Crosstalk in the darkness: bulb vernalization activates meristem transition via circadian rhythm and photoperiodic pathway

Abstract

Background: Geophytes possess specialized storage organs - bulbs, tubers, corms or rhizomes, which allow their survival during unfovarable periods and provide energy support for sprouting and sexual and vegetative reproduction. Bulbing and flowering of the geophyte depend on the combined effects of the internal and external factors, especially temperature and photoperiod. Many geophytes are extensively used in agriculture, but mechanisms of regulation of their flowering and bulbing are still unclear.

Results: Comparative morpho-physiological and transcriptome analyses and quantitative validation of gene expression shed light on the molecular regulation of the responses to vernalization in garlic, a typical bulbous plant. Long dark cold exposure of bulbs is a major cue for flowering and bulbing, and its interactions with the genetic makeup of the individual plant dictate the phenotypic expression during growth stage. Photoperiod signal is not involved in the initial nuclear and metabolic processes, but might play role in the later stages of development, flower stem elongation and bulbing. Vernalization for 12 weeks at 4 °C and planting in November resulted in flower initiation under short photoperiod in December-January, and early blooming and bulbing. In contrast, non-vernalized plants did not undergo meristem transition. Comparisons between vernalized and non-vernalized bulbs revealed ~ 14,000 differentially expressed genes.

Conclusions: Low temperatures stimulate a large cascades of molecular mechanisms in garlic, and a variety of flowering pathways operate together for the benefit of meristem transition, annual life cycle and viable reproduction results.The circadian clock appears to play a central role in the transition of the meristem from vegetative to reproductive stage in bulbous plant, serving as integrator of the low-temperature signals and the expression of the genes associated with vernalization, photoperiod and meristem transition. The reserved photoperiodic pathway is integrated at an upstream point, possibly by the same receptors. Therefore, in bulb, low temperatures stimulate cascades of developmental mechanisms, and several genetic flowering pathways intermix to achieve successful sexual and vegetative reproduction.

Keywords: Allium sativum; Bulbing; Flowering; Low temperature; Reproductive meristem.

Fig. 1

Effect of pre-planting vernalization on the development of #87 garlic plants. Planting and harvest dates were November 11th, 2015 and June–July 2016, respectively. a Plant and bulb weight and the number of flowers/inflorescence at the end of the growing season. b Images of intact plants in mid-season 140 DAP, March 30th and newly produced mature bulbs after harvest

Fig. 2

Effect of pre-planting vernalization treatments on mRNA expression in garlic meristems. Numbers indicate the number of differentially expressed genes (DEGs) and their share among the ~ 14,000 DEGs in all comparisons. a Venn diagram. Numbers represent common and specific DEGs in each comparison between treatments; b Number of up- and downregulated genes in three pairwise comparisons at the end of storage treatments

Fig. 3

GO term distribution of 547 DEGs shared by all comparisons between vernalization treatments, as revealed by Blast2GO and REVIGO algorithms. Represented by circles, GO terms were plotted in agreement with semantic similarities to other GO terms (adjoining circles are most closely related). Main patterns are related to the chromatin organization, RNA replication, regulation of gene expression and methylation. Circle sizes are proportional to the frequency of the GO terms within each cluster, and circles’ color indicates the log10 corrected P-value for enrichment

Fig. 4

Overview of the GO enrichment analysis obtained from the transcriptome data of the effect of vernalization in garlic. Output GO enrichment analysis was performed by comparing data from NV – no vernalization, SV – short vernalization, LV – long vernalization of #87 garlic. The GO terms listed on the left-hand side, in red, are specifically overrepresented in the up-regulated genes. On the right-hand side, in blue, GO terms specifically overrepresented in the down-regulated genes. a and b represent corrected P-value < 4.8e^− 8, c and d corrected P-value 7.5e^− 6 to 0.05 (at least in one comparison). Corrected P-values were log10 transformed

Fig. 5

Positive co-expression of garlic genes associated with vernalization (green), photoperiod (blue) pathway and meristem transition (purple). Data from three vernalization treatments were analyzed using the network-drawing software Cytoscape [81]. Pearson correlation value higher than 0.9

Fig. 6

Effect of vernalization treatments on the expression of the genes associated with vernalization pathway. a in silico analysis of 12 vernalization-annotated genes, grouped into three clusters; The expression values (FPKM; average of the replications) were log2-transformed. b qRT-PCR validation of PRMT5, VIN3, and AGL19 relative expression

Fig. 7

Effect of vernalization treatments on the expression of the genes associated with photoperiodic pathway. a in silico analysis of 30 annotated genes (Additional file 4: Table S1), grouped into four clusters; The expression values (FPKM; average of the replications) were log2-transformed. b a relative expression of four genes, as validated by qRT-PCR

Fig. 8

Effect of vernalization treatments on the expression of genes associated with meristem identity. a in silico analysis of 34 annotated genes (Additional file 4: Table S1), grouped into four clusters by their expression pattern; The expression values (FPKM; average of the replications) were log2-transformed. b a relative expression of four meristem-identity genes, as validated by qRT-PCR

Fig. 9

In silico analyses of the effect of vernalization treatments on the specific DEGs involved in the circadian rhythm. The KEGG algorithm employed the pathway map ko04712. Relative expression is quantified by comparing transcriptome data from #87 garlic. Differentially expressed genes are highlighted according to the expression values (FPKM; average of the replications) which were log2-transformed

Fig. 10

The proposed scheme of integration between vernalization, circadian rhythms and photoperiod-associated genes in garlic. The circadian clock senses and integrates the low-temperature signal and induces the expression of genes associated with vernalization, photoperiod and meristem transition