CONSTANS is a photoperiod regulated activator of flowering in sorghum
© Yang et al.; licensee BioMed Central Ltd. 2014
Received: 10 February 2014
Accepted: 13 May 2014
Published: 28 May 2014
Sorghum genotypes used for grain production in temperate regions are photoperiod insensitive and flower early avoiding adverse environments during the reproductive phase. In contrast, energy sorghum hybrids are highly photoperiod sensitive with extended vegetative phases in long days, resulting in enhanced biomass accumulation. SbPRR37 and SbGHD7 contribute to photoperiod sensitivity in sorghum by repressing expression of SbEHD1 and FT-like genes, thereby delaying flowering in long days with minimal influence in short days (PNAS_108:16469-16474, 2011; Plant Genome_in press, 2014). The GIGANTEA (GI)-CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway regulates flowering time in Arabidopsis and the grasses (J Exp Bot_62:2453-2463, 2011). In long day flowering plants, such as Arabidopsis and barley, CONSTANS activates FT expression and flowering in long days. In rice, a short day flowering plant, Hd1, the ortholog of CONSTANS, activates flowering in short days and represses flowering in long days.
Quantitative trait loci (QTL) that modify flowering time in sorghum were identified by screening Recombinant Inbred Lines (RILs) derived from BTx642 and Tx7000 in long days, short days, and under field conditions. Analysis of the flowering time QTL on SBI-10 revealed that BTx642 encodes a recessive CONSTANS allele containing a His106Tyr substitution in B-box 2 known to inactivate CONSTANS in Arabidopsis thaliana. Genetic analysis characterized sorghum CONSTANS as a floral activator that promotes flowering by inducing the expression of EARLY HEADING DATE 1 (SbEHD1) and sorghum orthologs of the maize FT genes ZCN8 (SbCN8) and ZCN12 (SbCN12). The floral repressor PSEUDORESPONSE REGULATOR PROTEIN 37 (PRR37) inhibits sorghum CONSTANS activity and flowering in long days.
Sorghum CONSTANS is an activator of flowering that is repressed post-transcriptionally in long days by the floral inhibitor PRR37, contributing to photoperiod sensitive flowering in Sorghum bicolor, a short day plant.
Optimal regulation of the timing of floral transition is critically important for reproductive success and crop yield. The C4 grass Sorghum bicolor is widely adapted and grown as an annual crop from 0 to >40 degrees N/S latitude. Sorghum crops have been selected for a range of flowering times depending on growing location and use as a source of grain, sugar, forage, or biomass [1–3]. Grain sorghum is generally selected for early flowering (60–80 days) to enhance grain yield stability by avoiding drought, adverse temperatures, and insect pressure during the reproductive phase. In contrast, energy sorghum hybrids are designed with high photoperiod sensitivity in order to delay flowering and extend the duration of vegetative growth, resulting in more than 2-fold increases in biomass production [3, 4]. The stage of plant development, signals from photoperiod, temperature, gibberellins and other factors are integrated to regulate flowering time in sorghum .
The genetic architectures of photoperiod-responsive flowering-time regulatory pathways have been characterized in many plants [6–18]. In Arabidopsis, flowering is promoted in long-days (LD) by coincidence of light signaling and circadian clock output, thus allowing the plant to sense and respond to seasonal changes in photoperiod. Clock output to the flowering pathway is mediated in part by GIGANTEA (GI). GI is regulated by the central clock oscillator comprised of TIMING OF CAB EXPRESSION 1 (TOC1), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). In long days, GI activates CONSTANS (CO) expression in conjunction with FLAVIN-binding KELCH DOMAIN F BOX PROTEIN1 (FKF1) by inducing degradation of CDF1 repressors of CONSTANS transcription. CO accumulates in LD due to stabilization mediated by cryptochromes (CRY1/2), phytochrome A (PHYA) and SUPPRESSOR OF PHYA-105 (SPA1) that counteract degradation of CO mediated by phytochrome B (PHYB): CONSTITUTIVE PHOTOMORPHOGENIC 1(COP1) [6, 9]. Increased CO protein levels in long days leads to the activation of FLOWERING LOCUS T (FT) expression and production of florigen that moves from leaves to shoot apical meristems (SAM) where it binds to FD and induces floral transition.
The GI-CO-FT regulatory pathway identified in Arabidopsis, a long day (LD) plant, is also present in rice, a short day (SD) plant . When rice is exposed to inductive SD, HEADING DATE1 (Hd1), the ortholog of CO, activates expression of the FT-like gene Hd3a, one of two sources of florigen in rice. In non-inductive LD, Hd1 functions as a repressor of Hd3a and flowering . Thus, photoperiod sensitivity in rice depends in part on differences in the activity of CO (Hd1) in long days and short days. Two modulators of flowering time unique to grasses were identified in rice: EARLY HEADING DATE 1 (EHD1)  and GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (GHD7) [21, 22]. EHD1 activates the expression of Hd3a and RICE FLOWERING LOCUS T1 (RFT1), a source of florigen in long days. GHD7 represses flowering by down-regulating expression of EHD1 and Hd3a in LD in rice  and SbEHD1 and SbCN8 in sorghum .
The effect of photoperiod on flowering time varies extensively among and within grass species. Barley and wheat are LD plants, while rice and sorghum are SD plants. Most cultivated maize is photoperiod insensitive therefore plants flower after a set number of degree days; however tropical maize is a photoperiod sensitive short day plant . Sorghum is a short day plant, although grain sorghum is usually photoperiod insensitive, and forage and energy sorghum genotypes exhibit varying degrees of photoperiod sensitivity . More than 40 QTL for flowering time have been identified in sorghum . The Ma1-Ma4 loci were discovered while breeding for early flowering photoperiod insensitive grain sorghum in the U.S. (1920–1960) . Ma1 corresponds to PSEUDORESPONSE REGULATOR PROTEIN 37 (SbPRR37), a repressor of flowering in LD . Ma3 encodes PHYTOCHROME B (PhyB), a red-light photoreceptor that plays an important role in photoperiod sensing and repression of flowering [26–28]. Ma6 encodes SbGhd7, a repressor of SbEHD1 expression and flowering in long days . Ma2, Ma4, and Ma5 are flowering time loci that enhance photoperiod sensitivity in sorghum [25, 29].
CONSTANS (CO) was initially identified as a transcriptional activator of FT and flowering in Arabidopsis . CO belongs to a family of transcription factors unique to plants that contain one or two N-terminal zinc finger B-box domains and a C-terminal CCT domain. Two conserved cysteine and histidine amino acids in the Zn finger domain are essential for CO activity . Arabidopsis mutants with amino acid substitutions at these positions have late flowering phenotypes. Extensive gene duplication events have occurred in this gene family, resulting in ~17 CO family members in Arabidopsis, ~16 in rice and ~9 in barley [31, 32]. The ortholog of CONSTANS in rice, Hd1, plays a key role in photoperiod regulation of flowering, by activating flowering in SD and repressing flowering in LD . Alleles of Hd1 account for ~44% of the variation in flowering time observed in cultivated rice . Hd1 transcript and protein levels are similar in LD and SD, consistent with the finding that Hd1 activity is modulated post-transcriptionally by PHYB  and PRR37 [35, 36].
Identification of flowering time QTL
Parameters of flowering time QTLs in BTx642/Tx7000 RILs population
Greenhouse LD (14 h)
Field LD condition CS08
Greenhouse SD (10 h)
Three flowering time QTL were identified when RILs were screened in LD greenhouse conditions (Figure 1B). The QTL on SBI-01 (19.2-22.0 Mbp) explained 12.3% of the phenotypic variance for flowering time in this environment. SbEHD1, an activator of flowering in grasses located on SBI-01 (Sb01g019980, 21921315–21925396) was found in a one LOD interval spanning this QTL. SbEHD1 was previously identified as a floral activator in sorghum based on sequence similarity to rice EHD1 and observed changes of SbEHD1 expression in LD compared to SD, consistent with this function . There were no amino acid differences between the SbEhd1 protein sequences from Tx7000 and BTx623. BTx623 is a grain sorghum used extensively for breeding, genetic, and genomic research . However, comparison of SbEhd1 from BTx642 and Tx7000 revealed two amino acid substitutions, Asp144Asn and Thr157Ile (Additional file 1: Table S1). The differences in Ehd1 protein sequences occur in a GARP domain that is highly conserved among OsEHD1, SbEHD1 and ARABIDOPSIS RESPONSE REGULATOR 1/2 (ARR1/2). The SbEHD1 allele in BTx642 (tentatively designated Sbehd1-2) delays flowering in LD and SD relative to Tx7000 (SbEHD1-1), consistent with the hypothesis that the amino acid changes in Sbehd1-2 reduce the activity of this floral activator and explaining why a flowering time QTL was detected in this region of SBI-01.
A flowering time QTL located on SBI-10 (10.1-13.7 Mbp) was observed in all environments and spanned a region that encodes a homolog of CONSTANS and Hd1 (Sb10g010050, 12275128–12276617), an important regulator of flowering time in Arabidopsis and rice, respectively (Figure 1B-D). The QTL spanning the sorghum homolog of CONSTANS explained ~40% of the variance in flowering time in LD greenhouses, and 16-17% when plants were grown in the field or SD greenhouses (Table 1). A flowering time QTL located on SBI-08 (48.1-50.3 Mbp) was observed in LD, SD and under field conditions. This QTL explained 8-14% of the phenotypic variance in LD and SD and 18-22% of the variance in field environments. Additional analysis will be required to identify the gene corresponding to this flowering time QTL. A QTL located at the end of SBI-01(~7.2 Mbp) was observed only when the BTx642/Tx7000 RIL population was grown in the SD greenhouse (Figure 1D). A QTL on SBI-06 (~40.2 Mbp) explaining ~15% of the variance in flowering time was identified when RILs were grown in the field (Figure 1C). SbPRR37 (Ma1), a repressor of flowering in LD, was located in the flowering time QTL on SBI-06. Sequence analysis showed that BTx642 encodes Sbprr37-1 and a truncated version of PRR37, and that Tx7000 contains Sbprr37-2, encoding a full-length version of PRR37 containing a Lys162Asn change in the pseudo-response regulator domain . Genotypes containing Sbprr37-2 flowered later than genotypes encoding Sbprr37-1 (null) under field conditions, indicating that Sbprr37-2 is an active but weak allele of SbPRR37. This conclusion is consistent with analysis of a flowering time QTL aligned to PRR37 identified in a RIL population derived from crossing the genotypes Rio and BTx623 . Sequence analysis of SbPRR37 alleles showed that Rio encodes Sbprr37-2 and BTx623 contains Sbprr37-3, a null allele . The Ma1 allele from Rio (Sbprr37-2) delayed flowering relative to BTx623 in field conditions in College Station in a manner similar to the delayed flowering attributed to the same allele in Tx7000 compared to BTx642, which encodes the null allele Sbprr37-1.
Identification of sorghum CONSTANS
The sorghum homolog of CONSTANS (Sb10g010050) is located on SBI-10 and rice Hd1 (Os06g16370) is located on the homeologous rice chromosome 6, suggesting that these genes may be orthologs. The sequences of these genes and adjacent sequences in each chromosome were aligned to determine if SbCO and OsHd1 were in a region of gene colinearity. The sorghum sequences flanking Sb10g010050 were downloaded from Phytozome and aligned with sequences from rice chromosome 6 flanking Hd1 using GEvo . Three genes and Hd1 were aligned and in the same relative order in a 100 kbp region in the two chromosomes, consistent with the identification of Sb10g010050 as an ortholog of rice Hd1 (Additional file 2: Figure S1). Therefore, based on sequence similarity and colinearity, Sb10g010050 was designated as an ortholog of rice Hd1 and a probable ortholog of Arabidopsis CO and termed “SbCO”.
Characterization of SbCO alleles from BTx623, Tx7000 and BTx642
T > C
T > G
C > T
G > A
C > T
A > G
SbCO alleles modulate expression of genes in the flowering time pathway
The influence of SbCO alleles on the expression of other genes in the flowering-time regulatory pathway was analyzed to further understand how SbCO affects flowering time. RIL105 and RIL112 were identified that differ in alleles of SbCO but not at the other main loci that affect flowering time. RIL105 and RIL112 are homozygous for BTx642 alleles for the flowering time QTL on SBI-01 (spanning Sbehd1-2), SBI-06 (spanning Sbprr37-1), and SBI-08. BTx642 encodes a null allele of Ma1 (Sbprr37-1), a gene that contributes to photoperiod sensitivity. Tx7000 contains a weak allele of Ma1 (Sbprr37-2) that encodes a full-length protein that inhibits flowering based on QTL analysis [1, 41]. Therefore, RIL105 and RIL112 were selected for expression studies because both contain DNA from BTx642 on SBI-06 from 0-42 Mbp, ensuring that these genotypes are null for Ma1 (Sbprr37-1). In addition, both RILs encode a null allele of Ma6 (Sbghd7-1) located at the proximal end of SBI-06 . Therefore, comparison of gene expression in RIL105 and RIL112 caused by differences in SbCO alleles will not be influenced by Ma1 or Ma6 the main determinants of photoperiod sensitivity in sorghum [1, 2].
RIL105 (SbCO-2) and RIL112 (Sbco-3) were used to analyze how alleles of CONSTANS affect expression of other genes in the sorghum flowering time regulatory pathway. Expression of the clock genes TOC1, LHY and GI were similar in RIL105 and RIL112, indicating that these genes are not affected by SbCO alleles as expected for genes upstream of SbCO (Additional file 3: Figure S2). In Arabidopsis CO activates flowering by inducing expression of FT and in rice Hd1 activates Hd3a/RFT1, genes encoding PEBP (phosphatidylethanolamine-binding) domain protein ‘florigens’ that move from the leaf to the shoot apical meristem where they interact with FD and induce floral transition. In rice, two members of the PEBP gene family, Hd3a and RFT1 were identified as encoding florigens . In maize, ZCN8, a different member of the PEBP gene family, was identified as a source of florigen [46, 47]. In sorghum, SbCN8 and SbCN12 are potential sources of florigen because expression of both genes is regulated by photoperiod, modulated by Ma1 alleles, and induction of expression occurs coincident with floral initiation . The sorghum orthologs of maize ZCN8 (SbCN8), ZCN12 (SbCN12) and rice Hd3a (SbCN15) were identified and qRT-PCR primers specific to each gene were designed to enable analysis of gene expression (Additional file 4: Table S2). No ortholog of RFT1 is present in the sorghum genome.
In leaves of RIL105 (SbCO-2) grown in LD, SbEHD1 expression was high at dawn and then declined during the day before increasing in the evening approximately 15 h after dawn (Figure 3C, black solid line), with a pattern similar to SbEHD1 expression in 100 M (Ma1) in short days . During the 24 h LD cycle, SbEHD1 RNA was higher in RIL105 (SbCO-2) compared to RIL112 (Sbco-3) indicating that CO activates expression of SbEHD1 (Figure 3C, RIL112 = red dashed line). The average difference in SbEHD1 RNA level in the two RILs during the 24 h LD cycle was 20-fold (Figure 3G). In leaves of RIL105 (SbCO-2) grown in LD, SbCN8 and SbCN12 mRNA levels were highest at dawn, then decreased during the day with a second smaller peak of expression approximately 12-18 h after dawn (Figure 3D and E, black solid line). In RIL112 (Sbco-3), the same pattern of expression was observed; however, SbCN8 and SbCN12 mRNA levels were much lower (Figure 3D and E, red dashed line). Expression of SbCN8 was ~10-fold higher in RIL105 (SbCO-2) compared to RIL 112 (Sbco-3) (Figure 3C) and expression of SbCN12 was ~100-fold higher in RIL105 (SbCO-2) compared to RIL112 (Sbco-3) (Figure 3D) over a 24 h LD cycle in the SbCO-2 background (Figure 3G). In contrast, the mRNA level of SbCN15 (Hd3a) was similar in the two genotypes (Figure 3G), although the gene’s peak of expression was at dawn in RIL105 (SbCO-2) and at 18 h in RIL112 (Sbco-3) (Figure 3F). Together, these results are consistent with the hypothesis that SbCO promotes flowering by inducing expression of SbEHD1, SbCN8, and SbCN12, with SbCN12 showing the largest CO-mediated increase in expression in LD.
Regulation of SbCO floral promoting activity in SD and LD
Post-transcriptional inhibition of SbCO activity by PRR37 (Ma1)
In sorghum, Ma1 (SbPRR37) increases photoperiod sensitivity by repressing expression of SbEHD1 and SbCN8/12, resulting in delayed flowering in LD but with minimal effect in SD . The ability of SbPRR37 to inhibit expression of SbCN8 and SbCN12 could be due to inhibition of SbEHD1 or SbCO, activators of SbCN8 and SbCN12 expression, and/or by direct inhibition of SbCN8 and SbCN12. A flowering time QTL coincident with Ma1 was identified in the BTx642/Tx7000 RIL population grown under field conditions in 2008, 2009 and 2010 (e.g. Figure 1C) as well as in Lubbock, Texas (data not shown). This QTL was not observed in SD conditions, as expected, because SbPRR37 has minimal impact on flowering under these conditions. As noted above, BTx642 encodes a null allele Sbprr37-1, however, the Ma1 allele in Tx7000, Sbprr37-2, encodes a full-length protein with one amino acid substitution Lys62Asn with sufficient activity to delay flowering time under field conditions.
Sorghum accessions exhibit a wide range of flowering times when plants are grown in long days (i.e., 48d to >175d under field conditions in College Station, Texas) . A large extent of this variation is caused by differences in photoperiod sensitivity mediated by floral repressors encoded by Ma1 and Ma6 that inhibit flowering in long days [1, 29]. Much less is known about floral activators in sorghum. The grass specific floral activator SbEHD1 was previously identified based on the gene’s sequence similarity to rice EHD1 and activation of SbEHD1 expression coincident with floral initiation . In this study we identify and characterize a second activator of sorghum flowering SbCO, a homolog of the floral activator CONSTANS in Arabidopsis and an ortholog of Hd1 in rice. Coding alleles of CONSTANS were identified through analysis of a flowering time QTL on SBI-10. Results showed that SbCO functions as an activator of flowering in LD and SD in sorghum genotypes using RILs with null versions of Sbprr37-1 and Sbghd7-1. The Sbco-3 allele in BTx642 was remarkable because it contained a His106Tyr amino acid substitution that also inactivates CO function in Arabidopsis . Sorghum and Arabidopsis genotypes containing the inactive His106Tyr co-3 allele flower late in long days, as well as late in short days in sorghum, indicating that CONSTANS functions as an activator of flowering in both species. SbCO shares a conserved CCT (CO, CO-like, TOC1) domain with TOC1, PRR37, Ghd7, and HEME ACTIVATOR PROTEINS (HAP or NF-Y proteins). Yeast two-hybrid screens showed that CO can interact with HAP3 and HAP5 subunits through its CCT-domain, forming CCAAT-binding CBF-complexes that bind to FT promoters and activate transcription [48, 49]. In sorghum, SbCO was found to activate transcription of SbEHD1, SbCN8 and SbCN12, consistent with its role as an activator of flowering, presumably through formation of CBF-complexes, but possibly through direct binding to DNA .
The results indicate that there has been a significant change in the complement of FT-like genes that function as the main sources of florigen in sorghum (SbCN8, SbCN12) and rice (Hd3a = SbCN15; RFT1, no sorghum ortholog), therefore regulation of flowering time could also differ, even though both grass species are short day plants. SbCO activates expression of SbCN8 and SbCN12, although SbCN12 was induced to a significantly greater extent. SbCO also increased expression of SbEHD1, an activator of Hd3a expression in rice. SbEHD1 expression is repressed by SbPRR37 and SbGhd7 and induced when photoperiod sensitive sorghum grown in LD is transferred to SD [1, 2]. Increases in SbEHD1 expression occur in parallel with increases in SbCN8 and SbCN12 expression, suggesting that SbEhd1 can induce the expression of these genes as shown in Figure 6. However, the extent and specificity of this proposed activity of SbEhd1 will require further analysis in backgrounds where SbCO has minimal influence on the expression of these genes (Sbco-3 backgrounds). The results in this paper show that SbCO increases expression of SbEHD1 and it is proposed that SbEhd1 can activate expression of SbCN8 and SbCN12 and flowering. In contrast, rice Hd1 has not been reported to increase expression of EHD1[20, 33]. Of interest is the finding that SbCO activates expression of SbCN12 to a much greater extent than SbEHD1. Therefore we conclude that SbCO directly increases SbCN12 transcription and that this may be the most important way that SbCO activates flowering.
The finding that SbCO can activate flowering in LD and SD in sorghum genotypes that are null for Ma1 and Ma6 raised the question as to how the activity of this gene is regulated by photoperiod in this short day plant. SbCO expression is low during the day, then increases in the evening to a peak at ~15 h after dawn, followed by a decrease and a second peak of expression at dawn (Figure 3B). In Arabidopsis, a similar increase in CO expression in the evening is due to the interaction of GI and blue light-activated FKF1, resulting in degradation of CDF-factors that inhibit CO expression . This mechanism may also explain the evening peak of SbCO expression in sorghum. The second less prominent peak of SbCO expression at dawn is modulated by alleles of SbPRR37 and enhanced in LD . The function of the peak of SbCO expression at dawn is not currently understood, although production of SbCO at this time could help activate SbEHD1 expression in the morning.
Functional alleles of SbCO increased the expression of SbCN8 and SbCN12 to a greater extent in SD relative to LD (Figure 4). SbCO expression levels increase during the evening, helping to explain why SbCN8/12 expression increases at night. Since expression of SbCO was not altered significantly by photoperiod (Additional file 5: Figure S3), increased activity of SbCO in SD is most likely due to an increase in protein level or activity. In Arabidopsis, a long day plant, CO levels are higher in LD due to COP1-SPA1-Cry2 stabilization of the protein . This stabilization module may be missing or attenuated in sorghum. Reduced PhyB/C-mediated degradation of SbCO in SD, relative to LD, could result in greater SbCO-mediated activation of SbCN8/12 in SD. In sorghum genotypes containing active alleles of SbPRR37 the evening peak of SbCO expression is not altered, the peak of expression at dawn increases, but the activity of SbCO is strongly attenuated. Expression of SbPRR37 is high in the evening in plants grown in LD, but low in SD. Therefore, higher levels of SbPRR37 expression in LD, and SbPRR37 repression of SbCO activity under these conditions, is predicted to prevent SbCO from activating flowering in LD.
SbPRR37 is a CCT-domain protein that has been shown to interact with HAP3, the same CBF-subunit that interacts with SbCO . Therefore, SbPRR37 may be a competitive inhibitor of SbCO binding to the HAP complex. SbPRR37 may also directly bind to DNA in a fashion similar to TOC1 and other PRR-proteins . TOC1 binding to its cognate motif in the promoter of LHY/CCA1 is mediated by its CCT-domain, resulting in PRR-domain mediated repression of transcription. If PRR37 binds to the SbCN12 promoter in a similar manner, it could directly repress transcription, block SbCO binding to the HAP complex, and/or interact with CO or other proteins in order to repress SbCN12 transcription. Recent results on the PRR37 ortholog in rice (Hd2) indicated that PRR37 directly represses Hd3a transcription . Further genetic and biochemical analysis will be required to distinguish among these possibilities.
In barley, a long day plant, HvCO1 activates flowering in LD, and activation is dependent on Ppd-H1, an ortholog of SbPRR37. Overexpression of HvCO1 induced flowering in both LD and SD, but photoperiod sensitivity mediated by Ppd-H1 was still observed in this background . Ppd-H1 does not directly affect expression of HvCO1, but potentiates the ability of HvCO1 to activate HvFT1 expression in LD. It is interesting to note that Ppd-H1 increases HvCO1 activity in LD, whereas SbPRR37 inhibits the floral promoting activity of SbCO in LD. The expression and activity of SbPRR37 and Ppd-H1 increase in LD and both affect CO’s ability to modulate FT-gene expression, but in an opposite manner, consistent with barley being a long day plant and sorghum a short day plant. The difference in activity of PRR37 could be due to differences in direct binding of PRR37 to the promoters of SbCN12 and HvFT1 (homolog of Hd3a, SbCN15), or indirectly by interaction of PRR37/Ppd-H1 with activators, repressors, HAP subunits, HvCO1 and SbCO. In rice, it has been suggested that phosphorylation of PRR37 by Hd6 may cause PRR37, in conjunction with Hd1, to become a repressor of Hd3a expression in LD . The possibility that PRR37 can form a co-repression complex with CO is consistent with results in sorghum and rice. However in the absence of PRR37, CO functions as an activator of FT expression in sorghum and rice. While the biochemical basis of variation in PRR37 activity remains to be elucidated, taken together, the results suggest that interaction between PRR37 and CO on the promoters of specific florigen-related PEBP-genes result in fundamental differences in photoperiod-sensitive flowering between LD and SD grasses.
The flowering time model in Figure 6 shows that SbEhd1 and SbCO can independently induce flowering by activating SbCN8 and/or SbCN12. EHD1 and Hd1 have been shown to independently activate Hd3a (FT) and flowering in rice . In sorghum we show that there is cross-talk between these pathways because SbPRR37 activates SbCO expression at dawn in LD, while SbCO induces SbEHD1 expression in SD. Rice EHD1, a B-type response regulator, is controlled by several upstream modulators including the repressors GHD7, GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 8 (GHD8), OsLEC1 and FUSCA-LIKE1 (OsLFL1), OsMADS56 and the activators GI, EARLY HEADING DATE 2 (EHD2) and OsMADS50 [8, 53]. The existence of two parallel pathways that can activate flowering in sorghum provides for a wide range of responses to diverse environmental factors, contributing to sorghum’s wide geographical adaptation. Sorghum crop breeders are utilizing different alleles of key genes in these parallel pathways to generate early flowering grain sorghum hybrids and late flowering energy sorghum hybrids.
Alleles of sorghum CONSTANS (SbCO) were identified through analysis of a flowering time QTL on chromosome 10 identified in a RIL population derived from BTx642 and Tx7000. Genetic analysis and gene expression studies indicate that SbCO is an activator of flowering in long and short days in genotypes lacking active SbPRR37 and GHD7 alleles. PRR37 was found to block CO-mediated floral activation in long days.
The BTx642/Tx7000 RIL population (n = 90) and parental lines were grown under field conditions in a replicated randomized block design at Texas A&M Research Farm near College Station Texas in 2008, 2009 and 2010 with planting between April 1–14. Days to mid-anthesis (pollen shed) were determined as a measure of flowering time. In the field, day-lengths increased from ~12.6 h in April to 14.3 h in July, with an average daily maximum temperature of 31.7°C and an average daily minimum temperature of 20.0°C. Ten plants of each RIL and the parental lines were grown in a greenhouse in 10 h day lengths (SD, 2011) or 14 h day lengths (LD, 2009 and 2010) and phenotyped for flowering time in a similar manner as the populations grown in the field. RIL105 and RIL112 correspond to 4_6 and 12_14 in original BTx642/Tx7000 RIL population .
Genotyping by sequencing and QTL analysis
Genotyping by sequencing was carried out using Digital Genotyping (DG)  on the 90 RILs derived from BTx642 and Tx7000 . A genetic linkage map was constructed using data generated from 1462 polymorphic DG markers using Mapmaker/EXP ver. 3.0b where recombination frequency was calculated using the Kosambi mapping function. QTLs were detected using Composite Interval Mapping (CIM) in WinQTL Cartographer v2.5 . Significant LOD thresholds for QTL detection were determined based on experiment-specific permutations with 1000 repeats at α = 0.05 . In QTL-based epistasis analysis, the 90 RILs were categorized into sub-populations based on alleles of SbPRR37 or alleles of SbCO respectively. Sub-populations homozygous for each allele of SbPRR37 and each allele of SbCO were then subjected to QTL analysis.
Phylogenetic and Colinearity Analysis
The amino acid sequence of rice Hd1(Os06g16370) was used to search Phytozome v9.1  for homologs of CONSTANS in rice, maize, barley and sorghum. Multiple sequence alignment, alignment scores and phylogentic analysis were performed using ClustalW2  using protein sequences of Sb10g010050 (Sorghum CO), GRMZM2G405368_T01 (Maize conz1), Os06g16370 (Rice Hd1), AF490468 (Barley HvCO1) and AT5G15850 (Arabidopsis CO). Rice and sorghum genome sequences (Phytozome v9.1, 100 kbp) spanning homologs of CO were used for synteny/colinearity analysis. Colinearity was determined by GEvo , a high-resolution sequence analysis tool of genomic regions from CoGe (Accelerating Comparative Genomics) tool kit . A similar phylogenetic/colinearity analysis was performed for EHD1.
SNPs in candidate genes were identified by comparing DNA sequences derived from BTx623, BTx642 and Tx7000. The BTx623 Sbi1 assembly and Sbi1.4 gene annotation were used as the reference genome sequence (Phytozome). BTx642 and Tx7000 genome sequence assemblies used for analysis were obtained previously . SNPs were called using the SNP Detection function of CLC Genomics Workbench 4.9. Minimum coverage for a variant call was set at 5, and maximum was set at 150. Allele types were designated based on SNPs. The SIFT algorithm (sorting intolerant from tolerant)  was utilized to predict whether an amino acid substitution affects protein function based on the degree of conservation of amino acid residues in sequence alignments derived from closely related gene sequences. RIL105 and RIL112 were selected from the BTx642/Tx7000 RIL population using DG markers flanking each QTL peak and spanning each candidate gene. RIL112 contains BTx642 haplotypes spanning all of the flowering time QTL, including the QTL on SBI-10 (Sbco-3). RIL105 contains BTx642 haplotypes spanning QTL on SBI-01, SBI-06, SBI-08 and the Tx7000 haplotype spanning the QTL on SBI-10 (SbCO-2) (Additional file 6: Table S3).
LD, SD and circadian experiments
For circadian rhythm experiments, RIL105 and RIL112 were grown in the greenhouse in LD (14 h light) for 32 days. For entrainment, the plants were transferred to growth chambers set for LD (14 h light/10 h dark) or SD (10 h light/14 h dark) treatment for one week prior to collection of tissue for expression analysis. In the growth chamber, daytime temperature was 30°C at a light intensity of ~300 μmol · s−1 · m−2 and night (dark) temperature was 23°C with ~60% relative humidity. At day 39, the fully expanded portion of the top three leaves from three different plants were sampled from each genotype and photoperiod every 3 hours through a 24 h light–dark cycle followed by 24 h of continuous light (continuous 30°C) (LL). RNA was extracted from leaf tissues using TRI Reagent (MRC) using the protocol for tissues with high polysaccharide content. RNA was cleaned up using RNeasy Mini Kits (QIAGEN), including DNA removal by on-column DNase I digestion. RNA integrity was examined on 1% MOPS gels. First-strand cDNA synthesis was performed using the SuperScript® III First-Strand Synthesis System (Invitrogen) with oligo dT and random hexamer primer mix. After first-strand cDNA synthesis, the reactions were diluted to a final concentration of 10 ng/μl of the initial total RNA. Gene-specific qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems). 18S rRNA was selected as internal control and detected using the TaqMan Universal PCR Master Mix (Applied Biosystems), rRNA Probe (VIC® Probe) and rRNA Forward/Reverse Primer. All reactions were run on a 7900HT PCR System with SDS v2.3 software (Applied Biosystems). The specificity of each qRT-PCR primer set was validated using melting temperature curve analysis. Amplification efficiency of each primer set was determined by the series dilution method , which can be calculated by the slope of the curve made from each Ct value and the dilution factor (Additional file 7: Table S4). Relative expression was determined using the comparative cycle threshold (ΔΔCt) method with calibration using samples with the highest levels of RNA. The primer efficiency was employed to adjust data for relative quantification following the efficiency correction method . Each expression data point was derived from analysis of three technical replicates within three biological replicates.
Availability of supporting data
All the supporting data are included as additional files. BTx642 and Tx7000 whole genome sequence assemblies used for analysis were published previously in Evans J, McCormick RF, Morishige D, Olson SN, Weers B, et al. (2013) Extensive Variation in the Density and Distribution of DNA Polymorphism in Sorghum Genomes. PLoS ONE 8(11): e79192. doi:10.1371/journal.pone.0079192. Genome sequences were deposited with NCBI SRA under the following identifiers: BioProjectPRJNA189453, Accession SRP019171 (http://www.ncbi.nlm.nih.gov/sra/?term=PRJNA189453); SAMN01942195 contains sequence information for BTx642, and SAMN01942194 contains sequence information for Tx7000.
The authors thank William Rooney for growing the BTx642*Tx7000 RIL population in field plots in College Station. Funding for this research was provided by the Perry Adkisson Chair in Agricultural Biology (JEM), Ceres, Inc., and Pioneer Hi-Bred International, Inc.
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