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Overexpression of Ricinus communis L. malate synthase enhances seed tolerance to abiotic stress during germination
Industrial Crops & Products 145 (2020) 112110
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Overexpression of Ricinus communis L. malate synthase enhances seed tolerance to abiotic stress during germination
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Valdinei Carvalho Britoa,b, Catherine P. de Almeidaa, Rhaíssa R. Barbosaa, Maria G.A. Carosioc, Antônio G. Ferreirac, Luzimar G. Fernandezd, Renato D. de Castrod, Henk Hilhorste, Wilco Ligterinke, Paulo Roberto Ribeiroa,b,* a
Metabolomics Research Group, Departamento de Química Orgânica, Instituto de Química, Universidade Federal da Bahia, Rua Barão de Jeremoabo s/n, 40170-115, Salvador, Brazil b Programa de Pós-Graduação em Química Aplicada, Departamento de Ciências Exatas e Da Terra - Campus I, Universidade do Estado da Bahia, Salvador, Brazil c Laboratório de Ressonância Magnética Nuclear, Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil d Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100, Salvador, Brazil e Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University (WU), Droevendaalsesteeg 1, NL-6708 PB, Wageningen, The Netherlands
A R T I C LE I N FO
A B S T R A C T
Keywords: Abiotic stress Castor bean Functional characterization Temperature-responsive genes
Ricinus communis L. seeds can germinate at high temperatures, but further development of the seedlings is negatively affected. This mainly caused by impairment of energy-generating pathways when seeds are germinated at 35 °C. Ricinus communis malate synthase (RcMLS) is a key responsive gene in lipid mobilization and gluconeogenesis and as such might have a role in sustaining successful seed germination and seedling growth. Herein, we raised the question whether RcMLS might be involved in the biochemical and molecular mechanisms required for R. communis seed germination under unfavourable environmental conditions. For that, we used a robust approach that encompassed bioinformatics analysis, transgenic Arabidopsis thaliana (L.) Heynh seeds overexpressing RcMLS, along with phenotypical characterization of seed germination under abiotic stress. The phylogenetic tree revealed important evolutionary relationship amongst MLS sequences from R. communis and from other crop species/model plants. Overexpression of RcMLS enhanced A. thaliana seed germination under high temperature and salt stress. For example, wild-type A. thaliana Columbia seeds (Col-0) showed 37 % of maximum germination at 35 °C, whereas A. thaliana seeds overexpressing RcMLS showed up to 71%. When salt stress was applied (75 mM NaCl), maximum germination of Col-0 seeds reached 37%, whereas for A. thaliana seeds overexpressing RcMLS it reached up to 93%. Nuclear Magnetic Resonance (NMR) and Gas Chromatography coupled to Time-Of-Flight Mass Spectrometry (GC-TOF-MS) metabolomics analysis showed a robust metabolic signature of A. thaliana seeds overexpressing RcMLS in response to abiotic stress. They accumulated high levels of Met, Ile, fructose, glucose, and sucrose. Therefore, we suggested that overexpression of RcMLS has modulated the glyoxylate cycle and gluconeogenesis pathway in order to maintain cellular homeostasis under unfavorable environmental conditions. Our results provide important leads into the contribution of RcMLS to the underlying mechanism of R. communis seed germination under adverse environmental conditions. This might be helpful for breeding programs to develop more resistant R. communis cultivars which are more likely to sustain growth and high yield under the severe conditions found in arid and semi-arid areas worldwide.
1. Introduction Ricinus communis L. is a highly productive and resistant crop easily cultivated in hot and dry regions worldwide. The oil obtained from R. communis seeds possesses a diverse panel of applications, mainly because of the high levels of ricinoleic acid (up to 90% of the oil). This oil
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can reach up to three-times higher commercial value than Glycine max (L.) Merr. (soybean), Helianthus annuus L. (sunflower), and Brassica (canola) oils. Additionally, this species is able to grow and still yield as much as 900 kg of oil per hectare even under the harsh environmental conditions of arid and semi-arid regions worldwide where most other crops would not even survive (Salihu, 2014).
Corresponding author. E-mail addresses: [email protected], [email protected] (P.R. Ribeiro).
https://doi.org/10.1016/j.indcrop.2020.112110 Received 20 October 2019; Received in revised form 9 January 2020; Accepted 9 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.
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Molecular Evolutionary Genetics Analysis software (MEGA 7.0). Prediction of cellular locations were performed with Cello, whereas molecular weight and predicted isoelectric point (pI) were provided by ProtParam. Analysis of the conserved motifs were performed by the Multiple EM for Motif Elicitation (MEME). Then, the potential motif sequences candidates were loaded into the Pfam protein database. Further details are described in Gomes Neto et al. (2018).
Malate synthase (MLS) is an acyltransferase enzyme that catalyzes the Claisen-like condensation of acetyl-coenzyme A and glyoxylate to produce malate and coenzyme A. After its production, malate might be transported to the cytosol, where it is converted to oxaloacetate that in turn enters gluconeogenesis. Along with isocitrate lyase, MLS bypass the tricarboxylic acid cycle through the glyoxylate shunt in a sequence of anaplerotic reactions that enable plants and micro-organisms to use two-carbon compounds derived from lipids, such as acetate, to produce energy and support seed germination and seedling growth (González, 1990; Rodriguez et al., 1990). In R. communis, acetate is produced in the glyoxysome via the β-oxidation of fatty acids that are mobilized from lipid bodies in the endosperm upon seed imbibition and germination (Ribeiro et al., 2015b; Rodriguez et al., 1990). Malate synthase is highly active during early seed maturation and germination, but low activity was observed in mature dry seeds (Faraoni et al., 2019; González, 1990; Zhao et al., 2018). Although it has been suggested that in A. thaliana seedlings MLS is partially dispensable for lipid degradation and gluconeogenesis (Cornah et al., 2004), we have previously reported that RcMLS might be essential to sustain R. communis successful growth (Ribeiro et al., 2015c). Metabolite profiling techniques have provided important insights into the understanding of plant plasticity and adaptation to abiotic stress (Arbona et al., 2013; Ribeiro et al., 2018). Nuclear magnetic resonance and mass spectrometry are the most important techniques for metabolite detection in plant biology since they combine excellent sensitivity and high-throughput analysis to characterize the metabolome of a biological system. A combination of Gas and Liquid Chromatography coupled to Mass Spectrometry (GC–MS and LC–MS) were used to show that high temperatures decreased RcMLS expression leading to lower levels of carbohydrates in germinating R. communis seeds (Ribeiro et al., 2015a, d). Additionally, it has been suggested that high temperatures impair β-oxidation downstream pathways responsible to produce energy (Ribeiro et al., 2015c). This is supported by the fact that overexpression of RcMLS led to greater levels of starch in Nicotiana benthamiana Domin leaves (Ribeiro et al., 2015c). In cotton (Gossypium hirsutum L.), MLS activity increases during seed maturation, and it is kept at steady levels throughout desiccation and imbibition, but it increases again upon germination (Turley and Trelease, 1987). Malate synthase catalytic activity is highly dependent on the temperature: highest activity was observed at 35 °C for Gossypium hirsutum and at 28 °C for Helianthus annuus L., whereas optimal seedling emergence was observed at 31−37 °C for G. hirsutum and at 27−35 °C for H. annuus (Mahan, 2000). Overexpression of Escherichia coli MLS in Chlamydomonas reinhardtii led to greater microalgal biomass as compared with wild-type cells through enhancement of the heterotrophic metabolism (Paik et al., 2019). Nevertheless, there is no study that correlates MLS expression and activity with acquisition of tolerance to abiotic stresses during seed germination. Therefore, we addressed the question whether RcMLS might be involved in crucial mechanisms required for R. communis seed germination under harsh conditions.
2.2. Extraction of RNA, cDNA synthesis, cloning and plant transformation Ricinus communis samples were collected during seed germination at 25 °C. The procedure used for extraction of total RNA and for the synthesis of first strand cDNA is described in details in Gomes Neto et al. (2018). Ricinus communis malate synthase (RcMLS) gene was cloned by using the Gateway® technology (Ribeiro et al., 2015c). Primers used and additional information about the cloned genes are presented in Supplementary Table S1. For cloning purposes, we used the donor vector pDONr207 and the gateway binary expression vector pGD625 bearing the CaMV35S (CaMV-cauliflower mosaic virus). Further details are described in Ribeiro et al. (2015c). A. tumefaciens strains carrying the gene of interest (CaMV35S::RcMLS) were used for plant transformation in a modified floral‐dip protocol (Logemann et al., 2006). The expression vector pGD625 contained a kanamycin resistance selection marker and, therefore, transformants were selected by sowing seeds on 1/2 strength MS medium supplemented with 50 μg.mL−1 kanamycin. 2.3. Germination under abiotic stress Seeds of A. thaliana plants overexpressing RcMLS and seeds of Col-0 were germinated under abiotic stress conditions. They were sown on petri dishes with germination paper moistened with 6 mL water or saline solutions followed by incubation at 4 °C i for 72 h, for stratification purposes. High temperature stress assays were performed by incubating seeds at different temperatures (22 °C, 31 °C through 36 °C). Saline stress was performed by incubating stratified seeds at 0, 25, 50, and 75 mM NaCl. Germination percentage was counted daily and the data was processed using the GERMINATOR software (Gomes Neto et al., 2018; Ribeiro et al., 2015c). For metabolite profiling analysis at different temperatures seeds were imbibed in H2O for 30 h at 22 and 35 °C, whereas for metabolite profiling analysis under salt stress seeds were imbibed in water and in 75 mM NaCl for 30 h. 2.4. GC-TOF-MS and NMR-based metabolite profiling analysis Twenty mg of each sample were extracted in a system containing methanol, chloroform and water, derivatized to obtain trimethylsilyl (TMS) derivatives, and injected into a gas chromatographer coupled to a quadrupole time of flight mass spectrometry system (GC-TOF-MS). Further details are provided in Ribeiro et al. (2014). 1 H-NMR spectra were recorded at 20 °C using Bruker AVANCE III spectrometer (400 MHz). 3-(trimethylsilyl) propionic acid-D4 sodium salt (TMSP-d4) was used as internal standard. For 1D experiments, we used the cpmgpr1d (Bruker standard) pulse sequence, performed with 64 scans (ns) with 64k points during acquisition (td) in a spectral window (sw) of 15 ppm and with a receiver gain (rg) of 1290, 3.0 s between each acquisition (d1) and this had a total time (aq) of approximately 5.46 s. The power attenuation for the pre-saturation (PLdB9) was 45.00 dB. Total acquisition time of each spectrum was 11 min and 35 s. Spectra were processed with 64k (SI) using an exponential multiplication with a lb of 0.3 Hz and phase and baseline automatic correction. For HMBC experiments, we used the hmbcgplpndqf (Bruker standard) performed with 64 scans (ns) with 4k (F2) and 256 (F1) points during acquisition (td) in a spectral window (sw) of 15 ppm (F2) and 238 ppm (F1). The total acquisition time of each HMBC experiment was 6 h and 34 min.
2. Material and methods 2.1. Phylogenetic analysis and prediction of cellular locations Initially, Ricinus communis malate synthase protein sequence (30,147.m013773) was used as queries in a blastp search within the proteome of Arabidopsis thaliana (L.) Heynh, Brassica oleracea L., Cucumis sativus L., Glycine max (L.) Merr., Manihot esculenta Cranz, Oryza sativa L., Populus trichocarpa Torrey & A. Gray, Solanum lycopersicum L., Solanum tuberosum L., Sorghum bicolor L. Moench, and Zea mays L. These sequences were named by adding the initials of the plant species followed by MLS, for example AtMLS stands for Arabidopsis thaliana malate synthase. The identified MLS sequences were aligned at the online platform multiple sequence comparison by log-expectation (MUSCLE) and the phylogenetic analysis was performed by using 2
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evolutionary relationship between R. communis, M. esculenta, and P. trichocarpa that have been suggested by phylogenetic analysis of superoxide dismutase (Gomes Neto et al., 2018), papain-like cysteine protease (Zou et al., 2017), aquaporin (Zou et al., 2015), S-phase kinase-associated protein 1 (SKP1) (HajSalah El Beji et al., 2019), and argonaute (Mirzaei et al., 2014) proteins. MLS protein sequences from A. thaliana and B. oleracea, both belonging to the Brassicaceae family, clustered together in the same subclade, whereas MLS protein sequences from Solanum lycopersicum and S. tuberosum also clustered together in a different subclade (Fig. 1). Six putative motifs were found amongst MLS genes, with lengths ranging from 29 to 159 aa (Fig. 2, Supplementary Table S2). All MLS sequences contained six motifs from which five were identified as MLS protein domains (pfam code CL0151) (Supplementary Table S3). The MLS signature polypeptide is [K/R]-[D/E/N/Q]-[H]-[XeX]-GLNCGRWDY-[L/I/V/M]-[F] (Zambuzzi-Carvalho et al., 2009). The MLS signature polypeptide was found in motif-1 and identified as RDHSVGLNCGRWDYIF. Since MLS are biosynthesized in the cytoplasm and later transferred to the glyoxysomes, these sequences show a protein targeting signal (PTS) residing in the last three amino acids of the C-terminus. The PTS is defined by the consensus [A/C/S]-[K/R/H]-[L/M] (Zambuzzi-Carvalho et al., 2009). Ricinus communis malate synthase protein sequence (RcMLS) has the SRL tripeptide as the glyoxysomal PTS. This PTS was also observed for PtMLS, SlMLS, StMLS, AtMLS, and BoMLS. GmMLS, and CsMLS have the SKL glyoxysomal PTS, whereas OsMLS and ZmMLS have the CKL glyoxysomal PTS. Although acetylCoA is fairly common in enzymatic reactions the lack of conserved CoA binding motifs is still very intriguing. It seems that MLS does not possess a specific motif for recognition of CoA, but its recognition occurs through hydrogen bonds and van der Waals interactions with the polypeptide backbone (Anstrom et al., 2003). Nevertheless, these results suggest high protein evolutionary conservation amongst these organisms (De La Torre et al., 2017; Rahman et al., 2013).
2.5. Data processing, compound identification, and multivariate statistical analysis We used the online pipeline software NMRProcFlow to process 1HNMR spectra, which included the standard workflow (correction of the baseline, alignment and peak integration) (Jacob et al., 2017; Ribeiro et al., 2015b). Compound identification was performed by comparison of the NMR signals of authentic samples and data from the literature. GC-TOF-MS data were processed was described in Ribeiro et al. (2014). Compound identification was performed by comparison of the mass (MS) spectra and data from spectral libraries (NIST and Golm). Multivariate statistical analysis were performed at MetaboAnalyst 3.0, by using the default settings provided in the platform (Xia and Wishart, 2011). Further details are provided in Ribeiro et al. (2014) and in Santos et al. (2018). 3. Results and discussion 3.1. Identification and phylogenetic analysis of the MLS gene in different crop and model plant species Ricinus communis malate synthase protein sequence (30,147.m013773) possess 567 amino acids (Supplementary Figure S1), molecular formula of C2866H4467N815O816S27, molecular weight of 64,262.59 Da, and theoretical pI of 8.53. It possesses an instability index of 38.55, which classifies it as a stable protein. These results are supported by available data from other MLS protein sequences (Hoppe and Theimer, 1995; Jaunin et al., 1995; Köller and Kindl, 1980; Lewis and Doyle, 2001; Mori et al., 1988; Turley et al., 1990). Prediction of cellular location indicated that all MLS sequences are cytoplasmic with reliability index ranging from 3.997 to 4.673. At first, one might think that this does not fit with the well-known location of MLS in the glyoxysomes (special peroxisomes) (Ahn et al., 2016; Frevert et al., 1980; Guex et al., 1995; Ha et al., 2018; Kunze et al., 2006; Ohno et al., 2015; Trelease et al., 1974), but several studies have demonstrated that MLS is indeed produced in the cytosol, but subsequently transferred into the glyoxysomes (Gerdes et al., 1982; Kruse et al., 1981; Kruse and Kindl, 1983; Turley and Trelease, 1987; Wolfram and Helmut, 1980). As a matter of fact, the octameric structure of malate synthase present in glyoxysomes are assembled from its monomeric precursors which are biosynthesized in the cytoplasm. The monomeric precursor possesses identical subunit molecular weight, but differs from the mature enzyme by its oligomerization and aggregation state (Gerdes et al., 1982; Kruse and Kindl, 1983). Ricinus communis malate synthase protein sequence was used as queries in a blastp search against whole proteome of some important industrial crops and/or model plants to identify potential MLS proteins. We constructed a neighbor-joining (NJ) phylogenetic tree using their full-length amino acid sequences to investigate the evolutionary relationship between MLS sequences, (Fig. 1). For that, only well-annotated full sequences were considered. The retrieved MLS sequences showed similar lengths ranging from 534 aa for MeMLS and 573 aa for SbMLS. This included one MLS sequence from Arabidopsis thaliana, Brassica oleracea, Cucumis sativus, Manihot esculenta, Oryza sativa, Populus trichocarpa, Solanum tuberosum, Sorghum bicolor, and Zea mays. However, Glycine max and Solanum lycopersicum seem to possess multiple MLS copies in their genome and three sequences were retrieved. It is not clear, however, why and how genome duplication events may have taken place in G. max and S. lycopersicum and their relationship with plant adaptation to adverse land environments. There is a clear separation in the NJ phylogenetic tree between MLS sequences from monocots and eudicots species. Eudicots form a large clade, whereas the three monocots appear in an isolated clade. Nevertheless, important evolutionary insights can be obtained from a closer analysis of the eudicots’ clade: RcMLS clustered within the same subclade as PtMLS and MeMLS (Fig. 1). This supports a closer
3.2. Arabidopsis thaliana seeds overexpressing RcMLS showed enhanced tolerance to abiotic stresses during germination Arabidopsis thaliana seeds overexpressing RcMLS were germinated under high temperature and salt stress to ultimately address the contribution of RcMLS for the acquisition of tolerance under abiotic stress. For that, we used seeds from two A. thaliana overexpression independent lines (RcMLS1 and RcMLS2) and seeds from A. thaliana wild type (Col-0). Initially, a pilot germination assay was performed with Col-0 seeds at different temperatures (22 °C, 31−36 °C). Col-0 seeds germinated nearly 100% at temperature below 33 °C. At 34 °C maximum germination lowered to 92%, whereas at 35 °C maximum germination lowered to 36%. At 36 °C no germination was observed. Therefore, 34 °C was selected as the threshold temperature for further germination experiments. At 34 °C, Col-0 seeds showed 94% of maximum germination, whereas RcMLS1 seeds showed 97% and RcMLS2 seeds showed 88% of maximum germination (Fig. 3a). At 35 °C, Col-0 seeds showed 37% of maximum germination, whereas A. thaliana seeds overexpressing RcMLS showed up to 71% of maximum germination (Fig. 3a). Therefore, overexpression of RcMLS enhanced A. thaliana seed germination thermotolerance. Four NaCl concentrations (25, 50, and 75 mM) were used to impose salt stress during germination (Fig. 3b). Nearly 100% of maximum seed germination was observed in control conditions and at 25 mM NaCl for both Col-0 and A. thaliana seeds overexpressing RcMLS (Fig. 3b). At 50 mM NaCl, Col-0 seeds showed 83% of maximum germination, whereas RcMLS1 seeds showed 96% and RcMLS2 seeds showed 73% of maximum germination (Fig. 3a), but no significant statistical differences were observed whatsoever. At 75 mM NaCl, maximum germination of Col-0 seeds reached 37%, whereas A. thaliana seeds overexpressing RcMLS showed up to 93% of maximum germination 3
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Fig. 1. Neighbor-joining (NJ) phylogenetic tree of malate synthase protein sequences from different plant species. AtMLS (Arabidopsis thaliana), BoMLS (Brassica oleracea), CsMLS (Cucumis sativus), GmMLS (Glycine max), MeMLS (Manihot esculenta), OsMLS (Oryza sativa), PtMLS (Populus trichocarpa), RcMLS (Ricinus communis), SlMLS (Solanum lycopersicum), StMLS (Solanum tuberosum), SbMLS (Sorghum bicolor), and ZmMLS (Zea mays).
Fig. 2. Conserved motifs of malate synthase protein sequences from different plant species. AtMLS (Arabidopsis thaliana), BoMLS (Brassica oleracea), CsMLS (Cucumis sativus), GmMLS (Glycine max), MeMLS (Manihot esculenta), OsMLS (Oryza sativa), PtMLS (Populus trichocarpa), RcMLS (Ricinus communis), SlMLS (Solanum lycopersicum), StMLS (Solanum tuberosum), SbMLS (Sorghum bicolor), and ZmMLS (Zea mays).
3.3. Arabidopsis thaliana seeds overexpressing RcsMLS showed a specific metabolic signature under abiotic stress conditions
(Fig. 3b). Therefore, overexpression of RcMLS also enhanced A. thaliana seed germination performance under salt stress. Cornah et al. (2004) used seeds of two independent mls t-DNA lines (At5g03860) to assess the role of the AtMLS in important energy-generating pathways. Seedlings of the knock-out mutants showed inhibition of hypocotyl elongation, impairment of root development, and compromised seedling establishment during short days. Nevertheless, these phenotypes could be rescued by applying exogeneous succinate to the seedlings. They also showed that in the absence of the MLS enzyme, mutant seedlings were able to use [14C]-glycine and [14C]-serine as gluconeogenic precursors (Cornah et al., 2004). Unfortunately, the authors did not evaluate seed performance under abiotic stress. This is a pioneer study that assessed the possible relationship between the glyoxylate cycle enzyme MLS and the acquisition of tolerance to abiotic stress in the oilseed crop R. communis, which in turn can be further investigated in other industrial crops and model plants. Therefore, overexpression of RcMLS enhanced A. thaliana seed germination under high temperature and salt stress, highlighting the fact that a better understanding of RcMLS might be essential to enhance R. communis performance under unfavorable conditions.
Since the glyoxylate cycle allow plants to use acetate derived from the mobilization of storage lipids to produce carbohydrates, we raised the question whether overexpression of RcMLS gene would lead to a specific metabolic signature that would explain the enhanced germination performance under unfavorable conditions of A. thaliana seeds overexpressing RcMLS. Initially, we assessed the metabolome of Col-0 and A. thaliana seeds overexpressing RcMLS in response to different temperatures. We detected over 100 peaks by GC-TOF-MS, of which 17 could be annotated (Supplementary Table S4), whereas 16 metabolites were identified by NMR analysis (Supplementary Table S5). Partial least squares discriminant analysis (PLS-DA) was applied to the entire data set to assess variation in the metabolome in response to high temperatures (Fig. 4a). The goodness of fit (R2) and the predictability of the model (Q2) values suggests that this is a very strong PLSDA model that allowed us to extract metabolite changes in A. thaliana seeds overexpressing RcMLS in response to high temperatures (Supplementary Figure S3). There is a clear separation along principal component 1 amongst Col-0 and A. thaliana seeds overexpressing RcMLS, whereas principal component 2 (PC2) reflects a separation 4
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sucrose), twelve amino acids (Ala, Arg, Asn, Asp, Glu, Gly, Lys, Met, pyroglutamate, Ser, Thr, Val), seven organic acids (citrate, formate, fumarate, malate, succinate, threonate, t-sinnapic acid), along with allantoin, ethanolamine, galactinol, and phosphate (Supplementary Table S6). We set the variable importance in projection (VIP) threshold as 1 (Xia and Wishart, 2011) to discuss the possible contribution of each individual metabolites to the enhanced tolerance of A. thaliana seeds overexpressing RcMLS (Fig. 4b). Since we were mainly interested in differences that could explain the observed phenotype, we focused our attention on metabolites that varied between Col-0 and A. thaliana seeds overexpressing RcMLS seeds when both were imbibed at 35 °C. Seven metabolites showed higher levels in A. thaliana seeds overexpressing RcMLS than in Col-0 seeds: allantoin (up to 1.39-fold), Asn (up to 5.72-fold), ethanolamine (up to 9.68-fold), glucose (up to 10.27fold), Lys (up to 1.21-fold), Met (up to 2.66-fold), and sucrose (up to 1.09-fold) (Supplementary Table S7). It seems that higher temperatures increased the levels of these metabolites in A. thaliana seeds overexpressing RcMLS to a greater extent than at Col-0 seeds. Therefore, overexpression of RcMLS contributed to higher levels of these metabolites in response to temperature. Levels of five metabolites were higher in Col-0 seeds imbibed at 35 °C than in A. thaliana seeds overexpressing RcMLS imbibed at the same temperature: fumarate (up to 1.09-fold), Gly (up to 1.31-fold), malate (up to 1.60-fold), phosphate (up to 1.06-fold), and Val (up to 1.73-fold). Additionally, formate (up to 70.47-fold) and succinate (up to 5.04-fold) showed the same behavior, but to a greater extent (Supplementary Table S7). This suggests that higher temperatures increase the levels of these metabolites in Col0 seeds, but it decreases their levels in A. thaliana seeds overexpressing RcMLS imbibed at 35 °C. Therefore, seeds overexpressing RcMLS might show a different mechanism to cope with higher temperatures than Col0 seeds. We also assessed the metabolome of Col-0 and A. thaliana seeds overexpressing RcMLS in response to salt stress. For that, we used NMR analysis. Initially, PLS-DA was applied to the entire data set to assess variation in the metabolome in response to salt stress (Fig. 5a). R2 (0.88) and Q2 (0.63) values suggest that this is a strong PLS-DA model to extract metabolite changes in A. thaliana seeds overexpressing RcMLS in response to salt stress (Supplementary Figure S5). There is a clear separation along principal component 1 amongst Col-0 and A. thaliana seeds overexpressing RcMLS in response to salt stress. Based on the PLS-DA plot (Fig. 5a) and dendrogram analysis (Supplementary Figure S4b), A. thaliana seeds overexpressing RcMLS are closely related in terms of metabolome than Col-0 seeds. Once again, since we were mainly interested in differences that could explain
Fig. 3. Maximum germination percentage of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase under (a) high temperature and (b) salt stress. Results are presented as means and standard errors. Significant differences between samples was assessed by Tukey’s HSD (p < 0.05). Four replicates of 25 seeds each were used.
between Col-0 seeds imbibed at 22 and 35 °C. Based on the PLS-DA plot (Fig. 4a) and dendrogram analysis (Supplementary Figure S4a), A. thaliana seeds overexpressing RcMLS are closely related in terms of metabolome to each other than to Col-0 seeds. Levels of 24 metabolites varied significantly if we consider the comparison amongst Col-0 and A. thaliana seeds overexpressing RcMLS at 22 and 35 °C (Supplementary Figure S2a and Supplementary Table S6). This included four carbohydrates (fructose, glucose, raffinose,
Fig. 4. (a) PLS-DA and (b) VIP scores based on NMR and GC-TOF-MS metabolite profile of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase imbibed at different temperatures. 5
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Fig. 5. (a) PLS-DA and (b) VIP scores based on NMR metabolite profile of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase imbibed at 75 mM NaCl.
napus (Gzik, 1996) and in Beta vulgaris (Good and Zaplachinski, 1994), which is an indicative of its possible involvement in plant adaptation strategies in response to abiotic stresses. Under abiotic stress, methionine γ-lyase acts as an alternate route to fulfill the increased demand for isoleucine. Isoleucine is a branched-chain amino acid (BCAA) that belongs to the aspartate-derived pathway (Joshi et al., 2010). Branchedchain amino acid (BCAA) are involved in different mechanisms underlying plant to growth and defense (Xing and Last, 2017). For example, high temperature stress during Glycine max seed development led to accumulation of BCAAs (Chebrolu et al., 2016), whereas low- and high-temperature stress led to accumulation of isoleucine Zea mays (Sun et al., 2016). Therefore, increased levels of Met and Ile support the hypothesis that these amino acids play major role in enhancing abiotic stress tolerance in seeds of transgenic plants overexpressing RcMLS genes. Arabidopsis thaliana seeds overexpressing RcMLS showed a highly coordinated metabolic fingerprint in response to abiotic stresses in order to maintain cellular homeostasis during germination. These seeds showed higher levels of mono and disaccharides as compared with Col0 seeds. The accumulation of these metabolites is linked with reduced levels of succinate in response to high temperature and salt stress. R. communis seeds and seedlings possess a precise metabolic fingerprint to adjust germination and seedling growth under higher temperatures. This encompass a change in their C-N metabolism toward the accumulation of N-containing compounds in response to high temperatures (Ribeiro et al., 2015b). Herein, we hypothesized that overexpression of RcMLS has led to increased activity of the glyoxylate cycle, which has succinate as a by-product. Succinate levels were found to be reduced upon imbibition under abiotic stress, suggesting that this organic acid was possibly transferred to the mitochondria and transformed to oxaloacetate. Oxaloacetate, in turn fed the gluconeogenesis pathway in the cytosol explaining higher levels of fructose, glucose and sucrose (Fig. 6). Taken together, these results might explain the enhanced tolerance to abiotic stresses during germination of A. thaliana seeds overexpressing RcMLS.
the observed phenotype, we focused our attention on metabolites that varied between Col-0 and A. thaliana seeds overexpressing RcMLS when both were imbibed in 75 mM NaCl. Then, levels of 15 metabolites varied significantly, which included four carbohydrates (fructose, glucose, raffinose, sucrose), eight amino acids (Ala, Arg, Asn, Ile, Lys, Met, Phe, and Val), and three organic acids (acetate, lactate, and succinate) (Supplementary Figure S2b and Supplementary Table S8). Five metabolites showed higher in A. thaliana seeds overexpressing RcMLS than in Col-0 seeds imbibed under salt stress: acetate (up to 1.81-fold), glucose (up to 3.73-fold), Ile (up to 2.16-fold), lactate (up to 2.10-fold), and Met (up to 2.37-fold) (Supplementary Table S9). If we compare these results with those from Col-0 seeds imbibed in water, it seems that salt stress increased the levels of these metabolites in A. thaliana seeds overexpressing RcMLS to a greater extent than at Col-0 seeds. Curiously, levels of fructose (up to 3.90-fold), raffinose (up to 1.68-fold), and sucrose (up to 1.68-fold) increased in response to salt stress, but to a greater extent in Col-0 seeds than in A. thaliana seeds overexpressing RcMLS (Supplementary Table S9). Nevertheless, overexpression of RcMLS contributed to higher levels of these metabolites in response to salt stress. Levels of three metabolites were higher in Col-0 seeds than in A. thaliana seeds overexpressing RcMLS imbibed under salt stress: Asn (up to 1.57-fold), Phe (up to 1.41-fold), and succinate (up to 1.28-fold) (Supplementary Table S9). A. thaliana seeds overexpressing RcMLS seems to possess a robust and conserved biochemical reprogramming mechanism to cope with high temperature and salt stress (Fig. 6). We have observed accumulation of fructose, glucose and Met in A. thaliana seeds overexpressing RcMLS in response to high temperature and salt stress. Additionally, we have observed accumulation of isoleucine and fructose in A. thaliana seeds overexpressing RcMLS in response to salt stress. Methionine (Met) is a sulfur-containing amino acid usually found at low levels in seeds (Amir et al., 2019). It plays an important role in plant metabolism, not only as precursor for protein biosynthesis, but in the biosynthesis of important metabolites, such as ethylene and some polyamines. These metabolites act as plant hormones regulating metabolism and growth (Amir and Hacahm, 2013). Methionine is produced from homocysteine by the enzyme methionine synthase (MetSyn), whereas Met is a substrates for methionine γ-lyase during isoleucine biosynthesis (Joshi et al., 2010). The activity of MetSyn increases sharply upon imbibition reaching its peak at radicle protusion (Gallardo et al., 2002). Furthermore, seed germination is impaired in the presence of the MetSyn inhibitors and it can be rescued by Met supplementation in the germination medium (Gallardo et al., 2002). Met levels increased with the induction of drought stress in Brassica
4. Conclusion We assessed the possible role of RcMLS to the acquisition of tolerance in R. communis. The neighbor-joining phylogenetic tree allowed us to visualize important evolutionary relationship amongst MLS sequences from R. communis and other crop species and model plants. All MLS sequences showed high degree of conservation, including a highly conserved signature polypeptide found in motif-1 and identified as 6
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Fig. 6. Schematic representation of proposed metabolic signature of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase during germination under abiotic stress. Transgenic A. thaliana seeds overexpressing RcMLS genes showed increased levels of protective metabolites, along with specific modulation of gluconeogenesis.
References
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Author statement VCB carried out the physiological experiments. VCB, CPA, RRB, MGAC, and AGF conducted the metabolite profiling experiments. PRR coordinated the development of the study, carried out data processing, statistical analysis and wrote the manuscript. PRR, LGF, RDC, WL and HWMH participated in the design of the study and critical reading of the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments Funding was provided by UFBA, FAPESB, CNPq and CAPES. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112110. 7
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Overexpression of Ricinus communis L. malate synthase enhances seed tolerance to abiotic stress during germination
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Valdinei Carvalho Britoa,b, Catherine P. de Almeidaa, Rhaíssa R. Barbosaa, Maria G.A. Carosioc, Antônio G. Ferreirac, Luzimar G. Fernandezd, Renato D. de Castrod, Henk Hilhorste, Wilco Ligterinke, Paulo Roberto Ribeiroa,b,* a
Metabolomics Research Group, Departamento de Química Orgânica, Instituto de Química, Universidade Federal da Bahia, Rua Barão de Jeremoabo s/n, 40170-115, Salvador, Brazil b Programa de Pós-Graduação em Química Aplicada, Departamento de Ciências Exatas e Da Terra - Campus I, Universidade do Estado da Bahia, Salvador, Brazil c Laboratório de Ressonância Magnética Nuclear, Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil d Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100, Salvador, Brazil e Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University (WU), Droevendaalsesteeg 1, NL-6708 PB, Wageningen, The Netherlands
A R T I C LE I N FO
A B S T R A C T
Keywords: Abiotic stress Castor bean Functional characterization Temperature-responsive genes
Ricinus communis L. seeds can germinate at high temperatures, but further development of the seedlings is negatively affected. This mainly caused by impairment of energy-generating pathways when seeds are germinated at 35 °C. Ricinus communis malate synthase (RcMLS) is a key responsive gene in lipid mobilization and gluconeogenesis and as such might have a role in sustaining successful seed germination and seedling growth. Herein, we raised the question whether RcMLS might be involved in the biochemical and molecular mechanisms required for R. communis seed germination under unfavourable environmental conditions. For that, we used a robust approach that encompassed bioinformatics analysis, transgenic Arabidopsis thaliana (L.) Heynh seeds overexpressing RcMLS, along with phenotypical characterization of seed germination under abiotic stress. The phylogenetic tree revealed important evolutionary relationship amongst MLS sequences from R. communis and from other crop species/model plants. Overexpression of RcMLS enhanced A. thaliana seed germination under high temperature and salt stress. For example, wild-type A. thaliana Columbia seeds (Col-0) showed 37 % of maximum germination at 35 °C, whereas A. thaliana seeds overexpressing RcMLS showed up to 71%. When salt stress was applied (75 mM NaCl), maximum germination of Col-0 seeds reached 37%, whereas for A. thaliana seeds overexpressing RcMLS it reached up to 93%. Nuclear Magnetic Resonance (NMR) and Gas Chromatography coupled to Time-Of-Flight Mass Spectrometry (GC-TOF-MS) metabolomics analysis showed a robust metabolic signature of A. thaliana seeds overexpressing RcMLS in response to abiotic stress. They accumulated high levels of Met, Ile, fructose, glucose, and sucrose. Therefore, we suggested that overexpression of RcMLS has modulated the glyoxylate cycle and gluconeogenesis pathway in order to maintain cellular homeostasis under unfavorable environmental conditions. Our results provide important leads into the contribution of RcMLS to the underlying mechanism of R. communis seed germination under adverse environmental conditions. This might be helpful for breeding programs to develop more resistant R. communis cultivars which are more likely to sustain growth and high yield under the severe conditions found in arid and semi-arid areas worldwide.
1. Introduction Ricinus communis L. is a highly productive and resistant crop easily cultivated in hot and dry regions worldwide. The oil obtained from R. communis seeds possesses a diverse panel of applications, mainly because of the high levels of ricinoleic acid (up to 90% of the oil). This oil
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can reach up to three-times higher commercial value than Glycine max (L.) Merr. (soybean), Helianthus annuus L. (sunflower), and Brassica (canola) oils. Additionally, this species is able to grow and still yield as much as 900 kg of oil per hectare even under the harsh environmental conditions of arid and semi-arid regions worldwide where most other crops would not even survive (Salihu, 2014).
Corresponding author. E-mail addresses: [email protected], [email protected] (P.R. Ribeiro).
https://doi.org/10.1016/j.indcrop.2020.112110 Received 20 October 2019; Received in revised form 9 January 2020; Accepted 9 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.
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Molecular Evolutionary Genetics Analysis software (MEGA 7.0). Prediction of cellular locations were performed with Cello, whereas molecular weight and predicted isoelectric point (pI) were provided by ProtParam. Analysis of the conserved motifs were performed by the Multiple EM for Motif Elicitation (MEME). Then, the potential motif sequences candidates were loaded into the Pfam protein database. Further details are described in Gomes Neto et al. (2018).
Malate synthase (MLS) is an acyltransferase enzyme that catalyzes the Claisen-like condensation of acetyl-coenzyme A and glyoxylate to produce malate and coenzyme A. After its production, malate might be transported to the cytosol, where it is converted to oxaloacetate that in turn enters gluconeogenesis. Along with isocitrate lyase, MLS bypass the tricarboxylic acid cycle through the glyoxylate shunt in a sequence of anaplerotic reactions that enable plants and micro-organisms to use two-carbon compounds derived from lipids, such as acetate, to produce energy and support seed germination and seedling growth (González, 1990; Rodriguez et al., 1990). In R. communis, acetate is produced in the glyoxysome via the β-oxidation of fatty acids that are mobilized from lipid bodies in the endosperm upon seed imbibition and germination (Ribeiro et al., 2015b; Rodriguez et al., 1990). Malate synthase is highly active during early seed maturation and germination, but low activity was observed in mature dry seeds (Faraoni et al., 2019; González, 1990; Zhao et al., 2018). Although it has been suggested that in A. thaliana seedlings MLS is partially dispensable for lipid degradation and gluconeogenesis (Cornah et al., 2004), we have previously reported that RcMLS might be essential to sustain R. communis successful growth (Ribeiro et al., 2015c). Metabolite profiling techniques have provided important insights into the understanding of plant plasticity and adaptation to abiotic stress (Arbona et al., 2013; Ribeiro et al., 2018). Nuclear magnetic resonance and mass spectrometry are the most important techniques for metabolite detection in plant biology since they combine excellent sensitivity and high-throughput analysis to characterize the metabolome of a biological system. A combination of Gas and Liquid Chromatography coupled to Mass Spectrometry (GC–MS and LC–MS) were used to show that high temperatures decreased RcMLS expression leading to lower levels of carbohydrates in germinating R. communis seeds (Ribeiro et al., 2015a, d). Additionally, it has been suggested that high temperatures impair β-oxidation downstream pathways responsible to produce energy (Ribeiro et al., 2015c). This is supported by the fact that overexpression of RcMLS led to greater levels of starch in Nicotiana benthamiana Domin leaves (Ribeiro et al., 2015c). In cotton (Gossypium hirsutum L.), MLS activity increases during seed maturation, and it is kept at steady levels throughout desiccation and imbibition, but it increases again upon germination (Turley and Trelease, 1987). Malate synthase catalytic activity is highly dependent on the temperature: highest activity was observed at 35 °C for Gossypium hirsutum and at 28 °C for Helianthus annuus L., whereas optimal seedling emergence was observed at 31−37 °C for G. hirsutum and at 27−35 °C for H. annuus (Mahan, 2000). Overexpression of Escherichia coli MLS in Chlamydomonas reinhardtii led to greater microalgal biomass as compared with wild-type cells through enhancement of the heterotrophic metabolism (Paik et al., 2019). Nevertheless, there is no study that correlates MLS expression and activity with acquisition of tolerance to abiotic stresses during seed germination. Therefore, we addressed the question whether RcMLS might be involved in crucial mechanisms required for R. communis seed germination under harsh conditions.
2.2. Extraction of RNA, cDNA synthesis, cloning and plant transformation Ricinus communis samples were collected during seed germination at 25 °C. The procedure used for extraction of total RNA and for the synthesis of first strand cDNA is described in details in Gomes Neto et al. (2018). Ricinus communis malate synthase (RcMLS) gene was cloned by using the Gateway® technology (Ribeiro et al., 2015c). Primers used and additional information about the cloned genes are presented in Supplementary Table S1. For cloning purposes, we used the donor vector pDONr207 and the gateway binary expression vector pGD625 bearing the CaMV35S (CaMV-cauliflower mosaic virus). Further details are described in Ribeiro et al. (2015c). A. tumefaciens strains carrying the gene of interest (CaMV35S::RcMLS) were used for plant transformation in a modified floral‐dip protocol (Logemann et al., 2006). The expression vector pGD625 contained a kanamycin resistance selection marker and, therefore, transformants were selected by sowing seeds on 1/2 strength MS medium supplemented with 50 μg.mL−1 kanamycin. 2.3. Germination under abiotic stress Seeds of A. thaliana plants overexpressing RcMLS and seeds of Col-0 were germinated under abiotic stress conditions. They were sown on petri dishes with germination paper moistened with 6 mL water or saline solutions followed by incubation at 4 °C i for 72 h, for stratification purposes. High temperature stress assays were performed by incubating seeds at different temperatures (22 °C, 31 °C through 36 °C). Saline stress was performed by incubating stratified seeds at 0, 25, 50, and 75 mM NaCl. Germination percentage was counted daily and the data was processed using the GERMINATOR software (Gomes Neto et al., 2018; Ribeiro et al., 2015c). For metabolite profiling analysis at different temperatures seeds were imbibed in H2O for 30 h at 22 and 35 °C, whereas for metabolite profiling analysis under salt stress seeds were imbibed in water and in 75 mM NaCl for 30 h. 2.4. GC-TOF-MS and NMR-based metabolite profiling analysis Twenty mg of each sample were extracted in a system containing methanol, chloroform and water, derivatized to obtain trimethylsilyl (TMS) derivatives, and injected into a gas chromatographer coupled to a quadrupole time of flight mass spectrometry system (GC-TOF-MS). Further details are provided in Ribeiro et al. (2014). 1 H-NMR spectra were recorded at 20 °C using Bruker AVANCE III spectrometer (400 MHz). 3-(trimethylsilyl) propionic acid-D4 sodium salt (TMSP-d4) was used as internal standard. For 1D experiments, we used the cpmgpr1d (Bruker standard) pulse sequence, performed with 64 scans (ns) with 64k points during acquisition (td) in a spectral window (sw) of 15 ppm and with a receiver gain (rg) of 1290, 3.0 s between each acquisition (d1) and this had a total time (aq) of approximately 5.46 s. The power attenuation for the pre-saturation (PLdB9) was 45.00 dB. Total acquisition time of each spectrum was 11 min and 35 s. Spectra were processed with 64k (SI) using an exponential multiplication with a lb of 0.3 Hz and phase and baseline automatic correction. For HMBC experiments, we used the hmbcgplpndqf (Bruker standard) performed with 64 scans (ns) with 4k (F2) and 256 (F1) points during acquisition (td) in a spectral window (sw) of 15 ppm (F2) and 238 ppm (F1). The total acquisition time of each HMBC experiment was 6 h and 34 min.
2. Material and methods 2.1. Phylogenetic analysis and prediction of cellular locations Initially, Ricinus communis malate synthase protein sequence (30,147.m013773) was used as queries in a blastp search within the proteome of Arabidopsis thaliana (L.) Heynh, Brassica oleracea L., Cucumis sativus L., Glycine max (L.) Merr., Manihot esculenta Cranz, Oryza sativa L., Populus trichocarpa Torrey & A. Gray, Solanum lycopersicum L., Solanum tuberosum L., Sorghum bicolor L. Moench, and Zea mays L. These sequences were named by adding the initials of the plant species followed by MLS, for example AtMLS stands for Arabidopsis thaliana malate synthase. The identified MLS sequences were aligned at the online platform multiple sequence comparison by log-expectation (MUSCLE) and the phylogenetic analysis was performed by using 2
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evolutionary relationship between R. communis, M. esculenta, and P. trichocarpa that have been suggested by phylogenetic analysis of superoxide dismutase (Gomes Neto et al., 2018), papain-like cysteine protease (Zou et al., 2017), aquaporin (Zou et al., 2015), S-phase kinase-associated protein 1 (SKP1) (HajSalah El Beji et al., 2019), and argonaute (Mirzaei et al., 2014) proteins. MLS protein sequences from A. thaliana and B. oleracea, both belonging to the Brassicaceae family, clustered together in the same subclade, whereas MLS protein sequences from Solanum lycopersicum and S. tuberosum also clustered together in a different subclade (Fig. 1). Six putative motifs were found amongst MLS genes, with lengths ranging from 29 to 159 aa (Fig. 2, Supplementary Table S2). All MLS sequences contained six motifs from which five were identified as MLS protein domains (pfam code CL0151) (Supplementary Table S3). The MLS signature polypeptide is [K/R]-[D/E/N/Q]-[H]-[XeX]-GLNCGRWDY-[L/I/V/M]-[F] (Zambuzzi-Carvalho et al., 2009). The MLS signature polypeptide was found in motif-1 and identified as RDHSVGLNCGRWDYIF. Since MLS are biosynthesized in the cytoplasm and later transferred to the glyoxysomes, these sequences show a protein targeting signal (PTS) residing in the last three amino acids of the C-terminus. The PTS is defined by the consensus [A/C/S]-[K/R/H]-[L/M] (Zambuzzi-Carvalho et al., 2009). Ricinus communis malate synthase protein sequence (RcMLS) has the SRL tripeptide as the glyoxysomal PTS. This PTS was also observed for PtMLS, SlMLS, StMLS, AtMLS, and BoMLS. GmMLS, and CsMLS have the SKL glyoxysomal PTS, whereas OsMLS and ZmMLS have the CKL glyoxysomal PTS. Although acetylCoA is fairly common in enzymatic reactions the lack of conserved CoA binding motifs is still very intriguing. It seems that MLS does not possess a specific motif for recognition of CoA, but its recognition occurs through hydrogen bonds and van der Waals interactions with the polypeptide backbone (Anstrom et al., 2003). Nevertheless, these results suggest high protein evolutionary conservation amongst these organisms (De La Torre et al., 2017; Rahman et al., 2013).
2.5. Data processing, compound identification, and multivariate statistical analysis We used the online pipeline software NMRProcFlow to process 1HNMR spectra, which included the standard workflow (correction of the baseline, alignment and peak integration) (Jacob et al., 2017; Ribeiro et al., 2015b). Compound identification was performed by comparison of the NMR signals of authentic samples and data from the literature. GC-TOF-MS data were processed was described in Ribeiro et al. (2014). Compound identification was performed by comparison of the mass (MS) spectra and data from spectral libraries (NIST and Golm). Multivariate statistical analysis were performed at MetaboAnalyst 3.0, by using the default settings provided in the platform (Xia and Wishart, 2011). Further details are provided in Ribeiro et al. (2014) and in Santos et al. (2018). 3. Results and discussion 3.1. Identification and phylogenetic analysis of the MLS gene in different crop and model plant species Ricinus communis malate synthase protein sequence (30,147.m013773) possess 567 amino acids (Supplementary Figure S1), molecular formula of C2866H4467N815O816S27, molecular weight of 64,262.59 Da, and theoretical pI of 8.53. It possesses an instability index of 38.55, which classifies it as a stable protein. These results are supported by available data from other MLS protein sequences (Hoppe and Theimer, 1995; Jaunin et al., 1995; Köller and Kindl, 1980; Lewis and Doyle, 2001; Mori et al., 1988; Turley et al., 1990). Prediction of cellular location indicated that all MLS sequences are cytoplasmic with reliability index ranging from 3.997 to 4.673. At first, one might think that this does not fit with the well-known location of MLS in the glyoxysomes (special peroxisomes) (Ahn et al., 2016; Frevert et al., 1980; Guex et al., 1995; Ha et al., 2018; Kunze et al., 2006; Ohno et al., 2015; Trelease et al., 1974), but several studies have demonstrated that MLS is indeed produced in the cytosol, but subsequently transferred into the glyoxysomes (Gerdes et al., 1982; Kruse et al., 1981; Kruse and Kindl, 1983; Turley and Trelease, 1987; Wolfram and Helmut, 1980). As a matter of fact, the octameric structure of malate synthase present in glyoxysomes are assembled from its monomeric precursors which are biosynthesized in the cytoplasm. The monomeric precursor possesses identical subunit molecular weight, but differs from the mature enzyme by its oligomerization and aggregation state (Gerdes et al., 1982; Kruse and Kindl, 1983). Ricinus communis malate synthase protein sequence was used as queries in a blastp search against whole proteome of some important industrial crops and/or model plants to identify potential MLS proteins. We constructed a neighbor-joining (NJ) phylogenetic tree using their full-length amino acid sequences to investigate the evolutionary relationship between MLS sequences, (Fig. 1). For that, only well-annotated full sequences were considered. The retrieved MLS sequences showed similar lengths ranging from 534 aa for MeMLS and 573 aa for SbMLS. This included one MLS sequence from Arabidopsis thaliana, Brassica oleracea, Cucumis sativus, Manihot esculenta, Oryza sativa, Populus trichocarpa, Solanum tuberosum, Sorghum bicolor, and Zea mays. However, Glycine max and Solanum lycopersicum seem to possess multiple MLS copies in their genome and three sequences were retrieved. It is not clear, however, why and how genome duplication events may have taken place in G. max and S. lycopersicum and their relationship with plant adaptation to adverse land environments. There is a clear separation in the NJ phylogenetic tree between MLS sequences from monocots and eudicots species. Eudicots form a large clade, whereas the three monocots appear in an isolated clade. Nevertheless, important evolutionary insights can be obtained from a closer analysis of the eudicots’ clade: RcMLS clustered within the same subclade as PtMLS and MeMLS (Fig. 1). This supports a closer
3.2. Arabidopsis thaliana seeds overexpressing RcMLS showed enhanced tolerance to abiotic stresses during germination Arabidopsis thaliana seeds overexpressing RcMLS were germinated under high temperature and salt stress to ultimately address the contribution of RcMLS for the acquisition of tolerance under abiotic stress. For that, we used seeds from two A. thaliana overexpression independent lines (RcMLS1 and RcMLS2) and seeds from A. thaliana wild type (Col-0). Initially, a pilot germination assay was performed with Col-0 seeds at different temperatures (22 °C, 31−36 °C). Col-0 seeds germinated nearly 100% at temperature below 33 °C. At 34 °C maximum germination lowered to 92%, whereas at 35 °C maximum germination lowered to 36%. At 36 °C no germination was observed. Therefore, 34 °C was selected as the threshold temperature for further germination experiments. At 34 °C, Col-0 seeds showed 94% of maximum germination, whereas RcMLS1 seeds showed 97% and RcMLS2 seeds showed 88% of maximum germination (Fig. 3a). At 35 °C, Col-0 seeds showed 37% of maximum germination, whereas A. thaliana seeds overexpressing RcMLS showed up to 71% of maximum germination (Fig. 3a). Therefore, overexpression of RcMLS enhanced A. thaliana seed germination thermotolerance. Four NaCl concentrations (25, 50, and 75 mM) were used to impose salt stress during germination (Fig. 3b). Nearly 100% of maximum seed germination was observed in control conditions and at 25 mM NaCl for both Col-0 and A. thaliana seeds overexpressing RcMLS (Fig. 3b). At 50 mM NaCl, Col-0 seeds showed 83% of maximum germination, whereas RcMLS1 seeds showed 96% and RcMLS2 seeds showed 73% of maximum germination (Fig. 3a), but no significant statistical differences were observed whatsoever. At 75 mM NaCl, maximum germination of Col-0 seeds reached 37%, whereas A. thaliana seeds overexpressing RcMLS showed up to 93% of maximum germination 3
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Fig. 1. Neighbor-joining (NJ) phylogenetic tree of malate synthase protein sequences from different plant species. AtMLS (Arabidopsis thaliana), BoMLS (Brassica oleracea), CsMLS (Cucumis sativus), GmMLS (Glycine max), MeMLS (Manihot esculenta), OsMLS (Oryza sativa), PtMLS (Populus trichocarpa), RcMLS (Ricinus communis), SlMLS (Solanum lycopersicum), StMLS (Solanum tuberosum), SbMLS (Sorghum bicolor), and ZmMLS (Zea mays).
Fig. 2. Conserved motifs of malate synthase protein sequences from different plant species. AtMLS (Arabidopsis thaliana), BoMLS (Brassica oleracea), CsMLS (Cucumis sativus), GmMLS (Glycine max), MeMLS (Manihot esculenta), OsMLS (Oryza sativa), PtMLS (Populus trichocarpa), RcMLS (Ricinus communis), SlMLS (Solanum lycopersicum), StMLS (Solanum tuberosum), SbMLS (Sorghum bicolor), and ZmMLS (Zea mays).
3.3. Arabidopsis thaliana seeds overexpressing RcsMLS showed a specific metabolic signature under abiotic stress conditions
(Fig. 3b). Therefore, overexpression of RcMLS also enhanced A. thaliana seed germination performance under salt stress. Cornah et al. (2004) used seeds of two independent mls t-DNA lines (At5g03860) to assess the role of the AtMLS in important energy-generating pathways. Seedlings of the knock-out mutants showed inhibition of hypocotyl elongation, impairment of root development, and compromised seedling establishment during short days. Nevertheless, these phenotypes could be rescued by applying exogeneous succinate to the seedlings. They also showed that in the absence of the MLS enzyme, mutant seedlings were able to use [14C]-glycine and [14C]-serine as gluconeogenic precursors (Cornah et al., 2004). Unfortunately, the authors did not evaluate seed performance under abiotic stress. This is a pioneer study that assessed the possible relationship between the glyoxylate cycle enzyme MLS and the acquisition of tolerance to abiotic stress in the oilseed crop R. communis, which in turn can be further investigated in other industrial crops and model plants. Therefore, overexpression of RcMLS enhanced A. thaliana seed germination under high temperature and salt stress, highlighting the fact that a better understanding of RcMLS might be essential to enhance R. communis performance under unfavorable conditions.
Since the glyoxylate cycle allow plants to use acetate derived from the mobilization of storage lipids to produce carbohydrates, we raised the question whether overexpression of RcMLS gene would lead to a specific metabolic signature that would explain the enhanced germination performance under unfavorable conditions of A. thaliana seeds overexpressing RcMLS. Initially, we assessed the metabolome of Col-0 and A. thaliana seeds overexpressing RcMLS in response to different temperatures. We detected over 100 peaks by GC-TOF-MS, of which 17 could be annotated (Supplementary Table S4), whereas 16 metabolites were identified by NMR analysis (Supplementary Table S5). Partial least squares discriminant analysis (PLS-DA) was applied to the entire data set to assess variation in the metabolome in response to high temperatures (Fig. 4a). The goodness of fit (R2) and the predictability of the model (Q2) values suggests that this is a very strong PLSDA model that allowed us to extract metabolite changes in A. thaliana seeds overexpressing RcMLS in response to high temperatures (Supplementary Figure S3). There is a clear separation along principal component 1 amongst Col-0 and A. thaliana seeds overexpressing RcMLS, whereas principal component 2 (PC2) reflects a separation 4
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sucrose), twelve amino acids (Ala, Arg, Asn, Asp, Glu, Gly, Lys, Met, pyroglutamate, Ser, Thr, Val), seven organic acids (citrate, formate, fumarate, malate, succinate, threonate, t-sinnapic acid), along with allantoin, ethanolamine, galactinol, and phosphate (Supplementary Table S6). We set the variable importance in projection (VIP) threshold as 1 (Xia and Wishart, 2011) to discuss the possible contribution of each individual metabolites to the enhanced tolerance of A. thaliana seeds overexpressing RcMLS (Fig. 4b). Since we were mainly interested in differences that could explain the observed phenotype, we focused our attention on metabolites that varied between Col-0 and A. thaliana seeds overexpressing RcMLS seeds when both were imbibed at 35 °C. Seven metabolites showed higher levels in A. thaliana seeds overexpressing RcMLS than in Col-0 seeds: allantoin (up to 1.39-fold), Asn (up to 5.72-fold), ethanolamine (up to 9.68-fold), glucose (up to 10.27fold), Lys (up to 1.21-fold), Met (up to 2.66-fold), and sucrose (up to 1.09-fold) (Supplementary Table S7). It seems that higher temperatures increased the levels of these metabolites in A. thaliana seeds overexpressing RcMLS to a greater extent than at Col-0 seeds. Therefore, overexpression of RcMLS contributed to higher levels of these metabolites in response to temperature. Levels of five metabolites were higher in Col-0 seeds imbibed at 35 °C than in A. thaliana seeds overexpressing RcMLS imbibed at the same temperature: fumarate (up to 1.09-fold), Gly (up to 1.31-fold), malate (up to 1.60-fold), phosphate (up to 1.06-fold), and Val (up to 1.73-fold). Additionally, formate (up to 70.47-fold) and succinate (up to 5.04-fold) showed the same behavior, but to a greater extent (Supplementary Table S7). This suggests that higher temperatures increase the levels of these metabolites in Col0 seeds, but it decreases their levels in A. thaliana seeds overexpressing RcMLS imbibed at 35 °C. Therefore, seeds overexpressing RcMLS might show a different mechanism to cope with higher temperatures than Col0 seeds. We also assessed the metabolome of Col-0 and A. thaliana seeds overexpressing RcMLS in response to salt stress. For that, we used NMR analysis. Initially, PLS-DA was applied to the entire data set to assess variation in the metabolome in response to salt stress (Fig. 5a). R2 (0.88) and Q2 (0.63) values suggest that this is a strong PLS-DA model to extract metabolite changes in A. thaliana seeds overexpressing RcMLS in response to salt stress (Supplementary Figure S5). There is a clear separation along principal component 1 amongst Col-0 and A. thaliana seeds overexpressing RcMLS in response to salt stress. Based on the PLS-DA plot (Fig. 5a) and dendrogram analysis (Supplementary Figure S4b), A. thaliana seeds overexpressing RcMLS are closely related in terms of metabolome than Col-0 seeds. Once again, since we were mainly interested in differences that could explain
Fig. 3. Maximum germination percentage of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase under (a) high temperature and (b) salt stress. Results are presented as means and standard errors. Significant differences between samples was assessed by Tukey’s HSD (p < 0.05). Four replicates of 25 seeds each were used.
between Col-0 seeds imbibed at 22 and 35 °C. Based on the PLS-DA plot (Fig. 4a) and dendrogram analysis (Supplementary Figure S4a), A. thaliana seeds overexpressing RcMLS are closely related in terms of metabolome to each other than to Col-0 seeds. Levels of 24 metabolites varied significantly if we consider the comparison amongst Col-0 and A. thaliana seeds overexpressing RcMLS at 22 and 35 °C (Supplementary Figure S2a and Supplementary Table S6). This included four carbohydrates (fructose, glucose, raffinose,
Fig. 4. (a) PLS-DA and (b) VIP scores based on NMR and GC-TOF-MS metabolite profile of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase imbibed at different temperatures. 5
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Fig. 5. (a) PLS-DA and (b) VIP scores based on NMR metabolite profile of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase imbibed at 75 mM NaCl.
napus (Gzik, 1996) and in Beta vulgaris (Good and Zaplachinski, 1994), which is an indicative of its possible involvement in plant adaptation strategies in response to abiotic stresses. Under abiotic stress, methionine γ-lyase acts as an alternate route to fulfill the increased demand for isoleucine. Isoleucine is a branched-chain amino acid (BCAA) that belongs to the aspartate-derived pathway (Joshi et al., 2010). Branchedchain amino acid (BCAA) are involved in different mechanisms underlying plant to growth and defense (Xing and Last, 2017). For example, high temperature stress during Glycine max seed development led to accumulation of BCAAs (Chebrolu et al., 2016), whereas low- and high-temperature stress led to accumulation of isoleucine Zea mays (Sun et al., 2016). Therefore, increased levels of Met and Ile support the hypothesis that these amino acids play major role in enhancing abiotic stress tolerance in seeds of transgenic plants overexpressing RcMLS genes. Arabidopsis thaliana seeds overexpressing RcMLS showed a highly coordinated metabolic fingerprint in response to abiotic stresses in order to maintain cellular homeostasis during germination. These seeds showed higher levels of mono and disaccharides as compared with Col0 seeds. The accumulation of these metabolites is linked with reduced levels of succinate in response to high temperature and salt stress. R. communis seeds and seedlings possess a precise metabolic fingerprint to adjust germination and seedling growth under higher temperatures. This encompass a change in their C-N metabolism toward the accumulation of N-containing compounds in response to high temperatures (Ribeiro et al., 2015b). Herein, we hypothesized that overexpression of RcMLS has led to increased activity of the glyoxylate cycle, which has succinate as a by-product. Succinate levels were found to be reduced upon imbibition under abiotic stress, suggesting that this organic acid was possibly transferred to the mitochondria and transformed to oxaloacetate. Oxaloacetate, in turn fed the gluconeogenesis pathway in the cytosol explaining higher levels of fructose, glucose and sucrose (Fig. 6). Taken together, these results might explain the enhanced tolerance to abiotic stresses during germination of A. thaliana seeds overexpressing RcMLS.
the observed phenotype, we focused our attention on metabolites that varied between Col-0 and A. thaliana seeds overexpressing RcMLS when both were imbibed in 75 mM NaCl. Then, levels of 15 metabolites varied significantly, which included four carbohydrates (fructose, glucose, raffinose, sucrose), eight amino acids (Ala, Arg, Asn, Ile, Lys, Met, Phe, and Val), and three organic acids (acetate, lactate, and succinate) (Supplementary Figure S2b and Supplementary Table S8). Five metabolites showed higher in A. thaliana seeds overexpressing RcMLS than in Col-0 seeds imbibed under salt stress: acetate (up to 1.81-fold), glucose (up to 3.73-fold), Ile (up to 2.16-fold), lactate (up to 2.10-fold), and Met (up to 2.37-fold) (Supplementary Table S9). If we compare these results with those from Col-0 seeds imbibed in water, it seems that salt stress increased the levels of these metabolites in A. thaliana seeds overexpressing RcMLS to a greater extent than at Col-0 seeds. Curiously, levels of fructose (up to 3.90-fold), raffinose (up to 1.68-fold), and sucrose (up to 1.68-fold) increased in response to salt stress, but to a greater extent in Col-0 seeds than in A. thaliana seeds overexpressing RcMLS (Supplementary Table S9). Nevertheless, overexpression of RcMLS contributed to higher levels of these metabolites in response to salt stress. Levels of three metabolites were higher in Col-0 seeds than in A. thaliana seeds overexpressing RcMLS imbibed under salt stress: Asn (up to 1.57-fold), Phe (up to 1.41-fold), and succinate (up to 1.28-fold) (Supplementary Table S9). A. thaliana seeds overexpressing RcMLS seems to possess a robust and conserved biochemical reprogramming mechanism to cope with high temperature and salt stress (Fig. 6). We have observed accumulation of fructose, glucose and Met in A. thaliana seeds overexpressing RcMLS in response to high temperature and salt stress. Additionally, we have observed accumulation of isoleucine and fructose in A. thaliana seeds overexpressing RcMLS in response to salt stress. Methionine (Met) is a sulfur-containing amino acid usually found at low levels in seeds (Amir et al., 2019). It plays an important role in plant metabolism, not only as precursor for protein biosynthesis, but in the biosynthesis of important metabolites, such as ethylene and some polyamines. These metabolites act as plant hormones regulating metabolism and growth (Amir and Hacahm, 2013). Methionine is produced from homocysteine by the enzyme methionine synthase (MetSyn), whereas Met is a substrates for methionine γ-lyase during isoleucine biosynthesis (Joshi et al., 2010). The activity of MetSyn increases sharply upon imbibition reaching its peak at radicle protusion (Gallardo et al., 2002). Furthermore, seed germination is impaired in the presence of the MetSyn inhibitors and it can be rescued by Met supplementation in the germination medium (Gallardo et al., 2002). Met levels increased with the induction of drought stress in Brassica
4. Conclusion We assessed the possible role of RcMLS to the acquisition of tolerance in R. communis. The neighbor-joining phylogenetic tree allowed us to visualize important evolutionary relationship amongst MLS sequences from R. communis and other crop species and model plants. All MLS sequences showed high degree of conservation, including a highly conserved signature polypeptide found in motif-1 and identified as 6
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Fig. 6. Schematic representation of proposed metabolic signature of Arabidopsis thaliana seeds overexpressing Ricinus communis malate synthase during germination under abiotic stress. Transgenic A. thaliana seeds overexpressing RcMLS genes showed increased levels of protective metabolites, along with specific modulation of gluconeogenesis.
References
RDHSVGLNCGRWDYIF and moderate conserved targeting signal identified within the consensus [A/C/S]-[K/R/H]-[L/M]. Our results demonstrate that overexpression of RcMLS enhanced A. thaliana seed germination under a variety of abiotic stresses. Additionally, it was observed a specific metabolic signature that include increased levels of the Met, Ile, fructose, glucose, and sucrose, along with reduced levels of succinate upon imbibition under abiotic stress. We hypothesized that overexpression of RcMLS has led to increased activity of the glyoxylate cycle, which in turn fed the gluconeogenesis pathway in the cytosol explaining higher levels of mono and disaccharides. Our results might contribute toward the understanding of the molecular and biochemical mechanisms underlying R. communis seed germination and seedling establishment under abiotic stress conditions. This might aid breeding programs to develop more tolerant and high-yield genotypes that could be used by family farmers under the harsh environmental conditions in arid and semi-arid areas worldwide.
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Author statement VCB carried out the physiological experiments. VCB, CPA, RRB, MGAC, and AGF conducted the metabolite profiling experiments. PRR coordinated the development of the study, carried out data processing, statistical analysis and wrote the manuscript. PRR, LGF, RDC, WL and HWMH participated in the design of the study and critical reading of the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments Funding was provided by UFBA, FAPESB, CNPq and CAPES. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112110. 7
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