3.1. 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.
3.2. 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 fusion construct under the control of the ubiquitin gene promoter (Fig. 2a). Three independent T3 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.
An earlier phylogenetic analysis revealed that among the six family members, the greatest genetic diversity was between OsPIL11 and OsPIL16 (Nakamura et al. 2007). To further explore whether other PIF members are also involved in rice skotomorphogenesis, we added the SRDX domain to the C-terminal of OsPIL11 (Fig. 2a). Two independent T4 OsPIL11-SRDX transgenic lines (#2 and #3) were used to analyze the role of OsPIL11-SRDX based on the result of Southern blot analysis and their high expression levels (Fig. 2b and c). Similar to the OsPIL16-SRDX lines, the OsPIL11-SRDX lines had shorter coleoptiles (0.87 ± 0.03 cm and 1.72 ± 0.01 cm in lines #2 and #3, respectively) than the WT seedlings (3.76 ± 0.02 cm) in darkness (Fig. 2d and e). These observations suggest that OsPIL11-SRDX negatively regulates rice skotomorphogenesis.
3.3. Subcellular localization of OsPIL11-SRDX and OsPIL16-SRDX proteins
To examine the subcellular localization of OsPIL11-SRDX and OsPIL16-SRDX, we generated constructs for the expression of GFP-tagged 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. 3). In the control protoplasts expressing GFP alone, fluorescent signals were detected in the cytoplasm and nucleus (Fig. 3). These results indicate that OsPIL11-SRDX and OsPIL16-SRDX are localized in the nucleus.
3.4. 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 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 8,020 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 4,129 and 3,891 genes were up-regulated and down-regulated, respectively, in the OsPIL16-SRDX lines (Fig. S2, 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. 4a). 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 7,268 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. 4a). These results indicate that the dark-grown OsPIL11-SRDX and R -grown WT seedlings have similar gene expression patterns.
We also compared the DEGs between OsPIL16-SRDX(D) and WT(D) with the DEGs between OsPIL11-SRDX(D) and WT(D). Approximately 73.6% (5,903 of 8,020) of the DEGs between OsPIL16-SRDX(D) and WT(D) were also differentially expressed between OsPIL11-SRDX(D) and WT(D), whereas about 81.2% (5,903 of 7,268) of the DEGs between OsPIL11-SRDX(D) and WT(D) were also differentially expressed between OsPIL16-SRDX(D) and WT(D) (Fig. 4a). Accordingly, OsPIL11-SRDX and OsPIL16-SRDX seedlings grown in darkness have highly similar gene expression profiles.
3.5. 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 3,183 shared DEGs among the three DEG sets (Fig. 4a, Table S5). Of these DEGs, the expression levels of 1,239 and 1,320 genes were respectively up-regulated and down-regulated in the OsPIL11-SRDX(D), OsPIL16-SRDX(D), and WT(R) seedlings (Fig. S2, 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. 4b, 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. 4c, 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. 4c, Table 1). Moreover, 15 genes involved in chlorophyll biosynthesis were assigned to the porphyrin and chlorophyll metabolism pathway (Fig. 4c, 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. 4b, 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. 4b, 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. 4d, Table S9). Of the 21 genes assigned to the plant 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. 4e, 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 de-etiolation (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.
Table 1. Differentially expressed genes assigned to the photosynthesis, photosynthesis–antenna proteins, and porphyrin and chlorophyll metabolism KEGG pathways
Locus_ID
|
Gene Symbol/ Description
|
log2FoldChange
|
WTR vs WTD
|
R11#2 vs WTD
|
R11#3 vs WTD
|
R16 #8 vs WTD
|
R16#9 vs WTD
|
Photosynthesis
|
|
|
|
|
|
|
CAA33954
|
psaC/Photosystem I reaction centre
|
4.19
|
3.90
|
2.19
|
1.10
|
2.13
|
Os08g0560900
|
PsaD/Photosystem I reaction centre
|
4.14
|
3.24
|
2.42
|
2.44
|
3.10
|
Os07g0435300
|
PsaE/Photosystem I reaction centre
|
4.08
|
3.89
|
2.33
|
2.53
|
2.64
|
Os03g0778100
|
PsaF/Photosystem I reaction centre
|
4.30
|
3.94
|
2.69
|
2.80
|
3.07
|
Os09g0481200
|
PsaG/Photosystem I reaction centre
|
5.80
|
5.40
|
3.54
|
3.80
|
4.45
|
Os05g0560000
|
PsaH/Photosystem I reaction centre
|
4.06
|
3.65
|
2.61
|
3.10
|
3.33
|
Os12g0420400
|
PsaL/Photosystem I reaction centre
|
4.36
|
3.70
|
2.39
|
2.56
|
3.16
|
Os12g0189400
|
psaN/Photosystem I reaction centre
|
5.24
|
4.51
|
2.97
|
3.17
|
3.80
|
Os04g0414700
|
PsaO/Photosystem I reaction centre
|
5.91
|
4.86
|
3.74
|
3.36
|
4.23
|
Os07g0148900
|
PsaK/Photosystem I reaction centre
|
6.59
|
5.43
|
4.28
|
4.18
|
4.81
|
Os01g0501800
|
PsbO/Photosystem II reaction centre
|
3.11
|
2.93
|
0.94
|
1.09
|
1.45
|
Os07g0141400
|
PsbP/Photosystem II reaction centre
|
3.81
|
3.31
|
1.97
|
2.14
|
2.40
|
Os08g0347500
|
PsbP/Photosystem II reaction centre
|
2.37
|
2.21
|
0.98
|
1.18
|
1.28
|
Os07g0544800
|
PsbQ/Photosystem II reaction centre
|
3.76
|
3.18
|
1.79
|
2.06
|
2.41
|
Os07g0105600
|
PsbQ/Photosystem II reaction centre
|
2.67
|
2.34
|
0.86
|
1.44
|
1.15
|
Os08g0200300
|
PsbR/Photosystem II reaction centre
|
4.09
|
3.64
|
2.05
|
2.34
|
2.85
|
Os05g0508900
|
PsbW/Photosystem II reaction centre
|
5.14
|
5.70
|
4.13
|
4.63
|
4.70
|
Os01g0773700
|
PsbW/Photosystem II reaction centre
|
4.00
|
3.62
|
2.14
|
2.31
|
2.74
|
Os08g0119800
|
PsbY/Photosystem II reaction centre
|
5.88
|
5.83
|
4.39
|
4.46
|
4.13
|
Os03g0333400
|
Pbs27/Photosystem II reaction centre
|
4.41
|
4.39
|
2.30
|
2.58
|
2.80
|
Os01g0938100
|
Psb28/Photosystem II reaction centre
|
3.04
|
3.14
|
2.14
|
2.16
|
2.41
|
Os07g0556200
|
PetC/Cytochrome b6/f complex
|
3.26
|
3.02
|
0.80
|
1.14
|
1.44
|
Os06g0101600
|
PetE/Photosynthetic electron transport
|
4.35
|
3.87
|
2.55
|
2.88
|
3.40
|
Os08g0104600
|
PetF/Photosynthetic electron transport
|
3.86
|
3.67
|
1.97
|
2.22
|
2.52
|
Os03g0685000
|
PetF/Photosynthetic electron transport
|
2.51
|
2.71
|
1.51
|
1.68
|
1.54
|
Os03g0659200
|
PetF/Photosynthetic electron transport
|
1.94
|
2.39
|
1.21
|
1.31
|
1.35
|
Os07g0567400
|
PetJ/Photosynthetic electron transport
|
1.80
|
1.73
|
0.91
|
1.20
|
1.14
|
Os07g0513000
|
gamma/F-type ATPase
|
3.05
|
2.38
|
1.17
|
1.32
|
1.98
|
Os02g0750100
|
delta/F-type ATPase
|
3.84
|
3.50
|
2.00
|
2.07
|
2.59
|
Os03g0278900
|
b/F-type ATPase
|
3.42
|
3.24
|
1.50
|
1.90
|
2.12
|
Photosynthesis - antenna proteins
|
|
|
|
|
|
Os07g0577600
|
Lhca2 /Light-harvesting chlorophyll protein complex
|
4.49
|
3.93
|
2.62
|
2.24
|
2.88
|
Os09g0439500
|
Lhca6/Light-harvesting chlorophyll protein complex
|
3.26
|
2.79
|
1.50
|
1.69
|
2.04
|
Os08g0435900
|
Lhca4/Light-harvesting chlorophyll protein complex
|
8.31
|
7.45
|
5.97
|
5.57
|
6.39
|
Os06g0320500
|
Lhca1/Light-harvesting chlorophyll protein complex
|
6.22
|
5.17
|
3.75
|
3.55
|
4.12
|
Os02g0197600
|
Lhca3/Light-harvesting chlorophyll protein complex
|
5.96
|
5.18
|
3.60
|
2.93
|
3.88
|
Os02g0764500
|
Lhca5/Light-harvesting chlorophyll protein complex
|
3.56
|
3.27
|
1.85
|
1.83
|
2.37
|
Os01g0720500
|
Lhcb1/Light-harvesting chlorophyll protein complex
|
11.82
|
12.01
|
8.54
|
8.96
|
10.13
|
Os11g0242800
|
Lhcb5/Light-harvesting chlorophyll protein complex
|
6.19
|
5.10
|
4.08
|
3.71
|
4.39
|
Os09g0346500
|
Lhcb1/Light-harvesting chlorophyll protein complex
|
8.20
|
7.35
|
6.54
|
6.17
|
6.73
|
Os07g0558400
|
Lhcb4/Light-harvesting chlorophyll protein complex
|
6.71
|
6.10
|
4.64
|
4.50
|
5.26
|
Os07g0562700
|
Lhcb3/Light-harvesting chlorophyll protein complex
|
5.65
|
4.69
|
3.66
|
3.09
|
4.03
|
Os03g0592500
|
Lhcb2/Light-harvesting chlorophyll protein complex
|
6.83
|
6.10
|
4.98
|
4.36
|
5.47
|
Porphyrin and chlorophyll metabolism
|
|
|
|
|
|
Os03g0563300
|
CHLI, Magnesium Chelatase OsCHLI
|
2.89
|
2.67
|
2.02
|
2.20
|
2.50
|
Os10g0419600
|
OsCHL, chlorophyllase-2
|
4.91
|
3.41
|
1.68
|
1.95
|
2.40
|
Os01g0279100
|
YGL8, catalytic subunit of magnesium-protoporphyrin IX monomethyl ester cyclase
|
3.83
|
3.71
|
2.49
|
2.39
|
3.00
|
Os06g0132400
|
ChlM, Magnesium Chelatase
|
3.23
|
3.28
|
2.53
|
2.61
|
2.81
|
Os10g0567400
|
CAO, chlorophyll a oxygenase
|
3.31
|
3.36
|
1.95
|
1.33
|
2.15
|
Os03g0337600
|
UroD, uroporphyrinogen decarboxylase
|
2.55
|
2.89
|
1.74
|
1.93
|
1.87
|
Os01g0622300
|
HEME,uroporphyrinogen decarboxylase
|
1.77
|
2.00
|
1.14
|
1.55
|
1.59
|
Os02g0168800
|
HemC,porphobilinogen deaminase
|
2.08
|
3.19
|
2.23
|
2.48
|
2.07
|
Os02g0744900
|
LYL1, Geranylgeranyl Reductase
|
3.36
|
3.23
|
1.93
|
1.57
|
2.18
|
Os10g0496900
|
PORB, protochlorophyllide oxidoreductase B
|
2.90
|
2.75
|
1.76
|
1.70
|
2.04
|
Os02g0296800
|
PF01903: CbiX, sirohydrochlorin ferrochelatase
|
1.42
|
1.43
|
0.78
|
1.21
|
0.98
|
Os01g0286600
|
HemY, protoporphyrinogen oxidase
|
1.78
|
1.82
|
1.17
|
1.27
|
1.21
|
Os05g0349700
|
OsYGL1, Chlorophyll synthase
|
1.48
|
1.90
|
1.16
|
1.34
|
1.21
|
Os08g0532200
|
HemL,aminotransferase
|
1.86
|
1.61
|
0.93
|
1.01
|
1.10
|
Os03g0351200
|
OsDVR, DVR,Divinyl Reductase gene
|
2.51
|
2.29
|
1.23
|
1.31
|
1.47
|
All genes were detected as differentially expressed based on an adjusted P < 0.05. 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.
Table 2. Differentially expressed genes assigned to the plant hormone signal transduction KEGG pathway
Locus_ID
|
Gene Symbol
|
log2FoldChange
|
WTR vs WTD
|
R11#2 vs WTND
|
R11#3 vs WTD
|
R16 #8 vs WTD
|
R16#9 vs WTD
|
Auxin signal pathway
|
|
|
|
|
|
Os01g0856500
|
OsAUX1
|
-1.21
|
-1.46
|
-1.39
|
-1.71
|
-1.52
|
Os02g0805100
|
OsIAA9
|
-3.36
|
-5.35
|
-2.21
|
-4.56
|
-4.91
|
Os03g0633500
|
OsIAA11
|
-6.48
|
-5.09
|
-3.67
|
-3.41
|
-3.96
|
Os03g0633800
|
OsIAA12
|
-4.65
|
-4.66
|
-2.57
|
-3.57
|
-3.68
|
Os03g0742900
|
OsIAA13/OsIAA1
|
-3.56
|
-3.59
|
-2.49
|
-2.78
|
-3.43
|
Os05g0143800
|
OsGH3-6
|
-1.65
|
-2.20
|
-1.71
|
-2.54
|
-1.62
|
Os06g0166500
|
OsIAA20
|
-3.04
|
-5.58
|
-2.97
|
-4.57
|
-4.60
|
Os09g0437400
|
OsSAUR38
|
-4.53
|
-4.77
|
-2.83
|
-2.94
|
-2.65
|
Os12g0601300
|
OsIAA30
|
-1.36
|
-1.84
|
-1.19
|
-1.31
|
-1.41
|
Os12g0601400
|
OsIAA3
|
-3.20
|
-3.45
|
-2.59
|
-3.17
|
-3.04
|
Cytokinin signal pathway
|
|
|
|
|
|
Os11g0143300
|
OsRR9
|
-1.28
|
-1.36
|
-1.95
|
-1.60
|
-1.41
|
Ethylene signal pathway
|
|
|
|
|
|
Os02g0527600
|
OsCTR2
|
-1.62
|
-1.96
|
-1.80
|
-2.50
|
-2.32
|
Os08g0508700
|
OsEIL4
|
-2.10
|
-3.15
|
-1.53
|
-2.66
|
-2.36
|
Os06g0605900
|
OsFBL30
|
-1.79
|
-2.04
|
-1.13
|
-2.50
|
-1.94
|
Abscisic acid signal pathway
|
|
|
|
|
|
Os01g0859300
|
OsABI5/OREB1
|
-3.55
|
-3.47
|
-2.33
|
-2.51
|
-2.64
|
Os04g0432000
|
OsSAPK7
|
-1.40
|
-1.97
|
-1.66
|
-1.11
|
-1.24
|
Brassinosteroid signal pathway
|
|
|
|
|
|
Os01g0718300
|
d61/OsBRI1
|
-1.09
|
-1.50
|
-1.04
|
-1.21
|
-1.15
|
Os09g0459450
|
BKI1
|
-0.72
|
-1.43
|
-0.98
|
-1.74
|
-1.23
|
Jasmonic acid signal pathway
|
|
|
|
|
|
Os08g0428400
|
OsJAZ3/OsTIFY6a
|
-1.30
|
-2.02
|
-1.52
|
-1.95
|
-1.55
|
Salicylic acid signal pathway
|
|
|
|
|
|
Os12g0152900
|
OsbZIP83
|
-3.35
|
-2.83
|
-2.13
|
-3.15
|
-1.95
|
Os07g0125500
|
Cysteine-rich secretory protein family
|
-9.89
|
-5.87
|
-3.67
|
-3.83
|
-4.54
|
All genes were detected as differentially expressed based on an adjusted P < 0.05. 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.
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 using rice ACTIN gene as the reference gene. 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 (Fig. 5a, b, and c). 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 (Fig. 5d). Meantime, the same results were obtained in the qRT-PCR assay using eEF-1α as the reference gene (Fig. S3). 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 T4 lines (#4 and #26) were selected to functionally characterize OsPIL11 in rice because of their high OsPIL11 expression levels (Fig. 6a). 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. 6b 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. 6b 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 (Fig. 6d-f). Meantime, the same results were obtained in the qRT-PCR assay using eEF-1α as a reference gene (Fig. S4). 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. 6g). 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.