A unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating ethylene and the abscisic acid pathway.

The submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here, we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates GALACTOSYLTRANSFERASE 9 (GALT9) and their expression is inversely affected at 24 h of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild-type and MIM775 Arabidopsis shoot. A similar recovery phenotype in the galt9 mutant indicates the role of the miR775-GALT9 module in post-submergence recovery. We predicted that Golgi-localized GALT9 is potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29 and ORE1), ethylene signalling (EIN2 and EIN3) and abscisic acid (ABA) biosynthesis (NCED3) pathway genes occurs in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role for the miR775-GALT9 module in post-submergence recovery through a crosstalk between the ethylene signalling and ABA biosynthesis pathways.

Identification of hub genes involved in gibberellin-regulated elongation of coleoptiles of rice seeds germinating under submerged conditions.

Rapid elongation of coleoptiles from rice seeds to reach the water surface enables plants to survive submergence stress and therefore plays a crucial role in allowing direct seeding in rice cultivation. Gibberellin (GA) positively influences growth in rice, but the molecular mechanisms underlying its regulation of coleoptile elongation under submerged conditions remain unclear. In this study, we performed a weighted gene co-expression network analysis to conduct a preliminarily examination of the mechanisms. Four key modules were identified with high correlations to the GA regulation of submergence tolerance. The genes within these modules were mainly involved in the Golgi apparatus and carbohydrate metabolic pathways, suggesting their involvement in enhancing submergence tolerance. Further analysis of natural variation revealed that the specific hub genes Os03g0337900, Os03g0355600, and Os07g0638400 exhibited strong correlations with subspecies divergence of the coleoptile elongation phenotype. Consistent with this analysis, mutation of Os07g0638400 resulted in a lower germination potential and a stronger inhibition of coleoptile elongation under submerged conditions. The hub genes identified in this study provide new insights into the molecular mechanisms underlying GA-dependent tolerance to submergence stress in rice, and a potential basis for future modification of rice germplasm to allow for direct seeding.

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.

Transcriptomic Analysis of the Photosynthetic, Respiration, and Aerenchyma Adaptation Strategies in Bermudagrass (Cynodon dactylon) under Different Submergence Stress.

Submergence impedes photosynthesis and respiration but facilitates aerenchyma formation in bermudagrass. Still, the regulatory genes underlying these physiological responses are unclear in the literature. To identify differentially expressed genes (DEGs) related to these physiological mechanisms, we studied the expression of DEGs in aboveground and underground tissues of bermudagrass after a 7 d treatment under control (CK), shallow submergence (SS), and deep submergence (DS). Results show that compared with CK, 12276 and 12559 DEGs were identified under SS and DS, respectively. Among them, the DEGs closely related to the metabolism of chlorophyll biosynthesis, light-harvesting, protein complex, and carbon fixation were down-regulated in SS and DS. Meanwhile, a large number of DEGs involved in starch and sucrose hydrolase activities, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation were down-regulated in aboveground tissues of bermudagrass in SS and DS. Whereas in underground tissues of bermudagrass these DEGs were all up-regulated under SS, only beta-fructofuranosidase and α-amylase related genes were up-regulated under DS. In addition, we found that DEGs associated with ethylene signaling, Ca2+-ROS signaling, and cell wall modification were also up-regulated during aerenchyma formation in underground tissues of bermudagrass under SS and DS. These results provide the basis for further exploration of the regulatory and functional genes related to the adaptability of bermudagrass to submergence.

Flooding tolerance of forage legumes.

We review waterlogging and submergence tolerances of forage (pasture) legumes. Growth reductions from waterlogging in perennial species ranged from >50% for Medicago sativa and Trifolium pratense to <25% for Lotus corniculatus, L. tenuis, and T. fragiferum. For annual species, waterlogging reduced Medicago truncatula by ~50%, whereas Melilotus siculus and T. michelianum were not reduced. Tolerant species have higher root porosity (gas-filled volume in tissues) owing to aerenchyma formation. Plant dry mass (waterlogged relative to control) had a positive (hyperbolic) relationship to root porosity across eight species. Metabolism in hypoxic roots was influenced by internal aeration. Sugars accumulate in M. sativa due to growth inhibition from limited respiration and low energy in roots of low porosity (i.e. 4.5%). In contrast, L. corniculatus, with higher root porosity (i.e. 17.2%) and O2 supply allowing respiration, maintained growth better and sugars did not accumulate. Tolerant legumes form nodules, and internal O2 diffusion along roots can sustain metabolism, including N2 fixation, in submerged nodules. Shoot physiology depends on species tolerance. In M. sativa, photosynthesis soon declines and in the longer term (>10 d) leaves suffer chlorophyll degradation, damage, and N, P, and K deficiencies. In tolerant L. corniculatus and L. tenuis, photosynthesis is maintained longer, shoot N is less affected, and shoot P can even increase during waterlogging. Species also differ in tolerance of partial and complete shoot submergence. Gaps in knowledge include anoxia tolerance of roots, N2 fixation during field waterlogging, and identification of traits conferring the ability to recover after water subsides.

Lipids signaling and unsaturation of fatty acids participate in ramie response to submergence stress and hypoxia-responsive gene regulation.

Ramie is a valuable crop that produces high-quality fibers and holds promise in ecological management and potential therapeutic properties. The damage of submergence during the fertile period seriously affects the growth of ramie. This study used transcriptomics and UPLC-QTOF/MS-based lipidomics analysis to reveal the lipids remodeling and stress adaptation mechanism in ramie response to submergence. The results of subcellular distribution showed that lipids in ramie leaf cells mostly aggregate in the inter-chloroplast cytoplasm to form lipid droplets under submergence stress. High-performance thin-layer chromatography (HPTLC) and lipidomics analysis showed that the composition and content of lipids in ramie leaves significantly changed under submergence stress, and the content of fatty acids (FAs) gradually accumulated with the extension of the submergence treatment time. Further analysis revealed that the content of 18:3 (n3) Coenzyme A (C18:3-CoA) increased significantly with the prolongation of submergence stress, and the exogenous addition of C18:3-CoA activated the expression of hypoxia-responsive marker genes such as BnADH1, BnPCO2, BnADH1, and BnPDC1. These results suggest that the ramie lipid metabolism pathways were significantly affected under submergence, and the C18:3-CoA may act directly or indirectly on the hypoxia-responsive genes to activate their transcriptional activities, thereby enhancing the tolerance of ramie to submergence stress.

CiAP2/ERF65 and CiAP2/ERF106, a pair of homologous genes in pecan (Carya illinoensis), regulate plant responses during submergence in transgenic Arabidopsis thaliana.

When plants are entirely submerged, photosynthesis and respiration are severely restricted, affecting plant growth and potentially even causing plant death. The AP2/ERF superfamily has been widely reported to play a vital role in plant growth, development and resistance to biotic and abiotic stresses. However, no relevant studies exist on flooding stress in pecan. In this investigation, we observed that CiAP2/ERF65 positively modulated the hypoxia response during submergence, whereas CiAP2/ERF106 was sensitive to submergence. The levels of physiological and biochemical indicators, such as POD, CAT and among others, in CiAP2/ERF65-OE lines were significantly higher than those in wild-type Arabidopsis thaliana, indicating that the antioxidant capacity of CiAP2/ERF65-OE lines was enhanced under submergence. The RNA-seq results revealed that the maintenance of the expression levels of the antenna protein gene, different signaling pathways for regulation, as well as the storage and consumption of ATP, might account for the opposite phenotypes of CiAP2/ERF65 and CiAP2/ERF106. Furthermore, the expression of some stress-related genes was altered during submergence and reoxygenation. Overall, these findings enhance our understanding of submergence stress in pecan, providing important candidate genes for the molecular design and breeding of hypoxia resistant in plants.

Genome-wide characterization and identification of Trihelix transcription factors and expression profiling in response to abiotic stresses in Chinese Willow (Salix matsudana Koidz).

Trihelix transcription factors (TTF) are a class of light-responsive proteins with a typical triple-helix structure (helix-loop-helix-loop-helix). Members of this gene family play an important role in plant growth and development, especially in various abiotic stress responses. Salix matsudana Koidz is an allotetraploid ornamental forest tree that is widely planted for its excellent resistance to stress, but no studies on its Trihelix gene family have been reported. In this study, the Trihelix gene family was analyzed at the genome-wide level in S. matsudana. A total of 78 S. matsudana Trihelix transcription factors (SmTTFs) were identified, distributed on 29 chromosomes, and classified into four subfamilies (GT-1, GT-2, SH4, SIP1) based on their structural features. The gene structures and conserved functional domains of these Trihelix genes are similar in the same subfamily and differ between subfamilies. The presence of multiple stress-responsive cis-elements on the promoter of the S. matsudana Trihelix gene suggests that the S. matsudana Trihelix gene may respond to abiotic stresses. Expression pattern analysis revealed that Trihelix genes have different functions during flooding stress, salt stress, drought stress and low temperature stress in S. matsudana. Given that SmTTF30, as a differentially expressed gene, has a faster response to flooding stress, we selected SmTTF30 for functional studies. Overexpression of SmTTF30 in Arabidopsis thaliana (Arabidopsis) enhances its tolerance to flooding stress. Under flooding stress, the leaf cell activity and peroxidase activity (POD) of the overexpression strain were significantly higher than the leaf cell activity and POD of the wild type, and the malondialdehyde (MDA) content was significantly lower than the MDA content of the wild type. Thus, these results suggest that SmTTF30 enhances plant flooding tolerance and plays a positive regulatory role in plant flooding tolerance.

Biocuration of a Transcription Factors Network Involved in Submergence Tolerance during Seed Germination and Coleoptile Elongation in Rice (Oryza sativa).

Modeling biological processes and genetic-regulatory networks using in silico approaches provides a valuable framework for understanding how genes and associated allelic and genotypic differences result in specific traits. Submergence tolerance is a significant agronomic trait in rice; however, the gene-gene interactions linked with this polygenic trait remain largely unknown. In this study, we constructed a network of 57 transcription factors involved in seed germination and coleoptile elongation under submergence. The gene-gene interactions were based on the co-expression profiles of genes and the presence of transcription factor binding sites in the promoter region of target genes. We also incorporated published experimental evidence, wherever available, to support gene-gene, gene-protein, and protein-protein interactions. The co-expression data were obtained by re-analyzing publicly available transcriptome data from rice. Notably, this network includes OSH1, OSH15, OSH71, Sub1B, ERFs, WRKYs, NACs, ZFP36, TCPs, etc., which play key regulatory roles in seed germination, coleoptile elongation and submergence response, and mediate gravitropic signaling by regulating OsLAZY1 and/or IL2. The network of transcription factors was manually biocurated and submitted to the Plant Reactome Knowledgebase to make it publicly accessible. We expect this work will facilitate the re-analysis/re-use of OMICs data and aid genomics research to accelerate crop improvement.

Comparative transcriptomic analysis of the gene expression and underlying molecular mechanism of submergence stress response in orchardgrass roots.

Introduction: Submergence stress creates a hypoxic environment. Roots are the first plant organ to face these low-oxygen conditions, which causes damage and affects the plant growth and yield. Orchardgrass (Dactylis glomerata L.) is one of the most important cold-season forage grasses globally. However, their submergence stress-induced gene expression and the underlying molecular mechanisms of orchardgrass roots are still unknown. Methods: Using the submergence-tolerant 'Dianbei' and submergence-sensitive 'Anba', the transcriptomic analysis of orchardgrass roots at different time points of submergence stress (0 h, 8 h, and 24 h) was performed. Results: We obtained 118.82Gb clean data by RNA-Seq. As compared with the control, a total of 6663 and 9857 differentially expressed genes (DEGs) were detected in Dianbei, while 7894 and 11215 DEGs were detected in Anba at 8 h and 24 h post-submergence-stress, respectively. Gene Ontology (GO) enrichment analysis obtained 986 terms, while Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis obtained 123 pathways. Among them, the DEGs in plant hormones, mitogen-activated protein kinase (MAPK) and Ca2+ signal transduction were significantly differentially expressed in Dianbei, but not in Anba. Discussion: This study was the first to molecularly elucidate the submergence stress tolerance in the roots of two orchardgrass cultivars. These findings not only enhanced our understanding of the orchardgrass submergence tolerance, but also provided a theoretical basis 36 for the cultivation of submergence-tolerant forage varieties.

Comparative transcriptomic analysis reveals potential mechanisms for high tolerance to submergence in arbor willows.

BACKGROUND: Submergence threatens plant growth and survival by decreasing or eliminating oxygen supply. Uncovering the complex regulatory network underlying the tolerance of Salix to submergence and identifying the key regulators are important for molecular-assisted breeding of Salix. METHODS: In this study, we screened germplasm resources of arbor willows and discovered both submergence-tolerant and submergence-sensitive varieties. Then, by performing RNA-seq, we compared the differences between the transcriptomes of two varieties, i.e., the submergence-tolerant variety "Suliu 795" and the submergence-sensitive variety "Yanliu No. 1," and the different submergence treatment time points to identify the potential mechanisms of submergence in Salix and the unique approaches by which the variety "Suliu 795" possessed a higher tolerance compared to "Yanliu No. 1". RESULTS: A total of 22,790 differentially expressed genes were identified from 25 comparisons. Using gene ontology annotation and pathway enrichment analysis, the expression pattern of transcriptional factors, important players in hormone signaling, carbohydrate metabolism, and the anaerobic respiration pathway were found to differ significantly between the two varieties. The principal component analysis and qRT-PCR results verified the reliability of the RNA sequencing data. The results of further analysis indicated that "Suliu 795" had higher submergence tolerant activity than "Yanliu No. 1" because of three characteristics: (1) high sensitivity to the probable low oxygen stress and initiation of appropriate responding mechanisms in advance; (2) maintenance of energy homeostasis to prevent energy depletion under hypoxic stress; and (3) keep "quiescence" through fine-tuning the equilibrium between phytohormones GA, SA and ethylene.

Glutathione improves rice tolerance to submergence: insights into its physiological and biochemical mechanisms.

Complete submergence (Sub) imposes detrimental effects on growth and survival of crop plants, including rice. Here, we investigated the beneficial effects of reduced glutathione (GSH) in mitigating Sub-induced adverse effects in two high-yielding rice cultivars BRRI dhan29 and dhan52. Both cultivars experienced growth defects, severe yellowing, necrosis and chlorosis, when they were completely immersed in water for 14 days. The poor growth performance of these cultivars was linked to biomass reduction, decreased levels of photosynthetic pigments and proline, increased levels of H2O2 and malondialdehyde, and declined activities of enzymatic antioxidants like superoxide dismutase, ascorbate peroxidase, peroxidase, catalase, glutathione peroxidase and glutathione S-transferase. Pretreatment with exogenous GSH led to significant growth restoration in both cultivars exposed to Sub. The elevated Sub-tolerance promoted by GSH could partly be attributed to increased levels of chlorophylls, carotenoids, soluble proteins and proline. Exogenous GSH also mitigated Sub-induced oxidative damage, as evidenced from reduced levels of H2O2 and malondialdehyde in accordance with the increased activities of antioxidant enzymes. Results revealed that dhan52 was more tolerant to Sub-stress than dhan29, and GSH successfully rescued both cultivars from the damage of Sub-stress. Collectively, our findings provided an insight into the GSH-mediated active recovery of rice from Sub-stress, thereby suggesting that external supply of GSH may be an effective strategy to mitigate the adverse effects of Sub in rice.

Arabidopsis CALMODULIN-LIKE 38 Regulates Hypoxia-Induced Autophagy of SUPPRESSOR OF GENE SILENCING 3 Bodies.

During the energy crisis associated with submergence stress, plants restrict mRNA translation and rapidly accumulate stress granules that act as storage hubs for arrested mRNA complexes. One of the proteins associated with hypoxia-induced stress granules in Arabidopsis thaliana is the calcium-sensor protein CALMODULIN-LIKE 38 (CML38). Here, we show that SUPPRESSOR OF GENE SILENCING 3 (SGS3) is a CML38-binding protein, and that SGS3 and CML38 co-localize within hypoxia-induced RNA stress granule-like structures. Hypoxia-induced SGS3 granules are subject to turnover by autophagy, and this requires both CML38 as well as the AAA+-ATPase CELL DIVISION CYCLE 48A (CDC48A). CML38 also interacts directly with CDC48A, and CML38 recruits CDC48A to CML38 granules in planta. Together, this work demonstrates that SGS3 associates with stress granule-like structures during hypoxia stress that are subject to degradation by CML38 and CDC48-dependent autophagy. Further, the work identifies direct regulatory targets for the hypoxia calcium-sensor CML38, and suggest that CML38 association with stress granules and associated regulation of autophagy may be part of the RNA regulatory program during hypoxia stress.

Transcriptome profiling of two Dactylis glomerata L. cultivars with different tolerance in response to submergence stress.

Submergence is one of the environmental stresses that limit plant growth and development. Dactylis glomerata L. is an important cool-season forage grass globally. To investigate the genes related to submergence response and the molecular mechanism associated with submergence tolerance, the transcriptome of D. glomerata in response to waterlogging treatment was analyzed. RNA-sequencing was performed in two D. glomerata cultivars, submergence tolerant 'Dianbei' and submergence sensitive 'Anba'. A total of 50,045 unique genes matched the known proteins in the NCBI nr database by BLAST searches and 60.8% (30,418) of these genes were annotated with GO terms. Among these, 1395 genes only differentially expressed in 'Dianbei' and 18 genes shown different expression all the time were detected between the submergence tolerant 'Dianbei' and sensitive 'Anba'. Gene ontology (GO) and KEGG pathway enrichment analyses demonstrated that the DEGs were mainly implicated in oxidation-reduction system, nucleic acid binding transcription factor activity, and glycerol kinase activity. The D. glomerata assembled transcriptome provided substantial molecular resource for further genomic analysis of forage grasses in response to submergence stress. The significant difference in expression of specific unigenes may account for waterlogging tolerance or acclimation in the two different D. glomerata cultivars. This study provided new insights into the molecular basis of submergence tolerance in D. glomerata.

Comparative Transcriptional Profiling of Primed and Non-primed Rice Seedlings under Submergence Stress.

Submergence stress is a limiting factor for direct-seeded rice systems in rainfed lowlands and flood-prone areas of South and Southeast Asia. The present study demonstrated that submergence stress severely hampered the germination and seedling growth of rice, however, seed priming alleviated the detrimental effects of submergence stress. To elucidate the molecular basis of seed priming-induced submergence tolerance, transcriptome analyses were performed using 4-day-old primed (selenium-Se and salicylic acid-SA priming) and non-primed rice seedlings under submergence stress. Genomewide transcriptomic profiling identified 2371 and 2405 transcripts with Se- and SA-priming, respectively, that were differentially expressed in rice compared with non-priming treatment under submergence. Pathway and gene ontology term enrichment analyses revealed that genes involved in regulation of secondary metabolism, development, cell, transport, protein, and metal handling were over-represented after Se- or SA-priming. These coordinated factors might have enhanced the submergence tolerance and maintained the better germination and vigorous seedling growth of primed rice seedlings. It was also found that many genes involved in cellular and metabolic processes such as carbohydrate metabolism, cellular, and metabolic biosynthesis, nitrogen compound metabolic process, transcription, and response to oxidative stress were induced and overlapped in seed priming treatments, a finding which reveals the common mechanism of seed priming-induced submergence tolerance. Taken together, these results may provide new avenues for understanding and advancing priming-induced responses to submergence tolerance in crop plants.

Exogenous spermidine alleviates oxidative damage and reduce yield loss in rice submerged at tillering stage.

To figure out whether spermidine (Spd) can alleviate oxidative damage on rice (Oryza sativa L.) caused by submergence stress, Ningjing 3 was used in this study. The results showed that, spraying Spd on rice leaves at a concentration of 0.5 mM promoted the growth recovery of rice after drainage, such as green leaves, tillers, and aboveground dry mass. According to physiological analysis, Spd accelerate restored chlorophylls damage by submergence, and decreased the rate of [Formula: see text] generation and H2O2 content, inhibited submergence-induced lipid peroxidation. Spd also helped to maintain antioxidant enzyme activities after drainage, such as superoxide dismutase, peroxidase, and GR, which ultimately improved the recovery ability of submerged rice. With the effect of Spd, the rice yields increased by 12.1, 17.9, 13.5, and 18.0%, of which submerged for 1, 3, 5, 7 days, respectively. It is supposed that exogenous Spd really has an alleviate effect on submergence damage and reduce yield loss of rice.