Fusion of the SRDX motif to OsPIL11 or OsPIL16 causes rice constitutively photomorphogenic phenotypes in darkness

Dark-grown seedlings develop skotomorphogenesis. Because of the development of rice direct seeding cultivation systems, there is an increasing need for clarifying the molecular mechanism underlying rice skotomorphogenic development. It has been reported that SRDX motif, LDLDLELRLGFA, was able to convert a transcriptional activator into a strong repressor. In the present study, to explore the functions of PILs in rice skotomorphogenesis, we generated OsPIL11-SRDX and OsPIL16-SRDX transgenic lines by fusing the SRDX transcriptional repressor motif to the C-terminal of two members of the phytochrome interacting factor-like (OsPIL) family in rice (OsPIL11 and OsPIL16). The OsPIL11-SRDX and OsPIL16-SRDX seedlings grown in darkness had constitutively photomorphogenic phenotypes with short coleoptiles and open leaf blades. The results of an RNA sequencing analysis revealed that the dark-grown OsPIL11-SRDX and OsPIL16-SRDX lines had gene expression patterns similar to those of wild-type seedlings grown under red light. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that the expression levels of genes related to photosynthesis, photosynthesis–antenna proteins, and porphyrin and chlorophyll metabolism were up-regulated in the dark-grown OsPIL11-SRDX and OsPIL16-SRDX lines, whereas the expression of genes related to the auxin pathway was down-regulated. In contrast, the expression levels of these photosynthesis-related genes were down-regulated in dark-grown transgenic seedlings overexpressing OsPIL11 or OsPIL16, which had exaggerated skotomorphogenesis. Considered together, our data indicate that OsPIL11 and OsPIL16 primarily function as transcriptional activators, at least in regards to promoting skotomorphogenesis and repressing the expression of photosynthesis-related genes.


Introduction
Skotomorphogenesis in the absence of light and photomorphogenesis in the presence of light are two contrasting plant developmental programs. Dark-grown Arabidopsis thaliana (Arabidopsis) plants have etiolated phenotypes with long hypocotyls, apical hooks, and closed and yellowish cotyledons (Leivar et al. 2008;Pham et al. 2018). In darkness, skotomorphogenic plant growth is necessary for photoautotroph survival. Regarding Arabidopsis, rapid and exaggerated hypocotyl elongation helps seedlings seek light. The tightly folded apical hook allows easy passage through the soil and protects the small cotyledons and underlying meristematic region from damage (Josse and Halliday 2008). Thus, clarifying the molecular mechanisms underlying skotomorphogenesis is an important research objective. There has recently been rapid progress in the characterization of Arabidopsis phytochrome interacting factors (PIFs) in terms of their functions mediating the switch from skotomorphogenesis to photomorphogenesis.
Phytochrome interacting factors, which form a subset of the basic helix-loop-helix (bHLH) transcription factor superfamily, are reportedly essential for maintaining the skotomorphogenic state of etiolated Arabidopsis seedlings (Castillon et al. 2007; Leivar and Monte 2014;Leivar and Quail 2011). The Arabidopsis PIF family comprises eight members (PIF1-8) (Lee and Choi 2017). Previous studies revealed that in darkness, a quadruple mutant (pifQ) lacking PIF1, PIF3, PIF4, and PIF5 resembles a wild-type (WT) seedling exposed to light, with short hypocotyls and expanded cotyledons (Kim et al. 2011;Leivar et al. 2009;Shin et al. 2009). Microarray analyses of the pifQ mutant and the subsequent Gene Ontology analysis indicated that PIF1, PIF3, PIF4, and PIF5 repress the expression of genes involved in chlorophyll biosynthesis and photosynthesis in plants grown in darkness (Shin et al. 2009;Zhang et al. 2013). Similarly, PIF1 and/or PIF3 suppress the expression of genes associated with chlorophyll biosynthesis and photosynthesis during de-etiolation Liu et al. 2013).
Although the functions of Arabidopsis PIFs in skotomorphogenesis continue to be elucidated, the functions of rice PIF homologs affecting skotomorphogenesis remain unclear. Nakamura et al. (2007) identified six PIF-like (PIL) homologs in the rice genome (OsPIL11-16). In darkness, the Arabidopsis pifQ mutant has constitutively photomorphogenic phenotypes. However, monogenic mutations at each of the four loci result in no or minimal visible effects on skotomorphogenesis. Various double and triple pif mutations result in increasingly photomorphogenic phenotypes in darkness (Kim et al. 2011;Leivar et al. 2008Leivar et al. , 2012Lorrain et al. 2009;Shin et al. 2009;Stephenson et al. 2009). These results indicate that these four Arabidopsis PIFs functions in a highly redundant manner to control skotomorphogenic development.
It has been reported that ethylene-responsive elementbinding factor (ERF)-associated amphiphilic repression (EAR) motif was able to convert a transcriptional activator into a strong repressor (Hiratsu et al. 2002;Ohta et al. 2001). Hiratsu et al. maximized the potential repressive activity of the EAR motif repression domain, and the resultant repression domain, LDLDLELRLGFA, was designated as SRDX (Hiratsu et al. 2003). This technology was developed to study the consequences of silencing the target genes of individual transcription factors. Interestingly, SRDX dominant repressors may repress the transcription of their target genes as well as the expression of the genes targeted by other members of their respective gene families, thereby overcoming functional redundancy (Hiratsu et al. 2003;Mitsuda et al. 2007). In this study, to explore the functions of rice PILs in skotomorphogenesis, we fused the SRDXencoding DNA sequence to the 3′ end of OsPIL11 and OsPIL16. The OsPIL11-SRDX and OsPIL16-SRDX fusion constructs were expressed in WT Nipponbare plants. The resulting transgenic plants had constitutively photomorphogenic phenotypes in the absence of light. Molecular analyses revealed that genes related to photosynthesis and the auxin pathway were enriched among the differentially expressed genes (DEGs). Our findings suggest that either OsPIL11 or OsPIL16 is necessary for maintaining rice skotomorphogenesis.

Phenotypes of dark-grown seedlings
Rice seeds were surface-sterilized and then sown on 0.4% (w/v) agar. After an overnight incubation at 4 °C, the seeds were germinated at 28 ± 1 °C and grown in darkness for 9 days. The seedlings were then photographed.

Generation of transgenic lines
The previously described OsPIL11-overexpression (OsPIL11-OX) vector (Li et al. 2012) and OsPIL16-overexpression (OsPIL16-OX) vector (He et al. 2016) were used for generating transgenic lines. In these two vectors, OsPIL11 and OsPIL16 were subcloned between the maize ubiquitin gene promoter and the nos terminator. To obtain the SRDX sequence, two single-stranded DNA fragments (5′-P GAT CCA TGG CAC TGG ATC TGG ATC TGG AAC TGC  GCC TGG GCT TTG CGT AAG OH -3′ and 5′-P AAT TCT TAC  GCA AAG CCC AGG CGC AGT TCC AGA TCC AGA TCC AGT GCC ATG OH -3′) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). After denaturing at 98 °C for 3 min, the sequences were annealed at 55 °C for 5 min to produce double-stranded SRDX, which encodes a peptide containing the SRDX domain (LDLDLELRLGFA) (Hiratsu et al. 2003). The ends of the SRDX sequence included BamH I (-P GATCC-, underlined) and EcoR I (-G-and -AATTC-, underlined) sites for the subcloning of the double-stranded SRDX sequence into the pBluescript II SK ( +) vector to generate the pBS-SRDX vector.
The plant expression vectors were transformed into Agrobacterium tumefaciens strain EHA105 by electroporation and the resulting bacteria cultures were used for the transformation of rice (Oryza sativa cv Nipponbare).

DNA extraction and southern blot analysis
Genomic DNA was isolated and purified from the OsPIL16-SRDX lines, OsPIL11-SRDX lines, and WT plants at the three-leaf stage following Murray and Thompson (1980). DNA was digested with HindIII and transferred to a nylon membrane (Hybond-N + ; Amersham, Buckinghamshire, UK) after electrophoresis on a 0.8% agarose gel. Hpt II gene was selected as the marker gene. The detailed protocol was the same as described by He et al. (2016).

Subcellular localization of OsPIL16-SRDX-GFP and OsPIL11-SRDX-GFP
To construct the OsPIL11-SRDX-GFP and OsPIL16-SRDX-GFP fusion proteins, the OsPIL11-SRDX and OsPIL16-SRDX sequences were separately amplified by PCR using either pBS-OsPIL11-SRDX or pBS-OsPIL16-SRDX as the template. The primer pair used for amplifying OsPIL11-SRDX was OsPIL11 F2 (5′-gagaacacgggggactctagaATG AAC CAG TTC GTC CCT GATT-3′, 21 bases homologous to the ends of the linearized vector in lowercase letters) and SRDX R2 (5′-tcatgcctccgcggcggatccaCGC AAA GCC CAG GCG CAG -3′, 22 bases homologous to the ends of the linearized vector in lowercase letters). The primer pair used for amplifying OsPIL16-SRDX was OsPIL16 F2 (5′-gaga-acacgggggactctagaATG CTA CGC GGG AAC GAC A-3′, 21 bases homologous to the ends of the linearized vector in lowercase letters) and SRDX R2. The PCR products were subcloned between the cauliflower mosaic virus 35S promoter and the GFP start codon in the pBI221-GFP vector according to the In-Fusion® HD Cloning Kit User Manual (Clontech, CA, USA). The resulting plasmid DNA was transiently introduced into rice protoplasts isolated from 10-day-old etiolated NIP seedlings using the polyethylene glycol method. The OsGEN1-mCherry protein was used as a nuclear marker. After an overnight incubation in darkness, fluorescence was observed using the FluoView FV1000 fluorescence confocal microscope (Olympus, Tokyo, Japan).

Measurement of coleoptile and mesocotyl lengths
Surface-sterilized seeds of the NIP, OsPIL11-SRDX, OsPIL16-SRDX, OsPIL11-OX, and OsPIL16-OX lines were sown on 0.4% (w/v) agar and then incubated in darkness at 28 °C for 9 days. The seedlings were then photographed, after which the coleoptile and mesocotyl lengths were measured using a ruler.

Transcriptome sequencing
Surface-sterilized seeds of the WT control and the OsPIL11-SRDX (#2 and #3) and OsPIL16-SRDX (#8 and #9) transgenic lines were sown on 0.4% (w/v) agar and incubated overnight at 4 °C. The seeds were then incubated in darkness (D) or under red light (R) at 28 °C for 6 days. The above-ground plant parts were harvested for transcriptome sequencing analyses, which were completed by Novogene Biotech (Tianjin, China) using standard Illumina protocols. The clean reads were aligned to the assembled transcriptome using the Bowtie2 program (version 2.3.4.1) (Langmead and Salzberg 2012). Following the alignment, raw read counts for each transcript were determined using RSEM (version 1.2.31) (Li and Dewey 2011) and then normalized to FPKM (fragments per kilobase exon per million mapped fragments). Raw counts were analyzed using the DESeq2 package (Love et al. 2014) to identify DEGs, which were defined as genes with a false discovery rate adjusted P < 0.05 and an expression level fold-change ≥ 2. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using the clusterProfiler R package (version 3.4.4) (Yu et al. 2012) to identify the significantly enriched pathways among the DEGs.

Quantitative real-time (qRT)-PCR analysis
Total RNA was isolated from the seedlings grown in darkness for 6 days using RNAiso Plus (TaKaRa, Dalian, China). First-strand cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa). The qRT-PCR analysis was performed using the ABI Q5 PCR System (Applied Biosystems, Foster City, CA, USA) and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa). The expression level of each target gene was derived from its signals normalized against either eEF-1α (AK061464) or ACTIN (LOC4333919) using the ΔCT method (Jain et al. 2006;Livak and Schmittgen 2001). The qRT-PCR analysis was completed using three biological replicates. Primer details are listed in Table S1.

Data analysis of coleoptile length
All results are expressed as mean values ± standard error (SE) based on more than 20 seedlings. Statistical significance was assessed using Student's t-test. Probability values of less than 0.05 were considered to be statistically significant. A single asterisk (*) and double asterisks (**) represent significance at the levels of 0.05 and 0.01, respectively.

Characteristics of rice skotomorphogenic phenotypes
Dark-grown Arabidopsis seedlings have etiolated phenotypes with long hypocotyls, apical hooks, and closed and yellowish cotyledons (Leivar et al. 2008). In this study, we examined the phenotypes of dark-grown japonica and indica rice seedlings. The japonica rice varieties grown in darkness had long coleoptiles. In 9-day-old seedlings grown in darkness, yellowish and rolled leaves were either wrapped by coleoptiles or grew out of coleoptiles (Fig. 1a,upper panel). Although the morphological characteristics of the indica seedlings were similar to those of japonica seedlings, some indica varieties grown in darkness had longer mesocotyls (Fig. 1b,upper panel). However, seedlings grown under R conditions had shorter coleoptiles, green leaves, and expanded leaf blades ( Fig. 1a and b, lower panels). These results indicate that relatively long coleoptiles and mesocotyls as well as yellowish and rolled leaf blades are typical characteristics of dark-grown rice seedlings.

OsPIL16-SRDX and OsPIL11-SRDX lines grown in darkness had constitutively photomorphogenic phenotypes
In Arabidopsis, PIFs negatively regulate light responses by repressing photomorphogenesis and maintaining the skotomorphogenic state of etiolated seedlings in darkness (Leivar and Monte 2014;Leivar et al. 2008). To clarify the roles of rice PIFs in maintaining etiolated seedling development, we produced transgenic lines expressing the OsPIL16-SRDX Fig. 1 Phenotypes of etiolated rice seedlings grown under continuous light or in darkness for 9 days. a Representative phenotypes of japonica rice varieties grown in darkness (upper panel) or under continuous red light (lower panel) for 9 days. NIP Nipponbare, WYJ7 Red arrows indicate the coleoptile apex. Yellow arrows indicate the mesocotyl apex. Scale bar = 1 cm. (Color figure online) ◂ fusion construct under the control of the ubiquitin gene promoter (Fig. 2a). Three independent T 3 lines (#4, #8, and #9) were used to analyze the role of OsPIL16-SRDX based on the result of Southern blot analysis and their high expression levels ( Fig. 2b and c). A comparison of the WT and OsPIL16-SRDX lines grown in darkness for 9 days revealed that the coleoptiles of the OsPIL16-SRDX lines (0.58 ± 0.01 cm, 0.50 ± 0.01 cm, and 0.44 ± 0.01 cm in lines #4, #8, and #9, respectively) were shorter than the WT coleoptile (3.76 ± 0.02 cm) ( Fig. 2d and e). Moreover, the second leaf blades of the OsPIL16-SRDX lines were expanded, in contrast to the rolled leaf blades of the WT seedlings (Fig. 2d). Therefore, OsPIL16-SRDX lines had photomorphogenic phenotypes (i.e., short coleoptiles and expanded leaf blades) similar to the WT seedlings grown under R (Fig. S1). These results imply that OsPIL16-SRDX negatively regulates rice skotomorphogenesis.

3
The OsPIL16-SRDX and OsPIL11-SRDX seedlings grown in darkness have gene expression profiles similar to those of wild-type seedlings grown under red light To investigate the role of PIFs in maintaining rice skotomorphogenesis at the genome level, we firstly examined the subcellular localization of OsPIL11-SRDX and OsPIL16-SRDX, we generated constructs for the expression of GFPtagged fusion proteins under the control of the cauliflower mosaic virus 35S promoter. The constructs were transiently expressed in rice protoplasts. The OsPIL11-SRDX-GFP and OsPIL16-SRDX-GFP signals were detected in the nucleus (Fig. S2). In the control protoplasts expressing GFP alone, fluorescent signals were detected in the cytoplasm and nucleus (Fig. S2). These results indicate that OsPIL11-SRDX and OsPIL16-SRDX are localized in the nucleus.
We then performed an RNA sequencing (RNA-seq) analysis of dark-grown OsPIL16-SRDX (#8 and #9), R-grown WT, and dark-grown WT seedlings. The data revealed 8020 DEGs between the dark-grown OsPIL16-SRDX [OsPIL16-SRDX(D)] and the dark-grown WT [WT(D)] (Table S2). Of these DEGs, the expression levels of 4129 and 3891 genes were up-regulated and down-regulated, respectively, in the OsPIL16-SRDX lines (Fig. S3, Table S2), implying that OsPIL16-SRDX positively and negatively regulates gene expression. Furthermore, 10,526 DEGs were detected between WT(D) seedlings and WT(R) seedlings (i.e., WT seedlings exposed to R) (Table S3). A comparison of the two DEG sets indicated that approximately 49.8% of the DEGs between OsPIL16-SRDX(D) and WT(D) were also differentially expressed between WT(R) and WT(D) (Fig. 3a). These results indicate that dark-grown OsPIL16-SRDX seedlings have expression patterns that are similar to those of the R-grown WT control.
Because OsPIL11-SRDX lines had phenotypes that resembled those of OsPIL16-SRDX lines in darkness (Fig. 2), we also performed an RNA-seq analysis of OsPIL11-SRDX lines. We detected 7268 DEGs between the dark-grown OsPIL11-SRDX [OsPIL11-SRDX(D)] and the WT(D) seedlings (Table S4). A comparison of the DEGs between OsPIL11-SRDX(D) and WT(D) and the DEGs between WT(R) and WT(D) revealed that approximately 52.0% of the DEGs between OsPIL11-SRDX(D) and WT(D) were also differentially expressed between WT(R) and WT(D) (Fig. 3a). These results indicate that the dark-grown OsPIL11-SRDX and R -grown WT seedlings have similar gene expression patterns.
OsPIL11-SRDX and OsPIL16-SRDX promote the expression of photosynthesis-related genes in darkness, but overexpression of OsPIL11 and OsPIL16 has the opposite effect Because the photomorphogenic phenotypes of the OsPIL11-SRDX and OsPIL16-SRDX lines were similar to those of R-grown WT seedlings, we speculated that the shared DEGs among OsPIL11-SRDX(D)/WT(D), OsPIL16-SRDX(D)/ WT(D), and WT(R)/WT(D) affect rice skotomorphogenesis. We detected 3183 shared DEGs among the three DEG sets (Fig. 3a, Table S5). Of these DEGs, the expression levels of 1239 and 1320 genes were respectively up-regulated and down-regulated in the OsPIL11-SRDX(D), OsPIL16-SRDX(D), and WT(R) seedlings (Fig. S3, Tables S6 and S7). The enriched KEGG pathways among the shared DEGs were associated with photosynthesis. More specifically, photosynthesis, photosynthesis-antenna proteins, and porphyrin and chlorophyll metabolism were the most enriched KEGG pathways among the shared up-regulated DEGs (Fig. 3b, Table S8). Of the 30 genes assigned to the photosynthesis pathway, 10 encode components of the photosystem I reaction center complex, whereas 11 genes encode components of the photosystem II reaction center complex (Fig. 3c, Table 1). Additionally, one, five, and three genes assigned to the photosynthesis pathway are associated with the cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPases, respectively (Table 1). Twelve genes encoding light-harvesting chlorophyll proteins were assigned to the photosynthesis-antenna proteins pathway (Fig. 3c, Table 1). Moreover, 15 genes involved in chlorophyll biosynthesis were assigned to the porphyrin and chlorophyll metabolism pathway (Fig. 3c, Table 1). Other significantly enriched pathways among the up-regulated DEGs were glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, and carbon metabolism (Fig. 3b, Table S8). These findings imply that fusion of SRDX to OsPIL11 and OsPIL16 promotes multiple important photosynthesis-related processes, including light absorption, electron transfer, and carbon assimilation. Additionally, many of the up-regulated DEGs were associated with ribosome assembly (Fig. 3b, Table S8), implying that protein synthesis is likely regulated by OsPIL11 and OsPIL16.
Plant hormone signal transduction was the most enriched KEGG pathway among the shared down-regulated DEGs (Fig. 3d, Table S9). Of the 21 genes assigned to the plant 1 3 hormone signal transduction pathway, 10 are associated with the auxin pathway, including seven auxin-responsive Aux/IAA genes, one small auxin up-regulated (SAUR) gene (OsSAUR38), one auxin-responsive gene (OsGH3-6), and one auxin influx carrier-encoding gene (OsAUX1) (Fig. 3e, Table 2). In addition to auxin, the down-regulated DEGs assigned to the plant hormone signal transduction pathway were associated with other hormones, including cytokinin, ethylene, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid (Table 2). Auxin regulates almost all aspects of plant growth and development essentially by modulating cell division and elongation (Santner and Estelle 2009). In Arabidopsis, PIFs may regulate auxin signaling during deetiolation (Hornitschek et al. 2012;Nozue et al. 2011). Thus, OsPIL11-SRDX and OsPIL16-SRDX likely suppress rice skotomorphogenesis by promoting photosynthetic processes and repressing the auxin pathway in darkness.
We further confirmed the expression of genes related to photosynthesis and auxin signaling in the dark-grown OsPIL16-SRDX and OsPIL11-SRDX seedlings by qRT-PCR. Among 57 shared up-regulated DEGs, 36 genes related to photosynthesis, photosynthesis-antenna proteins, and porphyrin and chlorophyll metabolism pathways were analyzed in a qRT-PCR assay. The data indicated that the expression levels of all of these genes were up-regulated in the R-grown WT seedlings and in the dark-grown OsPIL16-SRDX and OsPIL11-SRDX seedlings (Figs. 4a, b, and c and S4). Of the shared down-regulated DEGs assigned to the plant hormone signal transduction pathway (Table 2), eight genes related to the auxin signaling pathway were analyzed by qRT-PCR, which revealed that the expression of all eight genes was down-regulated in the R-grown WT seedlings and in the dark-grown OsPIL16-SRDX and OsPIL11-SRDX seedlings (Figs. 4d and S4). The qRT-PCR results were consistent with the RNA-seq data. These findings suggest that OsPIL11-SRDX and OsPIL16-SRDX induce the expression of photosynthesis-related genes and repress the expression of genes responsive to the auxin pathway in seedlings grown in darkness.
To further assess how the shared DEGs are regulated by OsPIL11 and OsPIL16, we produced transgenic rice lines overexpressing OsPIL11. Two independent homozygous T 4 lines (#4 and #26) were selected to functionally characterize OsPIL11 in rice because of their high OsPIL11 expression levels (Fig. 5a). Previously reported OsPIL16-OX lines were also used in this study (He et al. 2016). A comparison of the OsPIL11-OX, OsPIL16-OX, and WT seedlings grown in darkness for 9 days revealed that the mesocotyls of the OsPIL11-OX and OsPIL16-OX seedlings were significantly longer than the WT mesocotyl ( Fig. 5b and c), which is consistent with the skotomorphogenic phenotypes of some of the indica rice varieties (Fig. 1b). Unexpectedly, the coleoptiles of the OsPIL11-OX and OsPIL16-OX lines were significantly shorter than the WT coleoptile ( Fig. 5b and c). We speculate that the limited seed reserves were mainly used by the elongating mesocotyls in the OsPIL11-OX and OsPIL16-OX lines. These results suggest that OsPIL11 and OsPIL16 are involved in promoting rice skotomorphogenesis. We further analyzed the expression patterns of DEGs related to photosynthetic processes and the auxin pathway in OsPIL16-OX and OsPIL11-OX seedlings grown in darkness. The expression of genes assigned to the photosynthesis, photosynthesis-antenna proteins, and porphyrin and chlorophyll metabolism KEGG pathways was significantly repressed in the OsPIL11-OX and OsPIL16-OX lines (Figs. 5d-f and S5). These observations imply that OsPIL11 and OsPIL16 repress the expression of genes related to several important photosynthesis-related processes. The opposite effects of overexpression vs expressing SRDX fusion proteins of OsPIL11 and OsPIL16 suggest that OsPIL11 and OsPIL16 primarily function as transcriptional activators, in regards to promoting skotomorphogenesis and repressing the expression of photosynthesis-related genes.
Unexpectedly, the expression levels of genes related to auxin signaling were also down-regulated in the OsPIL11-OX and OsPIL16-OX lines, which was consistent with the expression patterns of these genes in the OsPIL11-SRDX and OsPIL16-SRDX lines (Fig. 5g). These results indicate that OsPIL11 and OsPIL16 help maintain rice skotomorphogenesis by repressing the expression of genes involved in photosynthetic activities. However, whether and how the genes related to the auxin signaling pathway are regulated by OsPIL11 and OsPIL16 during rice skotomorphogenesis remains to be elucidated.

Fusion of the SRDX domain suppresses the functions of OsPIL11 and OsPIL16 in dark-grown rice seedlings
In the present study, OsPIL11-OX and OsPIL16-OX seedlings grown in darkness had substantially elongated mesocotyls and shortened coleoptiles (Fig. 5). Mesocotyl elongation is one of the skotomorphogenic characteristics of some of the analyzed indica rice varieties (Fig. 1). Thus, ecotopic expression of OsPIL11 and OsPIL16 causes seedlings an exaggeratedly skotomorphogenic phenotypes. However, OsPIL11-SRDX and OsPIL16-SRDX seedlings had typical photomorphogenic phenotypes, resembling WT seedlings grown under light (Fig. 2). These observations suggest that the addition of the SRDX domain repressed the functions of OsPIL11 and OsPIL16 related to the maintenance of rice skotomorphogenesis. Consistent with these phenotypes, OsPIL11 and OsPIL16 with and without the SRDX motif had the opposite effects on gene expression patterns. For example, the expression of photosynthesis-related DEGs was repressed in the OsPIL11-OX and OsPIL16-OX lines, but was up-regulated in the OsPIL11-SRDX and OsPIL16-SRDX lines (Figs. 3, 4, and 5). It has been reported that fusion of SRDX motif to transcriptional activators converts them into dominant repressors, exhibiting phenotypic changes similar to those of plants with the corresponding loss-of-function alleles even in the presence of the original activator domain (Hiratsu et al. 2003). Based on the opposite effects of overexpression vs expressing SRDX fusion proteins for OsPIL11 and OsPIL16, we speculate that OsPIL11 and OsPIL16 function primarily as transcriptional activators, at least in regards to promoting skotomorphogenesis and repressing the expression of photosynthesis-related genes.
Notably, SRDX dominant repressors may suppress the expression of their target genes as well as the target genes of other members of the respective gene families (Hiratsu et al. 2003;Mitsuda et al. 2007). On the basis of the similarities in the photomorphogenic phenotypes and gene expression profiles of the OsPIL11-SRDX and OsPIL16-SRDX lines grown in darkness (Figs. 2 and 3), we assumed that the phenotypes of OsPIL11-SRDX and OsPIL16-SRDX rice lines are probably the result of the combined effects of all or multiple OsPILs. In Arabidopsis, PIF1, PIF3, PIF4, and PIF5 function in a highly redundant manner to control skotomorphogenic development. For example, in contrast to the single, double, and triple pif mutants, the pifQ quadruple mutant has constitutively photomorphogenic phenotypes in darkness (Kim et al. 2011;Leivar et al. 2008Leivar et al. , 2009Leivar et al. , 2012Lorrain et al. 2009;Shin et al. 2009;Stephenson et al. 2009). In this context, our findings suggest that the fusion of the SRDX domain to OsPIL11 and OsPIL16 can overcome the functional redundancy of OsPIF family members.

Fusion of SRDX to OsPIL16 and OsPIL11 caused constitutively photomorphogenic phenotypes partly via promoting expression of photosynthetic genes
In the present study, the expression levels of a set of genes associated with multiple steps of the photosynthetic process, including light absorption, electron transfer, carbon assimilation as well as chlorophyll biosynthesis, were up-regulated in dark-grown OsPIL11-SRDX and OsPIL16-SRDX lines, consistent with the corresponding expression levels in the R-grown WT seedlings, but were down-regulated in darkgrown OsPIL11-OX and OsPIL16-OX lines (Table 1; Figs. 4 and 5). Similar results were obtained for Arabidopsis. The expression levels of most chlorophyll biosynthesis-related genes and light harvesting-related genes are up-regulated in dark-grown Arabidopsis pifQ plants, similar to the expression levels in R-grown WT controls (Shin et al. 2009). These observations imply that PIFs probably repress the expression of photosynthesis-related genes in darkness. Several reports revealed the molecular mechanism by which PIFs directly regulate the photosynthesis genes in Arabidopsis (Huq et al. 2004;Liu et al. 2013;Tang et al. 2012). In Arabidopsis, 24 of 103 photosynthesis-related genes are directly targeted by PIF1, PIF3, PIF4, and PIF5, but most photosynthesisrelated genes are indirectly regulated by PIFs (Kim et al. 2016). REPRESSOR OF PHOTOSYNTHETIC GENES1 directly acts downstream of PIF1 in the endodermis to repress photosynthetic genes and regulate plastid development (Kim et al. 2016). In rice, OsPIL14 directly bound to the FLUORESCENT1 (FLU1) promoter and activated its expression to regulate chlorophyll biosynthesis in rice (Li et al. 2019). However, in the present study, the expression of FLU1 gene was up-regulated in dark-grown OsPIL11-SRDX and OsPIL16-SRDX lines and in the R-grown WT seedlings but were down-regulated in dark-grown OsPIL11-OX and OsPIL16-OX lines based on the data of RNA-seq (Fig. S6). These results suggest that OsPIL11 and OsPIL16 have the opposite effects on regulating FLU1 expression compared with OsPIL14. Thus, how OsPIL11 and OsPIL16 regulate the expression photosynthesis genes is waiting for being elucidated.

Auxin signaling pathway is probably involved in constitutively photomorphogenic phenotypes that OsPIL16-SRDX and OsPIL11-SRDX lines exhibited
The down-regulated genes in the dark-grown OsPIL11-SRDX and OsPIL16-SRDX lines and in the R-grown WT seedlings included 10 genes associated with auxin signaling (Table 2; Figs. 4d and 5g), implying that the auxin pathway may influence rice photomorphogenesis. Several PIFs regulate auxin signaling by inducing the expression of several AUX/IAA and SAUR genes during de-etiolation, diurnal growth, and an exposure to shade (Hornitschek et al. 2012;Nozue et al. 2011). In this study, the expression levels of eight DEGs associated with auxin signaling were down-regulated not only in the dark-grown OsPIL11-SRDX and OsPIL16-SRDX lines but also in the OsPIL11-OX and OsPIL16-OX lines (Figs. 4 and 5). It has been reported that when a transcription factor encodes a repressor, the phenotype caused by fusion of SRDX motif is similar to that of plants that overexpress the repressor but different from those with the corresponding loss-of-function alleles (Matsui et al. 2008). In this context, we speculate that OsPIL11 and OsPIL16 probably function as transcriptional repressor in regards to repressing the expression of genes related to auxin signal pathway. A potential relationship between auxin signals and skotomorphogenesis has not been established yet. However, published reports on Arabidopsis suggest that PIFs have a nonlinear relationship with auxin signaling that likely involves feedback regulatory mechanisms (Leivar and Monte 2014). As mentioned earlier, PIFs induce auxin synthesis and responses, implying they function upstream of auxin signaling. However, Chapman et al. (2012) suggested that auxin might promote growth partly through PIF-dependent pathways, implying auxin functions upstream of PIFs. Because auxin regulates almost every aspect of plant growth and development by modulating cell division and elongation (Santner and Estelle 2009;Wu et al. 2020), we speculate that the auxin signaling pathway is important for both skotomorphogenic and photomorphogenic development.
The KEGG pathway enrichment analysis indicated that the down-regulated DEGs were associated with hormones other than auxin (Table 2). Brassinosteroid is reportedly a skotomorphogenesis-promoting hormone that induces hypocotyl elongation in darkness and negatively regulates photomorphogenesis (Leivar and Monte 2014). In rice, D61/ BRI1 is a putative brassinosteroid receptor kinase involved in brassinosteroid signaling. Moreover, the loss-of-function mutant d61 exhibits defective skotomorphogenesis, implying D61/BRI1 is a positive regulator of skotomorphogenesis (Yamamuro et al. 2000). In accordance with this observation, D61 expression was significantly repressed in the dark-grown OsPIL11-SRDX and OsPIL16-SRDX seedlings (Table S2). However, whether and how other plant hormone pathways (e.g., cytokinin, ethylene, abscisic acid, jasmonic acid, and salicylic acid pathways) affect skotomorphogenesis in rice remain to be determined. Although the relationship between PIFs and plant hormones has been reported for Arabidopsis (Jeong and Choi 2013;Leivar and Monte 2014), there is relatively little information regarding this relationship in rice.
The observed phenotypes of the OsPIL16-SRDX and OsPIL11-SRDX seedlings were likely influenced by other rice PIF family members in addition to OsPIL11 and OsPIL16. Therefore, future studies should investigate the effects of various combinations of loss-of-function mutations to rice PIL genes on the phenotypes of plants grown in darkness. In addition, although we demonstrate that OsPIL11-SRDX and OsPIL16-SRDX suppress rice skotomorphogenesis and promotes the expression of photosynthesis-related genes in darkness in this study, the connection between photosynthesis-related gene expression and the morphogenesis characters is waiting for being established. Clearly, mis-expression of the photosynthesis-related genes in the SRDX and over-expression lines of OsPIL11 and OsPIL16 is unlikely to account for all changes in growth and development related to photomorphogenesis and skotomorphogenesis that we observed in this study. Considering that the direct seeding of rice is increasingly being used for rice production worldwide, elucidating the molecular mechanism underlying rice skotomorphogenic development is warranted. The genes responsible for maintaining rice skotomorphogenesis and the genes directly targeted by rice PIFs should be thoroughly investigated in future experiments. Skotomorphogenic phenotypes and analysis of selected differentially expressed genes (DEGs) by quantitative real-time (qRT)-PCR in dark-grown OsPIL11-OX and OsPIL16-OX seedlings. a OsPIL11 transcript levels in two independent OsPIL11-OX lines (#4 and #26). b Visible phenotypes of two independent OsPIL11-OX lines (#4 and #26), two independent OsPIL16-OX lines (#1 and #13), and wild-type (WT) seedlings grown in darkness for 9 days. Red arrows indicate the coleoptile apex. Yellow arrows indicate the mesocotyl apex. Scale bar = 1 cm. c Mesocotyl and coleoptile lengths of the WT, OsPIL11-OX, and OsPIL16-OX seedlings grown in darkness for 9 days. Data are presented as the mean ± standard error for 20-30 seedlings. **P < 0.01 compared with the WT control (Student's t-test). d-f qRT-PCR analysis of the up-regulated DEGs assigned to the photosynthesis (d), photosynthesis-antenna proteins (e), and porphyrin and chlorophyll metabolism (f) KEGG pathways. g qRT-PCR analysis of the down-regulated DEGs related to the auxin signaling pathway. Total RNA was isolated from WT, OsPIL11-OX, and OsPIL16-OX seedlings grown in darkness for 6 days. The ACTIN gene was used as an internal control. Bars indicate the standard deviation in three replicates. (Color figure online) ◂  WTR wild-type seedlings grown under red light, WTD wild-type seedlings grown in darkness, R11#2 and R11#3 dark-grown OsPIL11-SRDX lines #2 and #3, respectively, R16#8 and R16#9 dark-grown OsPIL16-SRDX lines #8 and #9, respectively All genes were detected as differentially expressed based on an adjusted P < 0.05