Cloning Of MfbHLH38 And MfPIF1 Of Myrothamnus Flabellifolia And Function Analysis In Respect To Abiotic Stress Tolerance

Plants as sessile species are vulnerable to environmental stress conditions.The transcription factors from various families play important roles in the gene regulation network under unfavorable environments to enhance tolerance to different abiotic stresses.Resurrection plant Myrothamnus flabellifolia Welw.is a dwarf shrub mainly distributing in South Africa.A long period of evolution and adaption to extremely drought environments make M.flabellifolia rich in genes for abiotic stress tolerance.In this study,two-dehydration induced genes,MfbHLH38 and MfPIF1,encoding putative bHLH transcription factors,were cloned fromM.flabellifolia.Through overexpression in Arabidopsis thaliana,the functions of two genes in regulating response to drought and salt stress were analyzed,and the underlying molecular mechanism was further explored.The main results obtained are as follows:1.Two bHLH genes,MfbHLH38 and MfPIF1,were isolated from the cDNA of M.flabellifolia by two pairs of specific primers.The length of the open reading frame（ORF）of the obtained nucleotide sequence is 720bp and 1188bp,which encoding 293 and 395 amino acids,respectively.MfbHLH38 has a typical basic helix-loop-helix（bHLH）domain,which belongs to the Ib group of bHLH family and displays the highest homology with grape VvORG2.MfPIF1 contains a typical bHLH domain and a typical APB domain,as well as a predicted APA domain,which belongs to the PIF subfamily of the bHLH family members.It shows the highest homology with grape（Vitis vinifera）VvPIF1.MfbHLH38 and MfPIF1were localized in the nucleus,thus they possible play basic biological functions as transcription factors（TFs）.2.The plant expression vectors were constructed and transformed into Arabidopsis plants.T₃ homozygous transgenic lines were obtained after being selected by the medium with kanamycin（Kan）.The expression level of MfbHLH38 and MfPIF1 in each transgenic line was detected by Quantitative Real-time PCR（qRT-PCR）analysis.For seedling stage assays,transgenic and wild-type（WT）Arabidopsis were subjected to drought（0,200,250,300 mM mannitol）and salinity（0,50,100,150 mM NaCl）stresses.MfbHLH38 and MfPIF1transgenic plants had significantly longer main roots than WT plants,which were more significant under moderate and mild stress conditions,respectively.For mature plant assays,the transgenic plants showed a lower degree of chlorisis and wilt of the leaves under natural drought or salinity（300 mM NaCl）stress conditions and recovered rapidly after rewatering upon drought treatment.3.Under drought and salinity stresses,the malondialdehyde（MDA）content and water loss rate of detached leaves in transgenic plants were lower than those of WT plants,while the chlorophyll content and osmoregulation substances including proline（Pro）,soluble protein,and soluble sugar in transgenic plants were higher than WT plants.Under stress conditions,compared with WT plants,transgenic plants showed lower accumulation of reactive oxygen species（ROS）indicating by milder stain intensity in histochemical staining（DAB and NBT）,lower hydrogen peroxide（H₂O₂）content,and higher anti-superoxide anion activity.Meanwhile,the activity of superoxide dismutase（SOD）,peroxidase（POD）,and catalase（CAT）in transgenic plants were significantly higher than those of WT plants under drought and salinity stresses.The stomatal movement of MfbHLH38 and MfPIF1overexpression plants was more sensitive to drought and exogenous abscisic acid（ABA）,and the stomatal aperture was notably lower than WT plants.4.To further study the molecular regulation mechanism of MfbHLH38 and MfPIF1genes in response to drought and salinity stresses,one ABA biosynthesis gene NCED3 and two ABA-responsive genes P5CS and RD29A related to stress were selected for determination.After the simulated drought with 10%polyethylene glycol（PEG-6000）and300 mM NaCl treatments for 1d or 4d,the relative expression levels of these target genes in transgenic and WT plants were determined using qRT-PCR.In comparison with WT plants,NCED3,P5CS and RD29A in transgenic plants showed significantly up-regulated expression to various degrees under drought and salinity stresses.In conclusion,two genes encoding bHLH transcription factor,MfbHLH38 and MfPIF1,were cloned fromM.flabellifolia.The results of morphological,physiological,biochemical and molecular studies showed that MfbHLH38 and MfPIF1 could regulate plant water retention capacity,maintain osmotic balance,enhance ROS scavenging ability,and may be involved in regulating ABA signaling pathway to significantly enhance tolerance of Arabidopsis to drought and salinity.This is also the first report that bHLH38 is involved in regulating plant response to drought and salinity stresses.This study will lay a foundation for further understanding of the molecular mechanism of drought adaptation in M.flabellifolia.