Genome-wide association study uncovers major genetic loci associated with seed flooding tolerance in soybean

Background Seed flooding stress is one of the threatening environmental stressors that adversely limits soybean at the germination stage across the globe. The knowledge on the genetic basis underlying seed-flooding tolerance is limited. Therefore, we performed a genome-wide association study (GWAS) using 34,718 single nucleotide polymorphism (SNPs) in a panel of 243 worldwide soybean collections to identify genetic loci linked to soybean seed flooding tolerance at the germination stage. Results In the present study, GWAS was performed with two contrasting models, Mixed Linear Model (MLM) and Multi-Locus Random-SNP-Effect Mixed Linear Model (mrMLM) to identify significant SNPs associated with electrical conductivity (EC), germination rate (GR), shoot length (ShL), and root length (RL) traits at germination stage in soybean. With MLM, a total of 20, 40, 4, and 9 SNPs associated with EC, GR, ShL and RL, respectively, whereas in the same order mrMLM detected 27, 17, 13, and 18 SNPs. Among these SNPs, two major SNPs, Gm_08_11971416, and Gm_08_46239716 were found to be consistently connected with seed-flooding tolerance related traits, namely EC and GR across two environments. We also detected two SNPs, Gm_05_1000479 and Gm_01_53535790 linked to ShL and RL, respectively. Based on Gene Ontology enrichment analysis, gene functional annotations, and protein-protein interaction network analysis, we predicted eight candidate genes and three hub genes within the regions of the four SNPs with Cis-elements in promoter regions which may be involved in seed-flooding tolerance in soybeans and these warrant further screening and functional validation. Conclusions Our findings demonstrate that GWAS based on high-density SNP markers is an efficient approach to dissect the genetic basis of complex traits and identify candidate genes in soybean. The trait associated SNPs could be used for genetic improvement in soybean breeding programs. The candidate genes could help researchers better understand the molecular mechanisms underlying seed-flooding stress tolerance in soybean.

Understanding a Mechanistic Basis of ABA Involvement in Plant Adaptation to Soil Flooding: The Current Standing

Soil flooding severely impairs agricultural crop production. Plants can cope with flooding conditions by embracing an orchestrated set of morphological adaptations and physiological adjustments that are regulated by the elaborated hormonal signaling network. The most prominent of these hormones is ethylene, which has been firmly established as a critical signal in flooding tolerance. ABA (abscisic acid) is also known as a “stress hormone” that modulates various responses to abiotic stresses; however, its role in flooding tolerance remains much less established. Here, we discuss the progress made in the elucidation of morphological adaptations regulated by ABA and its crosstalk with other phytohormones under flooding conditions in model plants and agriculturally important crops.

Proteomic and Biochemical Analyses of the Mechanism of Tolerance in Mutant Soybean Responding to Flooding Stress

To investigate the mechanism of flooding tolerance of soybean, flooding-tolerant mutants derived from gamma-ray irradiated soybean were crossed with parent cultivar Enrei for removal of other factors besides the genes related to flooding tolerance in primary generated mutant soybean. Although the growth of the wild type was significantly suppressed by flooding compared with the non-flooding condition, that of the mutant lines was better than that of the wild type even if it was treated with flooding. A two-day-old mutant line was subjected to flooding for 2 days and proteins were analyzed using a gel-free/label-free proteomic technique. Oppositely changed proteins in abundance between the wild type and mutant line under flooding stress were associated in endoplasmic reticulum according to gene-ontology categorization. Immunoblot analysis confirmed that calnexin accumulation increased in both the wild type and mutant line; however, calreticulin accumulated in only the mutant line under flooding stress. Furthermore, although glycoproteins in the wild type decreased by flooding compared with the non-flooding condition, those in the mutant line increased even if it was under flooding stress. Alcohol dehydrogenase accumulated in the wild type and mutant line; however, this enzyme activity significantly increased and mildly increased in the wild type and mutant line, respectively, under flooding stress compared with the non-flooding condition. Cell death increased and decreased in the wild type and mutant line, respectively, by flooding stress. These results suggest that the regulation of cell death through the fermentation system and glycoprotein folding might be an important factor for the acquisition of flooding tolerance in mutant soybean.

Leaf morphology and chlorophyll fluorescence characteristics of mulberry seedlings under waterlogging stress

Because of its high flooding tolerance, in recent years, mulberry has become a tree species that is used in plant restoration in impact zones in reservoir areas. Therefore, 3-year-old potted forage mulberry seedlings were used to investigate the mechanism of mulberry adaptation to flooding stress. An indoor simulated flooding method was adopted to study the morphology of mulberry seedling leaves and the changes in leaf chlorophyll fluorescence parameters and fluorescence imaging under different flooding times and depths. The results showed that the leaves of mulberry seedlings treated with shallow submergence remained healthy during the flooding period, while the leaves of mulberry seedlings treated with half submergence and full submergence showed different degrees of waterlogging symptoms in the middle and late flooding periods and formed adventitious roots at the base of the stem. Most of the chlorophyll fluorescence parameters decreased at the beginning of flooding, but the steady-state degree of closure of PS II reaction centres (1-qP_Lss) increased significantly. In the later stage of flooding, the fluorescence parameters showed relatively stable trends. Based on these results, we conclude that mulberry has high flooding tolerance due to a combination of morphological and physiological responses.

Antioxidant metabolites from riparian fungal endophytes improve the tolerance of rice seedlings to flooding

Endophytic fungi have the potential to enhance plant resistance to various stresses and promote the ecological adaptation of the hosts. To evaluate the effects of the riparian endophytes on rice seedlings to flooding tolerance, here we screened out two endophytes from the plant Myricaria laxiflora growing in the Yangtze River zone. Through morphological characteristics and rDNA ITS (internal transcriber region) sequence, the two strains were respectively identified as Aspergillus fumigatus and Chaetomium globosum. Metabolites derived from both fungi were capable of increasing tolerance to flooding. Systematic separation and purification coupled to bioassays revealed that two natural antioxidants, Z-N-4-hydroxystyryl formamide (NFA) and chaetoglobosin A (CheA), were effective for alleviating flooding stress. Both NFA and CheA inhibited the production of factors such as malondialdehyde, superoxide dismutase, ethanol dehydrogenase, and NADPH oxidase that are associated with oxidative stress induced by flooding. Gene expression analyses of NADPH oxidase families indicated that OsRbohB could be involved in conferring flooding tolerance mediated by the two natural antioxidants. These findings contribute to understanding the role of the natural antioxidants in riparian endophytic fungi, and providing a basis for improvement of flooding tolerance of rice and other crop plants through the antioxidants of the fungal endophytes.

Involvement of the miR156/SPL module in flooding response in Medicago sativa

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

Identification and Functional Analysis of ThADH1 and ThADH4 Genes Involved in Tolerance to Waterlogging Stress in Taxodium hybrid ‘Zhongshanshan 406’

The Taxodium hybrid ‘Zhongshanshan 406’ (T. hybrid ‘Zhongshanshan 406’) [Taxodium mucronatum Tenore × Taxodium distichum (L.). Rich] has an outstanding advantage in flooding tolerance and thus has been widely used in wetland afforestation in China. Alcohol dehydrogenase genes (ADHs) played key roles in ethanol metabolism to maintain energy supply for plants in low-oxygen conditions. Two ADH genes were isolated and characterized—ThADH1 and ThADH4 (GenBank ID: AWL83216 and AWL83217—basing on the transcriptome data of T. hybrid ‘Zhongshanshan 406’ grown under waterlogging stress. Then the functions of these two genes were investigated through transient expression and overexpression. The results showed that the ThADH1 and ThADH4 proteins both fall under ADH III subfamily. ThADH1 was localized in the cytoplasm and nucleus, whereas ThADH4 was only localized in the cytoplasm. The expression of the two genes was stimulated by waterlogging and the expression level in roots was significantly higher than those in stems and leaves. The respective overexpression of ThADH1 and ThADH4 in Populus caused the opposite phenotype, while waterlogging tolerance of the two transgenic Populus significantly improved. Collectively, these results indicated that genes ThADH1 and ThADH4 were involved in the tolerance and adaptation to anaerobic conditions in T. hybrid ‘Zhongshanshan 406’.