Integrated analyses of rice dark response and leaf color control reveal links with porphyrin and chlorophyll metabolism

Background: Light is a key regulatory signal for rice growth and development. Under dark stress, rice shows leaf yellowing. Whole genome transcriptomic analysis will identify differentially expressed genes (DEG) in dark-treated rice seedlings and DEG-enriched metabolic pathways. Rice leaf color is an essential agronomic trait. Traditional genetic experiments have reported over a hundred of leaf color control (LCC) genes and some of them were also regulated by light signal. Thus, an integrated analysis for the two set of data will be helpful for illustration of the mechanism for both dark-response and leaf color control. Results: Transcriptome changes in response to dark treatment were surveyed by RNA-Seq analysis. About 13,115 differentially expressed genes (DEGs) were identified. One hundred and fifty rice LCC genes were collected. It was found that 102 LCC genes (68.0%) were also dark-response DEGs, which suggests an overlap between dark response and LCC networks. Fifty DEG overlapped LCC genes was associated with chloroplast development. KEGG analysis revealed enrichment of LCC genes in porphyrin and chlorophyll metabolism (PCM) (18/44, 40.9%). Of the 18 LCC genes in the PCM pathway, 15 were dark-response DEGs (83.3%). More interestingly, all of them are involved in a central PCM sub-pathway, chlorophyll biosynthesis. Conclusions: Integrated analysis for dark stress-response and leaf color control identified the correlation between the two processes and mutually supported evidences were obtained. It was found that PCM pathway, particularly chlorophyll biosynthesis process, plays important roles in rice LCC and dark stress-response. This study provides important clues for understanding the mechanisms of dark response and leaf color control and identifying additional LCC genes. were carried out, producing supporting, supplementary evidence for this hypothesis. This study provides important clues for improving mechanistic understanding of dark response and leaf color control and identifying additional LCC genes.

Improved understanding of functions and cross-talk of genes involved in chlorophyll synthesis and degradation and chloroplast development would help clarify mechanisms controlling leaf color and facilitate improvements of photosynthesis efficiency. used to reveal differential gene transcription at varying locations and times or under various stresses.
It is useful for identifying clues that enable subsequent investigations of molecular mechanisms.
Using oligonucleotide arrays, transcriptional profiling was performed under four light treatments (blue, green, red and white), as well as dark treatment, in rice. The results showed that the expression of transcription factors, such as bHLH, MYB, C2H2, ERF, NAC and WRKY, changed significantly, and carbohydrate degradation decreased under dark treatment (Lakshmanan et al., 2015). It was also revealed that both Phy A and Phy C cooperatively regulate transient gene expression in red-light treated rice seedlings (Kiyota et al., 2012). It has been observed that middle mesocotyl elongation was almost completely inhibited when germinating seeds were exposed to lowintensity light. RNA-Seq analysis revealed that most of the differential expressed genes (DEGs) were associated with hormone changes that occur in response to light exposure (Feng et al., 2017).
Comparative transcriptome profiling for low-light tolerant and sensitive rice varieties was carried out via RNA-Seq analysis, and DEGs induced by low-intensity light at the tillering stage were identified (Sekhar et al., 2019). Rice leaf color mutant accessions were analyzed by whole-genome resequencing and transcriptomic approaches, and the identified DEGs were classified into different categories, including genes related to macronutrient (e.g. magnesium and sulfur) transport and genes related to flavonoid biosynthesis .
In this study, transcriptomic analysis using an RNA-Seq approach was carried out for yellow leaves of dark-treated rice seedlings. DEGs were identified and DEG-enriched metabolic pathway analysis was performed with KEGG. Furthermore, data for previously reported LCC genes were collected for KEGG analysis, and metabolic pathways that include both LCCs and dark-response DEGs were identified. It was found that the transcript abundance of most LCC genes changed under dark treatment, suggesting an overlap between leaf color control and dark response. Therefore, integrated analyses were carried out, producing supporting, supplementary evidence for this hypothesis. This study provides important clues for improving mechanistic understanding of dark response and leaf color control and identifying additional LCC genes. 100. 0% and 70.4%,respectively (Fig. 1C,1D and 1E). Taken together, yellowed leaves, increased plant height and decreased chlorophyll content are typical characteristics of plant skotomorphogenesis, suggesting that CD stress resulted in deficient rice growth and development.
RNA-Seq transcriptome analysis was carried out for CK, CD_3d and CD_6d rice seedlings. The results revealed 33,755 non-redundant transcripts, accounting for 59.2% of all predicted rice genes (about 57,023). A total of 13,115 DEGs were identified, which accounts for 38.9% of all non-redundant transcripts (Additional file: Table S1). Approximately 8,242 DEGs were detected between CK and CD_3d, of which 4,467 were up-regulated and 3,775 were down-regulated. Additionally, there were 8,266 DEGs between CK and CD_6d, of which 3,834 were up-regulated and 4,432 were downregulated. There were more up-regulated DEGs than down-regulated DEGs for CD_3d, while there were fewer up-regulated DEGs than down-regulated DEGs for CD_6d ( Fig. 2A). There were 4,849 CD_3d only and 4,873 CD_6d only DEGs. A total of 3,393 genes were DEGs in both CD_3d and CD_6d ( Fig. 2B). It can be inferred that the early stages of dark treatment induced the transcription of many genes, while longer periods of dark treatment led to increased numbers of down-regulated genes.
This switch from up-regulation to down-regulation is an indication that as the CD treatment is extended, rice seedling growth becomes more inhibited and the expression of additional genes is down regulated.

DEG-enriched KEGG metabolic pathway analysis
KEGG metabolic pathway analysis of dark-response DEGs was performed by ClusterProfiler R. DEGenriched pathways and corresponding rich factors (RF) for CD_3d and CD_6d are listed in Additional file: Table S2. The 20 most significantly enriched pathways are shown in Fig. 3. In CD_3d, the term with the highest RF was cutin, suberine and wax biosynthesis. There were 11 up-regulated DEGs and 6 down-regulated DEGs. The second highest RF term was glyoxylate and dicarboxylate metabolism with 13 up-regulated DEGs and 22 down-regulated DEGs. The third highest RF term was amino sugar and nucleotide sugar metabolism with 49 up-regulated DEGs and 13 down-regulated DEGs. In CD_6d, the pathway with highest RF was photosynthesis-antenna proteins with 13 down-regulated DEGs, the second highest RF term was monobactam biosynthesis with 7 down-regulated DEGs, and the third highest RF term was photosynthesis with 1 up-regulated DEG and 28 down-regulated DEGs.
Comparison the terms from 3d and 6d, 7 out of 20 were overlapped, which supported the unique dark-stress response at different time points.
Next, the CD_3d and CD_6d DEGs were combined, and RF was recalculated for each pathway using non-redundant DEGs. The 20 pathways with the highest RF are shown in Fig. 4, and the highest RF pathways include photosynthesis-antenna proteins; carotenoid biosynthesis; cutin, suberine and wax biosynthesis; monobactam biosynthesis; porphyrin and chlorophyll metabolism (PCM); other glycan degradation; one carbon pool by folate; glyoxylate and dicarboxylate metabolism; and photosynthesis and biotin metabolism. Data for additional pathways are listed in Additional file: Table S3.

Identification of transcription factors among dark-response DEGs
Because transcription factors (TFs) play important roles in many biological processes, transcription factors among the dark-response DEGs were identified from matches with the Oryza sativa subsp.
Combined, 661 non-redundant TFs, representing 52 TF families, were identified as DEGs at the two dark treatment time points. Note, as shown in Table 1, the TF families that were most affected by CD were the bHLH, MYB, C2H2 and WRKY TF families. In particular, there were 47 bHLH among 449 TFs

Collection of rice leaf color control (LCC) genes
Rice leaves showed green color under normal growth condition. Numerous leaf color mutants have been identified via traditional forward genetics and breeding programs in rice. Information on 150 LCC genes was collected from literature, and loc#, gene name and references are summarized in Table 2 and Additional file: Table S6. As shown in Table 2, the phenotypes of known LCC mutants are albino (26), yellow (52), temperature-sensitive (14), stay green (16), stripe (15), spot (9), purple (1) and turning green (17). On the basis of their annotation, LCC genes mainly participate in chlorophyll synthesis, chloroplast development and photosynthesis.

Transcription analysis of LCC genes in dark-treated rice
RNA-Seq measurements of CD stress transcript levels for 150 LCC genes were surveyed. This analysis revealed that 102 of 150 LCC genes (68.0%) were also dark-response DEGs, of which 18 were upregulated at both 3d and 6d, 77 were down-regulated at both 3d and 6d, and 7 had opposing up-and down-regulation at the two time points (Additional file: Table S6). Thus, the transcript abundance of most LCC genes changed in response to dark stress.
The 102 LCC DEGs were functionally classified on the basis of annotations. A heat map was drawn showing log2 (fold change) values under 3 d and 6 d dark treatments (Fig. 5). In Fig. 5, it can be seen that there are 50 chloroplast development related genes, including three that were up-regulated and 46 that were down-regulated at both time points. There were 15 chlorophyll synthesis-related LCC genes, including three that were up-regulated and eight that were down-regulated at both time points. There were 10 photosynthesis-related LCC genes and five chlorophyll degradation-related LCC genes.
These data support that chloroplast development, chlorophyll synthesis and photosynthesis LCC genes play important roles in leaf yellowing under CD treatment and, in particular, down-regulated genes in these pathways. Moreover, aging, active oxygen scavenging, light signal transduction and carotenoid synthesis are also involved in the yellowing of leaves. Significant down-regulated expression was observed for most LCC genes under constant dark treatment, which implies that these LCC genes play a positive role in light signal responses.

KEGG analysis of LCC genes
Although hundreds of LCC genes have been cloned through traditional genetic approaches, largerscale understanding of molecular mechanisms controlling leaf color remains poor. Thus, LCC genes were functionally classified and assigned to KEGG Oryza sativa japonica (Japanese rice) pathways.
Similar to the calculation of rich factor in KEGG enrichment analysis, the number of LCC genes as a proportion of the total number of genes in a specific metabolic pathway (#GMP) was calculated as mutant rich factor (mRF). Fig. 6 shows pathway names and mRF values. The highest mRF pathway was PCM, accounting for 40.9% (18/44), followed by betalain biosynthesis (16.7%; 1/6) and photosynthesis-antenna proteins (6.7%; 1/15). The mRF for other metabolic pathways was less than 6% (Additional file: Table S7). Based on this result, it can be postulated that the PCM pathway plays important roles in regulating rice leaf color. KEGG analysis of the collection of LCC genes brought focus to specific metabolic pathways, providing a broader understanding of leaf color mechanisms and clues for identifying novel LCC genes.

Integrated analysis of dark-response DEGs and LCC genes
Among the 150 LCC genes, the transcript abundance of 102 genes changed under CD treatment, indicating an overlap between dark-response and leaf color control. Thus, it is reasonable and necessary to carry out integrated analysis of the two biological networks. Fig. 7 highlights pathways that include both dark-enriched DEGs and LCC genes, and the name or locus number of each gene is shown. A correlation between the two sets of data can be seen. As shown in Fig. 7, there was an overlap between dark-response DEGs and LCCs for a number of metabolic pathways. In the PCM pathway (Dosa00860), there are 27 dark response DEGs and 18 are LCC genes. Fifteen of these are both LCC genes and dark-response DEGs: OsNYC1, OsGGR2,OsCRD1,OsHY2,OsLYL1,OsDVR,OsChlI,OsChlD,OsPORA,OsSGRL,OsYGL1,OsYGL18,OsSGR,OsRCCR1 and OsPGL. In other words, the number of LCC genes overlapped with dark-response DEGs normalized to the total number of darkresponse DEGs is 55.6% (15/27) and it is 83.3% when normalized to total number of LCC genes (15/18), which provides strong evidence supporting a connection between PCM and both dark response and LCC. Taken together, integrated analysis provided a tool for specific examination of genome-wide transcriptome data within the context of metabolic pathways established using traditional forward genetics data. The complementary sets of data mutually support one another.

The chlorophyll biosynthesis process in the PCM pathway plays important roles in rice leaf color control
To further examine the function of LCC genes and DEGs in PCM, a flow chart was drawn, on the basis of the KEGG data, to highlight the position and distribution of the 15 LCC dark-response DEGs (Fig. 8).  Table S8 shows that the LCC overlapped dark-response DEGs were neither evenly nor randomly distributed throughout the metabolic pathway. Instead, almost all of them were concentrated to chlorophyll biosynthesis, which is conserved in higher plants. Thus, it can be postulated that this process is the key part of the PCM pathway responsible for both LCC and dark response, and additional genes that participate in this process are potential LCC genes.

Discussion
To understand the effect of light on rice growth, RNA-Seq analysis was carried out for rice leaves treated with constant dark, and DEG-enriched KEGG pathways with high rich factors were identified.
Meanwhile, a collection of rice LCC genes identified and cloned by traditional genetics were analyzed for mutant-enriched pathways with KEGG. It was found that 102 of 150 LCCs (68.0%) were DEGs under CD treatment, suggesting an overlap between dark response and leaf color control networks.
An integrated analysis of the two sets of data found that 83.3% of the LCCs in PCM pathways were also dark-response DEGs. More importantly, most of the LCC genes participate in chlorophyll synthesis, which suggests that chlorophyll synthesis, as a central part of the PCM pathway, plays an important role in both leaf color control and dark response.

The mechanisms of dark response in rice
Darkness is a severe stress. Rice leaves become yellow and unhealthy. Results obtained in this study and reported literature have shown that chlorophyll a, chlorophyll b and carotenoid contents are significantly reduced under CD treatment.
RNA-Seq analysis of dark-treated leaves revealed down-regulation of several genes involved in chlorophyll and carotenoid synthesis, including OsChlI, OsChlH and OsYGL1. Their transcript abundances decreased 13.3-, 5.5-and 5.30-fold, respectively. It has been reported that OsChlI and OsChlH encode the CHLI and CHLH subunits of Mg 2+ -protoporphyrin IX chelatase (Mg 2+ -chelatase) (Zhang et al., 2006a;Inagaki et al., 2015), and OsYGL1 encodes chlorophyll synthase (Wu et al., 2007). These are key enzymes for chlorophyll synthesis and leaf color control. In addition, the expression of β-OsLCY, encoding lycopene β-cyclase, decreased by 2.4-fold. The expression of OsPDS, encoding phytoene desaturase, decreased by 3.5-fold, and the expression of OsZDS, encoding ζcarotene desaturase, decreased by 2.9-fold. These three genes have been identified as key enzymes in carotenoid synthesis, with β-OsLCY also being a known LCC gene (Fang et al., 2008).
CD-enriched DEGs in photosynthesis-related pathways were detected by RNA-Seq analysis in this study. Four of the 10 most highly DEG-enriched metabolic pathways in dark response are associated with photosynthesis. For the photosynthesis-antenna proteins pathway, all 13 DEGs were downregulated, including OsLhca4, which was down-regulated approximately 217-fold. It has been reported that this gene encodes a light-harvesting complex I (LHCI) subunit, and it is also an LCC gene (Yamatani et al., 2018). In PCM, 21 out of the 27 DEGs were down-regulated under dark treatment, among which, LOC_Os10g28370 was down-regulated 69-fold. In glyoxylate and dicarboxylate metabolism, OsRBCS4, which encodes a small subunit of rubisco, was down-regulated 3001-fold and is also an LCC gene (OGAWA et al., 2012). In the photosynthesis pathway, 30 out of 33 DEGs were down-regulated, among which, LOC_Os12g10570 was down-regulated 90-fold. RNA-Seq analysis has previously been carried out for different light treatments (white, red, blue and green). It was found that photosynthesis-related genes were significantly down-regulated and carbohydrate degradation was pronounced in darkness (Lakshmanan et al., 2015), consistent with the results obtained in this study.

The mechanisms of leaf color control
Leaf color is an important agronomic trait that is directly related to rice growth and grain yield. In this study, data on 150 LCC genes were consolidated from the literature. According to LCC gene transcription data, most LCC genes were down-regulated under constant dark treatment, which demonstrates that light signals can play positive roles in regulating LCC gene expression. Light is the upstream initiator of signal transduction in plants. PIFs (phytochrome interacting factors) are a class of bHLH transcription factors that can interact with phytochrome (Phy). Light signals regulate PIFs protein stability; that is, the protein is stable in the dark and degrades in the light (Demarsy et al., 2017). In rice, six PIF genes have been identified and designated as OsPIL11 to OsPIL16 (Nakamura et al., 2007;Piao et al., 2015). It was reported that OsPIL13 is an LCC gene, with expression controlled by circadian rhythms (Nakamura et al., 2007). OsPIL13 binds to the promoters of two Chl biosynthetic genes, OsPORB and OsCAO1, and induces the transcription of downstream genes (Sakuraba et al., 2017). OsPIL15 is responsible for regulating rice tiller angle in response to light and gravity, and OsPIL15 expression is negatively regulated in etiolated seedlings exposed to light (Xie et al., 2019). In this study, up-regulated expression for OsPIL11, OsPIL13 and OsPIL16 was detected, and bHLH TFs were found to be CD-response DEGs at a higher percentage than any other TF family, suggesting that bHLH TFs play important roles to propagate light signal transduction in rice.
Darkness can lead to extensive stress responses, including loss of green leaf color. Direct correlations between light and leaf color mutation have been reported. For example, OsLYL1, encoding a geranylgeranyl reductase, was induced by light and suppressed by dark (Zhou et al., 2013b).

OsYGL18 encodes a putative magnesium protoporphyrin IX methyltransferase (ChlM). When an
OsYGL18 deletion mutant (ygl18) was transferred from dark to light, chlorophyll content increased and its expression was up-regulated (Wang et al., 2017c). OsZN encodes a thylakoid-bound protein of unknown function, and its mRNA level in constant light is higher than that in CD, indicating that OsZN transcription is controlled by light (Li et al., 2010). In addition, an OsOTP51 mutant, encoding a pentatricopeptide repeat protein, showed dramatic changes in PSI structure and function, which led to severe photoinhibition (Ye et al., 2012). In this study, it was found that expression of these genes was down-regulated after CD treatment, which suggests that light signals positively regulate LCC genes.
Although more than 100 LCC genes have been cloned by traditional genetic methods, it has been difficult to assess the role of specific LCC genes in the context of metabolic pathways. In this study, LCC genes were surveyed with KEGG metabolic pathway analysis. LCC gene-enriched pathways were identified by calculating the mutant rich factor (mRF). It was found that the mRF was highest for PCM, which suggested that PCM plays an important role in leaf color control, and additional genes from the PCM pathway may be LCC genes.

The feasibility and importance of integrated analysis of transcriptome and genetic data
Genome-wide transcriptome analysis can detect stress-related DEGs, and KEGG enrichment analysis can further identify DEG-enriched metabolic pathways. However, clues obtained by transcriptomic analysis require verification from genetic analyses and functional studies. Traditional forward genetics approaches can efficiently identify genes related to specific mutant phenotypes, but it is difficult to integrate a group of mutant-related genes into specific metabolic pathways. In this circumstance, it was possible to obtain mutual supporting, complementary evidence through integration of two sets of data. The integrated analysis can get focused view from transcriptomic data and a magnified view from genetic data.
In this study, RNA-Seq analysis of CD-treated rice seedlings was carried out and DEGs were identified.
Data on 150 LCC genes previously cloned by traditional genetic means were collected, and 102 LCC genes were found to be dark-response DEGs, which suggests an overlap between dark response and leaf color control networks. Furthermore, KEGG analysis of LCC genes showed that the mRF of LCC genes was the highest for PCM, and most PCM LCC genes (83.3%) were dark-response DEGs.
Meanwhile, the rich factor for PCM was among the highest revealed by RNA-Seq analysis of dark stress. Thus, it can be concluded that PCM plays an important role not only in dark response but also in leaf color control. RNA-Seq data provided understanding of leaf color mutations, and traditional genetic research provided complementary data towards clarifying the mechanism of dark-stress response.
Genome-wide analysis is aimed at investigating all genes. When facing different biological questions in the same organism, data from different sources can be integrated for coordinated analysis. With the rapid accumulation of transcriptome data, which can be combined with genetic data that has been collected over many years, integrated analysis can now be carried out to a greater extent, which will contribute to improved understanding of rice biology.

Dissection of the PCM pathway
DEGs can be allocated to specific metabolic pathways via KEGG analysis; however, a metabolic pathway may contain dozens or even hundreds of genes that are connected with multiple interrelated processes. Analysis of the location and distribution of DEGs is helpful for making functional associations with specific processes. In other words, more accurate and precise results can be obtained if metabolic pathways are dissected. In this study, DEGs derived from whole-genome transcriptomic analysis and LCC genes collected from traditional genetics literature were associated with the PCM pathway. Further, the DEGs and LCC genes were neither randomly nor evenly distributed throughout the pathway. Instead, they were concentrated in the chlorophyll biosynthesis part of the pathway (Nagata et al., 2005). On this basis, it can be postulated that chlorophyll biosynthesis is the key process for leaf color regulation. Analyzing dissected metabolic pathways is important because it helps narrow the range of target genes and leads to comprehensive understanding of gene functions.

Conclusions
In this study, transcriptomic analysis using an RNA-Seq approach was carried out for yellow leaves of dark-treated rice seedlings. DEGs and DEG-enriched metabolic pathways were analyzed. Data for reported LCC genes were collected. Metabolic pathways that included both LCCs and dark-response DEGs were identified. It was found that the transcript abundance of most LCC genes changed under dark treatment, suggesting an overlap between leaf color control and dark response. KEGG analysis revealed enrichment of LCC genes in porphyrin and chlorophyll metabolism (PCM). Interestingly, all of the overlapped LCC genes and DEGs were concentrated at chlorophyll biosynthesis in the central of PCM, indicating that PCM pathway, particularly chlorophyll biosynthesis process, plays important roles in rice LCC and dark stress-response. This study provides important clues for understanding mechanisms of dark response and leaf color control and identifying additional LCC genes.

Supplementary Information
The datasets supporting the conclusions of this article are included within the article and its additional file. experiments; WT, CY and ZZ analyzed the data; LG, LL, CY and WT wrote and edited the manuscript.  7:1698. Su N, Hu M, Wu D, Wu F, Fei G, Lan Y, Chen X, Shu X, Zhang X, Guo X (2012 Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production. Plant Physiol 159 (1)  Rich factors for DEG-enriched metabolic pathways identified by KEGG analysis of darktreated rice. Twenty pathways with highest rich factor value were listed. More pathways were listed in additional file: Table S3.  Integrated analysis of dark response and leaf color control. Gene names in red (italic) are LCC genes that were also dark-response DEGs. Dosa# indicated the number of pathways in KEGG database. The percentages at the bottom represent the number of LCC genes overlapped with dark-response DEGs normalized to the total number of dark-response DEGs. Ten pathways are listed here, and detailed information for additional pathways is listed in additional file: Table S8.

Supplementary Files
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