The Regulatory Mechanism Of The Interaction Of PAS2 And GAAPs Under Stress In Arabidopsis Thaliana

Various stress stimuli can cause a large number of unfolded or misfolded proteins accumulating in the endoplasmic reticulum(ER),named as ER stress.The unfolded protein response(UPR)will be activated when facing to ER stress to recover the homeostasis of endoplasmic reticulum.And programmed cell death(PCD)will be induced when the stress intensity exceeds the recovery capability of cells.BI-1suppresses mammalian cell death through association with IRE1,one of UPR sensors.Arabidopsis BI-1 modulates stress-mediated PCD by interacting with FAHs via Cb5to promote sphingolipid synthesis or positively regulates autophagy by interacting with ATG6.Arabidopsis GAAP is one of subfamilies of BI-1.At GAAPs negatively regulated UPR and suppressed cell death under ER stress.At PAS2 was isolated as one of the interactors of GAAPs by Y2H screen assay.As a key enzyme of fatty acid elongation complex,PAS2 is involved in the synthesis of VLCFA and also functions in anti-phosphatase.PAS2 has been shown to play an important role in both development and signal transduction in eukaryotes.To determine further the function of PAS2 and its interaction with GAAPs,we analyzed their roles under ER stress and biotic stress and explored the mechanism preliminarily.The main results are as follows:1.PAS2 and its interaction with GAAPs positively regulated the resistance of ER stress and suppressed cell death induced by ER stress.Mutations in PAS2 and GAAPs enhanced the sensitivity of seedlings to ER stress induced by DTT or TM.The development of the mutant pas2-5 and the double mutants pas2-5gaap1-1 and pas2-5gaap2-2 which grew in soil for 25 days was severely inhibited upon DTT treatment and the photosynthesis was also influenced in the mutants.The cotyledons and roots of pas2-5,pas2-5gaap1-1 and pas2-5gaap2-2seedlings were inhibited significantly under TM.Compared with Col at the same concentration of TM,the percentage of healthy plants with fully expanded cotyledons and root length were significantly lower in pas2-5,pas2-5gaap1-1 and pas2-5gaap2-2.Conversely,the relative conductivity of all mutants was higher than that of Col.The detached leaves of pas2-5gaap1-1 were etiolated more severely in dark.These results suggest that the mutants of PAS2 and GAAPs are hypersensitive under ER stress.Both trypan blue and PI staining assay showed that the cell death was enhanced in pas2-3,pas2-7,pas2-5gaap1-1 and pas2-5gaap2-2 mutants while the cell death was inhibited in PAS2-OX#7 and PAS2-OX#8.These data suggested that PAS2 functions in plant resistance to ER stress along with GAAPs and resists to ER-stress-induced cell death.2.PAS2 and its interaction with GAAPs inhibited the accumulation of ROS induced by ER stress.Compared with Col,more ROS accumulated in the transition and elongation region of roots and in cotyledons in the PAS2 mutants but less ROS in the PAS2 overexpressing lines in response to TM for 36 h assayed by DAB staining.Real-time PCR tested the expression level of ROS-related genes under TM treatment for 24 h and showed that RBOHD,RBOHF and ERO1 was up-regulated in Col but not significantly changed in the mutants of PAS2.These results showed that the mutation of PAS2 promoted the accumulation of ROS under chronic ER stress but inhibited the production of ROS from plasma membrane at the transcriptional level.H₂DCF-DA fluorescence signal in pas2-5gaap1-1 and pas2-5gaap2-2 was stronger than Col under TM treatment for 6 h and showed that the production of ROS induced by ER stress was activated in PAS2and GAAPs double mutants quicker than Col.When ER stress prolonged to 36 h,DAB staining showed that degree and extent of staining was significantly enhanced in pas2-5gaap1-1 and pas2-5gaap2-2,which was consistent with the most serious cell death.These results showed that PAS2 and its interaction with GAAPs suppressed the accumulation of ROS under ER stress.3.The interaction of PAS2,GAAPs and IRE1 positively regulated ER Stress-induced autophagy.The expression level of UPR-related genes was not significantly influenced in double mutants of PAS2 and GAAPs under chronic and acute ER stress assayed by q-PCR.Only a fewer of autophagosomes were observed in pas2-5,pas2-5gaap1-1 and pas2-5gaap2-2 mutants after DTT treatment by MDC staining.RNA-seq data showed that ATG6,ATG8,ATG5 and some other autophagy-related genes were significantly down-regulated in pas2-5gaap1-1.And the protein level of ATG8,detected by western blot,was decreased in mutants but recovered to Col when PAS2 expression level was recovered.These data indicated that the mutation of PAS2 or PAS2 and GAAPs inhibited autophagy induced by ER stress.PAS2 was located in punctuated structure,ER and vacuolar membrane.Bi FC assay showed that PAS2 interacted with IRE1A and IRE1B,which suggested that PAS2 might be involved in function in the ER stress-induced autophagy-dependent on IRE1.These data showed that PAS2 and GAAPs positively regulated autophagy induced by ER stress mediated by IRE1signaling.4.PAS2 was involved in regulating autophagy induced by nutrition-deficient.Mutants of PAS2 which grew on the medium with low or without nitrogen showed more anthocyanin accumulation,less chlorophyll and more serious albefaction of cotyledons,whereas the overexpressing lines of PAS2 grew well.The hypersensitivity of single PAS2 mutant to nitrogen deficient suggested that both proteins also participated in autophagy induced by auxotrophs.5.PAS2 and its interaction with GAAPs resisted Botrytis cinerea.Botrytis cinerea spores marked by GFP tag were inoculated in leaves and we observed that more spores germinated into hypha in the leaves of pas2-5,pas2-5gaap1-1 and pas2-5gaap2-2,but much fewer in PAS2.The expression level of PDF1.2A,PDF1.3,PR-4 and ORA59 was significantly decreased in the mutants but up-regulated in PAS2obviously.Meanwhile,ICS1 and NPR1,SA pathway marker genes,were not significantly different between mutants and other lines.These results showed that interaction of PAS2 and GAAPs up-regulated resistant genes of JA pathway at the transcription level in response Botrytis cinerea at early stage.When Botrytis cinerea infected persistently,etiolated leaves were more serious and chlorophyll content decreased in pas2-5,pas2-5gaap1-1 and pas2-5gaap2-2 seedlings.Whereas,PAS2overexpression lines were resistant to biotic signals and harmful hurts induced by Botrytis cinerea.These results suggested that PAS2 and its interaction with GAAPs resisted Botrytis cinerea and its side effects.In summary,Arabidopsis PAS2 and its interaction with GAAPs function in resistance to ER stress and cell death by regulating ROS and autophagy dependent on IRE1.In addition,PAS2 and its interaction with GAAPs were not only involved in regulating autophagy under nitrogen deficient but also function in biotic stress induced by Botrytis cinerea.PAS2 and its interaction with GAAPs might promote activitating JA pathway to suppress the germination of spores and enhanced the resistance of Botrytis cinerea.Further study on how the interaction of PAS2 and GAAPs regulate autophagy under stress will help people understand autophagy induction and the mechanism of plant resistance,which can supply more strategies to improve crops characters.