Study On The Regulatory Mechanism Of The GA Pathway Involved In Scrambling Stem Elogation In Jasminum Sambac (L.) Aiton

Plant architecture are one of the important traits for commercial ornamental plants.The plant architecture is usually determined by plant height（elongation growth）and branching（lateral growth）,among which plant height is mainly regulated and controlled by the hormone gibberellin（GA）and the related pathway.GA is involved in many plant physiological processes,including flowering,seed germination and cell elongation.Although the molecular mechanism of GA pathway in plant growth and development has been deeply understood in model plants such as Arabidopsis（Arabidopsis thaliana）and rice（Oryza sativa）,the corresponding mechanism in many ornamental plants is little explored.Additionally,there exists a complex interaction between the molecular genetic regulatory network that affects plant height and branching traits,resulting in the diversity of regulatory genes among different plant species due to the habitat and genetic evolution differences.Further investigation using different plants with specific growing habits would enable the identification of unique mechanism underlying plant of special architectures.Jasminum sambac（L.）Aiton（Oleaceae）is a climbing shrub of the Jasminum genus that is important worldwide for ornamental value and perfume industries.Because of its typical spindling characteristic growth that directly leads to the poor plant architecture and the decrease of the number of flowers,maintenance of the plant architecture in the garden often requires large investments of labor and even chemicals,which limits its horticultural utilization.Research on the regulation of scrambling stem elogation of Jasmine can be beneficial for its architecture control in plantation.Moreover,due to the fact that Jasmine almost does not make branching under natural conditions,it is also in line with an ideal model for studying the growth mechanism of simple elongation.Therefore,we screened and detected the expression changes of GA pathway related genes and the SPL family genes from the aspects of the relationship between internode elongation and endogenous hormones,light and dark conditions,and the effect of exogenous hormones on internode elongation,and analyzed the potential role of these genes in regulating internode elongation in Jasmine shoots.In particular,we found that three GA receptor encoding genes,JsGID1b1,JsGID1b2 and JsGID1b3,were simultaneously expressed in the branches of Jasmine.The functional characteristics of these three GA receptor genes were analyzed by Arabidopsis heterologous expression system and yeast two hybrid test.The main results are listed as follows:⑴Under natural conditions,especially in the case of insufficient light,the shoots of Jasmine have unlimited growth potential,and only transition of flower bud will terminate the growth.GA content in vegetative branches was significantly higher than that in flowering branches with elongation terminated.The length of epidermal cells in different parts of the branch was uniform,which indicates that the elongation of cells was synchronous,but the division of epidermal cells at the base and top seemed to be more frequent than that in the middle.The distribution of different hormones in leaves and internodes showed that only GA accumulates in vegetative branches,but not in flowering branches without elongation.Therefore,GA was closely related to branches reproductive growth.Accordingly,exogenous GA₃ treatment significantly promoted the vegetative growth of branches,while the application of GA synthesis inhibitor UNI caused elongation to stop and promoted the transformation from vegetative buds to flower buds.The results showed that GA played a leading role in the growth of the reproductive branches,and also revealed that GA pathway played a certain role in the balance between vegetative growth and reproductive growth.⑵By local blast of transcriptome data from stem,leaf and flower of Jasmine previously constructed in our laboratory,we screened the homologous genes of GA synthesis,reaction and downstream regulation,which include three GA receptor genes（JsGID1b1,JsGID1b2 and JsGID1b3）and two closed JsCXE genes,one negative regulator JsDELLA gene,eight GA synthetase genes（four JsGA3ox genes and four JsGA20ox genes）,three GA inactivating enzyme JsGA2ox genes,and 13downstream growth regulation SPL family genes.A total of 7 gene overexpression vectors including 35S::JsGID1b1,35S::JsGID1b2,35S::JsGID1b3,35S::JsCXE2,35S::JsDELLA,35S::JsGA2ox-110140and 35S::JsGA20ox-67189 were constructed by Gateway technology and successfully transformed into A.thaliana plants.⑶RT-qPCR was used to further analyze the expression of genes related to GA synthase,receptor protein and signal transduction molecules.The results showed that the expression of GA receptor JsGID1b1 in vegetative branches was significantly higher than that in flowering branches.The expression of GA synthase JsGA20ox-c67189and GA receptor JsGID1b1 increased continuously in the top stem and top leaf of Jasmine during the vegetative shoot growth.On the other hand,there was no significant difference in the expression of JsDELLA in both branches.Moreover,the expression of JsDELLA（GA response inhibitor gene）in the top stem and top leaf of Jasmine was significantly changed during the growth of vegetative branches of Jasmine,and continued to decrease.（4）JsGID1b1,JsGID1b2 and JsGID1b3 genes responded differently to photoperiod and light.The expression of JsGID1b1 in the top stem node and top leaf of the vegetative branch was changed under light,but no response of JsGID1b2.The expression of JsGID1b3,JsCXE1,JsCXE2and JsDELLA only responded to light in the top stem node,but not in the top leaves.（5）The interaction between three GA receptor proteins（JsGID1b1,JsGID1b2 and JsGID1b3）and JsDELLA were analyzed by yeast two hybridization（Y2H）test.The results showed that there were significant differences in GA₃ dependence among the three GA receptor proteins.The interaction between JsGID1b1and JsDELLA was not affected by GA₃,and the interaction between JsGID1b2 and JsDELLA required GA₃of more than 0.01μM,but JsGID1b3 did not interact with JsDELLA within the range of GA₃ concentration（0.001μM～150μM）.Heterologous overexpression of three Jasmine GA receptors（JsGID1b1,JsGID1b2 and JsGID1b3）in transgenic Arabidopsis thaliana plants can promote the GA response of transgenic plants and have a significant effect on plant height.Taken together,we concluded that the vine growth or overgrowth of vegetative branches of Jasmine was dominated by the GA pathway.GA synthesis and receptor response are two important nodes of gene regulatory network.Three GA receptor genes were expressed in the elongating reproductive shoots of Jasmine simultaneously and showed different dependence on light and GA₃.The discovery of this mechanism opens up a potential line of research for the molecular design of the plant architecture of the ornamental plants in the future.