The Molecular Mechanism Of Transcription Factor PtoMYB142 Involved In Drought Resistance In Poplar

Climate extremes such as drought have become the most serious threat to global forest yield.In response to the lethal stress,regulation at tissue or organ level to active plant’s ability to adapt to water scarcity is the main way that plants cope with drought.As perennial plants,trees are exposed to repeated and prolonged periods of drought stress and therefore need to work more effectively to balance water uptake,water transport and water dissipation in a water-stressed environment.These adjustments depend on the plants’perception of drought signals and cross-integration with endogenous signal transduction systems.Under drought stress,reduced xylem pressure which occurs from the low transpiration rate and leads to the unstable stem sap flow in vessel sand triggers embolism.This will severely weaken the water transport capacity of xylem vessels.Normally,trees are able to mitigate embolism-induced barriers to water transfer by forming new vessels and adapting to their morphological structure.Consequently,the developmental plasticity of xylem vessels is a key strategy for trees to cope with the resistance of long-distance water transport and thus improve drought adaptation,and it is an ideal model for resolving the molecular mechanisms of drought-induced phenotypic changes in trees.However,so far it is not clear which endogenous signaling molecules are involved in the integration of xylem vessel developmental remodeling with drought signaling.Leaf size and the deposition of epidermal waxes are two of the main strategies in which plants alter their water transpiration rates under long term drought conditions.In order to increase the water retention capacity,plants reduce water loss by reducing the leaf area and increasing the deposition of waxes on the leaf epidermal cells.There are still few studies on the regulation of leaf development and epidermal wax synthesis in poplar under drought conditions at present.For the past few years,several Transcription factors including Ptr AREB1、Pd NF-YB21、Pd STZ1 mediated mechanisms of drought tolerance in poplar have been elucidated,suggesting that there exists a complex transcriptional regulatory network in response to drought in trees.MYB transcription factor superfamily is one of the largest families in plants.The MYB transcription factor family is characterized by the presence of one or several conserved MYB domains.It has been proved that numerous R2R3 MYB transcription factors are involved in the regulation of stem secondary growth,and serve as master switches in xylem formation and secondary cell wall deposition.Meanwhile,previous studies showed that several MYB TFs could directly regulate the expression of cuticular wax biosynthesis genes.In this study,we constructed a Full-Length c DNA Over-Expressing（FOX）Library from the Chinese white poplar,Populus tomentosa,and screened out a drought-induced R2R3 MYB transcription factor,PtoMYB142.PtoMYB142 positively regulates drought resistance of poplar under dehydration.Further morphology and anatomy studies demonstrate that overexpressing PtoMYB142 reduces leaf area and xylem vessel diameter and increased vessel density by modulating gibberellin catabolism.On the other hand,PtoMYB142 decreases leaf water loss by regulating cuticular wax biosynthesis.This study eventually put forward a molecular mechanism of the key role of PtoMYB142 in drought resistance of poplar through the regulation of GA levels,specifically as follows:（1）A Full-Length c DNA Populus-FOX-Arabidopsis library was constructed and identification of PtoMYB142In this study,the poplar c DNA library was constructed into the plant expression vector and further transformed into Arabidopsis thaliana.In total,we identified 2098Basta-resistant plants,and 1749 of them were positive transgenic plants,which was confirmed by PCR determination.593 single PCR bands from different transgenic lines were randomly selected for sequencing,and 402 diverse sequences of poplar genes were isolated.Most of these genes were involved in photosynthesis,environmental adaptation,and ribosome biogenesis based on Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway annotation.We observed and identified the phenotypes of the T₂ generation from sequenced positive seedlings.From the 402 FOX lines,262 individuals showed obvious phenotypes,including lodging,dwarfing,increased branching,glabrous leaves,and so on,suggesting that these genes play important roles in plant growth and development.For the purpose of screening genes that are conducive to drought stress tolerance,we planted the seeds of T₂ transgenic plants and conducted dehydration treatment.A transgenic line K108 which was insensitive to dehydration was screened out from the library.BLAST searching suggested that the inserted gene was a homolog of Potri.012G055600.Amino acid sequence alignment and phylogenetic tree analysis showed that this gene shares a high degree of homology with Ptr MYB142,so we named it PtoMYB142.To investigate the function of PtoMYB142,we further overexpressed PtoMYB142in Arabidopsis which led to abnormal growth,including delayed development,dark green leaves.Drought and re-watered treatment confirmed that PtoMYB142 significantly enhances the plant drought tolerance（2）PtoMYB142 is a transcription activator,and primarily induced by droughtAdditionally,transcriptional self activation analysis in yeast suggested suggested thatPtoMYB142 possessed a transcription al activation activity.Tissue expression profile analysis showed that PtoMYB142 predominantly expresses in stem secondary xylem and mature leaves by using RT-q PCR and pro PtoMYB142:GUS reporter lines.In addition,expression of PtoMYB142 was also induced significantly by PEG and drought treatment in both of these organs.These results suggest that PtoMYB142 possibly integrates leaf or xylem development with plants response to drought.（3）PtoMYB142 positively regulates drought tolerance by modulating leaf and xylem development of Populus tomentosaTo identify the function of PtoMYB142 in plants response to drought stress,WT lines,PtoMYB142-OE and PtoMYB142-Cas9 mutant lines were used for investigating their drought tolerance.Phenotype investigation found that overexpression of PtoMYB142 inhibited growth of transgenic poplar plants,but the growth situation of OE lines under drought treat was much better than WT lines.Alterations in morphology and physiological responses of WT and transgenic poplars to drought stress were examined and the results showed that overexpressing PtoMYB142 reduces the leaf water loss and enhances xylem water transport efficiency,while PtoMYB142-Cas9 lines displayed opposite phenotype.Taken together,these results suggest that PtoMYB142 positively regulates drought resistance in poplar.To elucidate the cause of reduced leaf water loss and enhanced xylem water transport,we compared the phenotypic differences of leaves and stems of PtoMYB142-OE and PtoMYB142-Cas9 plants relative to WT,respectively.PtoMYB142 negatively regulates leaf epidermal cell expansion and thus leads to leaf area reduction,meanwhile it alters stem secondary growth including increased vessel density and attenuated vessel lumen area.（4）PtoMYB142 directly regulates PtoGA2ox4 which is a GA2-oxidase geneTo determine downstream genes targeted by PtoMYB142 in poplar,comparative transcriptomic profiling via RNA-seq experiments was performed with WT and transgenic PtoMYB142-OE lines.As multiple GA-deficiency phenotypes were observed in PtoMYB142-OE plants,we further evaluated expression levels of GA metabolism-and signal transduction-related genes.Transcriptome sequencing results revealed that GA pathway genes were significantly changed in PtoMYB142-OE lines,the GA catabolism gene（PtoGA2ox4）was up-regulated remarkably.RT-q PCR analysis further confirmed the expression of PtoGA2ox4 was incuded in stem and leaves under drought conditon.Meanwhile,PtoGA2ox4 was significantly upregulated in PtoMYB142-OE lines and downregulated in PtoMYB142-Cas9 plants.We next measured the contents of the bioactive forms of GA（GA1 and GA4）in WT,PtoMYB142-OE and PtoMYB142-Cas9using liquid chromatography-coupled mass spectrometry（LC-MS）.The overall level of GAs was decreased in OE lines,but increased in PtoMYB142-Cas9 lines.We speculate that PtoGA2ox4 might be a downstream target gene of PtoMYB142 and also involved in drought tolerance.Promoter sequence analysis revealed that four putative MYB binding sites（MBS）were predicted in the 2kb upstream regions of PtoGA2ox4.To examine that PtoMYB142could directly bind to the MBS motif sequence in the PtoGA2ox4 promoter,chromatin immunoprecipitation Ch IP-PCR using an anti-GFP antibody EMSA and Y1H assay was performed.The results indicating that PtoMYB142 could bind to the PtoGA2ox4promoter regions around the P2 and P4 sites in vivo and in vitro.Dual luciferase assay showed that PtoMYB142 could significantly activate PtoGA2ox4 expression via directly binding to two MBS sites in its promoter.（5）PtoGA2ox4 mediates the regulation of PtoMYB142 on gibberellin content in Populus tomentosa under drought stressPtoMYB142 directly regulates the transcription of PtoGA2ox4,which in turn affects the content of active gibberellins GA1 and GA4 in poplar.Exogenous GA3 treatment can block the PtoMYB142-mediated GA-deficient phenotypes under drought stress.The functional complementation of PtoGA2ox4 in PtoMYB142-OE plants by conducting the RNA interfering（RNAi）-based knockdown of PtoGA2ox4 gene was constructed for phenotypic analysis.PtoGA2ox4-RNAi/PtoMYB142-OE plants were more sensitive to dehydration stress than PtoMYB142-OE trees while the leaf size,epidermal and palisade cell size in transgenic PtoGA2ox4-RNAi/PtoMYB142-OE plants was almost restored to that of WT.Taken together,drought stress induced the expression of PtoMYB142,which activated PtoGA2ox4 expression,resulting in reducing GA content and retarding the poplar growth including reducing leaf area and vessel lumen as well as increasing vessel density,thus resulting in improving drought tolerance.（6）PtoMYB142 positively regulates epidermal wax biosynthesis in Populus leavesChlorophyll leaching assay,leaf water-loss assay and Scanning electron microscope observation were used to prove that the glossy trait is involved in changes in epicuticular wax biosynthesis.We measured the cuticular wax contents and compositions of WT and PtoMYB142-OE leaves by gas chromatography（GC-MS）.Total wax load was elevated2.88-4.59 folds in the PtoMYB142-OE leaves in comparison with WT,the contents of fatty acids,alcohols and alkanes in PtoMYB142-OE plants were also changed.（7）PtoMYB142 activates the expression of PtoCER4 and PtoKCS6 directly to trigger epidermal waxes biosynthesis.For the purpose of illuminating the molecular mechanism of PtoMYB142 regulating wax synthesis.Several wax synthase genes were found to be up-regulated by RT-q PCR.Transient activation experiment showed that PtoMYB142 remarkable activates the expression of PtoCER3,PtoCER4 and PtoKCS6.Y1H assay and Ch IP-q PCR demonstrate the direct binding of PtoMYB142 to the promoter of PtoCER4 and PtoKCS6 in vitro and in vivo.PtoCER4 and PtoKCS6 were significantly induced by PEG treatment.In brief,drought stress induces the expression of PtoMYB142,which increases the expression levels of PtoCER4 and PtoKCS6,resulting in epicuticular wax deposition and enhancing resistance to drought.