The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare).

To decipher the molecular bases governing seed germination, this study presents the pivotal role of the cap-binding complex (CBC), comprising CBP20 and CBP80, in modulating the inhibitory effects of abscisic acid (ABA) in barley. Using both single and double barley mutants in genes encoding the CBC, we revealed that the double mutant hvcbp20.ab/hvcbp80.b displays ABA insensitivity, in stark contrast to the hypersensitivity observed in single mutants during germination. Our comprehensive transcriptome and metabolome analysis not only identified significant alterations in gene expression and splicing patterns but also underscored the regulatory nexus among CBC, ABA, and brassinosteroid (BR) signaling pathways.

Comprehensive mapping and modelling of the rice regulome landscape unveils the regulatory architecture underlying complex traits.

Unraveling the regulatory mechanisms that govern complex traits is pivotal for advancing crop improvement. Here we present a comprehensive regulome atlas for rice (Oryza sativa), charting the chromatin accessibility across 23 distinct tissues from three representative varieties. Our study uncovers 117,176 unique open chromatin regions (OCRs), accounting for ~15% of the rice genome, a notably higher proportion compared to previous reports in plants. Integrating RNA-seq data from matched tissues, we confidently predict 59,075 OCR-to-gene links, with enhancers constituting 69.54% of these associations, including many known enhancer-to-gene links. Leveraging this resource, we re-evaluate genome-wide association study results and discover a previously unknown function of OsbZIP06 in seed germination, which we subsequently confirm through experimental validation. We optimize deep learning models to decode regulatory grammar, achieving robust modeling of tissue-specific chromatin accessibility. This approach allows to predict cross-variety regulatory dynamics from genomic sequences, shedding light on the genetic underpinnings of cis-regulatory divergence and morphological disparities between varieties. Overall, our study establishes a foundational resource for rice functional genomics and precision molecular breeding, providing valuable insights into regulatory mechanisms governing complex traits.

Cold tolerance SNPs and candidate gene mining in the soybean germination stage based on genome-wide association analysis.

KEY MESSAGE: Three QTLs associated with low-temperature tolerance were identified by genome-wide association analysis, and 15 candidate genes were identified by haplotype analysis and gene expression analyses. Low temperature is a critical factor affecting the geographical distribution, growth, development, and yield of soybeans, with cold stress during seed germination leading to substantial productivity loss. In this study, an association panel comprising 260 soybean accessions was evaluated for four germination traits and four cold tolerance index traits, revealing extensive variation in cold tolerance. Genome-wide association study (GWAS) identified 10 quantitative trait nucleotides (QTNs) associated with cold tolerance, utilizing 30,799 single nucleotide polymorphisms (SNPs) and four GWAS models. Linkage disequilibrium (LD) analysis positioned these QTNs within three cold-tolerance quantitative trait loci (QTL) and, with QTL19-1, was positioned by three multi-locus models, underscoring its importance as a key QTL. Integrative haplotype analysis, supplemented by transcriptome analysis, uncovered 15 candidate genes. The haplotypes within the genes Glyma.18G044200, Glyma.18G044300, Glyma.18G044900, Glyma.18G045100, Glyma.19G222500, and Glyma.19G222600 exhibited significant phenotypic variations, with differential expression in materials with varying cold tolerance. The QTNs and candidate genes identified in this study offer substantial potential for marker-assisted selection and gene editing in breeding cold-tolerant soybeans, providing valuable insights into the genetic mechanisms underlying cold tolerance during soybean germination.

Genetic variation for tolerance to pre-harvest sprouting in mungbean (Vigna radiata) genotypes.

Pre-harvest sprouting (PHS) is one of the important abiotic stresses in mungbean which significantly reduces yield and quality of the produce. This study was conducted to evaluate the genetic variability for tolerance to pre-harvest sprouting in diverse mungbean genotypes while simultaneously deciphering the association of yield contributing traits with PHS. Eighty-three diverse mungbean genotypes (23 released varieties, 23 advanced breeding lines and 37 exotic germplasm lines) were investigated for tolerance to PHS, water imbibition capacities by pods, pod and seed physical traits. Wide variation in PHS was recorded which ranged between 17.8% to 81% (mean value 54.34%). Germplasm lines exhibited higher tolerance to PHS than the high-yielding released varieties. Correlation analysis revealed PHS to be positively associated with water imbibition capacity by pods (r = 0.21) and germinated pod % (r = 0.78). Pod length (r = -0.13) and seeds per pod (r = -0.13) were negatively influencing PHS. Positive associations between PHS and water imbibition capacity by pods, germinated pod % and 100-seed weight was further confirmed by multivariate analysis. Small-seeded genotypes having 100-seed weight <3 g exhibited higher tolerance to PHS compared to bold-seeded genotypes having 100-seed weight more than 3.5 g. Fresh seed germination among the selected PHS tolerant and susceptible genotypes ranged from 42% (M 204) to 98% (Pusa 1131). A positive association (r = 0.79) was recorded between fresh seed germination and PHS. Genotypes M 1255, M 145, M 422, M 1421 identified as potential genetic donors against PHS could be utilized in mungbean breeding programs.

Detection of closely linked QTLs and candidate genes controlling germination indices in response to drought and salinity stresses in barley.

The aim of current study was to identify closely linked QTLs and candidate genes related to germination indices under control, salinity and drought conditions in barley. A total of nine (a major), 28 (eight major) and 34 (five major) closely linked QTLs were mapped on the seven chromosomes in response to control, drought and salinity conditions using genome-wide composite interval mapping, respectively. The major QTLs can be used in marker-assisted selection (MAS) projects to increase tolerance to drought and salinity stresses during the germination. Overall, 422 unique candidate genes were associated with most major QTLs. Moreover, gene ontology analysis showed that candidate genes mostly involved in biological process related to signal transduction and response to stimulus in the pathway of resistance to drought and salinity stresses. Also, the protein-protein interaction network was identified 10 genes. Furthermore, 10 genes were associated with receptor-like kinase family. In addition, 16 transcription factors were detected. Three transcription factors including B3, bHLH, and FAR1 had the most encoding genes. Totally, 60 microRNAs were traced to regulate the target genes. Finally, the key genes are a suitable and reliable source for future studies to improve resistance to abiotic stress during the germination of barley.

Genetic, Epigenetic, and Environmental Control of Seed Dormancy and Germination.

Seed germination is controlled by a combination of the seed dormancy level and environmental conditions such as light, temperature, moisture, and nitrate levels. Seed dormancy is programed genetically, but it is also sensitive to maternal environmental conditions before and after anthesis. Recent developments in molecular genetics and bioinformatics have greatly enhanced our understanding of the molecular mechanisms of seed dormancy and germination in model plants and economically important crop species. This chapter focuses on temperature as an environmental factor and discusses the genetic and epigenetic mechanisms of dormancy and germination.

Elucidating the regulatory role of long non-coding RNAs in drought stress response during seed germination in leaf mustard.

Leaf mustard (Brassica juncea L. Czern & Coss), an important vegetable crop, experiences pronounced adversity due to seasonal drought stress, particularly at the seed germination stage. Although there is partial comprehension of drought-responsive genes, the role of long non-coding RNAs (lncRNAs) in adjusting mustard's drought stress response is largely unexplored. In this study, we showed that the drought-tolerant cultivar 'Weiliang' manifested a markedly lower base water potential (-1.073 MPa vs -0.437 MPa) and higher germination percentage (41.2% vs 0%) than the drought-susceptible cultivar 'Shuidong' under drought conditions. High throughput RNA sequencing techniques revealed a significant repertoire of lncRNAs from both cultivars during germination under drought stress, resulting in the identification of 2,087 differentially expressed lncRNAs (DELs) and their correspondingly linked 12,433 target genes. It was noted that 84 genes targeted by DEL exhibited enrichment in the photosynthesis pathway. Gene network construction showed that MSTRG.150397, a regulatory lncRNA, was inferred to potentially modulate key photosynthetic genes (Psb27, PetC, PetH, and PsbW), whilst MSTRG.107159 was indicated as an inhibitory regulator of six drought-responsive PIP genes. Further, weighted gene co-expression network analysis (WGCNA) corroborated the involvement of light intensity and stress response genes targeted by the identified DELs. The precision and regulatory impact of lncRNA were verified through qPCR. This study extends our knowledge of the regulatory mechanisms governing drought stress responses in mustard, which will help strategies to augment drought tolerance in this crop.

A CAM-Related NF-YB Transcription Factor Enhances Multiple Abiotic Stress Tolerance in Arabidopsis.

Abiotic stresses often occur simultaneously, and the tolerance mechanisms of plants to combined multiple abiotic stresses remain poorly studied. Extremophytes, adapted to abiotic stressors, might possess stress-adaptive or -responsive regulators that could enhance multiple abiotic stress resistance in crop plants. We identified an NF-YB transcription factor (TF) from the heat-tolerant obligate Crassulacean acid metabolism (CAM) plant, Kalanchoe fedtschenkoi, as a potential regulator of multiple abiotic stresses. The KfNF-YB3 gene was overexpressed in Arabidopsis to determine its role in multiple abiotic stress responses. Transgenic lines exhibited accelerated flowering time, increased biomass, larger rosette size, higher seed yield, and more leaves. Transgenic lines had higher germination rates under combined NaCl, osmotic, and water-deficit stress treatments compared to control plants. They also showed enhanced root growth and survival under simultaneous NaCl, osmotic, water-deficit, and heat stress conditions in vitro. Interestingly, potted transgenic lines had higher survival rates, yield, and biomass under simultaneous heat, water-deficit, and light stresses compared to control plants. Altogether, these results provide initial insights into the functions of a CAM-related TF and its potential roles in regulating multiple abiotic stress responses. The CAM abiotic stress-responsive TF-based approach appears to be an ideal strategy to enhance multi-stress resilience in crop plants.

Comparative Cytological and Gene Expression Analysis Reveals That a Common Wild Rice Inbred Line Showed Stronger Drought Tolerance Compared with the Cultivar Rice.

Common wild rice (Oryza rufipogon Griff.) is an important germplasm resource containing valuable genes. Our previous analysis reported a stable wild rice inbred line, Huaye3, which derives from the common wild rice of Guangdong Province. However, there was no information about its drought tolerance ability. Here, we assessed the germination characteristics and seedling growth between the Dawennuo and Huaye3 under five concentrations of PEG6000 treatment (0, 5%, 10%, 15%, and 20%). Huaye3 showed a stronger drought tolerance ability, and its seed germination rate still reached more than 52.50% compared with Dawennuo, which was only 25.83% under the 20% PEG6000 treatment. Cytological observations between the Dawennuo and Huaye3 indicated the root tip elongation zone and buds of Huaye3 were less affected by the PEG6000 treatment, resulting in a lower percentage of abnormalities of cortical cells, stele, and shrinkage of epidermal cells. Using the re-sequencing analysis, we detected 13,909 genes that existed in the genetic variation compared with Dawennuo. Of these genes, 39 were annotated as drought stress-related genes and their variance existed in the CDS region. Our study proved the strong drought stress tolerance ability of Huaye3, which provides the theoretical basis for the drought resistance germplasm selection in rice.

Comparative Phenotypic and Transcriptomic Analyses Provide Novel Insights into the Molecular Mechanism of Seed Germination in Response to Low Temperature Stress in Alfalfa.

Low temperature is the most common abiotic factor that usually occurs during the seed germination of alfalfa (Medicago sativa L.). However, the potential regulatory mechanisms involved in alfalfa seed germination under low temperature stress are still ambiguous. Therefore, to determine the relevant key genes and pathways, the phenotypic and transcriptomic analyses of low-temperature sensitive (Instict) and low-temperature tolerant (Sardi10) alfalfa were conducted at 6 and 15 h of seed germination under normal (20 °C) and low (10 °C) temperature conditions. Germination phenotypic results showed that Sardi10 had the strongest germination ability under low temperatures, which was manifested by the higher germination-related indicators. Further transcriptome analysis indicated that differentially expressed genes were mainly enriched in galactose metabolism and carbon metabolism pathways, which were the most commonly enriched in two alfalfa genotypes. Additionally, fatty acid metabolism and glutathione metabolism pathways were preferably enriched in Sardi10 alfalfa. The Weighted Gene Co-Expression Network Analysis (WGCNA) suggested that genes were closely related to galactose metabolism, fatty acid metabolism, and glutathione metabolism in Sardi10 alfalfa at the module with the highest correlation (6 h of germination under low temperature). Finally, qRT-PCR analysis further validated the related genes involved in the above pathways, which might play crucial roles in regulating seed germination of alfalfa under low temperature conditions. These findings provide new insights into the molecular mechanisms of seed germination underlying the low temperature stress in alfalfa.

Germination Assays for Comparable Seed Dormancy Depth and Distinction of Seed Color by Multiplex PCR in Wheat.

Seed dormancy is an important trait in cereal breeding, as it prevents preharvest sprouting (PHS). Although seed dormancy is a multifactorial trait, seed color has been demonstrated to be a major dormancy-related factor controlled by few genes. The R-1 gene is a seed color regulator that encodes a MYB-type transcription factor in wheat. A set of genetic markers designed against R-1 can provide a powerful tool for swift wheat breeding. Depth of seed dormancy varies not only among lines but also during seed development in each line. In this chapter, we describe how developmental seeds can be collected to perform germination tests, how seed color can be observed after NaOH staining, and how to genotype wheat R-1 genes using multiplex PCR.

Functional Analysis of Seed Dormancy Genes by Biolistic Transient Gene Expression in Immature Embryos of Wheat.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.

Genome-Wide Association Study of Preharvest Sprouting in Wheat.

Genome-wide association study (GWAS) is widely used to characterize genes or quantitative trait loci (QTLs) associated with preharvest sprouting and seed dormancy. GWAS can identify both previously discovered and novel QTLs across diverse genetic panels. The high-throughput SNP arrays or next-generation sequencing technologies have facilitated the identification of numerous genetic markers, thereby significantly enhancing the resolution of GWAS. Although various methods have been developed, the fundamental principles underlying these techniques remain constant. Here, we provide a basic technological flow to perform seed dormancy assay, followed by GWAS using population structure control, and compared it with previous identified QTLs and genes.

Evaluation Methods for Preharvest Sprouting Resistance in Japanese Wheat Breeding Programs.

Different methodologies have been applied for the selection of preharvest sprouting resistance in cereal breeding programs. We describe here a series of methods used in practical wheat breeding programs in Japan, including phenotyping based on germination score after artificial rain treatments and genotyping using DNA markers. These methods can be modified and applied to breeding programs in which preharvest sprouting is a problem during cereal cultivation.

MYB4 in Lilium pumilum affects plant saline-alkaline tolerance.

Lilium pumilum DC (L. pumilum DC) plays an important role in the rational utilization of salinized soil. To explore the molecular mechanism of salt-tolerant L. pumilum, the LpMYB4 was cloned. LpMYB4 close relationship with Bambusa emeiensis and Zea mays MYB4 throughout the phylogenetic tree construction. LpMYB4 protein was found to be localized in the nucleus. Prokaryotic and eukaryotic bacterial solution resistance experiments proved that the exogenous introduction of LpMYB4 made the overexpression strains obtain better survival ability under saline-alkaline stress. Compared with wild-type plants, tobacco plants overexpressing LpMYB4 had better growth and lower leaf wilting and lodging, the content of chlorophyll was higher, the content of hydrogen peroxide and superoxide anion was lower, the activity of peroxidase and superoxide dismutase was higher and the relative conductivity was lower under saline-alkaline stress. The analysis of seed germination and seedling resistance of transgenic plants under salt stress showed that LpMYB4 transgenic seeds were more tolerant to salt stress during germination and growth. Yeast two-hybrid and two-luciferase complementation experiments showed that LpMYB4 interacted with yeast two-hybrid and LpGPX6. The analysis of the role of LpMYB4 in improving plant saline-alkali resistance is helpful to the transformation of plant germplasm resources and has great significance for agriculture and sustainable development.

SlWRKY37 targets SlLEA2 and SlABI5-like7 to regulate seed germination vigor in tomato.

Seed germination is a critical phase for the life cycle and propagation of higher plants. This study explores the role of SlWRKY37, a WRKY transcription factor in tomato, in modulating seed germination. We discovered that SlWRKY37 expression is markedly downregulated during tomato seed germination. Through CRISPR/Cas9-mediated editing, we demonstrate that SlWRKY37 knockout enhances germination, while its overexpression results in a delay compared to the wild type. Transcriptome analysis revealed 679 up-regulated and 627 down-regulated genes in Slwrky37-CRISPR deletion mutants relative to the wild type. Gene ontology (GO) enrichment analysis indicated these differentially expressed genes are linked to seed dormancy, abscisic acid homeostasis, and protein phosphorylation pathways. Bioinformatics and biochemical assays identified SlABI5-like7 and SlLEA2 as key transcriptional targets of SlWRKY37, integral to tomato seed dormancy regulation. Additionally, SlWRKY37 was found to be post-translationally phosphorylated at Ser65, a modification crucial for its transcriptional activation. Our findings elucidate the regulatory role of SlWRKY37 in seed dormancy, suggesting its potential as a target for gene editing to reduce seed dormancy in tomato breeding programs.

Genome-wide identification of the GDSL-type esterase/lipase protein (GELP) gene family in Ricinus communis and its transcriptional regulation during germination and seedling establishment under different abiotic stresses.

GDSL-type esterase/lipase protein (GELP) genes are crucial in the specialized lipid metabolism, in the responses to abiotic stresses, and in the regulation of plant homeostasis. R. communis is an important oilseed crop species that can sustain growth and productivity when exposed to harsh environmental conditions. Herein, we raised the question of whether the GELP gene family could be involved in the acquisition of R. communis tolerance to abiotic stresses during seed germination and seedling establishment. Thus, we used bioinformatics and transcriptomics to characterize the R. communis GELP gene family. R. communis genome possesses 96 GELP genes that were characterized by extensive bioinformatics, including phylogenetic analysis, subcellular localization, exon-intron distribution, the analysis of regulatory cis-elements, tandem duplication, and physicochemical properties. Transcriptomics indicated that numerous RcGELP genes are readily responsive to high-temperature and salt stresses and might be potential candidates for genome editing techniques to develop abiotic stress-tolerant crops.

Expression analysis of genes involved in teliospores germination of Tilletia indica inciting Karnal bunt of wheat.

BACKGROUND: Karnal bunt of wheat is an important quarantine disease, incited by Tilletia indica. It limits India's trade in wheat export. The teliospores are major source of inoculum to initiate and spread the Karnal bunt disease. The study aimed to identify the germination-related genes in the teliospores of T. indica. METHODS AND RESULTS: The candidate genes in the teliospores germination were identified through the differential gene expression analysis with suitable bioinformatics analysis. Keeping in soil-borne nature of fungi, the teliospores of T. indica (2015 and 2018) were subjected to the qPCR analysis. 20 candidate genes were identified having role in germination of teliospores of T. indica. Twenty genes, viz. Ti9297 (9.31, 7.87-fold), Ti8696 (5.13, 6.54-fold), Ti7699 (8.9, 7.7-fold), Ti7858 (10.33, 6.21-fold), Ti7954 (7.46, 5.54-fold), Ti7739 (5.46, 6.46-fold), Ti9665 (10.74, 7.64-fold), Ti9335 (6.75, 4.36-fold), Ti8396 (9.35, 7.72-fold), Ti8126 (8.87, 11.31-fold), Ti7326 (6.04, 7.7-fold), Ti10208 (13.83, 5.81-fold), Ti12356 (7.83, 8.02-fold), Ti14271 (9.98, 6.32-fold), Ti9234 (11.2, 8.72-fold), Ti 8876 (6.47, 3.55-fold), Ti 10,606 (4.97, 2.35-fold), Ti7758 (10.33, 8.78-fold), Ti4692 (6.89, 9.88-fold), and Ti3932 (5.77, 4.5-fold) were found highly expressed in the germinating teliospores of 2015 and 2018, respectively. Eight genes (Ti508, Ti4152, Ti5346, Ti2375, Ti3739, Ti1134, Ti4399, and Ti4422) were downregulated in the germinating teliospores but these eight genes were showed higher expression in the dormant teliospores. CONCLUSIONS: Twenty candidate genes were upregulated in the germinating teliospores are supposed to be involved in the process of germination. Eight genes were downregulated which were related to the process of the dormancy of teliospores. The study will be helpful to devise the newer management strategies for Karnal bunt disease of wheat.

Microsatellite marker-assisted backcross breeding for improvement of wheat salt tolerance using Kharchia 65.

BACKGROUND: Salinity is a significant abiotic stress that affects plants from germination through all growth stages. This study was aimed to determine the morpho-physiological and genetic variations in BC1F2, BC2F1 and F3 generations resulting from the cross combination WH1105 × Kharchia 65. RESULTS: A significant reduction in germination percentage was observed under salt stress in BC1F2 and F3 seeds. Correlation, heritability in the broad sense, phenotypic coefficient of variability (PCV) and genotypic coefficient of variability (GCV) were measured for all traits. The presence of both Nax1 and Nax2 loci was confirmed in twenty-nine plants using the marker-assisted selection technique. Genetic relationships among the populations were assessed using twenty-four polymorphic SSR markers. CONCLUSION: Cluster analysis along with two and three-dimensional PCA scaling (Principal Component Analysis) revealed the distinct nature of WH 1105 and Kharchia 65. Six plants closer to the recurrent parent (WH1105) selected through this study can serve as valuable genetic material for salt-tolerant wheat improvement programs.

Assessing Falling Number Stability Increases the Genomic Prediction Ability of Pre-Harvest Sprouting Resistance in Common Winter Wheat.

Pre-harvest sprouting (PHS) resistance is a complex trait, and many genes influencing the germination process of winter wheat have already been described. In the light of interannual climate variation, breeding for PHS resistance will remain mandatory for wheat breeders. Several tests and traits are used to assess PHS resistance, i.e., sprouting scores, germination index, and falling number (FN), but the variation of these traits is highly dependent on the weather conditions during field trials. Here, we present a method to assess falling number stability (FNS) employing an after-ripening period and the wetting of the kernels to improve trait variation and thus trait heritability. Different genome-based prediction scenarios within and across two subsequent seasons based on overall 400 breeding lines were applied to assess the predictive abilities of the different traits. Based on FNS, the genome-based prediction of the breeding values of wheat breeding material showed higher correlations across seasons (r=0.505-0.548) compared to those obtained for other traits for PHS assessment (r=0.216-0.501). By weighting PHS-associated quantitative trait loci (QTL) in the prediction model, the average predictive abilities for FNS increased from 0.585 to 0.648 within the season 2014/2015 and from 0.649 to 0.714 within the season 2015/2016. We found that markers in the Phs-A1 region on chromosome 4A had the highest effect on the predictive abilities for FNS, confirming the influence of this QTL in wheat breeding material, whereas the dwarfing genes Rht-B1 and Rht-D1 and the wheat-rye translocated chromosome T1RS.1BL exhibited effects, which are well-known, on FN per se exclusively.