Functional Analysis Of SHR Gene In The Development Of Vascular Bundles In Rice And Green Foxtail

Leaves are the main organs for photosynthesis in higher plants.The transport tissues in leaves are regularly distributed in the form of vascular bundles(leaf veins),which play a role in the transport of water,mineral nutrients,organic matter and signal molecules.Vascular bundles,vascular bundle sheath cells and mesophyll cells of C4 plants form a unique Kranz structure,which is the main anatomical characteristics of C4 plants and the structural basis of high light efficiency.The study on the formation of leaf veins in rice can not only reveal the development and regulation mechanism of leaf veins,but also have important reference significance for elucidating the Kranz structure of C4 plants,but also provide a theoretical and practical basis for the creation of C4-like anatomical structures in rice.The main conclusions of this paper are as follows:1.Our laboratory screened a material with reduced leaf vein density in large-scale C4 maize transcription factor transgenic rice,which was caused by overexpression of ZmSHR2(SHORT ROOT 2).The overexpression of ZmSHR2 in rice results in plant type dwarfing,significantly positively curved leaves,and short roots.The anatomical structure of the leaves is characterized by an increase in the number of mesophyll cells between adjacent minor veins,a decrease in the total number of major and minor veins,and a decrease in the volume and number of bulliform cells,indicating that ZmSHR2 regulates the density of leaf veins by inhibiting the development of vascular bundles.2.Evolutionary analysis showed that ZmSHR2 was linearly homologous to SHR1(SHORT ROOT 1)of rice and green foxtail.In this paper,the function of SHR1 was studied in rice and green foxtail respectively.It was found that overexpressing OsSHR1 in rice and Sv SHR1 in green foxtail results in a thinning of leaf vein density and an increase in the number of mesophyll cells between adjacent minor veins,while the shr1 mutant results in a densification of leaf vein density and a decrease in the number of mesophyll cells between adjacent minor veins.Subcellular localization showed that OsSHR1 was located in the nucleus.Tissue expression pattern analysis showed that SHR1 was specifically expressed in vascular bundles of roots and young leaves.In situ hybridization showed that OsSHR1 was specifically expressed in the vascular bundles at the P2-P5 stage of the leaf primordium of seedlings.3.The interacting proteins OsIDD12(INDETERMINATE DOMAIN 12)and OsIDD13(INDETERMINATE DOMAIN 13)of OsSHR1 were obtained by screening yeast library.Pull-down also proved that OsSHR1 and OsIDD12/13 interact directly in vitro.The specific interaction between OsSHR1 and OsIDD12/13 in yeast,rice protoplasts and tobacco was verified by yeast two hybrid,bimolecular fluorescence complementarity and immunoprecipitation.Yeast peer-to-peer experiment also showed that there was a specific interaction between OsIDD12 and OsIDD13.Anatomical structure observation and statistical analysis showed that the overexpression of OsIDD12 and OsIDD13 and single mutant of rice leaf vein density were normal,but the leaf veins of Osidd12/13 double mutant rice became denser and the number of mesophyll cells between adjacent minor veins decreased.Tissue expression pattern analysis showed that OsIDD12 and OsIDD13 were expressed in vascular bundles and root stele at the base of leaves and stems at seedling stage.In situ hybridization showed that OsIDD12 and OsIDD13 were specifically expressed near the vascular bundles of P4-P5 leaf primordia.Studies have fully shown that OsSHR1 interacts with OsIDD12/13 and participates in the development of leaf veins.4.Furthermore,the downstream possible target gene OsPIN5c(PIN-FORMED 5c)was obtained by RNA-seq analysis.Yeast one hybrid and Ch IP-q PCR methods showed that OsIDD12 and OsIDD13 could bind to the conserved motif bound to the IDD protein of the third intron of OsPIN5 c in yeast and rice.EMSA confirmed the direct binding of OsIDD12 and OsIDD13 to conservative motif in vitro.Subsequent anatomical structural observation and statistical analysis showed that the leaf veins of rice overexpressing OsPIN5 c became denser,the number of mesophyll cells between adjacent minor veins decreased and secondary minor veins of C4-like plants appeared.Double luciferase experiments and genetic experiments(secondary transformation)showed that OsSHR1 and OsIDD12/13 as a transcriptional complex inhibited the expression of OsPIN5 c.Synthesize the functions of OsSHR1 regulating the downstream target gene OsPIN5 c by forming a complex with OsIDD12/13.As an auxin transport carrier,OsPIN5 c affects the distribution of auxin by controlling the transport of auxin,thus affecting the formation of vascular bundles and controlling the proliferation of mesophyll cells to determine the density of rice leaf veins.