The Pseudo-Response Regulator Ppd-H1 Provides Adaptation to Photoperiod in Barley

Plants have evolved sophisticated controls to ensure that flowering occurs when there is the greatest chance of pollination, seed development, and seed dispersal. Usually this involves restricting flowering to a specific time of year. To achieve this, many plants use photoperiod as an environmental cue to regulate development. The timing of flowering has important impacts on crop yield, and the modification of responses to environmental cues by human selection has been central to the success and spread of agriculture.

The control of flowering by photoperiod is understood best in the long-day (LD) dicot Arabidopsis and the short-day (SD) monocot cereal rice. In Arabidopsis, expression of GIGANTEA (GI) and CONSTANS (CO) is regulated by the circadian clock such that coincidence of the CO expression peak with light only occurs in LD conditions. Light-stabilized CO protein is a transcription factor inducing downstream genes, including FLOWERING LOCUS T (FT) (1, 2).

In rice, analyses of natural variation showed that Heading date1 (Hd1), a major determinant of photoperiod response, is an ortholog of CO (3), that Hd3a is an ortholog of FT (4), and that GI is also conserved (5). However, the interaction of Hd1 with FT is altered such that FT expression is inhibited in LDs (2, 5). The rice Ehd1 gene also controls photoperiod response but has no direct counterpart in Arabidopsis and regulates FT independently of Hd1 (6). Photoperiod response in rice therefore has conserved and novel aspects compared with Arabidopsis, but in both species increased FT expression is crucial to the induction of flowering. Genes controlling photoperiod response in temperate cereals such as barley (Hordeum vulgare) have not been identified previously.

Barley varieties can be broadly classified as winter or spring types. Winter (fall-sown) barleys require vernalization and usually show strong promotion of flowering in response to LDs. This is typical of H. spontaneum, the wild progenitor of barley, suggesting that this is the ancestral condition. Spring (spring-sown) barleys lack vernalization requirement and show weak or strong response to LDs depending on whether they have been selected for long or short growing seasons, respectively. In long growing seasons, as in Western Europe and much of North America, reduced response to photoperiod allows spring-sown plants to extend the period of vegetative growth and accumulate additional biomass that supports higher yields.

The major determinant of LD response in barley is the Photoperiod-H1 (Ppd-H1) locus (7, 8). The late-flowering ppd-H1 allele is recessive (Fig. 1A), suggesting that reduced response results from a mutation that impairs gene function. Ppd-H1 does not correspond to either of the barley CO-like genes (HvCO1 and HvCO2) (9), showing that different major determinants of photoperiod adaptation have been selected in barley and rice.

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We identified Ppd-H1 by positional cloning, using colinearity of the barley Ppd-H1 region with rice and Brachypodium (10). Fine-scale mapping using lines derived from an Igri (Ppd-H1) and Triumph (ppd-H1) cross (Fig. 1, B and C) enabled a physical map of the Ppd-H1 region to be developed (Fig. 1D). Recombinants defined a region containing a single gene that was a pseudo-response regulator (PRR) most similar overall to Arabidopsis PRR7 (fig. S1). PRR proteins are characterized by two conserved regions, a pseudoreceiver domain with similarities to bacterial two-component signaling systems and a CO, CO-like, and TOC1 (CCT) domain that is also found in the CO family (11). The barley PRR gene was amplified by polymerase chain reaction (PCR) from Igri and two H. spontaneum accessions (JIC-1894 and JIC-1947) crossed with Igri and shown to have the Ppd-H1 allele. Morex, which provided the bacteria artificial chromosome (BAC) sequence, was crossed with Igri and shown to have the ppd-H1 allele. Other ppd-H1 lines sequenced were Triumph, Golden Promise, and Optic. This revealed 23 polymorphisms, of which 7 were single nucleotide polymorphisms (SNPs) that produced amino acid changes distinguishing Ppd-H1 and ppd-H1 alleles (1, 12, 15, 20, 21, 22, and 23 in Fig. 1E). Regions containing these SNPs were sequenced from a further eight H. spontaneum accessions known to be early flowering in LDs and nine barley varieties previously classified as early or late flowering in LDs (table S3). In the extended set, four SNPs (1, 15, 22, and 23) remained completely associated with Ppd-H1 or ppd-H1 alleles (Fig. 2). Three were in regions of low conservation with rice and Arabidopsis (fig. S1), but the fourth produced a Gly-to-Trp change in the CCT domain affecting a residue that is conserved in all CCT domain genes identified to date (fig. S2) and that is the most likely causal basis of the ppd-H1 mutation. The CCT domain mutation was a G-to-T change, which removed a BstUI restriction site, providing a simple PCR-based assay for the ppd-H1 allele (fig. S3).

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Arabidopsis prr7 mutants showed delayed flowering in LDs but showed no significant effect in SDs (12, 13), similar to the effect of ppd-H1 (7, 8). prr7 mutants also lengthen the period of clock-mediated leaf movement (14) and affect the expression of clock components CCA1 and LHY, implicating the gene in the phasing of the clock in relation to light (15, 16). These results suggested that ppd-H1 might affect flowering by altering the expression of photoperiod pathway genes that have circadian control. To test this, we compared gene expression in Triumph (ppd-H1) with a Triumph line into which the Ppd-H1 allele from Igri had been introgressed.

In LDs Ppd-H1 was expressed predominantly in the early part of the day (Fig. 3A), similar to the expression patterns of Arabidopsis PRR7 and related genes in rice. An entrainment experiment confirmed that the barley gene was under circadian control, as previously shown for Arabidopsis and rice PRR genes (17, 18). Although PRR genes are implicated in clock function we detected no significant difference between Ppd-H1 and ppd-H1 plants in the expression of Ppd-H1 itself or the barley homolog of GI (HvGI) (Fig. 3B). However, two barley CO-like genes (HvCO1 and HvCO2) were affected. ppd-H1 plants showed reduced expression of HvCO1 at 8 and 12 hours (Fig. 3C), and HvCO2 was more significantly affected with reduced expression throughout the light period and a delay in the expression peak of about 4 hours (Fig. 3D). By analogy with Arabidopsis, the reduced expression of HvCO1 and HvCO2 during the latter part of the light period in ppd-H1 plants should reduce FT expression. We first tested whether barley CO genes behaved like CO in Arabidopsis by analyzing their expression under SDs [8 hours of light (fig. S4)]. HvCO2 expression was lower at the start of the day but peaked at a similar time in SD and LD, whereas HvCO1 peaked at 20 hours in SDs. The later peak of HvCO1 expression in SDs and the higher expression of both genes at dawn in LDs were similar to CO in Arabidopsis (19). We then isolated a barley FT (HvFT) gene that is orthologous to rice Hd3a (10). Expression of HvFT was consistently very low in SDs (figs. S4 and S5) and was markedly lower in ppd-H1 in LDs (Fig. 3E). The late-flowering phenotype of ppd-H1 can therefore be explained through known photoperiod mechanisms by a reduction in FT expression resulting from altered circadian timing of CO expression. The lack of effect on HvGI expression suggests that the ppd-H1 mutation does not have a strong disruptive effect on clock function or that the barley mutation affects an output linking the circadian clock to the HvCO genes. However, additional effects such as a direct role in HvFT expression cannot be ruled out.

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Previous work (14) surveying 150 Arabidopsis accessions identified PRR genes as candidates for quantitative trait loci that provide adaptive variation by modulating circadian timing. Clock period length was correlated with latitude of origin, suggesting that these genes provide adaptive variation in photoperiod response. The identification of Ppd-H1 as a PRR gene shows that the PRR family is of general importance for adaptation to natural and agricultural settings. Notably, comparative mapping shows that the major wheat photoperiod response genes are in colinear regions on the group 2 chromosomes (20) and that Hd2 is in the colinear region of rice chromosome 7 (21), making these attractive targets for further analysis. The availability of Ppd-H1 will provide greater understanding of the ways in which cereal development is regulated by environmental cues, allowing plant breeders to tailor crops to specific environments and to adjust varieties to new conditions arising from climate change.