Anatomical Study And Mechanism Of ZlARF5 Regulating Swelling Of Zizania Latifolia

Zizania latifolia is a widely consumed aquatic vegetable in southern China.It is a gall formed by the interaction between Ustilago esculenta and plant Zizania latifolia,which causes the stem tip of Zizania latifolia to expand at a specific time point.Currently,the research on Zizania latifolia mainly focuses on the life cycle of Ustilago esculenta and the expression of related genes in the symbiosis process between Ustilago esculenta and Zizania latifolia,but the research on developmental biology between them is rarely reported.This study used Xiang Ya Jiao as the experimental material.Firstly,the anatomical changes at different developmental stages of Zizania latifolia were analyzed through paraffin sections,and the three-dimensional structure of the fungal cavity was visualized using 3D reconstruction technology;Then,in situ hybridization and RT-q PCR were used to analyze the spatiotemporal expression pattern of the key gene Zl ARF5,and the subcellular localization of Zl ARF5 was analyzed by constructing an over expression vector,in order to clarify the origin of dividing cells in the process of Zizania latifolia expansion and the important functions that auxin responsive factor Zl ARF5 can participate in.The main results are as follows:1.Anatomical analysis found that,unlike previous studies on the pregnancy stage of Zizania latifolia,changes in the stem tips of Zizania latifolia had already occurred earlier,and the lifestyle of Ustilago esculenta in the stem tips had also undergone significant changes;The swelling of Zizania latifolia begins with the appearance of yellow secretion on the tissue section,and the microscopic structure shows that the Ustilago esculenta starts to form clusters of hyphae in the basic meristem area,and then expands to form the lumen;At the same time,a new vascular tissue was formed around the mycelium cluster in the stem of Zizania latifolia,establishing a channel for the lumen to obtain nutrition;3D reconstruction shows that mycelium extends outward from the fungal cavity to obtain nutrients.2.Through tissue specific expression analysis of cell division specific gene Zl CDKB2,2,it was found that during the expansion of Zizania latifolia,the source of cell division came from the cell Reprogramming formed by the basic meristem at the stem tip under the influence of mycelial clusters and lumens,and the cells that actively split were mainly distributed around the vascular tissue and lumen.The enlargement of the stem is due to the parenchyma cells growing by division,the enlarged lumen structure,the variant vascular tissue and the gap generated by cell separation.3.RT-q PCR showed that the expression of Zl ARF5 at the early stage of swelling was about 20 times higher than that in wild jiao and late swollen.The results of in situ hybridization of Zl ARF5 showed that it was mainly expressed around the lumen and around vascular tissue,and overlapped with the expression of Zl CDKB2,2 in the lumen and vascular tissue.In Zizania latifolia and callus,it was also mainly expressed in vascular tissue and stem cell formation;At the same time,elements related to meristem were also found at the promoter of Zl ARF5,so it was speculated that Zl ARF5 might be involved in regulating the induction and differentiation of vascular tissue during the expansion of Zizania latifolia,and had an important relationship with the formation of stem cells in the basic meristem.4.Subcellular localization of tobacco leaves shows that Zl ARF5 is located in the cytoplasm of tobacco leaves in micrometer sized particles.Based on the highly disordered regions contained in the Zl ARF5 protein sequence,it is speculated that Zl ARF5 may have intracellular protein aggregation activity,thus being strictly regulated at the protein level.