Received: 9 April 2016 DOI: 10.1111/jpi.12359
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Accepted: 29 July 2016
ORIGINAL ARTICLE
Melatonin enhances thermotolerance by promoting cellular protein protection in tomato plants Wen Xu1,2* | Shu-Yu Cai1* | Yun Zhang1 | Yu Wang1 | Golam Jalal Ahammed1 | Xiao-Jian Xia1,3 | Kai Shi1,3 | Yan-Hong Zhou1,3 | Jing-Quan Yu1,3,4 | Russel J. Reiter5 | Jie Zhou1,3 1
Department of Horticulture, Zhejiang University, Hangzhou, China 2
Department of Horticulture, Guizhou University, Guiyang, China 3
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou, China 4
Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Agricultural Ministry of China, Hangzhou, China 5
University of Texas Health Science Center, San Antonio, TX, USA Correspondence Jie Zhou, Department of Horticulture, Zhejiang University, Hangzhou, China. Email:
[email protected]
Abstract Melatonin is a pleiotropic signaling molecule that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms for melatonin-mediated thermotolerance remain largely unknown. Here, we report that endogenous melatonin levels increased with a rise in ambient temperature and that peaked at 40°C. Foliar pretreatment with an optimal dose of melatonin (10 μmol/L) or the overexpression of N-acetylserotonin methyltransferase (ASMT) gene effectively ameliorated heat-induced photoinhibition and electrolyte leakage in tomato plants. Both exogenous melatonin treatment and endogenous melatonin manipulation by overexpression of ASMT decreased the levels of insoluble and ubiquitinated proteins, but enhanced the expression of heat-shock proteins (HSPs) to refold denatured and unfolded proteins under heat stress. Meanwhile, melatonin also induced expression of several ATG genes and formation of autophagosomes to degrade aggregated proteins under the same stress. Proteomic profile analyses revealed that protein aggregates for a large number of biological processes accumulated in wild-type plants. However, exogenous melatonin treatment or overexpression of ASMT reduced the accumulation of aggregated proteins. Aggregation responsive proteins such as HSP70 and Rubisco activase were preferentially accumulated and ubiquitinated in wild-type plants under heat stress, while melatonin mitigated heat stress-induced accumulation and ubiquitination of aggregated proteins. These results suggest that melatonin promotes cellular protein protection through induction of HSPs and autophagy to refold or degrade denatured proteins under heat stress in tomato plants. KEYWORDS autophagy, heat-shock proteins, melatonin, N-acetylserotonin methyltransferase, protein protection, thermotolerance, tomato
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Heat stress is well recognized as a major abiotic stress that severely limits crop production worldwide.1 During heat *These authors contributed equally to this work.
stress, a series of metabolic alterations occur, including overproduction of reactive oxygen species (ROS), photoinhibition, denaturation of some proteins such as chloroplast proteins, damage to biofilm structure and functions, and inhibition in protein synthesis.2 Heat- shock- induced oxidative stress inhibits photosynthesis by impairing structural organization
Journal of Pineal Research 2016; 1–13 wileyonlinelibrary.com/journal/jpi
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© 2016 John Wiley & Sons A/S. 1 Published by John Wiley & Sons Ltd
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of photosystem II (PSII) and decreasing its activity.3 In addition to being a crucial component of plant cell structure, proteins are directly implicated in plant stress metabolism. Heat stress causes the oxidation and misfolding of proteins in plant cells.4 Therefore, enhancement of thermotolerance in plant preferentially involves the adoption strategies that protect proteins from oxidation and misfolding. Plants have evolved multiple defense mechanisms that precisely modulate their transcriptome, proteome, and metabolome to adapt and survive under heat stress.5 Moreover, their responses to heat stress involve numerous molecular pathways and relevant physiological processes such as activation of calcium and ROS signaling, regulation of the cellular redox status, production of plant hormones, and the induction of defensive genes.6,7 Phytohormones play critical roles in the response of plants to high temperatures.8 For instance, elevated temperatures promote abscisic acid (ABA) synthesis, leading to a ABA- mediated stomatal closure that ultimately reduces transpiration losses.9 Salicylic acid (SA), which is well characterized as a biotic stress-responsive hormone, can also promote photosynthesis under heat stress by influencing various physiological processes and biochemical reactions.10 Furthermore, jasmonic acid (JA) or jasmonates are among the important cellular regulators that critically control a spectrum of physiological process and mediate stress responses.11 Exogenous application of JA as well as its enhancement of endogenous levels by the overexpression of JA biosynthetic gene confers thermotolerance in Arabidopsis thaliana.12 Moreover, biosynthesis of plant metabolites, such as soluble sugars, amino acids, organic acids, and lipids, is also affected by high temperature; nevertheless, they are also crucial for plant adaptation to high-temperature stress.9,13,14 To monitor heat stress-induced misfolded and damaged proteins, plant cells have evolved an elaborate protein quality control system consisting of molecular chaperones and protein degradation systems.15,16 Heat-shock proteins (HSPs), the well- known molecular chaperones, bind to denatured or non-native conformation proteins to prevent them from forming polymers,17 while the protein degradation systems clear the damaged proteins.15,16 The ubiquitin proteasome system (UPS) is the main pathway for degrading soluble misfolded proteins via the 26S proteasome.18 However, heat stress-accumulated denatured proteins that are prone to aggregation are not efficiently dissociated by the 26S proteasome,19,20 but can be degraded by autophagy, a self-degradation system of cellular components through an autophagosomal–lysosomal pathway.21 Melatonin is a pleiotropic signaling molecule that provides physiological protection against a variety of environmental stresses such as heat stress, chilling, drought, salt, heavy metals, and ultraviolet radiation.22–25 Byeon and Back26 found that the endogenous melatonin synthesis is upregulated in high-temperature-exposed rice (Oryza sativa) seedlings; this is associated with the enhanced activities of serotonin N-acetyltransferase and N-acetylserotonin methyltransferase
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(ASMT), two enzymes involved in melatonin synthesis. In Arabidopsis, heat stress response is closely related to the melatonin-induced transcriptional upregulation of heat shock factor A1s (HSFA1s).23 Furthermore, the inhibitory effects of light and high temperature in photosensitive and thermosensitive genotypes of Phacelia tanacetifolia are well regulated by the application of melatonin.27 Melatonin is also reported to stimulate antioxidant enzymes activities and germination of Arabidopsis seeds under heat stress.28 The involvement of melatonin in thermotolerance and the underlying molecular mechanisms related to the protein quality control system in plants are still elusive. This study unveiled the involvement of cellular protein protection in melatonin-induced thermotolerance. This work also examined the function of endogenous melatonin in tomato tolerance to heat stress by overexpressing ASMT gene that encodes the enzyme ASMT involved in the conversion of N-acetylserotonin to melatonin. We found that pretreatment with 10 μmol/L melatonin or overexpression of ASMT substantially increased the values of maximum quantum efficiency of photosystem II (Fv/Fm), while melatonin reduced the level of relative electrolyte leakage through cellular membranes. Melatonin-induced heat stress mitigation was closely associated with decreased levels of insoluble and ubiquitinated proteins that were resulted from heat stress. The expression of HSPs and formation of autophagosomes were further enhanced in melatonin-pretreated or ASMT-overexpressing plants compared with those of wild-type plants under heat stress. Through proteomic profiling, we found that exogenous application of melatonin as well as enhancement of endogenous melatonin levels by overexpression of ASMT gene both alleviated the accumulation of aggregate-prone proteins.
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M ATERIAL S AND M ETHOD S
2.1 | Plant materials, growth conditions, and experimental design Tomato (Solanum lycopersicum L. cv Ailsa Craig) seeds were germinated in a growth medium containing a mixture of peat and vermiculite (2:1, v:v) in a greenhouse. When the second true leaves were fully expanded, seedlings were transferred into 15-cm plastic pots containing a mixture as described above. The growth conditions were as follows: photoperiod of 14/10 hour (day/night), temperature of 25/22°C (day/ night), and a photosynthetic photon flux density (PPFD) of 800 μmol/m2/s. Plants were fertilized with Hoagland’s nutrient solution every 2 days. For heat tolerance experiment, 8-week-old tomato plants were placed in growth chambers at 25, 35, 40, and 45°C single temperature treatment for 9 hours and then analyzed for the melatonin content, Fv/Fm, and malondialdehyde (MDA) content. To select the optimal dose of melatonin that could
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induce thermotolerance effectively, tomato plants were pretreated with different concentrations of melatonin (1, 10, 50, 100, 500 μmol/L). Foliar portion of each plant was sprayed with 10 mL of melatonin or water (control) 8 hours prior to the imposition of high-temperature treatment. Based on the preliminary dose trial, 10 μmol/L melatonin was selected for the rest of the experiments considering its effect as the best in the induction of thermotolerance (Fig. S1). Four replicates were used for each treatment, and each replicate consisted of 12 plants.
2.2 | Generation of ASMT- overexpressing plants To obtain the tomato ASMT-overexpressing (ASMT OE) construct, the 1074-bp full-length coding DNA sequence (CDS) was amplified with the primers ASMT OE-F (5′-TTGGCGC GCCATGGGTTCAACAAGCCTAAC-3′) and ASMT OE-R (5′-A CGCGTCGACCTTGGTGAATTCCATAATC-3 ′) using tomato cDNA as the template. The PCR product was digested with AscI and SalI and inserted behind the CaMV 35S promoter in the plant transformation vector pFGC1008HA. The resulting ASMT OE-HA plasmid was transformed into Agrobacterium tumefaciens strain EHA105. Tomato genotype Ailsa Craig was used for transformation, as described previously by Wang et al.29 Transgenic plants overexpressing the ASMT transgene were identified by Western blotting using an anti- HA (Pierce, 26183, Thermo Scientific, Rockford, IL, USA) monoclonal antibody (Fig. S2). Two independent homozygous lines of the F2 progeny were used in this study.
2.3 | Quantification of endogenous melatonin levels by HPLC Endogenous melatonin in tomato leaves was extracted according to Arnao and Hernández-Ruiz.30 Fresh leaf blades (0.3 g) were cut into small sections and placed into a 15-mL tube containing 6 mL of chloroform, which was shaken overnight at 4°C in the dark. After centrifugation at 4000 g for 5 minutes, the supernatants were transferred to a C18 solid-phase extraction (SPE) cartridge (Waters, Milford, MA, USA) for the purification of melatonin. The extract was then dried under nitrogen. The residue was dissolved in 2 mL of the mobile phase for HPLC analysis as described by Korkmaz et al.31 All the processes in the procedures were carried out in dim artificial light.
2.4 | Chlorophyll fluorescence and relative electrolyte leakage measurements The maximum quantum efficiency of photosystem II (PSII), expressed as the Fv/Fm, was determined in the fifth leaves after 30 minutes of dark adaptation using an Imaging-PAM Chlorophyll Fluorometer equipped with computer-operated PAM-control unit (IMAG-MAXI, Heinz Walz, Effeltrich,
Germany), as described elsewhere.32 Relative electrolyte leakage was determined in leaves measuring the electric conductivity as described previously.32
2.5 | RNA isolation and quantitative real-time PCR Total RNA was extracted from tomato leaves using TRIzol reagent (Tiangen, Shanghai, China) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from the isolated total RNA using reverse transcriptase (Toyobo, Osaka, Japan) following the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was performed with the diluted cDNA and SYBR Green PCR Master Mix (Takara, Tokyo, Japan). Each reaction (20 μL) consisted of 10 μL SYBR Green PCR Master Mix, 2 μL diluted cDNA, and 0.1 μmol/L forward and reserve primers. The cycling conditions were as follows: 95°C for 3 minutes followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds. Gene-specific qRT-PCR primers were designed based on their cDNA sequences and are listed in Table S1. Tomato Actin2 was used as an internal control. Relative gene expression was calculated according to Livak and Schmittgen.33
2.6 | Protein extraction and Western blotting For protein extraction, tomato leaves were ground in liquid nitrogen and homogenized in an extraction buffer (100 mmol/L Tris/HCl, pH 8, 10 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.2% β-mercaptoethanol). The soluble and insoluble proteins were detected as described previously.32 Protein fractionations were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then, the proteins on the SDS-PAGE gel were transferred to a nitrocellulose membrane. Ubiquitinated proteins were detected by protein blotting using an anti- ubiquitin monoclonal antibody as previously mentioned.32 HSP70 genes encode abundant heat-inducible 70-kDa HSPs (HSP70s). In tomato plants, HSP70 genes family has more than 20 members. The genes show a high degree of conservation, having at least 50% identity. In previous work, HSP70 accumulation was detected by protein blotting using rabbit polyclonal antibody HSP70 (Agrisera, Vännäs, Sweden) according to Li et al.34 Rubisco activity (RCA) was detected with commercial monoclonal antibody (Agrisera).35
2.7 | Protein profiles analysis of insoluble proteins Insoluble proteins were extracted and separated by SDS- PAGE. Mass spectrometer (LTQ- Orbitrap Elite, Thermo
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Fisher Scientific, Waltham, MA, USA) was used to analyze the insoluble proteins.36 In-gel proteins were digested overnight in 12.5 ng/mL trypsin in 25 mmol/L NH4HCO3. The peptide mixtures were injected onto the trap column with a flow rate of 10 μL/min for 2 minutes using a Thermo Scientific Easy nanoLC 1000. The trap was equilibrated at a maximum pressure of 500 bar for 12 μL followed by column equilibration at a maximum of 500 bar for 3 μL before starting gradient elution of column. The samples were subsequently eluted with a five-step linear gradient (A: ddH2O with 0.1% formic acid, B: ACN with 0.1% formic acid): 0–10 minutes, 3%–8% B; 10–120 minutes, 8%–20% B; 120–137 minutes, 20%–30% B; 137–143 minutes, 30%–90% B; 143–150 minutes, 90% B. The column flow was maintained as 250 nL/ min. The chromatographic system was composed of a trapping column (75 μm × 2 cm, nanoviper, C18, 3 μmol/L, 100 Å) and an analytical column (50 μm × 15 cm, nanoviper, C18, 2 μmol/L, 100 Å). Data collection was performed using Thermo LTQ-Orbitrap Velos Pro equipped Nanospray Flex ionization source and FTMS (Fourier transform ion cyclotron resonance mass analyzer) analyzer combined with Thermo LTQ-Orbitrap Elite equipped Ion Trap analyzer. The parameters for FTMS were as follows: data collection was at 60K for the full scan MS, positive as polarity, profile as data type, and then proceeded to isolate the top 20 ions for MS/MS by CID (1.0 m/z isolation width, 35% collision energy, 0.25 activation Q, 10 ms activation time). Scan range was set as 300 m/z First Mass and 2000 m/z Last Mass. The parameters for ion trap analyzer were normal mass range, rapid scan rate, and centroid data type. Three independent replicates were used for mass spectrometric analysis.
2.8 | Observation of autophagic activity using MDC staining To observe the accumulation of autophagosomes, tomato leaves were excised and vacuum infiltrated with 100 mmol/L MDC (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes,
2.9 | Accession numbers Sequence data for the genes described in this study can be found in the Sol genomics network under the accession numbers shown in parentheses: Actin2 (Sl), ATG5 (Sl02g036380), ATG6 (Sl05g050390), ATG8a (Sl02g036380), ATG8f (Sl07g 064680), ATG12 (Sl12g049310), ATG18f (Sl01g099400), HSP17.4 (Sl03g123540), HSP20 (Sl06g076570), HSP20-1 (Sl06g076570), HSP21 (Sl03g082420), HSP70 (Sl11g020040), HSP90 (Sl06g036290).
2.10 | Statistical analyses At least three independent replicates were used for each experiment, and the mean values of all the data are presented for each treatment. A statistical analysis of the bioassays was performed with the SAS statistical software package. The Tukey’s test (P
