Arabidopsis MACP2 contributes to autophagy induction by modulating starvation-induced reactive oxygen species homeostasis

Abstract

In plants, autophagy is a conserved recycling system essential for development and stress responses by targeting cellular components for massive degradation in the vacuole. Our previous work suggested that autophagy contributes to Arabidopsis (Arabidopsis thaliana) stress responses by modulating NADPH-oxidase-mediated reactive oxygen species (ROS) homeostasis; however, the molecular link between extracellular ROS and autophagy remains unknown. We performed a yeast two-hybrid screen to identify components involved in autophagy, using the central autophagy component ATG8e as a bait. We identified MEMBRANE ATTACK COMPLEX/PERFORIN-LIKE 2 (MACP2) as an interactor of ATG8e via its the ATG8-interacting motif and confirmed this interaction by co-immunoprecipitation and bimolecular fluorescence complementation assays. MACP2-overexpressing lines showed enhanced sensitivity to nutritional starvation, accelerated leaf senescence, and increased hydrogen peroxide (H₂O₂) levels, resembling the phenotypes of atg mutants defective in autophagy. Conversely, macp2 knockouts exhibited diminished starvation-induced H₂O₂ accumulation and attenuated autophagosome formation and fully suppressed the starvation-hypersensitive phenotypes of the atg5-1 mutant. In particular, MACP2 was degraded through the autophagy machinery during prolonged starvation, suggesting a feedback regulatory mechanism for maintaining MACP2 homeostasis. Our findings suggest that MACP2 acts as a key regulator in autophagy induction by controlling influx of extracellular H₂O₂ in Arabidopsis. 

Keywords

Autophagy / MACP2 / ROS / Arabidopsis

References

[1] AcheampongAK, ShanksC, ChengCY, SchallerGE, DagdasY, KieberJJ. EXO70D isoforms mediate selective autophagic degradation of type-A ARR proteins to regulate cytokinin sensitivity. Proc Natl Acad Sci U S A, 2020, 117: 27034-27043

[2] AsadaY, YamamotoM, TsutsuiT, YamaguchiJ. The Arabidopsis NSL2 negatively controls systemic acquired resistance via hypersensitive response. Plant Biotechnol, 2011, 28: 9-15

[3] Avila-OspinaL, MoisonM, YoshimotoK, Masclaux-DaubresseC. Autophagy, plant senescence, and nutrient recycling. J Exp Bot, 2014, 65: 3799-3811

[4] BakopoulosD, WhisstockJC, JohnsonTK. Control of growth factor signalling by MACPF proteins. Biochem Soc Trans, 2019, 47: 801-810

[5] BasshamDC, LaporteM, MartyF, MoriyasuY, OhsumiY, OlsenLJ, YoshimotoK. Autophagy in development and stress responses of plants. Autophagy, 2006, 2: 2-11

[6] CastroB, CittericoM, KimuraS, StevensDM, WrzaczekM, CoakerG. Stress-induced reactive oxygen species compartmentalization, perception and signalling. Nat Plants, 2021, 7: 403-412

[7] ChenL, LiaoB, QiH, XieLJ, HuangL, TanWJ, ZhaiN, YuanLB, ZhouY, YuLJ, ChenQF, ShuWS, XiaoS. Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana. Autophagy, 2015, 11: 2233-2246

[8] ChenQF, XuL, TanWJ, ChenL, QiH, XieLJ, ChenMX, LiuBY, YuLJ, YaoN, ZhangJH, ShuW, XiaoS. Disruption of the Arabidopsis defense regulator genes SAG101, EDS1, and PAD4 confers enhanced freezing tolerance. Mol Plant, 2015, 8: 1536-1549

[9] ChenP, JianH, WeiF, GuL, HuT, LvX, GuoX, LuJ, MaL, WangH, WuA, MaoG, YuS, WeiH. Phylogenetic analysis of the membrane attack complex/perforin domain-containing proteins in Gossypium and the role of GhMACPF26 in cotton under cold stress. Front Plant Sci, 2021, 12684227

[10] ChungT, PhillipsAR, VierstraRD. ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A and ATG12B loci. Plant J, 2010, 62: 483-493

[11] CloughSJ, BentAF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16: 735-743

[12] EssickEE, FloraS. Oxidative stress and autophagy in cardiac disease, neurological disorders, aging and cancer. Oxid Med Cell Longev, 2010, 3: 168-177

[13] FukunagaS, SogameM, HataM, Singkaravanit-OgawaS, Pislewska-BednarekM, Onozawa-KomoriM, NishiuchiT, HirumaK, SaitohH, TerauchiR, KitakuraS, InoueY, BednarekP, Schulze-LefertP, TakanoY. Dysfunction of Arabidopsis MACPF domain protein activates programmed cell death via tryptophan metabolism in MAMP-triggered immunity. Plant J, 2017, 89: 381-393

[14] GrondinA, RodriguesO, VerdoucqL, MerlotS, LeonhardtN, MaurelC. Aquaporins contribute to ABA-triggered stomatal closure through OST1-mediated phosphorylation. Plant Cell, 2015, 27: 1945-1954

[15] GuanB, LinZ, LiuD, LiC, ZhouZ, MeiF, LiJ, DengX. Effect of waterlogging-induced autophagy on programmed cell death in Arabidopsis roots. Front Plant Sci, 2019, 10468

[16] GuoY, RenG, ZhangK, LiZ, MiaoY, GuoH. Leaf senescence: progression, regulation, and application. Mol Hortic, 2021, 15

[17] HalliwellB. Biochemistry of oxidative stress. Biochem Soc Trans, 2007, 35: 1147-1150

[18] HanaokaH, NodaT, ShiranoY, KatoT, HayashiH, ShibataD, TabataS, OhsumiY. Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol, 2002, 129: 1181-1193

[19] He Y, Gao J, Luo M, Gao C, Lin Y, Wong HY, Cui Y, Zhuang X, Jiang L. VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana. Autophagy. 2023;13:19(5):1406–1423.

[20] Hernández-BarreraA, QuintoC, JohnsonEA, WuHM, CheungAY, CárdenasL. Using hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: a method with virtually unlimited potential in plant biology. Methods Enzymol, 2013, 527: 275-290

[21] Holmes DR, Bredow M, Thor K, Pascetta SA, Sementchoukova I, Siegel KR, Zipfel C, Monaghan J. A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling. Genetics. 2021;217:iyab022.

[22] HuangL, GuoH. Acetylation modifcation in the regulation of macroautophagy. Adv Biotech, 2024, 219

[23] HuangL, YuLJ, ZhangX, FanB, WangFZ, DaiYS, QiH, ZhouY, XieLJ, XiaoS. Autophagy regulates glucose-mediated root meristem activity by modulating ROS production in Arabidopsis. Autophagy, 2019, 15: 407-422

[24] KaufmannA, WollertT. Scaffolding the expansion of autophagosomes. Autophagy, 2014, 10: 1343-1345

[25] KaufmannA, BeierV, FranquelimHG, WollertT. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell, 2014, 156: 469-481

[26] KellnerR, de la ConcepcionJC, MaqboolA, KamounS, DagdasYF. ATG8 expansion: a driver of selective autophagy diversification?. Trends Plant Sci, 2017, 22: 204-214

[27] KrügerC, Waldeck-WeiermairM, KaynertJ, PokrantT, KomaragiriY, OttoO, MichelT, ElsnerM. AQP8 is a crucial H₂O₂ transporter in insulin-producing RINm5F cells. Redox Biol, 2021, 43101962

[28] LiF, VierstraRD. Autophagy: a multifaceted intracellular system for bulk and selective recycling. Trends Plant Sci, 2012, 17: 526-537

[29] LiJF, ChungHS, NiuY, BushJ, Mc CormackM, SheenJ. Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell, 2013, 25: 1507-1522

[30] LiX, LiuQ, FengH, DengJ, ZhangR, WenJ, DongJ, WangT. Dehydrin MtCAS31 promotes autophagic degradation under drought stress. Autophagy, 2020, 16: 862-877

[31] LiaoCY, BasshamDC. Combating stress: the interplay between hormone signaling and autophagy in plants. J Exp Bot, 2020, 71: 1723-1733

[32] LinZ, WangYL, ChengLS, ZhouLL, XuQT, LiuDC, DengXY, MeiFZ, ZhouZQ. Mutual regulation of ROS accumulation and cell autophagy in wheat roots under hypoxia stress. Plant Physiol Biochem, 2021, 158: 91-102

[33] LiuY, BasshamDC. Autophagy: pathways for self-eating in plant cells. Annu Rev Plant Biol, 2012, 63: 215-237

[34] LukoyanovaN, HoogenboomBW, SaibilHR. The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins. J Cell Sci, 2016, 129: 2125-2133

[35] MarshallRS, LiF, GemperlineDC, BookAJ, VierstraRD. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol Cell, 2015, 58: 1053-1066

[36] MarshallRS, HuaZ, MaliS, McLoughlinF, VierstraRD. ATG8-binding UIM proteins define a new class of autophagy adaptors and receptors. Cell, 2019, 177: 766-781

[37] MittlerR, ZandalinasSI, FichmanY, Van BreusegemF. Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol, 2022, 23: 663-679

[38] Moreno-HagelsiebG, VitugB, Medrano-SotoA, SaierMHJr. The membrane attack complex/perforin superfamily. J Mol Microb Biotech, 2017, 27: 252-267

[39] MorganBP. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem J, 1989, 264: 1-14

[40] Morita-YamamuroC, TsutsuiT, SatoM, YoshiokaH, TamaokiM, OgawaD, MatsuuraH, YoshiharaT, IkedaA, UyedaI, YamaguchiJ. The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain. Plant Cell Physiol, 2005, 46: 902-912

[41] NakatogawaH, OhbayashiS, Sakoh-NakatogawaM, KakutaS, SuzukiSW, KirisakoH, Kondo-KakutaC, NodaNN, YamamotoH, OhsumiY. The autophagy-related protein kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family interacting motif to facilitate autophagosome formation. J Biol Chem, 2012, 287: 28503-28507

[42] NazioF, CarinciM, ValaccaC, BielliP, StrappazzonF, AntonioliM, CiccosantiF, RodolfoC, CampelloS, FimiaGM, SetteC, BonaldoP, CecconiF. Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation. J Cell Biol, 2016, 215: 841-856

[43] NazioF, CarinciM, CecconiF. ULK1 ubiquitylation is regulated by phosphorylation on its carboxy terminus. Cell Cycle, 2017, 16: 1744-1747

[44] NiT, GilbertRJC. Repurposing a pore: highly conserved perforin-like proteins with alternative mechanisms. Philos Trans R Soc Lond B Biol Sci, 2017, 37220160212

[45] NoutoshiY, KuromoriT, WadaT, HirayamaT, KamiyaA, ImuraY, YasudaM, NakashitaH, ShirasuK, ShinozakiK. Loss of necrotic spotted lesions 1 associates with cell death and defense responses in Arabidopsis thaliana. Plant Mol Biol, 2006, 62: 29-42

[46] PanX, WeltiR, WangX. Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry. Nat Protoc, 2010, 5: 986-992

[47] PucciarielloC, ParlantiS, BantiV, NoviG, PerataP. Reactive oxygen species-driven transcription in Arabidopsis under oxygen deprivation. Plant Physiol, 2012, 159: 184-196

[48] QiH, XiaFN, XieLJ, YuLJ, ChenQF, ZhuangXH, WangQ, LiF, JiangL, XieQ, XiaoS. TRAF family proteins regulate autophagy dynamics by modulating AUTOPHAGY PROTEIN6 stability in Arabidopsis. Plant Cell, 2017, 29: 890-911

[49] QiH, XiaFN, XiaoS. Autophagy in plants: Physiological roles and post-translational regulation. J Integr Plant Biol, 2021, 63: 161-179

[50] QiH, LeiX, WangY, YuS, LiuT, ZhouSK, ChenJY, ChenQF, QiuRL, JiangL, XiaoS. 14-3-3 proteins contribute to autophagy by modulating SINAT-mediated degradation of ATG13. Plant Cell, 2022, 34: 4857-4876

[51] QiH, WangY, BaoY, BasshamDC, ChenL, ChenQF, HouSW, HwangI, HuangL, LaiZB, LiF, LiuY, QiuR, WangH, WangP, XieQ, ZengY, ZhuangX, GaoC, JiangL, XiaoS. Studying plant autophagy: challenges and recommended methodologies. Adv Biotechnol, 2023, 12

[52] QuX, ChattyPR, RoederAHK. Endomembrane trafficking protein SEC24A regulates cell size patterning in Arabidopsis. Plant Physiol, 2014, 166: 1877-1890

[53] RosadoCJ, KondosS, BullTE, KuiperMJ, LawRH, BuckleAM, VoskoboinikI, BirdPI, TrapaniJA, WhisstockJC, DunstoneMA. The MACPF/CDC family of pore-forming toxins. Cell Microbiol, 2008, 10: 1765-1774

[54] Scherz-ShouvalR, ShvetsE, FassE, ShorerH, GilL, ElazarZ. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J, 2007, 26: 1749-1760

[55] ShahJ. The salicylic acid loop in plant defense. Curr Opin Plant Biol, 2003, 6: 365-371

[56] ShahJ, TsuiF, KlessigDF. Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 gene. Mol Plant Microbe Interact, 1997, 10: 69-78

[57] Sharma P, Jha AB, Dubey RS, Pessarakli M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot. 2012;2012:217037. https://doi.org/10.1155/2012/217037.

[58] ShiW, WangL, YaoL, HaoW, HanC, FanM, WangW, BaiMY. Spatially patterned hydrogen peroxide orchestrates stomatal development in Arabidopsis. Nat Commun, 2022, 1315040

[59] ShinJH, YoshimotoK, OhsumiY, JeonJS, AnG. OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice. Mol Cell, 2009, 27: 67-74

[60] SignorelliS, TarkowskiŁP, Van den EndeW, BasshamDC. Linking autophagy to abiotic and biotic stress responses. Trends Plant Sci, 2019, 24: 413-430

[61] SmalleJ, KurepaJ, YangP, EmborgTJ, BabiychukE, KushnirS, VierstraRD. The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-s pecific function in abscisic acid signaling. Plant Cell, 2003, 2003(15): 965-980

[62] SuttangkakulA, LiF, ChungT, VierstraRD. The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis. Plant Cell, 2011, 23(10): 3761-3779

[63] TangJ, BasshamDC. Autophagy during drought: function, regulation, and potential application. Plant J, 2022, 109: 390-401

[64] TianS, WangX, LiP, WangH, JiH, XieJ, QiuQ, ShenD, DongH. Plant aquaporin AtPIP1;4 links apoplastic H₂O₂ induction to disease immunity pathways. Plant Physiol, 2016, 171(3): 1635-1650

[65] TsutsuiT, Morita-YamamuroC, AsadaY, MinamiE, ShibuyaN, IkedaA, YamaguchiJ. Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis. Biosci Biotechnol Biochem, 2006, 70: 2042-2048

[66] ValkaiI, KénesiF, DomonkosI, AyaydinF, TarkowskáD, StrnadM, FaragóA, BodaiL, FehérA. The Arabidopsis RLCK VI_A2 kinase controls seedling and plant growth in parallel with gibberellin. Int J Mol Sci, 2020, 217266

[67] WadaS, IshidaH, IzumiM, YoshimotoK, OhsumiY, MaeT, MakinoA. Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol, 2009, 149: 885-893

[68] WaszczakC, CarmodyM, KangasjarviJ. Reactive oxygen species in plant signaling. Annu Rev Plant Biol, 2018, 69: 209-236

[69] WeindelCG, MartinezEL, ZhaoX, MabryCJ, BellSL, VailKJ, ColemanAK, VanPortflietJJ, ZhaoB, WagnerAR, AzamS, ScottHM, LiP, WestAP, KarpacJ, PatrickKL, WatsonRO. Mitochondrial ROS promotes susceptibility to infection via gasdermin D-mediated necroptosis. Cell, 2022, 185: 3214-3231

[70] XiaFN, ZengB, LiuHS, QiH, XieLJ, YuLJ, ChenQF, LiJF, ChenYQ, JiangL, XiaoS. SINAT E3 ubiquitin ligases mediate FREE1 and VPS23A degradation to modulate abscisic acid signaling. Plant Cell, 2020, 32: 3290-3310

[71] XiaoS, GaoW, ChenQF, ChanSW, ZhengSX, MaJ, WangM, WeltiR, ChyeML. Overexpression of Arabidopsis Acyl-CoA binding protein ACBP3 promotes starvation-induced and age-dependent leaf senescence. Plant Cell, 2010, 22: 1463-1482

[72] XiongY, ContentoAL, NguyenPQ, BasshamDC. Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiol, 2007, 143: 291-299

[73] XuL, LiuF, LechnerE, GenschikP, CrosbyWL, MaH, PengW, HuangD, XieD. The SCF (COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell, 2002, 14: 1919-1935

[74] YooSD, ChoYH, SheenJ. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc, 2007, 2: 1565-1572

[75] YoshimotoK, HanaokaH, SatoS, KatoT, TabataS, NodaT, OhsumiY. Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell, 2004, 16: 2967-2983

[76] YoshimotoK, JikumaruY, KamiyaY, KusanoM, ConsonniC, PanstrugaR, OhsumiY, ShirasuK. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell, 2009, 21: 2914-2927

[77] YuL, LiuD, ChenS, DaiY, GuoW, ZhangX, WangL, MaS, XiaoM, QiH, XiaoS, ChenQF. Evolution and expression of the membrane attack complex and perforin gene family in the Poaceae. Int J Mol Sci, 2020, 215736

[78] YuanLB, DaiYS, XieLJ, YuLJ, ZhouY, LaiYX, YangYC, XuL, ChenQF, XiaoS. Jasmonate regulates plant responses to postsubmergence reoxygenation through transcriptional activation of antioxidant synthesis. Plant Physiol, 2017, 173: 1864-1880

[79] Zhang X, Dai YS, Wang YX, Su ZZ, Yu LJ, Zhang ZF, Xiao S, Chen QF. Overexpression of the Arabidopsis MACPF protein AtMACP2 promotes pathogen resistance by activating SA signaling. Int J Mol Sci. 2022;23:8784. https://doi.org/10.3390/ijms23158784.

[80] ZhouJ, LiXY, LiuYJ, FengJ, WuY, ShenHM, LuGD. Full-coverage regulations of autophagy by ROS: from induction to maturation. Autophagy, 2022, 18: 1240-1255

[81] ZhuangX, ChungKP, LuoM, JiangL. Autophagosome biogenesis and the endoplasmic reticulum: a plant perspective. Trends Plant Sci, 2018, 23: 677-692

[82] ZouJJ, LiXD, RatnasekeraD, WangC, LiuWX, SongLF, ZhangWZ, WuWH. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling and H₂O₂ homeostasis in stomatal guard cells under drought stress. Plant Cell, 2015, 27: 1445-1460

Funding

32321163646

32000215

32370293

2023A1515011779

2023A1515012038

RIGHTS & PERMISSIONS

The Author(s)