The Functions Of Protein Phosphatase Genes MePP2C24/26 In ABA-Mediated Drought Response In Cassava(Manihot Esculenta Crantz)

During their entire life cycles,crops are often subjected to adversity stresses,including drought,high salinity,low temperature and disease.These unfavorable environmental factors often affect the growth and development of crops,and even cause the decline in yield and quality.Cassava is widely planted in Asia,Africa and South America.It is the sixth largest food crop in the world,the third largest food crop in tropical and subtropical regions,and also the staple food for 700 million people in the world.Cassava has the characteristics of drought tolerance and can be used as a typical material to study the drought tolerance mechanism of crops.Abscisic acid(ABA)plays a crucial role in regulating drought tolerance of plants.However,the current research on the mechanism of ABA-mediated drought tolerance in tropical drought-tolerant crops is still very limited.In this study,based on the analyses of long non-coding RNAs(lnc RNAs)in cassava,two regulatory genes Me PP2C24/26(protein phosphatase 2Cs)in the ABA signaling pathway were identified.Using research methods such as bioinformatics,molecular biology,genetics,and physiology,the upstream and downstream interaction components of Me PP2C24/26 were identified,and the function of Me PP2C24/26 in ABA-mediated drought response was also analyzed.The main results are as follows:1)Through the physiological analysis,the contents of hydrogen peroxide(H₂O₂)in cassava leaves under PEG and ABA treatments were clarified,and the enzyme activities of peroxidase(POD)and catalase(CAT)were significantly increased.Using the technology of ss RNA-seq,the differential long non-coding RNAs(lnc RNAs)in cassava leaves under either PEG or ABA treatment were analyzed,and their regulations via cis-acting,trans-acting and mi RNA were dissected.It was also discovered that lnc RNAs could respond to drought stress through ABA-dependent signaling pathway and ABA-independent signaling pathway.Meanwhile,Me PP2C24/26,the key regulators of the ABA signaling pathway,were identified in response to drought and ABA treatments.2)In total,13 ABA receptors(PYLs),80 PP2Cs and 10 protein kinases(Sn RK2s)were identified in the cassava genome.Evolutionary analysis revealed that PYLs were divided into 3 subfamilies,PP2Cs were divided into 13 subfamilies,and Sn RK2s were divided into 3 subfamilies.Among them,Me PP2C24/26 belonged to subfamily A and were subsequently cloned c DNA from cassava by RT-PCR technique.Subcellular localization analysis showed that Me PP2C24/26 were located in the nucleus,and expression analysis demonstrated that Me PP2C24/26 were induced by ABA,mannitol,Na Cl,and Me JA.3)The yeast two-hybrid experiment confirmed that Me PP2C24 and Me PP2C26 can respectively interact with Me PYL1,Me Sn RK2.1 and Me PYL-1,-5,-6,-7,-8,-11,-13,Me Sn RK2.1 on the yeast culture medium without ABA.While on the yeast culture medium containing ABA,Me PP2C24 and Me PP2C26 interacted with Me PYL-1,-4,-7,-8,-9,-11,-12,-13,Me Sn RK2.1 and Me PYL-1,-4,-5,-6,-7,-8,-9,-11,-12,-13,Me Sn RK2.1,respectively.At the same time,Me Sn RK2.1 can interact with the downstream Meb ZIP11 and Meb ZIP67.The bimolecular fluorescence complementation method was used to further verify the interaction of 7 pairs of proteins in the nucleus.These results indicated that Me PP2C24/26 interacted with more Me PYL members in the presence of exogenous ABA than that without ABA.However,the interactions between Me PP2C24/26 and Me Sn RK2.1,Me Sn RK2.1 and Meb ZIP-11,-67 were not affected by exogenous ABA.The double-luciferase experiment was performed to detect the promoter activities of Me PP2C24/26,and the results showed that their full-length promoters had high activities.The yeast one-hybrid experiment identified that Meb ZIP11/27 could interact with Me PP2C24 promoter in cassava.Besides,the interactions between Meb ZIP11/27 and the Me PP2C24 promoter region with ABRE cis-acting elements were confirmed by yeast one-hybrid experiment with missing different fragments of promoters.4)Using Agrobacterium-mediated genetic transformation to express Me PP2C24/26 in Arabidopsis thaliana could reduce the sensitivity of transgenic lines to ABA in terms of seed germination and root length;Moreover,Me PP2C24/26 expressing transgenic lines reduced the tolerance of transgenic lines to drought stress.Physiological analysis found that the activities of antioxidant enzymes in Me PP2C24/26 expressing lines increased the membrane damage and the intracellular oxidative damage.By analyzing the expressions of ABA-dependent drought-stress-response genes,it was found that the expression levels of positive regulatory genes in response to drought stress were lower in the Me PP2C24/26expressing lines than that in the wild-type.In this study,through systematic analyses of lnc RNA in response to PEG and ABA treatments in cassava,Me PP2C24/26,the key regulators of the ABA signaling pathway,were identified and revealed their interactive proteins.After Me PP2C24/26 expressed in Arabidopsis,they were found to negatively regulate ABA signaling and drought stress tolerance.