Abstract: Hemin can improve the stress resistance of plants through the heme oxygenase system. Additionally, substances contained in plants, such as secondary metabolites, can improve stress resistance. However, few studies have explored the effects of hemin on secondary metabolite content. Therefore, the effects of hemin on saponin synthesis and the mechanism of plant injury relief by hemin in Conyza blinii were investigated in this study. Hemin treatment promoted plant growth and increased the antioxidant enzyme activity and saponin content of C. blinii under osmotic stress and cold stress. Further study showed that hemin could provide sufficient precursors for saponin synthesis by improving the photosynthetic capacity of C. blinii and increasing the gene expression of key enzymes in the saponin synthesis pathway, thus increasing the saponin content. Moreover, the promotion effect of hemin on saponin synthesis is dependent on heme oxygenase-1 and can be reversed by the inhibitor Zn-protoporphyrin-IX (ZnPPIX). This study revealed that hemin can increase the saponin content of C. blinii and alleviate the damage caused by abiotic stress, and it also broadened the understanding of the relationship between hemin and secondary metabolites in plant abiotic stress relief.

氯化血红素通过血红素加氧酶-1依赖方式诱导皂甙含量增加，有助于减轻渗透和冷胁迫对金龙胆草的损伤

[1]BaconCW, PalenciaER, HintonDM, 2015. Abiotic and biotic plant stress-tolerant and beneficial secondary metabolites produced by endophytic Bacillus species. In: Arora NK (Ed.), Plant Microbes Symbiosis: Applied Facets. Springer, >New Delhi,p.163-177.

[2]BaudouinE, FrendoP, le GleuherM, et al., 2004. A Medicago sativa haem oxygenase gene is preferentially expressed in root nodules. J Exp Bot, 55(394):43-47.

[3]BeckerEM, CardosoDR, SkibstedLH, 2011. Quenching of excited states of red-pigment zinc protoporphyrin IX by hemin and natural reductors in dry-cured hams. Eur Food Res Technol, 232(2):343-349.

[4]ChenH, ChengZJ, MaXD, et al., 2013. A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Rep, 32(12):1855-1867.

[5]ChenQ, GongCY, JuX, et al., 2018. Hemin through the heme oxygenase 1/ferrous iron, carbon monoxide system involved in zinc tolerance in Oryza sativa L. J Plant Growth Regul, 37(3):947-957.

[6]DenneryPA, VisnerG, WengYH, et al., 2003. Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron. Free Radic Biol Med, 34(1):124-133.

[7]EkhtariS, RazeghiJ, HasanpurK, et al., 2019. Different regulations of cell-type transcription by UV-B in multicellular green alga Volvox carteri. Plant Signal Behav, 14(11):1657339.

[8]FuGQ, ZhangLF, CuiWT, et al., 2011. Induction of heme oxygenase-1 with β-CD-hemin complex mitigates cadmium-induced oxidative damage in the roots of Medicago sativa. Plant Soil, 345(1-2):271-285.

[9]GengDS, ZhaoLP, ShiRM, et al., 2013. A study on antitussive, expectorant and anti-inflammatory effects of total saponins from Uyghur Herb Nigella glandulifera Freyn. J Xinjiang Med Univ, 36(7):908-911 (in Chinese).

[10]GhasemzadehA, JaafarHZE, 2011. Effect of CO₂ enrichment on synthesis of some primary and secondary metabolites in ginger (Zingiber officinale Roscoe). Int J Mol Sci, 12(2):1101-1114.

[11]GheisariHR, MøllerJKS, AdamsenCE, et al., 2010. Sodium chloride or heme protein induced lipid oxidation in raw, minced chicken meat and beef. Czech J Food Sci, 28(5):364-375.

[12]GitelsonAA, GritzY, MerzlyakMN, 2003. Relationships between leaf chlorophyll content and spectral reflectance and algorithms for non-destructive chlorophyll assessment in higher plant leaves. J Plant Physiol, 160(3):271-282.

[13]IshikawaK, SugawaraD, WangXP, et al., 2001. Heme oxygenase-1 inhibits atherosclerotic lesion formation in LDL-receptor knockout mice. Circ Res, 88(5):506-512.

[14]JiangY, SunFF, ZhanYG, et al., 2019. Effect of carbon monoxide on growth and triterpenoid production in Betula platyphylla suspension cells. Chin Tradit Herb Drugs, 50(15):3681-3686 (in Chinese).

[15]JinQJ, ZhuKK, XieYJ, et al., 2013. Heme oxygenase-1 is involved in ascorbic acid-induced alleviation of cadmium toxicity in root tissues of Medicago sativa. Plant Soil, 366(1-2):605-616.

[16]JinQJ, CuiWT, DaiC, et al., 2016. Involvement of hydrogen peroxide and heme oxygenase-1 in hydrogen gas-induced osmotic stress tolerance in alfalfa. Plant Growth Regul, 80(2):215-223.

[17]JungHY, KimDW, YimHS, et al., 2016. Heme oxygenase-1 protects neurons from ischemic damage by upregulating expression of Cu,Zn-superoxide dismutase, catalase, and brain-derived neurotrophic factor in the rabbit spinal cord. Neurochem Res, 41(4):869-879.

[18]LiH, JiangM, CheLL, et al., 2012. BjHO-1 is involved in the detoxification of heavy metal in India mustard (Brassica juncea). BioMetals, 25(6):1269-1279.

[19]LiuHY, HuCX, SunNN, et al., 2017. A triterpenoidal saponin fraction of Conyza blinii H.‍Lév. is a dual-targeting autophagy inhibitor for HeLa cells. RSC Adv, 7(39):24291-24297.

[20]LiuP, ZhouLJ, SuYF, et al., 2011. Effects of total saponins from Conyza blinii on the apoptosis of Hela cells and SPC-A1 cells. China Pharmacy, 22(35):3288-3291 (in Chinese).

[21]LiuYY, ChenQ, LiN, et al., 2016. Heme oxygenase-1 (HO-1) promote anthocyanin accumulation in the hypocotyl of radish sprouts. Acta Horticult Sin, 43(3):507-514 (in Chinese).

[22]LuoWW, WangY, YangHW, et al., 2018. Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling. Aging, 10(7):1722-1744.

[23]MaL, LiuJG, 2014. The protective activity of Conyza blinii saponin against acute gastric ulcer induced by ethanol. J Ethnopharmacol, 158:358-363.

[24]MaL, LiuHY, MengLP, et al., 2017a. Evaluation of the anti-cancer activity of the triterpenoidal saponin fraction isolated from the traditional Chinese medicine Conyza blinii H. Lév. RSC Adv, 7(6):3408-3412.

[25]MaL, LiuHY, QinP, et al., 2017b. Saponin fraction isolated from Conyza blinii H.Lév. demonstrates strong anti-cancer activity that is due to its NF-κB inhibition. Biochem Biophys Res Commun, 483(1):779-785.

[26]MainesMD, PanahianN, 2001. The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different functions? In: Roach RC, Wagner PD, Hackett PH (Eds.), Hypoxia: From Genes to the Bedside. Springer, Boston, p.249-272.

[27]MatthusE, WilkinsKA, SwarbreckSM, et al., 2019. Phosphate starvation alters abiotic-stress-induced cytosolic free calcium increases in roots. Plant Physiol, 179(4):1754-1767.

[28]MeiYQ, SongSQ, 2010. Response to temperature stress of reactive oxygen species scavenging enzymes in the cross-tolerance of barley seed germination. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 11(12):965-972.

[29]MüllebnerA, MoldzioR, RedlH, et al., 2015. Heme degradation by heme oxygenase protects mitochondria but induces ER stress via formed bilirubin. Biomolecules, 5(2):679-701.

[30]NoriegaG, CruzDS, BatlleA, et al., 2012. Heme oxygenase is involved in the protection exerted by jasmonic acid against cadmium stress in soybean roots. J Plant Growth Regul, 31(1):79-89.

[31]NoriegaGO, BalestrasseKB, BatlleA, et al., 2004. Heme oxygenase exerts a protective role against oxidative stress in soybean leaves. Biochem Biophys Res Commun, 323(3):1003-1008.

[32]NzowaLK, BarboniL, TeponnoRB, et al., 2010. Rheediinosides A and B, two antiproliferative and antioxidant triterpene saponins from Entada rheedii. Phytochemistry, 71(2-3):254-261.

[33]OrreniusS, KassGEN, NicoteraP, 1992. Oxygen free radicals and cell death. Neurochem Int, 21(Suppl):S13.

[34]OtterbeinLE, SoaresMP, YamashitaK, et al., 2003. Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol, 24(8):449-455.

[35]OzdenerY, AydinBK, 2010. The effect of zinc on the growth and physiological and biochemical parameters in seedlings of Eruca sativa (L.) (Rocket). Acta Physiol Plant, 32(3):469-476.

[36]PodkalickaP, MuchaO, JózkowiczA, et al., 2018. Heme oxygenase inhibition in cancers: possible tools and targets. Contemp Oncol, 2(1A):23-32.

[37]ShahK, NahakpamS, 2012. Heat exposure alters the expression of SOD, POD, APX and CAT isozymes and mitigates low cadmium toxicity in seedlings of sensitive and tolerant rice cultivars. Plant Physiol Biochem, 57:106-113.

[38]ShamloulR, WangR, 2006. Increased intracavernosal pressure response in hypertensive rats after chronic hemin treatment. J Sex Med, 3(4):619-627.

[39]SuYF, KoikeK, GuoDA, et al., 2001. New apiose-containing triterpenoid saponins from Conyza blinii. Tetrahedron, 57(31):6721-6726.

[40]SunR, LiuS, GaoJL, et al., 2014. Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthase gene from the medicinal plant Conyza blinii H.Lév. Turk J Biol, 38(5):664-670.

[41]SunWJ, ZhanJY, ZhengTR, et al., 2018. The jasmonate-responsive transcription factor CbWRKY24 regulates terpenoid biosynthetic genes to promote saponin biosynthesis in Conyza blinii H. Lév. J Genet, 97(5):1379-1388.

[42]TroxlerRF, 1972. Synthesis of bile pigments in plants.Formation of carbon monoxide and phycocyanobilin in wild-type and mutant strains of the alga, Cyanidium caldarium. Biochemistry, 11(23):4235-4242.

[43]VeliniED, TrindadeMLB, AlvesE, et al., 2005. Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides. Genet Mol Biol, 28(3 Suppl):548-554.

[44]WagenerFADTG, VolkHD, WillisD, et al., 2003. Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev, 55(3):551-571.

[45]WangJ, DoréS, 2007. Heme oxygenase-1 exacerbates early brain injury after intracerebral haemorrhage. Brain, 130(6):1643-1652.

[46]WangSQ, WangXM, SunFF, et al., 2019. Carbon monoxide: effect on growth and triterpene accumulation of phellinus linteus mycelium. Chin Agric Sci Bull, 35(21):90-95 (in Chinese).

[47]XiongCW, LiXW, XuDJ, et al., 2017. Determination of total saponins in quinoa by spectrophotometry. J Anhui Agric Sci, 45(26):96-98,121 (in Chinese).

[48]XuanW, ZhuFY, XuS, et al., 2008. The heme oxygenase/carbon monoxide system is involved in the auxin-induced cucumber adventitious rooting process. Plant Physiol, 148(2):881-893.

[49]YangCR, HeZT, LiXC, et al., 1989. Blinin, a neoclerodane diterpene from Conyza blinii. Phytochemistry, 28(11):3131-3134.

[50]YannarelliGG, NoriegaGO, BatlleA, et al., 2006. Heme oxygenase up-regulation in ultraviolet-B irradiated soybean plants involves reactive oxygen species. Planta, 224(5):1154-1162.

[51]ZhanJY, YangQ, LinZY, et al., 2021. Enhanced antioxidant capacity and upregulated transporter genes contribute to the UV-B-induced increase in blinin in Conyza blinii. Environ Sci Pollut Res, 28(11):13275-13287.

[52]ZhengTR, WangMJ, ZhanJY, et al., 2020. Ferrous iron-induced increases in capitate glandular trichome density and upregulation of CbHO-1 contributes to increases in blinin content in Conyza blinii. Planta, 252(5):81.

[53]ZhuZB, HuangYF, WuX, et al., 2019. Increased antioxidative capacity and decreased cadmium uptake contribute to hemin-induced alleviation of cadmium toxicity in Chinese cabbage seedlings. Ecotoxicol Environ Saf, 177:47-57.