MicroRNA And Related Genes Mediating H₂S To Mitigate Alkaline Salt Stress Of Malus Hupehensis

Soil salinization seriously affects apple production.Salt stress of fruit trees simulated by NaCl treatment has been paid more and more attention,However,the salt in salinized soil is actually a mixture of NaCl,Na₂SO₄,Na HCO₃and Na₂CO₃and othe salts,This mixture is alkaline,which is called alkaline salt in soil science.Roots are in directly contact with the soil,and are the first to experience and directly suffer from saline-alkali stress.Apple roots come from the root stock,Malus hupehensis Rehd.var.pingyiensis Jiang(PYTC)is an excellent apple rootstock,but it grows poorly in saline alkali soil.Micro RNAs(mi RNAs)are evolutionally conserved endogenous non-coding RNAs and participate in plant growth and development and response to various abiotic stresses.However,little is known about the regulatory mechanism of mi RNAs and their target genes in response to alkaline salt stress.Hydrogen sulfide(H₂S),as a gas signaling molecule,the donor sodium hydride sulfide(Na HS)can improve the tolerance of plants to salt stress.In this study,we used seedling of PYTC as material,on the basis of confirming that H₂S pretreatment alleviated the alkaline salt stress(Na Cl:Na₂SO₄:Na HCO₃:Na₂CO₃=1:9:9:1)of PYTC.The specific mi RNAs and their target genes in response to H₂S pretreatment and alkaline salt stress were identified,correlation analysis of mi RNA and its target genes with transcriptome was performed.By comprehensively analyzing the regulation function of mi RNA-mediated differentially expressed genes.Meanwhile,the role of mhp-mi R408a and target gene Mh BBP in relieving alkaline salt stress of H₂S was also discussed.The main results are as follows:1.Effects of exogenous H₂S on alleviating alkaline salt stress of PYTC and its physiological mechanism.H₂S pretreatment could partially alleviate the decrease of the number of absorbed roots induced by alkaline salt stress and the inhibition of root activity.At the same time,H₂S pretreatment significantly increased the endogenous H₂S content in leaves and roots.Under alkaline salt stress,the activities of antioxidant enzymes and and the content of H₂O₂was decreased.Na⁺content in leaves and roots increased significantly,K⁺content in roots decreased,and Na⁺/K⁺in leaves and roots increased under alkaline salt stress.H₂S pretreatment significantly reduced the content of Na⁺and the ratio of Na⁺/K⁺in leaves and roots.Under alkaline salt stress,the transcription levels of SOS1 and SKOR in the roots of PYTC were significantly increased,and H₂S pretreatment significantly inhibited the increase of SOS1 and SKOR transcription levels induced by alkaline salt.H₂S pretreatment can alleviate the inhibition of alkaline salt stress on the growth of PYTC.2.Identification of different micro RNAs involved in H₂S mitigation of alkaline salt stress in roots of PYTC.Twelve s RNA libraries(Control-1/2/3,H₂S-1/2/3,AS-1/2/3,H₂S+AS-1/2/3)were constructed using the roots of PYTC as total RNA templates.A total of115 known mi RNAs(belonging to 37 mi RNA families)and 15 new mi RNAs were identified using the BGISEQ500 sequencing platform.Under alkaline salt stress,the expression of mhp-mi R394a,mhp-mi R395d-5p and mhp-mi R160a were significantly down-regulated,suggesting that these mi RNAs may be completely induced or inhibited by alkaline salt stress.H₂S induced the expression of mhp-mi R477a and mhp-mi R827 upregulated while significantly inhibited the expression of mhp-mi R408a.Through the prediction and analysis of mi RNAs and their target genes under H₂S and alkaline salt stress,the differentially expressed genes are mainly involved in a variety of biological pathways,including plant hormone signal transduction(mhp-mi R160),DNA replication(mhp-mi R11011a),MAPK signaling pathway(mhp-mi R156),etc.3.Analysis of DEGs in response to H₂S and alkaline salt stress in roots of PYTC.Twelve c DNA libraries were constructed using the root RNA template of PYTC and RNA-seq sequencing was performed.In Control/H₂S+AS,Control/AS,Control/H₂S and AS/H₂S+AS comparison groups,5775 up-regulated genes(103434431)and 10800 down regulated genes(103434431),5847 up-regulated genes(103408663)and 12805 down regulated genes(103425512),726 up-regulated genes(103454388,103428875)and 892down regulated genes(103439436),2316 up-regulated genes(103405837)and 1998 down regulated genes(103442560)were identified,respectively.4.By associating mi RNA with transcriptional expression profiles,we analyzed the target genes predicted by mi RNA at the transcriptome level.The results showed that q RT-PCR was positively correlated with the transcriptome,while mi RNA was negatively correlated with the transcriptome,further confirming the reliability of the transcriptome data.Among them,in the H₂S+AS group,the expression of mhp-mi R169 was down-regulated,while the expression of its target gene NF-YA was up-regulated;in the AS group,the expression of mhp-mi R160 and its target gene ARF18 and SPL were up-regulated,which was consistent with the expression of transcriptome level.H₂S inhibits the expression of mhp--mi R408a and its target gene BBP expression is up-regulated,which is the same as the expression pattern of transcriptome.In addition,there were some differential gene expressions that were not mediated by mi RNAs,and they were found to be involved in S-containing compounds,ROS defense cell wall metabolism,carbohydrate and energy metabolism and osmotic regulation.Therefore,we speculated that H₂S pretreatment in the roots of PYTC could specifically induce the expression of S-containing compounds,change the expression level of cell wall density,maintain root elongation,and alleviate the damage to PYTC roots caused by alkaline salt stress by increasing primary energy metabolism and soluble sugar osmotic protection.5.The role of mi R408a and its target gene in mitigating alkaline salt stress by H₂S in PYTC.The Mh BBP gene and the primary transcript of mhp-mi R408a were cloned from PYTC,and the targeting relationship between mhp-mi R408a and Mh BBP was verified by histochemical staining.The primary transcripts of mhp-mi R408a and Mh BBP gene were overexpressed in apple calli.It was found that the expression of Mh BBP was down-regulated when mhp-mi R408a and Mh BBP were co-transformed,which indirectly proved the negative correlation targeting relationship between mhp-mi R408a and Mh BBP.And analyzed their expression characteristics under salt stress and found that 35S::mi R408a transgenic calli was sensitive to 150 m M Na Cl treatment,while 35S::Mh BBP transgenic calli showed improved salt tolerance.According to the above results,H₂S pretreatment inhibited the expression of mhp-mi R408a relieved the inhibitory effect on Mh BBP,and increased the expression level of Mh BBP,thus alleviating the damage of alkaline salt stress on PYTC.