Phytochrome controls alternative splicing to mediatelight responses in ArabidopsisHiromasa Shikataa,1,2, Kousuke Hanadab,1, Tomokazu Ushijimaa, Moeko Nakashimaa, Yutaka Suzukic,and Tomonao Matsushitaa,d,3

aFaculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan; bFrontier Research Academy for Young Researchers, Kyushu Institute of Technology,Fukuoka 820-8502, Japan; cDepartment of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan;and dPRESTO, JST, Saitama 332-0012, Japan

Edited by Steve A. Kay, University of Southern California, Los Angeles, CA, and approved November 17, 2014 (received for review April 18, 2014)

Plants monitor the ambient light conditions using several infor-mational photoreceptors, including red/far-red light absorbingphytochrome. Phytochrome is widely believed to regulate thetranscription of light-responsive genes by modulating the activityof several transcription factors. Here we provide evidence thatphytochrome significantly changes alternative splicing (AS) pro-files at the genomic level in Arabidopsis, to approximately thesame degree as it affects steady-state transcript levels. mRNA se-quencing analysis revealed that 1,505 and 1,678 genes underwentchanges in their AS and steady-state transcript level profiles, re-spectively, within 1 h of red light exposure in a phytochrome-dependent manner. Furthermore, we show that splicing factorgenes were the main early targets of AS control by phytochrome,whereas transcription factor genes were the primary direct targetsof phytochrome-mediated transcriptional regulation. We experi-mentally validated phytochrome-induced changes in the AS ofgenes that are involved in RNA splicing, phytochrome signaling,the circadian clock, and photosynthesis. Moreover, we show thatphytochrome-induced AS changes of SPA1-RELATED 3, the nega-tive regulator of light signaling, physiologically contributed topromoting photomorphogenesis. Finally, photophysiological experi-ments demonstrated that phytochrome transduces the signal fromits photosensory domain to induce light-dependent AS alterationsin the nucleus. Taking these data together, we show that phyto-chrome directly induces AS cascades in parallel with transcriptionalcascades to mediate light responses in Arabidopsis.

phytochrome | alternative splicing | light signaling |posttranscriptional regulation | photomorphogenesis

Phytochromes are the red/far-red light (R/FR) receptors bywhich plants monitor their surrounding light environment

and modulate their growth, development, and metabolism ac-cordingly. Upon absorption of R, phytochromes are convertedfrom the biologically inactive Pr form into the active Pfr form,whereas FR irradiation converts Pfr back to Pr. Arabidopsis hasfive molecular species of phytochrome, phyA to phyE, amongwhich phyA and phyB play predominant roles in seedling de-etiolation, a critical process during which the plant switches fromheterotrophic to autotrophic growth (1). PhyA and phyB displaydistinct light responsiveness; whereas light-stable phyB mediatesR/FR reversible responses, light-labile phyA is responsible forsensing continuous FR (high irradiance response) and very weaklight of various wavelengths (very low fluence response) (1).A phytochrome molecule consists of an N-terminal chromo-

phoric domain and a C-terminal dimerization domain (2). TheN-terminal domain is sufficient for transducing light signals inthe nucleus to induce photomorphogenesis (3, 4). Pfr is trans-located from the cytoplasm to the nucleus (5, 6), where it inhibitstwo major negative regulators of photomorphogenesis—that is,phytochrome-interacting factors (PIFs) and CONSTITUTIVEPHOTOMORPHOGENIC 1 (COP1)—to elicit light responsesin plants (7). PIFs are basic helix–loop–helix transcription factorsthat directly interact with Pfr (8). This R-dependent interaction

results in the phosphorylation and proteasome-mediated degra-dation of PIFs (9, 10). In contrast, COP1 is an E3 ubiquitin ligasethat mediates the ubiquitination and proteasomal degradation ofphotomorphogenesis-promoting transcription factors, includingLONG HYPOCOTYL 5 (HY5) (11). Therefore, phytochromeregulates the transcription of light-responsive genes by mod-ulating the protein stability of negatively acting (such as PIFs)and positively acting (such as HY5) transcription factors tomediate photomorphogenesis.Accumulating evidence suggests that light regulates not only

transcription but also other aspects of gene expression, such aschromatin reorganization (12), translation (13, 14), posttrans-lational modification (11, 15), and pre-mRNA splicing (16–18).Pre-mRNA splicing is a posttranscriptional event that is carriedout by a macromolecular complex called the spliceosome. Al-ternative splicing (AS) produces multiple transcripts from a sin-gle gene by using different splice sites. AS qualitatively expandstranscriptome diversity, whereas transcriptional regulation quanti-tatively controls the transcriptome. Because conventional micro-array experiments do not detect qualitative alterations in thetranscriptome, AS has been investigated to a lesser extent than hastranscriptional regulation. Selection of alternative splice sites ismediated by trans-acting splicing factors, such as serine/arginine-rich

Significance

Plants adapt to their fluctuating environment by monitoringsurrounding light conditions through several photoreceptors,such as phytochrome. It is widely believed that upon absorbingred light, phytochrome induces plant light responses by regu-lating the transcription of numerous target genes. In this study,we provide clear evidence that phytochrome controls not onlytranscription, but also alternative splicing in Arabidopsis. Wereveal that 6.9% of the annotated genes in the Arabidopsisgenome undergo rapid changes in their alternative splicingpatterns in a red light- and phytochrome-dependent manner.Our results demonstrate that phytochrome simultaneouslyregulates two different aspects of gene expression, namelytranscription and alternative splicing to mediate light respon-ses in plants.

Author contributions: H.S., K.H., and T.M. designed research; H.S., K.H., T.U., M.N., Y.S.,and T.M. performed research; H.S., K.H., and T.M. analyzed data; and H.S., K.H., and T.M.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence reported in this paper has been deposited in the GenBankdatabase (accession no. DRP002301).1H.S. and K.H. contributed equally to this work.2Present address: Department of Plant Systems Biology, Technische Universität München,85354 Freising, Germany.

3To whom correspondence should be addressed. Email: mat@agr.kyushu-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1407147112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1407147112 PNAS | December 30, 2014 | vol. 111 | no. 52 | 18781–18786

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Oct

ober

18,

202

0

(SR) proteins. These factors bind to cis elements on the pre-mRNAto promote or inhibit the recruitment of spliceosome components tothe adjacent alternative splice sites (19). Therefore, the regulationof AS depends on the expression level and posttranslational mod-ification of SR proteins and other splicing factors (20). It has beenreported that light affects the AS of several genes in plants (16–18).However, the physiological significance of this finding and theidentity of the photoreceptors that mediate light-regulated ASremain unclear. Recently, we identified rrc1 (reduced red-lightresponses in cry1cry2 background 1), an Arabidopsis mutant that isimpaired in phyB-mediated light responses (21). Because RRC1encodes a novel SR-like protein and rrc1 mutants show aberrantAS profiles, we hypothesized that RRC1 controls AS to mediatephyB signaling. Furthermore, we proposed that phyB is involvedin the R-dependent AS changes of several genes (21).Here, we present clear evidence that phytochrome induces

genome-wide AS alterations in Arabidopsis. mRNA sequencing(mRNA-seq) analysis revealed that over 1,500 genes underwentchanges in their AS patterns within 1 h of R exposure, in aphytochrome-dependent manner. Gene Ontology (GO) analysissuggested that RNA splicing-related genes were the most enrichedamong those genes that showed rapid phytochrome-dependentchanges in AS in response to continuous R (cR), whereas tran-scription factor genes were the main early targets of transcrip-tional regulation by phytochrome, as previously reported (22). Ourresults demonstrate that phytochrome controls not only tran-scription but also AS to mediate R responses in Arabidopsis.

Results and DiscussionGenome-Wide Analysis of AS Control by Phytochrome During De-Etiolation. In Arabidopsis, phyA and phyB play predominantand partially redundant roles in every step of seedling de-etio-lation, from changes in gene expression to changes in morphol-ogy (1, 22). To identify the genes that show phytochrome-dependent AS changes in the Arabidopsis genome, we performedmRNA-seq analysis and determined a series of time-coursetranscriptome profiles of 4-d-old, dark-grown WT and phyAphyBdouble-mutant (phyAB) etiolated seedlings that were exposed tocR (8.3 μmol·m−2·s−1) for 1 h (R1h) or 3 h (R3h) (Fig. 1A). Forthe dark control, transcriptome profiles were also determined for4-d-old WT etiolated seedlings with or without additional growthin darkness for 3 h (D3h and D0h) (Fig. 1A).AS patterns can be determined based on the number of splice-

junction reads within a gene. Although 45,372 splice junctionshave been annotated in TAIR10 (The Arabidopsis InformationResource), it is likely that many other splice junctions exist. Tocharacterize AS events at the genomic scale, we additionallyidentified 37,545 novel splice junctions in 32,398 annotatedgenes (SI Text). For each of these 82,917 previously known andnovel splice junctions, the number of junction reads was countedand normalized by reads per kilobase of exon model per millionmapped reads (RPKM) for the corresponding genes under thefollowing conditions (genotype_treatment): WT_D0h, WT_D3h,WT_R1h, WT_R3h, phyAB_D0h, phyAB_R1h, and phyAB_R3h(Fig. 1A). Then, AS was defined as being regulated by phyto-chrome at R1h if the relative number of junction reads inWT_R1h significantly differed, in the same direction, from thatin WT_D0h and from that in phyAB_R1h (Fig. 1A). Using thesame criteria, but with R1h being replaced with R3h, we iden-tified phytochrome-regulated AS at R3h; however, in this case,we added another criterion, namely that the number of junctionreads should not differ between WT_D0h and WT_D3h, becausesuch a difference might indicate that AS is controlled by thecircadian clock and not by phytochrome (Fig. 1A).The mRNA-seq coverage was high enough (×236 to ×546)

(Table S1) to allow robust statistical analyses that are likely tocover genome-wide light-responsive AS changes specifically con-trolled by phyA and/or phyB. Using this method, we found that 2,230

genes (∼6.9% of the 32,398 annotated genes in the Arabidopsisgenome) displayed phytochrome-regulated AS patterns at R1hand/or R3h (Pearson’s χ2 test, P < 0.05) (Fig. 1B and Dataset S1A–C). To determine the steady-state transcript level (TX), wedetermined the RPKM value for each of the 32,398 annotatedgenes in TAIR10. We next established the phytochrome-regu-lated TX using the same criteria as for the AS analysis (Fig. 1A),and found that TX was regulated by phytochrome in 5,096 genes[false discovery rate (FDR) < 0.01] (Fig. 1B and Dataset S1 A, D,and E). Interestingly, 1,505 genes rapidly altered AS patternswithin 1 h of transfer to cR in a phytochrome-dependent man-ner, and 88% of these genes did not show any significant phy-tochrome-mediated changes in TX at this time point (Fig. 1B).This result suggests that phytochrome is directly involved in thegenome-wide regulation of AS.

Functional Categorization of Genes Under Phytochrome-Mediated ASRegulation. We next performed GO analysis of the genes thatdisplayed phytochrome-dependent changes only in AS (ASonly),only in TX (TXonly), or in both AS and TX (AS&TX). At R1h,GO terms related to RNA splicing were significantly enrichedamong ASonly genes (Fig. 1C and Dataset S1F), whereas those

A

B C

Fig. 1. Red light triggers global transcriptomic changes that are mediatedby phytochrome in Arabidopsis. (A) Flowchart for the analysis of red light-and phytochrome-dependent changes in AS or TX. For transcriptomic pro-filing with mRNA-seq, total RNA was extracted from 4-d-old WT andphyAphyB double-mutant (phyAB) etiolated seedlings that were exposed tocontinuous red light (8.3 μmol·m−2·s−1) (pink shading) for 1 h (R1h) or 3 h(R3h). For the dark control, total RNA was also extracted from 4-d-old WTetiolated seedlings with or without additional growth in darkness (grayshading) for 3 h (D3h and D0h). (B) Venn diagrams showing the number ofgenes that displayed phytochrome-dependent changes in either AS (ASonly)or TX (TXonly), or in both AS and TX (AS&TX), at R1h and R3h. AS, P < 0.05(Pearson’s χ2 test); TX, FDR < 0.01 (FDR by LIMMA). (C) GO analysis of genesthat displayed phytochrome-induced changes in AS or TX. The bars representthe proportion of phytochrome-regulated genes in each category at R1h orR3h, or that of all of the genes in each category to all of the annotatedgenes in the genome (Genome). Asterisks indicate the statistical significance(FDR < 0.05 by LIMMA) that was calculated based on relative abundance inthe genome.

18782 | www.pnas.org/cgi/doi/10.1073/pnas.1407147112 Shikata et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

18,

202

0

related to transcription were overrepresented in TXonly genes atR1h, as previously reported (Fig. 1C and Dataset S1G) (22).Consistent with this result, phytochrome-mediated AS wasfound in 15% (58 of 395) of all splicing-related genes known inArabidopsis at R1h (Table S2 and Dataset S1I) (23). These 58genes include splicing regulators involved in splice site selectionand spliceosome recruitment, such as 7 of the 18 SR proteins(RS31, RS40, RS41, RSZ33, RSZ34, SR34a, and SR34b), U1small nuclear ribonucleoprotein 70 kDa, and U2 auxiliary factor65a (U2AF65a) (Dataset S1I). Interestingly, 56 (81%) of the 69splicing-related genes that were subject to phytochrome-medi-ated AS and/or TX control at R1h were ASonly genes (Table S2and Dataset S1I). In contrast, the transcription factor genes thatwere under phytochrome-mediated gene expression control atR1h were mostly TXonly genes (210 of 358 = 59%), and thisproportion is significantly higher than that of ASonly genes (124of 358 = 35%) (Fisher’s exact test, P = 0.0001) (Table S2 andDataset S1J). These results strongly suggest that RNA splicing-related genes and transcription factor genes are the main earlytargets of phytochrome-mediated AS and transcriptional regulation,respectively (Fig. 2). The finding that the phytochrome-mediatedcontrol of the expression of splicing-related genes at R3h was moreprominent at the TX level than at the AS level (Table S2) suggeststhat phytochrome also controls AS through transcriptional regula-tion of splicing factors at a later time point after the onset of cR.We further investigated the results of GO analysis to infer the

physiological significance of phytochrome-mediated AS, andfound that a number of photosynthesis- or plastid/chloroplast-related GO terms were overrepresented, not only in TXonly genesas previously reported (22), but also in ASonly genes and AS&TXgenes (Fig. 1C and Dataset S1 F–H), suggesting that phyto-chrome-mediated AS is also involved in light-induced chloro-plast differentiation during seedling de-etiolation.Because GO analysis is not sufficient to fully infer the physio-

logical effects of phytochrome-mediated AS control, we manuallygenerated a list of Arabidopsis genes reported to be important forlight signaling in vivo (Dataset S1K). Among the 184 genesidentified, 38 (21%) and 88 (48%) showed phytochrome-regulatedAS and TX alterations, respectively, at R1h and/or R3h (Table S3and Dataset S1K). Phytochrome-regulated AS and TX genes weresignificantly enriched in this list relative to the whole Arabidopsisgenome (Fisher’s exact test, P = 8.82 × 10−10 for AS and P =1.10 × 10−15 for TX) (Table S3), suggesting that not onlyphytochrome-mediated TX, but also AS was enriched amongthe light signaling genes. Furthermore, we found that genesunder phytochrome-mediated AS control were enriched in cate-gories such as “COP/DET,” “clock,” “phy-, cry-, or phot-signaling,”“shade avoidance,” and “photoreceptor” (Table S3). These findingsstrongly suggest that both phytochrome-mediated TX and ASplay important and specific roles in light signaling in Arabidopsis.

Experimental Validation of mRNA-seq Data. To validate the mRNA-seq data, we manually selected 19 genes from the categories thatwere overrepresented among the ASonly and AS&TX genes,and confirmed phytochrome-dependent and cR-responsive ASalterations for 10 of the 19 genes (validation rate, 10 of 19 =53%) using semiquantitative RT-PCR (sqRT-PCR) analysis(Fig. 3 and Fig. S1). Because sqRT-PCR is intrinsically lesssensitive than mRNA-seq, this result suggests that phytochrome-dependent AS regulation occurs in at least 53% of the 2,230genes that were detected in our mRNA-seq analysis. The vali-dated genes were related to splicing (RS31, SR30, SR34a, SR34b,and U2AF65a), phytochrome signaling [SPA1-RELATED 3 (SPA3)and PSEUDO-RESPONSE REGULATOR 7 (PRR7)], the circadianclock [LATE ELONGATEDHYPOCOTYL (LHY) and PRR7], andphotosynthesis [PHOTOSYNTHETIC NDH SUBCOMPLEX B 4(PnsB4)], strongly suggesting that phytochrome-induced AS alter-ations contribute to these functional categories.

Physiological Contribution of Phytochrome-Mediated AS Control. Togain insight into the roles of phytochrome-mediated AS reg-ulation during de-etiolation, we determined the sequences ofthe splice variants of the 10 validated genes, including SPA3(Fig. S1A). SPA3 is a member of the SPA family proteins,which interact with COP1 to form the COP1–SPA complex andfunction as negative regulators of photomorphogenesis in con-cert with COP1. The COP1–SPA complex is part of a multimericE3 ubiquitin ligase containing CULLIN 4 and DAMAGEDDNA-BINDING PROTEIN 1 (DDB1) (11). Both COP1 andSPA proteins possess a central coiled-coil domain, followed byC-terminal seven WD40 repeats. COP1 and SPA proteins in-teract with each other through their coiled-coil domains (24).SPA proteins are known to associate with DDB1 through thefourth repeat of the WD40 repeats (25). Interestingly, sqRT-PCR analysis revealed that phytochrome promoted the retentionof intron 4 and selection of the alternative 5′ splice site withinintron 4 of SPA3, both of which produce premature terminationcodons and result in the loss of most of the WD40 repeats, in-cluding the fourth repeat (Fig. S1A). These splice variants,named mRNA-3 and mRNA-2, respectively, likely encodetruncated SPA3 proteins that have dominant-negative effects onthe function of the endogenous COP1–SPA complex, becausethey are unable to bind to DDB1 but still retain the interactionwith COP1. Thus, it was suggested that the phytochrome-inducedAS of SPA3 promotes photomorphogenesis by increasing theamount of dominant-negative variants of SPA3.To investigate this possibility, we overexpressed mRNA-2 and

mRNA-3 of SPA3, as well as the full-spliced mRNA-1 that likelyencodes a functional full-length SPA3 (Fig. S1A) in the WTbackground to obtain overexpression (OX) lines of each isoform.In the mRNA-3 OX construct, all of the splice sites within intron4 were mutated to retain an unspliced intron 4 in the transcript(Fig. 4A). sqRT-PCR analysis showed that each isoform wasspecifically overaccumulated in each line, as expected (Fig. 4B).Then we examined the seedling de-etiolation phenotype of theselines and found that, although no clear phenotype was observedin darkness, obviously shorter- and longer-hypocotyl individualsthan WT seedlings were segregated under cR (11 μmol·m−2·s−1),at reasonable ratios, from T2 populations of the mRNA-2 andmRNA-3 OX lines and from those of the mRNA-1 OX lines,respectively (Fig. 4C). The R-dependent short-hypocotyl phe-notypes of the mRNA-2 and mRNA-3 OX lines are reminiscentof those of spa3 mutants (26), demonstrating that the proteinsencoded by mRNA-2 and mRNA-3 have dominant negativeeffects at least on the endogenous SPA3. Taken together, thesedata indicate that light-regulated, phytochrome-mediated AScontrol indeed contributes to promoting seedling de-etiolation.

Fig. 2. A model depicting the early signaling cascades of phytochrome thatregulate genome-wide gene expression in response to red light. Red arrowsindicate pathways that are regulated by AS; blue arrows indicate pathwayssubjected to transcriptional regulation.

Shikata et al. PNAS | December 30, 2014 | vol. 111 | no. 52 | 18783

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Oct

ober

18,

202

0

PhyA and PhyB Are Responsible for R-Dependent AS Control. Wepreviously showed that two SR protein genes, RS31 andSR34b, display cR-induced changes in AS, and that this re-sponse is partially reduced in phyB mutants (21). The mRNA-seq analysis conducted in this study revealed that RS31 andSR34b undergo phytochrome-mediated AS control (DatasetS1). These results strongly support the notion that at leastphyB is involved in the cR-induced AS changes. To further ex-amine the photophysiological properties and involvement ofspecific isoforms of phytochrome in the light-regulated ASevents identified in this study, we subjected RS31 and SR34b tosqRT-PCR analysis using samples obtained from seedlings grownunder various light conditions. These two SR protein genes werechosen as representatives, because the changes in the AS profilesof these genes are thought to be reflected in the AS of theirnumerous downstream target genes. We first changed the ex-posure time of etiolated seedlings to cR (8.3 μmol·m−2·s−1), andfound that the alterations in the AS patterns were dependent onthe length of cR irradiation (Fig. S2A). We found that even a2-min pulse of R (pR) could induce the response (Fig. S2A).Time-course analysis revealed that the AS changes were detectedwithin 1 h and peaked 3 h after pR irradiation (Fig. S2E), aswas the case for cR irradiation (21). Next, we exposed etiolatedseedlings to various intensities of 2-min pR or 3-h cR to confirmthat the AS responses were dependent on the fluence rate of R(Fig. S2 B and C). We then converted the unit on the x axes inFig. S2 A–C into fluence, which is the product of fluence rate andexposure time, and these plots fit closely to one another (Fig.S2D), indicating that the AS responses to R obey the Bunsen–Roscoe reciprocity law, where the extent of a certain response isproportional to the total number of photons, irrespective of thefluence rate or irradiation time (27). In this case, the responsewas dependent on R fluence of 1–106 μmol·m−2, a range thatcorresponds to the fluence required for phyB-dependent seedgermination in lettuce (Lactuca sativa) and Arabidopsis (28, 29),further supporting the notion that phyB is the major photore-ceptor involved in this AS response.

The most distinguishing feature of phytochrome-mediatedresponses is R/FR reversibility, in which R-induced responsescan be cancelled by subsequent FR irradiation. To examinewhether the light-induced AS changes exhibit R/FR reversibility,we exposed WT etiolated seedlings to a 2-min pR, immediatelyfollowed by a 2-min pulse of FR (pFR) (Fig. 5 A and B). Wefound that the pR-induced AS alterations of RS31 were sub-stantially suppressed by the subsequent pFR, indicating that thisAS response is indeed controlled by a light-stable phytochrome,such as phyB. However, SR34b did not show a clear R/FR re-versible response (Fig. 5B), probably because of the very lowfluence response of phyA where even pFR can induce the re-sponse (30).To further confirm that phytochrome is required for the light-

responsive AS changes, we examined the pR-induced AS alter-ations in the mutants that lacked phyA and/or phyB, which arethe major molecular species of phytochrome in etiolated seed-lings. Although the pR-induced AS changes were marginallyreduced in both phyA and phyB single-mutants, the response wasalmost completely abolished in phyAB double-mutants (Fig. 5C).These results clearly demonstrate that phyA and phyB arephotoreceptors that play dominant and partially redundant roles inmediating R-responsive AS changes during seedling de-etiolation.Furthermore, we observed that pFR induced smaller AS

changes than did pR (Fig. 5 B and C). Because phyA is thoughtto be the sole receptor for FR in Arabidopsis etiolated seedlings(1), we investigated whether the pFR-induced AS response isdependent on phyA and on the fluence of FR. As expected, al-though the AS patterns of RS31 and SR34b in WT plants werealtered in a FR fluence-dependent manner, the phyA mutant didnot show any significant alterations in AS even under variousfluences of FR (Fig. S2 F and G). These results demonstrate thatphyA mediates AS alterations in response to FR.Finally, we investigated which domain in the phytochrome

molecule underlies the signaling that induces the light-responsiveAS alterations. The N-terminal domain of phyB fused to GFP(NG), when dimerized and targeted to the nucleus by the ac-tivities of β-glucuronidase (GUS) and a nuclear localization

Fig. 3. Validation of phytochrome-regulated alternative splicing. Total RNA from 4-d-old WT and phyAphyB (phyAB) etiolated seedlings exposed to con-tinuous red light (8.3 μmol·m−2·s−1) for 0 h (D0h), 1 h (R1h), and 3 h (R3h) was analyzed by sqRT-PCR. The regions that were predicted to display differential ASpatterns were amplified with gene-specific primers, and the DNA concentration of each PCR product of different sizes was quantified. AS events in each gene,which were designated as mRNA-x (where x represents 1–6), are shown as diagrams above the bar graphs. mRNA-1 represents splice variants that areconsidered to encode functional full-length proteins in each gene. Boxes and lines indicate exons (numbered) and spliced-out introns, respectively. The whiteand gray regions in the boxes indicate untranslated regions and coding regions, respectively. AS patterns were expressed as ratios of the indicated splicevariants to the total transcripts, and the values are shown as the mean ± SE (n = 3). Asterisks indicate statistical significance relative to D0h samples, asdetermined using Student t test (*P < 0.05; **P < 0.01).

18784 | www.pnas.org/cgi/doi/10.1073/pnas.1407147112 Shikata et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

18,

202

0

signal (NLS), respectively (NG-GUS-NLS), is biologically fullyfunctional in all of the phyB-mediated responses examined (3).When NG-GUS-NLS and full-length phyB fused to GFP (PBG)were overexpressed in the phyAB double-mutant background,we found that NG-GUS-NLS was as active as PBG and the en-dogenous phyB present in phyA single-mutants in mediating pR-responsive AS changes and seedling de-etiolation under cR(Fig. 5D and Fig. S3). These results indicate that phyB trans-duced the signal from its N-terminal domain to induce the light-dependent AS alterations in the nucleus. Taken together,through detailed photophysiological experiments, we have clearlydemonstrated that phyA and phyB are photoreceptors that me-diate light-induced AS alterations in Arabidopsis.

Concluding Remarks. In this study, using a combination of high-throughput mRNA-seq analysis and detailed photophysiologicalsqRT-PCR experiments, we have provided clear evidence thatphytochrome controls genome-wide AS alterations in Arabidopsis.We extracted junction reads from high-coverage mRNA-seq dataand systematically compared the relative frequency of use ofeach splice site among WT and phyAB double-mutant etiolatedseedlings with or without cR irradiation, which enabled us toconduct robust statistical analyses of R-responsive AS changesthat were specifically controlled by phytochrome. As a result, wewere able to obtain a highly reliable list of 2,230 Arabidopsis genesthat are under phytochrome-mediated AS regulation, which hada validation rate of more than 50% (Fig. 3). We also found that

1,722 genes displayed R-responsive but phytochrome-independentAS alterations at R1h or R3h, indicating that 56.4% of the genesthat showed R-induced AS changes were under phytochrome-mediated AS control (Table S4). It is well known that the relativeamount of splice variants could be affected by differential mRNAdegradation such as nonsense-mediated decay (NMD), where al-ternatively spliced transcripts containing premature termina-tion codons are recognized for degradation (31). However, theR-dependent changes in the ratio among splice variants that weobserved in this study are considered to be because of AS but notto differential mRNA degradation, because the responses toR were still observed in the NMD-impaired mutant upf1-5 (32)(Fig. S4A), and because inhibiting transcription by the treatmentwith α-amanitin completely abolished the response to R(Fig. S4B).Light-regulated AS has been reported not only in different

species in higher plants (16–18) but also in a chlorophyteChlamydomonas (33). Moreover, recently, phytochrome was sug-gested to be involved in R-dependent AS alterations in the mossPhyscomitrella (34). Therefore, phytochrome-mediated AS regu-lation that we demonstrated here seems to be widely conserved inplants. It has been reported very recently that light affects AS ofa subset of Arabidopsis genes through a chloroplast retrogradesignal, but not through photoreceptor signaling (35). We thus

A B

C

Fig. 4. Functional analysis of SPA3 splice variants in transgenic Arabidopsis.(A) Schematic illustration of constructs for generating OX lines of each SPA3splice variant in the WT background. In the mRNA-3 OX construct, all of thesplice sites within intron 4 were mutated, as indicated by the arrows. 35S,Cauliflower mosaic virus 35S promoter; boxes, spliced exons; horizontal lines,unspliced intron 4; asterisks, premature termination codons. (B) Ratios ofeach SPA3 isoform to the total transcripts in the OX lines. For each constructshown in A, two independent representative lines that segregated about 3:1for kanamycin-resistance in the T2 generation were chosen for analysis.These lines, shown on the top, were used in B and C. Total RNA from WTplants and kanamycin-resistant T2 plants for each line, grown under con-tinuous white light (35 μmol·m−2·s−1) for 10 d, was analyzed by sqRT-PCR. (C)Box plots of hypocotyl lengths of the SPA3 isoform OX lines. Seedlings of WTand T2 segregating populations of mRNA-1 OX, mRNA-2 OX, and mRNA-3OX lines were grown under cR (11 μmol·m−2·s−1) or in darkness (Dark) for 5 d,and hypocotyl lengths were determined. The top, middle, and bottom of thebox indicate the 25th, 50th, and 75th percentiles, respectively. Dots andwhiskers represent individual values and the spread of the data, respectively.

A

C

B

D

Fig. 5. PhyA and phyB control the red light-dependent alternative splicingof RS31 and SR34b. (A) Illustration of the experimental procedure. Four-day-old etiolated seedlings were exposed to a 2-min pR (30 μmol·m−2·s−1), 2-minpFR (60 μmol·m−2·s−1), pR followed by subsequent pFR (pR/pFR), or were keptin darkness (D). After the exposure, the seedlings were placed in the darkfor 3 h and then harvested for extraction of total RNA. (B) AS alterations inresponse to pR, pR/pFR, or pFR. sqRT-PCR was performed for RS31 andSR34b, and each PCR product was quantified. The ratios of mRNA-1 (forRS31) or mRNA-1 and mRNA-2 (for SR34b) to the total transcripts werecompared with those of the D control, and the relative values are shown asthe mean ± SE (RS31, n = 4; SR34b, n = 3). (C) pR-induced AS alterationsdepend on both phyA and phyB. For sqRT-PCR analysis, total RNA wasextracted from pR, pFR, and D samples of WT, phyA, phyB, and phyAphyB(phyAB). Details are as in B. Data are shown as the mean ± SE (RS31, n = 5;SR34b, n = 4). (D) The N-terminal domain of phyB is sufficient to bring aboutthe phyB-dependent AS changes. Total RNA for sqRT-PCR was extractedfrom pR and D samples of phyA, phyAB, and transgenic lines expressing PBGor NG-GUS-NLS in the phyAB mutant background. Details are as in B. Dataare shown as the mean ± SE (n = 3). Asterisks show statistical significancecompared with pR samples (B), WT (C), and phyAB (D), as determined usingStudent t test (*P < 0.05; **P < 0.01).

Shikata et al. PNAS | December 30, 2014 | vol. 111 | no. 52 | 18785

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Oct

ober

18,

202

0

analyzed the overlapping target genes that are regulated by bothphytochrome and chloroplast retrograde signaling pathways andfound that there is basically no correlation between these twopathways (Table S5), although they are not mutually exclusiveand may coexist and act coordinately in certain conditions, sharingcommon target genes, such as RS31 (35).GO and other statistical analyses suggested that phytochrome-

mediated control of not only TX, but also AS, have significantroles in light signaling (Fig. 1C and Table S3). We experimentallyvalidated the phytochrome-dependent and cR-responsive ASchanges of several genes, including SPA3, which has a pivotalrole in light signaling, and demonstrated that phytochrome-mediated AS control of SPA3 actually contributed to promotingphotomorphogenesis (Figs. 3 and 4 and Fig. S1). Taken together,these data indicate that phytochrome induces genome-wide ASalterations to mediate light responses in Arabidopsis.Intriguingly, our GO analysis revealed that splicing factor

genes are the main early targets of phytochrome-mediated ASregulation, whereas transcription factor genes are the primarydirect targets of transcriptional regulation mediated by phyto-chrome, as previously reported (22) (Fig. 2). This finding sug-gests that phytochrome initiates both AS and transcriptioncascades, by regulating the AS of splicing factor genes and thetranscription of transcription factor genes, respectively, in re-sponse to R. It remains to be elucidated how phytochromeregulates the AS of splicing factor genes within 1 h of cR irra-diation. Phytochrome probably regulates the expression level(mRNA and protein level) or posttranslational modification ofmore upstream splicing regulators. RRC1, an SR-like splicingfactor that is required for phyB signaling, is among these can-didate upstream factors, because the R-responsive AS alterationsof RS31 an SR34b have been shown to be partially dependenton RRC1 (21). Moreover, we found that some of the earlyR-responsive and phytochrome-dependent AS changes weresubstantially reduced in rrc1 mutants, whereas others were

totally independent of RRC1 (Fig. S4C). Therefore, RRC1 isresponsible for mediating phytochrome-induced AS alterationsonly in a subset of the target genes, which may account for thefact that phyB-mediated light responses are diminished only par-tially in rrc1 mutants (21).Recently, it has been shown that phytochrome also regulates

the translation of protochlorophyllide reductase mRNA by di-rectly interacting with the RNA-binding protein PENTA1 in thecytosol (13). Therefore, phytochrome has emerged as a directregulator of various aspects of gene expression that interacts withdifferent partners in different subcellular locations. It would beof particular interest to establish if a common initial mecha-nism underlies these apparently distinct modes of phytochromesignal transduction.

Materials and MethodsPlant materials and growth conditions, α-amanitin treatment, RNA prepa-rations and RNA-sequencing analysis, read mapping to Arabidopsis genes,comparison of splicing events, statistical tests for determining overrepresentedGO categories, sqRT-PCR analysis of alternative splicing patterns, quantita-tive RT-PCR analysis, and generation and analysis of transgenic plants aredescribed in SI Text.

ACKNOWLEDGMENTS. We thank Satoru Kuhara for valuable discussions;Naomi Koike and Mami Shibata for technical assistance; Yasuomi Tada andNorihito Nakamichi for technical suggestions; and the National Institute ofGenetics of the Research Organization of Information and Systems for pro-viding excellent supercomputer services. This work was supported by MEXTKAKENHI 25291064, 25120720, and 23012033 (to T.M.), 25710017 and24114713 (to K.H.), and 221S0002 (to Y.S.); JST PRESTO (T.M.) and CREST(K.H.) in the research area “Creation of essential technologies to utilizecarbon dioxide as a resource through the enhancement of plant productivityand the exploitation of plant products”; Kyushu University InterdisciplinaryPrograms in Education and Projects in Research Development (T.M.); theProgram for Promotion of Basic and Applied Researches for Innovations inBio-oriented Industry (K.H.); and a grant for Basic Science Research Projectsfrom The Sumitomo Foundation (to T.M.).

1. Franklin KA, Quail PH (2010) Phytochrome functions in Arabidopsis development.J Exp Bot 61(1):11–24.

2. Nagatani A (2010) Phytochrome: Structural basis for its functions. Curr Opin Plant Biol13(5):565–570.

3. Matsushita T, Mochizuki N, Nagatani A (2003) Dimers of the N-terminal domain ofphytochrome B are functional in the nucleus. Nature 424(6948):571–574.

4. Palágyi A, et al. (2010) Functional analysis of amino-terminal domains of the photo-receptor phytochrome B. Plant Physiol 153(4):1834–1845.

5. Kircher S, et al. (1999) Light quality-dependent nuclear import of the plant photo-receptors phytochrome A and B. Plant Cell 11(8):1445–1456.

6. Yamaguchi R, Nakamura M, Mochizuki N, Kay SA, Nagatani A (1999) Light-dependenttranslocation of a phytochrome B-GFP fusion protein to the nucleus in transgenicArabidopsis. J Cell Biol 145(3):437–445.

7. Li J, Li G, Wang H, Deng XW (2011) Phytochrome signaling mechanisms. Arabidopsis Book9:e0148.

8. Ni M, Tepperman JM, Quail PH (1999) Binding of phytochrome B to its nuclear sig-nalling partner PIF3 is reversibly induced by light. Nature 400(6746):781–784.

9. Shen H, Moon J, Huq E (2005) PIF1 is regulated by light-mediated degradationthrough the ubiquitin-26S proteasome pathway to optimize photomorphogenesis ofseedlings in Arabidopsis. Plant J 44(6):1023–1035.

10. Al-Sady B, Ni W, Kircher S, Schäfer E, Quail PH (2006) Photoactivated phytochromeinduces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. MolCell 23(3):439–446.

11. Lau OS, Deng XW (2012) The photomorphogenic repressors COP1 and DET1: 20 yearslater. Trends Plant Sci 17(10):584–593.

12. van Zanten M, et al. (2010) Photoreceptors CRYTOCHROME2 and phytochrome Bcontrol chromatin compaction in Arabidopsis. Plant Physiol 154(4):1686–1696.

13. Paik I, Yang S, Choi G (2012) Phytochrome regulates translation of mRNA in the cy-tosol. Proc Natl Acad Sci USA 109(4):1335–1340.

14. Liu MJ, Wu SH, Chen HM, Wu SH (2012) Widespread translational control contributesto the regulation of Arabidopsis photomorphogenesis. Mol Syst Biol 8(1):566.

15. Hoecker U (2005) Regulated proteolysis in light signaling. Curr Opin Plant Biol 8(5):469–476.

16. Mano S, Hayashi M, Nishimura M (1999) Light regulates alternative splicing of hy-droxypyruvate reductase in pumpkin. Plant J 17(3):309–320.

17. Simpson CG, et al. (2008) Monitoring changes in alternative precursor messenger RNAsplicing in multiple gene transcripts. Plant J 53(6):1035–1048.

18. Jung KH, et al. (2009) Analysis of alternatively spliced rice transcripts using microarraydata. Rice 2(1):44–55.

19. Kornblihtt AR, et al. (2013) Alternative splicing: A pivotal step between eukaryotictranscription and translation. Nat Rev Mol Cell Biol 14(3):153–165.

20. Syed NH, Kalyna M, Marquez Y, Barta A, Brown JW (2012) Alternative splicing inplants—Coming of age. Trends Plant Sci 17(10):616–623.

21. Shikata H, et al. (2012) The RS domain of Arabidopsis splicing factor RRC1 is requiredfor phytochrome B signal transduction. Plant J 70(5):727–738.

22. Tepperman JM, Hwang YS, Quail PH (2006) phyA dominates in transduction of red-light signals to rapidly responding genes at the initiation of Arabidopsis seedling de-etiolation. Plant J 48(5):728–742.

23. Wang BB, Brendel V (2004) The ASRG database: Identification and survey of Arabi-dopsis thaliana genes involved in pre-mRNA splicing. Genome Biol 5(12):R102.

24. Hoecker U, Quail PH (2001) The phytochrome A-specific signaling intermediate SPA1interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis.J Biol Chem 276(41):38173–38178.

25. Chen H, et al. (2010) Arabidopsis CULLIN4-damaged DNA binding protein 1 interactswith CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes toregulate photomorphogenesis and flowering time. Plant Cell 22(1):108–123.

26. Laubinger S, Hoecker U (2003) The SPA1-like proteins SPA3 and SPA4 repress pho-tomorphogenesis in the light. Plant J 35(3):373–385.

27. Bunsen R, Roscoe H (1862) Photochemische Untersuchungen. Ann Phys 193(12):529–562.28. Borthwick HA, Hendricks SB, Parker MW, Toole EH, Toole VK (1952) A reversible

photoreaction controlling seed germination. Proc Natl Acad Sci USA 38(8):662–666.29. Shinomura T, et al. (1996) Action spectra for phytochrome A- and B-specific photo-

induction of seed germination in Arabidopsis thaliana. Proc Natl Acad Sci USA 93(15):8129–8133.

30. Mancinelli AL (1994) The physiology of phytochrome action. Photomorphogenesis inHigher Plants, eds Kendrick RE, Kronenberg GHM (Kluwer Academic, Dordt, TheNetherlands), 2nd Ed, pp 211–269.

31. Brogna S, Wen J (2009) Nonsense-mediated mRNA decay (NMD) mechanisms. NatStruct Mol Biol 16(2):107–113.

32. Arciga-Reyes L, Wootton L, Kieffer M, Davies B (2006) UPF1 is required for nonsense-mediated mRNA decay (NMD) and RNAi in Arabidopsis. Plant J 47(3):480–489.

33. Falciatore A, et al. (2005) The FLP proteins act as regulators of chlorophyll synthesis inresponse to light and plastid signals in Chlamydomonas. Genes Dev 19(1):176–187.

34. Wu HP, et al. (2014) Genome-wide analysis of light-regulated alternative splicingmediated by photoreceptors in Physcomitrella patens. Genome Biol 15(1):R10.

35. Petrillo E, et al. (2014) A chloroplast retrograde signal regulates nuclear alternativesplicing. Science 344(6182):427–430.

18786 | www.pnas.org/cgi/doi/10.1073/pnas.1407147112 Shikata et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

18,

202

0