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Gene expression profile of the LanFTc1 gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying different LanFTc1 alleles (Ku, Pal, and ku). (A) expression under an 8-h photoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3 technical replicates. A logarithmic scale was used to accommodate observed large differences in gene expression values.
Source publication
Narrow-leafed lupin (Lupinus angustifolius L.) is a moderate-yielding legume crop known for its high grain protein content and contribution to soil improvement. It is cultivated under photoperiods ranging from 9 to 17 h, as a spring-sown (in colder locations) or as an autumn-sown crop (in warmer regions). Wild populations require a prolonged cold p...
Citations
... In legumes, the mechanisms of flowering induction in response to environmental signals appear to be more complex than in Arabidopsis for several reasons. First, orthologs of the FLC and CO integrator genes are absent or inactive in vernalization-sensitive legume species [20,21]. Secondly, the genomes of temperate legumes contain four to six FT-like genes, grouped into three subclades, namely FTa, FTb, and FTc [22]. ...
... Two deletions in cultivated varieties encompassing 1423 bp and 5162 bp of the LanFTc1 promoter region were named as Ku and Jul, respectively, while the wild allele having intermediate phenology and carrying 1208 bp deletion was named as Pal. The wild allele (ku) without a mutation retained vernalization responsiveness and flowered late [21,26]. ...
... The recently published L. angustifolius dataset [21] includes the expression of four FT-like genes and their putative target AGL8, whose protein sequence revealed the highest similarity to A. thaliana FUL/AGL8 (AT5G60910) and AP1 (AT1G69120) genes [21]. The expression data varied with respect to vernalization, the photoperiod, and circadian clock. ...
Flowering is initiated in response to environmental cues, with the photoperiod and ambient temperature being the main ones. The regulatory pathways underlying floral transition are well studied in Arabidopsis thaliana but remain largely unknown in legumes. Here, we first applied an in silico approach to infer the regulatory inputs of four FT-like genes of the narrow-leafed lupin Lupinus angustifolius. We studied the roles of FTc1, FTc2, FTa1, and FTa2 in the activation of meristem identity gene AGL8 in response to 8 h and 16 h photoperiods, vernalization, and the circadian rhythm. We developed a set of regression models of AGL8 regulation by the FT-like genes and fitted these models to the recently published gene expression data. The importance of the input from each FT-like gene or their combinations was estimated by comparing the performance of models with one or few FT-like genes turned off, thereby simulating loss-of-function mutations that were yet unavailable in L. angustifolius. Our results suggested that in the early flowering Ku line and intermediate Pal line, the FTc1 gene played a major role in floral transition; however, it acted through different mechanisms under short and long days. Turning off the regulatory input of FTc1 resulted in substantial changes in AGL8 expression associated with vernalization sensitivity and the circadian rhythm. In the wild ku line, we found that both FTc1 and FTa1 genes had an essential role under long days, which was associated with the vernalization response. These results could be applied both for setting up new experiments and for data analysis using the proposed modeling approach.
... A prolonged cold period during germination and juvenile growth, that induces vernalization, usually accelerates the transition from vegetative to generative growth phase in white lupin landraces, except those adapted to spring sowing and drought escape by rapid flowering [8][9][10]. A similar observation was done also for two other Old World lupin crop species, narrow-leafed and yellow lupins, where Palestinian accessions gained significant vernalization independence [11][12][13]. In the latter two species, abolition of vernalization requirements is conferred by large deletions in regulatory regions of one of the four FLOWERING LOCUS T (FT) homologs present in their genomes: LanFTc1 in the narrow-leafed lupin, and LlutFTc1 in the yellow lupin [11,13,14]. ...
... All three Old World lupin crop species delay flowering under short day photoperiod (as compared to long days). Nevertheless, this response is much more significant in vernalization-requiring accessions than in the thermoneutral genotypes [9,11,12,[15][16][17]. ...
... Recent studies on the narrow-leafed and yellow lupin genomes highlighted the association between insertiondeletion (indel) polymorphism in the promoter region of FTc1 genes (LanFTc1 and LlutFTc1, respectively) and vernalization-independent early flowering [11][12][13][14]. Therefore, we investigated the potential structural polymorphism of the orthologous FTc1 region in white lupin. ...
Background
White lupin (Lupinus albus L.) is a high-protein Old World grain legume with remarkable food and feed production interest. It is sown in autumn or early spring, depending on the local agroclimatic conditions. This study aimed to identify allelic variants associated with vernalization responsiveness, in order to improve our knowledge of legume flowering regulatory pathways and develop molecular selection tools for the desired phenology as required for current breeding and adaptation to the changing climate.
Results
Some 120 white lupin accessions originating from a wide range of environments of Europe, Africa, and Asia were phenotyped under field conditions in three environments with different intensities of vernalization, namely, a Mediterranean and a subcontinental climate sites of Italy under autumn sowing, and a suboceanic climate site of France under spring sowing. Two hundred sixty-two individual genotypes extracted from them were phenotyped in a greenhouse under long-day photoperiod without vernalization. Phenology data, and marker data generated by Diversity Arrays Technology sequencing (DArT-seq) and by PCR-based screening targeting published quantitative trait loci (QTLs) from linkage map and newly identified insertion/deletion polymorphisms in the promoter region of the FLOWERING LOCUS T homolog, LalbFTc1 gene (Lalb_Chr14g0364281), were subjected to a genome-wide association study (GWAS). Population structure followed differences in phenology and isolation by distance pattern. The GWAS highlighted numerous loci significantly associated with flowering time, including four LalbFTc1 gene promoter deletions: 2388 bp and 2126 bp deletions at the 5’ end, a 264 bp deletion in the middle and a 28 bp deletion at the 3’ end of the promoter. Besides LalbFTc1 deletions, this set contained DArT-seq markers that matched previously published major QTLs in chromosomes Lalb_Chr02, Lalb_Chr13 and Lalb_Chr16, and newly discovered QTLs in other chromosomes.
Conclusions
This study highlighted novel QTLs for flowering time and validated those already published, thereby providing novel evidence on the convergence of FTc1 gene functional evolution into the vernalization pathway in Old World lupin species. Moreover, this research provided the set of loci specific for extreme phenotypes (the earliest or the latest) awaiting further implementation in marker-assisted selection for spring- or winter sowing.
... In order to make standard curves, a series of dilutions was prepared for each analyzed gene. Standard curves were developed following previously reported protocol [26]. ...
The main efforts in common wheat (Triticum aestivum L.) breeding focus on yield, grain quality, and resistance to biotic and abiotic stresses. One of the major threats affecting global wheat cultivation and causing significant crop production losses are rust diseases, including leaf rust caused by a bi-otrophic fungus Puccinia triticina Eriks. Genetically determined resistance to leaf rust has been characterized in young plants (seedling resistance) as well as in plants at the adult plant stage. At the seedling stage, resistance is controlled vertically by major R genes, conferring a race-specific re-sponse that is highly effective but usually short-lived due to the rapid evolution of potentially vir-ulent fungi. In mature plants, horizontal adult plant resistance (APR) was described, which pro-vides long-term protection against multiple races of pathogens. A better understanding of molecular mechanisms underlying the function of APR genes would enable the development of new strategies for resistance breeding in wheat. Therefore, in the present study we focused on early transcriptomic responses of two major wheat APR genes, Lr34 and Lr67, and three complementary miRNAs, tae-miR9653b, tae-miR9773 and tae-miR9677b, to inoculation with P. triticina. Plant material con-sisted of five wheat reference varieties, Artigas, NP846, Glenlea, Lerma Rojo and TX89D6435, containing the Lr34/Yr18 and Lr67/Yr46 resistance genes. Biotic stress was induced by inoculation with fungal spores under controlled conditions in a phytotron. Plant material consisted of leaf tissue sampled before inoculation as well as 6, 12, 24 and 48 h postinoculation (hpi). The APR gene ex-pression was quantified using real-time PCR with two reference genes, whereas miRNA was quantified using droplet digital PCR. This paper describes the resistance response of APR genes to inoculation with races of leaf rust-causing fungi that occur in central Europe. The study revealed high variability of expression profiles between varieties and time-points, with the prevalence of downregulation for APR genes and upregulation for miRNAs during the development of an early defense response. Nevertheless, despite the downregulation initially observed, the expression of Lr34 and Lr67 genes in studied cultivars was significantly higher than in a control line carrying wild (susceptible) alleles.
... The published results suggest that FT genes are the main targets of vernalization in legumes (Table 2) , although the mechanisms of FT activation are still unclear [44,45]. So far, the most intensive research of cold-induced flowering has been conducted in Medicago trancatula and narrow-leafed lupin Lupinus angustifolius [44][45][46][47]. Here, we summarize the available information for genetic bases of vernalization in eight legume species with a special emphasis on the role of FT genes. ...
... It has been shown that several natural mutations (Ku, Jul and Pal) provide vernalization independence in L. angustifolius. All these mutations are located in the promoter region of the LanFTc1 gene [45,47,90,110]. ...
... Besides LanFTc1, recent studies have shown the involvement of a number of novel candidate genes in the vernalization response [47,114]. The expression profiles of these genes were examined for vernalization responsiveness in three accessions, carrying domesticated allele Ku, intermediate allele Pal, and wild allele ku [47]. ...
Vernalization is the requirement for exposure to low temperatures to trigger flowering. The best knowledge about the mechanisms of vernalization response has been accumulated for Arabidopsis and cereals. In Arabidopsis thaliana, vernalization involves an epigenetic silencing of the MADS-box gene FLOWERING LOCUS C (FLC), which is a flowering repressor. FLC silencing releases the expression of the main flowering inductor FLOWERING LOCUS T (FT), resulting in a floral transition. Remarkably, no FLC homologues have been identified in the vernalization-responsive legumes, and the mechanisms of cold-mediated transition to flowering in these species remain elusive. Nevertheless, legume FT genes have been shown to retain the function of the main vernalization signal integrators. Unlike Arabidopsis, legumes have three subclades of FT genes, which demonstrate distinct patterns of regulation with respect to environmental cues and tissue specificity. This implies complex mechanisms of vernalization signal propagation in the flowering network, that remain largely elusive. Here, for the first time, we summarize the available information on the genetic basis of cold-induced flowering in legumes with a special focus on the role of FT genes.
... The present study revealed sub-functionalization of LlutFTc1 into vernalization pathway (Fig. 2-4), whereas LlutFTa1a into photoperiod response (Fig. 5). A similar observation was made for LanFTc1 (wild allele) and LanFTa1 (Palestinian allele) genes in narrow-leafed lupin 18,61 . ...
... Therefore, a general mechanism of vernalization response based on the FTc clade may have been established several million years before the ploidy event in the Lupinus lineage 58,65 . Following duplication, FTc1 orthologs retained basic functions whereas FTc2 differentiated in downstream lineages, resulting in the loss-of-function in narrow-leafed lupin 18,61 and partial sub-functionalization in yellow lupin ( Fig. 2-5). The other evidence supporting relatively recent evolution of vernalization trait is the observed lack of conservation of Arabidopsis FRIGIDA-FLC model in many species, including the above-mentioned Pooideae grasses 64,66 . ...
... Gene expression profiling was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad). Standard curves were developed following previously reported protocol 61 . R 2 and PCR efficiency values (Table S16) were calculated using Bio-Rad CFX Manager 3.1. ...
Ongoing climate change has considerably reduced the seasonal window for crop vernalization, concurrently expanding cultivation area into northern latitudes with long-day photoperiod. To address these changes, cool season legume breeders need to understand molecular control of vernalization and photoperiod. A key floral transition gene integrating signals from these pathways is the Flowering locus T (FT). Here, a recently domesticated grain legume, yellow lupin (Lupinus luteus L.), was explored for potential involvement of FT homologues in abolition of vernalization and photoperiod requirements.
Two FTa (LlutFTa1a and LlutFTa1b) and FTc (LlutFTc1 and LlutFTc2) homologues were identified and sequenced for two contrasting parents of a reference recombinant inbred line (RIL) population, an early-flowering cultivar Wodjil and a late-flowering wild-type P28213. Large deletions were detected in the 5′ promoter regions of three FT homologues. Quantitative trait loci were identified for flowering time and vernalization response in the RIL population and in a diverse panel of wild and domesticated accessions. A 2227 bp deletion found in the LlutFTc1 promoter was linked with early phenology and vernalization independence, whereas LlutFTa1a and LlutFTc2 indels with photoperiod responsiveness. Comparative mapping highlighted convergence of FTc1 indel evolution in two Old World lupin species, addressing both artificial selection during domestication and natural adaptation to short season environmental conditions. We concluded that rapid flowering in yellow lupin is associated with the de-repression of the LlutFTc1 homologue from the juvenile phase, putatively due to the elimination of all binding sites in the promoter region for the AGAMOUS-like 15 transcription factor.
... The set of genes analyzed by quantitative PCR included glucan endo-1,3-beta-glucosidase-like (TanjilG_23384), LlR18A (TanjilG_27015), acidic endochitinase (Tan-jilG_04706), HSP17.4 (TanjilG_05080), a candidate gene for the Lanr1 locus-disease resistance protein (TIR-NBS-LRR class) (TanjilG_05042), a candidate gene for the AnMan locus-a rho GTPase-activating protein (Tan-jilG_12861), and a legume-specific hypothetical protein significantly upregulated in the majority of line × time point combinations (TanjilG_10657). Reference genes validated in the previous NLL quantitative gene expression studies were selected, namely LanDExH7 (TanjilG_23733) and LanTUB6 (TanjilG_32899) 53,[109][110][111][112] . Primers were designed in Geneious Prime (Auckland, New Zealand) using Primer3 113,114 . ...
... Primers were designed in Geneious Prime (Auckland, New Zealand) using Primer3 113,114 . Standard curves were developed for all analyzed genes using the same method as in previous narrow-leafed lupin study 112 . R 2 and PCR efficiency values were calculated in Bio-Rad CFX Manager 3.1 (Supplementary Table S9). ...
Narrow-leafed lupin (NLL, Lupinus angustifolius L.) is a legume plant cultivated for grain production and soil improvement. Worldwide expansion of NLL as a crop attracted various pathogenic fungi, including Colletotrichum lupini causing a devastating disease, anthracnose. Two alleles conferring improved resistance, Lanr1 and AnMan, were exploited in NLL breeding, however, underlying molecular mechanisms remained unknown. In this study, European NLL germplasm was screened with Lanr1 and AnMan markers. Inoculation tests in controlled environment confirmed effectiveness of both resistance donors. Representative resistant and susceptible lines were subjected to differential gene expression profiling. Resistance to anthracnose was associated with overrepresentation of “GO:0006952 defense response”, “GO:0055114 oxidation–reduction process” and “GO:0015979 photosynthesis” gene ontology terms. Moreover, the Lanr1 (83A:476) line revealed massive transcriptomic reprogramming quickly after inoculation, whereas other lines showed such a response delayed by about 42 h. Defense response was associated with upregulation of TIR-NBS, CC-NBS-LRR and NBS-LRR genes, pathogenesis-related 10 proteins, lipid transfer proteins, glucan endo-1,3-beta-glucosidases, glycine-rich cell wall proteins and genes from reactive oxygen species pathway. Early response of 83A:476, including orchestrated downregulation of photosynthesis-related genes, coincided with the successful defense during fungus biotrophic growth phase, indicating effector-triggered immunity. Mandelup response was delayed and resembled general horizontal resistance.
... The subsequent breeding and release of early-flowering, vernalization-insensitive cultivars expanded the range of narrow-leafed lupins to warm winter-growing climates of southern Australia (Ku) and summer-growing continental climates in northern Europe (Jul). Almost all Australian and European varieties released since the 1970's carry one of these mutations (Cowling, 2020;Rychel-Bielska et al., 2020). Strong selection for Ku and Jul in breeding programmes over the course of the last 50 years has not been an entirely positive development, however. ...
... More recently, it was shown that Ku is caused by a 1,423 bp deletion in the promoter region of the floral integrator gene LanFTc1, which de-represses its expression and thereby permits early flowering in the absence of vernalization under controlled conditions (Nelson et al., 2017). Jul was found to be caused by an independent 5,162 bp deletion in the same promoter region of LanFTc1, which results in a similar flowering phenotype and expression profile as Ku (Nelson et al., 2017;Rychel-Bielska et al., 2020;Taylor et al., 2019). Sequencing of genetically diverse narrow-leafed lupins additionally revealed a third unique deletion of 1,208 bp overlapping the same promoter region of LanFTc1 (Taylor et al., 2019). ...
... (i) LanFTc1-5162 (Jul), which has been under selection in European varieties but has yet to be adopted in Australian narrow-leafed lupin breeding where LanFTc1-1423 (Ku) predominates (Cowling, 2020;Rychel-Bielska et al., 2020); and (ii) LanFTc1-1208, which has only been observed in wild germplasm originating from the eastern Mediterranean thus far Taylor et al., 2019). ...
We designed and validated a new multiplex PCR marker which discriminates between four insertion/deletion (INDEL) alleles in the 5' regulatory region of a major flowering time gene in Lupinus angustifolius, LanFTc1. The four INDEL alleles were the wild-type allele (ku) in variety Geebung (G), a 1208-bp deletion allele in accession P22660 (P), a 1423-bp deletion allele (Ku) in variety Tanjil (T), and a 5162-bp deletion allele (Jul) in variety Krasnolistny (K). F2 PCR marker genotypes segregated as expected in populations K×G, K×T, P×G and P×T. Heritability of flowering times based on F2:3 parent:offspring correlation was high in K×G (0.81 ± 0.09), P×G (0.76 ± 0.10) and P×T (0.81 ± 0.11), but low in K×T (-0.04 ± 0.15) due to similar early-flowering phenotypes produced by Ku and Jul. Progeny homozygous for the 1208-bp deletion allele resulted in unique array of mid-season phenology in the F2, F3 and F4 generations which may improve agronomic adaptation. This multiplex PCR marker will improve the efficiency of introgression of the new INDELs into future lupin varieties.
... The second L. angustifolius domesticated early phenology mutation, Jul, was recently revealed to be a 5162 bp deletion in the FTc1 promoter region, fully encompassing the Ku indel [44]. Recently, a fourth FTc1 allele, Pal, was found in a wild population originating from Palestine, carrying 1208 bp deletion partially overlapping with Ku [44,45]. Recent gene-based genome-wide association study confirmed that this series of indels in FTc1 has a major effect on time to flowering and maturity in diversified narrow-leafed germplasm collection [35]. ...
White lupin (Lupinus albus L.) is a pulse annual plant cultivated from the tropics to temperate regions for its high-protein grain as well as a cover crop or green manure. Wild populations are typically late flowering and have high vernalization requirements. Nevertheless, some early flowering and thermoneutral accessions were found in the Mediterranean basin. Recently, quantitative trait loci (QTLs) explaining flowering time variance were identified in bi-parental population mapping, however, phenotypic and genotypic diversity in the world collection has not been addressed yet. In this study, a diverse set of white lupin accessions (n = 160) was phenotyped for time to flowering in a controlled environment and genotyped with PCR-based markers (n = 50) tagging major QTLs and selected homologs of photoperiod and vernalization pathway genes. This survey highlighted quantitative control of flowering time in white lupin, providing statistically significant associations for all major QTLs and numerous regulatory genes, including white lupin homologs of CONSTANS, FLOWERING LOCUS T, FY, MOTHER OF FT AND TFL1, PHYTOCHROME INTERACTING FACTOR 4, SKI-INTERACTING PROTEIN 1, and VERNALIZATION INDEPENDENCE 3. This revealed the complexity of flowering control in white lupin, dispersed among numerous loci localized on several chromosomes, provided economic justification for future genome-wide association studies or genomic selection rather than relying on simple marker-assisted selection.
Flowering time is the most important agronomic trait which is used in breeding and determines the crop performance. Vernalization, or prolonged exposure to cold, accelerates flowering and increases yields in many crops. The molecular mechanisms of vernalization-induced flowering are well studied in Arabidopsis thaliana, but remain largely unknown for legumes. Mathematical modeling is a powerful tool to predict regulatory interactions in gene networks on the basis of gene expression patterns. This review concerns previously developed approaches to modeling gene regulatory networks of the flowering transition process and the prospects for their adaptation with the aim of conducting the analysis of the mechanisms of vernalization requirement in legumes.
Lupinus mutabilis is an under‐domesticated legume species from the Andean region of South America. It belongs to the New World lupins clade, which groups several lupin species displaying large genetic variation and adaptability to highly different environments. L. mutabilis is attracting interest as a potential multipurpose crop to diversify the European supply of plant proteins, increase agricultural biodiversity, and fulfill bio‐based applications. This study reports the first high‐quality L. mutabilis genome assembly, which is also the first sequenced assembly of a New World lupin species. Through comparative genomics and phylogenetics, the evolution of L. mutabilis within legumes and lupins is described, highlighting both genomic similarities and patterns specific to L. mutabilis , potentially linked to environmental adaptations. Furthermore, the assembly was used to study the genetics underlying important traits for the establishment of L. mutabilis as a novel crop, including protein and quinolizidine alkaloids contents in seeds, genomic patterns of classic resistance genes, and genomic properties of L. mutabilis mycorrhiza‐related genes. These analyses pointed out copy number variation, differential genomic gene contexts, and gene family expansion through tandem duplications as likely important drivers of the genomic diversity observed for these traits between L. mutabilis and other lupins and legumes. Overall, the L. mutabilis genome assembly will be a valuable resource to conduct genetic research and enable genomic‐based breeding approaches to turn L. mutabilis into a multipurpose legume crop.
