Molecular Mechanism Of Interaction Between ABA And TOR Signals In Plants

Plants suffer from a variety of biotic and abiotic stresses during growth and development.In order to adapt to environmental changes and resist the invasion of pathogens,plants have evolved a set of precise regulatory mechanisms to resist environmental stress.ABA is a key plant hormone for plant resistance to abiotic stress.ABA enhances plant resistance to abiotic stress by regulating the closure of stomata and increasing the expression of ABA-responsive genes.ABA signal interacts with TOR signal,BR signal and JA signal to regulate plant growth and stress resistance.The transcription factors ABI4 and ABI5 of ABA signaling pathway play key roles in the interaction between ABA signal and other plant hormone signals.TOR signaling pathway is a key regulator of plant growth and development.Interpreting the interaction between TOR signaling pathway and ABA signaling pathway is of great significance for understanding the dynamic balance between plant growth and resistance to environmental stresses.Whether ABI4 and ABI5 participate in TOR signaling pathway in plants requires further research.In this study,through genetics,biochemistry and molecular biology,and transcriptome sequencing,we reveal the interaction between ABI5 and TOR signaling and autophagy signals to regulate the balance between plant growth and stress response.(1)The mutant trin1,which is insensitive to the TOR inhibitor AZD,was first screened from the EMS mutant library by using the TOR inhibitor AZD.Fine mapping and sequencing results indicated that TRIN1 is the allele of ABI4.Further functional analysis indicated that ABI4 plays an important role in chloroplast development and chlorophyll metabolism.Transgenic Arabidopsis overexpressing ABI4 is hypersensitive to AZD,which inhibits the extension of hypocotyls and cotyledons in Arabidopsis seedlings,resulting in cotyledons failure in turning green.GUS staining results showed that inhibition of TOR activity by AZD resulted in a large accumulation of ABI4 protein,thereby inhibiting chloroplast development and plant growth.These results indicate that TOR positively regulates chloroplast development and plant growth by promoting the degradation of TRIN1/ABI4 in Arabidopsis.(2)The expression levels of ABA biosynthesis and ABA-responsive genes were significantly up-regulated by inhibiting the growth and development of Arabidopsis thaliana using the specific inhibitors RAP and AZD.In the case of TOR inhibition,the content of ABA was significantly increased in Arabidopsis thaliana,indicating that inhibition of TOR induces biosynthesis of ABA,which activates the expression of ABA-responsive genes.Further tested the CI value of the drug effect combined index,found that the CI of RAP+ABA was significantly less than 1,indicating that RAP and ABA can produce strong synergistic effect to inhibit the growth of BP12-2 Arabidopsis seedlings.Inhibition of TOR mimics abiotic stress in plants,and plants thus induce biosynthesis of ABA and activate the expression of ABA-responsive genes to against this stress.These results suggest that there may be crosstalk between TOR and ABA signals to synergistically regulate plant growth and stress responses.(3)Drug sensitivity tests found that abi5-1 mutant Arabidopsis was insensitive to AZD,but overexpression AtABI5 transgenic Arabidopsis lines were hypersensitive to AZD.The cotyledons of overexpressing AtABI5 transgenic Arabidopsis cannot turn green and hypocotyls and roots cannot elongate when inhibits the activity of TOR by AZD,which seriously hinder the growth and development of seedlings.GUS staining results showed that inhibition of TOR activity by AZD resulted in a large accumulation of ABI5 protein,thereby inhibiting the growth and development of Arabidopsis thaliana.Y2 H experiments indicated that AtS6K2 interacts with AtABI5 full length and AtABI5-C terminal containing bZIP domain.Bimolecular fluorescence complementation assay(BiFC)confirmed the interaction of AtS6K2 with AtABI5 in the nucleus.These results indicated that TOR and ABA signals regulate plant growth and development through the interaction between AtS6K2 and AtABI5.(4)In the dark,the abi5-1 mutant significantly slowed the senescence and yellowing of the leaves compared with the wild type,whereas the transgenic Arabidopsis overexpressing AtABI5 accelerated the senescence and yellowing process of the leaves.Western blot and MDC staining showed that the autophagosomes of the abi5-1 mutant were significantly less than the wild type control treated with dark,while overexpression AtABI5 lines showed significantly more autophagosomes than the wild type control group in the dark.Moreover,the expression levels of autophagy-related genes in overexpression AtABI5 lines was also significantly higher than that in wild-type controls,indicating that AtABI5 is involved in the regulation of autophagy in dark conditions.Yeast two-hybrid and BiFC experiments found AtABI5 interacts with AtKIN10/11.Additionally,the AtABI5 protein contains conserved SnRK1? phosphorylation sites,implying that AtKIN10/11 may regulate autophagy and ABA signaling through phosphorylating AtABI5.(5)A basic bioinformatics analysis of StABI5 found that StABI5 also contains conserved C1,C2,C3,bZIP and C4 domains as AtABI5.Subcellular localization analysis revealed that StABI5 is mainly localized in the nucleus of tobacco.Overexpression of StABI5 transgenic potato lines showed earlier maturity and senescence than wild-type Desiree potato,and transgenic potato leaves showed yellowing and premature senescence phenotypes.Furthermore,overexpression StABI5 transgenic potato accelerated the yellowing and senescence of the leaves under dark treatment,indicating that StABI5 is involved in the regulation of chloroplast and autophagy,which is consistent with the function of AtABI5 in Arabidopsis.(6)Transcriptome sequencing analysis of overexpressing StABI5 transgenic potato showed that StABI5 plays important roles in regulating potato photosynthesis,chloroplast development,protein synthesis and autophagy.Transcriptome data showed that most of the genes involved in photosynthesis and carbon fixation were significantly down-regulated in overexpressing StABI5 transgenic potato plants,indicating that StABI5 negatively regulates photosynthesis-related genes expression in potato.In addition,many genes involved in plant hormone signaling pathways such as auxin,CKs,GA,BRs,ET,and JA were reprogrammed,suggesting that StABI5 is involved in the regulation of these plant hormone signals in potato.These results indicate that there is a tight and complex regulatory network between StABI5 and other plant hormone signals to regulate plant growth and development in potato.