Molecular Mechanism Of Auxin Signaling In Regulating Lateral Root Development Under Salt Stress In Poplar

Poplar are fast-growing woody plants of great economical and ecological importance,which widely cultivated in the world.Soil salinity is a major threat to the yield and quality of wood in forestry production.In general,high salinity reduces the absorption of water and nutrients,causing ionic toxicity and oxidative damage to cells.Developmental plasticity in the root system is an important strategy allowing trees to cope with salt stress,as the root plays a vital role in water and nutrient uptake,anchorage to the soil,and is the first organ to sense high salinity.In order to balance between growth and stress response,trees need to finely regulate root development,especially lateral root development.Auxin is a well-known phytohormone that plays a key role in regulating lateral root development.Previous studies have shown that the auxin signaling-related miR390/TAS3/ARF module orchestrates lateral root formation in Arabidopsis.However,it is still unclear whether this module is also required for the development of the lateral root in trees,and their roles in salt response mechanism remain elusive.What are the specific mechanisms of auxin signaling in response to salt stress?In this study,a miRNA（miR390a）specifically expressed in lateral root and induced by salt stress was screened from poplar.We demonstrated that the role of miR390/TAS3/ARF4 module in regulating lateral root development in poplar under salt stress,and the IAA17.1-HSFA5a module in regulating lateral root development through auxin signaling in response to salt stress.The main findings are as follows:（1）Salt-induced expression of miR390 during lateral root development in poplarThrough bioinformatics analysis and reverse transcription-quantitative PCR experiments,high expression of miR390 induced by salt in poplar roots was screened.The 1509 bp promoter fragment upstream of miR390a was further cloned and fused with GUS gene and introduced into poplar.GUS tissue staining in transgenic poplar showed that the miR390a promoter was highly expressed in lateral root,and its expression activity was induced by salt stress.These results indicated that miR390 may be involved in the regulation of lateral root development under salt stress.（2）MiR390 positively regulates lateral root development and salt tolerance in poplarIn order to clarify the biological function of miR390,overexpressing（OE）and short tandem target mimic（STTM）-based knockdown expression vectors were constructed and transformed into wild-type respectively.Compared with the wild type,the lateral root length and density of miR390-OE four-week-old seedlings were significantly increased,while those of miR390-STTM seedlings were significantly decreased.Wild-type,miR390-OE and miR390-STTM seedlings were treated with salt stress.The results showed that the lateral root growth of both wild type and transgenic lines was inhibited under high salt concentration,but the decrease of lateral root biomass of miR390-OE line was smaller than that of wild type,indicating that the overexpression of miR390 gene enhanced the salt tolerance of poplar.The miR390-STTM line was more sensitive to salt,and its lateral root biomass decreased more significantly.These results indicated that miR390 positively regulates lateral root development and enhances salt tolerance.（3）MiR390 regulates lateral root development under salt stress through the auxin response factor ARF4 in poplarIn order to determine the downstream regulatory genes of miR390 in poplar,reverse transcription-quantitative PCR detection of predicted candidate genes ARF2,ARF3 and ARF4 in poplar,and ARF4 with high expression in root was selected for subsequent genetic related experiments.To verify whether ARF4 is the target gene of miR390,a tasi ARF-resistant ARF4（ARF4m）containing mutated tasi ARF-binding sites was constructed.When ARF4m was overexpressed in miR390-OE plants,found that introducing ARF4m compromised lateral root development of the miR390-OE plants.The lateral root development of the ARF4m-OE was suppressed,the length of lateral root was shortened,and the number of lateral roots was also significantly reduced.Lateral root development was significantly enhanced in ARF4-RNAi lines.Salt stress treatment showed that ARF4-RNAi plants improved salt tolerance and ARF4m-OE plants reduced salt tolerance.These results indicated that ARF4 negatively regulates the biomass accumulation and salt tolerance in poplar,which is completely opposite to miR390.It is suggested that miR390 regulates the growth and salt tolerance of poplar lateral root by targeting downstream ARF4 gene and inhibiting its expression.（4）MiR390/TAS3/ARF4 regulates lateral root development under salt stress through auxin signalingIn order to further elucidate the molecular mechanism of miR390/TAS3/ARF4module in regulating lateral root development under salt stress,the expression of early auxin response genes were analyzed in miR390 and ARF4 transgenic plants.The expression of IAA3.1,IAA3.2,GH3.2,GH3.5 and auxin transporter PIN were affected.When DR5-GFP was introduced into miR390-OE,miR390-STTM,ARF4m-OE,ARF4-RNAi and wild type plants.The GFP fluorescent signaling driven by the auxin-responsive DR5 promoter were stronger in the lateral root tip of the miR390-OE and ARF4-RNAi plants than in the wild type plants but were weaker in the miR390-STTM and ARF4m-OE plants than in the wild-type plants.Salt stress analysis showed that salt treatment significantly reduced the expression of DR5:GFP in all plants.Compared with wild-type plants,the effect was much stronger in the miR390-STTM and ARF4m-OE plants.Conversely,DR5:GFP expression was less affected in the miR390-OE and ARF4-RNAi plants.These results suggested that,in poplar plants,miR390 and ARF4 antagonistically regulate endogenous auxin signaling during lateral root development in response to salinity.The auxin signaling pathway of miR390-OE and ARF4-RNAi transgenic lines was further blocked by overexpression of IAA17.1m.Compared with the miR390-OE and ARF4-RNAi,IAA17.1m-OE/miR390-OE and IAA17.1m-OE/ARF4-RNAi transgenic plants were more sensitive to salt stress,and their lateral root development and salt tolerance were partly recovered.These results indicated that the miR390/TAS3/ARF4 module regulated of salt-responsive lateral root development in poplar plants is mediated by auxin signaling..（5）IAA17.1 negatively regulates flavonols accumulation and inhibits ROS scavenging under salt stressThe salt tolerance of IAA17.1m-OE plants decreased when the auxin signaling pathway was blocked by IAA17.1m.NBT staining and H₂O₂determination showed that H₂O₂content in the overexpressing lines was significantly higher than wild type.These results indicated that IAA17.1m-OE lines accumulated more H₂O₂under salt stress,resulting in more serious oxidative damage to plants and reduced salt tolerance.DPBA（flavonols staining）staining of wild type and IAA17.1m-OE lines showed that fluorescence intensity of IAA17.1m-OE lines was significantly reduced compared with wild type plants,indicating that flavonols content in IAA17.1m-OE lines was reduced.The expression levels of CHS1,F3′H2,FLS1,FLS4 in the flavonols biosynthesis pathway were decreased in IAA17.1m-OE lines.Exogenous application of quercetin（one of flavonols）could enhance the salt tolerance of IAA17.1m-OE lines.These results indicated that IAA17.1 regulates the salt stress by regulating the accumulation of flavonols and affecting ROS scavenging.（6）IAA17.1 interacts with HSFA5a to co-regulate flavonols biosynthesisIn order to explore how IAA17.1 negatively regulates flavonols biosynthesis,yeast expression vector PGBKT7-IAA17.1（BD-IAA17.1）was constructed using IAA17.1 as bait protein.The IAA17.1 interacting protein,heat shock transcription factor HSFA5a were screened by yeast two-hybrid.Further BIFC experiments demonstrated that IAA17.1 could interact with HSFA5a in tobacco leaf cells.In order to clarify the biological function of HSFA5a,the HSFA5a gene overexpression vector was constructed and transformed into wild type.The lateral root growth of both wild type and transgenic lines was inhibited under high salt concentration,but the decrease of lateral root biomass of HSFA5a-OE lines was smaller than that of the wild type,indicating that the overexpression of HSFA5a gene improved the salt tolerance.These results indicated that HSFA5a enhanced salt tolerance in poplar.DPBA staining of WT and HSFA5a-OE lines showed that the fluorescence intensity of HSFA5a-OE lines was significantly increased compared with WT plants,indicating that the flavonols content in HSFA5a-OE lines was increased.Furthermore,the expression levels of key enzymes in the flavonols biosynthesis pathway,such as CHS1,F3′H2,FLS1 and FLS4,were increased in HSFA5a-OE lines.By Ch IP quantitative PCR and yeast one hybrid assay,HSFA5a can directly bind to the binding sites of CHS1,F3’H2,FLS1 and FLS4 promoters to regulate gene expression.HSFA5a directly activated the expression of flavonols biosynthesis genes such as CHS1,F3′H2,FLS1and FLS4,and IAA17.1 could inhibit HSFA5a activation of these genes.These results indicated that IAA17.1 regulates the biosynthesis of flavonols through interaction with HSFA5a.（7）HSFA5a can enhance the salt tolerance of IAA17.1m-OE plantsIn order to verify the molecular mechanism of IAA17.1-HSFA5a on lateral root development of poplar under salt stress,HSFA5a-OE was transformed into IAA17.1m-OE line.The seedlings of IAA17.1m-OE and HSFA5a-OE/IAA17.1m-OE were treated with salt stress.The results showed that the lateral root growth of all seedlings was inhibited under high salt concentration.However,the decrease of lateral root biomass of HSFA5a-OE/IAA17.1m-OE was smaller than that of IAA17.1m-OE.These results indicated that HSFA5a overexpression enhanced the salt tolerance of IAA17.1m-OE plants.In summary,this study demonstrated that the miR390/TAS3/ARF4 module is involved in lateral root maintenance against salt toxicity by modulating auxin signaling,and demonstrated that the auxin signaling through the transcriptional activator HSFA5a interacts with IAA17.1 to regulate the biosynthesis of flavonols,affect the ROS scavenging,and ultimately improve salt tolerance.This study laid a foundation for understanding the adaptive mechanism of trees root development under salt stress,and provided theoretical support for improving the salt tolerance of woody plants in the future.