Abstract
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.