Epistatic interactions between PHOTOPERIOD1, CONSTANS1 and CONSTANS2 modulate the photoperiodic response in wheat

Abstract

In Arabidopsis, CONSTANS (CO) integrates light and circadian clock signals to promote flowering under long days (LD). In the grasses, a duplication generated two paralogs designated as CONSTANS1 (CO1) and CONSTANS2 (CO2). Here we show that in tetraploid wheat plants grown under LD, combined loss-of-function mutations in the A and B-genome homeologs of CO1 and CO2 (co1 co2) result in a small (3 d) but significant (P<0.0001) acceleration of heading time both in PHOTOPERIOD1 (PPD1) sensitive (Ppd-A1b, functional ancestral allele) and insensitive (Ppd-A1a, functional dominant allele) backgrounds. Under short days (SD), co1 co2 mutants headed 13 d earlier than the wild type (P<0.0001) in the presence of Ppd-A1a. However, in the presence of Ppd-A1b, spikes from both genotypes failed to emerge by 180 d. These results indicate that CO1 and CO2 operate mainly as weak heading time repressors in both LD and SD. By contrast, in ppd1 mutants with loss-of-function mutations in both PPD1 homeologs, the wild type Co1 allele accelerated heading time >60 d relative to the co1 mutant allele under LD. We detected significant genetic interactions among CO1, CO2 and PPD1 genes on heading time, which were reflected in complex interactions at the transcriptional and protein levels. Loss-of-function mutations in PPD1 delayed heading more than combined co1 co2 mutations and, more importantly, PPD1 was able to perceive and respond to differences in photoperiod in the absence of functional CO1 and CO2 genes. Similarly, CO1 was able to accelerate heading time in response to LD in the absence of a functional PPD1. Taken together, these results indicate that PPD1 and CO1 are able to respond to photoperiod in the absence of each other, and that interactions between these two photoperiod pathways at the transcriptional and protein levels are important to fine-tune the flowering response in wheat.

Fig 1. CO1 and CO2 gene structure and position of the selected TILLING mutations.

Both CO1 and CO2 genes have two exons, which encode two B-BOX domains at the N-terminus and a CCT domain at the C-terminus. The selected co-A1 and co-B2 mutations, located in the first exon after the two B-Box domains, are predicted to generate premature stop codons, whereas the co-B1 and co-A2 mutants have acceptor splice-site mutations that result in intron retention and premature stop codons that eliminate the complete exon 2 including the CCT domain. The T4 numbers are the mutant identification. The schematic gene structure is based on CO-A1 from Kronos (GenBank MT043302).

Fig 2. Heading time of wild type, co1, co2 and co1 co2 mutants under LD and SD in Kronos-PI (Ppd-A1a) and Kronos-PS (Ppd-A1b) backgrounds.

(A) Averages are based on 15–19 plants in LD-PI (2 experiments), 21–24 plants in LD-PS (3 experiments) and 8 to 14 plants in SD-PI and SD-PS (1 experiment). Red numbers indicate differences in heading time relative to the wild type (mut-WT), with P values from a Dunnett test. Vertical arrows above the bars indicate that spikes have not emerged at 180 d (end of the experiment). (B-C) Interaction graphs for CO1 and CO2 in Kronos-PS (Ppd-A1b) and Kronos-PI (Ppd-A1a) backgrounds. The interaction was significant in B (P = 0.0052) but not in C (S3 Table). Error bars are standard errors of the means (henceforth, s.e.m). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant.

Fig 3. Effect of co1 and co2 mutations on spike development in Kronos-PS under SD.

Kronos-PI was included as control. (A) Dissecting microscope images of developing apices at week 6 (double-ridge, W2.5) and week 11 with a detail of their floral organs. a = anthers, elongated in Kronos-PI and primordia in other genotypes, c = carpel and s = styles well developed in Kronos-PI and c&o = carpel extending around the ovule in other genotypes. (B) Developmental time course of SAM using the Waddington scale from 4- through 11-week-old plants (n = 3). W1 = vegetative apex, W2 = double ridge, W3 = glume primordium, W4 = stamen primordium, W5 = carpel extending round three sides of ovule, W6 = short style primordia and stylar canal open, W7 = stigmatic branches just differentiating as swollen cells on styles, W8 = stigmatic branches elongating, W9 = styles and stigmatic branches erect and stigmatic hairs differentiating.

Fig 4. Phenotypic effects of co1 and co2 mutations in the absence of functional PPD1 genes under LD.

(A) Average days to heading in ppd1 mutants carrying mutations in either co1 or co2. Kronos-PS wild type and co1 co2 double mutants are included as controls (arrow on top of the bar indicates heading time > 180 d). (B) Spike phenotypes at the end of the experiment. Floral transition and spike formation occurred in all genotypes, but in the ppd1 co1 double mutant floral development was delayed or arrested and spikes failed to emerge before the experiment was terminated. (C) Number of grains per spike recovered from the same genotypes as in A. Error bars are standard errors of the means calculated from 7 to 16 plants per genotype for flowering and from 4 to 8 plants for grain number.

Fig 5. Effect of loss-of-function mutations in CO1, CO2 and PPD1 on the transcriptional profiles of six wheat flowering genes in Kronos-PS and Kronos-PI (SD-only) plants grown under LD and SD.

(A) CO1, (B) CO2, (C) PPD1, (D) FT1, (E) VRN1, and (F) VRN2. LD experiments included Kronos-PS, co1, co2, co1 co2, and ppd1 mutants; whereas SD experiments included Kronos-PS, Kronos-PI, and their respective co1 co2 and ppd1 mutants. We grew the plants in a growth chamber under 16 h light at 22 °C and 8 h darkness at 17 °C for LD, and under 8 h light and 16 h darkness for SD (same temperatures). Fully expanded 4^th leaves (Zadoks scale = 14) were harvested at ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20. The ZT0 value is the same as at ZT24. Shaded boxes represent night. Values are averages of four biological replicates and bars are SE of the means. Expression values are relative to ACTIN using the delta Ct method. Primers and primer efficiencies are listed in S5 Table (all amplify both A and B homologs of the respective genes).

Fig 6. Effect of mutations in Kronos vrn-A1 (winter growth habit), vrn-B1 (spring growth habit) and vrn-A1 vrn-B1 (vrn1, very late heading) on the transcriptional profiles of CO1 and CO2.

All mutants are homozygous and in the Kronos-PI background. (A-B) CO1 and (C-D) CO2 transcription profiles. A & C) More recently expanded leaves from three weeks old plants were sampled before vernalization (0w), at 3w and 6w vernalization (4°C), 2 weeks after return to room temperature (RT = 22°C day and 18°C) and at heading time (HT), which was different for each genotype. B & D) Same genotypes but grown at RT without vernalization. Points are averages of eight plants and error bars are standard errors of the means. Means with different letters are significantly different in Tukey test for that time point (P < 0.05). Note that the upregulation of CO1 after the return of the plants from vernalization occurred only in the vrn1 mutant that has no functional copies of VRN1.

Fig 7. Protein interactions.

(A) Yeast-two-hybrid (Y2H) interactions between PPD1 and CO1 or CO2. Left panel: SD medium lacking Leucine and Tryptophan (SD-L-W) to select for yeast transformants containing both bait and prey. Right panel: interaction on SD-L-W-H-A medium (no L, W, H (Histidine) and A (Adenine)). Dilution factors = 1, 1:10 and 1:100. (B-D) Split-YFP in wheat protoplast. (B) Positive nuclear interactions between CO1 and CO2. (C-D) Positive nuclear interactions between CO1 and PPD1 in two different YFP-N/YFP-C configurations. (E) Summary of Y2H interactions among wheat CO1, CO2, PPD1, VRN2, PHYB, PHYC, and ELF3 from S5 Fig. Green lines are based on published results from Brachypodium [35] and violet lines on published results from wheat [24]. Thick lines indicate interactions validated by split-YFP in wheat protoplasts (blue) or in vitro coIP (violet). Curved arrows indicate self-dimerization.