Expression Profiles of Alkaloid-Related Genes across the Organs of Narrow-Leafed Lupin (Lupinus angustifolius L.) and in Response to Anthracnose Infection.
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ABSTRACT: The main restraint obstructing the wider adoption of lupins as protein crops is the presence of bitter and toxic quinolizidine alkaloids (QAs), whose contents might increase under exposure to stressful environmental conditions. A poor understanding of how QAs accumulate hinders the breeding of sweet varieties. Here, we characterize the expression profiles of QA-related genes, along with the alkaloid content, in various organs of sweet and bitter narrow-leafed lupin (NLL, Lupinus angustifolius L.). Special attention is paid to the RAP2-7 transcription factor, a candidate regulator of the QA pathway. We demonstrate the upregulation of RAP2-7 and other QA-related genes, across the aerial organs of a bitter cultivar and the significant correlations between their expression levels, thus supporting the role of RAP2-7 as an important regulatory gene in NLL. Moreover, we showed that the initial steps of QA synthesis might occur independently in all aerial plant organs sharing common regulatory mechanisms. Nonetheless, other regulatory steps might be involved in RAP2-7-triggered QA accumulation, given its expression pattern in leaves. Finally, the examination of QA-related gene expression in plants infected with Colletotrichum lupini evidenced no connection between QA synthesis and anthracnose resistance, in contrast to the important role of polyamines during plant-pathogen interactions.
Project description:BackgroundWhilst information regarding small RNAs within agricultural crops is increasing, the miRNA composition of the nutritionally valuable pulse narrow-leafed lupin (Lupinus angustifolius) remains unknown.ResultsBy conducting a genome- and transcriptome-wide survey we identified 7 Dicer-like and 16 Argonaute narrow-leafed lupin genes, which were highly homologous to their legume counterparts. We identified 43 conserved miRNAs belonging to 16 families, and 13 novel narrow-leafed lupin-specific miRNAs using high-throughput sequencing of small RNAs from foliar and root and five seed development stages. We observed up-regulation of members of the miRNA families miR167, miR399, miR156, miR319 and miR164 in narrow-leafed lupin seeds, and confirmed expression of miR156, miR166, miR164, miR1507 and miR396 using quantitative RT-PCR during five narrow-leafed lupin seed development stages. We identified potential targets for the conserved and novel miRNAs and were able to validate targets of miR399 and miR159 using 5' RLM-RACE. The conserved miRNAs are predicted to predominately target transcription factors and 93% of the conserved miRNAs originate from intergenic regions. In contrast, only 43% of the novel miRNAs originate from intergenic regions and their predicted targets were more functionally diverse.ConclusionThis study provides important insights into the miRNA gene regulatory networks during narrow-leafed lupin seed development.
Project description:Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes.
Project description:Selection for resistance against gray leaf spot (GLS) is a major objective in the lupin breeding programs. A segregation ratio of 1:1 (resistant:susceptible) in F8 recombinant inbred lines (RIL8) derived from a cross between a breeding line 83A:476 (resistant to GLS) and a wild accession P27255 (susceptible to GLS) indicated that GLS was controlled by a single major gene. To develop molecular markers linked to GLS, in the beginning, only 11 resistant lines and six susceptible lines from the 83A:476 and P27255 population were genotyped with MFLP markers, and three MFLP markers were identified to be co-segregated with GLS. This method was very efficient, but the markers were located outside of the gene, and could not be used in other germplasms. Then QTL analysis and fine mapping were conducted to identify the gene. Finally, the gene was narrowed down to a 241-kb region containing two disease resistance genes. To further identify the candidate gene, DNA variants between accessions Tanjil (resistant to GLS) and Unicrop (susceptible to GLS) were analyzed. The results indicated that only one SNP was detected in the 241 kb region. This SNP was located in the TMV resistance protein N-like gene region and also identified between 83A:476 and P27255. Genotyping the Tanjil/Unicrop RIL8 population showed that this SNP co-segregated with GLS resistance. The phylogenetic tree analysis of this gene among 18 lupin accessions indicates that Australian resistant breeding line/varieties were clustered into one group and carry two resistant alleles, while susceptible accessions were clustered into different groups.
Project description:The last century has witnessed rapid domestication of the narrow-leafed lupin (Lupinus angustifolius L.) as a grain legume crop, exploiting discovered alleles conferring low-alkaloid content (iucundus), vernalization independence (Ku and Julius), and reduced pod shattering (lentus and tardus). In this study, a L. angustifolius mapping population was subjected to massive analysis of cDNA ends (MACE). The MACE yielded 4185 single nucleotide polymorphism (SNP) markers for linkage map improvement and 30,595 transcriptomic profiles for expression quantitative trait loci (eQTL) mapping. The eQTL highlighted a high number of cis- and trans-regulated alkaloid biosynthesis genes with gene expression orchestrated by a regulatory agent localized at iucundus locus, supporting the concept that ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7 may control low-alkaloid phenotype. The analysis of Ku shed light on the vernalization response via FLOWERING LOCUS T and FD regulon in L. angustifolius, providing transcriptomic evidence for the contribution of several genes acting in C-repeat binding factor (CBF) cold responsiveness and in UDP-glycosyltransferases pathways. Research on lentus selected a DUF1218 domain protein as a candidate gene controlling the orientation of the sclerified endocarp and a homolog of DETOXIFICATION14 for purplish hue of young pods. An ABCG transporter was identified as a hypothetical contributor to sclerenchyma fortification underlying tardus phenotype.
Project description:Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future.
Project description:Low-alkaloid content is an important breeding target to improve the quality of lupin seeds. An APETALA2/ethylene response transcription factor, RAP2-7, is likely a candidate gene for the major alkaloid locus iucundus, and plays a crucial role in regulation of seed alkaloid content in narrow-leafed lupin (NLL; Lupinus angustifolius L.). Here, we exploited a single-nucleotide polymorphism within RAP2-7 credibly associated with seed alkaloid content, to develop the co-dominant derived cleaved amplified polymorphic sequence (dCAPS) marker iuc_RAP2-7. Marker validation in 202 NLL accessions demonstrated that seed alkaloid content ≥0.9% of the seed dry weight was associated with the high-alkaloid marker band (Iucundus genotypes), whereas alkaloid content up to 0.5% of the seed dry weight was associated with the low-alkaloid marker band (iucundus genotypes). Within a given detection limit, iuc_RAP2-7 unambiguously identified all but three low-alkaloid accessions. The latter accessions apparently have a different regulatory mechanism for seed alkaloid content because the RAP2-7 gene/putative promoter sequence and expression of alkaloid-associated genes in the leaves of the three ambiguous accessions were similar to those of bitter Iucundus lines. We consider the iuc_RAP2-7 marker is a powerful tool that will facilitate NLL marker-assisted selection by rapid rejection of bitter Iucundus genotypes and thus accelerate development of new low-alkaloid cultivars.
Project description:IntroductionLupins and other legumes have been considered as alternative plant-based protein sources to soybeans for both humans and livestock. Furthermore, they can contribute to more sustainable agricultural systems. The productivity and chemical composition of legumes is highly variable between species, cultivars, and with the edaphoclimatic conditions.MethodsThis work evaluated the adaptability of seven Lupinus cultivars in two different sowing locations, during two consecutive years, through the characterization of their seed, as a means of investigating their suitability to be used as a source of food and/or feed.Results and discussionLupinus angustifolius cv. Tango and Lupinus luteus cv. Acos were the most stable genotypes across the environments when considering the seed and protein production, while L. luteus cv. Alburquerque and L. luteus cv. Mister showed less variation in the total alkaloid content across the environments. The edaphoclimatic conditions affected seed and protein yields, as higher rainfall resulted in high productivity. The lower temperatures observed in the first year at both locations caused a reduction in the production of alkaloids in L. luteus cv. Acos and Cardiga. Due to the high alkaloid content of some of the studied cultivars their use as food or feed can pose some safety concerns. However, these cultivars can have high levels of resistance to herbivore and insect attacks, which can be of the utmost importance for the use of these crops for recovering poor or exhausted soils.
Project description:Lupins, like other legumes, have a unique biosynthesis scheme of 5-deoxy-type flavonoids and isoflavonoids. A key enzyme in this pathway is chalcone isomerase (CHI), a member of CHI-fold protein family, encompassing subfamilies of CHI1, CHI2, CHI-like (CHIL), and fatty acid-binding (FAP) proteins. Here, two Lupinus angustifolius (narrow-leafed lupin) CHILs, LangCHIL1 and LangCHIL2, were identified and characterized using DNA fingerprinting, cytogenetic and linkage mapping, sequencing and expression profiling. Clones carrying CHIL sequences were assembled into two contigs. Full gene sequences were obtained from these contigs, and mapped in two L. angustifolius linkage groups by gene-specific markers. Bacterial artificial chromosome fluorescence in situ hybridization approach confirmed the localization of two LangCHIL genes in distinct chromosomes. The expression profiles of both LangCHIL isoforms were very similar. The highest level of transcription was in the roots of the third week of plant growth; thereafter, expression declined. The expression of both LangCHIL genes in leaves and stems was similar and low. Comparative mapping to reference legume genome sequences revealed strong syntenic links; however, LangCHIL2 contig had a much more conserved structure than LangCHIL1. LangCHIL2 is assumed to be an ancestor gene, whereas LangCHIL1 probably appeared as a result of duplication. As both copies are transcriptionally active, questions arise concerning their hypothetical functional divergence. Screening of the narrow-leafed lupin genome and transcriptome with CHI-fold protein sequences, followed by Bayesian inference of phylogeny and cross-genera synteny survey, identified representatives of all but one (CHI1) main subfamilies. They are as follows: two copies of CHI2, FAPa2 and CHIL, and single copies of FAPb and FAPa1. Duplicated genes are remnants of whole genome duplication which is assumed to have occurred after the divergence of Lupinus, Arachis, and Glycine.
Project description: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 period, called vernalization, to induce flowering. The key achievement of L. angustifolius domestication was the discovery of two natural mutations (named Ku and Jul) conferring vernalization independence. These mutations are overlapping deletion variants in the promoter of LanFTc1, a homolog of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene. The third deletion, named here as Pal, was recently found in primitive germplasm. In this study, we genotyped L. angustifolius germplasm that differs in domestication status and geographical origin for LanFTc1 alleles, which we then phenotyped to establish flowering time and vernalization responsiveness. The Ku and Jul lines were vernalization-independent and early flowering, wild (ku) lines were vernalization-dependent and late flowering, whereas the Pal line conferred intermediate phenotype. Three lines representing ku, Pal, and Ku alleles were subjected to gene expression surveys under 8- and 16-h photoperiods. FT homologs (LanFTa1, LanFTa2, LanFTc1, and LanFTc2) and some genes selected by recent expression quantitative trait loci mapping were analyzed. Expression profiles of LanFTc1 and LanAGL8 (AGAMOUS-like 8) matched observed differences in flowering time between genotypes, highlighted by high induction after vernalization in the ku line. Moreover, these genes revealed altered circadian clock control in Pal line under short days. LanFD (FD) and LanCRLK1 (CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1) were negatively responsive to vernalization in Ku and Pal lines but positively responsive or variable in ku, whereas LanUGT85A2 (UDP-GLUCOSYL TRANSFERASE 85A2) was significantly suppressed by vernalization in all lines. Such a pattern suggests the opposite regulation of these gene pairs in the vernalization pathway. LanCRLK1 and LanUGT85A2 are homologs of A. thaliana genes involved in the FLOWERING LOCUS C (FLC) vernalization pathway. Lupins, like many other legumes, do not have any FLC homologs. Therefore, candidate genes surveyed in this study, namely LanFTc1, LanAGL8, LanCRLK1, and LanUGT85A2, may constitute anchors for further elucidation of molecular components contributing to vernalization response in legumes.
Project description:Unravelling the biosynthetic pathway of quinolizidine alkaloids (QAs), regarded as antinutritional compounds of narrow-leafed lupin (NLL) seeds, is fundamental to best exploit NLL as food or feed. We investigated 12 candidate genes connected to QA biosynthesis, selecting them by transcriptomic and genomic approaches, from the landscape of genes differentially expressed in leaves of the high- and low-alkaloid NLL accessions. Linkage analysis enabled the assessment of the location of the candidate genes in relation to iucundus, a major locus of unknown identity, that confers reduced QA content in seeds. The key finding was the identification of APETALA2/ethylene response transcription factor, RAP2-7, cosegregating with the iucundus locus and located within a region with highly significant QTLs that affect QA composition. We additionally identified a 4-hydroxy-tetrahydrodipicolinate synthase (DHDPS) gene involved in L-lysine biosynthesis as being closely linked to iucundus. The distributed location of other remaining candidates (including previously known QA genes) across different linkage groups, also indirectly supports the transcription factor as a possible regulator of lupin alkaloid biosynthesis. Our findings provide crucial insight into QA biosynthesis in NLL. Additionally, we evaluated and selected appropriate reference genes for qRT-PCRs to analyse the expression levels of QA genes in NLL.