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Proc Natl Acad Sci U S A. Sep 27, 2011; 108(39): 16469–16474.
Published online Sep 19, 2011. doi:  10.1073/pnas.1106212108
PMCID: PMC3182727
Plant Biology

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, Ma1, 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.

Keywords: circadian rhythm, pseudo-response regulator

Sorghum [Sorghum bicolor (L.) Moench] is a C4 grass native to Africa that provides an indispensable food source for over 300 million people inhabiting food-insecure regions worldwide (1). Although primarily grown for its grain and forage, high-biomass sorghum is also an excellent drought-tolerant energy crop for sustainable production of lignocellulosic-based biofuels (2). Forage and energy sorghums are selected for delayed flowering to increase biomass yield through longer duration of vegetative growth, whereas grain sorghums are selected for early flowering to ensure sufficient time for grain maturation and to avoid drought and frost. Optimal production of each of these sorghum crops requires the precise regulation of flowering time, which varies depending on planting location and climate. Differences in photoperiod sensitivity confer a wide range of flowering times on diverse accessions of the sorghum germplasm collection (3). Due to its critical importance to crop yield and hybrid seed production, photoperiodic regulation of flowering has been an important trait characterized by sorghum improvement programs dating back to the early 1900s (4).

In Arabidopsis, flowering is induced in long days (LD) that expose plants to light in the evening during a phase of circadian clock oscillation required for induction of floral genes, consistent with the external coincidence model (57). Rhythmic expression of the core circadian clock components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB 1 (TOC1) during each day–night cycle regulates expression of the clock output gene GIGANTEA (GI), which in turn regulates the floral-inducing gene CONSTANS (CO). The stability and activity of these floral regulators are further altered through light-mediated posttranslational modifications and protein–protein interactions, allowing FLOWERING LOCUS T (FT), a known florigen, to accumulate to critical levels in LD but not in short days (SD) (7). In rice, a species induced to flower in SD, orthologs of GI, CO (Hd1), and FT (Hd3a) also regulate flowering time (8). In contrast to Arabidopsis, Hd1 (CO) was found to repress flowering in LD in rice (9). In wheat and barley (LD-inductive grasses), an ortholog of AtPRR7, PHOTOPERIOD 1 (Ppd1), plays an important role in regulating flowering time in response to photoperiod (10, 11). Other key regulators of flowering time have been identified in grass species with no known orthologs in Arabidopsis, including EARLY HEADING DATE 1 and 2 (Ehd1, Ehd2) (12, 13), which activate FT expression, and VRN2 (Ghd7), a repressor of FT in wheat and rice (14, 15). These and other reports indicate that the pathway regulating flowering time diversified as grass species adapted to different latitudes and environments.

Sorghum genotypes show a wide range of photoperiod sensitivity and critical floral-inductive day lengths (4, 16). Historic genetic studies uncovered four flowering-time (maturity) loci, which were designated Ma1, Ma2, Ma3, and Ma4 (1719). More recently, two additional maturity genes, Ma5 and Ma6 (20), which increase photoperiod sensitivity and lengthen the duration of vegetative growth in forage and high-biomass sorghum hybrids, were described. Dominant alleles at each maturity locus contribute to late flowering in LD. Of the four original maturity loci, Ma1 has the largest impact on flowering time in sorghum (4). Mutations in Ma1 were critical for the early domestication and dispersal of sorghum from its center of origin during the migration of people across Africa and Asia (19). During the first 40 years of the 20th century, growers and plant breeders in the United States and elsewhere selected recessive alleles of Ma1 that resulted in the development of early flowering sorghum cultivars suitable for grain production in temperate regions worldwide (1, 4). More recently, the manipulation of flowering-time loci has been of fundamental importance to the production of high-biomass sorghum for biopower and lignocellulosic biofuels (2).

The overall importance of flowering time and the critical role that Ma1 plays in this regulatory pathway led to this large-scale effort to understand the molecular basis of Ma1 function and allelic variation. We report here the positional cloning of sorghum Ma1 and describe how coordinated regulation of Ma1 gene expression by light and the circadian clock provides a mechanism for regulating flowering time in response to photoperiod.

Results and Discussion

Map-Based Cloning of the Ma1 Gene.

The gene corresponding to Ma1 was cloned using three mapping populations derived from genotypes that vary in flowering time due to differences in Ma1 alleles (Fig. 1, SI Methods, and SI Discussion). Ma1 was initially mapped using a population created by crossing two early flowering genotypes, ATx623 (ma1, Ma5) and R.07007 (Ma1, ma5), to generate photoperiod-sensitive, late-flowering F1 hybrids that are useful for biomass production (Fig. 1A). Exposure of 65-d-old vegetative F1 plants to SD resulted in flowering 36 d later, whereas F1 hybrids kept in LD remained vegetative indefinitely (Fig. 1B). To create a population suitable for mapping Ma1, F1 plants were backcrossed to ATx623 to eliminate allelic effects of Ma5, a flowering locus recessive in R.07007 (20). Genetic mapping using 1,821 BC1F1 plants identified a flowering-time locus within a 700-kb interval on chromosome SBI-06 (Fig. 1D) that mapped coincidently with the reported location of Ma1 (21, 22). This region encodes 34 putative genes (Table S1), among which the most likely candidate gene was PSEUDORESPONSE REGULATOR 37 (Sb06g014570, SbPRR37). The Ma1 locus was fine-mapped using several historically important grain-producing cultivars that possess different Ma1 alleles. 100M and SM100 are nearly isogenic lines that contain dominant and recessive Ma1 alleles, respectively (19), and differ in flowering time by ~60 d when grown in LD (Fig. 1C). A mapping population was created by crossing 100M (Ma1) to the elite inbred BTx406, which derives its ma1 allele from the same source as SM100, but provides a level of polymorphism more suitable for mapping. BTx406 is also of historical importance as the genetic donor of the ma1 allele used to convert tropical late-flowering sorghum to photoperiod-insensitive cultivars useful for grain sorghum breeding (23). Genetic analysis of this F2 population refined the Ma1 locus to a 240-kb region (Fig. 1E). Further Ma1 fine mapping was accomplished using an F2 population (n = 1,925) derived by crossing 100M to the photoperiod-insensitive cultivar Blackhull Kafir (ma1), a founder genotype from an ancestral lineage different from 100M (17, 19). Analysis of F2 plants from this population, in combination with derived F3 families, allowed the Ma1 locus to be reduced to an 86-kb interval delimited by markers Xtxi62 and Xtxi58 (Fig. 1F). The best candidate for Ma1, SbPRR37, was the sole gene present among the stretches of repetitive DNA found in this region (Phytozome v5.0).

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 ...

Sequence Analysis of SbPRR37 Alleles in Historical Cultivars.

To substantiate the identity of the Ma1 gene as SbPPR37 and to characterize alleles of this locus that modify photoperiod sensitivity, full-length cDNAs were sequenced for a select set of founder and elite sorghum cultivars. The structure of SbPRR37 alleles was examined by aligning full-length cDNA sequences from photoperiod-sensitive (Ma1) and -insensitive (ma1) parental genotypes to genomic DNA sequences. The 3,165-nucleotide SbPRR37 mRNA from R.07007 and 100M contained three untranslated and eight protein-coding exons (Fig. 1G and Fig. S1). This transcript encodes a 739-amino-acid, ~93-kDa protein that contains a predicted N-terminal pseudoreceiver domain (residues 99–207) and a C-terminal CCT domain (residues 682–727), present in all known plant PRR proteins. Sorghum PRR37 was compared with other plant PRR proteins using the method described by Turner et al. (11) (Fig. S2). This analysis showed that sorghum PRR37 is most closely related to Arabidopsis PRR7, two maize PRR37-like proteins (encoded by GRMZM2G033962 and GRMZM2G005732), rice PRR37 (LOC_Os07g49460), and PRR proteins encoded by barley Ppd-H1 and wheat Ppd-D1a (10, 11).

The coding sequences from photoperiod-insensitive ma1 genotypes revealed mutations in the PRR37 protein that are predicted to disrupt function (Fig. 1 HJ) as well as other background nucleotide polymorphisms (Fig. S1 and Table S2). The nucleotide sequence of the coding region from the Sbprr37-1 allele (genotypes BTx406 and SM100) was identical to SbPRR37 except for a single nucleotide deletion upstream of the pseudoreceiver domain, resulting in the premature termination of the Sbprr37-1 protein (Fig. 1H). Allele Sbprr37-2 (from cultivar Blackhull Kafir) differs from SbPRR37 by three amino acid substitutions; two substitutions are present in regions of low conservation among PRR37 proteins (Fig. S1 and Table S2), but the third substitution occurs in the pseudoreceiver domain at Lys162, a highly conserved amino acid in all pseudoreceiver and receiver-domain proteins (Fig. 1I). The substitution of an uncharged Asn for a positively charged Lys at this position could alter the functionality of the pseudoreceiver domain. Recessive allele Sbprr37-3 from ATx623 harbored both the Lys162Asn substitution found in Sbprr37-2 and an additional nucleotide substitution, resulting in an in-frame stop codon (Q270 → Stop) before the CCT motif (Fig. 1J). Quinby (4) proposed more than 50 y prior that a series of unique recessive ma1 alleles have been preserved in sorghum germplasm from different temperate regions of the world, and the present results support this assertion. The Sbprr37-1 functional mutation occurring in tropical Standard Milo was introduced into the United States from Columbia in the mid-1800s (1, 19). By comparison, the Sbprr37-2 allele can be traced to Kafir cultivars from South Africa that were introduced in 1876 (4, 19). Sbprr37-3 represents a second Kafir allele originally present in the progenitor cultivar Combine Kafir-60 (1). The full extent of the Sbprr37 allelic series remains to be determined, but these results suggest that multiple independent mutation events in SbPRR37 have occurred during sorghum's adaptation to temperate climates worldwide.

Photoperiod and the Circadian Clock Modulate SbPRR37 and Floral Gene Expression.

In wheat, misexpression of Ppd-D1a is correlated with reduced photoperiod sensitivity, indicating the importance of PRR37 expression in photoperiodic regulation of flowering in this LD grass (10). Therefore, SbPRR37 mRNA levels were quantified in photoperiod-sensitive 100M and F1 plants under LD, SD, and circadian cycling conditions (Fig. 2). Analysis of cDNA revealed several PRR37 splice variants (Table S2 and Fig. S3F). The abundance of splice variants and full-length transcripts was regulated in a similar manner; therefore, overall SbPRR37 transcript abundance was quantified by quantitative RT-PCR (qRT-PCR). In LD, 100M and F1 plants show peaks of SbPRR37 mRNA abundance in the morning and in the evening ~3 and 15 h after lights were turned on, respectively (Fig. 2 A and B). The daily bimodal cycling pattern of SbPRR37 mRNA abundance persisted in continuous light and temperature (LL) (Fig. 2 A and B, days 2/3, star), indicating that the circadian clock modulates SbPRR37 expression under these free-running conditions. By contrast, in SD, 100M and F1 plants showed only the morning-phase peak of SbPRR37 mRNA abundance (Fig. 2 A and B, day 1). However, when plants grown in SD were transferred to LL, both the morning and evening-phase peaks of SbPRR37 mRNA abundance were observed (Fig. 2 A and B, days 2/3, star). This suggests that SbPRR37 expression is light dependent and that the disappearance of the evening peak of SbPRR37 expression in SD is caused by the lack of light during the evening. Expression of the core clock genes TOC1 and LHY continues to cycle in LD, SD, and LL (Fig. S3 A and B), indicating that decreased SbPRR37 expression in SD is likely due to lack of light during the evening rather than to disruption of clock function. The light dependence of SbPRR37 expression was further analyzed by transferring F1 plants grown in LD to continuous dark (DD) (Fig. 2C). In DD, neither peak of SbPRR37 expression was observed, consistent with a requirement for light for SbPRR37 expression.

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 ...

The results described above indicate that SbPRR37 expression is dependent on illumination of plants during times of the day when output from the circadian clock has the potential to activate SbPRR37 expression. This mode of regulation is consistent with the external coincidence model of flowering-time regulation (6). In LL or LD, output from the circadian clock activates SbPRR37 expression in the morning and evening, and the continuous production of PRR37 in LD is proposed to repress flowering. In SD, output from the clock increases SbPRR37 expression during the morning but not in the evening because the evening phase of potential clock activation of SbPRR37 expression occurs in darkness. In SD, lack of increased SbPRR37 expression during the evening phase is proposed to reduce the level of the repressor PRR37, allowing floral initiation.

The important contribution of the evening peak of SbPRR37 expression to floral repression in LD was supported by analysis of the genotype R.07007 (Fig. 2D). This genotype is photoperiod insensitive and flowers early in LD due to recessive ma5 (20), despite possessing a functional SbPRR37 allele. In LD, the morning-phase increase in SbPRR37 expression was observed in R.07007 and 100M (Fig. 2D, arrow). However, the increase of SbPRR37 expression in the evening that occurs in 100M was not observed in R.07007 (Fig. 2D, arrowhead). The evening peak is restored under LL conditions, although shifted three hours later than peaks of SbPRR37 mRNA abundance observed in 100M or the F1 (Fig. 2D, star). The molecular basis for altered SbPRR37 expression during the evening and under LL conditions in R.07007 is not known. However, these results show that evening-phase expression of SbPRR37 is correlated with repression of flowering in LD photoperiods in sorghum.

We next investigated the mechanism by which PRR37 represses flowering in LD in sorghum. Genes in the canonical Arabidopsis flowering pathway also contribute to the control of flowering time in rice and other grasses, and the most noted of these, CO, is a repressor of flowering in rice in LD (9). Therefore, to gain further understanding of how PRR37 modulates the floral induction pathway, we characterized the expression of the sorghum ortholog of CO over a 40-h time course in 100M and SM100 in LD and SD to determine if this gene was regulated by PRR37 as previously shown in barley (11). 100M grown in LD (Fig. 3A, solid line) showed two increases of CO mRNA abundance in leaves each day, similar to the daily bimodal CO expression pattern observed in maize in LD (25). The first peak of CO mRNA occurred in the evening ~15 h after lights on, and the second increase occurred during the last several hours of the night, peaking at dawn (24 h) (Fig. 3A, arrowhead). By contrast, in SD (Fig. 3A, red dashed line), the peak of CO expression at dawn was greatly reduced. In addition, the second peak of CO expression observed at dawn in 100M grown in LD is absent in SM100 (Sbprr37-1) (Fig. 3A, arrowhead). These results indicate that the reduction of CO mRNA abundance at dawn in SM100 plants grown in LD is due to prr37-1 and that PRR37 is required for differential expression of CO in response to photoperiod in sorghum. CO expression is regulated by the circadian clock through the action of GI in Arabidopsis and rice (2628). Therefore, it is possible that PRR37 alters CO expression through an indirect effect on clock gene expression. Small differences in the patterns of TOC1, LHY, and GI expression in 100M and SM100 were noted but could not be directly connected to the altered expression of CO in SM100 compared with 100M (Fig. S3 AC). We interpret these results to indicate that, although PRR37 may have an effect on clock gene expression, this protein also directly regulates CO. Regulation of CO expression by PRR7, an ortholog of SbPRR37, independent of the clock-GI pathway, was also proposed to occur in Arabidopsis (29).

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 ...

In rice, Ehd1 encodes a B-type response regulator transcription factor unique to grasses that has been shown to promote flowering (12). Because Ehd1 has a role in floral activation via a pathway separate from CO, we identified the sorghum ortholog of Ehd1 and found that 100M expression of this gene was strongly repressed in LD, whereas in SM100, LD and SD levels were similar (Fig. 3B). Moreover, when 100M plants grown in LD were transferred to SD, expression of Ehd1 at 15 h after lights on increased ~17-fold. In contrast, transfer of SM100 plants to SD increased Ehd1 mRNA levels only 2.4-fold (Fig. S3E, Upper). These results are consistent with the de-repression of Ehd1 in the Sbprr37-1 background (SM100).

FT is part of a 6-member PEBP-domain gene family in Arabidopsis and a >20-member gene family in maize (30). Several members of the PEBP-domain gene family encode florigens that modulate flowering in rice and maize (30, 31). In rice, Hd3a and RFT1 act synergistically to promote the transition from vegetative to reproductive growth (31). No ortholog of RFT1 was found in sorghum (31) (Phytozome v5.0). However, a collinear sorghum ortholog of rice FT (OsHd3a) was present in sorghum, and this gene was regulated by photoperiod and PRR37 (Fig. 3C). The sorghum ortholog of OsHd3a (SbFT) was expressed in 100M leaves at lower levels in LD compared with SD (Fig. 3C, Upper). In contrast, SbFT showed elevated expression in SM100 plants in LD and SD (Fig. 3C, Lower). When 100M plants were transferred from LD to SD for 1 wk, SbFT mRNA levels increased 7.1-fold during the evening phase (15 h after lights on). By contrast, transfer of SM100 plants from LD to SD for 1 wk increased SbFT levels in SM100 only 0.3-fold, indicating the absence of repression of SbFT expression in LD in the Sbprr37-1 background (Fig. S3E, Lower).

Sorghum ZCN8, the collinear ortholog of the maize florigen Zea mays CENTRORADIALIS 8 (30), was expressed at low levels in LD in 100M and at elevated levels in SD (Fig. 3D, Upper). Similar to Ehd1 and SbFT, SbZCN8 expression in SM100 plants was de-repressed regardless of photoperiod (Fig. 3D, Lower). In addition, expression analysis of ZCN12, a second florigen candidate gene in maize that responds to photoperiod (30), showed a pattern of expression in 100M and SM100 similar to SbZCN8 (Fig. S3D). In summary, expression of sorghum orthologs of genes that are involved in floral induction in other grasses, including Ehd1, FT, ZCN8, and ZCN12, is regulated by photoperiod in 100M (SbPRR37), but not in SM100 (Sbprr37-1), a genotype that lacks a functional PRR37.

This study demonstrates that SbPRR37 is a central repressor in a regulatory pathway that controls sorghum flowering in response to photoperiod. A working model for this regulatory network is shown in Fig. 4. In LD, light dependent, circadian-regulated increases in SbPRR37 expression in the morning and evening are proposed to result in a sufficient level of PRR37 throughout the day to repress FT, other genes encoding florigens, and floral initiation. In SD, the evening peak of SbPRR37 expression is reduced or eliminated, leading to floral induction consistent with the external coincidence model of flowering-time regulation (5, 6). By contrast, Arabidopsis PRR7, the ortholog of SbPRR37, shows only a single morning-phase peak of expression (32), indicating that evening-phase expression of this gene may be a special feature of grass species. The light-dependent induction of SbPRR37 expression and the clock-mediated evening-phase peak of expression enable SbPRR37 to regulate flowering time in response to photoperiod; PRR37 mRNA levels in the evening phase decrease as day length is reduced. The photoreceptor(s) that mediate light-induced SbPRR37 expression are currently under investigation; however, phytochrome B is likely involved because recessive alleles of this gene cause early flowering in LD in sorghum (ma3, ma3R) (33), barley (34), and rice (35). SbPRR37 is proposed to repress FT, SbZCN8, and SbZCN12 and flowering in LD in part by inhibiting expression of Ehd1, an activator of FT and flowering in rice (12); by increasing expression of CO, a repressor of flowering in rice in LD (26); and possibly by other mechanisms that modulate SbFT and SbZCN8 expression (Fig. 4).

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 ...

This study provides insight into the mechanism of photoperiodic regulation of flowering time in the SD grass sorghum. The importance of PRR37 in photoperiod regulation was first documented in LD barley (11) and wheat (10). In these grasses, PRR37 activates FT and flowering in LD whereas in sorghum PRR37 represses FT, ZCN8, and flowering in LD. The molecular basis of this difference in PRR37 activity in sorghum, a short-day plant, and the long-day grasses barley and wheat may relate to differences in CO activity in the formation of CCAAT-box–binding complexes involved in floral gene expression (26, 36). In addition to documenting how sorghum regulates flowering time in response to photoperiod, this study identified important alleles of SbPRR37 that were critical for the domestication and utilization of this tropical grass for grain production in temperate regions worldwide (1, 16). This information will allow plant breeders to more precisely control flowering time in grass species, thus increasing yield and sustainable production.

Methods

Genotyping and Phenotyping Mapping Populations.

All sorghum accessions used in this study, as well as relevant descriptors, are listed in Table S3. For flowering-date determinations, plants were grown in different LD environments in the greenhouse or in the field in the summer seasons of 2003–2010. Days to midanthesis were evaluated in the greenhouse under 14-h light/10-h dark photoperiods (30–34/20–25 °C), and in field locations in College Station, Vega, and Plainview, all in Texas. For genotyping, plant DNA was extracted with either the FastDNA Spin Kit (MP Biomedicals) or by disruption of a leaf punch using a GenoGrinder (BT&C/OPS Diagnostics). Plants from all three mapping populations were subjected to PCR-based marker analysis as previously described (37).

SbPRR37 Allele Sequencing.

To examine the SbPRR37 gene for functional mutations that contribute to the temperate-zone adaptation of sorghum, either the full 10-kb SbPRR37 genomic region or expressed cDNA was sequenced from six genotypes including historically prominent cultivars. PCR-amplified products from genomic DNA (Phusion High-Fidelity DNA Polymerase, New England BioLabs, Inc.) were isolated using the QIAquick PCR Purification and Gel Extraction Kits (QIAGEN). Sequencing of the purified PCR products was carried out in a reaction using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Capillary sequencing was performed on the Applied Biosystems 3130xl Genetic Analyzer. The results were assembled and analyzed using either Sequencher v4.8 (Gene Codes) or Phred/Phrap and Consed (http://www.phrap.org/phredphrapconsed.html).

Plant Materials for Gene Expression Studies.

Sorghum genotypes 100M, SM100, ATx623, R.07007, and ATx623 × R.07007 F1 plants were grown in a greenhouse in Metro-Mix 200 (Sunshine MVP; Sun Gro Horticulture Ltd.) under long-day conditions (14-h days) and were fertilized once after 2 wk using Peters Professional Allrounder fertilizer (The Scotts Company). After 32 d, the plants were transferred to a growth chamber for 1 wk under either long (14-h) days or short (10-h) days at a light intensity of ~300 μmol·s−1·m−2 at ~50% humidity with 30 °C day temperatures and 23 °C night temperatures. At day 39, 1 wk after SD or LD treatment, the three topmost leaves from three different plants (pooled) were harvested from each genotype every 3 h for one 24-h light–dark cycle and an additional 48-h constant light (constant 30 °C) or constant dark cycle (constant 23 °C), as indicated. Total RNA was extracted and used in downstream quantitative real time PCR (qRT-PCR) reactions. Relative expression was determined using the comparative cycle threshold (Ct) method, and relative transcript number was calculated using the standard curve method (24).

Supplementary Material

Supporting Information:

Acknowledgments

This research was supported by the Office of Science (Biological and Environmental Research), US Department of Energy, Grant DE-FG02-06ER64306, the Perry Adkisson Chair (J.E.M.), and US Department of Agriculture-Agricultural Research Service (R.R.K.).

Footnotes

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. JF801178JF801190).

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106212108/-/DCSupplemental.

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