Molecular Mechanism Of The Involvement Of Poplar ZFHD1-CML23 Module In The Regulation Of Adventitious Root Development And Pi And Aluminum Stresses

Calcium ion(Ca²⁺)is a crucial second messenger in plants,which affects plant growth,development,and stress response signaling in various ways.The calcium signaling system relies on calcium-binding proteins,such as calmodulin（Ca M）and calmodulin-like proteins（CMLs）.As downstream Ca²⁺receptors in the calcium signaling system,CMLs facilitate the completion of the entire calcium signaling system and regulate plant physiological metabolism and gene expression.Poplar（Populus）is one of the tree species widely used for afforestation in northern China,with characteristics such as rapid growth and easy asexual reproduction.Therefore,analyzing the related functions and molecular mechanisms of CML genes in poplar is of great significance for China’s wood production and ecological environment construction.In previous research,we cloned the Pde CML23-1 gene in poplar.For convenience,the CML23 gene described in this paper represents Pde CML23-1.The root length and average fresh weight of the overexpressed transgenic strain OE-CML23 were significantly higher than those of the wild-type strain（WT）,and the Ca²⁺content in the plant body was lower than that in the WT lines,indicating that the CML23 gene may be involved in the regulation of root-related traits in poplar.Based on this,we conducted in-depth research on this gene,and the main research results are as follows:1.We constructed a CML23 gene knockdown liens（RE-CML23）and obtained three lines:RE-CML23-1,RE-CML23-2,and RE-CML23-3.The root length of the three lines was significantly shorter than that of the WT plant（P<0.05）,further demonstrating the involvement of this gene in the regulation of adventitious root elongation in poplar.2.During the experiment,we found that the RE-CML23 strains showed obvious phosphate deficiency symptoms,such as reddening of the petiole,under low phosphate conditions.By finely determining the inorganic phosphorus（Pi）content in poplar apical buds and roots,we found that under low phosphorus conditions,the Pi content in OE-CML23 apical buds increased significantly（P<0.05）compared to WT,while the Pi content in roots decreased significantly（P<0.05）;the Pi content in RE-CML23 apical buds decreased significantly（P<0.05）,and the Pi content in roots increased significantly（P<0.05）,At the same time,the expression of the CML23 gene was up-regulated when poplar was subjected to low Pi stress.This experimental result proved that the CML23 gene responded to the low phosphate stress in plants and participated in the upward transport of Pi in the plant body.3.The CML23 gene responds to aluminum stress in transgenic materials,and fine measurements of Al³⁺content in the apical buds and roots of OE-CML23 and RE-CML23showed that under aluminum stress,Al³⁺content significantly increased in OE-CML23apical buds（P<0.05）but significantly decreased in its roots（P<0.05）,whereas RE-CML23apical buds showed a significant decrease in Al³⁺content（P<0.05）and a significant increase in its roots,which is consistent with the results of low-phosphate treatment.In addition,we observed that in a high-Al³⁺environment,the leaves of OE-CML23 fell off earlier than those of WT and RE-CML23,and its growth was better.These results indicate that the CML23 gene in poplar is involved in the regulation of aluminum ion stress.4.To screen for upstream transcription factors of the CML23 gene,we first determined its core regulatory region by promoter deletion experiments.Through the promoter deletion experiment and the characteristic of high expression in the root system,we found that the promoter fragment named CML23pro-A is the core regulatory region of the CML23 gene.Using this fragment,we screened a yeast one-hybrid library and ultimately identified a zinc finger domain transcription factor that may be an upstream transcription factor of the CML23 gene,which was named ZFHD1.Through yeast one-hybrid（Y1H）,tobacco transient co-expression experiments,and chromatin immunoprecipitation quantitative PCR（Ch IP-q PCR）,it was confirmed that poplar ZFHD1 can specifically bind to the CML23promoter and promote its expression.Analysis of the characteristics related to the ZFHD1gene revealed that the ZFHD1 protein is located in the nucleus.Expression analysis of the gene under different conditions such as in different tissues,low phosphate,and high aluminum showed that ZFHD1 responds to low phosphate and aluminum stress at the expression level,and the gene is also significantly expressed in stems,mature leaves,leaf stalks,and roots.5.Transgenic materials were created to overexpress（OE-ZFHD1）and suppress（RE-ZFHD1）the expression of the ZFHD1 gene.Two overexpression lines（OE-ZFHD1-1 and OE-ZFHD1-2）and two suppression lines（RE-ZFHD1-1 and RE-ZFHD1-2）were obtained after positive identification.Two strains of RE-ZFHD1 with sufficient plant numbers were phenotyped for adventitious root length and it was found that the adventitious root length of RE-ZFHD1 was significantly smaller than that of the WT strain,and the phenotype was consistent with that of the RE-CML23 strain.In addition,CML23 gene expression was inhibited in the RE-ZFHD1 transgenic lines.These experimental results indicate that ZFHD1 regulates the elongation and development of poplar adventitious roots by positively regulating downstream CML23 gene expression.6.Furthermore,the Pi content in poplar apical buds and roots was determined under low phosphate culture conditions in the two RE-ZFHD1 transgenic lines.The results showed that the Pi content in the apical buds of the two RE-ZFHD1 lines significantly decreased（P<0.05）,while the Pi content in the roots significantly increased（P<0.05）.The roots of RE-ZFHD1 plants grown in high Al³⁺hydroponic solution were stained with safranin,and the results showed that the Al³⁺content in the roots of the RE-ZFHD1 lines was higher than that in the wild type.These results indicate that the ZFHD1 transcription factor regulates the concentrations of phosphate and Al³⁺in plants by positively regulating the expression of CML23.In summary,through the present study,we have revealed a ZFHD1-CML23 molecular module involved in the regulation of adventitious root elongation and phosphate/aluminum stress in plants.Under low phosphate or high aluminum ion conditions,the expression of ZFHD1 is induced,which in turn activates the expression of CML23,promoting the upward transport of Pi or Al³⁺in plants and alleviating the effects of low phosphate stress or aluminum toxicity.