AINTEGUMENTA-LIKE genes regulate reproductive growth and bud dormancy in Platanus acerifolia.

KEY MESSAGE: Platanus acerifolia AIL genes PaAIL5a/b and PaAIL6b participate in FT-AP1/FUL-AIL pathways to regulate bud dormancy. In addition, PaAIL6a/b can promote flowering, and PaAIL5b and PaAIL6b affect floral development. Bud dormancy and floral induction are essential processes for perennial plants, they are both regulated by photoperiod, temperature, and hormones, indicating the existence of common regulators for both processes. AINTEGUMENTA-LIKE (AIL) genes regulate reproductive growth of annual plants, including floral induction and flower development, and their homologs in poplar and grape act downstream of the florigen gene FT and the floral meristem identity genes AP1/FUL and function to maintain growth and thus inhibit dormancy induction. However, it is not known whether AIL homologs participate in the reproduction processes in perennials and whether the Platanus acerifolia AIL genes are involved in dormancy. P. acerifolia is a perennial woody plant whose reproductive growth is strongly associated with dormancy. Here, we isolated four AIL homologs from P. acerifolia, PaAIL5a, PaAIL5b, PaAIL6a, and PaAIL6b, and systematically investigated their functions by ectopic-overexpression in tobacco. The findings demonstrate that PaAIL5a/b and PaAIL6b respond to short day, low temperature, and hormone signals and act as the components of the FT-AP1/FUL-AIL pathway to regulate the bud dormancy in P. acerifolia. Notably, PaAIL5a/b and PaAIL6b function downstream of PaFTL-PaFUL1/2/3 to inhibit the dormancy induction and downstream of PaFT-PaFUL2/3 to promote the dormancy release. In addition, PaAIL6a/b were found to accelerate flowering in transgenic tobacco, whereas PaAIL5b and PaAIL6b affected the flower development. Together, our results suggest that PaAIL genes may act downstream of different PaFT/PaFTL and PaFUL proteins to fulfill conservative and diverse roles in floral initiation, floral development, and dormancy regulation in P. acerifolia.

Dormancy regulator Prunus mume DAM6 promotes ethylene-mediated leaf senescence and abscission.

Leaf senescence and abscission in autumn are critical phenological events in deciduous woody perennials. After leaf fall, dormant buds remain on deciduous woody perennials, which then enter a winter dormancy phase. Thus, leaf fall is widely believed to be linked to the onset of dormancy. In Rosaceae fruit trees, DORMANCY-ASSOCIATED MADS-box (DAM) transcription factors control bud dormancy. However, apart from their regulatory effects on bud dormancy, the biological functions of DAMs have not been thoroughly characterized. In this study, we revealed a novel DAM function influencing leaf senescence and abscission in autumn. In Prunus mume, PmDAM6 expression was gradually up-regulated in leaves during autumn toward leaf fall. Our comparative transcriptome analysis using two RNA-seq datasets for the leaves of transgenic plants overexpressing PmDAM6 and peach (Prunus persica) DAM6 (PpeDAM6) indicated Prunus DAM6 may up-regulate the expression of genes involved in ethylene biosynthesis and signaling as well as leaf abscission. Significant increases in 1-aminocyclopropane-1-carboxylate accumulation and ethylene emission in DEX-treated 35S:PmDAM6-GR leaves reflect the inductive effect of PmDAM6 on ethylene biosynthesis. Additionally, ethephon treatments promoted autumn leaf senescence and abscission in apple and P. mume, mirroring the changes due to PmDAM6 overexpression. Collectively, these findings suggest that PmDAM6 may induce ethylene emission from leaves, thereby promoting leaf senescence and abscission. This study clarified the effects of Prunus DAM6 on autumn leaf fall, which is associated with bud dormancy onset. Accordingly, in Rosaceae, DAMs may play multiple important roles affecting whole plant growth during the tree dormancy induction phase.

Allelic variation of TaABI5-A4 significantly affects seed dormancy in bread wheat.

KEY MESSAGE: We identified a pivotal transcription factor TaABI5-A4 that is significantly associated with pre-harvest sprouting in wheat; its function in regulating seed dormancy was confirmed in transgenic rice. ABI5 is a critical transcription factor in regulation of crop seed maturation, dormancy, germination, and post-germination. Sixteen copies of homologous sequences of ABI5 were identified in Chinese wheat line Zhou 8425B. Cultivars of two haplotypes TaABI5-A4a and TaABI5-A4b showed significantly different seed dormancies. Based on two SNPs between the sequences of TaABI5-A4a and TaABI5-A4b, two complementary dominant sequence-tagged site (STS) markers were developed and validated in a natural population of 103 Chinese wheat cultivars and advanced lines and 200 recombinant inbred lines (RILs) derived from the Yangxiaomai/Zhongyou 9507 cross; the STS markers can be used efficiently and reliably to evaluate the dormancy of wheat seeds. The transcription level of TaABI5-A4b was significantly increased in TaABI5-A4a-GFP transgenic rice lines compared with that in TaABI5-A4b-GFP. The average seed germination index of TaABI5-A4a-GFP transgenic rice lines was significantly lower than those of TaABI5-A4b-GFP. In addition, seeds of TaABI5-A4a-GFP transgenic lines had higher ABA sensitivity and endogenous ABA content, lower endogenous GA content and plant height, and thicker stem internodes than those of TaABI5-A4b-GFP. Allelic variation of TaABI5-A4-affected wheat seed dormancy and the gene function was confirmed in transgenic rice. The transgenic rice lines of TaABI5-A4a and TaABI5-A4b had significantly different sensitivities to ABA and contents of endogenous ABA and GA in mature seeds, thereby influencing the seed dormancy, plant height, and stem internode length and diameter.

The MKK3 module integrates nitrate and light signals to modulate secondary dormancy in Arabidopsis thaliana.

Seed dormancy corresponds to a reversible blockage of germination. Primary dormancy is established during seed maturation, while secondary dormancy is set up on the dispersed seed, following an exposure to unfavorable factors. Both dormancies are relieved in response to environmental factors, such as light, nitrate, and coldness. Quantitive Trait Locus (QTL) analyses for preharvest sprouting identified MKK3 kinase in cereals as a player in dormancy control. Here, we showed that MKK3 also plays a role in secondary dormancy in Arabidopsis within a signaling module composed of MAP3K13/14/19/20, MKK3, and clade-C MAPKs. Seeds impaired in this module acquired heat-induced secondary dormancy more rapidly than wild-type (WT) seeds, and this dormancy is less sensitive to nitrate, a signal able to release dormancy. We also demonstrated that MPK7 was strongly activated in the seed during dormancy release, especially in response to light and nitrate. This activation was greatly reduced in map3k13/14/19/20 and mkk3 mutants. Finally, we showed that the module was not regulated and apparently did not regulate the genes controlling abscisic acid/gibberellin acid hormone balance, one of the crucial mechanisms of seed dormancy control. Overall, our work identified a MAPK module controlling seed germination and enlarged the panel of functions of the MKK3-related modules in plants.

Altered expression of a raspberry homologue of VRN1 is associated with disruption of dormancy induction and misregulation of subsets of dormancy-associated genes.

Winter dormancy is a key process in the phenology of temperate perennials. Climate change is severely impacting its course leading to economic losses in agriculture. A better understanding of the underlying mechanisms, as well as the genetic basis of the different responses, is necessary for the development of climate-resilient cultivars. This study aims to provide an insight into winter dormancy in red raspberry (Rubus idaeus L). We report the transcriptomic profiles during dormancy in two raspberry cultivars with contrasting responses. The cultivar 'Glen Ample' showed a typical perennial phenology, whereas 'Glen Dee' registered consistent dormancy dysregulation, exhibiting active growth and flowering out of season. RNA-seq combined with weighted gene co-expression network analysis identified gene clusters in both genotypes that exhibited time-dependent expression profiles. Functional analysis of 'Glen Ample' gene clusters highlighted the significance of the cell and structural development prior to dormancy entry as well the role of genetic and epigenetic processes such as RNAi and DNA methylation in regulating gene expression. Dormancy release in 'Glen Ample' was associated with up-regulation of transcripts associated with the resumption of metabolism, nucleic acid biogenesis, and processing signal response pathways. Many of the processes occurring in 'Glen Ample' were dysregulated in 'Glen Dee' and 28 transcripts exhibiting time-dependent expression in 'Glen Ample' that also had an Arabidopsis homologue were not found in 'Glen Dee'. These included a gene with homology to Arabidopsis VRN1 (RiVRN1.1) that exhibited a sharp decline in expression following dormancy induction in 'Glen Ample'. Characterization of the gene region in the 'Glen Dee' genome revealed two large insertions upstream of the ATG start codon. We propose that expression below detection level of a specific VRN1 homologue in 'Glen Dee' causes dormancy misregulation as a result of inappropriate expression of a subset of genes that are directly or indirectly regulated by RiVRN1.1.

Characterizing seed dormancy in Epimedium brevicornu Maxim.: Development of novel chill models and determination of dormancy release mechanisms by transcriptomics.

PURPOSE: Epimedium brevicornu Maxim. is a perennial persistent C3 plant of the genus Epimedium Linn. in the family Berberaceae that exhibits severe physiological and morphological seed dormancy.We placed mature E. brevicornu seeds under nine stratification treatment conditions and explored the mechanisms of influence by combining seed embryo growth status assessment with related metabolic pathways and gene co-expression analysis. RESULTS: We identified 3.9 °C as the optimum cold-stratification temperature of E. brevicornu seeds via a chilling unit (CU) model. The best treatment was variable-temperature stratification (10/20 °C, 12/12 h) for 4 months followed by low-temperature stratification (4 °C) for 3 months (4-3). A total of 63801 differentially expressed genes were annotated to 2587 transcription factors (TFs) in 17 clusters in nine treatments (0-0, 0-3, 1-3, 2-3, 3-3, 4-3, 4-2, 4-1, 4-0). Genes specifically highly expressed in the dormancy release treatment group were significantly enriched in embryo development ending in seed dormancy and fatty acid degradation, indicating the importance of these two processes. Coexpression analysis implied that the TF GRF had the most reciprocal relationships with genes, and multiple interactions centred on zf-HD and YABBY as well as on MYB, GRF, and TCP were observed. CONCLUSION: In this study, analyses of plant hormone signal pathways and fatty acid degradation pathways revealed changes in key genes during the dormancy release of E. brevicornu seeds, providing evidence for the filtering of E. brevicornu seed dormancy-related genes.

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.

Regulation of Seed Dormancy Genes in Triticeae Species.

Wild progenitors of Triticeae crops generally have long dormancy periods. Domesticated crops inherited these longer dormancy alleles from their wild progenitors, which have since been modified and selected during cultivation and utilization by humans. Thus, allelic combinations at different seed dormancy loci are currently represented in Triticeae germplasm preserved in seed repositories and gene banks as accessions and materials of breeding programs. Methods to evaluate seed dormancy are key to explore, analyze, and exploit optimal alleles in dormancy genes. Recent developments in genomics have accelerated the identification and analysis of seed dormancy loci in Triticeae species. Transgenic experiments have been conducted to validate if candidate genes affect seed dormancy and more recently have yielded an array of mutations derived from genome editing for practical applications. The information gathered on these seed dormancy loci provides a deeper knowledge of germplasm diversity and offers strategies to control seed dormancy in breeding programs in Triticeae crops.

Assessment of Dormancy Level and Germination Ability of Seeds with Physiological Dormancy.

Germination test is fundamental and commonly used technique for seed dormancy and germination studies, and proper assessment of dormancy level and germination ability of a given set of seeds is prerequisite for most of the studies. However, germination is very sensitive to imbibition conditions, and dormancy development is also sensitive to growth conditions of the mother plants. In this chapter, we describe tips for plant growth and germination test mainly for physiological and molecular genetic studies with Arabidopsis. This protocol can be applied for other plant species with relatively small seeds and for various studies to analyze the effect of light, phytohormones, and other chemicals in seed germination.

Map-Based Cloning of the Causal Gene for a Seed Dormancy Quantitative Trait Locus in Barley.

Seed dormancy is an important agronomic trait in cereal crops. Throughout the domestication of cereals, seed dormancy has been reduced to obtain uniform germination. However, grain crops must retain moderate levels of seed dormancy to prevent problems such as preharvest sprouting in wheat (Triticum aestivum) and barley (Hordeum vulgare). To produce modern cultivars with the appropriate seed dormancy levels, it is important to identify the genes responsible for seed dormancy. With recent advances in sequencing technology, several causal genes for seed dormancy quantitative trait loci (QTLs) have been identified in barley and wheat. Here, we present a method to identify causal genes for seed dormancy QTLs in barley, a method that is also applicable to other cereals.

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.

Targeted Modification of Grain Dormancy Genes in Barley.

Transgenesis technologies, such as overexpression or RNA interference-mediated suppression, have often been used to alter the activity of target genes. More recently developed targeted genome modification methods using customizable endonucleases allow for the regulation or knockout mutation of target genes without the necessity of integrating recombinant DNA. Such approaches make it possible to create novel alleles of target genes, thereby significantly contributing to crop improvement. Among these technologies, the Cas9 endonuclease-based method is widely applied to several crops, including barley (Hordeum vulgare). In this chapter, we describe an Agrobacterium-based approach to the targeted modification of grain dormancy genes in barley using RNA-guided Cas9 nuclease.

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.

Genome Editing to Produce Knockout Mutations of Seed Dormancy Genes in Wheat.

Knockout mutants provide definitive information about the functions of genes related to agronomic traits, including seed dormancy. However, it takes many years to produce knockout mutants using conventional techniques in polyploid plants such as hexaploid wheat. Genome editing with sequence-specific nucleases is a promising approach for obtaining knockout mutations in all targeted homoeologs of wheat simultaneously. Here, we describe a procedure to produce a triple recessive mutant in wheat via genome editing. This protocol covers the evaluation of gRNA and Agrobacterium-mediated transformation to obtain edited wheat seedlings.

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.

Seed production determines the entrance to dormancy of the inflorescence meristem of Pisum sativum and the end of the flowering period.

Flowering plants adjust their reproductive period to maximize the success of the offspring. Monocarpic plants, those with a single reproductive cycle that precedes plant senescence and death, tightly regulate both flowering initiation and flowering cessation. The end of the flowering period involves the arrest of the inflorescence meristem activity, known as proliferative arrest, in what has been interpreted as an evolutionary adaptation to maximize the allocation of resources to seed production and the viability of the progeny. Factors influencing proliferative arrest were described for several monocarpic plant species many decades ago, but only in the last few years studies performed in Arabidopsis have allowed to approach proliferative arrest regulation in a comprehensive manner by studying the physiology, hormone dynamics, and genetic factors involved in its regulation. However, these studies remain restricted to Arabidopsis and there is a need to expand our knowledge to other monocarpic species to propose general mechanisms controlling the process. In this work, we have characterized proliferative arrest in Pisum sativum, trying to parallel available studies in Arabidopsis to maximize this comparative framework. We have assessed quantitatively the role of fruits/seeds in the process, the influence of the positional effect of these fruits/seeds in the behavior of the inflorescence meristem, and the transcriptomic changes in the inflorescence associated with the arrested state of the meristem. Our results support a high conservation of the factors triggering arrest in pea and Arabidopsis, but also reveal differences reinforcing the need to perform similar studies in other species.

A comprehensive phenotypic and genotypic evaluation of Spanish groundnuts from diverse crosses to identify superior and stable donors for fresh seed dormancy.

Breeding high yielding groundnut cultivars with 2-3 weeks of fresh seed dormancy, particularly in Spanish-type cultivars, enhances the sustainability of agriculture in groundnuts. In this context, we conducted a comprehensive phenotypic and genotypic evaluation of advanced breeding lines developed in the genetic background of Spanish types. By employing multi-phenotyping and marker data, we identified PBS 15044, 16004, 16013, 16015, 16016, 16017, 16020, 16021, 16026, 16031, 16035, 16037, 16038, 16039, 16041, and 16042 with 2-3 weeks dormancy (> 90%).The various parametric and non-parametric estimates identified the stable fresh dormant genotypes with one or more superior economic trait. PBS 16021, 15044, 16038, and 16039 identified with high hundred pod weight (HPW) were also reported having high intensity of dormancy (> 90% for up to 3 weeks); PBS 15044, 16016, PBS 16038 and PBS 16039 with high hundred kernel weight (HKW) also reported with up to 3 weeks fresh seed dormancy; and PBS 16013, 16031, and 16038 with up to 3 weeks fresh seed dormancy had high shelling percentage (SP). They can be used to develop lines with the desired level of dormancy, and high yields, by designing appropriate breeding strategies.

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.

In Planta Genome Editing in Commercial Wheat Varieties: Use of TaQsd1 to Lengthen Seed Dormancy.

Dependency on in vitro culture and regeneration limits the ability to use genome editing on elite wheat (Triticum aestivum L.) varieties. We recently developed an in planta particle bombardment (iPB) technique for gene editing in wheat that utilizes shoot apical meristems (SAMs) as a target tissue. Since the method does not require in vitro culture, it can therefore be used on recalcitrant varieties. In this chapter, we describe in detail the steps used in the iPB method. With this protocol, 3% to 5% of T0 plants grown from bombarded SAMs typically carry mutant alleles and approximately 1% to 2% of the T0 plants inherit mutant alleles in the next generation.

The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination.

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.