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First published online May 20, 2005; 10.1104/pp.104.059071 Plant Physiology 138:1126-1135 (2005) © 2005 American Society of Plant Biologists New Arabidopsis Recombinant Inbred Lines (Landsberg erecta x Nossen) Reveal Natural Variation in Phytochrome-Mediated Responses1IFEVA, Facultad de Agronomía, Universidad de Buenos Aires y Consejo Nacional de Investigaciones Científicas y Técnicas, 1417 Buenos Aires, Argentina (T.M.A.M., J.F.B., J.J.C.); and University of Texas, Department of Molecular, Cell, and Developmental Biology, Austin, Texas 78712 (A.V.G., V.V.S., A.M.L.)
We used 52 Arabidopsis (Arabidopsis thaliana) accessions and developed a new set of 137 recombinant inbred lines between Landsberg erecta (Ler) and Nossen (No-0) to explore the genetic basis of phytochrome-mediated responses during deetiolation. Unexpectedly, most accessions showed weak or moderate hypocotyl growth and cotyledon unfolding responses to pulses of far-red light (FR). Crosses between Columbia and No-0, two accessions with poor response, segregated seedlings with unfolded cotyledons under pulsed FR, suggesting the occurrence of accession-specific loci in the repression of morphological responses to weak light signals. Confirming the latter expectation, mapping of responses to pulsed FR in the Ler x No-0 lines identified novel loci. Despite its weak response to pulsed FR, No-0 showed a response to continuous FR stronger than that observed in Ler. By mapping the differential effect of pulsed versus continuous FR, we identified two high-irradiance response loci that account for the steeper response to continuous FR in No-0. This underscores the potential of the methodology to identify loci involved in the regulation of the shape of signal input-output relationships. Loci specific for a given phytochrome-mediated response were more frequent than pleiotropic loci. Segregation of these specific loci is predicted to yield different combinations of seedling responsivity to light. Such flexibility in combination of responses is observed among accessions and could aid in the adjustment to different microenvironments.
Some fluctuations of the light environment tightly correlate with the occurrence of conditions that impose a challenge to plant survival such as seasonal changes that result in extreme temperatures, organ emergence out of the soil, or competition with neighbor individuals. Subtle light signals, including small changes in photoperiod throughout the year, the transition between darkness and very low fluences of light reaching the top millimeters of the soil, and small reductions in the red light (R) to far-red light (FR) ratio caused by light reflected by neighbors, actually anticipate stressful conditions. Plants are able to perceive these signals, which are translated into regulation of developmental plasticity (Casal et al., 2004  ). Not surprisingly, fitting developmental decisions to these signals requires an intricate network of molecular players.
The use of Arabidopsis (Arabidopsis thaliana) mutants has been the primary approach in the search for players in light signaling. Mutant screens have led to the discovery of the photoreceptors phytochrome A (phyA; Whitelam et al., 1993
phyA mediates two different types of response, the VLFR and the high-irradiance response (HIR). The VLFR is saturated by a brief exposure to R or FR, which establishes a very small proportion of phyA in its Pfr form (Botto et al., 1996
Here, we report on the genetic variability among Arabidopsis accessions in VLFR and on the construction of a new set of RILs derived from a cross between Ler and Nossen (No-0). Ler was originally collected from Poland and No-0 from Germany. There are several reasons for this choice. First, while exploring variability among different accessions, we observed that Ler and No-0 differed not only in VLFR but also in HIR. This provided an excellent opportunity to identify novel QTLs, particularly those controlling HIR. Second, since we had already used Ler x Col and Ler x Cvi RILs (Yanovsky et al., 1997
Variability among Arabidopsis Accessions in the Response to FR Pulses
We investigated the variability of the response of hypocotyl growth and cotyledon unfolding to hourly pulses of FR in 52 Arabidopsis accessions available at the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus). This light treatment induces weak responses mediated by phyA (Yanovsky et al., 1997
We investigated in further detail the differences between Ler and No-0, an accession frequently used as wild-type background and phenotypically representative of the majority under pulses of FR. No-0 seedlings are taller than those of Ler in darkness (Fig. 2A) and show reduced inhibition of hypocotyl growth and cotyledon unfolding in response to pulses of FR (calculated Pfr/P = 10%; Fig. 2, B and C). The difference between Ler and No-0 increased with the Pfr/P provided by hourly R/FR pulses, reaching a maximum for a calculated Pfr/P = 10% (Fig. 2, B and C). This corresponds to the phyA-mediated VLFR (Yanovsky et al., 1997 ). At higher Pfr/P, the response curves remained parallel, indicating no obvious differences in the phyB-mediated low-fluence response (LFR).
Most accessions show reduced cotyledon unfolding under pulses of FR, suggesting that common and/or accession-specific loci could be involved in repression of this VLFR. Before initiating the effort to obtain Ler x No-0 RILs, we evaluated the potential of No-0 as a source of QTLs involved in VLFR different from those already identified as polymorphic between Ler and Col. We observed seedlings with higher cotyledon angle under pulses of FR in the F2 generation of Col x No-0 than in the parental lines (Fig. 3). This pattern of segregation suggests that at least partially different loci could be responsible for the reduced cotyledon unfolding in Col and No-0 compared to Ler.
In addition to the VLFR, phyA also mediates the HIR, which requires sustained (continuous or very frequent) excitation with FR at higher fluence rates than those required to saturate the VLFR phase with hourly light pulses (Casal et al., 2000 ). The accessions showing stronger VLFR did not necessarily exhibit stronger HIR (Fig. 1A, inset). Actually, although No-0 showed a reduced VLFR of hypocotyl growth inhibition (Fig. 2B), its HIR was stronger (note steeper slope, Fig. 4A). The different slope was detectable in the linear phase of the log fluence rate response curve (i.e. below 10 µmol m–2 s–1), and this argues against a physically constrained HIR as a consequence of enhanced VLFR in Ler compared to No-0. Ler reached maximum cotyledon unfolding at 10 µmol m–2 s–1 of continuous FR, and at lower fluence rates the average slope of the cotyledon angle-fluence rate response was similar for both accessions (Fig. 4B). The analysis of additional accessions showed that the enhanced HIR of hypocotyl growth in lines with reduced VLFR of the same process was neither exclusive to No-0 (see Eppenheim, Ep-0, in Fig. 4C) nor obligatory (see Achkarren, Ak-1, in Fig. 4C). Taken together, these observations suggest that at least partially independent loci control VLFR and HIR.
Generation of Ler x No-0 RILs
To investigate in further detail some of the aforementioned differences among accessions, we generated a mapping population of 137 F9 lines obtained by the single-seed descent method after a cross between the parental lines. A linkage map of polymorphisms between Ler and No-0 was constructed with 46 microsatellite markers and Mapmaker (Lander et al., 1987
To illustrate the potential of these RILs, we mapped several QTLs that reflect variability in vegetative growth. In plants grown under short days in a glasshouse, we identified three QTLs controlling leaf dimensions, all in chromosome II (Fig. 5). LNG affects petiole length and leaf lamina length and apparently overlaps with ju-LSI2/ad-PSI1, mapped in RILs derived from a Ler x Col-4 cross (Pérez-Pérez et al., 2002 ). WID1 and WID2 affect leaf lamina width (Fig. 5).
Seedlings of the Ler x No-0 RILs were grown under hourly pulses of FR, hourly pulses of R, or continuous FR (2 µmol m–2 s–1), or in full darkness. In 4-d-old seedlings, we identified a QTL that collocates with HYP2, a QTL that accounts for the longer hypocotyl in No-0 compared to Ler under different light or dark conditions, previously mapped using Ler x Cvi RILs (Borevitz et al., 2002
Mapping of VLF, LFR, and HIR QTLs
We identified two QTLs affecting VLFR, two affecting HIR, and one affecting LFR (Fig. 7). One of the alleles affecting VLFR maps close to marker nga225 in chromosome V, a region where we had identified VLF2 in Ler x Col RILs (Yanovsky et al., 1997
The HIR1 QTL mapped close to the marker ACC2 at the top of chromosome I and caused enhanced HIR of cotyledon unfolding in the lines carrying the No-0 allele (Fig. 7). The HIR2 allele mapped close to the nga1126 marker in chromosome II and enhanced the HIR of hypocotyl growth in the lines carrying the No-0 allele (Fig. 7). The LFR1 locus mapped close to the marker nga1126 in chromosome II and enhanced cotyledon unfolding in the lines bearing Ler alleles (Fig. 7). Mapping of HIR1, HIR2, and LFR1 is based on the difference between seedlings grown under FR pulses and seedlings grown under continuous FR or pulsed R. Since the FR pulses induce VLFR, we evaluated the possibility that these derived QTLs were mathematical artifacts created as a reaction to the presence of VLF loci. We conclude that HIR1, HIR2, and LFR1 are genuine QTLs for the following reasons. First, HIR1 and LFR1 do not overlap with any locus involved in VLFR (maximum log of the odds [LOD] scores for cotyledon unfolding VLFR in the 2-LOD support interval were 0.1 and 0.5, respectively). Second, HIR2 partially overlaps with VLF8 but VLF1, another QTL with comparable LOD score for hypocotyl growth (3.3 and 3.0, respectively) and based on the same set of data (Fig. 7), had no associated QTL involved in HIR (maximum LOD score for HIR in the 2-LOD support interval of VLF1 = 0.2). Thus, the occurrence of an HIR locus is not a necessary consequence of the presence of a VLF locus. Third, to minimize (dilute) the contribution of a VLFR component to the response to continuous FR without having to subtract the effect of FR pulses, we used 10 rather than 2 µmol m–2 s–1 of continuous FR. We mapped the difference in hypocotyl length between the seedlings grown in darkness and those grown under 10 µmol m–2 s–1 FR. Following this alternative procedure, any contribution of VLF8 should play against finding HIR2 because their effects have opposite signal and are not discriminated by the calculation. Despite this unfavorable protocol, a QTL indicating stronger inhibition by continuous FR in No-0 than Ler was mapped close to the previous HIR2 (Fig. 8). The small shift in location is likely to reflect the fact that the difference between darkness and 10 µmol m–2 s–1 FR still includes the diluted VLFR component playing against HIR2 in the vicinity of VLF8. Thus, HIR2 can be identified by a protocol that does not require subtracting the effect of hourly FR pulses.
To investigate the genetic basis of sensitivity to light, we compared the response of Arabidopsis accessions to pulses of FR and mapped QTL for light responses in a newly generated set of RILs between the Ler and No-0 accessions. RILs derived from parents of divergent locations have allowed the identification of loci that denote adaptation to divergent geographical locations such as photoperiod (El-Assal et al., 2001 ). Ler and No-0 are of relatively close geographic origins but they still contain significant variability as revealed by the leaf morphology, hypocotyl growth, and cotyledon unfolding QTLs reported here (Figs. 5 and 7) and the trichrome density and auxin response QTLs that will be reported elsewhere. Interestingly, Ler and No-0 also occupied distant positions in the analysis of hypocotyl growth response to continuous light of different wavelengths (Maloof et al., 2001) and in microarray gene expression studies (Wang et al., 2002). The Ler x No-0 RILs have already been donated and will become available through the ABRC stocks.
Previous reports describe the variability of Arabidopsis accessions in hypocotyl length under continuous light (Maloof et al., 2001
We mapped one QTL affecting LFR and two QTLs affecting HIR (Fig. 7). LFR1 maps to the 39-to-51-cM region of chromosome II. Compared to No-0, the Ler allele at this locus enhances the difference in cotyledon unfolding induced by pulses of R and pulses of FR. HIR1 locates to the 0-to-12-cM region of chromosome I, and No-0 alleles at this locus enhanced the HIR of cotyledon unfolding compared to Ler. HIR2 locates to the 24-to-43-cM region of chromosome II and No-0, compared to Ler, alleles at this locus enhanced the HIR or hypocotyl growth. The contrasting Ler/No-0 allelic effects of VLF8 and HIR2 account, at least in principle, for the differential FR fluence-rate response curves of the parental accessions (Fig. 4A). Many genes respond differentially to continuous FR in Ler and No-0 (Wang et al., 2002 We have successfully mapped QTLs based on the differential response observed under two light conditions that differ either in the duration of the light exposure (for HIR loci) or the established proportion of Pfr (for the LFR locus). This indicates that RILs may be a powerful tool to map loci involved in regulating the shape of signal input-output relationships. Such loci could be difficult to identify if a single input at a time (a single light treatment in the case reported here) was mapped. Mutant screening protocols are based on the analysis of the phenotype under a given environment. Loci involved in the regulation of dose response curves may be more readily identified with the use of RILs, where a given genotype can be characterized under different conditions.
After the analysis of three sets of RILs (Ler x Col, Ler x Cvi, and Ler x No-0), we have found no locus with significant effects in the same direction for more than one response mode (VLFR, LFR, HIR). For VLF6/CRY2, the alleles that enhance VLFR reduce LFR, and this locus is considered to operate upstream of the phyA VLFR-signaling branch that down-regulates phyB signaling (Botto et al., 2003
Generation of RILs
A set of 137 RILs were generated by single-seed descent to the F8 generation from a segregating F2 population derived from a cross between the laboratory strain Ler (kindly provided by Maarteen Koornneef) and the accession No-0 (ABRC CS1394). Plants were grown in a growth chamber at 22°C under continuous fluorescent-white light. Seeds were bulked at the F9 generation. Ler originates from Northern Europe (Rédei, 1992
Fifteen seeds of each of the accessions (ABRC) or RIL were sown on 0.8% agar-water in clear plastic boxes (42 x 35 mm2 x 20 mm to compare Ler and No-0, and 215 x 85 mm2 x 20 mm when multiple lines were involved) and incubated in darkness at 6°C for 3 d. Chilled seeds were given a saturating pulse of R and incubated in darkness at 22°C for 24 h. One-day-old seedlings were exposed to hourly pulses of R, FR, or R plus FR mixtures (3 min, 15–40 µmol m–2 s–1; these fluence rates saturate the response to the pulses) or to continuous FR (fluence rates between 0.1 and 200 µmol m–2 s–1) for 3 d, whereas control seedlings remained in darkness. Details of light sources, spectral distribution, and Pfr/P calculations were as described earlier (Yanovsky et al., 2000
We used 46 PCR-based markers (Fig. 5) to detect simple sequence length polymorphisms between Ler and No-0 found in The Arabidopsis Information Resource database (http://www.arabidopsis.org) or in Loudet et al. (2002) Thirty-nine markers were screened in similar PCR reactions (10 µL reaction mix in 96-well plates containing 4 µL of DNA; 200 µM of each dNTP, 1.5 mM MgCl2; 0.6 µM primer, 1x PCR reaction buffer [Invitrogen, Carlsbad, CA], and 1 unit Taq Polymerase [Invitrogen]). For some markers, 2.0 mM Mg2Cl was used. Amplification was performed using an Eppendorph Mastercycler gradient (1 cycle, 2 min at 95°C; 30 cycles, 45 s denaturation at 94°C, 45 s annealing at 57°C; 1 min elongation at 72°C; the cycles were followed by a final elongation at 72°C for 7 min). Ten microliters of loading buffer were added to the amplification reactions. Five microliters of this mix were loaded on 3% or 4% high-resolution agarose gels (MetaPhor, Bio Whittaker, Walkersville, MD) containing 50 µg/mL ethidium bromide and run for 3 h at 100 V. Markers ACC2, CIW5, NGA8, NT204, MSAT4.37, MSAT4.41, and MSAT 4.7 were screened by using a tailed-PCR scheme. Each forward primer was 5' tailed with the M13 forward sequence and used in conjunction with a standard reverse primer and a FAM-6-labeled M13 primer. The M13 and reverse primers were equimolar and the forward primer was used at 15-fold lower concentration. Cycling was the same as described above, except that the annealing temperature was 52°C. Following confirmation of amplification success on standard agarose gels, 0.5 µL of each reaction was combined with 9.5 µL formamide and 0.5 µL size standard, and separated and sized on an ABI 3100 DNA analyzer.
Mapmaker/EXP 3.0 (Lander et al., 1987 All RILs were grown simultaneously in each box. The experiments were repeated on three to five occasions (one box per experiment) and QTL analysis was based on the phenotypic mean of each RIL. The S statistic (Qstats package of QTL Cartographer; S. Wang, C.J. Basten, and Z.-B. Zeng [2001–2004] Windows QTL Cartographer 2.0. Department of Statistics, North Carolina State University, Raleigh, NC [http://statgen.ncsu.edu/qtlcart/WQTLCart.htm]) was used to test the normality of the distributions.
The composite-interval mapping (Zeng, 1994 Received December 28, 2004; returned for revision February 1, 2005; accepted February 2, 2005.
1 This work was supported by the University of Buenos Aires (grant nos. G021 to J.J.C. and G013 to J.F.B), by the Agencia Nacional de Promoción Científica y Tecnológica (grant nos. PICT 11631 to J.J.C and PICT 10765 to JFB), and by the National Science Foundation (grant no. 0114976 to A.M.L.). 
2 Present address: INIBIOLP, Instituto de Investigaciones Bioquímicas La Plata, Facultad de Ciencias Médicas, Universidad de La Plata, calle 60 y 120, 1900 La Plata, Argentina.
3 Present address: Universidad Nacional de Mar del Plata, Instituto de Investigaciones Biológicas, 7600 Mar del Plata, Argentina. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.059071. * Corresponding author; casal{at}ifeva.edu.ar; fax 5411–4514–8730.
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-mercaptoethanol) was added following vortexing for 15 s. Another 150 µL of extraction buffer was added and the sample was incubated 2 h at 65°C. Next, 300 µL of chloroform/isoamyl alcohol (24:1) was added, and the sample was well shaken and centrifuged 2 min. The aqueous phase was transferred to a clean Eppendorf tube. DNA was precipitated, washed, and dissolved in 100 µL of Tris-EDTA. DNA was diluted 10 times before being used for PCR reactions. 
