Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs

Abstract

Genes controlling the transition to flowering have been studied in several species, including Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but have not yet received much attention in legumes. Here, we describe a new allelic series of late-flowering, photoperiod-insensitive mutants in the pea (Pisum sativum) LATE BLOOMER1 (LATE1) gene and show that LATE1 is an ortholog of Arabidopsis GIGANTEA. Mutants display defects in phytochrome B-dependent deetiolation under red light and in the diurnal regulation of pea homologs of several Arabidopsis circadian clock genes, including TIMING OF CAB1, EARLY FLOWERING4, and CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL. LATE1 itself shows strongly rhythmic expression with a small but distinct acute peak following dark-to-light transfer. Mutations in LATE1 prevent the induction of a FLOWERING LOCUS T (FT) homolog FTL in long days but cause only minor alteration to the rhythmic expression pattern of the only known group Ia CONSTANS homolog COLa. The late-flowering phenotype of late1 mutants can be completely rescued by grafting to the wild type, but this rescue is not associated with a significant increase in FTL transcript level in shoot apices. Genetic interactions of late1 with the photoperiod-insensitive, early-flowering sterile nodes (sn) mutant and impairment of the LATE1 diurnal expression rhythm in sn plants suggest that SN may also affect the circadian clock. These results show that several functions of Arabidopsis GIGANTEA are conserved in its pea ortholog and demonstrate that genetic pathways for photoperiodic flowering are likely to be conserved between these two species. They also suggest that in addition to its role in the floral transition, LATE1 also acts throughout reproductive development.

Figure 1.

Isolation of photoperiod-insensitive late1 mutants. A, Effect of photoperiod on the phenotype of the wild-type line NGB5839 (‘Torsdag’ le). Left, Plant grown in LD; right, plant grown in SD. Bar = 10 cm. B, Phenotypes of representative mutant alleles for several late-flowering pea mutants in comparison to their isogenic wild-type line NGB5839. Plants were grown in LD. Arrows indicate the branching from basal nodes in photoperiod-insensitive late1, late2, and phyA mutants. Mutants gigas and veg2 do not affect photoperiod sensitivity and do not show basal branching. Bar = 10 cm. C, Comparison of the LD phenotypes of three late1 mutant alleles. Lateral branches have been removed from the late1 mutants for clarity. Note the later flowering and extended reproductive phase (dashed vertical lines) of all three late1 mutants in comparison to the wild type. Bar = 10 cm. D, Effect of late1 mutations on the initiation of flowering and the duration of the reproductive phase under LD conditions. Plants were sown in autumn (19 May). Left, Node of flowering initiation; right, number of reproductive nodes. E, Effect of photoperiod on initiation of flowering in wild-type ‘Torsdag’ (TOR), NGB5839 (5839), and late1 mutants. Plants were sown in spring (15 September). Plants were grown under an 8-h photoperiod of natural daylight in the phytotron, either with (LD) or without (SD) a 16-h extension with white fluorescent light of 10 μmol m⁻² s⁻¹. D and E, n = 8 to 12 and error bars represent se.

Figure 2.

Light responses of late1 mutants and genetic interactions with phyA and phyB. A, Effect of the late1-1 mutation on spectral sensitivity for deetiolation responses. Top, Internode length; bottom, leaflet area. B, Deetiolation responses to R of different late1 mutant alleles. Top, Internode length; bottom, leaflet area. C, Interaction of late1 (late1-2) and phyA (phyA-1) in control of internode elongation under R. A similar interaction was also observed for phyA-1 and the late1-1 allele. D, Interaction of late1 (late1-1) and phyB (phyB-5) in control of internode elongation under R. E, Interaction of late1 (late1-2) and phyA (phyA-1) in the control of the initiation of flowering and the duration of the reproductive phase under LD. Left, Node of flower initiation; right, number of reproductive nodes. F, Interaction of late1-1 and phyB (phyB-5) in the control of the initiation of flowering under SD and LD. Plants were sown in autumn (14 April). A to D, Seedlings were grown from sowing under continuous R, B, or FR light (all 15 μmol m⁻² s⁻¹) or in complete darkness for 14 d at 20°C. Internode length was measured between nodes 1 and 3, and leaf area was estimated as the product of the length and width of the larger leaflet from leaf 3. E and F, Plants were grown under an 8-h photoperiod of natural daylight in the phytotron, either with (LD) or without (SD) a 16-h extension with white fluorescent light at 10 μmol m⁻² s⁻¹. In all segments, n = 8 to 12 and error bars represent se.

Figure 3.

Effects of LATE1 on rhythmic expression of clock-related genes. Diurnal rhythms of MYB1 (A), TOC1 (B), and ELF4 (C) transcript accumulation in wild-type (black symbols) and late1-1 mutant seedlings (white symbols) grown for 14 d under LD (16 h L:8 h D) and harvested every 4 h over the next 48 h. Tissue harvests consisted of the entire shoot apex above and including the uppermost expanded leaf. All plants were grown in growth cabinets at 20°C under a photoperiod of white fluorescent light at 120 to 140 μmol m⁻² s⁻¹. Relative transcript levels were analyzed by RT-qPCR and quantified based on nonequal efficiencies (Pfaffl, 2001). For each data point, n = 3 and bars represent se.

Figure 4.

Molecular characterization of the LATE1 locus. A, Diagram of the LATE1 cDNA showing the location and nature of mutations in late1 mutants. Intron positions are marked by black arrows. B, Diurnal rhythms of LATE1 transcript accumulation in wild-type seedlings grown for 14 d under LD (16 h L:8 h D; black symbols) or SD (8 h L:16 h D; white symbols) and harvested every 4 h over the next 48 h. Tissue harvests consisted of the entire shoot apex above and including the uppermost expanded leaf. LD material analyzed was the same as in Figure 3A. C, Diurnal rhythms of LATE1 transcript accumulation in wild-type seedlings grown for 14 d under 16 h L:8 h D and harvested every 2 h for 8 h. Tissue harvests were comprised of the entire shoot apex above and including the uppermost expanded leaf. D, LATE1 transcript accumulation in 7-d-old dark-grown wild-type seedlings following transfer to continuous B, R, or FR light (15 μmol m⁻² s⁻¹) or maintained in complete darkness (D). Relative transcript levels were analyzed by RT-qPCR and quantified based on nonequal efficiencies (Pfaffl, 2001). In all segments, n = 3 and bars represent se.

Figure 5.

Effects of LATE1 on rhythmic expression of CO and FT homologs under LD. Wild-type (black symbols) and late1-2 (white symbols) seedlings were grown for 28 d under LD (16 h L:8 h D) and leaflets from the sixth leaf were harvested at 4-h intervals over the next 48 h. Relative transcript levels of COLa (A) and FTL (B) were analyzed by RT-qPCR and quantified based on nonequal efficiencies (Pfaffl, 2001). For each data point, n = 3 and bars represent se.

Figure 6.

LATE1 regulates a graft-transmissible flowering stimulus. A, Representative grafted plant 2 weeks after grafting. Arrow indicates site of graft. Bar = 1 cm. B, Representative examples of self-grafted plants and reciprocal graft combinations between the wild type and the late1-1 mutant at approximately 7 weeks after grafting. The late1/late1 self-graft has not yet flowered, whereas all other graft combinations have flowered and terminated apical growth. Arrow indicates site of graft. Bar = 10 cm. C, Node of flower initiation in ungrafted controls, self-grafts, and reciprocal grafts between the wild type and late1-1. For each data point, n = 12 to 16 and bars represent se. D, Relative transcript levels of FTL in the uppermost stock leaf at d 21 and in dissected apices of self-grafted late1-1 scions and in late1-1 scions grafted to the wild type. Harvests took place at 17, 21, and 28 d after grafting to span the estimated time of flower initiation at around day 20 after grafting. Relative transcript levels were analyzed by RT-qPCR and quantified based on nonequal efficiencies (Pfaffl, 2001). For each data point, n = 3 and bars represent se. All plants were grown in the phytotron under LD conditions (8-h photoperiod of natural daylight extended for 16 h with white fluorescent light at 10 μmol m⁻² s⁻¹).

Figure 7.

Interaction of LATE1 and SN. A, Interaction of late1 (late1-1) and sn (sn-4) mutations in the control of initiation of flowering and the duration of the reproductive phase under LD conditions. Left, Node of flowering initiation; right, number of reproductive nodes. Plants were grown in the phytotron where they received an 8-h photoperiod of natural daylight extended for 16 h with white fluorescent light (10 μmol m⁻² s⁻¹). For each data point, n = 8 to 12 and bars represent se. B, Interaction of late1-1 and sn-4 mutations in the control of internode elongation under monochromatic R (left) and B (right). For each data point, n = 8 to 12 and bars represent se. C, Diurnal rhythms of LATE1 transcript accumulation in the wild type (black symbols) and sn-4 (white symbols) mutant seedlings under LD. Growth conditions and tissue harvests were the same as in Figure 3A, and data for the wild type are replotted from Figure 4B. Relative transcript levels were analyzed by RT-qPCR and quantified based on nonequal efficiencies (Pfaffl, 2001). For each data point, n = 3 and bars represent se.