Coincident light and clock regulation of pseudoresponse regulator protein 37 (PRR37) controls photoperiodic flowering in sorghum

Abstract

Optimal flowering time is critical to the success of modern agriculture. Sorghum is a short-day tropical species that exhibits substantial photoperiod sensitivity and delayed flowering in long days. Genotypes with reduced photoperiod sensitivity enabled sorghum's utilization as a grain crop in temperate zones worldwide. In the present study, Ma(1), the major repressor of sorghum flowering in long days, was identified as the pseudoresponse regulator protein 37 (PRR37) through positional cloning and analysis of SbPRR37 alleles that modulate flowering time in grain and energy sorghum. Several allelic variants of SbPRR37 were identified in early flowering grain sorghum germplasm that contain unique loss-of-function mutations. We show that in long days SbPRR37 activates expression of the floral inhibitor CONSTANS and represses expression of the floral activators Early Heading Date 1, FLOWERING LOCUS T, Zea mays CENTRORADIALIS 8, and floral induction. Expression of SbPRR37 is light dependent and regulated by the circadian clock, with peaks of RNA abundance in the morning and evening in long days. In short days, the evening-phase expression of SbPRR37 does not occur due to darkness, allowing sorghum to flower in this photoperiod. This study provides insight into an external coincidence mechanism of photoperiodic regulation of flowering time mediated by PRR37 in the short-day grass sorghum and identifies important alleles of SbPRR37 that are critical for the utilization of this tropical grass in temperate zone grain and bioenergy production.

Fig. 1.

Phenotypic and genetic analysis of Ma₁. (A) LD-entrained ATx623 and R.07007 flower by 54 and 68 d, respectively; ATx623 × R.07007 F₁ plants remain vegetative for >150 d. (B) Flowering is induced in LD-entrained ATx623 × R.07007 F₁ hybrids exposed to SD; continued exposure to LD represses flowering. (C) In LD, SM100 flowers in 54 d; 100M in 120 d. (Scale bar, 0.5 m.) (D) Ma₁ locus delimited to an ∼700-kb region between Xtxsn1 and Xtxi20 in a BC₁F₁ population (n = 1,821) derived from ATx623 and R.07007 (E) Ma₁ mapped to an ∼240-kb region delimited by Xtxp696 and Xtxp711 using a population of F₂ plants (n = 122) derived from 100M and BTx406. (F) The Ma₁ locus was refined between markers Xtxi62 and Xtxi58. Recombination events are shown in parentheses, physical coordinates are at the end of each chromosome segment, and the Ma₁ locus is shaded in blue. (G) Functional SbPRR37 allele in 100M and R.07007. (H) Recessive Sbprr37-1 allele from SM100 and BTx406 with a single nucleotide deletion and frameshift upstream of the PRR domain. (I) Sbprr37-2 allele from Blackhull Kafir with a missense mutation in the PRR domain at conserved Lys¹⁶² residue. (J) Sbprr37-3 allele from ATx623 containing both the Lys¹⁶²Asn substitution and a nonsense mutation at Gln²⁷⁰ resulting in premature termination. Exons are shown as boxes, and introns as solid lines. Yellow boxes, protein coding sequence; blue boxes, pseudoreceiver domain; red boxes, CCT domain; light blue boxes, missense coding post frameshift.

Fig. 2.

Clock-regulated SbPRR37 expression is light dependent. Plants were grown in 14-h light:10-h dark LD (solid line) or 10-h light:14-h dark SD (red dashed line) and then released into LL at time 24 h. Relative expression of SbPRR37 was analyzed at 3-h intervals by quantitative RT-PCR. In 100M (A) and ATx623 × R.07007 F₁ plants (B), SbPRR37 expression increased in the morning (arrow) and evening (arrowhead) of long days. (C) ATx623 × R.07007 F₁ plants grown in 14-h:10-h LD and then released into DD at time 24 h. (D) R.07007 plants grown in LD and then transferred to LL at time 24 h. The Sbprr37-1 mutation in SM100 results in a nonfunctional protein whereas its expression profiles remain similar to 100M; therefore, these data are not shown. The ordinate represents normalized expression relative to a calibrator sample and is based on three biological replicates ± SEM (. The black bar at the top of the figure indicates the dark period for LD-treated plants, and the gray bars indicate subjective dark during LL conditions. The red bar indicates darkness for SD-treated plants; pink indicates subjective dark during LL conditions. Open bars denote light periods. The light gray shading within the plot area indicates darkness for SD-treated plants only, and the dark gray shading indicates darkness for both LD- and SD-treated plants.

Fig. 3.

SbPRR37 modulates expression of downstream flowering genes. Plants were treated under14-h light:10-h dark (LD, solid line) or 10-h light:14-h dark (SD, red dashed line) conditions. (A) Relative CO expression in 100M peaks at dawn (arrowhead) in plants treated in LD, but not in SD. This peak is absent in SM100 under either condition. (B) Relative Ehd1 expression is repressed under LD in 100M, but is activated under both LD and SD in SM100. (C) Expression of FT is repressed in LD in 100M, but SM100 expression levels are equivalent in LD and SD. (D) Expression of ZCN8 is elevated in SD-treated 100M plants but is repressed to near undetectable levels in LD. In SM100, expression is de-repressed in LD. The ordinate represents expression normalized to 18S ribosomal RNA expression and relative to a calibrator sample and is based on three biological replicates ± SEM (24). The black bar above the plot indicates the dark period for LD-treated plants; gray bars indicate subjective dark during LL conditions. Red bars indicate darkness for SD-treated plants; pink indicates subjective dark during LL conditions. Open bars denote light periods. Light-gray shading within the plot area indicates darkness for SD-treated plants only; dark-gray shading indicates darkness for both LD- and SD-treated plants.

Fig. 4.

Model of photoperiodic flowering-time regulation in sorghum. PRR37 is a central floral repressor that blocks transition from the vegetative phase to flowering in LD. PRR37 represses FT, ZCN8, and flowering by activating expression of CO, a repressor of FT in rice, and by inhibiting Ehd1, a grass-specific inducer of FT. SbPRR37 expression is regulated by the circadian clock and light in a manner consistent with the external coincidence model. It is proposed that photoreceptors (PHOT) such as phytochromes mediate light activation of SbPRR37 expression coincident with output from the circadian clock, resulting in increased SbPRR37 expression in the morning and evening in LD. In SD, SbPRR37 expression is not activated in the evening, leading to floral induction.