Molecular Mechanism Of Fraxinus Mandshurica SPL2 In Drought Adaptation And Xylem Development

Fraxinus mandshurica is a large deciduous tree of the genus Ash in the family Lignanaceae.It is one of the three most valuable hardwoods in the northeast of China.It is an important afforestation species in the national reserve forest plan.SPL（SQUAMOSA promoter binding protein-like）is a family of transcription factors unique to plants.A previous study showed that the transcriptional expression of Fraxinus mandshurica SPL2 was induced by drought,and the FmSPL2 gene was cloned and genetically transformed into tobacco to obtain FmSPL2 transgenic tobacco FmSPL2-OE1 and FmSPL2-OE2.In this study,FmSPL2-OE1 and FmSPL2-OE2 were used as materials to investigate the molecular mechanism of FmSPL2involvement in drought stress and xylem development.To investigate the molecular mechanism of FmSPL2 involved in drought stress and xylem development.The details of the study are as follows.1.Functions of FmSPL2 in response to adversity stress.FmSPL2 transgenic tobacco was treated with drought and salt stresses.It was shown that FmSPL2-OE was more drought and salt tolerant.26.07%and 25.93%higher xylem thickness between the sixth internodes of FmSPL2-OE1 and FmSPL2-OE2 than WT strains,respectively.The lignin content was 19.87%and 18.48%higher than that of the WT strain,respectively.After drought stress,the xylem thickness and lignin content of FmSPL2-OE were still higher than those of WT.The stomatal opening of FmSPL2-OE1 and FmSPL2-OE2 were 12.05%and19.23%smaller than that of WT,respectively.Stomatal density was 11.54%and 26.09%smaller than that of WT,respectively.Under normal growth conditions,FmSPL2-OE1 and FmSPL2-OE2 pollen grains were 75%and 60.34%more numerous than WT,respectively.After salt stress,FmSPL2-OE1 and FmSPL2-OE2 pollen grains were 2.17 and 2.15 times more numerous than WT pollen grains,respectively,and WT showed necrosis of pollen grains.52.17%and 51.57%higher stomatal density of WT than FmSPL2-OE1 and FmSPL2-OE2,respectively.The SOD and POD activities of FmSPL2-OE increased and the MDA,H₂O₂and DFR contents decreased compared with WT strain after drought and salt stress treatments.After drought stress treatment,the expression of FmSPL2 was up-regulated.The expression levels of drought-related genes（Sn RK2.6 and AREB1）were up-regulated after transient transformation of FmSPL2.The results indicated that the FmSPL2 gene could improve the tolerance to drought and salt stress.2.Analysis of downstream gene regulation mechanisms by FmSPL2 in drought stress.The transcriptomes of FmSPL2-OE1 and wild-type tobacco were sequenced after drought stress.KEGG annotation and enrichment results showed that the differentially expressed genes were mainly enriched in phytohormone signaling,plant-pathogen interaction pathway,starch and sucrose metabolic pathway and benzyl propane synthesis pathway.Among the phytohormone signaling pathways,the analysis revealed that FmSPL2 was involved in activating growth hormone signaling and promoting plant growth.It is involved in activating cytokinin signaling to promote cell division and root development.Activates gibberellin signaling pathway through the degradation of DELLA protein by ubiquitination to regulate stem growth and plant germination.Activates the abscisic acid signaling pathway by encoding the abscisic acid receptor gene（PYR/PYL）,which downregulates PP2C,thereby affecting seed dormancy and stomatal closure and enhancing tolerance to adversity.In the phenylpropane synthesis pathway,by regulating key genes for phenylpropane synthesis（4CL,CCR,CAD）at the transcriptional level,it upregulates pineal alcohol glucosyltransferase（UGT72E）,which in turn catalyzes the synthesis of lignin.FmSPL2 can up-regulate the expression of drought-related genes（MYB167,NAC6,NAC120）.For WGCNA analysis,FmSPL2 has a higher correlation in the brown module.It may have functions such as regulating plant development,root formation and seed germination.Taken together,these analyses suggest that FmSPL2 may enhance adaptation to drought by participating in the abscisic acid signaling pathway and promoting the synthesis of lignin.3.Fraxinus mandshurica miR156a negatively regulates the transcriptional expression of FmSPL2.Bioinformatic analysis showed that Fraxinus mandshurica miR156 precursor sequences have low affinity to Arabidopsis and poplar.There were four miR156 precursor sequences in Fraxinus mandshurica,and all of them constituted a mature sequence.Fraxinus mandshurica miR156 is predicted to target 14 FmSPLs genes.Transcript expression analysis confirmed that FmSPL2,FmSPL9 and FmSPL13 were downstream target genes of miR156 and were down-regulated by miR156 expression.Fraxinus mandshurica miR156 primary transcript sequences were cloned,overexpression vectors were constructed,and Fraxinus mandshurica hypocotyls were used as explants for genetic transformation,and more buds emerged from FmmiR156-OE compared with FmSPL2-OE.The expression of shoot-associated genes（WUS）was detected,and it was clear that FmmiR156 could up-regulate the expression of WUS genes.The results indicate that miR156 can target and negatively regulate the FmSPLs gene.