Molecular Mechanism Of Pu-miR172d Regulated Stomatal Development Response To Drought Stress In Populus Ussuriensis

The stoma are small pores formed by the surrounding of the guard cells by the two protective cells,which have a great influence on regulating the balance of photosynthetic carbon fixation and water dispersion.The occurrence of stoma is a complex biological process regulated by a variety of genes.The development of stoma can not only affect the growth of plants,but also the ability of plants to cope with environmental stress.At present,some reports have found that miRNAs can participate in the process of stoma development,but the molecular mechanism is still not very clear.Therefore,this study aims to preliminarily clarify the molecular mechanism of poplar miR172 regulating stomatal development in response to drought stress.In this study,the molecular functions of Pu-miRl 72d and its target gene PuGTLl were analyzed and identified by biochemistry,molecular biology and genetic engineering.The main results are as follows:(1)Target gene prediction results of small RNA transcriptome data showed that transcription factor PuGTLl of Trihelix gene family is a target gene of Pu-miR172d.Using 5’RLM-RACE technology and Agrobacterium transient transformation experiments,it was verified that PuGTLl is indeed the target gene of Pu-miR172d.It was first discovered that miRl 72 can bind to the transcription factor of Trihelix family.(2)The classification and functional identification of the Populus Trihelix gene family were carried out using bioinformatics methods and qRT-PCR technology.The Trihelix family can be divided into five subfamilies composed of 56 members.Among them,most genes can respond to osmotic stress.The results of eximage analysis indicated that PuGTLl was mainly expressed in young leaves.QRT-PCR showed that PuGTLl can increase plant stress resistance to some extent.(3)QRT-PCR results showed that Pu-miRl 72d was expressed in the first 1-4 young leaves,and it could respond to osmotic stress.The full-length promoter sequence of the Populus ussuriensis Pu-miR172d was cloned and the Pu-miR172dpro::GUS expression vector was constructed.After GUS staining,it was found that Pu-miR172d was mainly expressed in the stoma of leaves,suggesting that Pu-miR172d may be involved in stomatal movement or development.(4)The full-length.ussuriensis Pu-miR172d sequence was cloned,and the Pu-miRl 72d overexpression vector and the inhibited expression vector were constructed.After osmotic stress on tissue cultured seedlings,it was found that Pu-miR172d overexpressed transgenic P.ussuriensis growed better and had higher rooting rate than WT,while Pu-miR172d inhibit expressed of transgenic P.ussuriensis and WT had little difference.Histochemical staining of DAB,NBT,Evans bule;SOD,POD,MDA,conductivity and chlorophyll content showed that Pu-miR172d overexpressed plants had stronger abilities of resistance to stress,indicating that Pu-miR172dhas certain resistance to osmotic stress.(5)Compared with WT,Pu-miR172d overexpressed.ussuriensis soil cultured seedlings became shorter and the stomatal density was significantly reduced.The photosynthetic rate,stomatal conductance and transpiration rate of Pu-miR172d overexpressed plants were lower than WT,and the instantaneous water use efficiency was higher than WT.After drought stress,the growth of Pu-miR172d overexpressed plants was better than that of WT and the water content of the leaves was higher.After drought and rehydration,Pu-miR172d overexpressed.ussuriensis still growed normally,while WT and Pu-miRl 72d inhibit expressed plant growth is severely inhibited.The results of MDA,conductivity and H2O2 content also showed that Pu-miR172d can significantly improve the drought tolerance of plants.The above results show for the first time that Pu-miR172d can affect stomatal development,which can improve plant drought resistance by reducing stoma density,thereby reducing transpiration rate and increasing instantaneous water use efficiency,thus increasing the resistance to drought.(6)The transcriptome data of WT and Pu-miR172d overexpressed.ussuriensis showed that before and after drought treatment,there are a large number of genes differentially expressed related photosynthesis,water deficit response and osmotic stress which indicated that Pu-miR172d caused a change in the expression level of photosynthesis genes after reducing the stoma density.After drought stress,Pu-miRl 72d promoted some drought-tolerant gene.The results of qRT-PCR showed that the transcription level of PuGTLl in Pu-miRl 72d overexpressed plants decreased significantly,indicating that PuGTLl is the target gene of Pu-miR172d.However,PuGTLl expressed specificly in tissues and only differentially expressed the top 5 leaves.(7)The full-length PuGTLl coding region of PuGTLl was cloned and the PuGTLl inhibitory expression vector(PuGTLl-SRDX)was constructed.Under normal growth conditions,PuGTLl-SRDX inhibited the expression of transgenic P.ussuriensis and Pu-miR172d overexpressed plants in the regulation of stomatal development.PuGTL1-SRDX inhibited the expression of P.ussuriensis significantly compared to WT,and the stoma density was significantly reduced.It is lower than WT in terms of photosynthetic rate,stomatal conductance and transpiration rate,and the instantaneous water use efficiency is higher than WT.After stopping watering,PuGTLl-SRDX inhibited the expression of.ussuriensis with good growth and leaf water content higher than WT,while growth of WT was significantly inhibited.After rehydration,PuGTL1-SRDX inhibited the expression of P.ussuriensis and maintained normal growth,while WT was difficult to return to normal growth.The determination of physiological indicators MDA,conductivity and H2O2 content further indicated that PuGTL1 has strong drought resistance potential.QRT-PCR showed that the expression level of PuSDDl was significantly up-regulated in PuGTLl-SRDX inhibited plants and Pu-miR172d overexpressed plants.These results indicate that PuGTLl and Pu-miR1 72d function similarly,which can reduce the stomat density to reduce the transpiration rate,and increase the instantaneous water use efficiency,so that the plant has enhanced drought resistance.Based on the above results,this study demonstrated the molecular mechanism of Pu-miR172d regulating plant stomatal development by inhibiting the expression of its target gene PuGTL1.This will lay a theoretical foundation for the future study of miRNA on the study of stomatal development regulation and drought-resistant breeding in forest trees.