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Molecular cloning and expression analysis of three ThERFs involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products.

ABSTRACT: As a subfamily of the APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factor superfamily, the ethylene response factor (ERF) is widely involved in the regulation of growth and response to various abiotic stresses in plants, and has been shown to be the main transcription factor regulating transcription of the genes related to hypoxia and waterlogging stress. In this study, three ThERF genes, with significant differences in expression profile in response to flooding stress, were identified from the transcriptomics data acquired from Taxodium hybrid 'Zhongshanshan 406' (T. mucronatum Tenore × T. distichum (L.) Rich) under waterlogging stress: ThERF15, ThERF39 and ThRAP2.3 (GenBank ID: KY463467, KY463468 and KY463470, respectively).The full-length cDNA of each of the three ERFs was obtained using the RACE (rapid amplification cDNA ends) method, and all three were intron-free. Multiple protein sequence alignments indicated that ThERF15, ThERF39 and ThRAP2.3 proteins all had only one AP2-ERF domain and belonged to the ERF subfamily. A transient gene expression assay demonstrated that ThERF15, ThERF39 and ThRAP2.3 were all localized to the nucleus. Real-time quantitative PCR (qPCR) revealed that the expression of ThERF15, ThERF39 and ThRAP2.3 exhibited significant differences, compared with the control, in response to two levels of flooding treatment (half-flooding or total-submergence) of 'Zhongshanshan 406'. Quantification of ethylene concentration revealed that ethylene was more relevant to the level of expression than the period of flooding treatment. Based on the experimental results above, ThERF15, ThERF39 and ThRAP2.3 were identified as being related to the regulation of downstream flooding- responsive gene expression in 'Zhongshanshan 406'. ThRAP2.3 is most likely to be a key downstream-response ERF gene to respond to the output of the ethylene signal generated by flooding stress.

SUBMITTER: Fan W

PROVIDER: S-EPMC5853676 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Taxodium is renowned for its strong tolerance to waterlogging stress, thus it has great ecological and economic potential. However, the scant genomic resources in genus Taxodium have greatly hindered further exploration of its underlying flood-tolerance mechanism. Taxodium 'Zhongshansa' is an interspecies hybrid of T. distichum and T. mucronatum, and has been widely planted in southeastern China. To understand the genetic basis of its flood tolerance, we analyzed the transcriptomes of Taxodium 'Zhongshansa' roots and shoots in response to short-term waterlogging. RESULTS: RNA-seq was used to analyze genome-wide transcriptome changes of Taxodium 'Zhongshansa 406' clone root and shoot treated with 1 h of soil-waterlogging stress. After de novo assembly, 108,692 unigenes were achieved, and 70,260 (64.64%) of them were annotated. There were 2090 differentially expressed genes (DEGs) found in roots and 394 in shoots, with 174 shared by both of them, indicating that the aerial parts were also affected. Under waterlogging stress, the primary reaction of hypoxic-treated root was to activate the antioxidative defense system to prevent cells experiencing reactive oxygen species (ROS) poisoning. As respiration was inhibited and ATP decreased, another quick coping mechanism was repressing the energy-consuming biosynthetic processes through the whole plant. The glycolysis and fermentation pathway was activated to maintain ATP production in the hypoxic root. Constantly, the demand for carbohydrates increased, and carbohydrate metabolism were accumulated in the root as well as the shoot, possibly indicating that systemic communications between waterlogged and non-waterlogged tissues facilated survival. Amino acid metabolism was also greatly influenced, with down-regulation of genes involvedin serine degradation and up-regulation of aspartic acid degradation. Additionally, a non-symbiotic hemoglobin class 1 gene was up-regulated, which may also help the ATP production. Moreover, the gene expression pattern of 5 unigenes involving in the glycolysis pathway revealed by qRT-PCR confirmed the RNA-Seq data. CONCLUSIONS: We conclude that ROS detoxification and energy maintenance were the primary coping mechanisms of 'Zhongshansa' in surviving oxygen deficiency, which may be responsible for its remarkable waterlogging tolerance. Our study not only provided the first large-scale assessment of genomic resources of Taxodium but also guidelines for probing the molecular mechanism underlying 'Zhongshansa' waterlogging tolerance.

Project description:Alfalfa (Medicago sativa) is the most widely grown and most important forage crop in the world. However, alfalfa is susceptible to waterlogging stress, which is the major constraint for its cultivation area and crop production. So far, the molecular mechanism of alfalfa response to the waterlogging is largely unknown. Here, comparative transcriptome combined with proteomic analyses of two cultivars (M12, tolerant; M25, sensitive) of alfalfa showing contrasting tolerance to waterlogging were performed to understand the mechanism of alfalfa in response to waterlogging stress. Totally, 748 (581 up- and 167 down-regulated) genes were differentially expressed in leaves of waterlogging-stressed alfalfa compared with the control (M12_W vs. M12_CK), whereas 1193 (740 up- and 453 down-regulated) differentially abundant transcripts (DATs) were detected in the leaves of waterlogging-stressed plants in comparison with the control plants (M25_W vs. M25_CK). Furthermore, a total of 187 (122 up- and 65 down-regulated) and 190 (105 up- and 85 down-regulated) differentially abundant proteins (DAPs) were identified via isobaric tags for relative and absolute quantification (iTRAQ) method in M12_W vs. M12_CK and M25_W vs. M25_CK comparison, respectively. Compared dataset analysis of proteomics and transcriptomics revealed that 27 and eight genes displayed jointly up-regulated or down-regulated expression profiles at both mRNA and protein levels in M12_W vs. M12_CK comparison, whereas 30 and 27 genes were found to be co-up-regulated or co-down-regulated in M25_W vs. M25_CK comparison, respectively. The strongly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for co-up-regulated genes at mRNA and protein levels in M12_W vs. M12_CK comparison were 'Amino sugar and nucleotide sugar metabolism', 'Arginine and proline metabolism' and 'Starch and sucrose metabolism', whereas co-up-regulated protein-related pathways including 'Arginine and proline metabolism' and 'Valine, leucine and isoleucine degradation' were largely enriched in M25_W vs. M25_CK comparison. Importantly, the identified genes related to beta-amylase, Ethylene response Factor (ERF), Calcineurin B-like (CBL) interacting protein kinases (CIPKs), Glutathione peroxidase (GPX), and Glutathione-S-transferase (GST) may play key roles in conferring alfalfa tolerance to waterlogging stress. The present study may contribute to our understanding the molecular mechanism underlying the responses of alfalfa to waterlogging stress, and also provide important clues for further study and in-depth characterization of waterlogging-resistance breeding candidate genes in alfalfa.

Project description:The overall health of a plant is constantly affected by the changing and hostile environment. Due to climate change and the farming pattern of rice (Oryza sativa) and rapeseed (Brassica napus L.), stress from waterlogging poses a serious threat to productivity assurance and the yield of rapeseed in China's Yangtze River basin. In order to improve our understanding of the complex mechanisms behind waterlogging stress and identify waterlogging-responsive proteins, we firstly conducted iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic analysis of rapeseed roots under waterlogging treatments, for both a tolerant cultivar ZS9 and sensitive cultivar GH01. A total of 7736 proteins were identified by iTRAQ, of which several hundred showed different expression levels, including 233, 365, and 326 after waterlogging stress for 4H, 8H, and 12H in ZS9, respectively, and 143, 175, and 374 after waterlogging stress for 4H, 8H, and 12H in GH01, respectively. For proteins repeatedly identified at different time points, gene ontology (GO) cluster analysis suggested that the responsive proteins of the two cultivars were both enriched in the biological process of DNA-dependent transcription and the oxidation⁻reduction process, and response to various stress and hormone stimulus, while different distribution frequencies in the two cultivars was investigated. Moreover, overlap proteins with similar or opposite tendencies of fold change between ZS9 and GH01 were observed and clustered based on the different expression ratios, suggesting the two genotype cultivars exhibited diversiform molecular mechanisms or regulation pathways in their waterlogging stress response. The following qRT-PCR (quantitative real-time polymerase chain reaction) results verified the candidate proteins at transcription levels, which were prepared for further research. In conclusion, proteins detected in this study might perform different functions in waterlogging responses and would provide information conducive to better understanding adaptive mechanisms under environmental stresses.

Project description:IntroductionPlants respond to water stress with a variety of physiological and biochemical changes, but their response varies among species, varieties and cultivars. Waterlogging in tomato reduces plant growth, degrade chlorophyll and increase concentration of oxidative parameters. Priming can alleviate stress in plants caused by waterlogging enabling plants to be more tolerant to an additional stress in the current or even subsequent generation. The aim of this study was to evaluate tomato genotypes for their sensitivity to waterlogging stress applied during early vegetative growth and at full flowering stage.Materials and methodsThe study included two local genotypes, Trebinjski sitni (GB1126) and Žuti (GB1129), and the reference variety Novosadski jabučar (NJ), which is the variety most commonly used in Serbia and Bosnia and Herzegovina. The activity of class III peroxidase (POX), hydrogen peroxide (H2O2) content and malondialdehyde (MDA) content were measured spectrophotometrically, and for quantification of individual phenolic compounds, targeted approach was adopted, using UHPLC/DAD/(-)HESI-MS2 instrument (Dionex UltiMate 3000 UHPLC system with a DAD detector, configured with a triple quadrupole mass spectrometer TSQ Quantum Access Max (Thermo Fisher Scientific, Germany)).Results and discussionOxidative parameters (H2O2 and MDA) exhibited an increase in content in leaves of tomato plants that underwent waterlogging stress compared to control plants. Moreover, oxidative parameters showed positive correlation with proteins and phenolics content. The obtained correlations can indicate that one of the response strategies of tomato plants to waterlogging is the increased synthesis of proteins and phenolic compounds. The POX activity was not correlated with other parameters except with the polyphenols. A positive correlation was shown between POX activity and the content of phenolic compounds, indicating their independent roles in the removal of ROS. Changes in the phenolic profiles after the exposure of plants to waterlogging stress are recorded, and these changes were more severe in leaves and fruits of GB1129 and NJ genotypes than in GB1126. Thus, genotype GB1126 is the most efficient in maintaining the phenolic profiles of leaves and fruits, and therefore of the nutritive and organoleptic qualities of fruits following the exposure to waterlogging. Also, genotype GB1126 exhibited the ability to maintain the content of oxidative parameters during waterlogging at certain growth stages, implying certain waterlogging tolerance.ConclusionWaterlogging triggered stress memory but not at all growth stages. The most pronounced stress memory was obtained in fruit samples in the phase of full fruit maturity on the 1st truss. This study shed light on the defense mechanisms of tomato plants to repeated waterlogging stress from the perspectives of the changes in the composition of major phenolics, and pointed to the 5-O-caffeoylquinic acid and rutin as the chemical markers of the waterlogging stress tolerance in tomato. However, it remains to be determined whether this modulation has a positive or negative effect on the overall plant metabolism. Further investigations are needed to fully elucidate the benefits of waterlogging pretreatment in this context.

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Molecular cloning and expression analysis of three ThERFs involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products.

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