Vernalization response in Medicago truncatula

Vernalization is the acquisition of the competence to flower upon exposure to prolonged winter cold. The physical conditions that promote vernalization have been analyzed here in the model legume, Medicago truncatula. The length of cold treatment correlates with the promotion of floral transition. Vernalization is optimum within the 4C- 10C range for two weeks and tends to saturate at longer duration. Whole plants are equally responsive to vernalization treatment as imbibed seeds. FT is a key flowering time gene that has been shown in a range of plants to encode a floral signal molecule that is produced in the leaves and moves to the apex to induce flowering. There are five copies of FT-like genes in Medicago (MtFTLa-e), but only MtFTLa, MtFTLb, and MtFTLc were shown to be upregulated by vernalization. Among the MtFTLs, MtFTLa has been shown to be the major flowering time gene whose expression is regulated both by long-day (LD) photoperiod and vernalization. The duration of cold has a quantitative effect on MtFTLa expression and induction of flowering. However, vernalization-induced upregulation of MtFTLa does not occur right after the prolonged cold treatment but takes place after growth in LD photoperiods. This finding and the observation that MtFTLa is expressed only in leaves that expanded after vernalization but not in differentiated ones present prior to the treatment, suggest that vernalization is an epigenetic process that requires dividing or undifferentiated cells. Further analysis of the MtFTLa locus using chromatin immunoprecipitation techniques revealed that the epigenetic regulation of MtFTLa expression is indeed associated with changes in chromatin modification. High transcript levels of MtFTLa following vernalization are consistent with enrichment of the H3Ac mark and concurrent loss of the H3K27me3 at part of the promoter (promoter B) of MtFTLa, modifications that are linked with chromatin structure permissive for transcription. Changes in histone marks were also observed at the more distal promoter region (promoter A) of MtFTLa. To identify components that might be involved in vernalization response, sequences homologous to the PHD-finger containing VIN3 and the MADS-box genes AGL19, AGL24, and SVP were identified in Medicago. Although, MtVIN3 has relatively conserved PHD and VID domains typical of the VIN3/VEL family, unlike in Arabidopsis, its expression was not significantly induced by vernalization. Ectopic expression of MtVIN3 in Arabidopsis did not alter the flowering time. MtAGL19, MtAGL24, MtSVP1 and MtSVP2 are highly-expressed in apical buds and leaves during the vegetative stage of development but minimally detected in floral organs. These MADS-box genes are not responsive to vernalization, which is expected for MtSVPs. The flowering time of Arabidopsis was not hastened by the ectopic expression of either MtAGL19 or MtAGL24. However, 35S:MtAGL24 plants exhibited floral abnormalities that phenocopy 35S:AtAGL24 plants, such as enlarged sepals, elongated carpel, greenish/leaf-like petals, stunted siliques, and delayed maturation rate and senescence of siliques. Overexpression of both MtSVP1 and MtSVP2 genes in Arabidopsis delayed flowering with accompanying floral defects including alterations in floral organ number and symmetry, elongated carpel, larger sepals and pale green petals. The severity of the floral defects also correlated with the delay in flowering time. Apical bud genes regulated by short term cold and by vernalization treatment were identified using Affymetrix GeneChip Medicago Genome Arrays. The microarray data showed that the “cold-shock” transcriptome of Medicago resembles that of Arabidopsis, which mainly involves genes implicated in the cold-acclimation pathway and putative transcription factors (TFs). Among the genes stably upregulated by prolonged cold include three genes with predicted chromatin-related functions and one uncharacterized gene annotated as “cold-responsive”. There are significantly more genes downregulated than upregulated by prolonged cold in apical buds, among of which encode products homologous to the chromatin modifier SNF-2, a FYVE/PHD zinc finger-containing protein, and a putative novel TF with annotated transcriptional repressor activity. Analysis of the leaf microarray data also showed that among the genes stably upregulated by prolonged cold treatment following growth in LD photoperiod, MtFTLa exhibited the highest fold change in expression level, consistent with its important role in vernalization response

Gene-edited Mtsoc1 triple mutant Medicago plants do not flower

Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. SOC1-like genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model Arabidopsis thaliana (Arabidopsis), where SOC1 promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. Medicago truncatula (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one SOC1 gene, multiple gene duplication events have given rise to three MtSOC1 paralogs within the Medicago genus in legumes: one Fabaceae group A SOC1 gene, MtSOC1a, and two tandemly repeated Fabaceae group B SOC1 genes, MtSOC1b and MtSOC1c. Previously, we showed that MtSOC1a has unique functions in floral promotion in Medicago. The Mtsoc1a Tnt1 retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, Mtsoc1b Tnt1 single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of Mtsoc1a Mtsoc1b Mtsoc1c triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three MtSOC1 genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single Mtsoc1a mutant and the Arabidopsis soc1 mutant; implicating multiple MtSOC1 genes in critical overlapping roles in the transition to flowering in Medicago

Fine mapping links the FTa1 flowering time regulator to the dominant spring1 locus in Medicago.

To extend our understanding of flowering time control in eudicots, we screened for mutants in the model legume Medicago truncatula (Medicago). We identified an early flowering mutant, spring1, in a T-DNA mutant screen, but spring1 was not tagged and was deemed a somaclonal mutant. We backcrossed the mutant to wild type R108. The F1 plants and the majority of F2 plants were early flowering like spring1, strongly indicating that spring1 conferred monogenic, dominant early flowering. We hypothesized that the spring1 phenotype resulted from over expression of an activator of flowering. Previously, a major QTL for flowering time in different Medicago accessions was located to an interval on chromosome 7 with six candidate flowering-time activators, including a CONSTANS gene, MtCO, and three FLOWERING LOCUS T (FT) genes. Hence we embarked upon linkage mapping using 29 markers from the MtCO/FT region on chromosome 7 on two populations developed by crossing spring1 with Jester. Spring1 mapped to an interval of ∼0.5 Mb on chromosome 7 that excluded MtCO, but contained 78 genes, including the three FT genes. Of these FT genes, only FTa1 was up-regulated in spring1 plants. We then investigated global gene expression in spring1 and R108 by microarray analysis. Overall, they had highly similar gene expression and apart from FTa1, no genes in the mapping interval were differentially expressed. Two MADS transcription factor genes, FRUITFULLb (FULb) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1a (SOC1a), that were up-regulated in spring1, were also up-regulated in transgenic Medicago over-expressing FTa1. This suggested that their differential expression in spring1 resulted from the increased abundance of FTa1. A 6255 bp genomic FTa1 fragment, including the complete 5' region, was sequenced, but no changes were observed indicating that the spring1 mutation is not a DNA sequence difference in the FTa1 promoter or introns

Flowering time of plants from the Test cross.

<p><i>Spring1</i>, an early flowering mutant, was crossed with Jester plants and the resulting F1 plants were then crossed with Jester in the Testcross (♂(♂“<i>spring1</i> x ♀Jester”) x ♀Jester). The Testcross progeny were grown in long day conditions and scored for flowering time. Graph showing the distribution of flowering time of plants that were classified as early flowering (n = 83) and late flowering (n = 95) compared with Jester (n = 6) and F1 plants (n = 32). The class with ≥11 nodes includes plants that had up to 21 nodes, but had not flowered by the time scoring was terminated at 69 days after germination. Plants that were “unclassified” or died young are not included. As parental and progeny plants grew at different rates, flowering was measured as the node number on the main axis at flowering.</p

Flowering time of plant populations derived from crosses with the <i>spring1</i> mutant.

<p>Flowering time was scored and plants were classified for flowering time. Plants were classified as early flowering if they had ≤7 nodes at flowering or had flowered rapidly (≤28 days after germination), similar to <i>spring1</i>. They were classified as late flowering if they had ≥11 nodes at flowering or had not yet flowered but had ≥11 nodes, similar to Jester. They were scored as unclassified due to not falling into our two classes, or being difficult to score due to their tiny size or altered aerial architecture.</p>a, b<p>F2 plants were grown in six groups and scoring was terminated after 39, 46, 73, 77, 83 or 87 days.</p>a<p>A total of 27 plants had not yet flowered when scoring was terminated. None of the plants classified as late had flowered by 39 days. Four plants had not flowered by 87 days and had up to 25 nodes on the primary axis.</p>b<p>In total 10 plants had not flowered by the time scoring was terminated.</p>c<p>F2 plants were grown up and scoring was terminated after 65 days. Two plants had not yet flowered and had up to 19 nodes on the primary axis.</p>d/e<p>Scoring was terminated at 69 days.</p>d<p>A total of 33 plants had not yet flowered and had up to 21 nodes on the primary axis.</p>e<p>Two plants had not yet flowered, one had 10 nodes and one was unscoreable due to its small size and architecture.“Test Cross” is (♂(♂<i>spring1</i> x ♀Jester) x ♀Jester). Plants classified as Dead, usually died as very young seedlings. nd is not done.</p

<i>FTa1</i> is up-regulated in <i>spring1</i> plants.

<p>Accumulation of <i>FTa1</i> and <i>FTa2</i> transcript in <i>spring1</i> and R108 in long day conditions was measured using qRT-PCR on 12–14 day old seedlings with two trifoliate leaves.Relative transcript abundance of <i>FTa1</i> (a) and <i>FTa2</i> (b), over a diurnal timecourse in the aerial parts of seedlings. Levels were normalised to <i>TUBULIN (TUB)</i> and calibrated relative to the expression of <i>FTa1</i> (second biological rep) at Zeitgeber 20 (ZT0 is the time of lights on). The mean +/− SE of 2 biological replicates is shown for the <i>spring1</i> samples. For R108, the two cDNA samples from each biological replicate were pooled and the mean +/− SE of the 3 technical replicates are presented. c) Accumulation of <i>FTa1</i> transcript in the first trifoliate leaf of homozygous (after two backcrosses to R108) and heterozygous <i>spring1</i> plants (F1 plants from a backcross to R108) with levels normalised to <i>PROTODERMAL FACTOR 2</i> (<i>PDF2)</i>. The mean +/− SE of 3 biological replicates is shown.</p

Flowering time of plants from “<i>spring1</i> x Jester” and from “R108 x Jester”.

<p><i>Spring1</i>, an early flowering mutant in the R108 accession was crossed with Jester plants and the F1 and F2 progeny were grown in long day conditions and scored for flowering time (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053467#pone-0053467-t001" target="_blank">Table 1</a>). A Control cross “R108 x Jester” was also performed. a) Flowering time of the F1 progeny from the Backcross “<i>spring1</i> x Jester” (n = 32) and from the Control cross “R108 x Jester” (n = 12) was compared to <i>spring1</i> (n = 12), Jester (n = 6) and R108 (n = 12). Flowering time was scored using two methods; the number of days after germination to flowering, or the number of nodes on the primary axis at flowering. The F1 plants from the Mapping cross flowered much more rapidly than the F1 plants from the Control cross by either measure, indicating that <i>spring1</i> confers dominant early flowering in crosses to Jester. b) Distribution of the flowering time of the F2 progeny from the Mapping cross and the Control cross compared to parental lines. Plants that were scored as “unclassified” or died young are not included. The F2 population from “<i>spring1</i> x Jester” segregated 421 early flowering and 57 late flowering plants as scored by nodes at flowering. The class with ≥11 nodes includes plants that had up to 25 nodes, but had not flowered by the time scoring was terminated at 87 days. The Control cross produced only late flowering F2 plants, with some having up to 19 nodes, but not having flowered by the time scoring was terminated at 65 days. c) Photographs of F2 plants from the “<i>spring1</i> x Jester” Mapping cross; a typical early flowering plant with flowers (left), plants that have not flowered that are either very small, pale and slow growing, or small with an altered morphology (middle), and a typical late flowering plant (right). All plants were photographed at 26 days old.</p

<i>FULb</i> and <i>SOC1a</i> are up-regulated in <i>spring1</i> and in transgenic Medicago plants over expressing <i>FTa1.</i>

<p>Accumulation of <i>FTa1, FULb and SOC1a</i> in <i>spring1, 35S::FTa1</i> transgenic Medicago plants and R108 in long daylength conditions was measured using qRT-PCR on the first trifoliate leaf from 12–14 day old seedlings. The mean +/− SE of 3 biological replicates is shown relative to <i>PDF2</i>. Relative transcript abundance of <i>FTa1</i> (a), <i>FULb</i> (c) and <i>SOC1a</i> (e) in <i>spring1.</i> Relative transcript abundance of <i>FTa1</i> (b), <i>FULb</i> (d) and <i>SOC1a</i> (f) in <i>35S::FTa1</i> lines.</p

DNA markers used for fine mapping <i>spring1</i> on chromosome 7.

<p>DNA markers from the <i>MtCO</i> region of chromosome 7 were analysed for linkage to <i>spring1</i> in the mapping crosses (see Text). The interval containing <i>spring1</i> is flanked by Medtr7g084170.1 and Medtr7g085190.1 shown in bold; each marker is separated from <i>spring1</i> by one recombination event.</p>a<p>DNA marker taken from the University of Minnesota (UMN) Integrated Genetic Map of <i>Medicago truncatula</i><a href="http://www.medicago.org/genome/map.php" target="_blank">http://www.medicago.org/genome/map.php</a>.</p>b<p>DNA marker from Pierre <i>et al</i> (2010).</p>c<p>Size of PCR fragment predicted from Mt3.5 Genome assembly from <a href="http://medicagohapmap.org/" target="_blank">http://medicagohapmap.org/</a>.</p>d<p>Numbers of recombinants detected by the marker in the late or early flowering classes from the F2 of the “<i>spring1</i> x Jester” cross, or from the Test cross (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053467#pone-0053467-t001" target="_blank">Table 1</a> and Text). “Test Cross” is (♂(♂<i>spring1</i> x ♀Jester) x ♀Jester). NT is not tested. Indel is insertion/deletion; SSR is Simple Sequence Repeat. The column entitled Mt3.5 Genome assembly gives the position of the marker on the pseudomolecule from Mt3.5 Genome assembly from <a href="http://medicagohapmap.org/after" target="_blank">http://medicagohapmap.org/after</a> a Chromosome Visualisation Tool (CViT) BLAST search <a href="http://medicagohapmap.org/was" target="_blank">http://medicagohapmap.org/was</a> carried out.</p