Genomics of the origin and evolution of Citrus.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.

Differential expression of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like genes in Nicotiana benthamiana during corolla abscission, stem growth and water stress.

BACKGROUND: IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plant Arabidopsis thaliana (Arabidopsis) to be deeply involved in the regulation of abscission. Actually, IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission in economically important crops is scarce. In this work, we determined the conservation and phylogeny of the IDA-like and HAE-like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in the allopolyploid Nicotiana benthamiana, in order to identify members involved in abscission, stem growth and in the response to drought conditions. RESULTS: The phylogenetic relationships among the IDA-like members of the Solanaceae studied, grouped the two pairs of NbenIDA1 and NbenIDA2 protein homeologs with the Arabidopsis prepropeptides related to abscission. Analysis of promoter regions searching for regulatory elements showed that these two pairs of homeologs contained both hormonal and drought response elements, although NbenIDA2A lacked the hormonal regulatory elements. Expression analyses showed that the pair of NbenIDA1 homeologs were upregulated during corolla abscission. NbenIDA1 and NbenIDA2 pairs showed tissue differential expression under water stress conditions, since NbenIDA1 homeologs were highly expressed in stressed leaves while NbenIDA2 homeologs, especially NbenIDA2B, were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of the IDA-like family and their putative HAE-like receptors. CONCLUSION: Our results suggest that the pair of NbenIDA1 homeologs are involved in the natural process of corolla abscission while both pairs of NbenIDA1 and NbenIDA2 homeologs are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results provide additional evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

Genome-wide association study of agronomic traits in rice cultivated in temperate regions.

BACKGROUND: Rice plants are sensitive to the agro-climate conditions, being photoperiod one of main factor contributing to their adaptation to the region where they are grown. Dissecting the genetic bases underlying diversity in rice populations adapted to specific environmental conditions is a fundamental resource for breeding. In this study we have analysed a collection of japonica varieties adapted to temperate regions to perform association studies with traits of high agronomical interest such as heading date, plant height, number of panicles, panicle length and number of grains per panicle. RESULTS: We have performed a genome wide association study using a panel of 1713 SNPs that, based on previous linkage disequilibrium estimations, provides a full coverage of the whole genome. We have found a total of 43 SNPs associated with variations in the different traits. The identified SNPs were distributed across the genome except in chromosome 12, where no associated SNPs were found. The inspection of the vicinity of these markers also revealed a set of genes associated with physiological functions strongly linked to agronomic traits. Of special relevance are two genes involved in gibberellin homeostasis that are associated with plant height and panicle length. We also detected novel associated sites with heading date, panicle length and number of grain per panicle. CONCLUSION: We have identified loci associated with important agronomic traits among cultivars adapted to temperate conditions. Some of these markers co-localized with already known genes or QTLs, but the association also provided novel molecular markers that can be of help to elucidate the complicated genetic mechanism controlling important agronomic traits, as flowering regulation in the non-dependent photoperiod pathway. The detected associated markers may provide important tools for the genetic improvement of rice cultivars in temperate regions.

Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.).

KEY MESSAGE: Copper deficiency and excess differentially affect iron homeostasis in rice and overexpression of the Arabidopsis high-affinity copper transporter COPT1 slightly increases endogenous iron concentration in rice grains. Higher plants have developed sophisticated mechanisms to efficiently acquire and use micronutrients such as copper and iron. However, the molecular mechanisms underlying the interaction between both metals remain poorly understood. In the present work, we study the effects produced on iron homeostasis by a wide range of copper concentrations in the growth media and by altered copper transport in Oryza sativa plants. Gene expression profiles in rice seedlings grown under copper excess show an altered expression of genes involved in iron homeostasis compared to standard control conditions. Thus, ferritin OsFER2 and ferredoxin OsFd1 mRNAs are down-regulated whereas the transcriptional iron regulator OsIRO2 and the nicotianamine synthase OsNAS2 mRNAs rise under copper excess. As expected, the expression of OsCOPT1, which encodes a high-affinity copper transport protein, as well as other copper-deficiency markers are down-regulated by copper. Furthermore, we show that Arabidopsis COPT1 overexpression (C1 OE ) in rice causes root shortening in high copper conditions and under iron deficiency. C1 OE rice plants modify the expression of the putative iron-sensing factors OsHRZ1 and OsHRZ2 and enhance the expression of OsIRO2 under copper excess, which suggests a role of copper transport in iron signaling. Importantly, the C1 OE rice plants grown on soil contain higher endogenous iron concentration than wild-type plants in both brown and white grains. Collectively, these results highlight the effects of rice copper status on iron homeostasis, which should be considered to obtain crops with optimized nutrient concentrations in edible parts.

Diversity of floral regulatory genes of japonica rice cultivated at northern latitudes.

BACKGROUND: Rice is considered a short day plant. Originally from tropical regions rice has been progressively adapted to temperate climates and long day conditions in part by modulating its sensitivity to day length. Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) that code for florigens, are known as major regulatory genes of floral transition in rice. Both Hd3a and RFT1 are regulated by Early heading date 1 (Ehd1) and Days to heading on chromosome 2 (DTH2) while Heading date 1 (Hd1) also governs Hd3a expression. To investigate the mechanism of rice adaptation to temperate climates we have analyzed the natural variation of these five genes in a collection of japonica rice representing the genetic diversity of long day cultivated rice. RESULTS: We have investigated polymorphisms of Hd3a, RFT1, Ehd1, Hd1 and DTH2 in a collection of 57 japonica varieties. Hd3a and RFT1 were highly conserved, displaying one major allele. Expression analysis suggested that RFT1 rather than Hd3a could be the pivotal gene controlling flowering under long day conditions. While few alleles were found in the Ehd1 promoter and DTH2 coding region, a high degree of variation in Hd1, including non-functional alleles, was observed. Correlation analysis between gene expression levels and flowering periods suggested the occurrence of other factors, additionally to Ehd1, affecting RFT1 regulation in long day adapted cultivars. CONCLUSIONS: During domestication, rice expansion was accompanied by changes in the regulatory mechanism of flowering. The existence of non-functional Hd1 alleles and the lack of correlation of their presence with flowering times in plants grown under long day conditions, indicate a minor role of this branch in this process and the existence of an alternative regulatory pathway in northern latitudes. Expression analysis data and a high degree of conservation of RFT1 suggested that this gene could be the main factor regulating flowering among japonica cultivars adapted to northern areas. In the absence of inhibition exerted by Hd1 through repression of Hd3a expression, the role of Ehd1 as a regulator of RFT1 and Hd3a appears to be reinforced. Data also indicated the occurrence of additional regulatory factors controlling flowering.

Deciphering the Genetic Basis of Allelopathy in japonica Rice Cultivated in Temperate Regions Using a Genome-Wide Association Study.

Allelopathy has been considered as a natural method of weed control. Despite the nature of allelochemical compounds has been studied, little is known about the genetic basis underlying allelopathy. However, it is known that rice exhibits diverse allelopathic potentials across varieties, and breeding for rice plants exhibiting allelopathic potential conferring an advantage against weeds in paddy fields would be highly desirable. Knowledge of the gene factors and the identification of the genomic regions responsible for allelopathy would facilitate breeding programs. Taking advantage of the existing genetic diversity in rice, particularly in temperate japonica rice, we conducted a comprehensive investigation into the genetic determinants that contribute to rice allelopathy. Employing Genome-Wide Association Study, we identified four Quantitative Trait Loci, with the most promising loci situated on chromosome 2 and 5. Subsequent inspection of the genes located within these QTLs revealed genes associated with the biosynthesis of secondary metabolites such as Phenylalanine Ammonia Lyase (PAL), a key enzyme in the synthesis of phenolic compounds, and two genes coding for R2R3-type MYB transcription factors. The identification of these two QTLs associated to allelopathy in rice provides a useful tool for further exploration and targeted breeding strategies.

HKT2;2/1, a K⁺-permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism.

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na⁺ concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na⁺ and K⁺, even at high external Na⁺ concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K⁺ uptake under saline conditions.

Bioinformatics approach for developing a minimum set of SNP markers for identification of temperate japonica rice varieties cultivated in Spain.

The use of molecular markers for plant variety identification and protection is increasing. For this purpose, SNP markers have provided a reliable and stable tool for plant genotyping. The availability of small and low-cost SNP panels to accelerate the identification of the cultivated rice varieties should be beneficial for breeders, seed certification entities and rice industry. With the intention of providing of such a facility, we first developed a simple and easy-handle bioinformatics tool based on the widely used and freely available software R to generate small sets of SNPs that can discriminate varieties, by selecting markers from a larger genotyping dataset. By applying this algorithm to data from a previously genotyped collection of temperate japonica varieties from different countries, we identified a minimal set of 31 SNPs markers to distinguish 210 varieties. In addition, we used this algorithm to discriminate the 43 most cultivated in Spain rice varieties with minimal sets of 8 SNPs. We then developed and tested 22 Kompetitive Allele-Specific PCR (KASP) assays for the markers included in these panels, and obtained reliable genotype patterns for rice varieties identification. The complete 22 markers panel and the rice genotypes data could offer a useful and low-cost tool for rice breeders and industry to identify varieties and therefore to guarantee the quality of rice. The provided R-based algorithm can be applied to other genomic resources to develop core sets of discriminating markers.

Development and Genome-Wide Analysis of a Blast-Resistant japonica Rice Variety.

Rice is one of the most important crops in the world, and its production is severely affected by the rice blast disease caused by the fungus Magnaporthe oryzae. Several major blast resistance genes and QTLs associated with blast resistance have been described and mostly identified in indica rice varieties. In this work, we report the obtention of a blast-resistant rice breeding line derived from crosses between the resistant indica variety CT13432 and the japonica elite cultivar JSendra (highly susceptible to blast). The breeding line, named COPSEMAR9, was found to exhibit resistance to leaf blast and panicle blast, as demonstrated by disease assays under controlled and field conditions. Furthermore, a high-quality genome sequence of the blast-resistant breeding line was obtained using a strategy that combines short-read sequencing (Illumina sequencing) and long-read sequencing (Pacbio sequencing). The use of a whole-genome approach allowed the fine mapping of DNA regions of indica and japonica origin present in the COPSEMAR9 genome and the identification of parental gene regions potentially contributing to blast resistance in the breeding line. Rice blast resistance genes (including Pi33 derived from the resistant parent) and defense-related genes in the genome of COPSEMAR9 were identified. Whole-genome analyses also revealed the presence of microRNAs (miRNAs) with a known function in the rice response to M. oryzae infection in COPSEMAR9, which might also contribute to its phenotype of blast resistance. From this study, the genomic information and analysis methods provide valuable knowledge that will be useful in breeding programs for blast resistance in japonica rice cultivars.

The G123 rice mutant, carrying a mutation in SE13, presents alterations in the expression patterns of photosynthetic and major flowering regulatory genes.

Day length is a determinant of flowering time in rice. Phytochromes participate in flowering regulation by measuring the number of daylight hours to which the plant is exposed. Here we describe G123, a rice mutant generated by irradiation, which displays insensitivity to the photoperiod and early flowering under both long day and short day conditions. To detect the mutation responsible for the early flowering phenotype exhibited by G123, we generated an F2 population, derived from crossing with the wild-type, and used a pipeline to detect genomic structural variation, initially developed for human genomes. We detected a deletion in the G123 genome that affects the PHOTOPERIOD SENSITIVITY13 (SE13) gene, which encodes a phytochromobilin synthase, an enzyme implicated in phytochrome chromophore biosynthesis. The transcriptomic analysis, performed by RNA-seq, in the G123 plants indicated an alteration in photosynthesis and other processes related to response to light. The expression patterns of the main flowering regulatory genes, such as Ghd7, Ghd8 and PRR37, were altered in the plants grown under both long day and short day conditions. These findings indicate that phytochromes are also involved in the regulation of these genes under short day conditions, and extend the role of phytochromes in flowering regulation in rice.

Constitutive expression of OsGH3.1 reduces auxin content and enhances defense response and resistance to a fungal pathogen in rice.

GH3 genes are main components of the hormonal mechanism regulating growth and development and, hence, are deeply involved in a broad range of physiological processes. They are implicated in hormonal homeostasis through the conjugation to amino acids of the free form of essential plant growth regulators such as indoleacetic and jasmonic acids. In this work, we showed that OsGH3.1 overexpression in rice caused dwarfism and significantly reduced both free auxin content and cell elongation. Functional classification of the transcriptomic profiling revealed that most genes involved in auxin biosynthesis and auxin signaling inhibition were induced and repressed, respectively. Many genes related to cell organization and biogenesis were also significantly downregulated. The survey also showed that, although the response to abiotic stresses was not clearly stimulated, OsGH3.1 overexpression did activate a significant number of defense-related genes. In successive bioassays, it was demonstrated that the resistance of rice plants to pathogen infection, evaluated with two different Magnaporthe grisea strains, was higher in the transformants overexpressing OsGH3.1. Taken together, these results indicate that OsGH3.1 overexpression reduces auxin content, inhibits cell growth and cell wall loosening, and enhances resistance to a fungal pathogen. Our results provide evidence that auxin homeostasis can regulate the activation of the defense response in rice.

Genome wide association analysis of cold tolerance at germination in temperate japonica rice (Oryza sativa L.) varieties.

A pool of 200 traditional, landraces and modern elite and old cultivars of rice, mainly japonica varieties adapted to temperate regions, have been used to perform a genome wide association study to detect chromosome regions associated to low temperature germination (LTG) regulation using a panel of 1672 SNP markers. Phenotyping was performed by determining growth rates when seeds were germinated at 25° and 15°C in order to separate the germination vigorousness from cold tolerance effects. As expected, the ability to produce viable seedlings varied widely among rice cultivars and also depended greatly on temperature. Furthermore, we observed a differential response during seed germination and in coleoptile elongation. Faster development at 15°C was observed in seeds from varieties traditionally used as cold tolerant parents by breeders, along with other potentially useful cultivars, mainly of Italian origin. When phenotypic data were combined with the panel of SNPs for japonica rice cultivars, significant associations were detected for 31 markers: 7 were related to growth rate at 25°C and 24 to growth rates at 15°. Among the latter, some chromosome regions were associated to LTG while others were related to coleoptile elongation. Individual effects of the associated markers were low, but by combining favourable alleles in a linear regression model we estimated that 27 loci significantly explained the observed phenotypic variation. From these, a core panel of 13 markers was selected and, furthermore, two wide regions of chromosomes 3 and 6 were consistently associated to rice LTG. Varieties with higher numbers of favourable alleles for the panels of associated markers significantly correlated with increased phenotypic values at both temperatures, thus corroborating the utility of the tagged markers for marker assisted selection (MAS) when breeding japonica rice for LTG.

Corrigendum: Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus.

[This corrects the article on p. 126 in vol. 8, PMID: 28228766.].

Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus.

Abscission is a cell separation process by which plants can shed organs such as fruits, leaves, or flowers. The process takes place in specific locations termed abscission zones. In fruit crops like citrus, fruit abscission represents a high percentage of annual yield losses. Thus, understanding the molecular regulation of abscission is of capital relevance to control production. To identify genes preferentially expressed within the citrus fruit abscission zone (AZ-C), we performed a comparative transcriptomics assay at the cell type resolution level between the AZ-C and adjacent fruit rind cells (non-abscising tissue) during ethylene-promoted abscission. Our strategy combined laser microdissection with microarray analysis. Cell wall modification-related gene families displayed prominent representation in the AZ-C. Phylogenetic analyses of such gene families revealed a link between phylogenetic proximity and expression pattern during abscission suggesting highly conserved roles for specific members of these families in abscission. Our transcriptomic data was validated with (and strongly supported by) a parallel approach consisting on anatomical, histochemical and biochemical analyses on the AZ-C during fruit abscission. Our work identifies genes potentially involved in organ abscission and provides relevant data for future biotechnology approaches aimed at controlling such crucial process for citrus yield.

The GH3 family in plants: genome wide analysis in rice and evolutionary history based on EST analysis.

The GH3 gene family in Arabidopsis, implicated in hormonal homeostasis through the conjugation of indolacetic and jasmonic acids to amino acids, is involved in a broad range of plant growth and development processes. In this work, the analysis of the GH3 family in the genome of Oryza sativa identified 13 hypothetical ORFs. EST analysis and RT-PCR assays demonstrated that 12 of them were active genes. An extensive EST analysis of the GH3 family performed on 26 plant species was used to estimate the minimum number of GH3 genes en each one. The data indicated that the members of the GH3 family progressively increased in the different plant divisions from Chlorophyta (0), Bryophyta (3), and Coniferophyta (4), to Magnoliophyta (7-19). Phylogenetic analyses showed a high degree of conservation between Arabidopsis and rice GH3 proteins and, in general, in the plant kingdom. The data revealed a homology clustering consistent with the functional classification of the Arabidopsis proteins, since most of the 110 sequences analyzed grouped into 2 main clusters, corresponding to the Arabidopsis functional groups I (jasmonic acid adenylation) and II (indolacetic acid adenylation). And additional cluster including group III (non-adenylation ability) was exclusively composed of proteins from Arabidopsis thaliana, Brassica napus and Gossypium hirsutum.

Physiological Basis and Transcriptional Profiling of Three Salt-Tolerant Mutant Lines of Rice.

Salinity is a complex trait that affects growth and productivity in many crops, including rice. Mutation induction, a useful tool to generate salt tolerant plants, enables the analysis of plants with similar genetic background, facilitating the understanding of the salt tolerance mechanisms. In this work, we generated three salt tolerant mutant lines by irradiation of a salt-sensitive cultivar plants and screened M2 plants at seedling stage in the presence of high salinity. These three lines, SaT20, SaS62, and SaT58, showed different responses to salinity, but exhibited similar phenotype to wild type plants, except SaT20 that displayed shorter height when grown in the absence of salt. Under salt conditions, all three mutants and the parental line showed similar reduction in yield, although relevant differences in other physiological parameters, such as Na+ accumulation in healthy leaves of SaT20, were registered. Microarray analyses of gene expression profiles in roots revealed the occurrence of common and specific responses in the mutants. The three mutants showed up-regulation of responsive genes, the activation of oxido-reduction process and the inhibition of ion transport. The participation of jasmonate in the plant response to salt was evident by down-regulation of a gene coding for a jasmonate O-methyltransferase. Genes dealing with lipid transport and metabolism were, in general, up-regulated except in SaS62, that also exhibited down-regulation of genes involved in ion transport and Ca2+ signal transduction. The two most tolerant varieties, SaS62 and SaT20, displayed lower levels of transcripts involved in K+ uptake. The physiological study and the description of the expression analysis evidenced that the three lines showed different responses to salt: SaT20 showed a high Na+ content in leaves, SaS62 presented an inhibition of lipid metabolism and ion transport and SaT58 differs in both features in the response to salinity. The analysis of these salt tolerant mutants illustrates the complexity of this trait evidencing the breadth of the plant responses to salinity including simultaneous cooperation of alternative or complementary mechanisms.

Genetic Diversity and Population Structure of Rice Varieties Cultivated in Temperate Regions.

BACKGROUND: After its domestication, rice cultivation expanded from tropical regions towards northern latitudes with temperate climate in a progressive process to overcome limiting photoperiod and temperature conditions. This process has originated a wide range of diversity that can be regarded as a valuable resource for crop improvement. In general, current rice breeding programs have to deal with a lack of both germplasm accessions specifically adapted to local agro-environmental conditions and adapted donors carrying desired agronomical traits. Comprehensive maps of genome variability and population structure would facilitate genome-wide association studies of complex traits, functional gene investigations and the selection of appropriate donors for breeding purposes. RESULTS: A collection of 217 rice varieties mainly cultivated in temperate regions was generated. The collection encompasses modern elite and old cultivars, as well as traditional landraces covering a wide genetic diversity available for rice breeders. Whole Genome Sequencing was performed on 14 cultivars representative of the collection and the genomic profiles of all cultivars were constructed using a panel of 2697 SNPs with wide coverage throughout the rice genome, obtained from the sequencing data. The population structure and genetic relationship analyses showed a strong substructure in the temperate rice population, predominantly based on grain type and the origin of the cultivars. Dendrogram also agrees population structure results. CONCLUSIONS: Based on SNP markers, we have elucidated the genetic relationship and the degree of genetic diversity among a collection of 217 temperate rice varieties possessing an enormous variety of agromorphological and physiological characters. Taken together, the data indicated the occurrence of relatively high gene flow and elevated rates of admixture between cultivars grown in remote regions, probably favoured by local breeding activities. The results of this study significantly expand the current genetic resources available for temperate varieties of rice, providing a valuable tool for future association mapping studies.

Analysis of PHOTOPERIOD SENSITIVITY5 sheds light on the role of phytochromes in photoperiodic flowering in rice.

A great number of plants synchronize flowering with day length. In rice (Oryza sativa), photoperiod is the primary environmental cue that triggers flowering. Here, we show that the s73 mutant, identified in a gamma-irradiated Bahia collection, displays early flowering and photoperiodic insensitivity due to a null mutation in the PHOTOPERIOD SENSITIVITY5 (SE5) gene, which encodes an enzyme implicated in phytochrome chromophore biosynthesis. s73 mutant plants show a number of alterations in the characteristic diurnal expression patterns of master genes involved in photoperiodic control of flowering, resulting in up-regulation of the floral integrator Heading date3a (Hd3a). Early heading date1 (Ehd1), an additional rice floral activator, was also highly expressed in the s73 mutant, suggesting that SE5 represses Ehd1 in wild-type plants. Silencing of Ehd1 in both Bahia and s73 backgrounds indicated that SE5 regulates Ehd1 expression. The data also indicate that SE5 confers photoperiodic sensitivity through regulation of Hd1. These results provide direct evidence that phytochromes inhibit flowering by affecting both Hd1 and Ehd1 flowering pathways.