BACKGROUND
optimal regulation of the timing of floral transition is critically important for reproductive success and crop yield.
the c <dig> grass sorghum bicolor is widely adapted and grown as an annual crop from  <dig> to > <dig> 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  <cit> .
grain sorghum is generally selected for early flowering  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  <cit> .
the stage of plant development, signals from photoperiod, temperature, gibberellins and other factors are integrated to regulate flowering time in sorghum  <cit> .
the genetic architectures of photoperiod-responsive flowering-time regulatory pathways have been characterized in many plants  <cit> .
in arabidopsis, flowering is promoted in long-days  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 is regulated by the central clock oscillator comprised of timing of cab expression  <dig> , circadian clock associated  <dig>  and late elongated hypocotyl .
in long days, gi activates constans  expression in conjunction with flavin-binding kelch domain f box protein <dig>  by inducing degradation of cdf <dig> repressors of constans transcription.
co accumulates in ld due to stabilization mediated by cryptochromes , phytochrome a  and suppressor of phya- <dig>  that counteract degradation of co mediated by phytochrome b : constitutive photomorphogenic  <dig>  <cit> .
increased co protein levels in long days leads to the activation of flowering locus t  expression and production of florigen that moves from leaves to shoot apical meristems  where it binds to fd and induces floral transition.
the gi-co-ft regulatory pathway identified in arabidopsis, a long day  plant, is also present in rice, a short day  plant  <cit> .
when rice is exposed to inductive sd, heading date <dig> , the ortholog of co, activates expression of the ft-like gene hd3a, one of two sources of florigen in rice.
in non-inductive ld, hd <dig> functions as a repressor of hd3a and flowering  <cit> .
thus, photoperiod sensitivity in rice depends in part on differences in the activity of co  in long days and short days.
two modulators of flowering time unique to grasses were identified in rice: early heading date  <dig>   <cit>  and grain number, plant height and heading date  <dig>   <cit> .
ehd <dig> activates the expression of hd3a and rice flowering locus t <dig> , a source of florigen in long days.
ghd <dig> represses flowering by down-regulating expression of ehd <dig> and hd3a in ld in rice  <cit>  and sbehd <dig> and sbcn <dig> in sorghum  <cit> .
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  <cit> .
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  <cit> .
more than  <dig> qtl for flowering time have been identified in sorghum  <cit> .
the ma1-ma <dig> loci were discovered while breeding for early flowering photoperiod insensitive grain sorghum in the u.s.   <cit> .
ma <dig> corresponds to pseudoresponse regulator protein  <dig> , a repressor of flowering in ld  <cit> .
ma <dig> encodes phytochrome b , a red-light photoreceptor that plays an important role in photoperiod sensing and repression of flowering  <cit> .
ma <dig> encodes sbghd <dig>  a repressor of sbehd <dig> expression and flowering in long days  <cit> .
ma <dig>  ma <dig>  and ma <dig> are flowering time loci that enhance photoperiod sensitivity in sorghum  <cit> .
constans  was initially identified as a transcriptional activator of ft and flowering in arabidopsis  <cit> .
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  <cit> .
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 ~ <dig> co family members in arabidopsis, ~ <dig> in rice and ~ <dig> in barley  <cit> .
the ortholog of constans in rice, hd <dig>  plays a key role in photoperiod regulation of flowering, by activating flowering in sd and repressing flowering in ld  <cit> .
alleles of hd <dig> account for ~44% of the variation in flowering time observed in cultivated rice  <cit> .
hd <dig> transcript and protein levels are similar in ld and sd, consistent with the finding that hd <dig> activity is modulated post-transcriptionally by phyb  <cit>  and prr <dig>  <cit> .
RESULTS
identification of flowering time qtl
flowering time qtl were mapped in a ril population derived from a cross of btx <dig> and tx <dig>  genotypes used in u.s. grain sorghum breeding programs as sources of drought tolerance  <cit> .
a ril population  derived from these genotypes was previously used to map qtl for flowering time and the stay-green drought tolerance trait using a genetic map based on rflp markers  <cit> .
the genomes of btx <dig> and tx <dig> were recently sequenced and analyzed for variation in dna polymorphisms that distinguish these genotypes  <cit> .
digital genotyping was used to create a high-resolution genetic map aligned to the genome sequence based on this ril population  <cit> .
digital genotyping identified  <dig>  snp markers segregating in the ril population and data on recombination frequency was used to create a 1139 cm genetic map spanning the  <dig> sorghum chromosomes  <cit> .
flowering time qtl were mapped in this population by phenotyping the ril population for days to half pollen shed in greenhouses in 14 h long days , 10 h short days , and under field conditions where day length increases following plant emergence in mid-april from  <dig>  h to  <dig>  h in july.
tx <dig> flowered in 73 days and btx <dig> flowered approximately 4 days later under field conditions in college station, texas.
when grown in a greenhouse at constant 14 h day lengths  during the summer, tx <dig> flowered in 84 days and btx <dig> flowered ~19 days later .
when tx <dig> and btx <dig> were grown in a greenhouse under 10 h day lengths  during the winter, tx <dig> flowered in 54 days whereas btx <dig> flowered ~11 days later.
the btx642/tx <dig> ril population was grown and assayed for days to flowering under field conditions in 2008– <dig>  in ld greenhouses in  <dig> and  <dig>  and in a sd greenhouse during the winter of  <dig>  winqtl cartographer was used to identify flowering time qtl using flowering time data collected from each location/year and the genetic map generated by evans et al.
<cit> .
three qtl for flowering time were observed in every environment and two additional qtl were identified in only one environment .
a
b
c
2d
e
a
position of likelihood peak .
b
peak coordinate: physical coordinate of the likelihood peak.
c
additive effect: a positive value means the delay of flowering time due to tx <dig> allele.
a negative value means the delay of flowering time due to btx <dig> allele.
d
r <dig> : percentage of phenotypic variance explained by the qtl.
e
nd: candidate gene is not determined.
three flowering time qtl were identified when rils were screened in ld greenhouse conditions .
the qtl on sbi- <dig>  explained  <dig> % of the phenotypic variance for flowering time in this environment.
sbehd <dig>  an activator of flowering in grasses located on sbi- <dig>  was found in a one lod interval spanning this qtl.
sbehd <dig> was previously identified as a floral activator in sorghum based on sequence similarity to rice ehd <dig> and observed changes of sbehd <dig> expression in ld compared to sd, consistent with this function  <cit> .
there were no amino acid differences between the sbehd <dig> protein sequences from tx <dig> and btx <dig>  btx <dig> is a grain sorghum used extensively for breeding, genetic, and genomic research  <cit> .
however, comparison of sbehd <dig> from btx <dig> and tx <dig> revealed two amino acid substitutions, asp144asn and thr157ile .
the differences in ehd <dig> protein sequences occur in a garp domain that is highly conserved among osehd <dig>  sbehd <dig> and arabidopsis response regulator 1/ <dig> .
the sbehd <dig> allele in btx <dig>  delays flowering in ld and sd relative to tx <dig> , consistent with the hypothesis that the amino acid changes in sbehd1- <dig> reduce the activity of this floral activator and explaining why a flowering time qtl was detected in this region of sbi- <dig>
a flowering time qtl located on sbi- <dig>  was observed in all environments and spanned a region that encodes a homolog of constans and hd <dig> , an important regulator of flowering time in arabidopsis and rice, respectively .
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 .
a flowering time qtl located on sbi- <dig>  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- <dig> was observed only when the btx642/tx <dig> ril population was grown in the sd greenhouse .
a qtl on sbi- <dig>  explaining ~15% of the variance in flowering time was identified when rils were grown in the field .
sbprr <dig> , a repressor of flowering in ld, was located in the flowering time qtl on sbi- <dig>  sequence analysis showed that btx <dig> encodes sbprr37- <dig> and a truncated version of prr <dig>  and that tx <dig> contains sbprr37- <dig>  encoding a full-length version of prr <dig> containing a lys162asn change in the pseudo-response regulator domain  <cit> .
genotypes containing sbprr37- <dig> flowered later than genotypes encoding sbprr37- <dig>  under field conditions, indicating that sbprr37- <dig> is an active but weak allele of sbprr <dig>  this conclusion is consistent with analysis of a flowering time qtl aligned to prr <dig> identified in a ril population derived from crossing the genotypes rio and btx <dig>  <cit> .
sequence analysis of sbprr <dig> alleles showed that rio encodes sbprr37- <dig> and btx <dig> contains sbprr37- <dig>  a null allele  <cit> .
the ma <dig> allele from rio  delayed flowering relative to btx <dig> in field conditions in college station in a manner similar to the delayed flowering attributed to the same allele in tx <dig> compared to btx <dig>  which encodes the null allele sbprr37- <dig>
identification of sorghum constans
the hypothesis that the flowering time qtl on sbi- <dig> was caused by alleles of constans/hd <dig> was investigated further through gene sequence alignment and analysis of colinearity.
the amino acid sequence of rice hd <dig> was used to identify homologs in sorghum, maize, barley and arabidopsis using data from phytozome v <dig>   <cit> .
sb10g <dig> , grmzm2g405368_t <dig> , af <dig>  and at5g <dig>  had the highest similarity to hd <dig> in each species.
grmzm2g405368_t <dig> and af <dig> were previously identified as the maize constans-like gene, conz <dig> <cit>  and barley constans-like gene, hvco <dig> <cit> , respectively, while at5g <dig> encodes co in arabidopsis  <cit> .
multiple sequence alignment of the co homologs showed that sb10g <dig> has all of the characteristic protein domains found in constans-like gene families , including an n-terminal b-box <dig> , b-box <dig>  domains and a c-terminal cct domain .
the sorghum homolog of constans  is located on sbi- <dig> and rice hd <dig>  is located on the homeologous rice chromosome  <dig>  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 oshd <dig> were in a region of gene colinearity.
the sorghum sequences flanking sb10g <dig> were downloaded from phytozome and aligned with sequences from rice chromosome  <dig> flanking hd <dig> using gevo  <cit> .
three genes and hd <dig> were aligned and in the same relative order in a  <dig> kbp region in the two chromosomes, consistent with the identification of sb10g <dig> as an ortholog of rice hd <dig> .
therefore, based on sequence similarity and colinearity, sb10g <dig> was designated as an ortholog of rice hd <dig> and a probable ortholog of arabidopsis co and termed “sbco”.
the hypothesis that the flowering time qtl on sbi- <dig> was caused by different alleles of sbco in btx <dig> and tx <dig> was investigated further by comparing the sbco sequences from these genotypes.
the comparison revealed one difference in intron sequence and four differences in the coding region, three of which cause changes in amino acid sequence .
the amino acid change val60ala, occurs in b-box <dig> , a conservative change in amino acid sequence that is expected to be tolerated based on sift analysis  <cit> .
the amino acid change glu318gly occurs outside the b-boxes and cct-domain  and was also predicted to be tolerated based on sift analysis.
while the val60ala and glu318gly changes in protein sequence may not disrupt co function, it is possible that other aspects of co could be modified by these differences.
the his106tyr change in btx <dig> co protein sequence located in b-box <dig>  is predicted to disrupt co function.
in the wild type version of constans, his <dig> is required for zinc coordination and protein activity  <cit> .
the btx <dig> allele of constans was designated sbco- <dig> because the arabidopsis allele co- <dig> has the same his106try substitution that disrupts function  <cit> .
the wild type alleles of co in btx <dig> and tx <dig> had identical co protein sequences except for a ser177asn substitution in tx <dig> , a modification that does not affect the b-boxes or the cct domain, and is predicted by sift to have minimal impact on co function.
based on this analysis, the constans alleles in btx <dig> and tx <dig> were designated as sbco- <dig> and sbco- <dig>  respectively, and the allele in btx <dig> as sbco- <dig>  btx <dig>  flowers later than tx <dig>  in both long and short days.
sbco
alleles from btx <dig>  tx <dig> and btx642
*n/a: not applicable.
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.
ril <dig> and ril <dig> were identified that differ in alleles of sbco but not at the other main loci that affect flowering time.
ril <dig> and ril <dig> are homozygous for btx <dig> alleles for the flowering time qtl on sbi- <dig> , sbi- <dig> , and sbi- <dig>  btx <dig> encodes a null allele of ma <dig> , a gene that contributes to photoperiod sensitivity.
tx <dig> contains a weak allele of ma <dig>  that encodes a full-length protein that inhibits flowering based on qtl analysis  <cit> .
therefore, ril <dig> and ril <dig> were selected for expression studies because both contain dna from btx <dig> on sbi- <dig> from 0- <dig> mbp, ensuring that these genotypes are null for ma <dig> .
in addition, both rils encode a null allele of ma <dig>  located at the proximal end of sbi- <dig>  <cit> .
therefore, comparison of gene expression in ril <dig> and ril <dig> caused by differences in sbco alleles will not be influenced by ma <dig> or ma <dig> the main determinants of photoperiod sensitivity in sorghum  <cit> .
when grown in a ld greenhouse, ril <dig>  flowered in ~75 days, whereas ril <dig>  flowered in ~113 days consistent with the hypothesis that sbco functions as an activator of flowering .
sbco expression in ril <dig>  was analyzed using qrt-pcr during a 24 h ld cycle followed by 24 h of continuous light and temperature .
sbco expression decreased at dawn and remained at low levels during most of the day and then increased to a peak in the evening, approximately 15 h after dawn, followed by a decline and second smaller peak at dawn .
the peaks of sbco expression in the evening and near dawn were previously observed in sorghum  <cit>  and for conz <dig> in maize  <cit> .
the increase in sbco expression in the evening also occurred in continuous light , consistent with prior studies showing that light and the circadian clock modulate this peak of co expression.
the pattern of sbco expression in ril <dig>  was similar to ril <dig>  although with slightly higher  levels of expression .
ril <dig>  and ril <dig>  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 toc <dig>  lhy and gi were similar in ril <dig> and ril <dig>  indicating that these genes are not affected by sbco alleles as expected for genes upstream of sbco .
in arabidopsis co activates flowering by inducing expression of ft and in rice hd <dig> activates hd3a/rft <dig>  genes encoding pebp  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 rft <dig> were identified as encoding florigens  <cit> .
in maize, zcn <dig>  a different member of the pebp gene family, was identified as a source of florigen  <cit> .
in sorghum, sbcn <dig> and sbcn <dig> are potential sources of florigen because expression of both genes is regulated by photoperiod, modulated by ma <dig> alleles, and induction of expression occurs coincident with floral initiation  <cit> .
the sorghum orthologs of maize zcn <dig> , zcn <dig>  and rice hd3a  were identified and qrt-pcr primers specific to each gene were designed to enable analysis of gene expression .
no ortholog of rft <dig> is present in the sorghum genome.
in leaves of ril <dig>  grown in ld, sbehd <dig> expression was high at dawn and then declined during the day before increasing in the evening approximately 15 h after dawn , with a pattern similar to sbehd <dig> expression in 100 m  in short days  <cit> .
during the 24 h ld cycle, sbehd <dig> rna was higher in ril <dig>  compared to ril <dig>  indicating that co activates expression of sbehd <dig> .
the average difference in sbehd <dig> rna level in the two rils during the 24 h ld cycle was 20-fold .
in leaves of ril <dig>  grown in ld, sbcn <dig> and sbcn <dig> mrna levels were highest at dawn, then decreased during the day with a second smaller peak of expression approximately 12-18 h after dawn .
in ril <dig> , the same pattern of expression was observed; however, sbcn <dig> and sbcn <dig> mrna levels were much lower .
expression of sbcn <dig> was ~10-fold higher in ril <dig>  compared to ril  <dig>   and expression of sbcn <dig> was ~100-fold higher in ril <dig>  compared to ril <dig>   over a 24 h ld cycle in the sbco- <dig> background .
in contrast, the mrna level of sbcn <dig>  was similar in the two genotypes , although the gene’s peak of expression was at dawn in ril <dig>  and at 18 h in ril <dig>  .
together, these results are consistent with the hypothesis that sbco promotes flowering by inducing expression of sbehd <dig>  sbcn <dig>  and sbcn <dig>  with sbcn <dig> showing the largest co-mediated increase in expression in ld.
regulation of sbco floral promoting activity in sd and ld
comparison of flowering time and flowering pathway gene expression in ril <dig>  and ril <dig>  showed that sbco activates sbcn8/ <dig> expression and flowering in ld.
the next question addressed was whether photoperiod alters sbco activity in sorghum.
when grown in a sd greenhouse, ril <dig>  flowered in ~55 days, whereas ril <dig>  flowered in ~72 days consistent with the hypothesis that sbco functions as an activator of flowering in short days in sorghum.
a comparison of the relative expression of sbco in sd and ld showed that sbco expression was not altered significantly in response to day-length .
however, differences in the relative ability of sbco to activate expression of sbcn <dig> and sbcn <dig> in sd and ld were observed in comparisons of ril <dig>  and ril <dig>  .
in ril <dig>  sbcn <dig> and sbcn <dig> had higher expression in sd compared to ld, especially during the night when both genes showed their highest expression .
the difference between sbcn <dig> mrna levels in sd and ld varied depending on time of day, with the largest differences occurring during the night, peaking at 18 h .
a similar pattern was observed for sbcn <dig> where expression in sd was 20– <dig> fold higher during the night in sd, peaking between 18-21 h .
when a comparison of sbcn8/ <dig> expression in sd/ld was done using ril <dig> , ~10-fold differences in expression in sd vs. ld were observed during the night .
taken together, these results indicate that co has greater activity in sd compared to ld causing up to 10-fold higher expression of sbcn8/ <dig> during the night in genetic backgrounds that contain null alleles of ma <dig> and ma <dig>
post-transcriptional inhibition of sbco activity by prr <dig>
in sorghum, ma <dig>  increases photoperiod sensitivity by repressing expression of sbehd <dig> and sbcn8/ <dig>  resulting in delayed flowering in ld but with minimal effect in sd  <cit> .
the ability of sbprr <dig> to inhibit expression of sbcn <dig> and sbcn <dig> could be due to inhibition of sbehd <dig> or sbco, activators of sbcn <dig> and sbcn <dig> expression, and/or by direct inhibition of sbcn <dig> and sbcn <dig>  a flowering time qtl coincident with ma <dig> was identified in the btx642/tx <dig> ril population grown under field conditions in  <dig>   <dig> and  <dig>  as well as in lubbock, texas .
this qtl was not observed in sd conditions, as expected, because sbprr <dig> has minimal impact on flowering under these conditions.
as noted above, btx <dig> encodes a null allele sbprr37- <dig>  however, the ma <dig> allele in tx <dig>  sbprr37- <dig>  encodes a full-length protein with one amino acid substitution lys62asn with sufficient activity to delay flowering time under field conditions.
if sbprr <dig> delays flowering by inhibiting sbco, and sbco increases expression of sbehd <dig> and sbcn8/ <dig>  then epistatic interaction between sbprr <dig> and sbco may be detected in the ril population.
sbprr <dig> and sbco allelic interactions were examined by first sorting the rils into lines that contain sbprr37- <dig>  or sbprr37- <dig>  and then analyzing the influence of sbco and sbehd <dig> alleles on flowering time in each background.
in the portion of the population containing the null version of sbprr37- <dig>  the qtl corresponding to sbco/sbco- <dig>  explained 48% of the phenotypic variance for flowering time in the field .
in contrast, in the portion of the ril population containing the active allele of ma <dig> , no qtl corresponding to sbco was observed.
in this portion of the ril population , the qtl corresponding to sbehd1-1/sbehd1- <dig> explained ~20% of the phenotypic variance .
this result indicates that sbprr37- <dig> inhibits sbco-mediated induction of flowering.
if this hypothesis is correct, then the ability of sbprr37- <dig> to inhibit flowering could be dependent on an active allele of sbco.
to test this hypothesis, the ril population was sorted into lines that contained sbco- <dig> and lines that contained sbco- <dig>  and flowering time qtl were identified in each background .
this analysis showed that sbprr <dig> alleles affected flowering time in the sbco- <dig> background but not in the genetic background containing sbco- <dig> alleles, indicating that the ability of sbprr <dig> to inhibit flowering is dependent on sbco.
discussion
sorghum accessions exhibit a wide range of flowering times when plants are grown in long days   <cit> .
a large extent of this variation is caused by differences in photoperiod sensitivity mediated by floral repressors encoded by ma <dig> and ma <dig> that inhibit flowering in long days  <cit> .
much less is known about floral activators in sorghum.
the grass specific floral activator sbehd <dig> was previously identified based on the gene’s sequence similarity to rice ehd <dig> and activation of sbehd <dig> expression coincident with floral initiation  <cit> .
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 hd <dig> in rice.
coding alleles of constans were identified through analysis of a flowering time qtl on sbi- <dig>  results showed that sbco functions as an activator of flowering in ld and sd in sorghum genotypes using rils with null versions of sbprr37- <dig> and sbghd7- <dig>  the sbco- <dig> allele in btx <dig> was remarkable because it contained a his106tyr amino acid substitution that also inactivates co function in arabidopsis  <cit> .
sorghum and arabidopsis genotypes containing the inactive his106tyr co- <dig> 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  domain with toc <dig>  prr <dig>  ghd <dig>  and heme activator proteins .
yeast two-hybrid screens showed that co can interact with hap <dig> and hap <dig> subunits through its cct-domain, forming ccaat-binding cbf-complexes that bind to ft promoters and activate transcription  <cit> .
in sorghum, sbco was found to activate transcription of sbehd <dig>  sbcn <dig> and sbcn <dig>  consistent with its role as an activator of flowering, presumably through formation of cbf-complexes, but possibly through direct binding to dna  <cit> .
the ability of sbco alleles to induce flowering pathway gene expression and flowering was examined in ril genetic backgrounds that contained null alleles of ma <dig>  and ma <dig>  to eliminate the influence of these ld floral repressors.
in this null genetic background, sbco promoted early flowering in ld and sd and increased the expression of sbehd <dig> , sbcn <dig> , sbcn <dig>  and sbcn <dig>  relative to their expression in lines carrying the inactive sbco- <dig> allele.
this information is summarized in a flowering time regulatory model shown in figure  <dig>  the model includes three members of the pebp-gene family that could be sources of florigen in sorghum, sbcn <dig>  sbcn <dig> and sbcn <dig>  sbcn <dig> is an ortholog of maize zcn <dig>  with a pattern of gene expression consistent with the demonstrated role of zcn <dig> as a source of florigen in maize  <cit> .
sbcn <dig> expression is repressed by prr <dig>  induced in leaves in sd, and induced by sbco , indicating that this gene is also a likely source of florigen in sorghum.
in rice, hd3a and rft <dig> have been identified as sources of florigen; therefore expression of sbcn <dig>  the ortholog of hd3a, was analyzed.
sbcn <dig> showed relatively small changes in gene expression in response to photoperiod and mutations in sbprr <dig> and sbco.
the sorghum genome does not encode an ortholog of rft <dig>  a source of florigen in rice in ld.
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  and rice , therefore regulation of flowering time could also differ, even though both grass species are short day plants.
sbco activates expression of sbcn <dig> and sbcn <dig>  although sbcn <dig> was induced to a significantly greater extent.
sbco also increased expression of sbehd <dig>  an activator of hd3a expression in rice.
sbehd <dig> expression is repressed by sbprr <dig> and sbghd <dig> and induced when photoperiod sensitive sorghum grown in ld is transferred to sd  <cit> .
increases in sbehd <dig> expression occur in parallel with increases in sbcn <dig> and sbcn <dig> expression, suggesting that sbehd <dig> can induce the expression of these genes as shown in figure  <dig>  however, the extent and specificity of this proposed activity of sbehd <dig> will require further analysis in backgrounds where sbco has minimal influence on the expression of these genes .
the results in this paper show that sbco increases expression of sbehd <dig> and it is proposed that sbehd <dig> can activate expression of sbcn <dig> and sbcn <dig> and flowering.
in contrast, rice hd <dig> has not been reported to increase expression of ehd <dig> <cit> .
of interest is the finding that sbco activates expression of sbcn <dig> to a much greater extent than sbehd <dig>  therefore we conclude that sbco directly increases sbcn <dig> 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 ma <dig> and ma <dig> 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 .
in arabidopsis, a similar increase in co expression in the evening is due to the interaction of gi and blue light-activated fkf <dig>  resulting in degradation of cdf-factors that inhibit co expression  <cit> .
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 sbprr <dig> and enhanced in ld  <cit> .
the function of the peak of sbco expression at dawn is not currently understood, although production of sbco at this time could help activate sbehd <dig> expression in the morning.
functional alleles of sbco increased the expression of sbcn <dig> and sbcn <dig> to a greater extent in sd relative to ld .
sbco expression levels increase during the evening, helping to explain why sbcn8/ <dig> expression increases at night.
since expression of sbco was not altered significantly by photoperiod , 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-cry <dig> stabilization of the protein  <cit> .
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/ <dig> in sd.
in sorghum genotypes containing active alleles of sbprr <dig> 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 sbprr <dig> is high in the evening in plants grown in ld, but low in sd.
therefore, higher levels of sbprr <dig> expression in ld, and sbprr <dig> repression of sbco activity under these conditions, is predicted to prevent sbco from activating flowering in ld.
sbprr <dig> is a cct-domain protein that has been shown to interact with hap <dig>  the same cbf-subunit that interacts with sbco  <cit> .
therefore, sbprr <dig> may be a competitive inhibitor of sbco binding to the hap complex.
sbprr <dig> may also directly bind to dna in a fashion similar to toc <dig> and other prr-proteins  <cit> .
toc <dig> binding to its cognate motif in the promoter of lhy/cca <dig> is mediated by its cct-domain, resulting in prr-domain mediated repression of transcription.
if prr <dig> binds to the sbcn <dig> 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 sbcn <dig> transcription.
recent results on the prr <dig> ortholog in rice  indicated that prr <dig> directly represses hd3a transcription  <cit> .
further genetic and biochemical analysis will be required to distinguish among these possibilities.
in barley, a long day plant, hvco <dig> activates flowering in ld, and activation is dependent on ppd-h <dig>  an ortholog of sbprr <dig>  overexpression of hvco <dig> induced flowering in both ld and sd, but photoperiod sensitivity mediated by ppd-h <dig> was still observed in this background  <cit> .
ppd-h <dig> does not directly affect expression of hvco <dig>  but potentiates the ability of hvco <dig> to activate hvft <dig> expression in ld.
it is interesting to note that ppd-h <dig> increases hvco <dig> activity in ld, whereas sbprr <dig> inhibits the floral promoting activity of sbco in ld.
the expression and activity of sbprr <dig> and ppd-h <dig> 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 prr <dig> could be due to differences in direct binding of prr <dig> to the promoters of sbcn <dig> and hvft <dig> , or indirectly by interaction of prr37/ppd-h <dig> with activators, repressors, hap subunits, hvco <dig> and sbco.
in rice, it has been suggested that phosphorylation of prr <dig> by hd <dig> may cause prr <dig>  in conjunction with hd <dig>  to become a repressor of hd3a expression in ld  <cit> .
the possibility that prr <dig> can form a co-repression complex with co is consistent with results in sorghum and rice.
however in the absence of prr <dig>  co functions as an activator of ft expression in sorghum and rice.
while the biochemical basis of variation in prr <dig> activity remains to be elucidated, taken together, the results suggest that interaction between prr <dig> 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  <dig> shows that sbehd <dig> and sbco can independently induce flowering by activating sbcn <dig> and/or sbcn <dig>  ehd <dig> and hd <dig> have been shown to independently activate hd3a  and flowering in rice  <cit> .
in sorghum we show that there is cross-talk between these pathways because sbprr <dig> activates sbco expression at dawn in ld, while sbco induces sbehd <dig> expression in sd.
rice ehd <dig>  a b-type response regulator, is controlled by several upstream modulators including the repressors ghd <dig>  grain number, plant height and heading date  <dig> , oslec <dig> and fusca-like <dig> , osmads <dig> and the activators gi, early heading date  <dig>  and osmads <dig>  <cit> .
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.
CONCLUSIONS
alleles of sorghum constans  were identified through analysis of a flowering time qtl on chromosome  <dig> identified in a ril population derived from btx <dig> and tx <dig>  genetic analysis and gene expression studies indicate that sbco is an activator of flowering in long and short days in genotypes lacking active sbprr <dig> and ghd <dig> alleles.
prr <dig> was found to block co-mediated floral activation in long days.

 optimal regulation of the timing of floral transition is critically important for reproductive success and crop yield. the c grass sorghum bicolor is widely adapted and grown as an annual crop from to > 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. grain sorghum is generally selected for early flowering 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. 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. in arabidopsis, flowering is promoted in long-days 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 is regulated by the central clock oscillator comprised of timing of cab expression, circadian clock associated and late elongated hypocotyl. in long days, gi activates constans expression in conjunction with flavin-binding kelch domain f box protein by inducing degradation of cdf repressors of constans transcription. co accumulates in ld due to stabilization mediated by cryptochromes, phytochrome a and suppressor of phya- that counteract degradation of co mediated by phytochrome b: constitutive photomorphogenic. increased co protein levels in long days leads to the activation of flowering locus t expression and production of florigen that moves from leaves to shoot apical meristems where it binds to fd and induces floral transition. the gi-co-ft regulatory pathway identified in arabidopsis, a long day plant, is also present in rice, a short day plant. when rice is exposed to inductive sd, heading date, the ortholog of co, activates expression of the ft-like gene hd3a, one of two sources of florigen in rice. in non-inductive ld, hd functions as a repressor of hd3a and flowering. thus, photoperiod sensitivity in rice depends in part on differences in the activity of co in long days and short days. two modulators of flowering time unique to grasses were identified in rice: early heading date and grain number, plant height and heading date. ehd activates the expression of hd3a and rice flowering locus t, a source of florigen in long days. ghd represses flowering by down-regulating expression of ehd and hd3a in ld in rice and sbehd and sbcn 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 qtl for flowering time have been identified in sorghum. the ma1-ma loci were discovered while breeding for early flowering photoperiod insensitive grain sorghum in the u.s.. ma corresponds to pseudoresponse regulator protein, a repressor of flowering in ld. ma encodes phytochrome b, a red-light photoreceptor that plays an important role in photoperiod sensing and repression of flowering. ma encodes sbghd a repressor of sbehd expression and flowering in long days. ma ma and ma are flowering time loci that enhance photoperiod sensitivity in sorghum. constans 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 ~ co family members in arabidopsis, ~ in rice and ~ in barley. the ortholog of constans in rice, hd plays a key role in photoperiod regulation of flowering, by activating flowering in sd and repressing flowering in ld. alleles of hd account for ~44% of the variation in flowering time observed in cultivated rice. hd transcript and protein levels are similar in ld and sd, consistent with the finding that hd activity is modulated post-transcriptionally by phyb and prr.  identification of flowering time qtl flowering time qtl were mapped in a ril population derived from a cross of btx and tx genotypes used in u.s. grain sorghum breeding programs as sources of drought tolerance. a ril population derived from these genotypes was previously used to map qtl for flowering time and the stay-green drought tolerance trait using a genetic map based on rflp markers. the genomes of btx and tx were recently sequenced and analyzed for variation in dna polymorphisms that distinguish these genotypes. digital genotyping was used to create a high-resolution genetic map aligned to the genome sequence based on this ril population. digital genotyping identified snp markers segregating in the ril population and data on recombination frequency was used to create a 1139 cm genetic map spanning the sorghum chromosomes. flowering time qtl were mapped in this population by phenotyping the ril population for days to half pollen shed in greenhouses in 14 h long days, 10 h short days, and under field conditions where day length increases following plant emergence in mid-april from  h to  h in july. tx flowered in 73 days and btx flowered approximately 4 days later under field conditions in college station, texas. when grown in a greenhouse at constant 14 h day lengths during the summer, tx flowered in 84 days and btx flowered ~19 days later. when tx and btx were grown in a greenhouse under 10 h day lengths during the winter, tx flowered in 54 days whereas btx flowered ~11 days later. the btx642/tx ril population was grown and assayed for days to flowering under field conditions in 2008– in ld greenhouses in and and in a sd greenhouse during the winter of winqtl cartographer was used to identify flowering time qtl using flowering time data collected from each location/year and the genetic map generated by evans et al.. three qtl for flowering time were observed in every environment and two additional qtl were identified in only one environment. a b c 2d e a position of likelihood peak. b peak coordinate: physical coordinate of the likelihood peak. c additive effect: a positive value means the delay of flowering time due to tx allele. a negative value means the delay of flowering time due to btx allele. d r: percentage of phenotypic variance explained by the qtl. e nd: candidate gene is not determined. three flowering time qtl were identified when rils were screened in ld greenhouse conditions. the qtl on sbi- explained % of the phenotypic variance for flowering time in this environment. sbehd an activator of flowering in grasses located on sbi- was found in a one lod interval spanning this qtl. sbehd was previously identified as a floral activator in sorghum based on sequence similarity to rice ehd and observed changes of sbehd expression in ld compared to sd, consistent with this function. there were no amino acid differences between the sbehd protein sequences from tx and btx btx is a grain sorghum used extensively for breeding, genetic, and genomic research. however, comparison of sbehd from btx and tx revealed two amino acid substitutions, asp144asn and thr157ile. the differences in ehd protein sequences occur in a garp domain that is highly conserved among osehd sbehd and arabidopsis response regulator 1/. the sbehd allele in btx delays flowering in ld and sd relative to tx, consistent with the hypothesis that the amino acid changes in sbehd1- reduce the activity of this floral activator and explaining why a flowering time qtl was detected in this region of sbi- a flowering time qtl located on sbi- was observed in all environments and spanned a region that encodes a homolog of constans and hd, an important regulator of flowering time in arabidopsis and rice, respectively. 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. a flowering time qtl located on sbi- 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- was observed only when the btx642/tx ril population was grown in the sd greenhouse. a qtl on sbi- explaining ~15% of the variance in flowering time was identified when rils were grown in the field. sbprr, a repressor of flowering in ld, was located in the flowering time qtl on sbi- sequence analysis showed that btx encodes sbprr37- and a truncated version of prr and that tx contains sbprr37- encoding a full-length version of prr containing a lys162asn change in the pseudo-response regulator domain. genotypes containing sbprr37- flowered later than genotypes encoding sbprr37- under field conditions, indicating that sbprr37- is an active but weak allele of sbprr this conclusion is consistent with analysis of a flowering time qtl aligned to prr identified in a ril population derived from crossing the genotypes rio and btx. sequence analysis of sbprr alleles showed that rio encodes sbprr37- and btx contains sbprr37- a null allele. the ma allele from rio delayed flowering relative to btx in field conditions in college station in a manner similar to the delayed flowering attributed to the same allele in tx compared to btx which encodes the null allele sbprr37- identification of sorghum constans the hypothesis that the flowering time qtl on sbi- was caused by alleles of constans/hd was investigated further through gene sequence alignment and analysis of colinearity. the amino acid sequence of rice hd was used to identify homologs in sorghum, maize, barley and arabidopsis using data from phytozome v. sb10g, grmzm2g405368_t, af and at5g had the highest similarity to hd in each species. grmzm2g405368_t and af were previously identified as the maize constans-like gene, conz and barley constans-like gene, hvco, respectively, while at5g encodes co in arabidopsis. multiple sequence alignment of the co homologs showed that sb10g has all of the characteristic protein domains found in constans-like gene families, including an n-terminal b-box, b-box domains and a c-terminal cct domain. the sorghum homolog of constans is located on sbi- and rice hd is located on the homeologous rice chromosome 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 oshd were in a region of gene colinearity. the sorghum sequences flanking sb10g were downloaded from phytozome and aligned with sequences from rice chromosome flanking hd using gevo. three genes and hd were aligned and in the same relative order in a kbp region in the two chromosomes, consistent with the identification of sb10g as an ortholog of rice hd. therefore, based on sequence similarity and colinearity, sb10g was designated as an ortholog of rice hd and a probable ortholog of arabidopsis co and termed “sbco”. the hypothesis that the flowering time qtl on sbi- was caused by different alleles of sbco in btx and tx was investigated further by comparing the sbco sequences from these genotypes. the comparison revealed one difference in intron sequence and four differences in the coding region, three of which cause changes in amino acid sequence. the amino acid change val60ala, occurs in b-box, a conservative change in amino acid sequence that is expected to be tolerated based on sift analysis. the amino acid change glu318gly occurs outside the b-boxes and cct-domain and was also predicted to be tolerated based on sift analysis. while the val60ala and glu318gly changes in protein sequence may not disrupt co function, it is possible that other aspects of co could be modified by these differences. the his106tyr change in btx co protein sequence located in b-box is predicted to disrupt co function. in the wild type version of constans, his is required for zinc coordination and protein activity. the btx allele of constans was designated sbco- because the arabidopsis allele co- has the same his106try substitution that disrupts function. the wild type alleles of co in btx and tx had identical co protein sequences except for a ser177asn substitution in tx, a modification that does not affect the b-boxes or the cct domain, and is predicted by sift to have minimal impact on co function. based on this analysis, the constans alleles in btx and tx were designated as sbco- and sbco- respectively, and the allele in btx as sbco- btx flowers later than tx in both long and short days. sbco alleles from btx tx and btx642 *n/a: not applicable. 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. ril and ril were identified that differ in alleles of sbco but not at the other main loci that affect flowering time. ril and ril are homozygous for btx alleles for the flowering time qtl on sbi-, sbi-, and sbi- btx encodes a null allele of ma, a gene that contributes to photoperiod sensitivity. tx contains a weak allele of ma that encodes a full-length protein that inhibits flowering based on qtl analysis. therefore, ril and ril were selected for expression studies because both contain dna from btx on sbi- from 0- mbp, ensuring that these genotypes are null for ma. in addition, both rils encode a null allele of ma located at the proximal end of sbi-. therefore, comparison of gene expression in ril and ril caused by differences in sbco alleles will not be influenced by ma or ma the main determinants of photoperiod sensitivity in sorghum. when grown in a ld greenhouse, ril flowered in ~75 days, whereas ril flowered in ~113 days consistent with the hypothesis that sbco functions as an activator of flowering. sbco expression in ril was analyzed using qrt-pcr during a 24 h ld cycle followed by 24 h of continuous light and temperature. sbco expression decreased at dawn and remained at low levels during most of the day and then increased to a peak in the evening, approximately 15 h after dawn, followed by a decline and second smaller peak at dawn. the peaks of sbco expression in the evening and near dawn were previously observed in sorghum and for conz in maize. the increase in sbco expression in the evening also occurred in continuous light, consistent with prior studies showing that light and the circadian clock modulate this peak of co expression. the pattern of sbco expression in ril was similar to ril although with slightly higher levels of expression. ril and ril 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 toc lhy and gi were similar in ril and ril indicating that these genes are not affected by sbco alleles as expected for genes upstream of sbco. in arabidopsis co activates flowering by inducing expression of ft and in rice hd activates hd3a/rft genes encoding pebp 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 rft were identified as encoding florigens. in maize, zcn a different member of the pebp gene family, was identified as a source of florigen. in sorghum, sbcn and sbcn are potential sources of florigen because expression of both genes is regulated by photoperiod, modulated by ma alleles, and induction of expression occurs coincident with floral initiation. the sorghum orthologs of maize zcn, zcn and rice hd3a were identified and qrt-pcr primers specific to each gene were designed to enable analysis of gene expression. no ortholog of rft is present in the sorghum genome. in leaves of ril grown in ld, sbehd expression was high at dawn and then declined during the day before increasing in the evening approximately 15 h after dawn, with a pattern similar to sbehd expression in 100 m in short days. during the 24 h ld cycle, sbehd rna was higher in ril compared to ril indicating that co activates expression of sbehd. the average difference in sbehd rna level in the two rils during the 24 h ld cycle was 20-fold. in leaves of ril grown in ld, sbcn and sbcn mrna levels were highest at dawn, then decreased during the day with a second smaller peak of expression approximately 12-18 h after dawn. in ril, the same pattern of expression was observed; however, sbcn and sbcn mrna levels were much lower. expression of sbcn was ~10-fold higher in ril compared to ril and expression of sbcn was ~100-fold higher in ril compared to ril over a 24 h ld cycle in the sbco- background. in contrast, the mrna level of sbcn was similar in the two genotypes, although the gene’s peak of expression was at dawn in ril and at 18 h in ril. together, these results are consistent with the hypothesis that sbco promotes flowering by inducing expression of sbehd sbcn and sbcn with sbcn showing the largest co-mediated increase in expression in ld. regulation of sbco floral promoting activity in sd and ld comparison of flowering time and flowering pathway gene expression in ril and ril showed that sbco activates sbcn8/ expression and flowering in ld. the next question addressed was whether photoperiod alters sbco activity in sorghum. when grown in a sd greenhouse, ril flowered in ~55 days, whereas ril flowered in ~72 days consistent with the hypothesis that sbco functions as an activator of flowering in short days in sorghum. a comparison of the relative expression of sbco in sd and ld showed that sbco expression was not altered significantly in response to day-length. however, differences in the relative ability of sbco to activate expression of sbcn and sbcn in sd and ld were observed in comparisons of ril and ril. in ril sbcn and sbcn had higher expression in sd compared to ld, especially during the night when both genes showed their highest expression. the difference between sbcn mrna levels in sd and ld varied depending on time of day, with the largest differences occurring during the night, peaking at 18 h. a similar pattern was observed for sbcn where expression in sd was 20– fold higher during the night in sd, peaking between 18-21 h. when a comparison of sbcn8/ expression in sd/ld was done using ril, ~10-fold differences in expression in sd vs. ld were observed during the night. taken together, these results indicate that co has greater activity in sd compared to ld causing up to 10-fold higher expression of sbcn8/ during the night in genetic backgrounds that contain null alleles of ma and ma post-transcriptional inhibition of sbco activity by prr in sorghum, ma increases photoperiod sensitivity by repressing expression of sbehd and sbcn8/ resulting in delayed flowering in ld but with minimal effect in sd. the ability of sbprr to inhibit expression of sbcn and sbcn could be due to inhibition of sbehd or sbco, activators of sbcn and sbcn expression, and/or by direct inhibition of sbcn and sbcn a flowering time qtl coincident with ma was identified in the btx642/tx ril population grown under field conditions in and as well as in lubbock, texas. this qtl was not observed in sd conditions, as expected, because sbprr has minimal impact on flowering under these conditions. as noted above, btx encodes a null allele sbprr37- however, the ma allele in tx sbprr37- encodes a full-length protein with one amino acid substitution lys62asn with sufficient activity to delay flowering time under field conditions. if sbprr delays flowering by inhibiting sbco, and sbco increases expression of sbehd and sbcn8/ then epistatic interaction between sbprr and sbco may be detected in the ril population. sbprr and sbco allelic interactions were examined by first sorting the rils into lines that contain sbprr37- or sbprr37- and then analyzing the influence of sbco and sbehd alleles on flowering time in each background. in the portion of the population containing the null version of sbprr37- the qtl corresponding to sbco/sbco- explained 48% of the phenotypic variance for flowering time in the field. in contrast, in the portion of the ril population containing the active allele of ma, no qtl corresponding to sbco was observed. in this portion of the ril population, the qtl corresponding to sbehd1-1/sbehd1- explained ~20% of the phenotypic variance. this result indicates that sbprr37- inhibits sbco-mediated induction of flowering. if this hypothesis is correct, then the ability of sbprr37- to inhibit flowering could be dependent on an active allele of sbco. to test this hypothesis, the ril population was sorted into lines that contained sbco- and lines that contained sbco- and flowering time qtl were identified in each background. this analysis showed that sbprr alleles affected flowering time in the sbco- background but not in the genetic background containing sbco- alleles, indicating that the ability of sbprr to inhibit flowering is dependent on sbco. discussion sorghum accessions exhibit a wide range of flowering times when plants are grown in long days. a large extent of this variation is caused by differences in photoperiod sensitivity mediated by floral repressors encoded by ma and ma that inhibit flowering in long days. much less is known about floral activators in sorghum. the grass specific floral activator sbehd was previously identified based on the gene’s sequence similarity to rice ehd and activation of sbehd 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 hd in rice. coding alleles of constans were identified through analysis of a flowering time qtl on sbi- results showed that sbco functions as an activator of flowering in ld and sd in sorghum genotypes using rils with null versions of sbprr37- and sbghd7- the sbco- allele in btx 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- 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 domain with toc prr ghd and heme activator proteins. yeast two-hybrid screens showed that co can interact with hap and hap subunits through its cct-domain, forming ccaat-binding cbf-complexes that bind to ft promoters and activate transcription. in sorghum, sbco was found to activate transcription of sbehd sbcn and sbcn consistent with its role as an activator of flowering, presumably through formation of cbf-complexes, but possibly through direct binding to dna. the ability of sbco alleles to induce flowering pathway gene expression and flowering was examined in ril genetic backgrounds that contained null alleles of ma and ma to eliminate the influence of these ld floral repressors. in this null genetic background, sbco promoted early flowering in ld and sd and increased the expression of sbehd, sbcn, sbcn and sbcn relative to their expression in lines carrying the inactive sbco- allele. this information is summarized in a flowering time regulatory model shown in figure  the model includes three members of the pebp-gene family that could be sources of florigen in sorghum, sbcn sbcn and sbcn sbcn is an ortholog of maize zcn with a pattern of gene expression consistent with the demonstrated role of zcn as a source of florigen in maize. sbcn expression is repressed by prr induced in leaves in sd, and induced by sbco, indicating that this gene is also a likely source of florigen in sorghum. in rice, hd3a and rft have been identified as sources of florigen; therefore expression of sbcn the ortholog of hd3a, was analyzed. sbcn showed relatively small changes in gene expression in response to photoperiod and mutations in sbprr and sbco. the sorghum genome does not encode an ortholog of rft a source of florigen in rice in ld. 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 and rice, therefore regulation of flowering time could also differ, even though both grass species are short day plants. sbco activates expression of sbcn and sbcn although sbcn was induced to a significantly greater extent. sbco also increased expression of sbehd an activator of hd3a expression in rice. sbehd expression is repressed by sbprr and sbghd and induced when photoperiod sensitive sorghum grown in ld is transferred to sd. increases in sbehd expression occur in parallel with increases in sbcn and sbcn expression, suggesting that sbehd can induce the expression of these genes as shown in figure  however, the extent and specificity of this proposed activity of sbehd will require further analysis in backgrounds where sbco has minimal influence on the expression of these genes. the results in this paper show that sbco increases expression of sbehd and it is proposed that sbehd can activate expression of sbcn and sbcn and flowering. in contrast, rice hd has not been reported to increase expression of ehd. of interest is the finding that sbco activates expression of sbcn to a much greater extent than sbehd therefore we conclude that sbco directly increases sbcn 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 ma and ma 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. in arabidopsis, a similar increase in co expression in the evening is due to the interaction of gi and blue light-activated fkf 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 sbprr 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 sbehd expression in the morning. functional alleles of sbco increased the expression of sbcn and sbcn to a greater extent in sd relative to ld. sbco expression levels increase during the evening, helping to explain why sbcn8/ expression increases at night. since expression of sbco was not altered significantly by photoperiod, 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-cry 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/ in sd. in sorghum genotypes containing active alleles of sbprr 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 sbprr is high in the evening in plants grown in ld, but low in sd. therefore, higher levels of sbprr expression in ld, and sbprr repression of sbco activity under these conditions, is predicted to prevent sbco from activating flowering in ld. sbprr is a cct-domain protein that has been shown to interact with hap the same cbf-subunit that interacts with sbco. therefore, sbprr may be a competitive inhibitor of sbco binding to the hap complex. sbprr may also directly bind to dna in a fashion similar to toc and other prr-proteins. toc binding to its cognate motif in the promoter of lhy/cca is mediated by its cct-domain, resulting in prr-domain mediated repression of transcription. if prr binds to the sbcn 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 sbcn transcription. recent results on the prr ortholog in rice indicated that prr directly represses hd3a transcription. further genetic and biochemical analysis will be required to distinguish among these possibilities. in barley, a long day plant, hvco activates flowering in ld, and activation is dependent on ppd-h an ortholog of sbprr overexpression of hvco induced flowering in both ld and sd, but photoperiod sensitivity mediated by ppd-h was still observed in this background. ppd-h does not directly affect expression of hvco but potentiates the ability of hvco to activate hvft expression in ld. it is interesting to note that ppd-h increases hvco activity in ld, whereas sbprr inhibits the floral promoting activity of sbco in ld. the expression and activity of sbprr and ppd-h 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 prr could be due to differences in direct binding of prr to the promoters of sbcn and hvft, or indirectly by interaction of prr37/ppd-h with activators, repressors, hap subunits, hvco and sbco. in rice, it has been suggested that phosphorylation of prr by hd may cause prr in conjunction with hd to become a repressor of hd3a expression in ld. the possibility that prr can form a co-repression complex with co is consistent with results in sorghum and rice. however in the absence of prr co functions as an activator of ft expression in sorghum and rice. while the biochemical basis of variation in prr activity remains to be elucidated, taken together, the results suggest that interaction between prr 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  shows that sbehd and sbco can independently induce flowering by activating sbcn and/or sbcn ehd and hd have been shown to independently activate hd3a and flowering in rice. in sorghum we show that there is cross-talk between these pathways because sbprr activates sbco expression at dawn in ld, while sbco induces sbehd expression in sd. rice ehd a b-type response regulator, is controlled by several upstream modulators including the repressors ghd grain number, plant height and heading date, oslec and fusca-like, osmads and the activators gi, early heading date and osmads. 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 were identified through analysis of a flowering time qtl on chromosome identified in a ril population derived from btx and tx genetic analysis and gene expression studies indicate that sbco is an activator of flowering in long and short days in genotypes lacking active sbprr and ghd alleles. prr was found to block co-mediated floral activation in long days. 
sorghum genotypes used for grain production in temperate regions are photoperiod insensitive and flower early avoiding adverse environments during the reproductive phase. quantitative trait loci that modify flowering time in sorghum were identified by screening recombinant inbred lines derived from btx and tx in long days, short days, and under field conditions. sbprr and sbghd contribute to photoperiod sensitivity in sorghum by repressing expression of sbehd and ft-like genes, thereby delaying flowering in long days with minimal influence in short days. genetic analysis characterized sorghum constans as a floral activator that promotes flowering by inducing the expression of early heading date and sorghum orthologs of the maize ft genes zcn and zcn. the floral repressor pseudoresponse regulator protein 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 prr contributing to photoperiod sensitive flowering in sorghum bicolor, a short day plant. in rice, a short day flowering plant, hd the ortholog of constans, activates flowering in short days and represses flowering in long days. in contrast, energy sorghum hybrids are highly photoperiod sensitive with extended vegetative phases in long days, resulting in enhanced biomass accumulation. in long day flowering plants, such as arabidopsis and barley, constans activates ft expression and flowering in long days. analysis of the flowering time qtl on sbi- revealed that btx encodes a recessive constans allele containing a his106tyr substitution in b-box known to inactivate constans in arabidopsis thaliana.
 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. sbprr and sbghd contribute to photoperiod sensitivity in sorghum by repressing expression of sbehd and ft-like genes, thereby delaying flowering in long days with minimal influence in short days. the gigantea -constans -flowering locus t pathway regulates flowering time in arabidopsis and the grasses. 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, hd the ortholog of constans, activates flowering in short days and represses flowering in long days.  quantitative trait loci that modify flowering time in sorghum were identified by screening recombinant inbred lines derived from btx and tx in long days, short days, and under field conditions. analysis of the flowering time qtl on sbi- revealed that btx encodes a recessive constans allele containing a his106tyr substitution in b-box 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 and sorghum orthologs of the maize ft genes zcn and zcn. the floral repressor pseudoresponse regulator protein 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 prr contributing to photoperiod sensitive flowering in sorghum bicolor, a short day plant. photoperiodsorghumflowering timeqtlconstansprr37 