Phytochromes are the sole photoreceptors forperceiving red/far-red light in riceMakoto Takanoa,1, Noritoshi Inagakia, Xianzhi Xiea,2, Seiichiro Kiyotaa, Akiko Baba-Kasaia, Takanari Tanabataa,b,and Tomoko Shinomurab

aDepartment of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan; and bHitachi Central Research Laboratory,Hatoyama, Saitama 350-0395, Japan

Communicated by Winslow R. Briggs, Carnegie Institution of Washington, Stanford, CA, July 7, 2009 (received for review November 17, 2008)

Phytochromes are believed to be solely responsible for red andfar-red light perception, but this has never been definitively tested.To directly address this hypothesis, a phytochrome triple mutant(phyAphyBphyC) was generated in rice (Oryza sativa L. cv. Nip-ponbare) and its responses to red and far-red light were moni-tored. Since rice only has three phytochrome genes (PHYA, PHYBand PHYC), this mutant is completely lacking any phytochrome.Rice seedlings grown in the dark develop long coleoptiles whileundergoing regular circumnutation. The phytochrome triple mu-tants also show this characteristic skotomorphogenesis, even un-der continuous red or far-red light. The morphology of the triplemutant seedlings grown under red or far-red light appears com-pletely the same as etiolated seedlings, and they show no expres-sion of the light-induced genes. This is direct evidence demonstrat-ing that phytochromes are the sole photoreceptors for perceivingred and far-red light, at least during rice seedling establishment.Furthermore, the shape of the triple mutant plants was dramati-cally altered. Most remarkably, triple mutants extend their inter-nodes even during the vegetative growth stage, which is a timeduring which wild-type rice plants never elongate their internodes.The triple mutants also flowered very early under long day con-ditions and set very few seeds due to incomplete male sterility.These data indicate that phytochromes play an important role inmaximizing photosynthetic abilities during the vegetative growthstage in rice.

internodal elongation � mutant � phytochrome � photoreceptor

L ight is one of the most important ambient signals for plants.To respond properly to environmental changes plants have

evolved multiple photoreceptor systems, including the phyto-chromes, cryptochromes, and phototropins, for perceiving lightsignals over a broad range of wavelength and intensity. Phyto-chromes mainly perceive red (R)/far-red light (FR), while thecryptochromes and phototropins recognize UV-A and blue light(1, 2). Among these groups, the phytochromes have a longhistory as a topic of research.

It had been known since the 19th century that light promotesgermination of certain seeds. Borthwick et al. (3) obtained thefirst quantitative action spectrum for light-induced germinationusing Grand Rapids lettuce seed. The curve had a shape withmaximum sensitivity in the red region (�660 nm). Furthermore,they discovered that the inducing effect of R was cancelled iffollowed by FR irradiation, and that even after alternatingbetween R and FR many times, the last treatment determinedthe outcome (3). Therefore, they predicted that the unidentifiedphotoreceptor was a photoreversible pigment. Using a specialspectrophotometer, Butler et al. (4) demonstrated the presenceof the photoreversible pigment, partially purified this substancefrom etiolated shoots of maize, and named it phytochrome. Thiswork tentatively brought an end to the argument concerning theidentity of phytochrome; however, further attempts to correlatephysiological responses of the plants to phytochrome contentwere frustratingly unsuccessful (5, 6). It was not until themid-1980s that immunochemical studies revealed that there was

more than one phytochrome protein (7–9). Around the sametime, Arabidopsis became a plant model for molecular geneticanalyses, and the identified sequences demonstrated that phy-tochromes are encoded by small gene families (10–12). Further-more, isolation and characterization of phytochrome mutantshave elucidated the distinct features of the individual phyto-chrome isoforms.

The completed whole genome sequences of Arabidopsis andrice made it clear that Arabidopsis contains five PHY genes,PHYA to PHYE, while rice has only three genes, PHYA, PHYB,and PHYC. Molecular phylogenetic analyses suggest that the firstgene duplication gave rise to the progenitors of PHYA andPHYC, along with the PHYB and PHYE subfamilies. The secondduplication is thought to have occurred before the formation ofgymnosperms, producing the separate PHYA and PHYC sub-families, because both monocotyledons and dicotyledons con-tain representatives of the PHYA, PHYB, and PHYC subfamilies.In dicotyledonous plants, the PHYB progenitors presumablyunderwent a further duplication, producing the PHYB and PHYEsubfamilies. In Arabidopsis, PHYD was further derived from anancestral PHYB gene by a recent gene duplication event (11).However, gramineous plants lack multiple members of PHYBsubfamily, and basically have three genes, PHYA, PHYB, andPHYC. Maize has two copies each of these three genes becauseof chromosomal duplication (13).

To date, many phytochrome mutants have been isolated anda variety of different mutants have been produced by crossingthese multiple mutants with each other. However, there havebeen no reports of phytochrome null mutants that lack allfunctional phytochromes. Phytochromes are considered as prin-cipal photoreceptors that perceive R and FR. But direct evi-dence demonstrating this fact by obtaining and analyzing mu-tants completely lacking all phytochrome proteins remains to bepresented. Since Arabidopsis has five different phytochromegenes (PHYA � PHYE), quintuple mutants would be needed togenerate phytochrome-free mutants, which would be ratherdifficult. To date, quadruple mutants (phyAphyBphyDphyE andphyBphyCphyDphyE) have been produced and analyzed (14, 15),but the quintuple mutant remains to be studied. The tomato alsohas five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE, andPHYF) and a triple mutant (phyAphyB1phyB2) is the most thathas been reported (16). In contrast, rice only has three phyto-chrome genes, which makes it simpler to produce the phyto-chrome null mutant (that is, the triple mutant).

Author contributions: M.T. designed research; M.T., N.I., X.X., and T.S. performed research;T.T. contributed new reagents/analytic tools; S.K., A.B.-K., and T.T. analyzed data; and M.T.wrote the paper.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: mtakano@affrc.go.jp.

2Present address, High-Tech Research Center, Shandong Academy of Agricultural Sciences,Jinan, Shandong, 250100 China.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907378106/DCSupplemental.

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There is another class of phytochrome-deficient mutantswhich have defects in the synthesis of chromophore. Mutationsin the chromophore biosynthesis pathway affect all phytochromespecies and, as such, have been widely used as phytochrome-deficient plants for photomorphogenetic studies, such as hy1 (17)and hy2 (18, 19) in Arabidopsis, yg-2 (20) and au (21, 22) intomato, pcd1 (23) and pcd2 (24) in pea, se5 (25) in rice, and soon. Strictly speaking, however, these mutants cannot be equatedto phytochrome-deficient mutants. They are considered leakybecause the heme oxygenase or phytochromobilin synthase genesthat are impaired in these mutants are both single members ofa small gene family (26). In addition, these chromophore-deficient mutants generally also have reduced levels of chloro-phyll (27). The phytochrome chromophore, phytochromobilin issynthesized from 5-aminorevulinic acid via the heme branch ofthe tetrapyrrole pathway. The early steps in the pathway, up toprotoporphyrin IX, are common to both heme and chlorophyllsynthesis. Thus, it is reasonably expected that defects in onebranch of the tetrapyrrole biosynthesis pathway might also affectthe other.

In our prior work, all of the phytochrome single mutants, andall combinations of double mutants, have been isolated and thedistinct features of the individual phytochrome species, and theinteractions between them, have been elucidated (28, 29). In thisstudy, we generated triple phytochrome mutants in rice anddemonstrated that these mutants are blind to R and FR in termsof the kinetics of coleoptile growth and the expression oflight-induced genes. Furthermore, the triple mutants grow wellbut the shape of the plant changes dramatically. For example,triple mutants extend their internodes even in the vegetativegrowth stage, a time when the internode is never elongated inwild-type plants. In addition, the lengths of the leaf bladesbecame shorter and, as a result, the shape of triple mutant plantis much different from that of the wild-type rice plants and moresimilar to that of gramineous weeds. These data indicate thatphytochromes play important roles during normal morphogen-esis in rice.

ResultsGeneration of Phytochrome Triple Mutants. The phyAphyC doublemutants (phyA-2phyC-1 and phyA-4phyC-1) were generated bycrossing phyA mutants (phyA-2 and phyA-4) with a phyC mutant(phyC-1) (29). Because the PHYA and PHYC genes are locatedin close proximity with each other on Chr.3 (10.1 cM apart), theprobability of getting phyAphyC double mutants was low. How-ever, once they were obtained, the phyA and phyC mutant allelesmostly behaved together like a single mutant allele. ThesephyAphyC double mutants were then crossed with phyB mutants(phyB-1 and phyB-2), and four different triple mutants wereproduced. This study examined all four of the triple mutants andobtained substantially the same results for all properties tested.The data obtained from the phyA-4phyB-1phyC-1 triple mutantare presented as representative results.

Seedlings of Triple Mutants are Blind to R and FR. In the dark, riceseedlings grow long coleoptiles with a regular circumnutation(30). An imaging system was developed to collect images ofgrowing rice seedlings every 10 min over a 1-week period (31).The big advantage of this monitoring system is that it usesinfra-red LED (peak at 950 nm) as a light source to monitor thegrowing behavior of rice seedlings under dark conditions. In Fig.1, the movements of the coleoptile tips and the first/secondleaves were traced. Orange lines show the traces of the coleoptiletips and green lines show the first/second leaves as they emergefrom the coleoptiles. The original time-lapse videos are includedin Movie S1. In the dark (Fig. 1, left of each pair), all seedlingstested (Nipponbare, phyB-1, phyB-1phyC-1, phyA-4phyB-1phyC-1, and se5) had long coleoptiles growing with circumnu-

tation (zigzag orange lines). This is a typical skotomorphogenesisfor rice. In contrast, under continuous R (Rc) (Fig. 1, right ofeach pair), coleoptiles of all except the triple mutant stoppedgrowing early without circumnutation (short, straight orangelines) and the leaves (green lines) emerged. The phytochrometriple mutant (phyA-4phyB-1phyC-1) showed the same growthpattern as seen in the dark (Fig. 1, phyAphyBphyC, surroundedby a red border). The responses of germinating seedlings to Rcwere also examined in the other phytochrome mutants and onlythe phyAphyB double mutant showed the same behavior as thetriple mutant. Since the PHYC protein level was greatly reducedin phyAphyB double mutants, the phyAphyB double mutants areessentially considered to be triple mutants (29). These resultsclearly show that the R-induced inhibition of coleoptile growthin rice is mediated by phytochromes alone. The same experi-ments were performed under FRc and the results were the same,indicating that phytochromes are also responsible for FR per-ception. Interestingly, the se5 mutant responded in the same wayas the wild type to Rc (Fig. 1, se5).

Expression of Light-Induced Genes in the Triple Mutants. Seedlings ofthe triple mutants that were grown under Rc looked the same asetiolated seedlings; no greening was observed. The chlorophylllevel was below detectable limits. To investigate this de-etiolation process at the level of gene expression, RNA wasisolated from the plants analyzed in Fig. 1B (grown for 7 daysunder Rc) and the expression of the LHCB and RBCS genes wasexamined by Northern hybridization. Since whole sequence ofLHCB (accession number, D0062) or RBCS (X07515) cDNAwas used to generate the probe, respectively, for the northernhybridization, detection of all members of each gene family wasexpected. As shown in Fig. 2, R induced both the LHCB andRBCS genes equally in wild-type (WT, Nipponbare), phyB-1, andphyB-1phyC-1 plants, but not at all in the phytochrome triplemutant (phyA-4phyB-1phyC-1). It has been already reported thatthe induction of the LHBC and RBCS genes in response to R wasalso completely diminished in phyAphyB double mutants (29).Thus, these results support the idea that the phyAphyB doublemutants can essentially be considered as a triple mutant.

The general expression pattern of light-induced genes wasexamined by microarray analysis. Four-day-old etiolated seed-lings of WT and triple mutants were exposed to an R pulse andharvested 30 min and 4 h after the pulse. Seedlings isolatedbefore the pulse treatment were used as a dark control. Genesthat were either induced or suppressed by a pulsed R wereidentified by two-dye microarray analysis. Wild type seedlingsshowed a total of �400 genes that increased or decreased theirexpression level more than twice or less than half, respectively,

Fig. 1. Traces of the shoot-top movement of growing rice seedlings. Fivelines of rice seedlings were grown in the monitoring system either in the dark(Left of each pair, labeled ‘‘Dark’’) or under the continuous red light (Right ofeach pair, labeled ‘‘Red’’) for 1 week. Only the top point pixels of the growingseedlings were extracted from each individual time-lapse image and overlaidonto the final picture. Orange lines, movement of coleoptile tops; green lines,movement of the first/second leaf tops. WT, Nipponbare; phyB, phyB-1 mu-tant; phyBC, phyB-1phyC-1 mutant; phyABC, phyA-4phyB-1phyC-1 mutant;se5, se5 mutant.

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at 30 min or 4 h after an R pulse treatment. By contrast, neitherinduction (more than twice) nor suppression (less than half) ofthe genes was detected by an R pulse treatment in the triplemutant (Fig. S1, SI Text, and Table S1).

Unusual Internode Elongation in the Phytochrome Triple Mutants. Theshape of the adult triple mutant plant (1 month old) is obviouslydifferent from that of the WT (Fig. 3A) or phytochrome singlemutants. In the vegetative phase, the rice leaves typically comeout from the basal part of the shoot where nonelongationinternodes are located in a multilayered stack (Fig. 3A, Center,Left). However, the triple mutant leaves appear to come outfrom the middle of the stems (Fig. 3A, Center, Right). To discoverthe reason for this difference, young WT and mutant plants, asshown in Fig. 3A, were dissected and found to have internodesthat were prematurely elongated in the triple mutant (Fig. 3A,Right, white arrow shows a shoot apex). This is in clear contrastto the WT plants, in which the internodes were all stackedwithout elongation (Fig. 3A, Left),

The unusual internode elongation in the triple mutants wasinvestigated more precisely by measuring the lengths of differentparts of the growing seedlings. Fig. 3B shows the growing profilesof each piece of WT (Left) and triple mutant seedlings (Right).A characteristic leaf growth pattern was observed, that is, newlyemerging leaves extend only leaf blades first (blue lines in Fig.3B) and then leaf sheaths (green lines) start growing a few dayslater. These growth patterns and the timing of the leaf extractionwere similar in both the WT and triple mutant, but the finallength of the leaf sheaths of the second, and the leaf blades andsheaths of the third and fourth leaves were clearly shorter in themutant than in WT. The biggest difference is that all internodeswere elongated in the triple mutant (purple lines) but not in theWT. As shown in the bottom right graph in Fig. 3B, the fourthinternode had elongated to 20 mm by 16 days after germination.The growth profiles of each part of the seedlings were examinedin other phytochrome mutants (phyA, phyB and phyAphyB) aswell (Fig. S2). The growth profiles of phyA and phyAphyBseedlings were almost the same as those of the WT and the triplemutant, respectively. The phyB seedlings showed an intermedi-ate pattern between the WT and the triple mutant, but nointernode elongation was detected in the phyB seedlings. There-

fore, phyB seems to play a dominant role, in conjunction withphyA, to expand leaf blades and sheaths in response to the lightstimuli. However, internode elongation is suppressed in thepresence of either phyA or phyB.

Characteristic Plant Shapes of the Mature Triple Mutants. Matureplants grown in the field for 66 days were compared among WTand phytochrome mutants (Fig. S3A). Plant shapes of phyto-chrome single and double mutants except for phyAphyB weresimilar to that of WT. The chromophore mutant, se5 also showeda normal shape, although color of leaves was lighter than theothers. The phyAphyB double mutants and triple mutants grewwell in the field but they were dwarf and their shapes were verydifferent from that of WT plants, leaves were short and bendedat a right angle. Protein levels of each phytochrome wereexamined in these plants to confirm the mutations (Fig. S3B).PHYA levels were lower even in WT because of the light-labileproperty of phyA, while PHYA apo-protein was stable in se5.Protein levels of PHYC were reduced in the phyB back groundcompared with WT or phyA single mutants, in line with previousreports (29, 32, 33).

To quantitatively evaluate differences in plant shape, thelengths of internodes and of leaf sheaths and blades remainingin the mature plants, which had stopped growing after heading,were measured. They were grown under short-day conditions tominimize the difference of their heading date. Only the last fiveinternodes are elongated in rice (japonica variety) (34). Intextbook fashion, the top four internodes were elongated andmeasurable, with the topmost being the longest in the WT(Nipponbare) (Fig. 4A). By contrast, internodes from the top tothe seventh were elongated in the triple mutant, and theelongated internodes all had relatively uniform lengths (Fig. 4A).

The distribution and length of the leaves were different in the

Fig. 2. Expression of light-induced genes in the triple mutants. Total RNA wasextracted from the 1-week-old seedlings used in the experiments shown in Fig. 1.The expression of the LHCB and RBCS genes was examined by northern hybrid-ization. D, dark-grown; Rc, grown under Rc; WT, Nipponbare; B, phyB-1 mutant;BC, phyB-1phyC-1 mutant; ABC, phyA-4phyB-1phyC-1 mutant.

Fig. 3. Distinct shape of triple mutant juvenile plants. (A) The appearance ofa WT and a triple mutant plant (Center, Left and Right, respectively) grownunder long day conditions for 1 month. Left and Right are anatomical displaysof the individual plants in the Center, respectively, in which all leaves wereseparated from the stem at the nodal portions. (B) The growing patterns ofeach portion of the WT and triple mutant plant during the first 3 weeks aftergermination. Green lines, leaf blade; blue, leaf sheath; orange, coleoptile;purple, internode. The mean � SE, n � 3 to 5.

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triple mutants than in the WT. The number of remaining leavesseemed to be related to the number of elongated internodes. Inthis stage (heading had been finished), a WT plant had only fiveleaves with photosynthetic activity because the lower leaves haddied due to nutrient-relocation. However, seven living leaveswere left in the triple mutants (Fig. 4 B and C). A clearcorrelation was also observed between the leaf length and thecorresponding internode length. The upper most leaf, except forthe flag leaf, had the longest sheath and blade. The leaf lengthsgradually decreased along with lower leaf position in the WTplants, whereas all leaves in the triple mutants had uniformly-sized leaf sheaths and blades (Fig. 4 B and C).

For reference, the shape of se5 plants grown under the sameday-length condition was also compared with that of the triplemutant. Basically, se5 plants showed a typical wild-type plantshape with respect to the number of elongated internodes andthe leaf distribution (Fig. 4 A and D).

Early Flowering and Reduced Fertility in the Triple Mutants. Undernatural day-length conditions [which were similar to the exper-imental long day (LD) conditions], the triple mutants (phyA-4phyB-1phyC-1) f lowered very early compared with the WT(Nipponbare) (52.9 � 0.35 days vs. 99.4 � 0.13 days at Tsukuba,Japan). However, the mutants flowered later than the WT undershort-day (10L/14D) conditions (60.1 � 0.68 days vs. 48.3 � 0.21days) in the growth chamber (Fig. S4A). As a result, the triplemutants flowered earlier in LD than in SD conditions, which isthe same response to the day length observed in the phyAphyBdouble mutants (29). These results suggest that light signalsmediated by the phytochromes promote flowering in rice inresponse to short day conditions, while they delay floweringunder long day conditions.

In addition to flowering early, another distinct feature is thattriple mutants have small panicles and set very few seeds (Fig.S4B). Both the number of grains per panicle and the filled-grainratio were lower in the triple mutants (27.9 � 1.3 and 22%,respectively) than in the phyAphyC double mutants (65.5 � 3.5and 87%, respectively). The early flowering itself does not causeformation of small panicles or premature anthesis because these5 and phyAphyC double mutants, which flowered as early as thetriple mutants, had normal sized panicles and fertility. Todetermine the reason for the lower fertility, the opened spikelets

were closely examined. Fig. S5 shows typical examples of thespikelets after flowering from the phyAphyC double mutant (Fig.S5A) and the triple mutant (Fig. S5B). During anthesis in normalrice, pollen is scattered just before the spikelets open andself-pollination is completed. The anthers protruding from thespikelet were empty (Fig. S5 A and C) and many pollen grainswere on the stigma in the phyAphyC double mutant (Fig. S5E).By contrast, in the triple mutants, the spikelets opened normally,but the anthers rarely split open and the pollen was still withinthe anther even after flowering (Fig. S5 B and D). Additionally,clean stigmas without pollen were predominantly observed in thetriple mutants (Fig. S5F). Pollen from the triple mutant was ableto germinate and a reciprocal cross with wild-type pollen re-stored fertility in the triple mutants. Therefore, the reducedfertility of the triple mutants seems to be caused by impaireddehiscence of the anther wall.

DiscussionPhytochromes are thought to be the sole photoreceptors for Rand FR, but until now, no direct evidence has been presented toprove this idea. Furthermore, the question of whether phyto-chromes are necessary has been around for decades (35). In thisstudy, phytochrome triple mutants are produced in rice, so thatthe plants possess no functional phytochrome protein, and clearevidence is presented showing that phytochromes are the solephotoreceptors for R and FR, at least for the de-etiolationresponse in rice. Moreover, it was also demonstrated thatphytochromes are essential for normal reproduction, but not forbasic plant development.

Germinating seedlings are most sensitive to light and showremarkable changes in response to light stimuli. The elongatedgrowth of coleoptiles with circumnutation is a characteristic ofskotomorphogenesis in rice (30). Red light inhibits the growthand causes the first leaves to emerge from coleoptiles. However,even under Rc, seedlings of the triple mutants grew in a mannersimilar to etiolated seedlings. These results present a directdemonstration that phytochromes are the sole photoreceptorsfor R, at least with respect to rice photomorphogenesis. If eitherphyA or phyB is functional, the seedlings exhibit normal pho-tomorphogenesis, suggesting redundancy in phytochrome func-tion during seedling establishment. It is noted that se5 mutantsalso show a normal response to Rc as far as seedling establish-ment is concerned. se5 is a hy1-type chromophore mutant in rice,in which a specific heme-oxygenase gene (OsHO1) is impaired(25). Rice has another HO gene (OsHO2) with 33% identity ofamino acid sequence to OsHO1, which may partially comple-ment the OsHO1 function (26).

Triple mutant seedlings did not respond to R either morpho-logically or by changes in gene expression levels. Red light-induced expression of the typical light-induced gene familiesLHCB and RBCS was examined by northern hybridization. Noinduction of these genes was observed in the triple mutantsgrown under R (Fig. 2). More comprehensive analysis of geneexpression using a 22 K microarray demonstrated no consistentchanges in the triple mutant by R-pulse treatment (Fig. S1 andTable S1).

Jiao et al. (36) have profiled global changes of gene expressionduring light-regulated seedling development in rice using a70-mer oligonucleotide microarray (37). The gene expressionlevels were compared between dark- and R-grown seedlings and853 genes were found to be regulated by R; 565 genes wereinduced and 288 genes were repressed by at least two-fold, witha P value �0.05. The total number of genes whose expressionlevel was affected by R is smaller in our experiments thanobserved by Jiao et al. (36), probably because our results mayonly contain early responses following a pulsed R treatment. Tocompare our results with those obtained by Jiao et al. (36), thetop 50 genes in the Jiao et al. (36) induced list are assigned to our

Fig. 4. Distinctive features of the adult triple mutant plants. (A) From theLower, lengths of panicle (P) and internodes (1�7, numbered beginning at theTop) of WT, triple mutant and se5 mutant after heading is completed. WT,Nipponbare; phyABC, phyA-4phyB-1phyC-1 mutant; se5, se5 mutant. Themean � SE obtained from 20 plants is displayed. Leaf lengths of Nipponbare(B), phyA-4phyB-1phyC-1 mutant (C), and se5 mutant (D). Gray bars, leafsheath; open bars, leaf blade. The mean � SE obtained from 20 plants isdisplayed. Leaves are numbered beginning at the Top (No. 1 is a flag leaf).

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list by BLAST search of the 70-mer oligonucleotide sequencesagainst the Rice Annotation Project database (RAP-DB; http://rapdb.dna.affrc.go.jp/). Since Jiao’s microarray represents36,929 genes, not all of the genes were assigned to our 22K array.The assigned genes were either induced or not changed byexposure to R in the WT, but no induction was observed in thetriple mutant by R treatment (Table S2 and Fig. S6).

Even under normal growth conditions, the triple mutants wereshaped very differently than the WT. In rice, the internodeelongation is closely related to the phase transition from vege-tative to reproductive growth. Coinciding with the time when theyoung panicle primordium is first initiated, the first elongationbegins at the fifth internode from the top. Thereafter, the topfive internodes initiate elongation in order from the fifth to thefirst, with the top-most (the first internode) extending last andlongest. The internodes below the top five never grow. Incontrast, no correlation was observed between the phase tran-sition and the internode elongation in the triple mutants becausethe internodes were elongated even in the seedling stage. Theseresults indicate that phytochromes suppress internode elonga-tion during the vegetative growth in the WT. Such regulation isimportant for the life cycle of rice because it is advantageous forthe plants to devote most of their resources to maximizephotosynthesis during vegetative growth.

The phyAphyB double mutants were compared with the triplemutants and found to show almost the same phenotype, from theseedling through to the adult plant. Therefore, phyC functionappears to be diminished in the phyAphyB double mutants,probably because phyB mutation leads to depletion of the PHYCprotein (29). It has been shown that Arabidopsis phyB and phyCform a heterodimer and that phyC is in fact present predomi-nantly as a phyB/phyC heterodimer (38, 39). Then, there isnothing strange about rice phyC also functioning as a het-erodimer with phyB.

In Arabidopsis, however, phyAphyBphyEphyD quadruple mu-tants respond to red light and exhibit photomorphogenesis (15).These observations indicate that phyC is able to function withoutphyB, although the phyC protein level is also reduced in the phyBmutants in Arabidopsis (32, 33). Light-regulated responses me-diated by phyC are different between rice and Arabidopsis. InArabidopsis, phyC is categorized as a type II phytochrome and itcontributes to R-induced de-etiolation (33, 40), whereas in rice,phyC functions more like phyA than phyB, mediating FR-HIRde-etiolation (29). Such functional differences may be associatedwith the differences in the dependence of phyC function onphyB.

The mechanism of immature internode elongation observedin the triple mutants is under investigation. Microarray experi-ments have revealed that the expression of the ACC Oxidase 1(ACO1) gene, which is highly expressed in the dark and issuppressed by light treatment, was greatly enhanced in thelight-grown triple mutants. It has been reported that partialsubmergence greatly stimulated internodal growth in deepwaterrice, which was, at least in part, mediated by ethylene (41).Mekhedov and Kende (42) found that the mRNA levels of ACO1gene and the ACC oxidase enzyme activity were increased in theelongating internodes of the deepwater rice. It has also beensuggested that ethylene levels are elevated by shade avoidanceresponses and that ethylene positively modulates phytochrome-mediated elongation responses in tobacco (43) and sorghum(44). These results suggest that the ectopic internodal growth inthe triple mutants may be mediated by ethylene. No differencein the amount of ethylene released from whole plants was foundbetween the triple mutants and the wild type. Measurement ofthe local concentration of ethylene is currently under way.

Triple mutants set very few seeds. However, they are notcompletely sterile and their fertility can be restored by cross-pollination with wild-type pollen, indicating incomplete male

sterility. As shown in left half of Fig. S5, pollen were still insideof the anthers even after f lowering was completed, and theanthers were out of the hulls. These observations suggest thatthe incomplete male sterility of the triple mutants is caused byimpaired dehiscence of the anther wall. The involvement ofjasmonic acid (JA) in dehiscence has been revealed by geneticanalyses of Arabidopsis mutants (45, 46). In JA-insensitive and-deficient mutants, cell organization and differentiation ofanther tissues appear normal, but dehiscence does not occur atf lower opening. A JA-deficient rice mutant, hebiba, also showsmale sterility (47). It has been reported that the synthesis of JAis induced by red light in rice and the induction is mediated byphytochromes (48). These lines of evidence suggest the pos-sibility that the regulation of JA synthesis is disrupted inthe triple mutant, which leads to impaired dehiscence ofanthers.

When effects of R on morphological and/or physiologicalchanges in plants are examined, it is often difficult to discrim-inate between the effects of photosynthesis and the R-mediatedresponses. The rice phytochrome triple mutant cannot perceivered light but appears to grow well under natural light conditions.Therefore, it might be possible to design experiments in whichtwo of the aspects of light, energy and signal, can be separatedby use of the triple mutants. Recently, Su and Lagarias (49) havefound that Arabidopsis plants with Tyr-to-His mutant alleles ofPHYB (PHYBY276H) showed a wide range of photomorphogenicresponses without light signals. This mutant also seems to beuseful for dissecting the phytochrome signal transduction path-ways without being affected by ambient light. Thus, these twomutant lines display a high potential to expand and deepen ourunderstanding of phytochrome function.

This study demonstrated that phytochromes perceive ambientlight signals to regulate whole vegetative growth in rice, includ-ing internode elongation and growth of leaves, in addition to thewell-known coleoptile growth. The importance of phytochromefunction for the normal architecture of rice plants was revealedbecause of the availability of the triple mutants. Additionally,because the light signals that are perceived by photoreceptors areeventually transduced to changes in the synthesis and/or signal-ing of plant hormones for final morphological changes, the triplemutants will also provide a potent tool for dissection of light-hormone interactions in these plants.

Materials and MethodsPlant Materials. All mutants, except for se5, are Oryza sativa L. cv. Nipponbare.se5 has a Norin 8 background. phyA-4 and phyC-1 mutants were obtainedfrom Tos17-insertinal lines by PCR-based screening. The phyC-1 has a reverseinsertion of Tos17 in the first exon of the PHYC gene. The insertion sites ofTos17 in the PHYA and PHYC genes correspond to the position of the 527th and244th amino acid, respectively. Both alleles are null mutants (29). phyB-1mutant was isolated from �-ray-mutagenized M2 plants by the phenotypescreening. The mutant has an insertion of one nucleotide at the position of481st amino acid and is null allele (29).

Construction of Triple Mutants. To obtain phyAphyBphyC triple mutants,phyA-2phyC-1 or phyA-4phyC-1 double mutants and phyB-1 or phyB-2 mu-tants (29) were crossed with each other. The F2 segregants were genotyped byPCR (29). F2 segregants with homozygous mutant alleles for two genes, PHYAand PHYC or PHYB and PHYC, and heterozygous for the remaining gene, wereselected and used to obtain F3 seedlings with the triple mutation. The triplemutation in the resulting F3 plants was confirmed by genotyping and bywestern blot analysis.

Imaging System. The growth of the rice seedlings was monitored by anautomatic digital imaging system (31). Details are in the legend of Movie S1and SI Text. Unique software was developed to semiautomatically detect theleaf tip position on each image by means of a space filter algorithm and plotthe extracted leaf tip points sequentially on the final picture.

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Growth Conditions. Seeds of Nipponbare WT and mutant lines were sown onMay 15, 2008, and the seedlings were transplanted in an irrigated rice fieldon June 18. Plants were grown under natural field conditions, and theheading (flowering) date was monitored for the appearance of the firstpanicle. The Nipponbare, se5 and the triple mutant line were also grown ina growth chamber in SD conditions (light cycle, 10 h of light/14 h of dark;28 °C by day/23 °C by night) for monitoring heading date as well as formeasuring lengths of plant parts. The light source was metal halide lamps(MLBOC400C-U, MITSUBISHI/OSRAM; 530 �mol photons m�2 s�1).

Measurements of Plant Parts. For the measurements of the different parts ofthe juvenile plants, Nipponbare and triple mutants were grown in a growthchamber under SD conditions (same as above) for 3 weeks. Approximately 100seeds of the WT and triple mutant plants were germinated at the same time.Three to five plants for each line were harvested daily at 10 AM for 3 weeks,and the indicated plant parts were measured.

For the measurements of mature plants, Nipponbare, triple mutant and se5

plants were grown in a growth chamber in SD (same as above) until floweringhad completely finished and the lengths of panicle, internodes, leaf sheathsand leaf blades were then measured.

RNA Analysis. To examine light-induced gene expression, Nipponbare WT andmutants seedlings were harvested after monitoring as above. Total RNA,extracted with the RNeasy Plant Mini Kit (QIAGEN, GmbH), was resolved by0.8% agarose gel electrophoresis and transferred to Hybond N� membranes(GE Healthcare). The LHCB (LHCP II, Accession No. D0062) and RBCS (AccessionNo.X07515) cDNAs from rice used for the northern analysis were provided byDr. N. Yamamoto (Ochanomizu Univ., Tokyo, Japan).

ACKNOWLEDGMENTS. We thank Dr. N. Yamamoto for the gift of the LHCBand RBCS cDNA; and Y. Iguchi, Y. Nemoto, and H. Shimizu for their technicalassistance. This work was supported by the Ministry of Agriculture, Forestry,and Fisheries of Japan Rice Genome Programs Grant SY1108, Integratedresearch project for plant, insect and animal using genome technology IP1006,and Genomics for Agricultural Innovation Grant GPN-0003.

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14710 � www.pnas.org�cgi�doi�10.1073�pnas.0907378106 Takano et al.

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