detection of spatial-specific phytochrome responses using

Detection of Spatial-Specific Phytochrome ResponsesUsing Targeted Expression of Biliverdin Reductasein Arabidopsis1[OA]

Sankalpi N. Warnasooriya and Beronda L. Montgomery*

Department of Energy Plant Research Laboratory (S.N.W., B.L.M.), Genetics Graduate Program (S.N.W.,B.L.M.), and Department of Biochemistry and Molecular Biology (B.L.M.), Michigan State University,East Lansing, Michigan 48824–1312

To regulate levels of holophytochrome in a spatial-specific manner and investigate the major sites of action of phytochromesduring seedling development, we constructed transgenic Arabidopsis (Arabidopsis thaliana) plant lines expressing plastid-targeted mammalian biliverdin IXa reductase (pBVR) under regulatory control of CAB3 and MERI5 promoters. Comparativephotobiological and phenotypic analyses indicated that spatial-specific expression of pBVR led to the disruption of distinctsubsets of phytochrome-regulated responses for different promoters. pBVR expression in photosynthetic tissues (CAB3::pBVRlines) had intermediate effects on chlorophyll accumulation, carotenoid production, anthocyanin synthesis, and leaf devel-opment responses in white-light conditions. CAB3::pBVR expression, however, resulted in distinctive phenotypes in far-red(FR) conditions. A number of FR high irradiance responses were disrupted in CAB::pBVR lines, including FR-dependentinhibition of hypocotyl elongation and stimulation of anthocyanin accumulation. By contrast, preferential expression of pBVRin the shoot apical meristem in MERI5::pBVR lines resulted in a phytochrome-deficient, leaf development phenotype undershort-day growth conditions. These results implicate leaf-localized phytochrome A as having a unique role in regulating FR-mediated hypocotyl elongation and meristem- and/or leaf primordia-localized phytochromes as having a novel role inphytochrome-dependent responses. Taken together, these studies demonstrate the efficacy of selectively inactivating distinctphytochrome-mediated responses by regulated expression of BVR in transgenic plants, a novel means to investigate the sites ofphytochrome photoperception and to regulate specifically light-mediated plant growth and development.

Numerous growth and developmental processes ofplants such as seed germination, inhibition of hypo-cotyl elongation, internode elongation, cotyledon andleaf expansion, plastid development and greening,and the induction of flowering are regulated by light(Franklin and Whitelam, 2004; Schepens et al., 2004;Mathews, 2006). To monitor and adapt to environ-mental light conditions, plants possess multiple pho-tomorphogenic photoreceptors, those which maximallyabsorb UV-B, blue/UV-A, and red (R)/far-red (FR)light (Kendrick and Kronenberg, 1994; Franklin et al.,2005; Schafer and Nagy, 2006). The most extensivelystudied of these photoreceptors are the R/FR revers-ible phytochromes. All higher plant species investi-gated possess multiple phytochrome isoforms, whichare encoded by a small nuclear gene family. Fiveindividual phytochrome genes, designated PHYA to

PHYE, have been isolated from Arabidopsis (Arabi-dopsis thaliana; Sharrock and Quail, 1989; Quail, 1994).These individual phytochromes mediate overlapping,yet discrete, aspects of photomorphogenesis.

All identified higher plant phytochromes utilize asingle linear tetrapyrrole chromophore precursor,phytochromobilin (Terry et al., 1993). Recent studieshave shown that there are multiple genes encodingkey enzymes of the plastid-localized chromophorebiosynthetic pathway in Arabidopsis (Muramotoet al., 1999; Kohchi et al., 2001; Emborg et al., 2006).Phytochromobilin-deficient plants exhibit defects inlight-mediated growth and development both as seed-lings and adults, corresponding to a lack of multiplephytochrome-regulatory activities (for review, seeTerry, 1997).

A number of recent studies have added significantlyto our understanding of the in vivo functions andintracellular signaling mechanisms of phytochromes.In these studies, researchers established that phyto-chromes translocate in a light-dependent fashion fromthe cytosol into the nucleus, where phytochrome mol-ecules interact with transcription factors to regulategene expression (Nagy et al., 2001; Nagatani, 2004; Jiaoet al., 2007; Kevei et al., 2007). In addition to nuclearfunctions, biochemical localization assays and injec-tion of signaling intermediates such as cGMP, cal-modulin, and calcium support additional cytosolic

1 This work was supported by the U.S. Department of Energy(grant no. DE–FG02–91ER20021 to B.L.M.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Beronda L. Montgomery ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

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functions of phytochromes (for review, see Nagy andSchafer, 2000). Furthermore, the analyses of mutantsharboring mutations in the genes FHY1 and FHL,whose gene products have been implicated in regu-lating nuclear import of phyA, provide convincingevidence for cytosolic functions of phyA (Rosler et al.,2007).Despite the progress that has been made in under-

standing phytochrome signaling in cells, we lack de-finitive molecular evidence about the distinct sites ofphytochrome photoperception and cellular mecha-nisms of localized pools of phytochromes that regulateintercellular and organ-specific phytochrome re-sponses in planta. Although the importance of suchspatial-specific responses is beginning to be recog-nized in the literature, much work remains to be doneto identify these responses and elucidate them fully atthe molecular level (Bou-Torrent et al., 2008; Josseet al., 2008; Montgomery, 2008). Early studies, whichaddressed intercellular and organ-specific phyto-chrome responses at the physiological level, activatedphytochromes in a localized region of the plant usingdetached plant parts or microbeam irradiation andassessed phytochrome-mediated responses at distinctsites in the plant (De Greef et al., 1971, 1975; De Greefand Verbelen, 1972; De Greef and Caubergs, 1972a,1972b; Black and Shuttleworth, 1974; Caubergs and DeGreef, 1975; Mandoli and Briggs, 1982; Nick et al.,1993). Results from these experiments suggest thatphytochromes exhibit cell autonomous and cell non-autonomous responses in plants. The most widelyrecognized of the cell nonautonomous phytochromeresponses is the photoperiodic induction of floweringin photoperiod-sensitive plant species that involvesthe perception of light in the leaves, followed bygeneration and subsequent movement of an inductivesubstance from leaves to the shoot apex (King andZeevaart, 1973; Parcy, 2005; Yu and Lin, 2005;Montgomery, 2006; Zeevaart, 2006; Kobayashi andWeigel, 2007; Turck et al., 2008). The perception of

light in cotyledons is also responsible for intercellularsignaling that results in the inhibition of hypocotylelongation and apical hook opening (Black andShuttleworth, 1974; Caubergs and De Greef, 1975), aswell as the pattern and level of accumulation ofanthocyanins (Nick et al., 1993). Results from suchstudies support the roles of cell- and tissue-specificphytochrome signaling pathways in controlling dis-crete aspects of light-dependent growth and devel-opment through intercellular and/or interorgancoordination. The implications of many of these earlyphysiological studies were confounded, however, bylight piping and light scattering, particularly in mi-crobeam irradiation assays. These conditions poten-tially result in light having a direct impact on tissues orcells far from the site of application (Mandoli andBriggs, 1982; Hiltbrunner et al., 2007).

In the present investigation, we utilize a novelexperimental approach to investigate the sites ofphotoperception for specific phytochrome-mediatedresponses at the molecular level. Based on previousexperiments showing that constitutive expression ofthe gene encoding the mammalian enzyme biliverdinIXa reductase (BVR) induces a phytochrome chromo-phore deficiency in transgenic plants (Lagarias et al.,1997; Montgomery et al., 1999, 2001), we used cell- andtissue-specific expression of BVR to probe the pheno-typic consequences of localized inactivation of phyto-chromes in transgenic Arabidopsis plants. We usedtwo Arabidopsis promoters, the –400 to –9 bp region ofthe CAB3 promoter (Mitra et al., 1989) and the 2.4-kbMERI5 promoter (Medford et al., 1991), to directexpression of BVR to photosynthetic and meristematictissues, respectively. These studies provide new in-sights into spatial-specific photoregulatory roles ofphytochromes.

Table I. BVR-specific activity for light-grown No-O wild-type andBVR transgenic plant extracts

Plant Linea BVR-Specific Activityb

(I.U. mg21) 3 103

No-O wild type 0.035S::pBVR3 13.4CAB3::pBVR1 2.3CAB3::pBVR2 9.0CAB3::pBVR3 2.9MERI5::pBVR1 0.3MERI5::pBVR2 0.2MERI5::pBVR3 0.8

aSeedlings (n = 50) were grown for 7 d at 25�C under continuouscool white illumination of 60 mmol m22 s21 and harvested at the sametime of day for each repetition. bBVR-specific activity was mea-sured for soluble protein extracts, as described in Lagarias et al. (1997).

Figure 1. BVR protein levels in soluble protein extracts from wild-typeand transgenic BVR seedlings. Transgenic 35S::pBVR3, representativeCAB3::pBVR (denoted CAB-1 and CAB-2), and wild-type (No-O WT)seedlings were grown at 25�C on Phytablend medium containing 1%Suc for 10 d.Whole-seedling soluble protein extracts (20 mg) were usedfor BVR immunoblot analysis with anti-BVR antibody. A, Analysis ofsoluble protein extracts from seedlings grown in darkness (D) or underWc illumination of 60 mmol m22 s-1 (L). B, Analysis of soluble proteinextracts from dissected cotyledons and true leaves (L) or dissectedhypocotyl and root tissues (H/R).

Spatial-Specific Phytochrome Responses

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RESULTS

Spatial-Specific Expression of BVR in Transgenic Plants

We isolated three independent homozygous CAB3::pBVR and MERI5::pBVR transgenic plant lines usingan approach described previously (Lagarias et al.,1997). BVR-specific activities were determined forsoluble protein extracts for each line and comparedwith the 35S::pBVR3 line (Montgomery et al., 1999), inwhich BVR was constitutively expressed and targetedto plastids. As shown in Table I, one of the CAB3::pBVR lines exhibited nearly as much BVR-specificactivity as the 35S::pBVR3 line. Although considerablylower, BVR activity was detectable in total solubleprotein extracts of seedlings for all three MERI5::pBVRlines.

We further assessed the spatial distribution of BVRaccumulation in pBVR lines to determine whetherBVR expression was light and tissue specific for theCAB3 promoter and restricted to the meristem and leafprimordia as directed by the MERI5 promoter. BVRaccumulation in representative CAB3::pBVR lines wasobserved to be light dependent by immunoblot anal-ysis (Fig. 1A). Additionally, BVR accumulated only in

the leaf tissue and not in the hypocotyl and root tissuesfor these CAB3::pBVR lines, whereas BVR was foundin leaf and hypocotyl/root tissues of the 35S::pBVR3line (Fig. 1B). For MERI5-driven expression, BVRaccumulation was detectable only in the plastids ofmeristematic and recently meristematic tissues ofMERI5::pBVR lines (Fig. 2).

Photomorphogenesis Is Selectively Impaired inCAB3::pBVR and MERI5::pBVR Plants

Previously, we showed that constitutive expressionof BVR in Arabidopsis altered numerous aspects oflight-mediated growth and development throughout

Figure 2. Whole-mount immunolocalization of BVR protein accumu-lation in MERI5::pBVR seedlings. A representative MERI5::pBVR line,MERI5::pBVR1, was grown at 22�C on Phytablend medium contain-ing 1% Suc for approximately 4 d under Wc illumination of 100 mmolm22 s21. A, Negative control, MERI5::pBVR1 seedlings incubatedwithout the anti-BVR primary antibody. B, DIC image of A. C, MERI5::pBVR1 seedling incubated with anti-BVR primary antibody at 1:2,500dilution. D, DIC image of C. Each image is a representative slice from aZ-series with 0.5-mm interval size andwas captured using 543-nm laserexcitation with a 203 lens objective. Fluorescence images werecollected using a 560- to 615-nm band pass filter. Bars = 50 mm.

Figure 3. Photomorphogenesis of Wc-grown wild-type and transgenicBVR plants. No-O wild-type (WT), 35S::pBVR3, CAB3::pBVR (CAB-1to CAB-3), and MERI5::pBVR (MERI-1 to MERI-3) transgenic lines weregrown at 20�C on Phytablend medium containing 1% Suc for 7 d underWc light (Grolux/Grolux wide spectrum) of 100 mmol m22 s21 (A), Rclight of 50 mmol m22 s21 (B), or FRc light of 5 mmol m22 s21 (C). B andC, Above each seedling, cotyledons are shown that were separated andarranged to display the full cotyledon surface area.

Warnasooriya and Montgomery

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the plant life cycle. Among the more pronouncedphenotypes observed in the transgenic plants wereelongated hypocotyls and petioles under all lightconditions tested (Lagarias et al., 1997; Montgomeryet al., 1999). Selective expression of pBVR under reg-ulatory control of the CAB3 and MERI5 promotersaffected hypocotyl growth and leaf morphology dis-tinctively. Qualitatively similar to 35S::pBVR trans-genic lines, continuous white light (Wc)-grown CAB3::pBVR lines display elongated hypocotyls comparedwith ecotype Nossen (No-O) wild-type seedlings,though not as severe as the 35S::pBVR3 line (Fig. 3A).The fluence rate dependence of hypocotyl growthinhibition shown in Figure 4 reflects the decreased,but not abolished, responsiveness of CAB3::pBVRplants to Wc light. By contrast with the elongatedhypocotyls ofWc-grown 35S::pBVR3 and CAB3::pBVRseedlings, MERI5::pBVR plants showed wild-type,light-dependent hypocotyl growth inhibition (Figs.3A and 4).To more fully assess the effect of spatial-specific

expression of pBVR on phytochrome-mediated hypo-cotyl growth inhibition, the fluence rate dependence ofhypocotyl lengths for MERI5::pBVR and CAB3::pBVR

lines under continuous R (Rc) and continuous FR (FRc)light was determined (Fig. 4). As was observed underWc, MERI5::pBVR lines responded to Rc and FRcsimilarly to wild-type seedlings (Fig. 4, D and F).CAB3::pBVR lineswere also deficient in light-mediatedhypocotyl growth inhibition under Rc (Fig. 4C). UnderRc conditions, CAB3::pBVR lines exhibited limitedresponsiveness, but 35S::pBVR lines were impairedmore significantly under these conditions. By contrast,CAB3::pBVR lines were at least as deficient in theirresponse to FRc as the 35S::pBVR3 transgenic seedlings(Fig. 4E). Furthermore, under FRc, CAB3::pBVR linesexhibited closed cotyledons similar to a null phyAmutant (Salk_014575; Ruckle et al., 2007), whereaswild-type and 35S::pBVR3 line cotyledons were openand yellow (Fig. 3C). Notably, approximately 1.5 h ofexposure to ambient white light during imaging re-sulted in enhanced greening of CAB3::pBVR lines, in-dicating that these lines are defective in the FR block togreening response similar to phyA seedlings (Fig. 3C).CAB3::pBVR lines also were the only lines to exhibit adeficiency in the FRc-mediated suppression of hypo-cotyl negative gravitropism (data not shown). Thus,CAB3::pBVR lines exhibit deficiencies in FR-high irra-

Figure 4. Fluence response curvesfor hypocotyl length of wild-typeand transgenic BVR seedlings.CAB3::pBVR and MERI5::pBVRlines are compared with No-Owild-type seedlings grown at 20�Con Phytablend medium containing1% Suc for 7 d under Wc, Rc, andFRc illumination of various fluencerates. Data points represent mean(6SD) of hypocotyl lengths mea-sured for 10 to 25 seedlings ineach of three independent experi-ments and correspond to No-Owild-type (n), 35S::pBVR3 (d),CAB3/MERI5::pBVR1 (¤, gray),CAB3/MERI5::pBVR2 (:, gray), andCAB3/MERI5::pBVR3 (n, gray) lines.





diance response (HIR) phenotypes that have beenpreviously observed for phyA and hfr1mutants (Barneset al., 1996; Fairchild et al., 2000, Yanovsky et al., 2002).

A distinctive phenotype associated with meristem-and/or leaf primordia-localized phytochrome defi-ciencies was that the average rosette diameter ofMERI5::pBVR lines was about 10% greater than wildtype only when plants were grown under short-day(SD) photoperiods (Fig. 5). This phenotype was notobserved in either CAB3::pBVR (Fig. 5) or 35S::pBVRlines (Montgomery et al., 1999). This increase in rosettediameter under SD photoperiod was determined toarise from an increase in the width to length ratios ofMERI5::pBVR leaves as compared to wild-type andCAB3::pBVR lines (data not shown).

Chlorophyll Accumulation Depends upon Bilin

Production in Photosynthetic Tissues

Previous findings showed that constitutive expres-sion of BVR in both Arabidopsis and tobacco (Nicoti-ana tabacum) significantly reduced chlorophyll andprotochlorophyll (PChl) accumulation when targeted

to plastids (Montgomery et al., 1999, 2001). Plants ofthe 35S::pBVR lines were notably intolerant of elevatedfluences of light and exhibited a striking reduction intotal chlorophyll and a coincident increase in thechlorophyll a/b (chla/chlb) ratio under these condi-tions. To assess the effect of site-specific BVR expres-sion on chlorophyll accumulation, we determined thefluence rate dependence of chlorophyll accumulationin light-grown CAB3::pBVR and MERI5::pBVR seed-lings. Like 35S::pBVR transgenics, CAB3::pBVR seed-lings showed a general decrease in total chlorophylllevels and a measurable increase in chla/chlb ratios(Fig. 6, A and C). By contrast to 35S::pBVR3, nodependence on light fluence rates was observed forchla/chlb ratios for the CAB3::pBVR lines in the flu-ence range tested. Total chlorophyll levels for light-grown MERI5::pBVR seedlings were slightly less thanfor wild type at low fluences yet comparable to wildtype at high fluences (Fig. 6B). Unlike the 35S::pBVRand CAB3::pBVR lines, chla/chlb ratios for MERI5::pBVR seedlings were indistinguishable from wild-type seedlings throughout the range of fluence ratesmeasured (Fig. 6D). We also determined the impact of

Figure 5. Rosette morphology of transgenic BVRplants under SD photoperiod. No-O wild-type (WT)and representative CAB3::pBVR and MERI5::pBVRlines were grown on a soil mixture for 40 d in acontrolled environment growth chamber at 25�Cunder Wc illumination of 100 mmol m22 s21 withan 8-h-light/16-h-dark SD photoperiod. Shown be-low are average rosette diameters (6SD, n = 20).

Figure 6. Fluence rate-dependentchlorophyll accumulation of wild-type and transgenic BVR seedlings.Transgenic CAB3::pBVR (CAB-1 toCAB-3), MERI5::pBVR (MERI-1 toMERI-3), and No-O wild-type seed-lings were grown at 22�C on Phyta-blend medium containing 1% Suc for7 d under Wc illumination of variousfluence rates. Data points represent themean obtained from three indepen-dent measurements. A and B, Totalchlorophyll. C and D, chla/chlb ratios.The symbol designations are the sameas those in Figure 4.





selective phytochrome inactivation on PChl accumu-lation in dark-grown seedlings. PChl accumulationwas largely unaffected in CAB3::pBVR and MERI5::pBVR lines (Fig. 7), with the exception of MERI5::pBVR3, which exhibited intermediate levels of PChl ascompared to wild type and 35S::pBVR3.

Anthocyanin Accumulation Is Altered inCAB3::pBVR-Expressing Seedlings

The previous finding that constitutive BVR expres-sion inhibits Suc-stimulated anthocyanin synthesis intransgenic Arabidopsis plants (Montgomery et al.,1999, 2001) supports the recognized regulatory roleof phytochromes in anthocyanin synthesis (Kim et al.,2003; Shin et al., 2007). To determine whether thelocalized phytochrome deficiencies distinctly affectedanthocyanin accumulation, we compared Suc-stimulatedanthocyanin accumulation in Wc-grown CAB3::pBVRand MERI5::pBVR plants with that of wild type and35S::pBVR3. Anthocyanin synthesis was reduced in allof the CAB3::pBVR lines to a level comparable to thatof the constitutive BVR-expressing 35S::pBVR3 line(Fig. 8A). Two of the CAB3::pBVR lines, CAB3::pBVR2and CAB3::pBVR3, also exhibited a reduced responseto Suc, compared to that observed for No-O wild type(see fold increase, Fig. 8A). The remaining CAB3::pBVR line, CAB3::pBVR1, showed an “enhanced”response to Suc, which might reflect the insertion ofthe CAB3::pBVR3 transgene near a Suc regulatorylocus. By contrast with the CAB3::pBVR lines, MERI5::pBVR lines did not exhibit significant changes inanthocyanin levels, nor did they show reduction inthe response to Suc (Fig. 8A).Given the marked disruption of FR-HIR responses

for hypocotyl elongation and the suppression of neg-ative gravitropism in CAB3::pBVR lines noted above,

we also investigated the impact of monochromatic Rcand FRc illumination on the accumulation of antho-cyanin in the presence of Suc using a representativeCAB3::pBVR line, 35S::pBVR3, null phyA and phyB(Salk_022035; Mayfield et al., 2007; Ruckle et al., 2007)

Figure 7. PChl accumulation of wild-type and transgenic BVR seed-lings. Transgenic CAB3::pBVR (CAB-1 to CAB-3), MERI5::pBVR (MERI-1to MERI-3), and No-O wild-type seedlings were grown at 22�C onPhytablend medium containing 1% Suc for 7 d in darkness. Barsindicate the relative fluorescence values calculated by normalizing tothe fluorescence value of No-O wild-type seedling extracts. Valuesrepresent the means (6SD) of three independent measurements.

Figure 8. Anthocyanin content of wild-type and transgenic BVR seed-lings. A, Transgenic 35S::pBVR3, CAB3::pBVR, and MERI5::pBVR linesand No-O wild-type seedlings were grown at 22�C on Phytablendmedium containing 1% Suc for 5 d under Wc illumination of 100 mmolm22 s21. Black bars (1% Suc) and white bars (no Suc) represent themean (6SD) of three independent measurements. Numbers belowgraph represent the fold increase in anthocyanin levels for seedlingsgrown in the presence of Suc versus those grown in the absence of Suc.B, No-O wild type, 35S::pBVR3, CAB3::pBVR2, Columbia-O (Col-O)wild type, and phyB (Salk_022035) were grown at 20�C on Phytablendmedium containing 1% Suc for 4 d under Rc illumination of 50 mmolm22 s21. Black bars (Rc) and white bars (dark) represent the mean (6SD)of three independent measurements. C, No-O wild type, 35S::pBVR3,CAB3::pBVR2, Columbia wild type, and phyA (Salk_014575) weregrown at 20�C on Phytablend medium containing 1% Suc for 4 d underFRc illumination of 5 mmol m22 s21. Black bars (FRc) and white bars(dark) represent the mean (6SD) of three independent measurements.





T-DNA insertion mutants, and the cognate wild-typeparents. Anthocyanin accumulation in Rc light waslow for all lines, with the CAB3::pBVR2 line havingless anthocyanin than all of the other lines, includingthe phyBmutant compared to its wild-type parent (Fig.8B). Notably, CAB3::pBVR lines completely lack theFR-HIR response of accumulating anthocyanins inresponse to FRc, identical to the phyA null mutant(Fig. 8C).

Carotenoid Accumulation Is Altered inCAB3::pBVR-Expressing Seedlings

Arabidopsis phytochromes, specifically phyA andlight-stable phytochromes other than phyB, have beenimplicated in the regulation of carotenoid biosyntheticgenes (von Lintig et al., 1997). To compare the impactof constitutive and localized phytochrome deficienciesinduced by BVR on carotenoid accumulation, wedetermined the carotenoid content of No-O wild-type, 35S::pBVR3, CAB3::pBVR, and MERI5::pBVRseedlings. These analyses indicated that constitutive(i.e. 35S-promoter driven) BVR expression yielded asignificant decrease in carotenoid levels. By compari-son, carotenoid accumulation was moderately re-duced in all three CAB3::pBVR lines but in only oneof the MERI5::pBVR lines (Fig. 9).

DISCUSSION

Directed Expression of BVR Reveals Spatial-SpecificRegulatory Roles for Individual Phytochrome Isoforms

These studies support the use of spatial-specificpromoters to drive BVR expression as an effective toolfor generating transgenic plants with distinct subsetsof phytochrome-deficient phenotypes. CAB3::pBVRexpression was regulated both spatially and by light,

whereas MERI5::pBVR expression was regulated spa-tially (Figs. 1 and 2). These diverse profiles of BVRexpression led to subsets of phytochrome-deficientphenotypes that differed substantially from each other,as well as those observed for constitutive 35S::pBVRexpression.

Specifically, CAB3::pBVR expression affected hypo-cotyl growth inhibition considerably under FRc illu-mination, to a lesser degree under Rc illumination, andmoderately under Wc illumination. As the analysisof publicly available gene expression data using theeFP browser (http://bbc.botany.utoronto.ca/efp/development/; Winter et al., 2007) indicated that ex-pression of CAB3 is relatively similar in Rc, FRc, Bc, orWc light, we ruled out the possibility that the pheno-typic differences observed for different monochro-matic light conditions reflect differences in the levelsof BVR expression; rather, these results point to uniqueregulatory roles for distinct phytochromes. For exam-ple, the complete inability of CAB3::pBVR lines toinhibit hypocotyl growth (Fig. 4E) and induce antho-cyanin accumulation (Fig. 8C) under FRc light sug-gests that CAB3::pBVR expression induces a severephytochrome deficiency in photosynthetic tissues.Previous studies have shown that both PHYA andPHYC are expressed abundantly in Arabidopsis coty-ledons (Toth et al., 2001). FRc conditions are those forwhich phyA has been assessed as the phytochromeresponsible for the inhibition of hypocotyl elongation(Nagatani et al., 1993; Parks and Quail, 1993; Whitelamet al., 1993), whereas phyC has not been shown to havea highly significant role in FR-mediated inhibition ofhypocotyl elongation (Monte et al., 2003; Balasubramanianet al., 2006). Thus, the elongated hypocotyls of CAB3::pBVR lines under FRc illumination, which corre-sponds to a phytochrome deficiency in photosynthetictissues, suggests that leaf-localized phyA is primarilyresponsible for initiating a signaling cascade thatresults in the FR-mediated inhibition of hypocotylelongation. This FR-dependent hypocotyl elongationphenotype is consistent with an early finding thatcotyledons are the site of a phytochrome-mediatedsignal that controls hypocotyl growth inhibition (Blackand Shuttleworth, 1974) and a more recent finding thatFR perception by cotyledons impacts gene expressionin the hypocotyl (Tanaka et al., 2002). Furthermore, thedistinct responses of CAB3::pBVR seedlings to Rcversus FRc illumination may be related to the distinctpatterns of expression of PHYA and PHYB observed incotyledons (Toth et al., 2001).

Careful analysis of the pattern of growth of thehypocotyls of CAB3::pBVR lines under FRc illumina-tion indicated that the hypocotyls of these lines arelonger than those of the 35S::pBVR3 line (Fig. 4E).When assessing these lines relative to their individualdark controls, the 35S:pBVR3 line is virtually blind toFR light and exhibits hypocotyls that are nearly iden-tical in length to dark-grown controls. Under theseconditions, CAB3::pBVR lines perceive FR light andinduce hypocotyl elongation. These observations sug-

Figure 9. Carotenoid content of wild-type and transgenic BVR plants.Transgenic CAB3::pBVR and MERI5::pBVR lines were compared withNo-O wild-type seedlings grown at 22�C for 7 d under white illumi-nation of 100 mmol m22 s21. Bars represent the mean (6SD) of threeindependent carotenoid measurements.





gested that apart from the disruption of FR-mediatedinhibition of hypocotyl elongation, CAB3::pBVR linesexhibit a FR-dependent induction of hypocotyl growth(Fig. 4E). As CAB3::pBVR lines do not accumulate thechromophore-degrading BVR enzyme in hypocotyl tis-sues and thus functional phytochromes persist in thehypocotyls of CAB3::pBVR plants, this FR-dependentinduction of hypocotyl growth is likely dependent onhypocotyl-localized phytochrome signaling. This re-sponse is distinct from that observed for CAB3::pBVRseedlings grown in Rc light, which exhibit extremelylimited responsiveness to Rc in regards to inhibitinghypocotyl elongation that is less than that observed forthe 35S::pBVR3 line (Fig. 4C). Notably, prior resultsdemonstrated that cotyledon-specific expression ofPHYB in a phyB-deficient background was nearly aseffective as expression throughout the seedling drivenby the native promoter in restoring Wc-dependentinhibition of hypocotyl elongation (Endo et al., 2005).Thus, ourobservations of CAB3::pBVR lines, in additionto suggesting novel insight into the role of cotyledon-localizedphyA, corroborate the suggestionof Endo et al.(2005) that phyB in the cotyledons is the major site ofR light perception for the regulation of seedling de-etiolation.CAB3::pBVR lines showed decreased levels of chlo-

rophyll and carotenoid levels. As leaves are the pri-mary site of synthesis for these pigments, reduction inthe levels of these pigments as a response to inactivat-ing phytochromes in photosynthetic tissues is antici-pated. As the levels of these pigments are furtherreduced in 35S::pBVR lines, phytochromes in photo-synthetic tissues are either not entirely responsible forthis response or the reduction of phytochrome levels inCAB3::pBVR lines was not sufficient to observe acomplete dampening of the accumulation of thesepigments, while being sufficient for disrupting FR-mediated inhibition of hypocotyl elongation completely.MERI5::pBVR transgenic lines also exhibit leaves thathave a larger surface area than either No-O wild-typeor CAB3::pBVR lines under SD photoperiods (Fig.5). These results may suggest that meristem- or leafprimordia-localized phytochromes affect primary leafexpansion.Anthocyanin accumulation was impacted uniquely

for the lines assessed. CAB3::pBVR lines had reducedanthocyanin in Rc growth conditions (Fig. 8B). Thisresponse was different from that observed for a phyBmutant and thus suggests that phyA or light-stablephytochromes other than phyB have a role in theinduction of anthocyanin accumulation in R light.CAB3::pBVR completely lacked the FR-HIR responseof inducing anthocyanin accumulation in response toFRc light (Fig. 8C). The response observed for CAB3::pBVR lines was identical to that of a phyA mutant.Other FR-HIR responses, including cotyledon separa-tion and FR block to greening, were also disrupted inCAB3::pBVR lines (Fig. 3C).In summary, our investigations indicate that spa-

tially distinct pools of phytochrome perceive light and

initiate intercellular and interorgan signaling path-ways that regulate distinct aspects of de-etiolation andSD-dependent responses. Meristematic- and/or leafprimordia-specific phytochromes have a role in plantresponsiveness that has not been previously reported.Our results also demonstrate a role for mesophyll-specific phyA in the regulation of numerous FR-HIRresponses, including the inhibition of hypocotyl elon-gation, suppression of negative gravitropism, and theinduction of anthocyanin accumulation. Our findingsalso support a potential role for hypocotyl-localizedphyA in the induction of cell elongation. The definitiveidentification of the molecular effectors involved inthese spatial-specific phytochrome responses awaitsadditional experimentation.

MATERIALS AND METHODS

Plasmid Constructions

The plasmid pCAB3/KS+ was obtained by subcloning a 1-kb EcoRI-PstI

fragment containing the CAB3 promoter (Mitra et al., 1989) from the plasmid

pUC13, a generous gift from J. Chory (Salk Institute, La Jolla, CA), into the

EcoRI and PstI sites of the pBluescript II KS+ vector (Stratagene). We

constructed the plant transformation vector pBIB/CAB3-TPBVR by perform-

ing a triple ligation of a 1.2-kb EcoRV-SstI TPBVR fragment consisting of a

translational fusion between a chloroplast-targeting signal from the small

subunit of Rubisco (Berry-Lowe et al., 1982) and BVR cDNA from the plasmid

pTPBVR (Lagarias et al., 1997), a HindIII-SmaI CAB3 promoter fragment from

pCAB3/KS+, and the HindIII-SstI-digested vector pBIB-KAN (Becker, 1990).

We obtained the plant transformation vector pMON/TPBVR by subcloning a

1.2-kb BamHI-SmaI TPBVR fragment into the BglII-StuI-digested vector

pMON672 (Monsanto), which contained the MERI5 promoter (Medford

et al., 1991).

Plant Transformation and Materials

Plasmid pBIB/CAB3-TPBVR was mobilized into the LBA4404 strain of

Agrobacterium tumefaciens and plasmid pMON/TPBVR into the ABI strain

(Monsanto) by triparental conjugation using the helper plasmid pRK2013

(Ditta et al., 1980). Following selection of transconjugants on M9 agar medium

containing 50 mg/mL kanamycin and 50 mg/mL streptomycin, we trans-

formed Arabidopsis (Arabidopsis thaliana) No-O with each construct using a

standard Agrobacterium-mediated transformation (Clough and Bent, 1998).

Kanamycin selection of transformants was performed in 100- 3 25-mm petri

dishes on media containing 13 Murashige and Skoog salts (Gibco-BRL), 1%

(w/v) Suc, 50 mg/mL kanamycin, and 0.7% agar. The transgenic 35S::pBVR3

line was previously isolated (Lagarias et al., 1997).

Plant Growth Conditions

Arabidopsis seeds were surface-sterilized for 15 min with 35% (v/v)

commercial bleach containing 0.025% (v/v) SDS and rinsed at least 3 times

with ultrapure water (Milli-Q, Millipore). Sterilized seeds were plated in

100- 3 25-mm petri dishes containing 13 Murashige and Skoog salts, 0.9%

Phytablend (Caisson Laboratories), and 0 or 1% (w/v) Suc. During imbibition,

seeds were cold stratified at 4�C in darkness for 3 d. Stratified seeds were

transferred to temperature- and humidity-controlled growth chambers under

defined light conditions. For flowering experiments, seeds sterilized as

described were germinated directly on pots containing Arabidopsis mix

and grown in controlled-environment chambers at 25�C under SD (8-h-light/

16-h-dark cycle).

Light Sources

Wc illumination was provided by cool-white lights (F48FT12/CW/VHO,

Sylvania) or Grolux lights (F20T12/GRO and F20T12/GRO/WS) as described





(Montgomery et al., 1999). Rc, FRc, and Bc illumination was provided by

R-, FR-, and B-emitting diode sources (Percival). Light spectral quality and

fluence rates weremeasured using a spectroradiometer (model 1800, LI-COR),

and fluence rates were altered for fluence rate curves using neutral-density

filters (Lux no. III, Rosco). All irradiation experiments were performed in

controlled-environment chambers at constant temperature and humidity.

Hypocotyl Length Measurements

We determined the hypocotyl lengths of seedlings grown under defined

light conditions by scanning the seedling images and quantifying them using

Image J software.

Protein Extraction and Assays, BVR Enzyme Assays, andImmunochemical Analysis

Soluble protein extracts for BVR enzyme assays and immunoblot analyses

were obtained as described (Lagarias et al., 1997). Biochemical fractionation

and subsequent protein and enzyme analyses were performed as previously

detailed (Montgomery et al., 1999). BVR-specific enzyme activity was mea-

sured spectrophotometrically as previously described (Lagarias et al., 1997)

and quantified using equations described by Kutty and Maines (1984). Protein

concentrations of soluble extracts were determined utilizing the bicinchoninic

acid method (Smith et al., 1985) using bovine serum albumin as a standard.

SDS-PAGE and immunoblot analyses were performed as previously de-

scribed (Lagarias et al., 1997; Montgomery et al., 1999) using a 1:5,000 dilution

of rabbit anti-BVR antibody (QED Bioscience).

Whole-Mount Immunohistochemical Analysis

Sterilized MERI5::pBVR1 seeds were treated with a RL pulse (approxi-

mately 75 mmol m22 s21) for 5 min prior to imbibition. Four-day-old MERI5::

pBVR1 seedlings grown at 22�C under Wc illumination of 100 mmol m22 s21

were subjected to whole-mount in situ protein localization as previously

described with limited modifications (Sauer et al., 2006). Seedlings were

treated with a paraformaldehyde-based fixative solution for 45 min. Cell walls

of fixed seedlings were digested with 3% Driselase (Sigma), followed by

washing 3 3 10 min with 13 phosphate buffered saline (PBS). Fixed and

permeated seedlings were incubated with rabbit anti-BVR antibody at 1:2,500

dilution in 13 PBS overnight at 4�C. Following incubation with the primary

antibody and washing, seedlings were incubated with goat anti-rabbit IgG

(H+L) conjugated to HiLyte Plus 555 (AnaSpec) at 0.005 mg/mL dilution in 13PBS for 6 h at 37�C. Seedlings were imaged on an inverted Axiovert 200 Zeiss

LSM 510 Meta confocal laser scanning microscope (Carl Zeiss MicroImaging)

using differential interference contrast (DIC) optics and fluorescence excitation/

emission filters. A 203/0.75 Plan Apochromat objective lens was used for

imaging. DIC imaging was performed using the 543-nm laser. Fluorescence

from the secondary antibody was collected using a 543-nm laser for excitation

and a 560- to 615-nm band pass filter for emission. Each optical slice of a Z series

was collected as an average of two individual scans. Images were acquired

using the LSM FCS Zeiss 510 Meta AIM imaging software.

Pigment Analyses

N,N-dimethylformamide chlorophyll extracts were obtained from excised

cotyledons of 7-d-old seedlings at 4�C (Moran, 1982) and chlorophyll concen-

trations determined using extinction coefficients and equations described by

Inskeep and Bloom (1985). Similarly, N,N-dimethylformamide PChl extracts

were obtained under green safe light using intact 7-d-old seedlings (Moran,

1982). Fluorescence spectroscopy was utilized to determine the PChl content

in the extracts as previously described (Montgomery et al., 1999). Carotenoid

content was determined essentially as described (Montgomery et al., 2001).

After freezing in liquid nitrogen, 7-d-old seedlings were ground to a fine

powder and homogenized in 80% acetone. Extracts were then incubated at

220�C for 1 h and centrifuged at high speed in a microcentrifuge for 5 min at

4�C. Supernatants were assayed spectrophotometrically and carotenoid con-

centrations determined using equations from Lichtenthaler and Wellburn

(1983). Anthocyanins were extracted from intact 4- or 5-d-old seedlings and

concentrations determined spectrophotometrically as previously described

(Montgomery et al., 1999).

ACKNOWLEDGMENTS

We thank Dr. Kuo-Chen Yeh for preparing the DNA constructs used in

these studies, Stephanie Costigan for technical assistance with some of the

anthocyanin assays, Dr. J. Clark Lagarias (supported by U.S. Department of

Agriculture grant AMD–9503140) for assistance with these studies, the

laboratory of Dr. Robert Larkin for providing phyA and phyB seeds, and Dr.

Wei Hu for critically reading the manuscript and providing comments.

Received July 25, 2008; accepted October 26, 2008; published October 29, 2008.

LITERATURE CITED

Balasubramanian S, Sureshkumar S, Agrawal M, Michael TP, Wessinger

C, Maloof JN, Clark R, Warthmann N, Chory J, Weigel D (2006) The

PHYTOCHROME C photoreceptor gene mediates natural variation in

flowering and growth responses of Arabidopsis thaliana. Nat Genet 38:

711–715

Barnes SA, Nishizawa NK, Quaggio RB, Whitelam GC, Chua NH (1996)

Far-red light blocks greening of Arabidopsis seedlings via a phytochrome

A-mediated change in plastid development. Plant Cell 8: 601–615

Becker D (1990) Binary vectors which allow the exchange of plant select-

able markers and reporter genes. Nucleic Acids Res 18: 203

Berry-Lowe SL, Mc Knight TD, Shah DM, Meagher RB (1982) The

nucleotide sequence, expression, and evolution of one member of a

multigene family encoding the small subunit of ribulose-1,5-bisphos-

phate carboxylase in soybean. J Mol Appl Genet 1: 483–498

Black M, Shuttleworth JE (1974) The role of the cotyledons in the photo-

control of hypocotyl extension in Cucumis sativus L. Planta 117: 57–66

Bou-Torrent J, Roig-Villanova I, Martinez-Garcia JF (2008) Light signal-

ing: back to space. Trends Plant Sci 13: 108–114

Caubergs R, De Greef JA (1975) Studies on hook-opening in Phaseolus

vulgaris L. by selective R/FR pretreatments of embryonic axis and

primary leaves. Photochem Photobiol 22: 139–144

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agro-

bacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:

735–743

De Greef JA, Caubergs R (1972a) Interorgan correlations and phyto-

chrome: hypocotyl hook opening. Arch Int Physiol Biochim 80: 959–960

De Greef JA, Caubergs R (1972b) Interorgan correlations and phyto-

chrome: leaf expansion. Arch Int Physiol Biochim 80: 961–962

De Greef JA, Caubergs R, Verbelen JP (1971) Localization of a light

reaction in the apex during the greening process in etiolated plants.

Arch Int Physiol Biochim 79: 826–827

De Greef JA, Verbelen JP (1972) Interorgan correlations and phytochrome:

changes in plastid ultrastructure during de-etiolation processes. Arch

Int Physiol Biochim 80: 962–963

DeGreef JA, Verbelen JP, Moereels E (1975) Preliminary studies on the site

of primary action of light during de-etiolation processes of Phaseolus

seedlings. Arch Int Physiol Biochim 83: 957–958

Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA

cloning system for Gram-negative bacteria: construction of a gene bank

of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351

Emborg TJ, Walker JM, Noh B, Vierstra RD (2006) Multiple heme oxy-

genase family members contribute to the biosynthesis of the phyto-

chrome chromophore in Arabidopsis. Plant Physiol 140: 856–868

Endo M, Nakamura S, Araki T, Mochizuki N, Nagatani A (2005) Phyto-

chrome B in the mesophyll delays flowering by suppressing FLOWER-

ING LOCUS T expression in Arabidopsis vascular bundles. Plant Cell 17:

1941–1952

Fairchild CD, Schumaker MA, Quail PH (2000) HFR1 encodes an atypical

bHLH protein that acts in phytochrome A signal transduction. Genes

Dev 14: 2377–2391

Franklin KA, Larner VS, Whitelam GC (2005) The signal transducing

photoreceptors of plants. Int J Dev Biol 49: 653–664

Franklin KA, Whitelam GC (2004) Light signals, phytochromes and cross-

talk with other environmental cues. J Exp Bot 55: 271–276

Hiltbrunner A, Nagy F, Schafer E (2007) Phytochromes. In GCWhitelam, K

Halliday, eds, Light and Plant Development, Vol 30. Blackwell Publish-

ing, Oxford, pp 3–16

Inskeep WP, Bloom PR (1985) Extinction coefficients of chlorophyll a and b

in N,N-dimethylformamide and 80% acetone. Plant Physiol 77: 483–485





Jiao YL, Lau OS, Deng XW (2007) Light-regulated transcriptional networks

in higher plants. Nat Rev Genet 8: 217–230

Josse EM, Foreman J, Halliday KJ (2008) Paths through the phytochrome

network. Plant Cell Environ 331: 667–678

Kendrick RE, Kronenberg GHM (1994) Photomorphogenesis in Plants, Ed

2. Martinus Nijhoff Publishers, Dordrecht, The Netherlands

Kevei E, Schafer E, Nagy F (2007) Light-regulated nucleo-cytoplasmic

partitioning of phytochromes. J Exp Bot 58: 3113–3124

Kim J, Yi H, Choi G, Shin B, Song PS, Choi G (2003) Functional

characterization of phytochrome interacting factor 3 in phytochrome-

mediated light signal transduction. Plant Cell 15: 2399–2407

King RW, Zeevaart JA (1973) Floral stimulus movement in Perilla and

flower inhibition caused by noninduced leaves. Plant Physiol 51:

727–738

Kobayashi Y, Weigel D (2007) Move on up, it’s time for change: mobile

signals controlling photoperiod-dependent flowering. Genes Dev 21:

2371–2384

Kohchi T, Mukougawa K, Frankenberg N, Masuda M, Yokota A, Lagarias

JC (2001) The Arabidopsis HY2 gene encodes phytochromobilin synthase,

a ferredoxin-dependent biliverdin reductase. Plant Cell 13: 425–436

Kutty RK, Maines MD (1984) Hepatic heme metabolism: possible role of

biliverdin in the regulation of heme oxygenase activity. Biochem

Biophys Res Commun 122: 40–46

Lagarias DM, Crepeau MW, Maines MD, Lagarias JC (1997) Regulation of

photomorphogenesis by expression of mammalian biliverdin reductase

in transgenic Arabidopsis plants. Plant Cell 9: 675–688

Lichtenthaler HK, Wellburn AR (1983) Determination of total carotenoids

and chlorophylls a and b in leaf extracts in different solvents. Biochem

Soc Trans 11: 591–592

Mandoli DF, Briggs WR (1982) The photoperceptive sites and the function

of tissue light-piping in photomorphogenesis of etiolated oat seedlings.

Plant Cell Environ 5: 137–145

Mathews S (2006) Phytochrome-mediated development in land plants: red

light sensing evolves to meet the challenges of changing light environ-

ments. Mol Ecol 15: 3483–3503

Mayfield JD, Folta KM, Paul AL, Ferl RJ (2007) The 14-3-3 Proteins mu and

upsilon influence transition to flowering and early phytochrome re-

sponse. Plant Physiol 145: 1692–1702

Medford JI, Elmer JS, Klee HJ (1991) Molecular cloning and characteriza-

tion of genes expressed in shoot apical meristems. Plant Cell 3: 359–370

Mitra A, Choi HK, An G (1989) Structural and functional analyses of

Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters.

Plant Mol Biol 12: 169–179

Monte E, Alonso JM, Ecker JR, Zhang Y, Li X, Young J, Austin-Phillips S,

Quail PH (2003) Isolation and characterization of phyC mutants in

Arabidopsis reveals complex crosstalk between phytochrome signaling

pathways. Plant Cell 15: 1962–1980

Montgomery BL (2006) Plant photoreceptors and the photoperiodic in-

duction of flowering. In JA Teixeira da Silva, ed, Floriculture, Orna-

mental and Plant Biotechnology: Advances and Topical Issues, Ed 1, Vol

1. Global Science Books Limited, Middlesex, UK, pp 256–262

Montgomery BL (2008) Right place, right time: spatiotemporal light regulation

of plant growth and development. Plant Signal Behav 3: 1053–1060

Montgomery BL, Franklin KA, Terry MJ, Thomas B, Jackson SD, Crepeau

MW, Lagarias JC (2001) Biliverdin reductase-induced phytochrome chro-

mophore deficiency in transgenic tobacco. Plant Physiol 125: 266–277

Montgomery BL, Yeh KC, CrepeauMW, Lagarias JC (1999) Modification of

distinct aspects of photomorphogenesis via targeted expression of

mammalian biliverdin reductase in transgenic Arabidopsis plants. Plant

Physiol 121: 629–639

Moran R (1982) Formulae for determination of chlorophyllous pigments

extracted with N,N-dimethylformamide. Plant Physiol 69: 1376–1381

Muramoto T, Kohchi T, Yokota A, Hwang I, Goodman HM (1999) The

Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome

chromophore biosynthesis as a result of a mutation in a plastid heme

oxygenase. Plant Cell 11: 335–348

Nagatani A (2004) Light-regulated nuclear localization of phytochromes.

Curr Opin Plant Biol 7: 708–711

Nagatani A, Reed JW, Chory J (1993) Isolation and initial characterization

of Arabidopsis mutants that are deficient in phytochrome A. Plant

Physiol 102: 269–277

Nagy F, Kircher S, Schafer E (2001) Intracellular trafficking of photore-

ceptors during light-induced signal transduction in plants. J Cell Sci 114:

475–480

Nagy F, Schafer E (2000) Nuclear and cytosolic events of light-induced,

phytochrome-regulated signaling in higher plants. EMBO J 19: 157–163

Nick P, Ehmann B, Furuya M, Schafer E (1993) Cell communication,

stochastic cell responses, and anthocyanin pattern in mustard cotyle-

dons. Plant Cell 5: 541–552

Parcy F (2005) Flowering: a time for integration. Int J Dev Biol 49: 585–593

Parks BM, Quail PH (1993) hy8, a new class of Arabidopsis long hypocotyl

mutants deficient in functional phytochrome A. Plant Cell 5: 39–48

Quail PH (1994) Photosensory perception and signal transduction in

plants. Curr Opin Genet Dev 4: 652–661

Rosler J, Klein I, Zeidler M (2007) Arabidopsis fhl/fhy1 double mutant

reveals a distinct cytoplasmic action of phytochrome A. Proc Natl Acad

Sci USA 104: 10737–10742

Ruckle ME, DeMarco SM, Larkin RM (2007) Plastid signals remodel light

signaling networks and are essential for efficient chloroplast biogenesis

in Arabidopsis. Plant Cell 19: 3944–3960

Sauer M, Paciorek T, Benkova E, Friml J (2006) Immunocytochemical

techniques for whole-mount in situ protein localization in plants. Nat

Protocols 1: 98–103

Schafer E, Nagy F, editors (2006) Photomorphogenesis in Plants and

Bacteria: Function and Signal Transduction Mechanisms, Ed 3. Springer,

Dordrecht, The Netherlands

Schepens I, Duek P, Fankhauser C (2004) Phytochrome-mediated light

signalling in Arabidopsis. Curr Opin Plant Biol 7: 564–569

Sharrock RA, Quail PH (1989) Novel phytochrome sequences in Arabi-

dopsis thaliana: structure, evolution, and differential expression of a

plant regulatory photoreceptor family. Genes Dev 3: 1745–1757

Shin J, Park E, Choi G (2007) PIF3 regulates anthocyanin biosynthesis in an

HY5-dependent manner with both factors directly binding anthocyanin

biosynthetic gene promoters in Arabidopsis. Plant J 49: 981–994

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano

MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measure-

ment of protein using bicinchoninic acid. Anal Biochem 150: 76–85

Tanaka S-I, Nakamura S, Mochizuki N, Nagatani A (2002) Phytochrome

in cotyledons regulates the expression of genes in the hypocotyl through

auxin-dependent and -independent pathways. Plant Cell Physiol 43:

1171–1181

Terry MJ (1997) Phytochrome chromophore-deficient mutants. Plant Cell

Environ 20: 740–745

Terry MJ, Wahleithner JA, Lagarias JC (1993) Biosynthesis of the plant

photoreceptor phytochrome. Arch Biochem Biophys 306: 1–15

Toth R, Kevei E, Hall A, Millar AJ, Nagy F, Kozma-Bognar L (2001)

Circadian clock-regulated expression of phytochrome and crypto-

chrome genes in Arabidopsis. Plant Physiol 127: 1607–1616

Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen:

FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 59:

573–594

von Lintig J, Welsch R, Bonk M, Giuliano G, Batschauer A, Kleinig H

(1997) Light-dependent regulation of carotenoid biosynthesis occurs at

the level of phytoene synthase expression and is mediated by phyto-

chrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J 12:

625–634

Whitelam GC, Johnson E, Peng J, Carol P, Anderson ML, Cowl JS,

Harberd NP (1993) Phytochrome A null mutants of Arabidopsis display a

wild-type phenotype in white light. Plant Cell 5: 757–768

Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007)

An “electronic Fluorescent Pictograph” browser for exploring and

analyzing large-scale biological data sets. PLoS ONE 2: e718

Yanovsky MJ, Luppi JP, Kirchbauer D, Ogorodnikova OB, Sineshchekov

VA, Adam E, Kircher S, Staneloni RJ, Schafer E, Nagy F, et al (2002)

Missense mutation in the PAS2 domain of phytochrome A impairs

subnuclear localization and a subset of responses. Plant Cell 14:

1591–1603

Yu X, Lin C (2005) Light regulation of flowering time in Arabidopsis. In M

Wada, K Shimazaki, M Iino, eds, Light Sensing in Plants. Springer-

Verlag, Tokyo, pp 325–332

Zeevaart JA (2006) Florigen coming of age after 70 years. Plant Cell 18:

1783–1789





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