Transcriptome analysis of an mvp mutant reveals important changes in global gene expression and a role for methyl jasmonate in vernalization and flowering in wheat

Abstract

The einkorn wheat mutant mvp-1 (maintained vegetative phase 1) has a non-flowering phenotype caused by deletions including, but not limited to, the genes CYS, PHYC, and VRN1. However, the impact of these deletions on global gene expression is still unknown. Transcriptome analysis showed that these deletions caused the upregulation of several pathogenesis-related (PR) and jasmonate-responsive genes. These results suggest that jasmonates may be involved in flowering and vernalization in wheat. To test this hypothesis, jasmonic acid (JA) and methyl jasmonate (MeJA) content in mvp and wild-type plants was measured. The content of JA was comparable in all plants, whereas the content of MeJA was higher by more than 6-fold in mvp plants. The accumulation of MeJA was also observed in vernalization-sensitive hexaploid winter wheat during cold exposure. This accumulation declined rapidly once plants were deacclimated under floral-inductive growth conditions. This suggests that MeJA may have a role in floral transition. To confirm this result, we treated vernalization-insensitive spring wheat with MeJA. The treatment delayed flowering with significant downregulation of both TaVRN1 and TaFT1 genes. These data suggest a role for MeJA in modulating vernalization and flowering time in wheat.

Keywords: Biotic stress; flowering; maintained vegetative phase; methyl jasmonate; vernalization; wheat..

Fig. 1.

Molecular characterization and apex stage development of the mvp mutant. (a) Total RNA was extracted from whole aerial parts and analysed by RT-PCR. Plants were acclimated at 4 ºC for 7 d under LD conditions. Each replicate (R1, R2, and R3) was obtained from three control wild-type plants (WT) or from five mutant plants (mvp). Relative expression level of gene markers of wild-type and mvp-mutant plants was analysed by RT-PCR to validate the mvp mutant. The 18S TaRNA was used for load control. The experiments were repeated three times and a representative result is shown. (b) Apex stage development of wild-type (WT) and mvp-mutant plants (mvp) used for molecular characterization and microarray analysis. The arrow indicates the double ridge and the scale bar is indicated.

Fig. 2.

Microarray analysis of the mvp-mutant and the wild-type seedlings. (a) Volcano plots illustrating the log₂ fold changes in gene expression differences between mvp mutants and wild-type plants. Probesets with statistically different expression (P≤0.05) and fold changes of ≥2-fold (263) are shown in the upper right corner and probesets with ≤–2-fold (105) are shown in the upper left corner. (b) Differentially regulated genes in mvp-mutant plants compared with wild-type plants derived from the microarray analysis: The differentially regulated genes were subdivided into six classes: Biotic-stress related genes (1), transcription factors (2), sugar metabolism (3), oxidative stress (4), miscellaneous genes (5), and unknown genes (6). The same RNA samples used in Fig. 1 were used for microarrays.

Fig. 3.

Relative expression level of several selected genes in mvp-mutant and wild-type control plants analysed by RT-PCR and PCR experiments. (a) Validation of TmUnG and TmCir expression by RT-PCR. The RNA samples are the same as those used in Fig. 1 and the microarray experiment. The 18S TaRNA was used as load control. (b) Genomic DNA was extracted from the same plant samples used for the microarray analysis and were tested for PCR amplification to analyse the possible deletion of the two most repressed genes from the array analysis (TmUnG repressed 114 times and TmCir repressed 169 times). The 18S DNA is used as control. The experiments were repeated with 3 different biological replicates with the same result. Each replicate (R1, R2, and R3) was obtained from five mutants plants (mvp) and from three control wild-type plants.

Fig. 4.

Relative expression level of TaVRN1 and TaFT1 and MeJA content during vernalization in hexaploid winter wheat seedlings analysed by qRT-PCR and HPLC/MS. Two weeks after germination at 20 ºC under LD conditions, non-vernalized winter wheat (cv Norstar) plants were vernalized under SD conditions at 4 ºC for 63 d and deacclimated for 14 d at 20 ºC under LD conditions. The aerial part was sampled around 4h after the beginning of the daylight period. The expression level of TaVRN-A1 (panel a) and TaFT-A1 (panel b) are expressed relative to the non-vernalized point (Ctrl-0). Data represent the mean ± SEM from three biological replicates. The expression levels are normalized with the TaRNA 18S. Plant samples from the same experiment were used to measure the MeJA content by HPLC / MS (panel c). Data represent the mean ± SEM from three biological replicates. AC-35D: cold-acclimated for 35 d; AC-63D: cold-acclimated for 63 d; DA-14D; plants cold-acclimated for 63 d and deacclimated under favourable growth conditions (LD and 20 ºC).

Fig. 5.

Effect of MeJA treatment on apex development in wild-type einkorn wheat (a–d) and spring wheat cv Manitou (e–h). Control and treated plants were grown at 20 ºC under long-day photoperiod (LD) conditions. Pictures of apex from dissected plants were taken before, during, and two weeks after MeJA treatment. (a, e) Apex of three-week-old wild-type einkorn wheat (a) or spring wheat Manitou (e) before treatment. (b–h) Control plants (sprayed with 0.1% tween 20 solution only) and treated plants (sprayed with 150 μM MeJA dissolved in 0.1% tween 20) were sprayed every day for one week (b, f) or two weeks (c, g); plants were then kept under the same growth conditions and apex pictures were taken two weeks (d, h) after the treatment. The scale bars are shown for each picture.

Fig. 6.

Effect of MeJA treatment on flowering and final leaf number in Triticum aestivum wheat plants cv Manitou. (a) Time course of flowering. Three-week-old plants were sprayed with 150 μM of MeJA dissolved in 0.1% tween 20 every day for 2 weeks at 20 ºC under LD conditions. Control plants were treated with 0.1% tween 20 solution only under the same growth conditions. The percentage of flowering plants was determined for both treated and control plants. (b) Final leaf number. Results were expressed as the mean ± SEM of six different experiments. Comparison between groups and analysis for differences between means of control and treated plants were performed using ANOVA followed by the post-hoc test Newman–Keuls. The threshold for statistical significance was: *: P<0.05.

Fig. 7.

Relative expression level of FUL2 and FUL3 analysed by qRT-PCR. (a, b) mvp-mutant and wild-type einkorn wheat plants. Plants were acclimated at 4 ºC for 7 d under LD conditions. The data represent the mean obtained from three biological replicates each using three control wild-type plants (WT) or five mvp-mutant plants (mvp). (c, d) During vernalization and de-acclimation conditions in hexaploid winter wheat seedlings (cv Norstar). After 2 weeks of germination at 20 ºC under LD conditions, non-vernalized winter wheat plants were vernalized under SD conditions at 4 ºC for 63 d and de-acclimated for 14 d at 20 ºC under LD conditions. The expression level of TaFUL2 (panel c) and TaFUL3 (panel d) are expressed relative to the non-vernalized plants (Ctrl-0). The aerial part was sampled around 4h after the beginning of the daylight period. (e, f) Effect of MeJA treatment in hexaploid spring wheat seedlings (Manitou). The expression level of TaFUL2 (panel e) and TaFUL3 (panel f) are expressed relative to the non-treated plants (Ctrl). Three weeks after germination at 20 ºC under LD conditions, control spring wheat (cv Manitou) plants (sprayed with 0.1% tween 20 solution only: Ctrl) were grown under LD conditions at 20 ºC for two weeks. Treated plants were sprayed with 150 μM MeJA dissolved in 0.1% tween solution every day for 2 weeks under the same growth conditions. Relative transcript abundance was calculated and normalized with respect to 18S TaRNA for the qRT-PCR experiment. Data represent the mean ± SEM from three biological replicates.

Fig. 8.

Effect of MeJA treatment on the expression of flowering-associated genes. Relative expression of flowering genes: (a) TaVRN-A1, (b) TaFT-A1, (c) TaPHYC-A. Three weeks after germination at 20 ºC under LD conditions, control spring wheat (cv Manitou) plants (treated with 0.1% tween 20 solution only: Ctrl) were grown under LD conditions at 20 ºC for two weeks. Treated plants were sprayed with 150 μM MeJA dissolved in 0.1% tween solution every day for 2 weeks under the same growth conditions. Total RNA was extracted from aerial parts and analysed by qRT-PCR. The 18S TaRNA was used as load control. Data represent the mean ± SEM from three biological replicates.