Hemin-induced increase in saponin content contributes to the alleviation of osmotic and cold stress damage to Conyza blinii in a heme oxygenase 1-dependent manner

Hemin-induced increase in saponin content contributes to the alleviation of osmotic and cold stress damage to Conyza blinii in a heme oxygenase 1-dependent manner 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 3 equal first-author 2 equal 1 1 1 1 1 corresp Hui CHEN, chenhui@sicau.edu.cn Hui CHEN, chenhui@sicau.edu.cn chenhui@sicau.edu.cn https://orcid.org/0000-0001-9185-1764 College of Life Science, Sichuan Agricultural University, Ya’an625014, China College of Life Science, Sichuan Agricultural University, Ya’an625014, China College of Life Science, Nanjing Agricultural University, Nanjing210095, China College of Life Science, Nanjing Agricultural University, Nanjing210095, China Chongqing Academy of Chinese Materia Medica, Chongqing400065, China Chongqing Academy of Chinese Materia Medica, Chongqing400065, China 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. 目的 2 研究氯化血红素对金龙胆草皂苷含量的影响，以及与植物非生物胁迫的相关性。 创新点 2 通过施加外源氯化血红素研究金龙胆草中皂苷的变化与冷胁迫或渗透胁迫的相关性。 方法 2 在渗透胁迫和低温胁迫下，施加外源氯化血红素及其抑制剂，检测并分析金龙胆草多项生理指标、皂苷含量差异和关键基因表达量差异。 结论 2 氯化血红素能通过依赖血红素加氧酶-1的调控方式，提高金龙胆草皂苷的含量，从而减轻非生物胁迫对金龙胆草的伤害。 Hemin Saponin Conyza blinii Heme oxygenase Abiotic stress 氯化血红素 皂苷 金龙胆草 血红素加氧酶 非生物胁迫 1 Introduction 1 Natural heme is a ubiquitous molecular complex of iron and tetrapyrrole to porphyrin IX ( Ishikawa et al., 2001 Ishikawa et al., 2001 ). Hemin is a purified natural heme found in animal blood ( Wang and Doré, 2007 Wang and Doré, 2007 ), which is nontoxic to organisms and the environment, and is widely used in pharmacological research and clinical treatment ( Shamloul and Wang, 2006 Shamloul and Wang, 2006 ; Luo et al., 2018 Luo et al., 2018 ). As a porphyrin, heme is similar in substance to chlorophyll ( Velini et al., 2005 Velini et al., 2005 ), and chloride has chemical properties similar to those of natural heme ( Gheisari et al., 2010 Gheisari et al., 2010 ). In the field of drug manufacturing, in addition to being used as one of the synthetic raw materials of bilirubin (BR), hemin also has applications in the field of new cancer drugs ( Müllebner et al., 2015 Müllebner et al., 2015 ). In the food manufacturing industry, as the natural pigment in red blood, which produces a ruddy and fresh appearance in meat, it can replace certain synthetic pigments and nitrite in order to reduce their use ( Becker et al., 2011 Becker et al., 2011 ). Hemin can be used as an exogenous additive in the process of plant cultivation. It can significantly enhance the antioxidant capacity of plants, regulate their basic metabolic intensity, and promote the accumulation of effective substances ( Fu et al., 2011 Fu et al., 2011 ). For example, when radish sprouts were treated with exogenous hemin and its different metabolites, the anthocyanin content increased and decreased to varying degrees ( Liu et al., 2016 Liu et al., 2016 ). Compared with the control group, the expression intensities of the regulatory genes polynucleotide adenylyltransferase ( PAP1 ), phenylalanine ammonia lyase ( PAL ), chalcone-flavonone isomerase ( CHI ), flavanone 3-hydroxylase ( F3H ), chalcone synthase ( CHS ), and dihydroflavonol-4-reductase ( DFR ) in the anthocyanin synthesis pathway were significantly enhanced, which promoted the accumulation of target substances ( Liu et al., 2016 Liu et al., 2016 ). Heme oxygenase (HO) is a general term for the initiation and rate-limiting enzymes of heme degradation in plants ( Troxler, 1972 Troxler, 1972 ). There are three subtypes of HO, HO-1, HO-2, and HO-3 ( Maines and Panahian, 2001 Maines and Panahian, 2001 ). Heme oxygenase-1 (HO-1, EC 1.14.14.18) is an oxidoreductase that can degrade heme to produce BR, carbon monoxide (CO), and ferrous ions (Fe 2+ ) in animals and plants ( Wagener et al., 2003 Wagener et al., 2003 ; Baudouin et al., 2004 Baudouin et al., 2004 ). Some studies have shown that HO-1 is the degradation product and performs the main functions. For example, suspended cells of Betula platyphylla and the mycelium culture system of Phellinus igniarius were treated with one of the bioproducts of hemin, CO, and it was found that the gene expression levels of key enzymes in triterpene synthesis were upregulated in both species ( Jiang et al., 2019 Jiang et al., 2019 ; Wang et al., 2019 Wang et al., 2019 ). CO in plants is mainly produced by the degradation of heme by HO-1 ( Otterbein et al., 2003 Otterbein et al., 2003 ), indicating that hemin can increase the accumulation of terpenoids. However, there are few experiments that can verify this. Conyza blinii , also known as bitter wormwood and bear gall grass, is a biennial deciduous herb of the licorice genus Compositae ( Sun et al., 2014 Sun et al., 2014 ). It is distributed in the Panxi area of Sichuan Province, central and southern Yunnan Province, and parts of Guizhou Province, China ( Liu et al., 2017 Liu et al., 2017 ). As a Chinese herbal medicine, C. blinii is widely used in clinical treatment because of its anti-inflammatory, antitussive, expectorant, and antiasthmatic effects ( Ma et al., 2017a Ma et al., 2017a ; Sun et al., 2018 Sun et al., 2018 ). C. blinii saponins are the main effective components of this plant. As early as 1989, an animal experiment with C. blinii in Chengdu City (China) showed that saponins had certain therapeutic effects in respiratory system diseases ( Yang et al., 1989 Yang et al., 1989 ). They promoted the clearance of foreign bodies in the tracheal ciliary mucus system of rabbits ( Geng et al., 2013 Geng et al., 2013 ). Further clinical experiments have shown that saponins are effective in the treatment of chronic bronchitis, with few side effects ( Su et al., 2001 Su et al., 2001 ). In addition, Liu et al. (2011) Liu et al. (2011) found that saponins inhibited the growth of cervical cancer and lung cancer cells. On this basis, Ma et al. (2017b) Ma et al. (2017b) studied the mechanism, by which saponins inhibit cancer. The results showed that saponins caused mitochondrial apoptosis by destroying mitochondrial membrane potential, inhibited the migration and invasion of cancer cells, caused S-phase arrest of cancer cells, and inhibited the nuclear factor-κB (NF-κB) signaling pathway. In addition to these four main effects, saponins have been reported to have preventive and therapeutic effects on gastric ulcers in rats ( Sun et al., 2014 Sun et al., 2014 ). Ma and Liu (2014) Ma and Liu (2014) found that saponins could protect gastric mucosa from ethanol-induced gastric ulcers; in addition, a C. blinii saponin aqueous solution inhibited the growth of Staphylococcus aureus and Staphylococcus albus . Our goal in this study was to investigate the effects of hemin treatment on saponin content in C. blinii , as well as its relationship to plant abiotic stress relief. 2 Materials and methods 1 2.1 Plant materials and hemin treatment 2 Seeds of C. blinii were obtained from Sichuan Agricultural University, Ya'an, China. The conditions of seed germination and seedling culture were described in our previous study ( Zhan et al., 2021 Zhan et al., 2021 ). When the seedlings were two months old, we put them into drought and low-temperature conditions and treated them with hemin. Suspension cells were cultured in the new configuration (with every 40 mL containing 0.1896 g Murashige-Skoog's (MS) medium, 1.2 g sugar, 4 μL 6-benzylamino purine, 40 μL 2,‍4-dichlorophenoxyacetic acid, 10 μL 6-furfurylamino-purine (kinetin), and 2.186 g mannitol, and the pH was adjusted to 5.5–5.8) in the mother liquor. Then, 1 mL of C. blinii suspension cells were blended with C. blinii suspension cell sap at 25 ℃ and 120 r/min and placed on a thermostatic cultivation table for 5 d. A series of 40 μL, 400 μL, 1 mL, and 2 mL of 1 mmol/L hemin were absorbed and added to 40 mL of C. blinii suspension cell solution so that the final concentrations were 1, 10, 25, and 50 μmol/L, respectively. All suspension cell solutions were incubated on a shaking table at 25 ℃ and 120 r/min for 4 d. The plants were treated with the same concentration of hemin and cultured in the greenhouse for 7 d, and the culture medium (supplemented with the corresponding concentration of hemin) was changed every 3 d. The control group (CK group) consisted of suspension cell culture medium and C. blinii plants with only the mother liquor and no hemin. Polyethylene glycol 6000 (PEG6000) was used to induce osmotic stress. PEG6000 was added to the culture medium for a final concentration of 10% (volume fraction). C. blinii was then treated with the culture medium containing 10% PEG6000 for 7 d. The culture medium was changed every 3 d, adding PEG6000 again. At 21:00 every day, C. blinii was put into a 4 ‍℃ refrigerator for cold-stress treatment, and the plants were removed from the refrigerator at 9:00 the next day. Then, the plants were placed at room temperature to alleviate the freezing damage to the plants so that they would not freeze to death. This process was repeated daily for one week, at which point the physiological indices were measured. The concentrations of the three metabolites of hemin were obtained by concentration gradient screening: 10 μmol/L hemin, BR, Fe 2+ , carbon monoxide-releasing molecule 3 (CORM3), and 100 μmol/L inhibitor Zn-protoporphyrin-IX (ZnPPIX) ( Chen et al., 2018 Chen et al., 2018 ). 2.2 Determination of physiological indices 2 Plant height, fresh weight, dry weight, photosynthetic pigment content, and photosynthetic parameters, including the net photosynthetic rate ( P n ), stomatal conductance ( G s ), intercellular CO 2 concentration ( C i ), and transpiration rate ( T r ), were measured via the same methods mentioned in our previous study ( Zheng et al., 2020 Zheng et al., 2020 ). The photosynthetic pigment content was detected by extraction with an acetone-ethanol mixture ( Gitelson et al., 2003 Gitelson et al., 2003 ). 2.3 Extraction of saponins and content analysis 2 Drawing the standard curve of total saponins: First, 20 mg of oleanolic acid standard was accurately weighed, and then 50 mL of a methanol reagent was used to dissolve the oleanolic acid solution to a concentration of 0.50 mg/mL. After reaching a constant volume, the standard solution was obtained by blending. Then, 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the oleanolic acid standard solutions were placed in test tubes. All of the solutions in the tubes were dried in a fume hood. Then, vanillin (5%, mass fraction)-glacial acetic acid solution (0.3 mL) and 0.8 mL of perchlorate were mixed into each tube. All test tubes were made airtight and shaken for 25 min at 65 ℃. The absorption value of the chromogenic solution was measured at a wavelength of 544 nm with an ultraviolet (UV) spectrophotometer. The mass was used as the abscissa and the absorbance was plotted as the ordinate to plot the standard curve of the total saponins with the measured data. 2.4 Preparation and content determination of total saponins to be measured 2 The C. blinii leaves from the experimental group and the CK group were washed after being treated with different concentrations of hemin and then dried at 55 ‍℃ to a constant weight. After grinding into a powder, 0.1 g of C. blinii powder was put into a 10-mL centrifuge tube filled with 1.5 mL of 60% (volume fraction) ethanol. Extraction was performed for half an hour under the extraction conditions of 65 ‍℃ and 350 W of ultrasonic power. After extraction, the solution was centrifuged at 12 000 r/min for 5 min at low temperature, and the supernatant was absorbed. The subsequent steps were consistent with the determination of the standard saponin solution mentioned above. The absorbance value was determined at a wavelength of 544 nm and measured in parallel three times. Then, the total saponins in the sample solutions treated with different concentrations of hemin were determined by comparison with the standard curve. The culture medium of the C. blinii suspension cells in the experimental group and the medium of the CK group treated with hemin at different concentrations were centrifuged at 12 000 r/min for 5 min ( Xiong et al., 2017 Xiong et al., 2017 ). The supernatant was poured out and then color development was carried out with the same method used to prepare the curve, and the corresponding absorption value was measured. 2.5 RNA extraction and qRT-PCR analysis 2 After treatment, all the leaves of the samples were immediately put into liquid nitrogen. Subsequently, RNA was extracted using an EasySpin plant RNA Rapid Extraction kit (Adlai Biological Co., Ltd., Beijing, China) ( Ekhtari et al., 2019 Ekhtari et al., 2019 ) and complementary DNA (cDNA) was synthesized using reverse transcription kits (HiScript III RT SuperMix for qPCR (+gDNA wiper), Vazyme, Nanjing, China). A PrimeScript ™ RT Reagent kit with genomic DNA (gDNA) Eraser (Vazyme) was used for reverse transcription. For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, three parallel assays were performed for each gene in each sample. All of the systems and conditions of RNA extraction and qRT-PCR are described in detail in our previous report ( Zheng et al., 2020 Zheng et al., 2020 ). Finally, the <math id="M1"><msup><mrow><mn mathvariant="normal">2</mn></mrow><mrow><mo>-</mo><mi mathvariant="normal">Δ</mi><mi mathvariant="normal">Δ</mi><msub><mrow><mi>C</mi></mrow><mrow><mi mathvariant="normal">T</mi></mrow></msub></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27610522&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27610529&type= 7.78933382 3.38666677 method was used to calculate gene expression and for analysis. 2.6 Statistical analysis 2 Statistical analysis was performed using SPSS statistics 17.0 software (SPSS Inc., Chicago, IL, USA). All data were analyzed by one-way analysis of variance (ANOVA) combined with Duncan's multiple range test, and statistical significance was determined as a P -value of < 0.05 using GraphPad Prism 7 statistical software (GraphPad Prism Inc., USA). 3 Results 1 3.1 Effects of hemin on the growth of C. blinii under osmotic stress and cold stress 2 To investigate the effects of different hemin concentrations on the growth of C. blinii under drought and cold stress, we used 1, 10, 25, and 50 μmol/L of hemin to treat C. blinii . As shown in Fig. 1 Fig. 1 , hemin treatment significantly promoted the growth of C. blinii under osmotic stress even at inappreciable doses. Under osmotic stress, 25 μmol/L hemin treatment best promoted the development of root length ( Fig. 1 Fig. 1 a), plant height ( Fig. 1 Fig. 1 b), and fresh weight ( Fig. 1 Fig. 1 c), increasing them by 46.67%, 9.37%, and 6.67%, respectively, compared with the CK group. As with osmotic stress, hemin could effectively alleviate the growth inhibition of C. blinii caused by low temperature. In the experiment with different hemin concentrations, the development of a promoted peak occurred at 25 μmol/L hemin, in which root length, plant height, and fresh weight increased by 38.46%, 17.24%, and 42.86%, respectively, compared with the CK group. 3.2 Effects of hemin on antioxidant enzyme activity of C. blinii under osmotic stress and cold stress 2 When plants are subjected to environmental stress, antioxidant enzymes perform key roles in the reactive oxygen-scavenging system and can clear reactive oxygen species (ROS) within and outside of the cells to protect tissues from oxidative damage. As shown in Fig. 2 Fig. 2 a, the catalase (CAT) enzyme activity of C. blinii under osmotic stress increased with the continuous increase in hemin concentration. When the hemin concentration reached 25 μmol/L, the maximum increase was 1.09% compared with the CK group. Similarly, as shown in Figs. 2b 2b and 2c 2c , peroxidase (POD) and superoxide dismutase (SOD) activity also significantly increased when the hemin concentration reached 25 μmol/L, with increases of 39.13% and 8.33%, respectively. Similar to the effects of hemin under cold stress, the CAT enzyme activity of C. blinii reached a maximum of approximately 2.12% at 25 μmol/L hemin ( Fig. 2 Fig. 2 a). Likewise, the activity of the POD and SOD enzymes increased by 66.67% and 30.76%, respectively (Figs. 2b 2b and 2c 2c ). 3.3 Effects of hemin on the saponin content of C. blinii under osmotic stress and cold stress 2 To explore the effects of hemin on the saponin content of C. blinii under osmotic stress and cold stress, we used different hemin concentrations to treat C. blinii . As expected, under drought and cold stress, the saponin content increased once C. blinii was subjected to imperceptible concentration (1 μmol/L) of hemin. To a certain extent, the increasing trend in saponin content was similar to that of hemin, regardless of drought or cold stress. When the hemin concentration reached 25 μmol/L, the saponin content increased 86.67% ( Fig. 3 Fig. 3 ). When the hemin concentration reached 50 μmol/L, the saponin content began to decrease; at its maximum, the increase was 84.61% compared with the CK group. The change in C. blinii saponin content after 25 μmol/L hemin treatment under cold stress was consistent with that under osmotic stress, with the greatest improvement effect of 85.29% ( Fig. 3 Fig. 3 ). 3.4 Effects of hemin on the biomass of C. blinii 2 The results showed that the fresh weight of C. blinii ( Fig. 4 Fig. 4 a) increased the most significantly in the 25 μmol/L hemin treatment group compared with the CK group, with an increase of 26.07%. However, when the hemin concentration continued to rise to 50 μmol/L, the improvement effect on the fresh weight decreased, with a slight increase of 11.58% compared with the CK group. Without exception, synchronous changes in the dry weight of C. blinii ( Fig. 4 Fig. 4 b) showed a similar trend and increased the most when the hemin concentration was 25 μmol/L. 3.5 Effects of hemin on the photosynthetic capacity of C. blinii 2 We studied the effects of hemin on the photosynthetic capacity of C. blinii with regard to the photosynthetic pigment content and photosynthetic capacity index. After the treatment of C. blinii with different hemin concentrations, a change in chlorophyll content was detected ( Fig. 5 Fig. 5 a). When the hemin concentration reached 25 μmol/L, the chlorophyll content showed the greatest increase, reaching 50.02% compared with the CK group. The change in carotenoid content was similar to that in the chlorophyll content ( Fig. 5 Fig. 5 b). When the hemin concentration reached 25 μmol/L, the carotenoid content increased by 44.60%, which was the highest increase. Subsequently, we explored the P n , T r , G s , and C i (Figs. 5c 5c – 5f 5f ). When the hemin concentration reached 25 μmol/L, P n , T r , G s , and C i synchronously reached their peak, increasing by 21.10%, 42.86%, 45.57%, and 26.05%, respectively, compared with the CK group. 3.6 Effects of hemin on the synthesis of C. blinii saponins 2 In order to explore the influence of hemin on saponin synthesis in C. blinii , different concentrations of hemin were used to treat the suspension cells and plants of C. blinii . The saponin content, in both the suspension cells and plants, significantly increased under 25 μmol/L hemin treatment, with increase rates of 47.69% and 87.09%, respectively, compared with the CK group (Figs. 6a 6a and 6b 6b ). Moreover, we detected the expression levels of key enzyme genes in the saponin synthesis pathway of C. blinii ( Fig. 6 Fig. 6 c) under different hemin concentrations, and the results showed that when the hemin concentration reached 25 μmol/L, the key enzyme genes in the saponin synthesis pathway, including CbHMGR , CbFPPS , CbSQS , CbSQE , and β‍‐amyrin synthase from C. blinii ( CbβAS ) ( Fig. 6 Fig. 6 d), were upregulated. We also tested the expression level of the heme oxygenase 1 ( CbHO-1 ) gene in C. blinii and found that hemin treatment increased the expression of the CbHO-1 gene; the enhancement effect was most significant when the hemin concentration was 25 μmol/L. 3.7 Effects of hemin and its decomposition products and inhibitors on the saponin content of C. blinii 2 The decomposition products of hemin in plants are BR, Fe 2+ , and CO. We investigated the effects of these different decomposition products on C. blinii saponin synthesis, and the results showed that BR had the most significant effect on C. blinii saponin synthesis; it also had the most similar effect to hemin ( Fig. 7 Fig. 7 ). Moreover, we found that when ZnPPIX, an inhibitor of the CbHO-1 gene, was added along with hemin treatment, the enhancement effect of hemin on saponin content and gene expression in the mevalonate (MVA) pathway disappeared. 4 Discussion 1 4.1 Hemin can alleviate the inhibitory effects of morphogenesis under osmotic stress and cold stress 2 Hemin affects plants in many ways, such as in the formation and elongation of roots and in supporting plant resistance to abiotic stress ( Xuan et al., 2008 Xuan et al., 2008 ; Chen et al., 2013 Chen et al., 2013 ; Jin et al., 2016 Jin et al., 2016 ). A large number of reports have shown that the tolerance of plants to a variety of abiotic stresses, including heavy metal stress and UV-B stress, can be greatly improved after exogenous hemin is applied ( Noriega et al., 2004 Noriega et al., 2004 ; Yannarelli et al., 2006 Yannarelli et al., 2006 ; Li et al., 2012 Li et al., 2012 ). The results of this experiment showed that the growth of C. blinii under osmotic stress and cold stress was not inhibited but instead promoted by the application of exogenous hemin. Moreover, the promotion of morphological development was efficient (even at a low dose of hemin) in a concentration-dependent manner. 4.2 Hemin, with dual status, is involved with ROS and in photosynthesis and metabolism 2 When plants are subjected to abiotic stress, a large amount of ROS is produced in the body, mainly in the form of superoxide anions and hydrogen peroxide ( Matthus et al., 2019 Matthus et al., 2019 ). These oxygen free radicals can cause significant damage to the organism and also cause wilt and plant death ( Orrenius et al., 1992 Orrenius et al., 1992 ). The antioxidant enzyme system plays a key role in the effective scavenging of ROS in plants ( Ozdener and Aydin, 2010 Ozdener and Aydin, 2010 ). The plant antioxidant enzyme system contains several components, such as SOD, POD, CAT, and ascorbate peroxidase (APX) ( Mei and Song, 2010 Mei and Song, 2010 ). These enzymes cooperate with each other and participate in the removal process of ROS in vivo in an orderly manner when plants are subjected to abiotic stress ( Shah and Nahakpam, 2012 Shah and Nahakpam, 2012 ). Hemin is degraded by HO-1 into BR, Fe 2+‍ , and CO, which function as secondary messengers to enhance antioxidant enzyme activity in plants and resist abiotic stress, a result that has been widely reported ( Dennery et al., 2003 Dennery et al., 2003 ). Of these, CO, which is as efficient as hemin, promotes the expression of POD , SOD , and other antioxidant enzyme genes ( Jin et al., 2013 Jin et al., 2013 ). In this study, the activity of antioxidant enzymes, including CAT, SOD, and POD, was upregulated, which was similar to the results of Noriega et al. (2012) Noriega et al. (2012) and Jung et al. (2016) Jung et al. (2016) . Therefore, our findings indicate that hemin could initiate the activity of the antioxidant enzymes in C. blinii under osmotic stress and cold stress, and then multiple antioxidant enzymes in C. blinii could participate in the process of scavenging ROS in an orderly manner. On the other hand, the stress adaptability of C. blinii was enhanced by hemin in addition to natural growth and development. Specifically, hemin treatment induced photosynthetic capability to an extraordinary degree and exhibited concentration dependence on the effects of enzyme activity. Photosynthesis is the basis of metabolism, providing energy and substrates for most redox reactions. Accordingly, we found that the saponin content of C. blinii increased after hemin treatment under osmotic stress and cold stress. As secondary metabolites, triterpenoid saponins have good antioxidant capacity ( Nzowa et al., 2010 Nzowa et al., 2010 ). Plant secondary metabolites are mostly synthesized based on primary metabolites to help plants resist multiple stresses, including abiotic stress and biotic stress ( Bacon et al., 2015 Bacon et al., 2015 ). Research has shown that hemin can promote the synthesis of anthocyanin under UV-B, thus avoiding further damage caused by UV-B ( Zhu et al., 2019 Zhu et al., 2019 ). Similar results in this study once again demonstrated that hemin can promote the synthesis of plant secondary metabolites, thereby participating in the process of ROS clearance and alleviating damage to plants. 4.3 Hemin promotes the synthesis of saponins by improving the photosynthetic capacity of C. blinii and the expression of key enzyme genes in the saponin synthesis pathway 2 We found that the content of saponins of C. blinii under normal conditions increased after hemin treatment, both in the plant and in its suspension cells. We also investigated the mechanism and found that the photosynthetic pigment content of C. blinii increased after hemin treatment; and the P n , G s , T r , and C i all showed an increasing trend, indicating that the photosynthetic capacity of C. blinii was significantly improved after hemin treatment. The synthesis of secondary metabolites requires a certain structural basis, mainly through photosynthetic synthesis of organic materials as the skeleton ( Ghasemzadeh and Jaafar, 2011 Ghasemzadeh and Jaafar, 2011 ). Therefore, the greatly improved photosynthetic capacity leads to the ability of C. blinii to synthesize more organic substances, providing sufficient precursors for saponin synthesis. This was verified by the increases in fresh weight and dry weight after hemin treatment. Moreover, we found that the expression levels of key enzyme genes in the saponin synthesis pathway were upregulated by exogenous hemin, which facilitated the synthesis of saponins. Therefore, C. blinii can be induced by hemin to synthesize more saponins by enhancing metabolic activity. 4.4 Increase in C. blinii saponins induced by hemin is dependent on CbHO-1 2 The relationship between hemin's effects on plants and the CbHO - 1 system in plants has been a hot research topic. Our study found that CbHO-1 gene expression was significantly upregulated in C. blinii after hemin treatment. After adding ZnPPIX, an inhibitor of CbHO-1 , to the hemin-treated group, the expression level of CbHO-1 was lower than that of the CK group; that is, the expression level of CbHO-1 was significantly reduced. Additionally, after ZnPPIX treatment, there was a decrease in saponin content and a decrease in the expression of key enzymes in the saponin synthesis pathway, which indicates that the increase in saponin synthesis induced by hemin is dependent on the expression of CbHO-1 . After inhibition of CbHO-1 expression, the positive effects of hemin on saponin synthesis disappeared or were even reversed. Furthermore, we found that BR played a dominant role among the hemin decomposition components in facilitating the synthesis of saponins and in the expression of key enzyme genes in the saponin synthesis pathway. Some studies have found that CO plays a key role as a signal molecule, and Fe 2+ is the major functioning component among the hemin decomposition components ( Podkalicka et al., 2018 Podkalicka et al., 2018 ). Here, BR played an equal and crucial role. 5 Conclusions 1 In this study, we found that after hemin treatment, the activity of the antioxidant enzymes of C. blinii increased as well as the synthesis of saponins, which improved the tolerance of C. blinii to osmotic stress and cold stress. Hemin can promote the synthesis of saponins by increasing the photosynthetic capacity of C. blinii and the expression of key enzyme genes in the saponin synthesis pathway, thus participating in resistance to abiotic stress. Overall, our study explored the mechanism of saponin synthesis in C. blinii in response to drought and cold stress induced by hemin and expanded the understanding of the effects of heme on plant secondary metabolites in response to abiotic stress. 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Planta , 252 ( 5 ): 81 . https://doi.org/10.1007/s00425-020-03492-1 https://doi.org/10.1007/s00425-020-03492-1 Ferrous iron-induced increases in capitate glandular trichome density and upregulation of CbHO-1 contributes to increases in blinin content in Conyza blinii Zhu ZB , Huang YF , Wu X , 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 . https://doi.org/10.1016/j.ecoenv.2019.03.113 https://doi.org/10.1016/j.ecoenv.2019.03.113 Increased antioxidative capacity and decreased cadmium uptake contribute to hemin-induced alleviation of cadmium toxicity in Chinese cabbage seedlings Junyi ZHAN and Tianrun ZHENG designed research; Junyi ZHAN, Tianrun ZHENG, Wenjun SUN, Ming YANG, and Maojia WANG performed research; Wenjun SUN, Ming YANG, and Maojia WANG analyzed data; Junyi ZHAN, Tianrun ZHENG, and Zhi SHAN wrote the paper. Hui CHEN is responsible for writing, reviewing, editing, and ensuring that the descriptions are accurate. All authors have read and approved the final manuscript, and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Sichuan Science and Technology Program (No. 2020YFH0136), China. 10.1631/jzus.B2000697 1673-1581（2021）08-0682-13 2020-10-28 2021-04-01 2021-08-15 2022-05-09 2021-06-18 © Zhejiang University Press 2021 Tianrun ZHENG, Junyi ZHAN, Ming YANG, Maojia WANG, Wenjun SUN, Zhi SHAN, and Hui CHEN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 8 22