Plant Cell, Vol. 12, 2299-2301, December 2000, Copyright © 2000, American Society of Plant Physiologists
Giving Rice the Time of Day: Molecular Identification of a Major Photoperiod Sensitivity Quantitative Trait Locus
Nancy A. Eckardt
News and Reviews Editor
Daylength controls flowering in many plant species. Early in the twentieth century, Garner and Allard 1920 
first described the phenomenon of plants that flower only when daylength is longer or shorter than a particular threshold, named long-day and short-day plants, respectively. Rice is a short-day plant; flowering (termed "heading" in rice and other cereals) is promoted by short daylength. However, there is a large degree of genetic variability in this trait among rice cultivars, from photoperiod insensitivity to strong photoperiod sensitivity. The manipulation of photoperiod sensitivity is an important breeding objective for rice grown in many tropical and temperate regions to optimize heading date and maturity for local environments (Poonyarit et al. 1989 ; Li et al. 1995 ).
Like many other important traits in plant breeding, heading date is a complex trait that shows continuous phenotypic variation among progeny and is controlled by multiple genes known as quantitative trait loci (QTLs). QTLs typically are difficult to identify because of the lack of discrete phenotypic segregation and because the phenotypic effects of each gene associated with a complex trait are relatively small (Yano and Sasaki 1997 ). QTL analysis involves selecting and hybridizing parental lines that differ in one or more quantitative traits and analyzing the segregating progeny to link the QTL to known DNA markers. Chromosomal QTL regions often are quite large and can include many open reading frames. This situation can exacerbate "linkage drag" in the application of QTL analysis to plant breeding or introgression into elite germplasm of undesirable characters that are linked to a desirable QTL (Tanksley and Nelson 1996 ). Thus, a principal objective of QTL analysis is defining QTLs to narrow chromosomal regions, ultimately leading to the identification and cloning of single genes that constitute major QTLs.
The concepts for detecting QTLs were developed more than 75 years ago (Sax 1923 ), and in recent years the availability of DNA markers and linkage maps has led to considerable progress in QTL mapping in plants and animals (Lander and Botstein 1989 ; Paterson et al. 1989 ). Plant QTLs that have been cloned include maize teosinte branched1, which exerts major effects on plant morphology (Doebley et al. 1997 ), and tomato fw2.2, which controls tomato fruit size (Frary et al. 2000 ). Nonetheless, the molecular identification of QTLs is still in its infancy. In this issue of The Plant Cell, Yano et al. (pp. 2473–2483) describe the use of fine-scale, high-resolution mapping to identify a single gene corresponding to Hd1, a major QTL controlling heading date in rice. The Hd1 gene is a homolog of CONSTANS (CO) in Arabidopsis, which functions in the photoperiodic control of flowering in this long-day plant (Putterill et al. 1995 ). This report marks the first demonstration of a QTL in a monocot corresponding to a known mutation in Arabidopsis and only the second flowering-time gene to be cloned from a monocot. The first was the INDETERMINATE gene of maize (Colasanti et al. 1998 ), which has no known ortholog in Arabidopsis. Furthermore, the work of Yano and colleagues shows that similar genes (i.e., putative orthologs) are involved in controlling the daylength response of flowering in short-day (rice) and long-day (Arabidopsis) plants.
THE CIRCADIAN CLOCK AND PHOTOPERIODIC CONTROL OF FLOWERING
The measurement of daylength in plants is controlled by the circadian clock, which controls circadian rhythms in gene expression and behavior that have been widely observed in eukaryotes, including insects, fungi, plants, and vertebrates. Circadian rhythms are 24-hr oscillations in gene expression and/or behavior that are characterized by persistence in the absence of external cues and entrainment by both light and temperature (Kreps and Simon 1997 ). The eukaryotic circadian clock has three general components: a "central oscillator" that creates periodicity, inputs that synchronize or entrain the oscillator to the prevailing day–night cycle, and outputs that are the resulting rhythms in gene expression and behavior (Dunlap 1999 ; Samach and Coupland 2000 ). Four genes in Arabidopsis have been described that are proposed to act within or in close association with the central oscillator: LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which encode similar MYB proteins, EARLY FLOWERING3 (ELF3), which encodes a novel protein, and TIMING OF CAB1 (TOC1). TOC1 was recently cloned and identified as an autoregulatory response regulator homolog (Strayer et al. 2000 ). Mutational analyses, overexpression studies, and mRNA expression patterns of these genes suggest that they perform functions central to the regulation of circadian rhythms (Samach and Coupland 2000 ).
The central oscillator is synchronized to the day–night cycle by the action of photoreceptors, namely, the cryptochromes (blue light receptors) and the phytochromes (red light receptors). CRY2 and PHYA may have a primary role in the promotion of flowering in Arabidopsis under long-day conditions (Samach and Coupland 2000 ). Phytochromes also have been found to play an important role in the photoperiodic control of flowering in rice (Izawa et al. 2000 ). Somers et al. 1998 examined various phy and cry mutants of Arabidopsis and concluded that photoreceptor diversity and redundancy are key features in the photocontrol of the circadian clock in higher plants. A diversity of photoreceptors that cover a wide range of fluence rates and spectral qualities may ensure accurate clock entrainment amid highly variable day-to-day light conditions (Somers et al. 1998 ). Temperature is another important input signal that affects clock entrainment (Kreps and Simon 1997 ).
Flowering time appears to be controlled by a large number of genes. A great many flowering-time mutants (Koornneef et al. 1998 ) and QTLs (Kuittinen et al. 1997 ) have been described in Arabidopsis, and numerous QTLs for heading date also have been reported in rice (Yano and Sasaki 1997 ). Despite the large number of genes involved, the two genes CO and GIGANTEA (GI) exert major control over the promotion of flowering under long days in Arabidopsis. These two genes are believed to act immediately downstream of the central oscillator and upstream of numerous other flowering-time genes whose activities they regulate (Nilsson et al. 1998 ; Samach and Coupland 2000 ). They do not appear to act within the central oscillator, because mutations in these genes affect flowering time (and expression of genes downstream in the long day pathway) but not circadian rhythms in general (although GI influences CCA1 expression and is believed to exert some degree of "backward" control on clock function; Samach and Coupland 2000 ). GI, originally described as a putative membrane protein (Fowler et al. 1999 ), was recently determined to be a nuclear protein involved in phytochrome signaling (Huq et al. 2000 ). GI is believed to function upstream of CO, because the late-flowering phenotype of gi mutants is corrected by CO overexpression (Fowler et al. 1999 ). A putative GI ortholog exists in rice, based on the similarity of the predicted GI amino acid sequence and rice expressed sequence tag sequences corresponding to a single rice gene. CO encodes a protein showing similarity to GATA1-type zinc finger transcription factors (Putterill et al. 1995 ). A family of CO-like regulatory or putative regulatory genes has been identified in Arabidopsis and other species (Lagercrantz and Axelsson 2000 ), members of which may function as transcriptional activators through DNA binding or protein–protein interactions.
CO AND Hd1 FUNCTION AND LONG-DAY VERSUS SHORT-DAY PLANTS
Flowering in the Arabidopsis co mutant occurs later than in the wild type under long days but is unaffected relative to the wild type under short days. In wild-type plants, CO mRNA is more abundant in long-day–grown than in short- day–grown seedlings, and overexpression of CO can cause early flowering under short days (Putterill et al. 1995 ). Thus, CO appears to be upregulated by long days (or short nights) and to function in the promotion of flowering, perhaps through the activation of floral meristem identity genes such as LEAFY (LFY) (Simon et al. 1996 ; Nilsson et al. 1998 ). Samach et al. 2000 have shown that early target genes of CO include SOC1 and FT, which activate floral meristem identity genes (LFY in the case of SOC1), and genes in the proline and ethylene biosynthetic pathways, which affect stem elongation and flowering, respectively.
Hd1 encodes a GATA1-type protein that exhibits a high degree of similarity to CO in the zinc finger domain and the C-terminal region (Yano et al. 2000 ). Yamamoto et al. 1998 identified the Hd1 QTL, along with two other QTLs controlling heading date (Hd2 and Hd3), as single Mendelian factors by crossing two rice varieties, Nipponbare (ecotype japonica) and Kasalath (ecotype indica), that differ in heading date. Using a combination of self-pollination of progeny and backcrossing to the Nipponbare parental line, plants were obtained that were homozygous for the recessive Kasalath allele at the Hd1 locus in a Nipponbare background (Yamamoto et al. 1998 ); such plants were found to be extremely early heading under field conditions (which were similar to long days in growth chamber experiments). Cloning and sequence analysis of Hd1 by Yano et al. 2000 , along with functional complementation experiments, confirmed that Nipponbare carries the functional Hd1 allele. The recessive Kasalath allele hd1 was found to contain numerous deletions and one insertion in the coding region compared with the Nipponbare allele. Experiments with various rice lines that are homozygous for either the functional Hd1 or the mutant hd1 allele showed that the presence of a functional Hd1 allele was associated with early heading under short days but, interestingly, significantly later heading under long days. Also, Hd1 expression appears to be unaffected by daylength, although this tentative conclusion needs to be confirmed with more detailed expression analyses (Yano et al. 2000 ). Thus, unlike CO in Arabidopsis, Hd1 appears to be bifunctional in rice, acting to promote heading under short days and to inhibit it under long days (Lin et al. 2000 ; Yano et al. 2000 ).
What distinguishes long-day plants from short-day plants at the molecular level? It is unlikely that the answer lies with a single gene. However, further characterization of the functional differences between CO and Hd1 should bring us closer to understanding the fundamental nature of daylength measurement and the photoperiodic control of flowering in higher plants. Characterization and molecular identification of the putative GI ortholog in rice also should prove to be highly illuminating in this regard.
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