Distinct actions in nonvascular plants revealed by targeted inactivation of phytobilin biosynthesis

Yu-Rong Chen, Yi-shin Su, and Shih-Long Tu1

Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan

Edited by J. Clark Lagarias, University of California, Davis, CA, and approved April 5, 2012 (received for review February 1, 2012)

The red/far-red light photoreceptor phytochrome mediates photo- Although their molecular properties are similar among all morphological responses in plants. For light sensing and signaling, plants, appear to mainly function in the cytosol of phytochromes need to associate with open-chain mol- nonvascular plants and chlorophytes, which contrast with their ecules as the chromophore. Biosynthesis of tetrapyrrole chromo- nuclear function in flowering plants. It is well established that phores requires members of ferredoxin-dependent reductases phototropic responses in mosses and ferns are mediated by phy- tochromes as well as by the blue-sensing phototropins and dual red- (FDBRs). It was shown that LONG HYPOCOTYL 2 (HY2) is the only – fi FDBR in flowering plants producing the phytochromobilin (PΦB) for and blue-sensing neochromes (10 13). The protonemal laments phytochromes. However, in the moss Physcomitrella patens,we sense red-light direction by phytochromes and neochromes to ad- just their growth. Data from gene-targeted phytochrome knockout found a second FDBR that catalyzes the formation of phycourobilin mutants in Physcomitrella patens and Ceratodon purpureus further (PUB), a tetrapyrrole pigment usually found as the protein-bound supports the role of these red-light sensors in phototropism (14, form in cyanobacteria and . Thus, we named the enzyme 15). Relocation of chloroplasts in nonvascular plants is also con- PUB synthase (PUBS). Severe photomorphogenic phenotypes, in- trolled by phytochromes, neochromes and phototropins (14, 16, cluding the defect of phytochrome-mediated phototropism, were 17). For these photoresponses, cytosolic photosensors are required observed in Physcomitrella patens when both HY2 and PUBS were to sense the light direction. Morphological controls usually require disrupted by gene targeting. This indicates HY2 and PUBS function changes in gene expression; it is therefore likely that phytochromes redundantly in phytochrome-mediated responses of nonvascular in nonvascular plants also play a gene regulatory role in the cytosol. plants. Our studies also show that functional PUBS orthologs are To further understand the function of phytochromes in crypto- found in selected lycopod and chlorophyte genomes. Using mRNA gams, generating knockout mutants or inactivating phytochromes sequencing for transcriptome profiling, we demonstrate that ex- is necessary. Physcomitrella possesses seven genes encoding plant- pression of the majority of red-light-responsive genes are misregu- type phytochromes, which raises the difficulty of studying phyto- lated in the pubs hy2 double mutant. These studies showed that chrome functions by this approach. Because phytochromes and moss phytochromes rapidly repress expression of genes involved neochromes share the same bilin chromophore, it should be pos- in cell wall organization, transcription, hormone responses, and pro- sible to inactivate the whole family by simply knocking out the gene tein phosphorylation but activate genes involved in for the chromophore biosynthesis enzyme, HY2. Surprisingly, we and stress signaling during deetiolation. We propose that, in non- found a second FDBR gene in the Physcomitrella genome that we name PUBS. Our studies show that, unlike HY2, which converts vascular plants, HY2 and PUBS produce structurally different but BV to PΦB, PUBS mediates the four-electron conversion of BV to functionally similar chromophore precursors for phytochromes. Hol- PUB. Although targeted knockout of the gene for either PUBS or ophytochromes regulate biological processes through light signal- fi HY2 alone yields no obvious phenotypic consequences, the double ing to ef ciently reprogram gene expression for vegetative growth knockout mutant showed severe photomorphogenesis-deficient in the light. phenotypes. Red-light-induced phototropic responses were also defective in the double mutant, indicating that both PUBS and hotosynthetic organisms develop sophisticated photoreceptor HY2 contribute to phytochrome functions. Using the mRNA se- Psystems to regulate their growth and development (1). Phyto- quencing technique for transcriptome profiling, we further iden- chromes are the major class of photoreceptors that mainly absorb tified phytochrome-regulated genes in moss protonema during red and far-red light, to display differential photosensory activities deetiolation by comparing the profiles of red-light-responsive for regulating physiological responses. Light perception of phyto- genes in wild type and double mutant. This approach provides chromes depends on the association with open-chain a global view of phytochrome-mediated gene regulation in a non- as the chromophore. A wide variety of open-chain tetrapyrroles vascular plant. are present in photosynthetic and nonphotosynthetic organisms. Results The majority of them are found in the of pelagic FDBR Physcomitrella phytoplankton, e.g., and , where they Second Gene in Encodes a Unique Enzyme. In Arabidopis thaliana, it was shown that HY2 is the only FDBR for function as light-harvesting chromophores (2, 3). Some of them, biosynthesis of the PΦB chromophore (9). Sequence searches for like phytochromobilin (PΦB), (PCB), and bili- verdin IXα (BV), can also act as the chromophores of phyto- chromes (4). Although phycoviolobilin (PVB) and phycourobilin fi Author contributions: S.-L.T. designed research; Y.-R.C. and Y.-s.S. performed research; (PUB) have been identi ed as the chromophore of cyanobacter- Y.-R.C. and S.-L.T. analyzed data; and Y.-R.C. and S.-L.T. wrote the paper. iochromes (5, 6), their occurrence as free bilin pigments has not The authors declare no conflict of interest. been reported. Biosynthesis of these diverse tetrapyrroles requires ferredoxin-dependent bilin reductases (FDBRs), which reduce BV This article is a PNAS Direct Submission. at different double bond positions (Fig. S1) (7). The greatest di- Data deposition: The sequence reported in this paper has been deposited in the GenBank database [accession no. JQ821349 (PpPUBS)] and the data have been deposited in the versity of the BV-metabolizing FDBR members is found in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. cyanobacteria. Land plants are thought to possess only HY2. GSE36274). Mutations in the HY2 gene lead to the loss of all photoactive 1To whom correspondence should be addressed. E-mail: [email protected]. phytochromes in plants, which results in the severe disruption in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. photomorphogenesis (8, 9). 1073/pnas.1201744109/-/DCSupplemental.

8310–8315 | PNAS | May 22, 2012 | vol. 109 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1201744109 Downloaded by guest on September 29, 2021 FDBR genes in the higher plant genomes sequenced also only 21). To confirm the intermediacy of 15,16-DHBV, we monitored return apparent HY2 orthologs. In the Physcomitrella genome, the time course of the PUBS reaction by HPLC. The two-electron- we found an additional FDBR gene. The encoded protein of this reduced intermediate 15,16-DHBV was indeed transiently pro- locus possesses a putative chloroplast transit peptide (tp) at the duced, followed by the formation of PUB (Fig. 1B). Consistent N terminus of a FDBR domain with strongest similarity to 15,16- with data from the spectroscopic assay, this result indicates that dihydrobiliverdin:ferredoxin oxidoreductase (PebA), a FDBR PUBS catalyzes the reduction of the C15,C16 double bond, fol- usually found in cyanobacteria and red algae. To confirm that the lowed by that at the C4,C5 position. pebA-like gene from Physcomitrella encodes a functional protein, the recombinant, tp-truncated protein was expressed in E. coli PUBS Genes Are Present in Chlorophyte and Lycophyte Genomes. A and purified. As shown in Fig. 1A, the PebA-like protein initially genome search was next performed to identify PUBS orthologs in converted BV to produce 15,16-DHBV that was subsequently other photosynthetic organisms. No PUBS-related sequence was converted to a new product. The absorption spectrum of the new foundinknownflowering plant genomes. The genome of the spike product was identical to that of phycourobilin (PUB) with moss Selaginella moellendorffii, an ancient plant grouped into the maximum absorbance at 492 nm (18, 19). In agreement with this lycophytes, does however possess a PUBS-like gene (SmPUBS)in interpretation, the molecular mass of the new product de- addition to its HY2 homolog. PUBS-related sequences were also termined from mass spectroscopy was 4 Da larger than BV (Fig. found in several chlorophyte species including Ostreococcus luci- S2). The mass difference between BV and the new product marinus, Ostreococcus tauri, Ostreococcus sp. RCC809, and Micro- suggests that the PebA-like protein reduces two double bonds. monas sp. RCC299 (Fig. S3). To establish the function of these gene fi Based on this evidence, we conclude that the product is PUB. products, the PUBS-like genes from Selaginella moellendorf i and The PebA-like protein was named PUBS for phycourobilin Ostreococcus lucimarinus were cloned and expressed in E. coli to synthase. assay the enzymatic activity of the gene products. Both Selaginella After phycocyanobilin:ferredoxin oxidoreductase (PcyA) and and Ostreococcus PUBS proteins converted BV to PUB, based on PEB synthase (PebS), PUBS is the third FDBR found to be ca- HPLC-based bilin reductase assay and by spectroscopic enzyme pable of catalyzing a four-electron reduction of BV (Fig. S1) (20, assays (Fig. S4). By comparison, as controls, PebA only formed 15,16-DHBV, whereas PebS yielded PEB under the same assay conditions. These results indicate that PUBS is present in chlorophytes, bryophytes, and lycophytes, implicating the loss of this gene during evolution of seed plants.

PUBS Is Localized in the Plastid. We further tested whether the PUBS gene is expressed in vivo. Gene expression of Physcomitrella PUBS (PpPUBS) and PpHY2 were measured and compared with an internal control ACTIN 2 by quantitative reverse transcription- PCR (qRT-PCR) (22). Both PpPUBS and PpHY2 were expressed in all developmental stages of mosses (Fig. S5A). Expression of both genes was significantly higher in the sporophyte, suggesting that both PpPUBS and PpHY2 may be important for spore ger- mination as well as development of the primary protonema. Because a tp-like sequence is located in the N terminus of PpPUBS, this protein appears to be targeted to the plastid. The subcellular localization of PpPUBS was therefore determined by transient expression in gametophore cells using particle bom- bardment and by stable transformation via gene targeting. Transient expression of full-length PpPUBS-YFP and PpHY2- YFP in gametophore cells showed that both proteins localize to chloroplasts (Fig. S5B). We also constructed a transgenic line specifically replacing the coding region of PpPUBS with the cDNA copy fused with sGFP, which was driven by the endoge- nous PpPUBS promoter (Fig. S6D). Imaging data confirmed that the PpPUBS protein was stably expressed and localized in chloroplasts (Fig. S5B, Bottom). Based on these data, we con- clude that both FDBRs function in the plastid.

HY2 and PUBS Are Redundant for Photomorphogenesis in Physcomitrella. The discovery of PUBS indicates that, unlike flowering plants, many cryptogams possess two FDBRs. In Physcomitrella,both PUBS and HY2 are in the plastid and metabolize BV to synthesize PUB and PΦB, respectively. As the function of PΦB in photo- morphogenesis is well known, these observations raised the pos- sibility that PUB is important for photomorphogenesis. Gene targeting was performed to disrupt PpPUBS and PpHY2 loci in Fig. 1. Time course of BV reduction activity of PUBS. (A) Spectrophotometric Physcomitrella, and single- and double-knockout mutants were time course of BV reduction by PUBS under anaerobic single turnover con- PLANT BIOLOGY ditions. Spectra at 1-min intervals are shown. The absorbance decrease at 690 obtained (Fig. S6). Both are single-copy genes; hence, these nm, corresponding to the disappearance of BV, and the increase at 492 nm, studies generated null mutants for loss-of-function analyses. representing the formation of PUB, are indicated by single arrows. The ab- Phenotypes of single and double pubs hy2 mutants grown sorbance change at 580 nm (corresponding to the appearance and disap- under white, red, and blue light conditions were analyzed at pearance of the two-electron reduced intermediate 15,16-DHBV) is indicated different developmental stages. As shown in Fig. 2A, protonemal by a double arrow. (B) Reaction mixture aliquots from the steady-state assay colonies of the wild type, pubs, and hy2 single mutants all had of PUBS were withdrawn every 150 s and analyzed by reverse phase HPLC. a similar diameter. By contrast, the double mutant grew more The integrated HPLC peak areas were plotted as a function of reaction time. slowly and exhibited shorter protonemata. Such phenotypes were Circles indicate IXα (BV), open triangles indicate 15,16-dihy- much stronger under red light, indicating that the double mutant drobiliverdin (15,16-DHBV) and squares indicate phycourobilin (PUB). is severely defective in red-light sensing. All mutants also showed

Chen et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 8311 Downloaded by guest on September 29, 2021 Fig. 2. Phenotypes of gene-targeted knockout mutants. (A) Twelve-day-old protonemata of wild type (WT), pubs, hy2 and pubs hy2 grown under white, red and blue light conditions are shown. Bar = 1 mm. (B) Thirty-day-old gametophores of wild type (WT), pubs, hy2 and pubs hy2 grown under white, red and blue light conditions are shown. Gametophores with 7, 9, and 11 recognizable leaves are placed from left to right. (Scale bar: 1 mm.) (C–E). Stem length of gametophores from WT and mutants grown under white (C), red (D), and blue (E) light conditions. The length of stems from the shoot apical meristem to the end of the stem was scored. The values shown are means ± SD (n = 10).

a pale green phenotype that reflected a decreased amount of FDBRs contribute to the red-light-phototropic response, we ana- (Fig. S7A). This decrease was similar in single and lyzed the red-light triggered phototropic response in single and double mutants, suggesting that the repression of chlorophyll double mutants. Under unilateral red light at different fluence biosynthesis may be due to a feedback inhibition of the tetra- rates, the orientation of primary protonemata growth was scored pyrrole biosynthesis pathway from an increased accumu- for filaments oriented toward (positive) and away from (negative) lation (23). light (Fig. S7B). Both pubs and hy2 single mutants behaved simi- We also characterized the gametophore phenotypes of single larly, with slightly weaker phototropic responses at both high and and double mutants. Although gametophores of pubs and hy2 were low fluence rates compared with the wild type. However, the pubs indistinguishable from that of the wild type, gametophores of the hy2 double mutant lost phototropism completely (Fig. 3). This re- double mutant grown under white light showed pale green and sult indicates that PUBS and HY2 are both required for phyto- stem elongation phenotypes (Fig. 2B, Top). Stems of the double chrome-mediated phototropic responses in Physcomitrella. mutant pubs hy2 were ∼2 times longer than that of wild type and both single mutants (Fig. 2C). Gametophores of the pubs hy2 Red-Light-Responsive Genes Require the Activity of both FDBRs. The double mutant showed severe growth arrest under red light (Fig. photomorphogenesis-deficient phenotypes of the pubs hy2 double 2B, Middle). There was little leaf development on pubs hy2 mutant strongly suggest that both FDBRs contribute phytobilin gametophores. Stem lengths were slightly shorter than that of wild chromophore biosynthesis required for red-light sensing. Because type and single mutants (Fig. 2D). In contrast, the morphology of phytochromes regulate gene expression in flowering plants, we wild type, single and double mutant were almost identical under compared the red-light regulated transcriptomes of wild type and blue light (Fig. 2 B, Middle, and E). These pubs hy2 double mutant the pubs hy2 double mutant. Next-generation sequencing tech- phenotypes resemble those of chromophore-deficient mutants in nology was chosen for the transcriptome profiling. higher plants, e.g., hy1 and hy2 in Arabidopsis (8). The data indicate For these experiments, wild-type and pubs hy2 protonema were that both FDBRs are required for leaf development and for in- grown in the dark for 3 d, followed by red-light irradiation for 1 h. hibition of stem elongation. RNA from three independent treatments were pooled and sub- It is well known that red light can trigger phototropic responses in jected for mRNA sequencing using Illumina/Solexa technology. mosses (24). The protonema grows toward unilateral red light Sequence reads were mapped to the Physcomitrella genome, and − − (positive phototropism) at high fluence rates (5–10 μmol m 2 s 1), the number and density of reads corresponding to cDNA from away from it (negative phototropism) at low fluence rates (below 1 each gene were determined. Transcript levels quantified as reads − − μmol m 2 s 1), and spreads randomly at intermediate fluence per kilobase of exon model per million mapped reads (RPKM) rates (25). Phytochromes have been implicated as the main pho- were then used to identify the red-light-responsive genes (26). toreceptor sensing light direction in control of protonemal cell Wild-type genes showing RPKM ≥twofold change following 1-h growth in Physcomitrella (14). To test the hypothesis that both red-light treatment were first identified. Examination of the same

8312 | www.pnas.org/cgi/doi/10.1073/pnas.1201744109 Chen et al. Downloaded by guest on September 29, 2021 the double mutant, revealing a severe deficiency in red-light sensing when both HY2 and PUBS were inactivated.

Red-Light-Regulated, FDBR-Dependent Genes in Physcomitrella Overlap with Those Regulated by Phytochromes in Flowering Plants. To further assess the biological functions of the red-light-regulated tran- scriptomes, the 1,860 genes were subjected to the functional en- richment analysis and classified according to Gene Ontology (GO) terms. Nearly 50% of these genes have no known functions. Among those functionally annotated, overrepresented GO terms in the red light up- and down-regulated genes were identified (Table 1). In the up-regulated genes, protein folding and stress- related genes were the most represented biological processes (Table 1 and Dataset S1 ). A similar result was observed by Tep- Fig. 3. Phototropism of wild type and mutants. The 4-d-old primary protonema perman et al. for red-light-regulated genes from Arabidopsis (27). fl By contrast with flowering plants, few transcription factors (TFs) germinated from spores was treated with different uence rates of unilaterally fi monochromatic red light for 6 d. Primary protonemata showing positive, neg- were rapidly induced by red light in Physcomitrella. This nding ative, or no phototropism were counted. For each sample, more than 100 pro- indicates that a transcription factor cascade does not dominate the tonemata were scored. Three biological repeats were performed. No primary early red-light-induced gene network in Physcomitrella or that such − − protonema was negatively phototropic at fluence rates above 1.5 μmol m 2 s 1. a cascade is more rapid in Physcomitrella. Our studies suggest that Conversely, no primary protonema was positively phototropic at fluence rates enhancement of protein dephosphorylation and repression of − − below 1.5 μmol m 2 s 1. The values shown are means ± SD. protein phosphorylation play the major role in red-light signaling (Table 1 and Datasets S1 and S2). Despite the paucity of TFs among the red-light-induced and red-light-mediated transcripts for the pubs hy2 double mutant was FDBR-dependent gene pool, the basic helix–loop–helix (bHLH), used to identify those genes, which required the activity of the two MYB, AP2/B3, and bZIP families were overrepresented among FDBRs. Such genes represent the pool of potential targets of the TFs rapidly repressed by red light (Dataset S2). Many of these moss phytochromes. are known to be involved in hormone-mediated signaling pathways A total of 2,202 genes in the wild type were differentially in flowering plants, such as the AP2/B3 and MYB TFs for ethylene, expressed after 1-h red-light exposure. Of these, approximately abscisic acid, and auxin signaling. Most notable of the down-reg- half were up-regulated and half were down-regulated (Datasets S1 ulated genes, those involved in cell wall organization were the most and S2). Expression patterns of some of these genes were con- enriched in the red-light-repressed and FDBR-dependent genes. firmed by qRT-PCR (Fig. S8). Among the 1,095 up-regulated These include xyloglucan endotransglucosylase/hydrolase (XTH), genes, 70% (766 genes) showed reduced expression under red light expansin, pectinesterase, and cellulose synthase genes (Table 1 in the pubs hy2 double mutant. Of the 1,107 down-regulated genes, and Dataset S2). The products of these genes are known to par- 90% (1,004 genes) were also misregulated in the double mutant ticipate in cell wall loosening and biosynthesis, which are required (Fig. 4 and Datasets S1 and S2). Overall, ∼80% (1,860 genes) of for the elongation and expansion of plant cells. Such processes red-light-responsive genes in the wild type showed misregulation in were not overrepresented in previous data from higher plants (27, 28), suggesting that immediate repression of cell wall organization upon red-light exposure is unique for the moss. It is noteworthy that known light-related genes, although they are not functionally enriched, are among those responding rapidly to red light and also FDBR-dependent in Physcomitrella (Dataset S3). These include two CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) homologs PpCCA1a and PpCCA1b were induced by 1-h red light. Of the two moss Golden2-like (GLK) genes encoding transcription factors important for photosynthetic gene expression that were up-regulated by red light, only PpGLK1 was dependent on the presence of functional FDBRs (29). GLKs are likely re- sponsible for the pronounced induction of photosynthetic genes, as significant amount of them were up-regulated by red light (Dataset S1). We also found transcript level of three moss phytochrome genes (PpPHY3, PpPHY5A,andPpPHY5C) were rapidly down- regulated by red light, with similar pattern as reported for phyto- chrome A in Arabidopsis (30). One of the phototropin genes PpPHOTB1 was also repressed. Together, these data reveal that FDBR-dependent light sensors mediate the response to red light in mosses in a manner analogous to flowering plant phytochromes. Discussion Phytobilins can be found in photosynthetic organisms ranging from aquatic prokaryotes to multicellular land plants (2). In cyanobac- teria, red algae and cryptomonads, FDBRs function not only to PLANT BIOLOGY synthesize the phytobilin precursors of the light harvesting phy- cobiliprotein antennae, but the light sensing phytochromes and cyanobacteriochromes as well. Because land plants and chlor- Fig. 4. Expression profiles of red-light-regulated genes in wild type and ophytes lack phycobiliproteins, the function of FDBRs appears

pubs hy2 double mutant. Log2-normalized fold changes of 1,095 red-light- restricted to the synthesis of phytobilin precursors of the chro- induced genes and 1,107 red-light-repressed genes obtained from wild-type mophores of their phytochrome light sensors. Our study shows that

datasets were calculated. Heat maps of log2-normalized fold changes of green algae and nonvascular plants possess a member of the these genes in wild type (WT) and pubs hy2 double mutant were shown. The FDBR family, PUBS, that catalyzes the conversion of BV to PUB. scale of expression is shown in the center. Organisms that contain PUBS also contain a second FDBR, either

Chen et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 8313 Downloaded by guest on September 29, 2021 Table 1. Functional enrichment of phytochrome up- and down-regulated genes in Physcomitrella Up-regulated Down-regulated

GO Term P value GO Term P value

BP: Response to stress 5.3E-13 CC: Cell wall 2.3E-13 MF: Unfolded protein binding 8.8E-08 BP: Cell wall organization 1.6E-12 BP: Protein folding 7.7E-06 CC: Extracellular region 4.1E-12 MF: Protein serine/threonine phosphatase activity 9.7E-04 MF: Hydrolase activity, hydrolyzing O-glycosyl compounds 4.3E-09 MF: Beta-amylase activity 9.7E-04 MF: Sequence-specific DNA binding transcription factor activity 2.4E-07 MF: 12-oxophytodienoate reductase activity 2.4E-03 CC: Plasma membrane 2.6E-06 BP: Protein dephosphorylation 3.2E-03 MF: Pectinesterase activity 1.8E-05 BP: Photosynthesis, light reaction 3.6E-03 BP: Regulation of transcription, DNA-dependent 5.2E-05 MF: Nucleotide kinase activity 5.1E-03 BP: Hormone-mediated signaling pathway 6.1E-04 BP: Sucrose transport 7.7E-03 MF: Protein kinase activity 1.2E-03 MF: Sucrose transmembrane transporter activity 7.7E-03 BP: Protein phosphorylation 1.5E-03 MF: Receptor activity 6.0E-03

GO terms with P value < 0.01 were shown. BP, biological process; CC, cellular component; MF, molecular function.

HY2 (nonvascular plants) or PcyA (chlorophytes). Whereas HY2 isomerized by unknown factors in the moss cell or by apophyto- and PcyA synthesize PΦB and PCB, respectively, which are both chrome itself to form both the C15,16 double bond needed for functional precursors of phytochrome chromophores (2), the photoisomerization and the A-ring ethylidene group needed for function of PUBS is unclear. The present study shows that PUBS covalent linkage. Indeed, an isomerization activity that converts and HY2 are both involved in photomorphogenesis of Physcomi- PEB to PCB already has been reported for the red algae Cyani- trella under red light. This finding suggests a functionally re- dium caldarium (33). Recent studies also showed cyanobacter- dundant role for PUB and PΦB in photomorphogenesis of iochromes can autocatalytically isomerize PCB chromophore into nonvascular plants. PVB (6, 34). Such isomerization activities in mosses remain to The pubs hy2 double mutant allows us to ascertain the pheno- be identified. type of a phytochrome null in a nonvascular plant. Transcriptome It is well established that phytochromes regulate gene ex- analysis showed that the majority of red-light-responsive genes pression in seed plants (1). In Arabidopsis, for example, the ex- were misregulated in the pubs hy2 double mutant, consistent with pression of many transcription factors is down-regulated by red the lack of photoactive phytochromes in pubs hy2. By comparing light; however, this mostly occurs after longer light exposure (27, transcript levels of red-light-responsive genes in wild type and pubs 28). Our data indicate that red-light sensing by Physcomitrella hy2, we identified putative target genes of moss phytochrome also leads to rapid transcriptional repression, particularly those signaling. Functional classification of these genes suggests that the of hormone-related transcriptional networks. Such repression rapid response to red light by mosses entails repression of specific might be beneficial to enhancing other metabolic activities, such transcriptional factors, kinase signaling, hormone-mediated sig- as photosynthesis. Gene repression could be executed either by naling and cell wall extension pathways. Like phytochrome-me- nucleus-localized phytochromes or signal transduction from the diated signaling in flowering plants, red light induces expression of cytoplasm, or both. We cannot exclude the possibility that genes involved in stress responses and photosynthesis in Phys- a portion of moss phytochromes are transported into the nucleus comitrella. This supports the hypothesis that phytochromes medi- that leads to the decrease of transcriptional activities for those ate fundamentally similar processes in flowering plants and down-regulated genes we identified. On the other hand, it is also Physcomitrella, i.e., optimize harvesting and use of the available possible that cytosolic signaling of phytochromes is dominated in light energy for photosynthesis. In cryptogams, phytochromes also nonvascular plants. One example is that phototropic responses play a critical role in phototropism, which is not observed in mediated by phytochromes in nonvascular plants require the flowering plants. We speculate that the retention of two FDBRs in directional signal transmitted to the nucleus. It is doubtful that cryptogams reflects their role to support bilin-based photo- the light-activated, soluble phytochrome itself moves to the nu- receptors that regulate gene expression and those regulate cleus for directional signal transmission. More likely, light-acti- phototropism. vated phytochromes interact with components in the cytosol or We also propose that PUB functions as an alternative chro- near the plasma membrane to trigger signal transduction for mophore agonist for phytochrome action. Because it lacks the A- nuclear gene regulation. Some phytochrome interacting proteins, ring ethylidene needed for a thioether linkage (31), PUB can bind like NUCLEOSIDE DIPHOSPHATE KINASE 2 (NDPK2) only reversibly to apo-phytochrome to induce signaling function. homologs, do exist in mosses that potentially work together with In this regard, BV feeding to the Arabidopsis hy2 mutant partially phytochromes in the cytoplasm for signaling (35). Our tran- rescued the long hypocotyl phenotype (8), suggesting that re- scriptome data also reveal that rapid repression of kinase and versible binding of BV to apo-phytochrome can partially restore enhanced expression of phosphatases accompany deetiolation of holo-phytochrome activities. Lamparter and colleagues also Physcomitrella, supporting the hypothesis that protein phos- reported that the noncovalent bilin adducts of phytochrome retain phorylation may play a more important role in red-light sensing photochemical activity (32). These studies support our hypothesis of nonvascular plants compared with seed plants. that reversible binding of PUB to apo-phytochromes in mosses Low red to far red-light treatment has been shown to positively may occur and contribute to the activities of phytochromes. The regulate expression of XTHs in Arabidopsis, which leads to the binding of PUB could potentially stabilize the phytochrome petiole elongation during shade avoidance (36). In Physcomitrella, structure, maintain the protein level of phytochrome, or even red light down-regulates expression of cell wall modifying enzymes generate the partially activated Pfr conformation in the cytosol. All that would decrease the dynamic change of cell wall architecture in of these can ensure moss phytochromes function in the cytosol for an analogous manner to attenuate the elongation of protonemal photomorphological control, such as phototropism and chloro- cells during deetiolation. Several genes encoding auxin trans- plast relocation. On the other hand, because we still observed the porters and responsive factors were also rapidly down-regulated by light-dependent phenotypes in hy2 mutant, which has only PUB red light (Dataset S2). This likely contributes to inhibition of available for moss phytochromes, PUB is therefore possibly protonemal elongation during light exposure. Stem elongation of

8314 | www.pnas.org/cgi/doi/10.1073/pnas.1201744109 Chen et al. Downloaded by guest on September 29, 2021 the pubs hy2 double mutant under white light may therefore reflect Materials and Methods the loss of repression of auxin-responsive genes. Similar results Plant Materials and Growth Conditions. Protonemata and gametophores of were reported for moss mutants of cryptochromes under blue light Physcomitrella patens subsp. patens were grown on solid Knop’s medium or ’ (37). In view of these results, we propose that FDBR-dependent cultured in liquid PPNH4. Spores were germinated on solid Knop s medium red-light sensors, e.g., phytochromes, rapidly inhibit protonemal supplemented with 10 mM CaCl2 and overlaid with cellophane. Plants were – μ −2 −1 growth via suppressing cell wall modification and auxin signaling. cultured at 25 °C under continuous white light (80 100 mol m s ). In gametophores, FDBR-dependent red-light sensors repress mRNA Sequencing. MID-mRNA sequencing was carried out as suggested by auxin responses that likely act together with cryptochrome sig- Illumina. Sequencing was performed on the Illumina Genome Analyzer IIx at naling to inhibit stem elongation. Yourgene Bioscience, Taiwan. Sequence reads were mapped to the Phys- In conclusion, our study supports the hypothesis that the pres- comitrella patens genome annotation V1.6. Detailed mRNA sequencing, ervation of PUBS from algae to nonvascular plants has been im- data analysis, other experimental procedures and related references are in SI portant for land colonization. The alternative PUB chromophore Materials and Methods. found in cryptograms might reflect the retention of an ancient redundant signaling system that was lost in the streptophyte line- ACKNOWLEDGMENTS. We thank Nicole Frankenberg-Dinkel, Shu-Hsing Wu, and Mitsuyasu Hasebe for pebA, pebS, 326GFP, and pTN80 constructs; age as the phytochrome lineage expanded to permit adaptation to Anthony H. C. Huang for Physcomitrella tissues; Hsou-min Li, Harry Wilson, the changing ecology of the land environment (38). During the and Shu-Hsing Wu for critically reading the manuscript; and L. Y. Kuang, evolution of flowering plants, signaling factors not found in the M. J. Fang, Y. C. Wu, and W. D. Lin in the Transgenic Plant, the Plant Cell genome of Physcomitrella patens were developed as the phyto- Biology, the Metabolomics and the Bioinformatics Core Laboratories of the Institute of Plant and Microbial Biology, Academia Sinica for technical assis- chrome family speciated. We envisage that HY2 became the only tance. This project was supported by the Career Development Award (to S.L.T.), FDBR for chromophore biosynthesis. Academia Sinica.

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