that some sucrose transporters are involved in the process of growth and seed filling,but there are few reports on how sucrose transporters affect the unloading and distribution of assimilation at the reservoir end.In this study,we obtained OsSUT4 gene modified mutants by CRISPR/Cas9 and explored them deeply.According to the nucleic acid sequence of OsSUT4?GenBank accession number:AB091673.1?,the target on CDS was designed and a vector was constructed to transform the callus induced by mature embryos of rice.OsSUT4 gene-modified plants were obtained by differentiation and regeneration.After three consecutive generations of selfing,the genetic background of Cas9 protein was removed and homozygous mutants were obtained.Recombinant OsSUT4-GFP plasmid was constructed and transiently expressed in onion leaf epidermal cells.The green fluorescence of OsSUT4-GFP fusion protein was observed on the vacuolar membrane under a laser confocal microscope,and it could be distinguished from the free green fluorescence of the vector.Therefore,OsSUT4 is a vacuolar mem-brane-localized rice sucrose transporter.The results of GUS staining experiments showed that OsSUT4 exhibited a higher expression level in the sink organs.Higher levels of OsSUT4promoter activity can be detected in the inflorescence,developing internodes,the pistil and stamen on the flowering day,and in the embryos of mature seeds.However,signals could hardly be detected in leaves,sheaths and roots at all stages.Real-time quantitative PCR detec-tion of OsSUT4 mRNA levels was consistent with GUS staining results.In view of the high-level expression of OsSUT4 in inflorescence,apical meristem samples at the jointing-booting stage were collected for scanning electron microscopy assay,and the primary branch,secondary branch and spikelet of ossut4 mutant were found to have a delayed development state compared with the wild type.The sugar content of inflorescence in the os-sut4 mutant and wild type was detected,and it was found that the soluble sugar content was significantly reduced.The sugar content and the sugar export rate in the young stem which were closely connected to the inflorescence were also detected.It was found that the sugar content in the young stem was increased,but the sugar transport capacity of the young stem was not affected.Under natural culture conditions,the accumulation of soluble sugar and starch in various tissues of the ossut4 mutant changed.The stable isotope 13CO2 was used to label the mutants and wild type.It was found that the distribution proportion of carbon assim-ilations in young stem increased and that in inflorescence decreased.It is suggested that Os-SUT4 is involved in the distribution of assimilates between sink tissues and influences the development of inflorescence.It was found that there was no significant change of expression level of other OsSUTs in rice inflorescence,but most of sucrose-substrate OsSWEETs were up-regulated.Therefore,the functional decrease caused by the loss of ossut4 might be compensated by the up-regulated expression of OsSWEETs.Statistics on field characteristics of ossut4 mutant and wild type showed that the plant height of ossut4 mutant became shorter,while panicle length,primary branch number,sec-ondary branch number,grain number per panicle,and spikelet number per panicle all de-creased significantly.Production statistics showed that the plant yield and plot yield of the mutants decreased significantly.The seeds of the ossut4 mutants were tested and found to have alittle more extended grain length and grain width compared with the wild type.Scanning electron microscope observa-tion of the cross section of the seeds revealed that the starch granules of the mutant seeds were spherical or ellipsoidal,and the endosperm structure was looser than the wild type;The rice quality test showed that the amylose content in the mutant brown rice was increased.The expression level of OsGBSS?was detected during the filling process,and found it was signif-icantly higher than that of the wild type,which indicated that it might synthesize excessive amylose and cause changes in the endosperm structure,which could cause the mutant rice quality to decline.ossut4-6 mutant and wild-type young inflerescence samples were collected for transcrip-tome sequencing.The COG database annotation results of differentially expressed genes in-dicated that the highest frequency annotation is the signal transduction mechanism.The KEGG pathway enrichment analysis in inflorescence found that the most reliable pathways were starch and sucrose metabolism.This result suggests that OsSUT4 may affect plant growth and development by participating in starch and sucrose metabolism.In this study,the expression pattern of rice sucrose transporter OsSUT4 and its possible role in the early development of inflorescence were explored by a variety of physiological means.It was found that the development of inflorescence was affected,which resulted in the de-crease of yield;moreover,its vegetative growth and rice quality were also influenced in the ossut4 mutant plants. |
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