EARLY FLOWERING 3 interactions with PHYTOCHROME B and PHOTOPERIOD1 are critical for the photoperiodic regulation of wheat heading time

Abstract

The photoperiodic response is critical for plants to adjust their reproductive phase to the most favorable season. Wheat heads earlier under long days (LD) than under short days (SD) and this difference is mainly regulated by the PHOTOPERIOD1 (PPD1) gene. Tetraploid wheat plants carrying the Ppd-A1a allele with a large deletion in the promoter head earlier under SD than plants carrying the wildtype Ppd-A1b allele with an intact promoter. Phytochromes PHYB and PHYC are necessary for the light activation of PPD1, and mutations in either of these genes result in the downregulation of PPD1 and very late heading time. We show here that both effects are reverted when the phyB mutant is combined with loss-of-function mutations in EARLY FLOWERING 3 (ELF3), a component of the Evening Complex (EC) in the circadian clock. We also show that the wheat ELF3 protein interacts with PHYB and PHYC, is rapidly modified by light, and binds to the PPD1 promoter in planta (likely as part of the EC). Deletion of the ELF3 binding region in the Ppd-A1a promoter results in PPD1 upregulation at dawn, similar to PPD1 alleles with intact promoters in the elf3 mutant background. The upregulation of PPD1 is correlated with the upregulation of the florigen gene FLOWERING LOCUS T1 (FT1) and early heading time. Loss-of-function mutations in PPD1 result in the downregulation of FT1 and delayed heading, even when combined with the elf3 mutation. Taken together, these results indicate that ELF3 operates downstream of PHYB as a direct transcriptional repressor of PPD1, and that this repression is relaxed both by light and by the deletion of the ELF3 binding region in the Ppd-A1a promoter. In summary, the regulation of the light mediated activation of PPD1 by ELF3 is critical for the photoperiodic regulation of wheat heading time.

Fig 1. Effect of phyB and elf3 mutations on wheat heading time.

(A-B) Long days (LD). (C-D) Short days (SD). (A and C) Kronos photoperiod insensitive (PI) background and (B and D) photoperiod sensitive (PS) background. (E) Effect of the different photoperiods and genetic backgrounds on heading time within each mutant combination. Error bars are s.e.m. Numbers in the base of the bars indicate the number of plants analyzed in each genotype / photoperiod combination. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 (Tukey tests). No statistical tests were performed for the plants that failed to head at 160 d when the experiment was terminated (indicated by gray arrows). Raw data and statistics are in Data A in S1 Data.

Fig 2. Transcript levels of PPD1 and FT1 in phyB and phyB elf3 mutants.

Samples were collected from leaves of 5-week-old Kronos photoperiod insensitive (PI) and sensitive (PS) plants grown under LD. (A-C) Wildtype vs. phyB in PI. (D-E) Wildtype vs. phyB in PS. (G-I) Wildtype vs. elf3 phyB in PI. (J-L) Wildtype vs. elf3 phyB in PS. (A & G) PPD-A1a allele with a deletion in the promoter. (D & J) PPD-A1b allele with intact promoter. (B, E, H, & K) PPD-B1 photoperiod sensitive allele. (C, F, I, & L) FT1 (both homoeologs). Expression in single elf3 mutants is discussed later in the ELF3 x PPD1 section. ACTIN was used as endogenous control. Error bars are s.e.m based on 5 biological replications. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001 based on t-tests between mutants and wildtype at the different time points. Raw data and statistics are available in Data B in S1 Data.

Fig 3. Yeast-two-hybrid interactions between ELF3 and phytochromes PHYB and PHYC.

SD medium lacking Leucine and Tryptophan (-L-W) was used to select for yeast transformants containing both bait and prey vectors. Interactions were determined on SD media lacking Leucine, Tryptophan, Histidine and Adenine (-L-W-H-A). Autoactivation was tested for ELF3 bait using the empty prey vector pLAW11, and for N-PHYB and C-PHYB preys using the empty bait vector pGBKT7. We did not add a chromophore, so we are likely seeing the interaction with the inactive Pr form.

Fig 4. ELF3-HA protein in UBI::ELF3-HA transgenic plants grown under LD, SD and SD interrupted night (SD-int.).

ELF3 protein levels were analyzed by immunoblotting using an anti-HA antibody (samples were harvested at the indicated ZT times). (A) Plants grown under LD. (B) Plants grown under SD. (C) Plants were grown under SD, but on the day when samples were collected, lights were turned on at ZT10, 2 h after the start of the night (SD-int.). (D) Plants under SD grown simultaneously with those in B but without turning on the light before sampling (collected at the same time points as in C). The black arrowhead indicates the higher and more diffuse band and the white arrowhead the sharper lower band detected in the dark. The bottom panel is a Coomassie Brilliant Blue (CBB) stained membrane used as a loading control. The white bar indicates lights on and the black bar lights off. The gray bar in C indicates that the lights were turned on during the subjective night. (E) Quantitative reverse transcription PCR (qRT-PCR) analysis of PPD-B1 expression in leaves collected at the same time points as in C. Raw data and statistics are available in Data C in S1 Data.

Fig 5. Effect of elf3 and ppd1 mutations on heading time under LD and SD conditions.

Bars are means and error bars are s.e.m. The blue arrow on top of the Elf3 ppd1 SD treatment indicates that plants did not head by the time the experiment was terminated at 150 d. Differences in heading times between SD and LD are indicated for each genotype above the bars with the corresponding t-tests (*** = P < 0.001). No t-test is provided for Elf3 ppd1 because plants failed to head under SD. Numbers at the base of the bars indicate the number of plants analyzed in each genotype / photoperiod combination. Raw data and statistics are available in Data D in S1 Data.

Fig 6. qRT-PCR analysis of transcript levels of PPD1 and FT1 in wildtype and elf3 mutants.

Leaf samples collected from 5-week-old Kronos photoperiod insensitive (PI) and sensitive (PS) plants grown under LD. (A-C) Wildtype vs. elf3 in PI. (D-F) Wildtype vs. elf3 in PS. (A) PPD-A1a allele with a deletion in the promoter associated with earlier heading under SD. (D) PPD-A1b allele with the intact promoter and late heading under SD. (B & E) PPD-B1 with intact promoter in PI and PS. (C & F) FT1 (both homoeologs combined). ACTIN was used as endogenous control. Error bars are s.e.m based on 5 biological replications. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001 based on t-tests between mutants and wildtype at the different time points. Raw data and statistics are available in Data E in S1 Data.

Fig 7. Transcript levels of flowering genes FT1, VRN1, VRN2, CO1, and CO2 in Kronos PI, elf3, ppd1 and elf3 ppd1 mutants.

(A-C) Flowering promoter gene FT1. (D-F) Flowering promoter gene VRN1. (G-I) Flowering repressor gene VRN2. (J-L) CO1. (M-O) CO2. (A, D, G, J, & M) Wildtype vs. elf3. (B, E, H, K, & N) Wildtype vs. ppd1. (C, F, I, L, & O) Wildtype vs. elf3 ppd1. Primers used for all genes amplify both homoeologs. The WT data is the same within each row but at different scales. Fig 7A is the same as Fig 6C, and is included again to facilitate comparisons. Error bars are s.e.m based on 5 biological replications. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001 based on t-tests between mutants and wildtype at the different time points. Raw data and statistics are available in Data F in S1 Data.

Fig 8. Chromatin immunoprecipitation (ChIP) analysis of the PPD1 promoter.

(A) Gene diagram of the promoter of PPD-B1 showing the regions -983 to -796, -460 to -313, -446 to -320, -142 to -21, and a control coding region at +1514 to +1904 analyzed by ChIP, followed by qRT-PCR. The grey dashed arrow demarks the location deleted within the Ppd-A1a promoter (PI). The red triangles mark the locations of predicted LUX binding sites (GATWCG). The PPD1 promoter is indicated by a horizontal red line and a rectangular box represent the first exon (ATG indicates the start codon). (B) Fold enrichment of ELF3 at the PPD1 promoter in the PS-elf3 mutant and transgenic UBI::ELF3-HA in a mutant PS-elf3 background. A FUL -promoter region between -1933 and -1750 that contained no LUX binding sites was used as a negative control. Bars represent the mean ± s.e.m. from four biological replicate experiments. ** = P < 0.01 and ns = not significant. Primer sequences are provided in Table D in S1 Text. Raw data and statistics are in Data G in S1 Data.

Fig 9. Working model for the regulation of heading time in wheat.

The model integrates photoperiod and vernalization signals into the regulation of FT1 expression in wheat leaves. FT1 is then transported to the shoot apical meristem (dotted green arrow) where it induces the transition from the vegetative to the reproductive phase. Blue arrows indicate promotion of gene expression or activity and red lines ending in a crossed-bar indicate repression. ELF3, LUX and ELF4 proteins form the evening complex, which binds to the PPD1 promoter and inhibits its transcription. The complex interactions between VRN2 and CO1, CO2 and PPD1 [5] are not included in the figure for clarity.