- Email: [email protected]
Melatonin facilitates lateral root development by coordinating PAO-derived hydrogen peroxide and Rboh-derived superoxide radical
Free Radical Biology and Medicine 143 (2019) 534–544
Contents lists available at ScienceDirect
Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed
Original article
Melatonin facilitates lateral root development by coordinating PAO-derived hydrogen peroxide and Rboh-derived superoxide radical
T
Jian Chena,∗,1, Hui Lia,1, Kang Yanga, Yongzhu Wanga, Lifei Yangb, Liangbin Huc, Ruixian Liud, Zhiqi Shia,∗∗ a
Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China c Department of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China d Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hydrogen peroxide Lateral root Melatonin Polyamine oxidase RBOH Reactive oxygen species Superoxide radical
Melatonin, a phytochemical, can regulate lateral root (LR) formation, but the downstream signaling of melatonin remains elusive. Here we investigated the roles of hydrogen peroxide (H2O2) and superoxide radical (O2•‾) in melatonin-promoted LR formation in tomato (Solanum lycopersicum) roots by using physiological, histochemical, bioinformatic, and biochemical approaches. The increase in endogenous melatonin level stimulated reactive oxygen species (ROS)-dependent development of lateral root primordia (LRP) and LR. Melatonin promoted LRP/ LR formation and modulated the expression of cell cycle genes (SlCDKA1, SlCYCD3;1, and SlKRP2) by stimulating polyamine oxidase (PAO)-dependent H2O2 production and respiratory burst oxidase homologue (Rboh)dependent O2•‾ production, respectively. Screening of SlPAOs and SlRbohs gene family combined with gene expression analysis suggested that melatonin-promoted LR formation was correlated to the upregulation of SlPAO1, SlRboh3, and SlRboh4 in LR-emerging zone. Transient expression analysis confirmed that SlPAO1 was able to produce H2O2 while SlRboh3 and SlRboh4 were capable of producing O2•‾. Melatonin-ROS signaling cassette was also found in the regulation of LR formation in rice root and lateral hyphal branching in fungi. These results suggested that SlPAO1-H2O2 and SlRboh3/4-O2•‾ acted as downstream of melatonin to regulate the expression of cell cycle genes, resulting in LRP initiation and LR development. Such findings uncover one of the regulatory pathways for melatonin-regulated LR formation, which extends our knowledge for melatonin-regulated plant intrinsic physiology.
1. Introduction Root branching resulted from LR (lateral root) development is vital for plants acquiring water and nutrients [1,2]. LR development initiates from the emergence and development of LRP (lateral root primordium) along the longitudinal axis of PR (primary root). LRP emergence is a dynamic process that involves several stages (I-VIII), starting from continuous division of different cell layers to just about emergence of LRP from parent PR [3,4]. This process is genetically controlled by cell cycle regulatory genes, such as CYCs (cyclins), CDKs (cyclin dependent kinases), and KRPs (Kip-related proteins). CYCD and CDKA are positive regulators for LR initiation [5,6]. KRPs, suppressors of CDK, are negative regulators for LR development [7–9]. These cell cycle regulators
(e.g. CYCD3;1, CDKA1, and KRP2) are always considered as marker genes to indicate LR initiation [10,11]. Auxin plays crucial roles in the regulation of cell cycle process and LR formation by integrating other plant hormones [12–14]. Apart from hormones, ROS (reactive oxygen species) have been considering as emerging regulators for LR development [15]. H2O2 (hydrogen peroxide) and O2•‾(superoxide radical) are two typical ROS, belonging to non-radical and free radical forms, respectively [16]. PAO (polyamine oxidase) catalyzes the metabolism of PAs (polyamines), including Spd (spermidine) and Spm (spermine), along with H2O2 production, which has been considered as an important biosynthetic pathway of H2O2 in plants [17]. PAO-dependent H2O2 generation contributes to LR development in soybean [18]. Rbohs
∗
Corresponding author. Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing, 210014, China. Corresponding author. Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing, 210014, China. E-mail addresses: [email protected] (J. Chen), [email protected] (Z. Shi). 1 These authors contribute equally to this work. ∗∗
https://doi.org/10.1016/j.freeradbiomed.2019.09.011 Received 19 August 2019; Received in revised form 10 September 2019; Accepted 10 September 2019 Available online 11 September 2019 0891-5849/ © 2019 Elsevier Inc. All rights reserved.
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
2.4. Determination of the content of H2O2 and O2•‾
(respiratory burst oxidase homologues) are important enzymes to produce O2•‾ for the regulation of plant physiology [19]. Rboh-derived ROS can facilitate LR emergence in Arabidopsis thaliana [20]. ROS facilitates LR development in either auxin-dependent or auxin-independent manners [20,21]. Melatonin, an important immune regulator in mammalian cells, has been proposed as a novel phytohormone with multiple physiological functions [22]. Melatonin-promoted LR formation has been found in several plant species, such as Lupinus albus [23], Arabidopsis [24], rice [25], and cucumber [26,27]. Auxin acts as a regulatory star for LR development, but melatonin facilitates LR development likely acting independently of auxin signaling in Arabidopsis [24]. Melatonin activates auxin-related genes during LR development in rice, but not in cucumber [25,26]. Thus, the detailed mechanism for melatonin-facilitated LR development remains elusive. Recent studies have implied that ROS signaling maybe involved in melatonin-induced LR development [26,28]. However, whether and how melatonin regulates ROS generation (including both H2O2 and O2•‾) to activate LR development remains unclear. In this work, we investigated the roles of PAO-generated H2O2 and Rbohs-generated O2•‾ during melatonin-facilitated LR development in tomato (Solanum lycopersicum) seedlings. Melatonin-ROS interaction cassette was also confirmed in LR formation in rice roots (monocot) and hyphal branching in fungi. This study may help reveal one of the important molecular mechanisms for melatonin-induced LR development.
Root tip without LR formation was removed from the entire root. The rest of root with branching sites was collected immediately for the measurement of ROS content (Fig. S1). H2O2 content was determined by using a commercial H2O2 assay kit (A064-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to our previously published method [33]. O2•‾ content was measured based on the hydroxylamine oxidation method described by Chen et al. [34]. 2.5. Histochemical analysis Intracellular H2O2 and O2•‾ in root were detected in situ using specific fluorescent probe HPF and DHE, respectively, based on our published method [35]. LRP were stained with methylene blue and observed under microscope as described by Wang et al. [36]. H2O2 and O2•‾ in tobacco leaves were detected histochemically by staining with DAB (3,3-diaminobenzidine) and NBT (nitro-blue tetrazolium), respectively, according to our published methods [37]. 2.6. Genomewide identification of SlPAOs and SlRbohs The protein sequences of PAOs in A. thaliana were obtained from TAIR (The Arabidopsis Information Resource). These sequences were used as baits for searching SlPAO homologues from Sol Genomics Network (SL2.50) by using BLAST (Basic Local Alignment Search Tool). The obtained SlPAOs were further confirmed by using Pfam database and NCBI Conserved Domain Database [38,39]. Genomewide identification of SlRbohs was performed by using the same strategy as SlPAOs. Then a set of tools were used to analyze the characteristics of the obtained sequences, which was demonstrated in detail in Supporting Methods.
2. Materials and methods 2.1. Plant culture and reagents Tomato (S. lycopersicum) seeds were surface-sterilized with 1% NaClO for 10 min and washed with distilled water for three times, followed by germination on a floating plastic net in darkness. The germinated seeds were transferred to 1/4-strength Hoagland nutrition [29]. After growing for 48 h, the identical seedlings with root length of 1.5 cm were selected and transferred to another container with treatment solution. The seedlings were placed in an incubator with photosynthetic active radiation of 200 μmol/m2/s, photoperiod of 16/8 h, and temperature at 25 ± 1 °C. Melatonin was added to treatment solution at concentrations of 50–600 μM. KI (potassium iodide) was applied as ROS scavenger. H2O2 was used as exogenous ROS donor. DPI (diphenylene iodonium), PY (pyridine), and IMZ (imidazole) were used as RBOH inhibitors. MDL (1,4-diamine dihydrochloride), GZT (guazatine acetate), and 2-HEH (2hydroxyethylhydrazine) were used as PAO inhibitors. All the above reagents and other ordinary chemicals were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China). DHE (dihydroethidium) and HPF (hydroxyphenyl fluorescein), used as fluorescent probes for the detection of endogenous O2•‾ and H2O2, respectively, were obtained from Beyotime Biotechnology (Shanghai, China).
2.7. Gene expression analysis Gene expression analysis was performed using qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction). The detailed procedure was described in Supporting Methods. Oligopeptide primers were listed in Table S1. 2.8. Transient expression of SlPAO1, SlRboh3, and SlRboh4 in tobacco leaves Transient expression of tomato genes in tobacco leaves was performed according to our previous method [33]. Primers with specific adaptors containing SamI restriction enzyme site (Table S2) were used to amplify the full length cDNA of designated tomato gene, which was further cloned into pCAMBIA-2300 vector. Then the recombined vector was transformed into Agrobacterium tumefaciens strain GV3101, which was further infiltrated into tobacco (Nicotiana benthamiana) leaves. Target genes in tobacco leaves were detected at DNA and mRNA level as described in Supporting Methods.
2.2. Measurements of LR development LR formation was evaluated based on several typical parameters, such as LR number, LR density (total LR number/PR length), LRP number, and LRP density (total LRP number/PR length) [30]. LRP development was evaluated by counting LRP at different developing stages (I-VIII) under microscopy [3].
2.9. Statistical analysis Each data was showed as mean ± standard deviation (SD) for at least three replicates. SD and ANOVA (one-way analysis of variance) were used to evaluate the significant differences between treatments by using SPSS 6.0 (Statistical Package for the Social Science, SPSS Inc., Chicago, IL, USA). For two designated treatments, the data were statistically compared by ANOVA, followed by F-test if the ANOVA result is significant at P < 0.5 or P < 0.1. For multiple comparison, least significant difference test (LSD) was used to test significance at P < 0.5 among any different treatments based on ANOVA and F-test.
2.3. Determination of melatonin content Melatonin in root samples was extracted using extraction buffer (acetone:methanol:water = 89:10:1) [31]. A plant melatonin ELISA (enzyme-linked immunosorbent assay) detection kit (Jiangu Baolai Biotechnology, Yancheng, China) was applied to determine melatonin content according to manufacturer's instructions [32]. 535
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 1. Melatonin promoted LR formation in tomato roots. Tomato roots were treated with melatonin at different concentrations (0–600 μM) for 4 days, followed by photographing phenotype (A), counting LR number (B), and determining LR density (C). Tomato roots were treated with melatonin at 200 μM for 1, 2, 3, 4, 5, and 6 days, respectively, followed by the determination of LR number (D) and LR density (E). Tomato roots were treated with melatonin at different concentrations (0–600 μM) for 5 days, followed by the determination of endogenous melatonin content in roots (F). Different lowercase letters in (B, C, F) indicated that the mean values of ten replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD). Two asterisks (**) in (D and E) indicated that the mean values of ten replicates were significantly different between control and melatonin treatment at each time point (P < 0.01, ANOVA).
3. Results 3.1. Melatonin promoted the development of LR and LRP Melatonin induced significant increases in LR number and LR density in dose-dependent manners. Melatonin at 200 μM exhibited the greatest effect (Fig. 1A–C). The continuous increases in LR number and LR density were also observed under treatment with 200 μM of melatonin in time-course experiments (Fig. 1D and E). Exogenous treatment with melatonin resulted in remarkable increase in endogenous melatonin level in root. Treatment with melatonin at 200 μM resulted in the highest level of endogenous melatonin in root (Fig. 1F). The remarkable increases in total LRP number and LRP density were observed after melatonin treatment, with maximal enhancement occurring at 200 μM of melatonin (Fig. 2A and B). In addition, melatonin stimulates LRP initiation and development as observed from LRP at different stages (Fig. 2C and D). These results suggested that exogenous melatonin treatment enhanced endogenous melatonin level, further promoting LRP initiation and accelerating LRP development to form more LR.
3.2. ROS was involved in melatonin-promoted LR formation Fig. 2. Melatonin promoted LRP formation in tomato roots. Tomato roots were treated with melatonin at different concentrations (0–600 μM) for 2 days, followed by counting the number of total LRP (A) and determining LRP density (B). Tomato roots were treated with melatonin at 200 μM for 2 days followed by counting the number of LRP at different developmental stages (C) and determining LRP proportion (D). Different lowercase letters in (A and B) indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD). One asterisk (*) and two asterisks (**) in (C) indicate that the mean values of six replicates were significantly different between control and melatonin treatment at P < 0.05 and P < 0.01, respectively.
Accumulation of O2•‾ and H2O2, detected in situ by DHE and HPF, respectively, were observed clearly in the sites where LRP emerged during the whole process of LRP formation (Fig. 3A and B). H2O2 treatment significantly stimulated the formation of LRP and LR, whereas treatment with ROS scavenger KI showed opposite effects (Fig. 3D–F). These results suggested that ROS was involved in the regulation of LR development. Then we found that KI compromised the promoting effect of melatonin on LR formation (Fig. 4A and B). Root tip without branch sites mainly functions for the regulation of the apical growth of PR. ROS has been found to be accumulated in root tip to control PR elongation [40,41]. Therefore, melatonin-treated roots without root tips were collected for the detection of ROS level in order to avoid the interference of ROS in root tips on the final results (Fig. S1). Melatonin significantly enhanced H2O2 and O2•‾ level in roots (Fig. 4C and D). These results suggested that H2O2 and O2•‾ might mediate the promoting effect of melatonin on LR development.
3.3. Melatonin promoted LR development by regulating Rboh-dependent O2•‾ generation and PAO-dependent H2O2 generation RBOH inhibitors (IMZ, PY, or DPI) significantly compromised the promoting effect of melatonin on LR, LRD, LRP, and LRPD (Fig. 5A–E), accompanying with significant decrease in O2•‾ content in melatonintreated root (Fig. 5F and Fig. S1). Similarly, application of PAO 536
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 3. Involvement of ROS in the formation of LRP and LR in tomato roots. (A) DHE-labelled O2•‾ in LRP at different developmental stages. (B) HPF-labelled H2O2 in LRP at different developmental stages. Tomato roots were treated with water (Control), H2O2 (1.2 mM), or KI (400 μM) for 2 days followed by the quantification of LRP number (C) and LRP density (D). Tomato roots were treated with water (Control), H2O2 (1.2 mM), or KI (400 μM) for 4 days followed by the quantification of LR number (E) and LR density (F). Different lowercase letters in (C-F) indicated that the mean values of seven replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
study the possible link between LR development and the expression levels of SlRboh2-5. More LRP were detected in part I than in part II (Fig. 9B), coinciding with similar patterns for the expression levels of SlRboh3 and SlRboh4, but not SlRboh2 and SlRboh5 (Fig. 9C–F). Intriguingly, melatonin induced similar changing patterns of LRP and the expression of SlRboh3 and SlRboh4 in different parts of root (Fig. 9G–I). These results suggested that the upregulation of SlRboh3 and SlRboh4 might be involved in melatonin-induced LRP formation.
inhibitors (MDL, GZT, or 2-HLH) inhibited the formation of LRP and LR (Fig. 6A–E) as well as H2O2 generation in melatonin-treated root (Fig. 6F and Fig. S1). Compared to control, treatment with DPI or 2HEH alone prohibited the formation of LR and LRP (Fig. S2). Melatonin upregulated the expression of SlCDKA1 and SlCYCD3;1 and downregulated the expression of SlKRP2. The effects of melatonin on these cell cycle genes were reversed by the addition of DPI or 2-HLH (Fig. 7). These results suggested that RBOH-dependent O2•‾ and PAO-dependent H2O2 contributed to melatonin-promoted LR development.
3.5. Identification of melatonin-targeted SlPAO during LRP development 3.4. Identification of melatonin-targeted SlRbohs during LRP development A total of eleven potential SlPAOs with typical amnio_acid domain were obtained from tomato genome (Table S5; Fig. S3; Fig. S5A). SlPAO9-11 (Solyc07g063450.1, Solyc07g063500.2, and Solyc04g081100.2) belonging to subfamily III had another SWIRM domain besides of amnio_acid domain (Fig. 10A; Fig. S7A). Thus SlPAO911 were characterized as histone lysine-specific demethylase rather than real PAO based on the classification of their homologues in rice (OsPAO9-11) and Arabidopsis (AtPAO6-9) (Fig. 10A) [42]. SlPAO1-8 were considered as typical PAOs that were distributed in three subfamilies (I, IIa, IIb) (Fig. 10A; Figs. S5B–C; Table S6). According to the identification of OsPAOs and AtPAOs in subfamily I [42], SlPAO1 with predicated apoplastic localization was supposed to catalyze the terminal catabolism of PAs. Subfamily IIa (SlPAO6-8) and IIb (SlPAO2-5) with predicated subcellular localization of cytoplasm and peroxisome, respectively, were supposed to catalyze the back-conversion of PAs (Fig. 10A; Table S5). These results suggested that SlRboh1-8 were PAO
A total of seven SlRboh genes with various chromosomal distributions were retrieved from tomato genome (Table S3; Fig. S3). All the SlRbohs were transmembrane proteins with typical conserved domains (Fig. S4A; Table S3). SlRbohs were found in subfamily I, II, III, and V, but not in subfamily IV, based on phylogenetic analysis, motif distribution, and gene structure diversity (Fig. 8A; Figs. S4B and C; Table S4). These results suggested that SlRboh1-7 were Rboh homologues with potential capability to produce O2•‾. Then we measured the effect of melatonin on the expression of SlRbohs in root sections with branch sites (Fig. S1). SlRboh2-5 showed significantly increased expression levels at 24 h under melatonin treatment (Fig. 8B). In time-course experiments, SlRboh3 and SlRboh4, but not SlRboh2 and SlRboh5, displayed enhanced expression levels, as compared to control groups (Fig. 8C–F). The different parts of root (part I and II in Fig. 9A) were harvested to 537
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
expression level of SlPAO1, but not SlPAO4, was correlated to LRP development (Fig. 10E and F; Fig. 9B). The expression level of SlPAO1 was positively correlated to LRP development in different parts of root under melatonin treatment (Fig. 10G; Fig. 9G). Thus the upregulation of SlPAO1 may be associated to melatonin-induced LRP formation. 3.6. Identification of the capability of BrRbohs3/4 and BrPAO1 in ROS production The capabilities of BrRbohs3, BrRbohs4 and BrPAO1 in ROS production were evaluated by using transient expression analysis (Fig. S6; Fig. 11A–C). DAB staining suggested that tobacco leaves expressing BrPAO1 showed higher H2O2 level than control or EV (empty vector) (Fig. 11D). NBT staining indicated that tobacco leaves with the expression of BrRbohs3 or BrRbohs4 showed remarkable increase in O2•‾ level (Fig. 11E). These results suggested that BrRbohs3 and BrRbohs4 had the ability to generate O2•‾ while BrPAO1 was able to produce H2O2. 3.7. Melatonin/ROS was involved in LR development in dicot and hyphal branching in fungi Since melatonin promoted ROS-dependent LR development in dicot (tomato), we wondered whether it acted similarly in monocot (e.g. rice). Melatonin promoted the formation of LR and LRP in the seminal root of rice seedlings, which was suppressed by the addition of KI (Fig. S7). ROS is also one of the important factors modulating hyphal branching, which is similar to root branching in plants [43]. Fusarium graminearum is a kind of model filamentous ascomycete in agriculture. Melatonin treatment stimulated the formation of lateral branches in the hyphae of F. graminearum, which could be inhibited by KI as well (Fig. S8). These results suggested that ROS might act as downstream of melatonin in the regulation of LR formation in monocot and lateral branching in fungal hyphal.
Fig. 4. Involvement of ROS in melatonin-promoted LR formation in tomato roots. Tomato roots were treated with water (Control), melatonin (200 μM), and melatonin (200 μM) + KI (400 μM) for 4 days, respectively, followed by photographing phenotype (A), counting LR number (B). Tomato roots were treated with melatonin at 200 μM for 12, 24, 36, 48, and 72 h, respectively, followed by the determination of H2O2 content (C) and O2•‾ content (D) in root sections with lateral branching sites. Different lowercase letters in (B) indicated that the mean values of ten replicates are significantly different among different treatments (P < 0.05, ANOVA, LSD). One asterisk (*) and two asterisks (**) in (C and D) indicated that the mean values of three replicates were significantly different between control and melatonin treatment at each time point at P < 0.05 and P < 0.01, respectively.
homologues with potential capability to produce H2O2 by metabolizing PAs. SlPAO1 and SlPAO4 showed the highest expression level in root sections with branch sites after melatonin treatment for 24 h (Fig. 10B). Compared to the control, SlPAO1, but not SlPAO4, showed enhanced expression levels in time-course measurements (Fig. 10C and D). The
4. Discussion 4.1. Melatonin promoted LR formation through ROS-dependent pathway Melatonin-induced increase in LR number resulted from the stimulation of LRP emergence. Auxin is a key regulator for LRP development, Fig. 5. Involvement of Rboh-dependent O2•‾ in melatonin-induced formation of LR and LRP in tomato roots. Tomato roots were treated with water (Control), melatonin (200 μM), melatonin (200 μM) + IMZ (400 μM), melatonin (300 μM) + PY (500 μM), and melatonin (200 μM) + DPI (200 μM) for 4 days, respectively, followed by photographing phenotype (A), counting LR number (B), and determining LR density (C). Tomato roots were treated with same solution mentioned above for 2 days, followed by counting LRP number (D), determining LRP density (E), and measuring O2•‾ content (LRP emerging zone) (F). Different lowercase letters in (B-F) indicated that the mean values of 3–10 replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
538
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 6. Involvement of PAO-dependent H2O2 in melatonin-induced formation of LR and LRP in tomato roots. Tomato roots were treated with water (Control), melatonin (200 μM), melatonin (200 μM) + MDL (1 μM), melatonin (300 μM) + GZT (1 μM), and melatonin (200 μM) + 2-HEH (0.4 μM) for 4 days, respectively, followed by photographing phenotype (A), counting LR number (B), and determining LR density (C). Tomato roots were treated with same solution mentioned above for 2 days, followed by counting LRP number (D), determining LRP density (E), and measuring H2O2 content (LRP emerging zone) (F). Different lowercase letters in (B-F) indicated that the mean values of 3–10 replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
but melatonin promotes LR formation likely acting independently of auxin signaling [24,26]. ROS functions as signaling molecules to regulate LR formation, paralleling to auxin signaling [21]. Therefore, we wandered whether melatonin regulated LR formation through ROS signaling. Indeed, we found that ROS located in LRP sites is required for LRP emergence in tomato root, in agreement with previous reports in the root of Arabidopsis [21]. Melatonin enhanced ROS level while the suppression of ROS level counteracted the promoting effect of melatonin on LR formation. Thus we revealed that melatonin promoted LR formation through ROS-dependent pathway in the root of tomato seedlings. LRP initiation started from the founder cell specification followed by cell division in PR [5]. LRP formation and development in Arabidopsis can be controlled by either auxin signaling or ROS signaling, likely through independent pathways [21]. Melatonin regulated the expression of cycle genes (SlCDKA1, SlCYCD3;1, and SlKRP2) in a ROS-dependent manner. Therefore, we suggest that melatonin triggers ROS accumulation in roots, which further enables the activation of cell division to start LRP formation program. Establishing the link between melatonin and ROS provides possible explanation for the fact that melatonin-promoted LR formation is auxin-independent in dicots. In monocot rice, transcriptome analysis suggests a possible link between auxin signaling responses and root architecture modulation (including LR formation) under melatonin treatment [25]. In the present study, we found that melatonin promoted rice LR formation was also ROS-dependent. Therefore, it seems that both auxin and ROS might be involved in melatonin-facilitated LR formation in monocot. Further studies are needed to compare the detailed downstream networks of melatonin in the regulation of LR formation between dicot and monocot. In Arabidopsis, peroxidase-modulated ROS level is important for LR development [21]. In addition, the regulation of the expression of several peroxidase genes has been found during melatonin-promoted LR formation in cucumber [26]. However, how melatonin activates ROS generation to regulate LR formation remains elusive. Here we identified that PAO and RBOH might be involved in the generation of H2O2 and O2•‾, respectively, during melatonin-promoted LR formation.
Fig. 7. RBOH inhibitor (DPI) or PAO inhibitor (2-HEH) affected the expression of cell cycle regulatory genes (SlCDKA1, SlCYCD3;1, and SlKRP2) in melatonin-treated roots. (A) SlCDKA1. (B) SlCDKA1YCD. Tomato roots were treated with water (Control), melatonin (200 μM), melatonin (300 μM) + DPI (200 μM), and melatonin (200 μM) + 2-HEH (0.4 μM), respectively, for 2 days. Then root sections with lateral branching sites were harvested for RNA extraction and gene expression analysis. Different lowercase letters indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
4.2. Melatonin promoted LR formation through SlPAO1-dependent H2O2 generation PAO metabolize various PAs with H2O2 as byproduct [44]. H2O2 539
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 8. Phylogenetic analysis of SlRboh family and melatonin-regulated expression of SlRbohs in tomato roots. (A) Neighbor-joining phylogenetic tree of the SlRbohs, A. thaliana Rbohs (AtRbohA-J), and rice Rbohs (OsRboh1-9). (B) The relative expression level of SlRboh1-7 in tomato root sections with branching sites after treatment with melatonin (200 μM) for 24 h. Tomato roots were treated with melatonin at 200 μM for 12, 24, 36, 48, and 72 h, respectively. Root sections with lateral branching sites were harvested for RNA extraction and the analysis of relative expression level of SlRboh2 (C), SlRboh3 (D), SlRboh4 (E), and SlRboh5 (F). One asterisk (*) and two asterisks (**) in (B-F) indicated that the mean values of three replicates were significantly different between control and melatonin treatment at P < 0.05 and P < 0.01, respectively.
Fig. 9. Involvement of the expression of SlRboh3 and SlRboh4 in melatonin-promoted LRP formation. (A) Schematic presentation for sampling section in the root of tomato seedling. Tomato seedlings were allowed to grow for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for counting LRP number (B) and analyzing the relative expression level of SlRboh2-5 (C-F). Tomato roots were treated with melatonin (200 μM) for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for counting LRP number (G) and analyzing the expression level of SlRboh3-4 (H-I). Different lowercase letters in (B-I) indicated that the mean values of 3–10 replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
540
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 10. Phylogenetic analysis of SlPAO family and the effect of melatonin on the expression of SlPAOs in tomato roots. (A) Neighbor-joining phylogenetic tree of the SlPAO family. (B) The relative expression level of SlPAO1-8 in tomato root sections with branching sites after treatment with melatonin (200 μM) for 24 h. Tomato roots were treated with melatonin at 200 μM for 12, 24, 36, 48, and 72 h, respectively. Root sections with lateral branching sites were harvested for RNA extraction and the analysis of relative expression level of SlPAO1 (C), and SlPAO4 (D). Tomato seedlings were allowed to grow for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for the analysis of relative expression level of SlPAO1 (E) and SlPAO4 (F). Tomato roots were treated with melatonin (200 μM) for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for the analysis of relative expression level of SlPAO1 (G). One asterisk (*) and two asterisks (**) in (B-D) indicated that the mean values of three replicates were significantly different between control and melatonin treatment at P < 0.05 and P < 0.01, respectively. Different lowercase letters in (E-G) indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
molecule to regulate various physiological processes. Melatonin can protect plant cells from stress conditions by suppressing PAO-dependent over-generation of H2O2 [50]. However, our results suggest that melatonin triggers SlPAO1-dependent H2O2 generation to facilitate LR formation under normal growth conditions. It will be interesting to investigate the role of melatonin-maintained PAO-H2O2 homeostasis in growth regulation and stress responses.
produced by PAO acts as signaling molecule to regulate multiple plant physiological processes, including LR formation [17,18]. Melatonin promotes LR and LRP development through PAO-dependent H2O2 production in tomato root. Eight typical SlPAO family genes consisted of three subfamilies with two types of predicted function (type I, terminal catabolism of PAs; type II, back-conversion of PAs). SlPAO1, AtPAO1, and OsPAO7 were grouped together in subfamily I. AtPAO1 and OsPAO7 are localized in cytoplasm and apoplast, respectively, belonging to type I PAO functioning for the terminal catabolism of Spm [45,46]. Therefore, SlPAO1 with predicted cytosolic localization may have similar functions to AtPAO1 and OsPAO7. SlPAO6-8 in subfamily IIa were proposed to work on the back-conversion of PAs (Spm→Spd) according to the already identified function of their homologues (AtPAO5 and OsPAO1) [47,48]. In subfamily IIb, SlPAO2-5 with predicted peroxisomal localization may also work on the back-conversion of PAs because their homologues AtPAO2/3 are two peroxisomal proteins that can oxidize both Spd and Spm through PA back-conversion pathway [45]. Collectively, it can be proposed that SlPAO1-8 may have potential to produce H2O2 by taking Spd or Spm as substrate to facilitate PAs metabolism. Melatonin induced the most significant increase in the expression of SlPAO1 and SlPAO4 in LR-emerging zone. AtPAO2, a homologue of SlPAO4, positively regulates LR growth [49], but only the expression of SlPAO1 was correlated to melatonin-induced LRP formation. Transient expression analysis further confirmed the capability of SlPAO1 in H2O2 production. Having linked SlPAO1 melatonin sensing and LRP formation into a regulatory module, this finding adds a critical piece of information to complete melatonin-H2O2 signaling for the regulation of LR development. High level of H2O2 poses threat to plant cells by inducing oxidative injury, but H2O2 at moderate level acts as a signaling
4.3. Melatonin promoted LR formation through SlRboh3-and SlRboh4dependent O2•‾ generation Rboh-derived O2•‾ plays vital roles in the regulation of plant development [19]. In the present study, Rboh-dependent O2•‾ contributed to melatonin-induced development of LRP and LR. SlRboh3 and SlRboh4 were able to generate O2•‾, which was closely linked to melatonin-induced formation of LRP. SlRboh3 and AtRbohB were found in the same clade. Genetic evidences demonstrate that PvRbohB (Phaseolus vulgaris RbohB), a functional homologue of AtRbohB, contributes to O2•‾ accumulation for LR development [51]. SlRboh4 and AtRbohE belong to the same clade. AtRbohE has been found to be expressed inside of the LRP and in the overlaying cells of epidermis. AtRbohE-derived extracellular O2•‾ not only modulates LRP initiation but also regulates cell wall remodeling to help LRP emergence [20]. Treatment with DPI compromised melatonin-induced O2•‾ production and expression of cell cycle genes, suggesting that melatonin-modulated Rboh/O2•‾ probably regulates LRP initiation. Rboh-generated O2•‾ can be utilized by peroxidase to produce H2O2 to facilitate LR formation [20,52]. The expressions of a set of peroxidase genes have been found to be upregulated during melatonin-promoted LR formation in cucumber [26]. Therefore, melatonin-Rboh-peroxidase signaling cascade is probably 541
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
antioxidant to protect mitochondria from ROS attack and oxidative stress in both plants and animals [55–57]. Melatonin can also be synthesized in chloroplast in plant cells, which help to scavenge ROS generated in chloroplast [16,55]. Here we found that melatonin promoted PAO- and Rboh-dependent ROS generation to induce LR development. Both PAO and Rboh are not localized to either mitochondria or chloroplast. Thus PAO-derived H2O2 and Rboh-derived O2•‾ induced by melatonin may act as signaling molecules, rather than oxidative attackers, to trigger LR development under normal growth conditions [21]. However, we cannot exclude the possibility that melatonin may regulate ROS level in different organelles in different ways, which needs to be studied in the future. 4.6. Possible regulators involved in melatonin-ROS cassette during LR development ROS are multifunctional signals that can regulate many signaling molecules [58]. In plant roots, H2O2 is able to facilitate Ca2+ (calcium) influx into cytosol by activating plasma membrane-located Ca2+ channel at either extracellular or intracellular membrane face [59–61]. And Ca2+ has been suggested as an important signal to regulate LR development by modulating cell cycle genes at transcriptional and posttranslational level [62,63]. The probable interaction between melatonin and Ca2+ has been found in plants [64]. Combining with the results of melatonin-ROS interplay obtained in this study, it could be speculated that Ca2+ might act downstream of ROS during melatoninregulated cell cycle genes and LR development.
Fig. 11. Assessment of the capability of SlPAO1, SlRboh3, and SlRboh4 in ROS production with transient expression analysis. (A-C) Relative expression level of SlPAO1 (A), SlRboh3 (B), and SlRboh4 (C) in tobacco leaves. (D) Detection of endogenous H2O2 with DAB staining in tobacco leaves expressing SlPAO1. (E) Detection of endogenous O2•‾ with NBT staining in tobacco leaves expressing SlRboh3 and SlRboh4. Contorl indicated infiltration with buffer. EV indicated infiltration with empty vector without carrying any tomato genes. Different lowercase letters in (A-C) indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
5. Conclusion Taken together, we propose a model for ROS generation during melatonin-induced LR formation in tomato roots (Fig. 12). Melatonin stimulates the expression of SlPAO1 to increase apoplastic H2O2 generation. Melatonin stimulates the expression of SlRboh3/4 to increase apoplastic O2•‾ generation. Then both H2O2 and O2•‾ enter into cytosol and/or nucleus and trigger signaling transduction, which further modulate the expression of cell cycle genes to initiate LRP development and LR formation. In addition, O2•‾ can be transformed to H2O2 that may activate Ca2+ channel to promote Ca2+ influx into cytosol. Then the increase in intracellular Ca2+ probably regulates cell cycle genes to stimulate LR development. However, the probable involvement of specific Ca2+ channels in this signaling cassette needs to be identified in future studies.
involved in the cell wall modification to facilitate LR formation, which needs to be elucidated further. 4.4. Possible role of PAO-Rboh cross-talk in ROS generation during melatonin-promoted LR formation PAO and Rboh contribute to the generation of H2O2 and O2•‾, respectively, but the cross-talk between PAO and Rboh can also occurred. In tobacco, Rboh can act as either upstream or downstream of apoplastic PAO (ZmPAO1 and NtPAO1) in order to amplify ROS generation cells upon salinity stress [53]. SlPAO1 was predicted to be an apoplastic protein. SlPAO1, ZmPAO1, and NtPAO1 were found in the same clade (Sub.I in Fig. S9), suggesting that these three proteins might have similar physiological functions. Therefore, it would be interesting to further investigate whether melatonin could trigger a PAO/Rboh feedback loop to generate ROS for LR regulation. 4.5. Balanced interaction between melatonin and ROS Abiotic stress conditions always cause over-generation of ROS in various plant organelles (e.g. mitochondrion, chloroplast, apoplast, and peroxisome), accompanied with inhibition of root growth. In these cases, the overall increased ROS in the whole cell tends to cause oxidative injury and cell death, with compromised role in signaling transduction [16,54]. Melatonin has been reported as a scavenger of excessive ROS in both plants and animals under stress conditions [55,56]. In the current study, melatonin could trigger ROS signaling to regulate cell cycle genes and LR development, which may be organellespecific effects. Mitochondrion is one of the major sites of ROS production due to ETC (electron transport chain) disturbance in cells upon stress [21]. Melatonin, synthesized in mitochondria, act as an important
Fig. 12. Schematic model for melatonin-promoted LR formation by coordinating ROS generation in tomato root. Melatonin induces SlRboh3/4derived O2•‾ generation and SlPAO1-derived H2O2 generation in apoplast. O2•‾ and H2O2 triggers signaling transduction to regulate the expression of cell cycle genes, which further stimulates LRP initiation and LRP formation. Ca2+ is a probable downstream signal of ROS to regulate cell cycle genes during melatonin-promoted LR development. 542
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Declarations of interest
165 (2014) 1105–1119. [22] R. Hardeland, Melatonin in plants – diversity of levels and multiplicity of functions, Front. Plant Sci. 7 (2016) 198. [23] M.B. Arnao, n.-R.J. Hernã, Melatonin promotes adventitious- and lateral root regeneration in etiolated hypocotyls of Lupinus albus L, J. Pineal Res. 42 (2010) 147–152. [24] R. Pelagioflores, E. Muñozparra, R. Ortizcastro, J. Lópezbucio, Melatonin regulates Arabidopsis root system architecture likely acting independently of auxin signaling, J. Pineal Res. 53 (2012) 279–288. [25] C. Liang, A. Li, H. Yu, W. Li, C. Liang, S. Guo, R. Zhang, C. Chu, Melatonin regulates root architecture by modulating auxin response in rice, Front. Plant Sci. 8 (2017). [26] N. Zhang, H.J. Zhang, B. Zhao, Q.Q. Sun, Y.Y. Cao, R. Li, X.X. Wu, S. Weeda, L. Li, S. Ren, The RNA-seq approach to discriminate gene expression profiles in response to melatonin on cucumber lateral root formation, J. Pineal Res. 56 (2013) 39–50. [27] N. Zhang, B. Zhao, H.J. Zhang, S. Weeda, C. Yang, Z.C. Yang, S. Ren, Y.D. Guo, Melatonin promotes water-stress tolerance, lateral root formation, and seed germination in cucumber (Cucumis sativus L.), J. Pineal Res. 54 (2012) 15–23. [28] Z. Chen, Q. Gu, X. Yu, L. Huang, S. Xu, R. Wang, W. Shen, W. Shen, Hydrogen peroxide acts downstream of melatonin to induce lateral root formation, Ann. Bot. 121 (2018) 1127–1136. [29] K. Guo, K. Xia, Z.M. Yang, Regulation of tomato lateral root development by carbon monoxide and involvement in auxin and nitric oxide, J. Exp. Bot. 59 (2008) 3443–3452. [30] I. De Smet, P.J. White, A.G. Bengough, L. Dupuy, B. Parizot, I. Casimiro, R. Heidstra, M. Laskowski, M. Lepetit, F. Hochholdinger, X. Draye, H. Zhang, M.R. Broadley, B. Peret, J.P. Hammond, H. Fukaki, S. Mooney, J.P. Lynch, P. Nacry, U. Schurr, L. Laplaze, P. Benfey, T. Beeckman, M. Bennett, Analyzing lateral root development: how to move forward, Plant Cell 24 (2012) 15–20. [31] C. Pape, K. Lüning, Quantification of melatonin in phototrophic organisms, J. Pineal Res. 41 (2010) 157–165. [32] Z. Chen, Y. Xie, Q. Gu, G. Zhao, Y. Zhang, W. Cui, S. Xu, R. Wang, W. Shen, The AtrbohF-dependent regulation of ROS signaling is required for melatonin-induced salinity tolerance in Arabidopsis, Free Radic. Biol. Med. 108 (2017) 465–477. [33] Y. Wang, X. Ye, K. Yang, Z. Shi, N. Wang, L. Yang, J. Chen, Characterization, expression, and functional analysis of polyamine oxidases and their role in seleniuminduced hydrogen peroxide production in Brassica rapa, J. Sci. Food Agric. 99 (2019) 4082–4093. [34] J. Chen, W.-H. Wang, F.-H. Wu, C.-Y. You, T.-W. Liu, X.-J. Dong, J.-X. He, H.L. Zheng, Hydrogen sulfide alleviates aluminum toxicity in barley seedlings, Plant Soil 362 (2013) 301–318. [35] W. Lv, L. Yang, C. Xu, Z. Shi, J. Shao, M. Xian, J. Chen, Cadmium disrupts the balance between hydrogen peroxide and superoxide radical by regulating endogenous hydrogen dulfide in the root tip of Brassica rapa, Front. Plant Sci. 8 (2017). [36] H. Wang, S. Taketa, A. Miyao, H. Hirochika, M. Ichii, Isolation of a novel lateralrootless mutant in rice (Oryza sativa L.) with reduced sensitivity to auxin, Plant Sci. 170 (2006) 70–77. [37] X. Ye, T. Ling, Y. Xue, C. Xu, W. Zhou, L. Hu, J. Chen, Z. Shi, Thymol mitigates cadmium stress by regulating glutathione levels and reactive oxygen species homeostasis in tobacco seedlings, Molecules 21 (2016) 1339. [38] R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangradorvegas, The Pfam protein families database: towards a more sustainable future, Nucleic Acids Res. 44 (2016) 279–285. [39] A. Marchlerbauer, M.K. Derbyshire, N.R. Gonzales, S. Lu, F. Chitsaz, L.Y. Geer, R.C. Geer, J. He, M. Gwadz, D.I. Hurwitz, CDD: NCBI's conserved domain database, Nucleic Acids Res. 43 (2015) 222–226. [40] C. Dunand, M. Crèvecoeur, C. Penel, Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases, New Phytol. 174 (2007) 332–341. [41] H. Tsukagoshi, W. Busch, P.N. Benfey, Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root, Cell 143 (2010) 606–616. [42] B. Chen, W. Li, Y. Gao, Z. Chen, W. Zhang, Q. Liu, Z. Chen, J. Liu, Involvement of polyamine oxidase-produced hydrogen peroxide during coleorhiza-limited germination of rice seeds, Front. Plant Sci. 7 (2016) 1219. [43] S.D. Harris, Branching of fungal hyphae: regulation, mechanisms and comparison with other branching systems, Mycologia 100 (2008) 823–832. [44] P. Rangan, R. Subramani, R. Kumar, A.K. Singh, R. Singh, Recent advances in polyamine metabolism and abiotic stress tolerance, BioMed Res. Int. 2014 (2014) 9. [45] P. Fincato, P.N. Moschou, V. Spedaletti, R. Tavazza, R. Angelini, R. Federico, K.A. Roubelakis-Angelakis, P. Tavladoraki, Functional diversity inside the Arabidopsis polyamine oxidase gene family, J. Exp. Bot. 62 (2011) 1155–1168. [46] T. Liu, D.W. Kim, M. Niitsu, S. Maeda, M. Watanabe, Y. Kamio, T. Berberich, T. Kusano, Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifically expressed in anthers, Plant Cell Physiol. 55 (2014) 1110–1122. [47] A. Ahou, D. Martignago, O. Alabdallah, R. Tavazza, P. Stano, A. Macone, M. Pivato, A. Masi, J.L. Rambla, F. Vera-Sirera, R. Angelini, R. Federico, P. Tavladoraki, A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines, J. Exp. Bot. 65 (2014) 1585–1603. [48] T. Liu, D. Wook Kim, M. Niitsu, T. Berberich, T. Kusano, POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5, Plant Signal. Behav. 9 (2014) e29773. [49] R. Wimalasekera, F. Schaarschmidt, R. Angelini, A. Cona, P. Tavladoraki, G.F.E. Scherer, POLYAMINE OXIDASE2 of Arabidopsis contributes to ABA mediated plant developmental processes, Plant Physiol. Biochem. 96 (2015) 231–240. [50] Q. Ke, J. Ye, B. Wang, J. Ren, L. Yin, X. Deng, S. Wang, Melatonin mitigates salt
None. Acknowledgements We are grateful to Dr. Yinghua Ji from Institute for Plant Protection, Jiangsu Academy of Agricultural Sciences for providing tobacco seeds. This work was supported by National Key Research and Development Program of China (2017YFD0201105), National Natural Science Foundation of China (31771705), The Program for Science & Technology Innovation Talents in Universities of Henan Province (18HASTIT037), and The Key Scientific and Technology Projects of Henan Province (182102110446). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.freeradbiomed.2019.09.011. References [1] J.A. Atkinson, A. Rasmussen, R. Traini, U. Voss, C. Sturrock, S.J. Mooney, D.M. Wells, M.J. Bennett, Branching out in roots: uncovering form, function, and regulation, Plant Physiol. 166 (2014) 538–550. [2] P. Yu, C. Gutjahr, C. Li, F. Hochholdinger, Genetic control of lateral root formation in cereals, Trends Plant Sci. 21 (2016) 951–961. [3] B. Péret, B. De Rybel, I. Casimiro, E. Benkova, R. Swarup, L. Laplaze, T. Beeckman, M.J. Bennett, Arabidopsis lateral root development: an emerging story, Trends Plant Sci. 14 (2009) 399–408. [4] J.E. Malamy, P.N. Benfey, Organization and cell differentiation in lateral roots of Arabidopsis thaliana, Development 124 (1997) 33–44. [5] I. De Smet, Lateral root initiation: one step at a time, New Phytol. 193 (2012) 867–873. [6] H. Nakagami, K. Kawamura, K. Sugisaka, M. Sekine, A. Shinmyo, Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco, Plant Cell 14 (2002) 1847–1857. [7] A. Verkest, C. Weinl, D. Inze, L. De Veylder, A. Schnittger, Switching the cell cycle. Kip-related proteins in plant cell cycle control, Plant Physiol. 139 (2005) 1099–1106. [8] H. Ren, A. Santner, J.C.D. Pozo, J.A.H. Murray, M. Estelle, Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated by two different ubiquitin E3 ligases, Plant J. 53 (2008) 705–716. [9] L. Sanz, W. Dewitte, C. Forzani, F. Patell, J. Nieuwland, B. Wen, P. Quelhas, S. De Jager, C. Titmus, A. Campilho, H. Ren, M. Estelle, H. Wang, J. Murray, The Arabidopsis D-type cyclin CYCD2;1 and the inhibitor ICK2/KRP2 modulate auxininduced lateral root formation, Plant Cell 23 (2011) 641–660. [10] T. Fang, J. Li, Z. Cao, M. Chen, W. Shen, L. Huang, Heme oxygenase-1 is involved in sodium hydrosulfide-induced lateral root formation in tomato seedlings, Plant Cell Rep. (2014) 1–10. [11] N. Correa-Aragunde, M. Graziano, C. Chevalier, L. Lamattina, Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato, J. Exp. Bot. 57 (2006) 581–588. [12] Y. Du, B. Scheres, Lateral root formation and the multiple roles of auxin, J. Exp. Bot. 69 (2018) 155–167. [13] H. Fukaki, M. Tasaka, Hormone interactions during lateral root formation, Plant Mol. Biol. 69 (2009) 437–449. [14] J. Gou, S.H. Strauss, C. Tsai, K. Fang, Y. Chen, X. Jiang, V. Busov, Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones, Plant Cell 22 (2010) 623–639. [15] H. Tsukagoshi, Control of root growth and development by reactive oxygen species, Curr. Opin. Plant Biol. 29 (2016) 57–63. [16] S.S. Gill, N. Tuteja, Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiol. Biochem. 48 (2010) 909–930. [17] R. Alcázar, A.F. Tiburcio, Plant polyamines in stress and development: an emerging area of research in plant sciences, Front. Plant Sci. 5 (2014) 319. [18] G.X. Su, W.H. Zhang, Y.L. Liu, Involvement of hydrogen peroxide generated by polyamine oxidative degradation in the development of lateral roots in soybean, J. Integr. Plant Biol. 48 (2006) 426–432. [19] D. Marino, C. Dunand, A. Puppo, N. Pauly, A burst of plant NADPH oxidases, Trends Plant Sci. 17 (2012) 9–15. [20] B. Orman-Ligeza, B. Parizot, R. de Rycke, A. Fernandez, E. Himschoot, F. Van Breusegem, M.J. Bennett, C. Périlleux, T. Beeckman, X. Draye, RBOH-mediated ROS production facilitates lateral root emergence in Arabidopsis, Development 143 (2016) 3328–3339. [21] C. Manzano, M. Pallero-Baena, I. Casimiro, B. De Rybel, B. Orman-Ligeza, G. Van Isterdael, T. Beeckman, X. Draye, P. Casero, J.C. del Pozo, The emerging role of reactive oxygen species signaling during lateral root development, Plant Physiol.
543
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
[51] [52] [53]
[54]
[55] [56]
[57]
[58] S. Bhattacharjee, The language of reactive oxygen species signaling in plants, J. Bot., Le 2012 (2012) 985298. [59] V. Demidchik, S. Shabala, S. Isayenkov, T.A. Cuin, I. Pottosin, Calcium transport across plant membranes: mechanisms and functions, New Phytol. 220 (2018) 49–69. [60] V. Demidchik, ROS-activated ion channels in plants: biophysical characteristics, physiological functions and molecular nature, Int. J. Mol. Sci. 19 (2018) 1263. [61] V. Demidchik, S.N. Shabala, J.M. Davies, Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels, Plant J. 49 (2007) 377–386. [62] Y.J. Li, J. Chen, M. Xian, L.G. Zhou, F.X. Han, L.J. Gan, Z.Q. Shi, In site bioimaging of hydrogen sulfide uncovers its pivotal role in regulating nitric oxide-induced lateral root formation, PLoS One 9 (2014) e90340. [63] S.K. Singh, C.-T. Chien, I.-F. Chang, The Arabidopsis glutamate receptor-like gene GLR3.6 controls root development by repressing the Kip-related protein gene KRP4, J. Exp. Bot. 67 (2016) 1853–1869. [64] R. Hardeland, Melatonin in plants and other phototrophs: advances and gaps concerning the diversity of functions, J. Exp. Bot. 66 (2014) 627–646.
stress in wheat seedlings by modulating polyamine metabolism, Front. Plant Sci. 9 (2018) 914. J. Montiel, M.-K. Arthikala, C. Quinto, Phaseolus vulgaris RbohB functions in lateral root development, Plant Signal. Behav. 8 (2013) e22694. M. Sagi, R. Fluhr, Production of reactive oxygen species by plant NADPH oxidases, Plant Physiol. 141 (2006) 336–340. K. Gémes, Y.J. Kim, K.Y. Park, P.N. Moschou, E. Andronis, C. Valassaki, A. Roussis, K.A. Roubelakis-Angelakis, An NADPH-oxidase/polyamine oxidase feedback loop controls oxidative burst under salinity, Plant Physiol. 172 (2016) 1418–1431. T. Karuppanapandian, M. Juncheol, K. Changsoo, K. Manoharan, K. Wook, Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms, Aust. J. Crop. Sci. 5 (2011) 709–725. M.K. Kanwar, J. Yu, J. Zhou, Phytomelatonin: recent advances and future prospects, J. Pineal Res. 65 (2018) e12526. R.J. Reiter, S. Rosales-Corral, D.X. Tan, M.J. Jou, A. Galano, B. Xu, Melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas, Cell. Mol. Life Sci. 74 (2017) 3863–3881. D.-X. Tan, R.J. Reiter, Mitochondria: the birth place, battle ground and the site of melatonin metabolism in cells, Melatonin Res 2 (2019) 44–46.
544
Contents lists available at ScienceDirect
Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed
Original article
Melatonin facilitates lateral root development by coordinating PAO-derived hydrogen peroxide and Rboh-derived superoxide radical
T
Jian Chena,∗,1, Hui Lia,1, Kang Yanga, Yongzhu Wanga, Lifei Yangb, Liangbin Huc, Ruixian Liud, Zhiqi Shia,∗∗ a
Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China c Department of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China d Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hydrogen peroxide Lateral root Melatonin Polyamine oxidase RBOH Reactive oxygen species Superoxide radical
Melatonin, a phytochemical, can regulate lateral root (LR) formation, but the downstream signaling of melatonin remains elusive. Here we investigated the roles of hydrogen peroxide (H2O2) and superoxide radical (O2•‾) in melatonin-promoted LR formation in tomato (Solanum lycopersicum) roots by using physiological, histochemical, bioinformatic, and biochemical approaches. The increase in endogenous melatonin level stimulated reactive oxygen species (ROS)-dependent development of lateral root primordia (LRP) and LR. Melatonin promoted LRP/ LR formation and modulated the expression of cell cycle genes (SlCDKA1, SlCYCD3;1, and SlKRP2) by stimulating polyamine oxidase (PAO)-dependent H2O2 production and respiratory burst oxidase homologue (Rboh)dependent O2•‾ production, respectively. Screening of SlPAOs and SlRbohs gene family combined with gene expression analysis suggested that melatonin-promoted LR formation was correlated to the upregulation of SlPAO1, SlRboh3, and SlRboh4 in LR-emerging zone. Transient expression analysis confirmed that SlPAO1 was able to produce H2O2 while SlRboh3 and SlRboh4 were capable of producing O2•‾. Melatonin-ROS signaling cassette was also found in the regulation of LR formation in rice root and lateral hyphal branching in fungi. These results suggested that SlPAO1-H2O2 and SlRboh3/4-O2•‾ acted as downstream of melatonin to regulate the expression of cell cycle genes, resulting in LRP initiation and LR development. Such findings uncover one of the regulatory pathways for melatonin-regulated LR formation, which extends our knowledge for melatonin-regulated plant intrinsic physiology.
1. Introduction Root branching resulted from LR (lateral root) development is vital for plants acquiring water and nutrients [1,2]. LR development initiates from the emergence and development of LRP (lateral root primordium) along the longitudinal axis of PR (primary root). LRP emergence is a dynamic process that involves several stages (I-VIII), starting from continuous division of different cell layers to just about emergence of LRP from parent PR [3,4]. This process is genetically controlled by cell cycle regulatory genes, such as CYCs (cyclins), CDKs (cyclin dependent kinases), and KRPs (Kip-related proteins). CYCD and CDKA are positive regulators for LR initiation [5,6]. KRPs, suppressors of CDK, are negative regulators for LR development [7–9]. These cell cycle regulators
(e.g. CYCD3;1, CDKA1, and KRP2) are always considered as marker genes to indicate LR initiation [10,11]. Auxin plays crucial roles in the regulation of cell cycle process and LR formation by integrating other plant hormones [12–14]. Apart from hormones, ROS (reactive oxygen species) have been considering as emerging regulators for LR development [15]. H2O2 (hydrogen peroxide) and O2•‾(superoxide radical) are two typical ROS, belonging to non-radical and free radical forms, respectively [16]. PAO (polyamine oxidase) catalyzes the metabolism of PAs (polyamines), including Spd (spermidine) and Spm (spermine), along with H2O2 production, which has been considered as an important biosynthetic pathway of H2O2 in plants [17]. PAO-dependent H2O2 generation contributes to LR development in soybean [18]. Rbohs
∗
Corresponding author. Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing, 210014, China. Corresponding author. Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing, 210014, China. E-mail addresses: [email protected] (J. Chen), [email protected] (Z. Shi). 1 These authors contribute equally to this work. ∗∗
https://doi.org/10.1016/j.freeradbiomed.2019.09.011 Received 19 August 2019; Received in revised form 10 September 2019; Accepted 10 September 2019 Available online 11 September 2019 0891-5849/ © 2019 Elsevier Inc. All rights reserved.
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
2.4. Determination of the content of H2O2 and O2•‾
(respiratory burst oxidase homologues) are important enzymes to produce O2•‾ for the regulation of plant physiology [19]. Rboh-derived ROS can facilitate LR emergence in Arabidopsis thaliana [20]. ROS facilitates LR development in either auxin-dependent or auxin-independent manners [20,21]. Melatonin, an important immune regulator in mammalian cells, has been proposed as a novel phytohormone with multiple physiological functions [22]. Melatonin-promoted LR formation has been found in several plant species, such as Lupinus albus [23], Arabidopsis [24], rice [25], and cucumber [26,27]. Auxin acts as a regulatory star for LR development, but melatonin facilitates LR development likely acting independently of auxin signaling in Arabidopsis [24]. Melatonin activates auxin-related genes during LR development in rice, but not in cucumber [25,26]. Thus, the detailed mechanism for melatonin-facilitated LR development remains elusive. Recent studies have implied that ROS signaling maybe involved in melatonin-induced LR development [26,28]. However, whether and how melatonin regulates ROS generation (including both H2O2 and O2•‾) to activate LR development remains unclear. In this work, we investigated the roles of PAO-generated H2O2 and Rbohs-generated O2•‾ during melatonin-facilitated LR development in tomato (Solanum lycopersicum) seedlings. Melatonin-ROS interaction cassette was also confirmed in LR formation in rice roots (monocot) and hyphal branching in fungi. This study may help reveal one of the important molecular mechanisms for melatonin-induced LR development.
Root tip without LR formation was removed from the entire root. The rest of root with branching sites was collected immediately for the measurement of ROS content (Fig. S1). H2O2 content was determined by using a commercial H2O2 assay kit (A064-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to our previously published method [33]. O2•‾ content was measured based on the hydroxylamine oxidation method described by Chen et al. [34]. 2.5. Histochemical analysis Intracellular H2O2 and O2•‾ in root were detected in situ using specific fluorescent probe HPF and DHE, respectively, based on our published method [35]. LRP were stained with methylene blue and observed under microscope as described by Wang et al. [36]. H2O2 and O2•‾ in tobacco leaves were detected histochemically by staining with DAB (3,3-diaminobenzidine) and NBT (nitro-blue tetrazolium), respectively, according to our published methods [37]. 2.6. Genomewide identification of SlPAOs and SlRbohs The protein sequences of PAOs in A. thaliana were obtained from TAIR (The Arabidopsis Information Resource). These sequences were used as baits for searching SlPAO homologues from Sol Genomics Network (SL2.50) by using BLAST (Basic Local Alignment Search Tool). The obtained SlPAOs were further confirmed by using Pfam database and NCBI Conserved Domain Database [38,39]. Genomewide identification of SlRbohs was performed by using the same strategy as SlPAOs. Then a set of tools were used to analyze the characteristics of the obtained sequences, which was demonstrated in detail in Supporting Methods.
2. Materials and methods 2.1. Plant culture and reagents Tomato (S. lycopersicum) seeds were surface-sterilized with 1% NaClO for 10 min and washed with distilled water for three times, followed by germination on a floating plastic net in darkness. The germinated seeds were transferred to 1/4-strength Hoagland nutrition [29]. After growing for 48 h, the identical seedlings with root length of 1.5 cm were selected and transferred to another container with treatment solution. The seedlings were placed in an incubator with photosynthetic active radiation of 200 μmol/m2/s, photoperiod of 16/8 h, and temperature at 25 ± 1 °C. Melatonin was added to treatment solution at concentrations of 50–600 μM. KI (potassium iodide) was applied as ROS scavenger. H2O2 was used as exogenous ROS donor. DPI (diphenylene iodonium), PY (pyridine), and IMZ (imidazole) were used as RBOH inhibitors. MDL (1,4-diamine dihydrochloride), GZT (guazatine acetate), and 2-HEH (2hydroxyethylhydrazine) were used as PAO inhibitors. All the above reagents and other ordinary chemicals were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China). DHE (dihydroethidium) and HPF (hydroxyphenyl fluorescein), used as fluorescent probes for the detection of endogenous O2•‾ and H2O2, respectively, were obtained from Beyotime Biotechnology (Shanghai, China).
2.7. Gene expression analysis Gene expression analysis was performed using qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction). The detailed procedure was described in Supporting Methods. Oligopeptide primers were listed in Table S1. 2.8. Transient expression of SlPAO1, SlRboh3, and SlRboh4 in tobacco leaves Transient expression of tomato genes in tobacco leaves was performed according to our previous method [33]. Primers with specific adaptors containing SamI restriction enzyme site (Table S2) were used to amplify the full length cDNA of designated tomato gene, which was further cloned into pCAMBIA-2300 vector. Then the recombined vector was transformed into Agrobacterium tumefaciens strain GV3101, which was further infiltrated into tobacco (Nicotiana benthamiana) leaves. Target genes in tobacco leaves were detected at DNA and mRNA level as described in Supporting Methods.
2.2. Measurements of LR development LR formation was evaluated based on several typical parameters, such as LR number, LR density (total LR number/PR length), LRP number, and LRP density (total LRP number/PR length) [30]. LRP development was evaluated by counting LRP at different developing stages (I-VIII) under microscopy [3].
2.9. Statistical analysis Each data was showed as mean ± standard deviation (SD) for at least three replicates. SD and ANOVA (one-way analysis of variance) were used to evaluate the significant differences between treatments by using SPSS 6.0 (Statistical Package for the Social Science, SPSS Inc., Chicago, IL, USA). For two designated treatments, the data were statistically compared by ANOVA, followed by F-test if the ANOVA result is significant at P < 0.5 or P < 0.1. For multiple comparison, least significant difference test (LSD) was used to test significance at P < 0.5 among any different treatments based on ANOVA and F-test.
2.3. Determination of melatonin content Melatonin in root samples was extracted using extraction buffer (acetone:methanol:water = 89:10:1) [31]. A plant melatonin ELISA (enzyme-linked immunosorbent assay) detection kit (Jiangu Baolai Biotechnology, Yancheng, China) was applied to determine melatonin content according to manufacturer's instructions [32]. 535
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 1. Melatonin promoted LR formation in tomato roots. Tomato roots were treated with melatonin at different concentrations (0–600 μM) for 4 days, followed by photographing phenotype (A), counting LR number (B), and determining LR density (C). Tomato roots were treated with melatonin at 200 μM for 1, 2, 3, 4, 5, and 6 days, respectively, followed by the determination of LR number (D) and LR density (E). Tomato roots were treated with melatonin at different concentrations (0–600 μM) for 5 days, followed by the determination of endogenous melatonin content in roots (F). Different lowercase letters in (B, C, F) indicated that the mean values of ten replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD). Two asterisks (**) in (D and E) indicated that the mean values of ten replicates were significantly different between control and melatonin treatment at each time point (P < 0.01, ANOVA).
3. Results 3.1. Melatonin promoted the development of LR and LRP Melatonin induced significant increases in LR number and LR density in dose-dependent manners. Melatonin at 200 μM exhibited the greatest effect (Fig. 1A–C). The continuous increases in LR number and LR density were also observed under treatment with 200 μM of melatonin in time-course experiments (Fig. 1D and E). Exogenous treatment with melatonin resulted in remarkable increase in endogenous melatonin level in root. Treatment with melatonin at 200 μM resulted in the highest level of endogenous melatonin in root (Fig. 1F). The remarkable increases in total LRP number and LRP density were observed after melatonin treatment, with maximal enhancement occurring at 200 μM of melatonin (Fig. 2A and B). In addition, melatonin stimulates LRP initiation and development as observed from LRP at different stages (Fig. 2C and D). These results suggested that exogenous melatonin treatment enhanced endogenous melatonin level, further promoting LRP initiation and accelerating LRP development to form more LR.
3.2. ROS was involved in melatonin-promoted LR formation Fig. 2. Melatonin promoted LRP formation in tomato roots. Tomato roots were treated with melatonin at different concentrations (0–600 μM) for 2 days, followed by counting the number of total LRP (A) and determining LRP density (B). Tomato roots were treated with melatonin at 200 μM for 2 days followed by counting the number of LRP at different developmental stages (C) and determining LRP proportion (D). Different lowercase letters in (A and B) indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD). One asterisk (*) and two asterisks (**) in (C) indicate that the mean values of six replicates were significantly different between control and melatonin treatment at P < 0.05 and P < 0.01, respectively.
Accumulation of O2•‾ and H2O2, detected in situ by DHE and HPF, respectively, were observed clearly in the sites where LRP emerged during the whole process of LRP formation (Fig. 3A and B). H2O2 treatment significantly stimulated the formation of LRP and LR, whereas treatment with ROS scavenger KI showed opposite effects (Fig. 3D–F). These results suggested that ROS was involved in the regulation of LR development. Then we found that KI compromised the promoting effect of melatonin on LR formation (Fig. 4A and B). Root tip without branch sites mainly functions for the regulation of the apical growth of PR. ROS has been found to be accumulated in root tip to control PR elongation [40,41]. Therefore, melatonin-treated roots without root tips were collected for the detection of ROS level in order to avoid the interference of ROS in root tips on the final results (Fig. S1). Melatonin significantly enhanced H2O2 and O2•‾ level in roots (Fig. 4C and D). These results suggested that H2O2 and O2•‾ might mediate the promoting effect of melatonin on LR development.
3.3. Melatonin promoted LR development by regulating Rboh-dependent O2•‾ generation and PAO-dependent H2O2 generation RBOH inhibitors (IMZ, PY, or DPI) significantly compromised the promoting effect of melatonin on LR, LRD, LRP, and LRPD (Fig. 5A–E), accompanying with significant decrease in O2•‾ content in melatonintreated root (Fig. 5F and Fig. S1). Similarly, application of PAO 536
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 3. Involvement of ROS in the formation of LRP and LR in tomato roots. (A) DHE-labelled O2•‾ in LRP at different developmental stages. (B) HPF-labelled H2O2 in LRP at different developmental stages. Tomato roots were treated with water (Control), H2O2 (1.2 mM), or KI (400 μM) for 2 days followed by the quantification of LRP number (C) and LRP density (D). Tomato roots were treated with water (Control), H2O2 (1.2 mM), or KI (400 μM) for 4 days followed by the quantification of LR number (E) and LR density (F). Different lowercase letters in (C-F) indicated that the mean values of seven replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
study the possible link between LR development and the expression levels of SlRboh2-5. More LRP were detected in part I than in part II (Fig. 9B), coinciding with similar patterns for the expression levels of SlRboh3 and SlRboh4, but not SlRboh2 and SlRboh5 (Fig. 9C–F). Intriguingly, melatonin induced similar changing patterns of LRP and the expression of SlRboh3 and SlRboh4 in different parts of root (Fig. 9G–I). These results suggested that the upregulation of SlRboh3 and SlRboh4 might be involved in melatonin-induced LRP formation.
inhibitors (MDL, GZT, or 2-HLH) inhibited the formation of LRP and LR (Fig. 6A–E) as well as H2O2 generation in melatonin-treated root (Fig. 6F and Fig. S1). Compared to control, treatment with DPI or 2HEH alone prohibited the formation of LR and LRP (Fig. S2). Melatonin upregulated the expression of SlCDKA1 and SlCYCD3;1 and downregulated the expression of SlKRP2. The effects of melatonin on these cell cycle genes were reversed by the addition of DPI or 2-HLH (Fig. 7). These results suggested that RBOH-dependent O2•‾ and PAO-dependent H2O2 contributed to melatonin-promoted LR development.
3.5. Identification of melatonin-targeted SlPAO during LRP development 3.4. Identification of melatonin-targeted SlRbohs during LRP development A total of eleven potential SlPAOs with typical amnio_acid domain were obtained from tomato genome (Table S5; Fig. S3; Fig. S5A). SlPAO9-11 (Solyc07g063450.1, Solyc07g063500.2, and Solyc04g081100.2) belonging to subfamily III had another SWIRM domain besides of amnio_acid domain (Fig. 10A; Fig. S7A). Thus SlPAO911 were characterized as histone lysine-specific demethylase rather than real PAO based on the classification of their homologues in rice (OsPAO9-11) and Arabidopsis (AtPAO6-9) (Fig. 10A) [42]. SlPAO1-8 were considered as typical PAOs that were distributed in three subfamilies (I, IIa, IIb) (Fig. 10A; Figs. S5B–C; Table S6). According to the identification of OsPAOs and AtPAOs in subfamily I [42], SlPAO1 with predicated apoplastic localization was supposed to catalyze the terminal catabolism of PAs. Subfamily IIa (SlPAO6-8) and IIb (SlPAO2-5) with predicated subcellular localization of cytoplasm and peroxisome, respectively, were supposed to catalyze the back-conversion of PAs (Fig. 10A; Table S5). These results suggested that SlRboh1-8 were PAO
A total of seven SlRboh genes with various chromosomal distributions were retrieved from tomato genome (Table S3; Fig. S3). All the SlRbohs were transmembrane proteins with typical conserved domains (Fig. S4A; Table S3). SlRbohs were found in subfamily I, II, III, and V, but not in subfamily IV, based on phylogenetic analysis, motif distribution, and gene structure diversity (Fig. 8A; Figs. S4B and C; Table S4). These results suggested that SlRboh1-7 were Rboh homologues with potential capability to produce O2•‾. Then we measured the effect of melatonin on the expression of SlRbohs in root sections with branch sites (Fig. S1). SlRboh2-5 showed significantly increased expression levels at 24 h under melatonin treatment (Fig. 8B). In time-course experiments, SlRboh3 and SlRboh4, but not SlRboh2 and SlRboh5, displayed enhanced expression levels, as compared to control groups (Fig. 8C–F). The different parts of root (part I and II in Fig. 9A) were harvested to 537
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
expression level of SlPAO1, but not SlPAO4, was correlated to LRP development (Fig. 10E and F; Fig. 9B). The expression level of SlPAO1 was positively correlated to LRP development in different parts of root under melatonin treatment (Fig. 10G; Fig. 9G). Thus the upregulation of SlPAO1 may be associated to melatonin-induced LRP formation. 3.6. Identification of the capability of BrRbohs3/4 and BrPAO1 in ROS production The capabilities of BrRbohs3, BrRbohs4 and BrPAO1 in ROS production were evaluated by using transient expression analysis (Fig. S6; Fig. 11A–C). DAB staining suggested that tobacco leaves expressing BrPAO1 showed higher H2O2 level than control or EV (empty vector) (Fig. 11D). NBT staining indicated that tobacco leaves with the expression of BrRbohs3 or BrRbohs4 showed remarkable increase in O2•‾ level (Fig. 11E). These results suggested that BrRbohs3 and BrRbohs4 had the ability to generate O2•‾ while BrPAO1 was able to produce H2O2. 3.7. Melatonin/ROS was involved in LR development in dicot and hyphal branching in fungi Since melatonin promoted ROS-dependent LR development in dicot (tomato), we wondered whether it acted similarly in monocot (e.g. rice). Melatonin promoted the formation of LR and LRP in the seminal root of rice seedlings, which was suppressed by the addition of KI (Fig. S7). ROS is also one of the important factors modulating hyphal branching, which is similar to root branching in plants [43]. Fusarium graminearum is a kind of model filamentous ascomycete in agriculture. Melatonin treatment stimulated the formation of lateral branches in the hyphae of F. graminearum, which could be inhibited by KI as well (Fig. S8). These results suggested that ROS might act as downstream of melatonin in the regulation of LR formation in monocot and lateral branching in fungal hyphal.
Fig. 4. Involvement of ROS in melatonin-promoted LR formation in tomato roots. Tomato roots were treated with water (Control), melatonin (200 μM), and melatonin (200 μM) + KI (400 μM) for 4 days, respectively, followed by photographing phenotype (A), counting LR number (B). Tomato roots were treated with melatonin at 200 μM for 12, 24, 36, 48, and 72 h, respectively, followed by the determination of H2O2 content (C) and O2•‾ content (D) in root sections with lateral branching sites. Different lowercase letters in (B) indicated that the mean values of ten replicates are significantly different among different treatments (P < 0.05, ANOVA, LSD). One asterisk (*) and two asterisks (**) in (C and D) indicated that the mean values of three replicates were significantly different between control and melatonin treatment at each time point at P < 0.05 and P < 0.01, respectively.
homologues with potential capability to produce H2O2 by metabolizing PAs. SlPAO1 and SlPAO4 showed the highest expression level in root sections with branch sites after melatonin treatment for 24 h (Fig. 10B). Compared to the control, SlPAO1, but not SlPAO4, showed enhanced expression levels in time-course measurements (Fig. 10C and D). The
4. Discussion 4.1. Melatonin promoted LR formation through ROS-dependent pathway Melatonin-induced increase in LR number resulted from the stimulation of LRP emergence. Auxin is a key regulator for LRP development, Fig. 5. Involvement of Rboh-dependent O2•‾ in melatonin-induced formation of LR and LRP in tomato roots. Tomato roots were treated with water (Control), melatonin (200 μM), melatonin (200 μM) + IMZ (400 μM), melatonin (300 μM) + PY (500 μM), and melatonin (200 μM) + DPI (200 μM) for 4 days, respectively, followed by photographing phenotype (A), counting LR number (B), and determining LR density (C). Tomato roots were treated with same solution mentioned above for 2 days, followed by counting LRP number (D), determining LRP density (E), and measuring O2•‾ content (LRP emerging zone) (F). Different lowercase letters in (B-F) indicated that the mean values of 3–10 replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
538
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 6. Involvement of PAO-dependent H2O2 in melatonin-induced formation of LR and LRP in tomato roots. Tomato roots were treated with water (Control), melatonin (200 μM), melatonin (200 μM) + MDL (1 μM), melatonin (300 μM) + GZT (1 μM), and melatonin (200 μM) + 2-HEH (0.4 μM) for 4 days, respectively, followed by photographing phenotype (A), counting LR number (B), and determining LR density (C). Tomato roots were treated with same solution mentioned above for 2 days, followed by counting LRP number (D), determining LRP density (E), and measuring H2O2 content (LRP emerging zone) (F). Different lowercase letters in (B-F) indicated that the mean values of 3–10 replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
but melatonin promotes LR formation likely acting independently of auxin signaling [24,26]. ROS functions as signaling molecules to regulate LR formation, paralleling to auxin signaling [21]. Therefore, we wandered whether melatonin regulated LR formation through ROS signaling. Indeed, we found that ROS located in LRP sites is required for LRP emergence in tomato root, in agreement with previous reports in the root of Arabidopsis [21]. Melatonin enhanced ROS level while the suppression of ROS level counteracted the promoting effect of melatonin on LR formation. Thus we revealed that melatonin promoted LR formation through ROS-dependent pathway in the root of tomato seedlings. LRP initiation started from the founder cell specification followed by cell division in PR [5]. LRP formation and development in Arabidopsis can be controlled by either auxin signaling or ROS signaling, likely through independent pathways [21]. Melatonin regulated the expression of cycle genes (SlCDKA1, SlCYCD3;1, and SlKRP2) in a ROS-dependent manner. Therefore, we suggest that melatonin triggers ROS accumulation in roots, which further enables the activation of cell division to start LRP formation program. Establishing the link between melatonin and ROS provides possible explanation for the fact that melatonin-promoted LR formation is auxin-independent in dicots. In monocot rice, transcriptome analysis suggests a possible link between auxin signaling responses and root architecture modulation (including LR formation) under melatonin treatment [25]. In the present study, we found that melatonin promoted rice LR formation was also ROS-dependent. Therefore, it seems that both auxin and ROS might be involved in melatonin-facilitated LR formation in monocot. Further studies are needed to compare the detailed downstream networks of melatonin in the regulation of LR formation between dicot and monocot. In Arabidopsis, peroxidase-modulated ROS level is important for LR development [21]. In addition, the regulation of the expression of several peroxidase genes has been found during melatonin-promoted LR formation in cucumber [26]. However, how melatonin activates ROS generation to regulate LR formation remains elusive. Here we identified that PAO and RBOH might be involved in the generation of H2O2 and O2•‾, respectively, during melatonin-promoted LR formation.
Fig. 7. RBOH inhibitor (DPI) or PAO inhibitor (2-HEH) affected the expression of cell cycle regulatory genes (SlCDKA1, SlCYCD3;1, and SlKRP2) in melatonin-treated roots. (A) SlCDKA1. (B) SlCDKA1YCD. Tomato roots were treated with water (Control), melatonin (200 μM), melatonin (300 μM) + DPI (200 μM), and melatonin (200 μM) + 2-HEH (0.4 μM), respectively, for 2 days. Then root sections with lateral branching sites were harvested for RNA extraction and gene expression analysis. Different lowercase letters indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
4.2. Melatonin promoted LR formation through SlPAO1-dependent H2O2 generation PAO metabolize various PAs with H2O2 as byproduct [44]. H2O2 539
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 8. Phylogenetic analysis of SlRboh family and melatonin-regulated expression of SlRbohs in tomato roots. (A) Neighbor-joining phylogenetic tree of the SlRbohs, A. thaliana Rbohs (AtRbohA-J), and rice Rbohs (OsRboh1-9). (B) The relative expression level of SlRboh1-7 in tomato root sections with branching sites after treatment with melatonin (200 μM) for 24 h. Tomato roots were treated with melatonin at 200 μM for 12, 24, 36, 48, and 72 h, respectively. Root sections with lateral branching sites were harvested for RNA extraction and the analysis of relative expression level of SlRboh2 (C), SlRboh3 (D), SlRboh4 (E), and SlRboh5 (F). One asterisk (*) and two asterisks (**) in (B-F) indicated that the mean values of three replicates were significantly different between control and melatonin treatment at P < 0.05 and P < 0.01, respectively.
Fig. 9. Involvement of the expression of SlRboh3 and SlRboh4 in melatonin-promoted LRP formation. (A) Schematic presentation for sampling section in the root of tomato seedling. Tomato seedlings were allowed to grow for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for counting LRP number (B) and analyzing the relative expression level of SlRboh2-5 (C-F). Tomato roots were treated with melatonin (200 μM) for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for counting LRP number (G) and analyzing the expression level of SlRboh3-4 (H-I). Different lowercase letters in (B-I) indicated that the mean values of 3–10 replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
540
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Fig. 10. Phylogenetic analysis of SlPAO family and the effect of melatonin on the expression of SlPAOs in tomato roots. (A) Neighbor-joining phylogenetic tree of the SlPAO family. (B) The relative expression level of SlPAO1-8 in tomato root sections with branching sites after treatment with melatonin (200 μM) for 24 h. Tomato roots were treated with melatonin at 200 μM for 12, 24, 36, 48, and 72 h, respectively. Root sections with lateral branching sites were harvested for RNA extraction and the analysis of relative expression level of SlPAO1 (C), and SlPAO4 (D). Tomato seedlings were allowed to grow for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for the analysis of relative expression level of SlPAO1 (E) and SlPAO4 (F). Tomato roots were treated with melatonin (200 μM) for 24 and 36 h, respectively. Root sections in different parts (I and II) were harvested, respectively, for the analysis of relative expression level of SlPAO1 (G). One asterisk (*) and two asterisks (**) in (B-D) indicated that the mean values of three replicates were significantly different between control and melatonin treatment at P < 0.05 and P < 0.01, respectively. Different lowercase letters in (E-G) indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
molecule to regulate various physiological processes. Melatonin can protect plant cells from stress conditions by suppressing PAO-dependent over-generation of H2O2 [50]. However, our results suggest that melatonin triggers SlPAO1-dependent H2O2 generation to facilitate LR formation under normal growth conditions. It will be interesting to investigate the role of melatonin-maintained PAO-H2O2 homeostasis in growth regulation and stress responses.
produced by PAO acts as signaling molecule to regulate multiple plant physiological processes, including LR formation [17,18]. Melatonin promotes LR and LRP development through PAO-dependent H2O2 production in tomato root. Eight typical SlPAO family genes consisted of three subfamilies with two types of predicted function (type I, terminal catabolism of PAs; type II, back-conversion of PAs). SlPAO1, AtPAO1, and OsPAO7 were grouped together in subfamily I. AtPAO1 and OsPAO7 are localized in cytoplasm and apoplast, respectively, belonging to type I PAO functioning for the terminal catabolism of Spm [45,46]. Therefore, SlPAO1 with predicted cytosolic localization may have similar functions to AtPAO1 and OsPAO7. SlPAO6-8 in subfamily IIa were proposed to work on the back-conversion of PAs (Spm→Spd) according to the already identified function of their homologues (AtPAO5 and OsPAO1) [47,48]. In subfamily IIb, SlPAO2-5 with predicted peroxisomal localization may also work on the back-conversion of PAs because their homologues AtPAO2/3 are two peroxisomal proteins that can oxidize both Spd and Spm through PA back-conversion pathway [45]. Collectively, it can be proposed that SlPAO1-8 may have potential to produce H2O2 by taking Spd or Spm as substrate to facilitate PAs metabolism. Melatonin induced the most significant increase in the expression of SlPAO1 and SlPAO4 in LR-emerging zone. AtPAO2, a homologue of SlPAO4, positively regulates LR growth [49], but only the expression of SlPAO1 was correlated to melatonin-induced LRP formation. Transient expression analysis further confirmed the capability of SlPAO1 in H2O2 production. Having linked SlPAO1 melatonin sensing and LRP formation into a regulatory module, this finding adds a critical piece of information to complete melatonin-H2O2 signaling for the regulation of LR development. High level of H2O2 poses threat to plant cells by inducing oxidative injury, but H2O2 at moderate level acts as a signaling
4.3. Melatonin promoted LR formation through SlRboh3-and SlRboh4dependent O2•‾ generation Rboh-derived O2•‾ plays vital roles in the regulation of plant development [19]. In the present study, Rboh-dependent O2•‾ contributed to melatonin-induced development of LRP and LR. SlRboh3 and SlRboh4 were able to generate O2•‾, which was closely linked to melatonin-induced formation of LRP. SlRboh3 and AtRbohB were found in the same clade. Genetic evidences demonstrate that PvRbohB (Phaseolus vulgaris RbohB), a functional homologue of AtRbohB, contributes to O2•‾ accumulation for LR development [51]. SlRboh4 and AtRbohE belong to the same clade. AtRbohE has been found to be expressed inside of the LRP and in the overlaying cells of epidermis. AtRbohE-derived extracellular O2•‾ not only modulates LRP initiation but also regulates cell wall remodeling to help LRP emergence [20]. Treatment with DPI compromised melatonin-induced O2•‾ production and expression of cell cycle genes, suggesting that melatonin-modulated Rboh/O2•‾ probably regulates LRP initiation. Rboh-generated O2•‾ can be utilized by peroxidase to produce H2O2 to facilitate LR formation [20,52]. The expressions of a set of peroxidase genes have been found to be upregulated during melatonin-promoted LR formation in cucumber [26]. Therefore, melatonin-Rboh-peroxidase signaling cascade is probably 541
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
antioxidant to protect mitochondria from ROS attack and oxidative stress in both plants and animals [55–57]. Melatonin can also be synthesized in chloroplast in plant cells, which help to scavenge ROS generated in chloroplast [16,55]. Here we found that melatonin promoted PAO- and Rboh-dependent ROS generation to induce LR development. Both PAO and Rboh are not localized to either mitochondria or chloroplast. Thus PAO-derived H2O2 and Rboh-derived O2•‾ induced by melatonin may act as signaling molecules, rather than oxidative attackers, to trigger LR development under normal growth conditions [21]. However, we cannot exclude the possibility that melatonin may regulate ROS level in different organelles in different ways, which needs to be studied in the future. 4.6. Possible regulators involved in melatonin-ROS cassette during LR development ROS are multifunctional signals that can regulate many signaling molecules [58]. In plant roots, H2O2 is able to facilitate Ca2+ (calcium) influx into cytosol by activating plasma membrane-located Ca2+ channel at either extracellular or intracellular membrane face [59–61]. And Ca2+ has been suggested as an important signal to regulate LR development by modulating cell cycle genes at transcriptional and posttranslational level [62,63]. The probable interaction between melatonin and Ca2+ has been found in plants [64]. Combining with the results of melatonin-ROS interplay obtained in this study, it could be speculated that Ca2+ might act downstream of ROS during melatoninregulated cell cycle genes and LR development.
Fig. 11. Assessment of the capability of SlPAO1, SlRboh3, and SlRboh4 in ROS production with transient expression analysis. (A-C) Relative expression level of SlPAO1 (A), SlRboh3 (B), and SlRboh4 (C) in tobacco leaves. (D) Detection of endogenous H2O2 with DAB staining in tobacco leaves expressing SlPAO1. (E) Detection of endogenous O2•‾ with NBT staining in tobacco leaves expressing SlRboh3 and SlRboh4. Contorl indicated infiltration with buffer. EV indicated infiltration with empty vector without carrying any tomato genes. Different lowercase letters in (A-C) indicated that the mean values of three replicates were significantly different among different treatments (P < 0.05, ANOVA, LSD).
5. Conclusion Taken together, we propose a model for ROS generation during melatonin-induced LR formation in tomato roots (Fig. 12). Melatonin stimulates the expression of SlPAO1 to increase apoplastic H2O2 generation. Melatonin stimulates the expression of SlRboh3/4 to increase apoplastic O2•‾ generation. Then both H2O2 and O2•‾ enter into cytosol and/or nucleus and trigger signaling transduction, which further modulate the expression of cell cycle genes to initiate LRP development and LR formation. In addition, O2•‾ can be transformed to H2O2 that may activate Ca2+ channel to promote Ca2+ influx into cytosol. Then the increase in intracellular Ca2+ probably regulates cell cycle genes to stimulate LR development. However, the probable involvement of specific Ca2+ channels in this signaling cassette needs to be identified in future studies.
involved in the cell wall modification to facilitate LR formation, which needs to be elucidated further. 4.4. Possible role of PAO-Rboh cross-talk in ROS generation during melatonin-promoted LR formation PAO and Rboh contribute to the generation of H2O2 and O2•‾, respectively, but the cross-talk between PAO and Rboh can also occurred. In tobacco, Rboh can act as either upstream or downstream of apoplastic PAO (ZmPAO1 and NtPAO1) in order to amplify ROS generation cells upon salinity stress [53]. SlPAO1 was predicted to be an apoplastic protein. SlPAO1, ZmPAO1, and NtPAO1 were found in the same clade (Sub.I in Fig. S9), suggesting that these three proteins might have similar physiological functions. Therefore, it would be interesting to further investigate whether melatonin could trigger a PAO/Rboh feedback loop to generate ROS for LR regulation. 4.5. Balanced interaction between melatonin and ROS Abiotic stress conditions always cause over-generation of ROS in various plant organelles (e.g. mitochondrion, chloroplast, apoplast, and peroxisome), accompanied with inhibition of root growth. In these cases, the overall increased ROS in the whole cell tends to cause oxidative injury and cell death, with compromised role in signaling transduction [16,54]. Melatonin has been reported as a scavenger of excessive ROS in both plants and animals under stress conditions [55,56]. In the current study, melatonin could trigger ROS signaling to regulate cell cycle genes and LR development, which may be organellespecific effects. Mitochondrion is one of the major sites of ROS production due to ETC (electron transport chain) disturbance in cells upon stress [21]. Melatonin, synthesized in mitochondria, act as an important
Fig. 12. Schematic model for melatonin-promoted LR formation by coordinating ROS generation in tomato root. Melatonin induces SlRboh3/4derived O2•‾ generation and SlPAO1-derived H2O2 generation in apoplast. O2•‾ and H2O2 triggers signaling transduction to regulate the expression of cell cycle genes, which further stimulates LRP initiation and LRP formation. Ca2+ is a probable downstream signal of ROS to regulate cell cycle genes during melatonin-promoted LR development. 542
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
Declarations of interest
165 (2014) 1105–1119. [22] R. Hardeland, Melatonin in plants – diversity of levels and multiplicity of functions, Front. Plant Sci. 7 (2016) 198. [23] M.B. Arnao, n.-R.J. Hernã, Melatonin promotes adventitious- and lateral root regeneration in etiolated hypocotyls of Lupinus albus L, J. Pineal Res. 42 (2010) 147–152. [24] R. Pelagioflores, E. Muñozparra, R. Ortizcastro, J. Lópezbucio, Melatonin regulates Arabidopsis root system architecture likely acting independently of auxin signaling, J. Pineal Res. 53 (2012) 279–288. [25] C. Liang, A. Li, H. Yu, W. Li, C. Liang, S. Guo, R. Zhang, C. Chu, Melatonin regulates root architecture by modulating auxin response in rice, Front. Plant Sci. 8 (2017). [26] N. Zhang, H.J. Zhang, B. Zhao, Q.Q. Sun, Y.Y. Cao, R. Li, X.X. Wu, S. Weeda, L. Li, S. Ren, The RNA-seq approach to discriminate gene expression profiles in response to melatonin on cucumber lateral root formation, J. Pineal Res. 56 (2013) 39–50. [27] N. Zhang, B. Zhao, H.J. Zhang, S. Weeda, C. Yang, Z.C. Yang, S. Ren, Y.D. Guo, Melatonin promotes water-stress tolerance, lateral root formation, and seed germination in cucumber (Cucumis sativus L.), J. Pineal Res. 54 (2012) 15–23. [28] Z. Chen, Q. Gu, X. Yu, L. Huang, S. Xu, R. Wang, W. Shen, W. Shen, Hydrogen peroxide acts downstream of melatonin to induce lateral root formation, Ann. Bot. 121 (2018) 1127–1136. [29] K. Guo, K. Xia, Z.M. Yang, Regulation of tomato lateral root development by carbon monoxide and involvement in auxin and nitric oxide, J. Exp. Bot. 59 (2008) 3443–3452. [30] I. De Smet, P.J. White, A.G. Bengough, L. Dupuy, B. Parizot, I. Casimiro, R. Heidstra, M. Laskowski, M. Lepetit, F. Hochholdinger, X. Draye, H. Zhang, M.R. Broadley, B. Peret, J.P. Hammond, H. Fukaki, S. Mooney, J.P. Lynch, P. Nacry, U. Schurr, L. Laplaze, P. Benfey, T. Beeckman, M. Bennett, Analyzing lateral root development: how to move forward, Plant Cell 24 (2012) 15–20. [31] C. Pape, K. Lüning, Quantification of melatonin in phototrophic organisms, J. Pineal Res. 41 (2010) 157–165. [32] Z. Chen, Y. Xie, Q. Gu, G. Zhao, Y. Zhang, W. Cui, S. Xu, R. Wang, W. Shen, The AtrbohF-dependent regulation of ROS signaling is required for melatonin-induced salinity tolerance in Arabidopsis, Free Radic. Biol. Med. 108 (2017) 465–477. [33] Y. Wang, X. Ye, K. Yang, Z. Shi, N. Wang, L. Yang, J. Chen, Characterization, expression, and functional analysis of polyamine oxidases and their role in seleniuminduced hydrogen peroxide production in Brassica rapa, J. Sci. Food Agric. 99 (2019) 4082–4093. [34] J. Chen, W.-H. Wang, F.-H. Wu, C.-Y. You, T.-W. Liu, X.-J. Dong, J.-X. He, H.L. Zheng, Hydrogen sulfide alleviates aluminum toxicity in barley seedlings, Plant Soil 362 (2013) 301–318. [35] W. Lv, L. Yang, C. Xu, Z. Shi, J. Shao, M. Xian, J. Chen, Cadmium disrupts the balance between hydrogen peroxide and superoxide radical by regulating endogenous hydrogen dulfide in the root tip of Brassica rapa, Front. Plant Sci. 8 (2017). [36] H. Wang, S. Taketa, A. Miyao, H. Hirochika, M. Ichii, Isolation of a novel lateralrootless mutant in rice (Oryza sativa L.) with reduced sensitivity to auxin, Plant Sci. 170 (2006) 70–77. [37] X. Ye, T. Ling, Y. Xue, C. Xu, W. Zhou, L. Hu, J. Chen, Z. Shi, Thymol mitigates cadmium stress by regulating glutathione levels and reactive oxygen species homeostasis in tobacco seedlings, Molecules 21 (2016) 1339. [38] R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangradorvegas, The Pfam protein families database: towards a more sustainable future, Nucleic Acids Res. 44 (2016) 279–285. [39] A. Marchlerbauer, M.K. Derbyshire, N.R. Gonzales, S. Lu, F. Chitsaz, L.Y. Geer, R.C. Geer, J. He, M. Gwadz, D.I. Hurwitz, CDD: NCBI's conserved domain database, Nucleic Acids Res. 43 (2015) 222–226. [40] C. Dunand, M. Crèvecoeur, C. Penel, Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases, New Phytol. 174 (2007) 332–341. [41] H. Tsukagoshi, W. Busch, P.N. Benfey, Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root, Cell 143 (2010) 606–616. [42] B. Chen, W. Li, Y. Gao, Z. Chen, W. Zhang, Q. Liu, Z. Chen, J. Liu, Involvement of polyamine oxidase-produced hydrogen peroxide during coleorhiza-limited germination of rice seeds, Front. Plant Sci. 7 (2016) 1219. [43] S.D. Harris, Branching of fungal hyphae: regulation, mechanisms and comparison with other branching systems, Mycologia 100 (2008) 823–832. [44] P. Rangan, R. Subramani, R. Kumar, A.K. Singh, R. Singh, Recent advances in polyamine metabolism and abiotic stress tolerance, BioMed Res. Int. 2014 (2014) 9. [45] P. Fincato, P.N. Moschou, V. Spedaletti, R. Tavazza, R. Angelini, R. Federico, K.A. Roubelakis-Angelakis, P. Tavladoraki, Functional diversity inside the Arabidopsis polyamine oxidase gene family, J. Exp. Bot. 62 (2011) 1155–1168. [46] T. Liu, D.W. Kim, M. Niitsu, S. Maeda, M. Watanabe, Y. Kamio, T. Berberich, T. Kusano, Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifically expressed in anthers, Plant Cell Physiol. 55 (2014) 1110–1122. [47] A. Ahou, D. Martignago, O. Alabdallah, R. Tavazza, P. Stano, A. Macone, M. Pivato, A. Masi, J.L. Rambla, F. Vera-Sirera, R. Angelini, R. Federico, P. Tavladoraki, A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines, J. Exp. Bot. 65 (2014) 1585–1603. [48] T. Liu, D. Wook Kim, M. Niitsu, T. Berberich, T. Kusano, POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5, Plant Signal. Behav. 9 (2014) e29773. [49] R. Wimalasekera, F. Schaarschmidt, R. Angelini, A. Cona, P. Tavladoraki, G.F.E. Scherer, POLYAMINE OXIDASE2 of Arabidopsis contributes to ABA mediated plant developmental processes, Plant Physiol. Biochem. 96 (2015) 231–240. [50] Q. Ke, J. Ye, B. Wang, J. Ren, L. Yin, X. Deng, S. Wang, Melatonin mitigates salt
None. Acknowledgements We are grateful to Dr. Yinghua Ji from Institute for Plant Protection, Jiangsu Academy of Agricultural Sciences for providing tobacco seeds. This work was supported by National Key Research and Development Program of China (2017YFD0201105), National Natural Science Foundation of China (31771705), The Program for Science & Technology Innovation Talents in Universities of Henan Province (18HASTIT037), and The Key Scientific and Technology Projects of Henan Province (182102110446). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.freeradbiomed.2019.09.011. References [1] J.A. Atkinson, A. Rasmussen, R. Traini, U. Voss, C. Sturrock, S.J. Mooney, D.M. Wells, M.J. Bennett, Branching out in roots: uncovering form, function, and regulation, Plant Physiol. 166 (2014) 538–550. [2] P. Yu, C. Gutjahr, C. Li, F. Hochholdinger, Genetic control of lateral root formation in cereals, Trends Plant Sci. 21 (2016) 951–961. [3] B. Péret, B. De Rybel, I. Casimiro, E. Benkova, R. Swarup, L. Laplaze, T. Beeckman, M.J. Bennett, Arabidopsis lateral root development: an emerging story, Trends Plant Sci. 14 (2009) 399–408. [4] J.E. Malamy, P.N. Benfey, Organization and cell differentiation in lateral roots of Arabidopsis thaliana, Development 124 (1997) 33–44. [5] I. De Smet, Lateral root initiation: one step at a time, New Phytol. 193 (2012) 867–873. [6] H. Nakagami, K. Kawamura, K. Sugisaka, M. Sekine, A. Shinmyo, Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco, Plant Cell 14 (2002) 1847–1857. [7] A. Verkest, C. Weinl, D. Inze, L. De Veylder, A. Schnittger, Switching the cell cycle. Kip-related proteins in plant cell cycle control, Plant Physiol. 139 (2005) 1099–1106. [8] H. Ren, A. Santner, J.C.D. Pozo, J.A.H. Murray, M. Estelle, Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated by two different ubiquitin E3 ligases, Plant J. 53 (2008) 705–716. [9] L. Sanz, W. Dewitte, C. Forzani, F. Patell, J. Nieuwland, B. Wen, P. Quelhas, S. De Jager, C. Titmus, A. Campilho, H. Ren, M. Estelle, H. Wang, J. Murray, The Arabidopsis D-type cyclin CYCD2;1 and the inhibitor ICK2/KRP2 modulate auxininduced lateral root formation, Plant Cell 23 (2011) 641–660. [10] T. Fang, J. Li, Z. Cao, M. Chen, W. Shen, L. Huang, Heme oxygenase-1 is involved in sodium hydrosulfide-induced lateral root formation in tomato seedlings, Plant Cell Rep. (2014) 1–10. [11] N. Correa-Aragunde, M. Graziano, C. Chevalier, L. Lamattina, Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato, J. Exp. Bot. 57 (2006) 581–588. [12] Y. Du, B. Scheres, Lateral root formation and the multiple roles of auxin, J. Exp. Bot. 69 (2018) 155–167. [13] H. Fukaki, M. Tasaka, Hormone interactions during lateral root formation, Plant Mol. Biol. 69 (2009) 437–449. [14] J. Gou, S.H. Strauss, C. Tsai, K. Fang, Y. Chen, X. Jiang, V. Busov, Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones, Plant Cell 22 (2010) 623–639. [15] H. Tsukagoshi, Control of root growth and development by reactive oxygen species, Curr. Opin. Plant Biol. 29 (2016) 57–63. [16] S.S. Gill, N. Tuteja, Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiol. Biochem. 48 (2010) 909–930. [17] R. Alcázar, A.F. Tiburcio, Plant polyamines in stress and development: an emerging area of research in plant sciences, Front. Plant Sci. 5 (2014) 319. [18] G.X. Su, W.H. Zhang, Y.L. Liu, Involvement of hydrogen peroxide generated by polyamine oxidative degradation in the development of lateral roots in soybean, J. Integr. Plant Biol. 48 (2006) 426–432. [19] D. Marino, C. Dunand, A. Puppo, N. Pauly, A burst of plant NADPH oxidases, Trends Plant Sci. 17 (2012) 9–15. [20] B. Orman-Ligeza, B. Parizot, R. de Rycke, A. Fernandez, E. Himschoot, F. Van Breusegem, M.J. Bennett, C. Périlleux, T. Beeckman, X. Draye, RBOH-mediated ROS production facilitates lateral root emergence in Arabidopsis, Development 143 (2016) 3328–3339. [21] C. Manzano, M. Pallero-Baena, I. Casimiro, B. De Rybel, B. Orman-Ligeza, G. Van Isterdael, T. Beeckman, X. Draye, P. Casero, J.C. del Pozo, The emerging role of reactive oxygen species signaling during lateral root development, Plant Physiol.
543
Free Radical Biology and Medicine 143 (2019) 534–544
J. Chen, et al.
[51] [52] [53]
[54]
[55] [56]
[57]
[58] S. Bhattacharjee, The language of reactive oxygen species signaling in plants, J. Bot., Le 2012 (2012) 985298. [59] V. Demidchik, S. Shabala, S. Isayenkov, T.A. Cuin, I. Pottosin, Calcium transport across plant membranes: mechanisms and functions, New Phytol. 220 (2018) 49–69. [60] V. Demidchik, ROS-activated ion channels in plants: biophysical characteristics, physiological functions and molecular nature, Int. J. Mol. Sci. 19 (2018) 1263. [61] V. Demidchik, S.N. Shabala, J.M. Davies, Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels, Plant J. 49 (2007) 377–386. [62] Y.J. Li, J. Chen, M. Xian, L.G. Zhou, F.X. Han, L.J. Gan, Z.Q. Shi, In site bioimaging of hydrogen sulfide uncovers its pivotal role in regulating nitric oxide-induced lateral root formation, PLoS One 9 (2014) e90340. [63] S.K. Singh, C.-T. Chien, I.-F. Chang, The Arabidopsis glutamate receptor-like gene GLR3.6 controls root development by repressing the Kip-related protein gene KRP4, J. Exp. Bot. 67 (2016) 1853–1869. [64] R. Hardeland, Melatonin in plants and other phototrophs: advances and gaps concerning the diversity of functions, J. Exp. Bot. 66 (2014) 627–646.
stress in wheat seedlings by modulating polyamine metabolism, Front. Plant Sci. 9 (2018) 914. J. Montiel, M.-K. Arthikala, C. Quinto, Phaseolus vulgaris RbohB functions in lateral root development, Plant Signal. Behav. 8 (2013) e22694. M. Sagi, R. Fluhr, Production of reactive oxygen species by plant NADPH oxidases, Plant Physiol. 141 (2006) 336–340. K. Gémes, Y.J. Kim, K.Y. Park, P.N. Moschou, E. Andronis, C. Valassaki, A. Roussis, K.A. Roubelakis-Angelakis, An NADPH-oxidase/polyamine oxidase feedback loop controls oxidative burst under salinity, Plant Physiol. 172 (2016) 1418–1431. T. Karuppanapandian, M. Juncheol, K. Changsoo, K. Manoharan, K. Wook, Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms, Aust. J. Crop. Sci. 5 (2011) 709–725. M.K. Kanwar, J. Yu, J. Zhou, Phytomelatonin: recent advances and future prospects, J. Pineal Res. 65 (2018) e12526. R.J. Reiter, S. Rosales-Corral, D.X. Tan, M.J. Jou, A. Galano, B. Xu, Melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas, Cell. Mol. Life Sci. 74 (2017) 3863–3881. D.-X. Tan, R.J. Reiter, Mitochondria: the birth place, battle ground and the site of melatonin metabolism in cells, Melatonin Res 2 (2019) 44–46.
544