Transcription Profile Analysis of Chlorophyll Biosynthesis in Leaves of Wild-Type and Chlorophyll b-Deficient Rice (Oryza sativa L.)

Photosynthesis is an essential biological process and a key approach for raising crop yield. However, photosynthesis in rice is not fully investigated. This study reported the photosynthetic properties and transcriptomic profiles of chlorophyll (Chl) b-deficient mutant (ch11) and wild-type rice (Oryza sativa L.). Chl b-deficient rice revealed irregular chloroplast development (indistinct membranes, loss of starch granules, thinner grana, and numerous plastoglobuli). Next-generation sequencing approach application revealed that the differential expressed genes were related to photosynthesis machinery, Chl-biosynthesis, and degradation pathway in ch11. Two genes encoding PsbR (PSII core protein), FtsZ1, and PetH genes, were found to be down-regulated. The expression of the FtsZ1 and PetH genes resulted in disrupted chloroplast cell division and electron flow, respectively, consequently reducing Chl accumulation and the photosynthetic capacity of Chl b-deficient rice. Furthermore, this study found the up-regulated expression of the GluRS gene, whereas the POR gene was down-regulated in the Chl biosynthesis and degradation pathways. The results obtained from RT-qPCR analyses were generally consistent with those of transcription analysis, with the exception of the finding that MgCH genes were up-regulated which enhance the important intermediate products in the Mg branch of Chl biosynthesis. These results indicate a reduction in the accumulation of both Chl a and Chl b. This study suggested that a decline in Chl accumulation is caused by irregular chloroplast formation and down-regulation of POR genes; and Chl b might be degraded via the pheophorbide b pathway, which requires further elucidation.


Introduction
Rice (Oryza sativa L.) is a major staple food supplying to 50% population worldwide [1]. Its production has recently increased to meet the growing demands for a rapid increase in world population. However, the rice yield is significantly affected by many factors including variety, climate, terrain, temperature, light intensity, soil properties, and polluted farmland, which are related to its development and photosynthetic capacity [2][3][4][5]. Therefore, there is an urgent need to investigate photosynthesis in rice under different environmental conditions.
Photosynthesis is an essential biological process in higher plants, which contributes to approximately 90% of biomass production in crops. Consequently, enhancing photosyn-in rice. However, the transcription profiles and photosynthetic characteristics of the Chl b-deficient mutant rice have not yet been reported.
In this study, next-generation sequencing (NGS) was used to determine the transcription profiles and photosynthetic characteristics of Chl b-deficient mutant rice type 2a (Chl a/b of~10). The characteristics, differentially expressed genes (DEGs), and transcription factors (TFs) related to Chl biosynthesis and degradation in wild-type and the Chl b-deficient rice type 2a were compared.

Plant Materials and Growth Condition
Seeds of wild-type (Norin No. 8, wt) and Chl b-deficient mutant rice type 2a (chlorina 11, ch11) were kindly provided by Dr. Tomio Terao (Department of Applied Physiology, National Institute of Agrobiological Resources, Tsukuba Science City, Japan). The seeds were sown in a growth chamber (Firstek Scientific Co Ltd., New Taipei, Taiwan), and the germinated seedling was subsequently grown for 6 weeks at a temperature of 25 • C, and relative humidity of ≥80%, under a 12/12 dark cycle at Biodiversity Research Center, Academia Sinica Institute, Taipei, Taiwan (25 • 02 34.1 N, 121 • 36 40.7 E). The leaves of these plants were then harvested, immediately frozen in liquid nitrogen, and stored at −80 • C for less than 2 weeks for further analysis.

Assessment of Pigment Contents
Approximately 0.1 g of wild-type and mutant leaf tissues were cut into pieces, immersed in 1 mL acetone (80%, v/v) at 4 • C, and homogenized using bullet blender tissue homogenizer (Next Advance Inc., New York, NY, USA) for 5 min. The extracts were then obtained through filtration. The absorption of the extracts was measured at 663.6, 646.6, and 440.5 nm using a Hitachi U2800 UV-Visible spectrophotometer (Hitachi, Tokyo, Japan) at room temperature to determine pigment contents (total Chl, Chl a, Chl b, and carotenoids (Car)) according to the method described by Yang et al. 1998. The pigment contents were calculated (on the basis of µg/mL Chl/g fresh weight [43]), as follows: Chl a = 12.25A 663.6 − 2.55A 646. 6 (1) Chl b = 20.31A 646.6 − 4.91A 663. 6 (2) Total Chl = 17.76A 646.6 + 7.34A 663. 6 (3) Car = 4.69A 440.5 − 4.91 Total Chl (4) where Chl a, Chl b, and total Chl are the Chl a, Chl b, and total Chl content, respectively; A 646.6 , A 646.6 , A 440. 5 are the absorbance of the extracts which was measured at 663.6, 646.6, and 440.5 nm, respectively. To validate the Chl content, soil-plant analysis development (SPAD) values were determined in situ on wt and ch11 plants using a SPAD-502Plus chlorophyll meter (Konica Minolta, Osaka, Japan).

cDNA Libraries Construction and Transcriptome Sequencing
Total RNA was extracted from the whole leaves of wt and ch11 rice using a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. The purity and quantity of RNA samples were evaluated using 1% formaldehyde agarose gel electrophoresis and determined with a NanoDropTM 2000 spectrophotometer (NanoDrop, Wilmington, NC, USA). RNA concentration and integrity were determined using an Agilent 2100 Bio-analyzer (Agilent RNA 6000 Nano Kit; Agilent Technologies, Inc., Santa Clara, CA, USA). Briefly, two sets of paired-end cDNA libraries were constructed from each wild-type and mutant rice sample, and the resulting cDNA libraries were sequenced using the BGISEQ-500 platform (Beijing Genomics Institute, Shenzhen, Guangdong, China) for transcriptome sequencing. As the result, the total raw reads of wt (30.60, 30.61 Mb) and ch11 (30.62, 30.63 Mb) were generated. Adapter sequences, unknown nucleotides, and low-quality sequences were removed using SOAPnuke v1.5.2 (https://github.com/ BGI-flexlab/SOAPnuke, accessed on 12 March 2021). Both cDNA library construction and transcriptome sequencing were conducted independently by a commercial service provider (Tri-I Biotech, Inc., The New Taipei City, Taiwan). Two biological replications of each sample in RNA-Seq were used to conduct a library.

Quantitative RT-qPCR
Quantitative RT-qPCR was performed to validate and analyze the basic expression levels of a subset of candidate genes involved in Chl biosynthesis and degradation pathways. Total RNA (µg) isolated from the leaves of wild-type and mutant plants was used to synthesize cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostic Systems, Branchburg, NJ, USA) and oligo (dT) primers. The sequences of primer sets used for amplification were designed using Primer Premiere 6 software (Premiere Biosoft, Palo Alto, Santa Clara, CA, USA) (

Statistical Analysis
The pigment contents, SPAD values, and relative gene expression (RT-qPCR results) of wild-type and mutant rice were statistically analyzed by the least significant difference (LSD) t-test at a p value ≤ 0.05, which was performed using the SAS v8.0 statistical package (SAS Institute, Cary, NC, USA).

Characterization of ch11 Rice
Seeds of wt and ch11 rice were grown in a growth chamber for 6 weeks under controlled conditions (25 • C, 12/12 dark cycle, RH ≥ 70%). After the cultivation, ch11 plantlets showed dwarf and unhealthy phenotypes (~25 cm in height) with green leaves, whereas wt plantlets showed normal and healthy development (~40 cm in height) with dark green leaves (Figure 1a,b). The total Chl content of wt (3.3 mg g −1 ) was almost twice that of the ch11 (1.7 mg g −1 ). A similar difference was detected for carotenoid (Car) content (0.5 and 0.3 mg g −1 for wt and ch11, respectively). Moreover, the content of Chl a in ch11 was approximate 64% that in the wt, whereas that of Chl b was only approximately 12.5%. Consequently, the ch11 mutant was characterized by a notably high Chl a/b of 11.25, which was almost three times higher than that of wt leaves (Figure 1c). Furthermore, soil-plant analysis development (SPAD) values were determined to validate the Chl accumulation in the leaves of wt and ch11 plants, obtaining values, 29.62 and 16.23, respectively. This result was consistent with our Chl assessments and indicative of abnormal Chl biosynthesis and degradation in Chl b-deficient rice. Differences were also observed with respect to the ultrastructure of wt and ch11 leaves, with the leaves of wt plants being characterized by healthy chloroplasts with distinct membranes, and stromal lamellae, small starch granules, and few plastoglobuli ( Figure 2a). In contrast, the chloroplasts in ch11 leaves showed abnormal development with indistinct membranes, indistinct or absent stromal lamellae, no starch granules, and numerous plastoglobuli (Figure 2b. Moreover, although stacked thylakoid grana were observed clearly in ch11 leaf sections, they were some two to five times thinner than the stacked grana in wt chloroplasts (Figure 2a  (LSD) t-test at a p value ≤ 0.05, which was performed using the SAS v8.0 statistical package (SAS Institute, Cary, NC, USA).

Characterization of ch11 Rice
Seeds of wt and ch11 rice were grown in a growth chamber for 6 weeks under controlled conditions (25 °C, 12/12 dark cycle, RH ≥ 70%). After the cultivation, ch11 plantlets showed dwarf and unhealthy phenotypes (~25 cm in height) with green leaves, whereas wt plantlets showed normal and healthy development (~40 cm in height) with dark green leaves (Figure 1a,b). The total Chl content of wt (3.3 mg g −1 ) was almost twice that of the ch11 (1.7 mg g −1 ). A similar difference was detected for carotenoid (Car) content (0.5 and 0.3 mg g −1 for wt and ch11, respectively). Moreover, the content of Chl a in ch11 was approximate 64% that in the wt, whereas that of Chl b was only approximately 12.5%. Consequently, the ch11 mutant was characterized by a notably high Chl a/b of 11.25, which was almost three times higher than that of wt leaves (Figure 1c). Furthermore, soil-plant analysis development (SPAD) values were determined to validate the Chl accumulation in the leaves of wt and ch11 plants, obtaining values, 29.62 and 16.23, respectively. This result was consistent with our Chl assessments and indicative of abnormal Chl biosynthesis and degradation in Chl b-deficient rice. Differences were also observed with respect to the ultrastructure of wt and ch11 leaves, with the leaves of wt plants being characterized by healthy chloroplasts with distinct membranes, and stromal lamellae, small starch granules, and few plastoglobuli ( Figure 2a). In contrast, the chloroplasts in ch11 leaves showed abnormal development with indistinct membranes, indistinct or absent stromal lamellae, no starch granules, and numerous plastoglobuli (Figure 2b. Moreover, although stacked thylakoid grana were observed clearly in ch11 leaf sections, they were some two to five times thinner than the stacked grana in wt chloroplasts (Figure 2a

Genome Mapping and Gene Expression Analysis
In this study, we sequenced samples from wt and ch11 seedlings using the BGISEQ-500 platform. The obtained raw sequencing reads were filtered by removing low-quality reads, adaptor sequences, and reads with a high content of unknown bases. The resulting 25.31-29.33 million clean reads were subsequently mapped to the rice reference genome (Oryza sativa 'Nipponbare', http://rapdb.dna.affrc.go.jp/, accessed on 12 March 2021) using HISAT (hierarchical indexing for spliced alignment of transcript), with match ratios in the range of 96.84-97.93% (Table S2, Supplementary Materials). Among the sequences in the wt and ch11 libraries, 88.93% and 89.67%, were respectively mapped to the reference transcript, and 79.56% and 80.42% of the uniquely mapped reads were mapped to a single locus in the reference genome (Table S3,

Genome Mapping and Gene Expression Analysis
In this study, we sequenced samples from wt and ch11 seedlings using the BGISEQ-500 platform. The obtained raw sequencing reads were filtered by removing low-quality reads, adaptor sequences, and reads with a high content of unknown bases. The resulting 25.31-29.33 million clean reads were subsequently mapped to the rice reference genome (Oryza sativa 'Nipponbare', http://rapdb.dna.affrc.go.jp/, accessed on 12 March 2021) using HISAT (hierarchical indexing for spliced alignment of transcript), with match ratios in the range of 96.84-97.93% (Table S2, Supplementary Materials). Among the sequences in the wt and ch11 libraries, 88.93% and 89.67%, were respectively mapped to the reference transcript, and 79.56% and 80.42% of the uniquely mapped reads were mapped to a single locus in the reference genome (Table S3, Supplementary Materials). Approximately 91.41% of the expressed genes were shared between wt and ch11 rice genome ( Figure 3).

Genome Mapping and Gene Expression Analysis
In this study, we sequenced samples from wt and ch11 seedlings using the BGISEQ-500 platform. The obtained raw sequencing reads were filtered by removing low-quality reads, adaptor sequences, and reads with a high content of unknown bases. The resulting 25.31-29.33 million clean reads were subsequently mapped to the rice reference genome (Oryza sativa 'Nipponbare', http://rapdb.dna.affrc.go.jp/, accessed on 12 March 2021) using HISAT (hierarchical indexing for spliced alignment of transcript), with match ratios in the range of 96.84-97.93% (Table S2, Supplementary Materials). Among the sequences in the wt and ch11 libraries, 88.93% and 89.67%, were respectively mapped to the reference transcript, and 79.56% and 80.42% of the uniquely mapped reads were mapped to a single locus in the reference genome (Table S3,

Detection and Annotation of Differentially Expressed Genes
Functional annotation was conducted to acquire information with respect to protein function, pathway involvement, and GO. Sequence orientation-driven genes were aligned based on the KEGG database. GO is a global criteria functional identification scheme for genes in which biological processes, cellular components, and molecular functions are assigned to transcripts. The distribution of detected genes among the GO categories is shown in Figure 4. A total of 5510 genes were mapped, with 1992, 2325, and 1193 genes being assigned to "Biological Process", "Cellular Component", and "Molecular Function", respectively. Notably, individual genes can be assigned to multiple GO terms. DEGs were identified using the fragments per kb per million reads method, and Poisson distribution, with false discovery rate (FDR) ≤ 0.001 and absolute log2 ratio ≤ 1 (Figure 5a,b). We accordingly identified 863 and 2007 DEGs, which were up-and down-regulated, respectively, in ch11 leaves, whereas 23,015 genes showed no significant differences in expression between wt and ch11 leaves (Figure 5b). Functional annotation was conducted to acquire information with respect to protein function, pathway involvement, and GO. Sequence orientation-driven genes were aligned based on the KEGG database. GO is a global criteria functional identification scheme for genes in which biological processes, cellular components, and molecular functions are assigned to transcripts. The distribution of detected genes among the GO categories is shown in Figure 4. A total of 5510 genes were mapped, with 1992, 2325, and 1193 genes being assigned to "Biological Process", "Cellular Component", and "Molecular Function", respectively. Notably, individual genes can be assigned to multiple GO terms. DEGs were identified using the fragments per kb per million reads method, and Poisson distribution, with false discovery rate (FDR) ≤ 0.001 and absolute log2 ratio ≤ 1 (Figure 5a,b). We accordingly identified 863 and 2007 DEGs, which were up-and down-regulated, respectively, in ch11 leaves, whereas 23,015 genes showed no significant differences in expression between wt and ch11 leaves (Figure 5b).

Role of Chlorophyll Metabolism Genes in Leaf Coloration
DEGs related to chloroplast development and cell division were identified based on KEGG pathway annotation. Genes related to the FtsZ1 cell division-related protein gene (Osa_4333567) and two PsbR chloroplast photosystem II protein genes (Osa_107276047 and Osa_4342395) were found to be down-regulated in ch11 rice (by 2.1, 1.7, and 4.5-fold,

Role of Chlorophyll Metabolism Genes in Leaf Coloration
DEGs related to chloroplast development and cell division were identified based on KEGG pathway annotation. Genes related to the FtsZ1 cell division-related protein gene (Osa_4333567) and two PsbR chloroplast photosystem II protein genes (Osa_107276047 and Osa_4342395) were found to be down-regulated in ch11 rice (by 2.1, 1.7, and 4.5-fold, respectively). Furthermore, the PetH gene (Osa_4334338) and Lhcb1 gene (Osa_4324705) encoding an FDX electron transfer protein and photosynthesis-antenna protein, respectively, was also down-regulated (Table 1). A total of 24 DEGs related to Chl biosynthesis and five DEGs related to Chl degradation were identified based on KEGG pathway annotation. The expression levels of the DEGs were determined using hierarchical cluster analysis ( Figure 6). GLuRS (Osa_4338118), and NCCR (Osa_4348519) were significantly up-regulated in ch11 (∆log2 = 1.4, and 1.7, respectively), whereas POR (Osa_4337415) was down-regulated (−1.7) compared with the wt (Figure 6). These results indicate that the processes of Chl biosynthesis and degradation may be detrimentally affected in the ch11 mutant. respectively). Furthermore, the PetH gene (Osa_4334338) and Lhcb1 gene (Osa_4324705) encoding an FDX electron transfer protein and photosynthesis-antenna protein, respectively, was also down-regulated (Table 1). A total of 24 DEGs related to Chl biosynthesis and five DEGs related to Chl degradation were identified based on KEGG pathway annotation. The expression levels of the DEGs were determined using hierarchical cluster analysis ( Figure 6). GLuRS (Osa_4338118), and NCCR (Osa_4348519) were significantly upregulated in ch11 (∆log2 = 1.4, and 1.7, respectively), whereas POR (Osa_4337415) was down-regulated (−1.7) compared with the wt (Figure 6). These results indicate that the processes of Chl biosynthesis and degradation may be detrimentally affected in the ch11 mutant.

RT-qPCR Validation of Differentially Expressed Chlorophyll Biosynthesis Genes
RT-qPCR expression analyses were performed to validate the identification of DEGs related to Chl biosynthesis and degradation. The expression patterns of 12 genes that code enzymes involving in 19 reactions in Chl biosynthesis were examined. The result revealed that these genes were similar to those observed in the RNA-Seq data (Figure 7). The RT-qPCR results were generally consistent with the transcription analysis data, with the exception of one gene (MgCH) showing an inconsistent expression pattern. POR was observed to be markedly down-regulated in ch11, whereas most of the genes showed non-significant changes. In contrast to the transcription analysis results, the qPCR results revealed that MgCH was up-regulated in ch11 (Figure 7). ception of one gene (MgCH) showing an inconsistent expression pattern. POR was observed to be markedly down-regulated in ch11, whereas most of the genes showed nonsignificant changes. In contrast to the transcription analysis results, the qPCR results revealed that MgCH was up-regulated in ch11 (Figure 7). Figure 7. Expression of levels of 12 differentially expressed genes from wild-type (wt) and chlorophyll b-deficient (ch11) rice. Gene expression was measured using quantitative RT-qPCR. Asterisks (*) indicate significant differences between the expression levels of the wild-type and ch11 rice (p ≤ 0.05).

Pigment Contents and Chloroplast Development
Chl-deficient mutants have been widely used to study Chl biosynthesis and the photosynthetic machinery properties of barley, maize, pea, sweet clover, wheat, rice, soybean, sugar beet, and the maiden-hair tree [32][33][34]47,48]. At the physiological level, pigment contents, the Chl a/b, and chloroplast development are associated with the coloring of the leaves in higher plants [49]. Accordingly, differences in leaf coloration have been observed between Chl-deficient mutants and the corresponding wild-type plants in species such as Arabidopsis thaliana, Gingko biloba, and Oryza sativa. Previous studies have identified two types of Chl-deficient rice mutant, which are based on the ratio of Chl a to Chl b, namely, type 1, in which Chl b is completely absent (Chl b-lacking mutants), and type 2, characterized by high Chl a/b ratios of between approximately −10 and −15 (Chl b-deficient mutant) [41,42,50]. Moreover, a study indicated that the Chl a/b in ch11 slightly fluctuated from 9 to 11 in 6 days incubation in various irradiances [50]. In our previous study, we observed reductions in Chl content, an absence of Chl b, and abnormal chloroplast development in Chl b-lacking mutants, which were characterized by light green leaves compared with the dark green pigmentation seen in the leaves of wild-type plants [26]. In the present study, we also noted visible differences in the leaf coloration of Chl b-deficient mutant and wildtype rice, with leaves of the mutant being a distinctly paler green compared with the dark green of the wild-type leaves. In addition, mutant plants showed a dwarf phenotype, together with marked reductions in the contents of total Chl and Chl b in leaves, as indicated Chl a/b of up to approximately 11.5, compared with the wild-type. Thus, we can assume that differences in Chl content and Chl a/b between the Chl b-deficient mutant and wildtype rice contributed to the observed variation in leaf coloration. Moreover, the presence of Chl b in ch11, albeit at a lower level, would account for the darker coloration compared with the light-green leaves characterizing Chl b-lacking mutant [26]. Given that the processes of photosynthesis and Chl biosynthesis occur in chloroplasts, organelles unique to higher plants [51,52], abnormal development in these structures can be expected to have Figure 7. Expression of levels of 12 differentially expressed genes from wild-type (wt) and chlorophyll b-deficient (ch11) rice. Gene expression was measured using quantitative RT-qPCR. Asterisks (*) indicate significant differences between the expression levels of the wild-type and ch11 rice (p ≤ 0.05).

Pigment Contents and Chloroplast Development
Chl-deficient mutants have been widely used to study Chl biosynthesis and the photosynthetic machinery properties of barley, maize, pea, sweet clover, wheat, rice, soybean, sugar beet, and the maiden-hair tree [32][33][34]47,48]. At the physiological level, pigment contents, the Chl a/b, and chloroplast development are associated with the coloring of the leaves in higher plants [49]. Accordingly, differences in leaf coloration have been observed between Chl-deficient mutants and the corresponding wild-type plants in species such as Arabidopsis thaliana, Gingko biloba, and Oryza sativa. Previous studies have identified two types of Chl-deficient rice mutant, which are based on the ratio of Chl a to Chl b, namely, type 1, in which Chl b is completely absent (Chl b-lacking mutants), and type 2, characterized by high Chl a/b ratios of between approximately −10 and −15 (Chl bdeficient mutant) [41,42,50]. Moreover, a study indicated that the Chl a/b in ch11 slightly fluctuated from 9 to 11 in 6 days incubation in various irradiances [50]. In our previous study, we observed reductions in Chl content, an absence of Chl b, and abnormal chloroplast development in Chl b-lacking mutants, which were characterized by light green leaves compared with the dark green pigmentation seen in the leaves of wild-type plants [26]. In the present study, we also noted visible differences in the leaf coloration of Chl b-deficient mutant and wild-type rice, with leaves of the mutant being a distinctly paler green compared with the dark green of the wild-type leaves. In addition, mutant plants showed a dwarf phenotype, together with marked reductions in the contents of total Chl and Chl b in leaves, as indicated Chl a/b of up to approximately 11.5, compared with the wild-type. Thus, we can assume that differences in Chl content and Chl a/b between the Chl b-deficient mutant and wild-type rice contributed to the observed variation in leaf coloration. Moreover, the presence of Chl b in ch11, albeit at a lower level, would account for the darker coloration compared with the light-green leaves characterizing Chl b-lacking mutant [26]. Given that the processes of photosynthesis and Chl biosynthesis occur in chloroplasts, organelles unique to higher plants [51,52], abnormal development in these structures can be expected to have a detrimental influence on the photosynthetic capacity and Chl synthesis of leaves [26,53]. Numerous studies have reported irregular chloroplast development (i.e., indistinct thylakoid membrane, non-existent granules, abundant vesicle, and numerous plastoglobuli) in Chl-deficient mutants, including those of green bamboo, maidenhair tree, and rice, which affects Chl contents, Chl a/b, and leaf coloration, compared with the corresponding wild-type [26,54,55]. Moreover, the integrity of the stacked grana of chloroplasts determines the efficiency of light absorption and energy conversion in the photosynthetic apparatus [56]. In this study, the chloroplast ultrastructure analysis revealed that wt rice was characterized by normally developed chloroplasts containing an abundance of thick stacked grana, whereas ch11 leaves showed distinctly abnormal chloroplast development (i.e., indistinct thylakoid membranes, numerous plastoglobuli, and absence of starch granules) with abundant thinner stacked grana. These observations indicate that in conjunction with a lower Chl content, irregular chloroplast development would contribute to deficiencies in the harvesting and conversion of light energy in ch11 compared with wt rice. A similar pattern has been observed in the leaves of Chl b-lacking rice (ch1), in which abnormal chloroplast development along with thinner grana was found to be associated with a reduction in pigmentation, an elevated Chl a/b, and reduced photosynthetic capacity [26]. Accordingly, at the physiological level, the dwarf phenotype and leaf color variation of the Chl-deficient rice are believed to be closely associated with a reduced Chl content, higher Chl a/b, and abnormal chloroplast development, which are presumed to contribute to a reduced photosynthetic capacity and thus have detrimental effects on the biomass and yield of rice.

Chloroplast-Related Differentially Expressed Genes and Photosynthetic Capacity
Next-generation sequencing and transcriptome profiling can provide valuable information regarding those genes that are differentially expressed between different interested genotypes [57,58]. In the present study, transcriptome profiling of wt and ch11 rice revealed a total of 863 and 2007 DEGs being up-and down-regulated, respectively. Among them, we identified a number of genes associated with photosynthetic machinery, chloroplast development, and pigment biosynthesis metabolism that may be associated with the observed differences in Chl a/b. The coordination of plastid and nuclear genes in plants plays an important role in normal chloroplast development. Differences in the levels of chloroplast-related gene expression could affect the biogenesis of chloroplast assembly and pigment contents, thus resulting in changes in Chl accumulation, Chl a/b, photosynthetic capacity, and leaf coloration [54,59]. In this regard, FtsZ1 and FtsZ2 in the FtsZ family of protein are key players in the initiation and progression of chloroplast division in plants and green algae [60,61]. The analysis of chloroplast-related DEGs, revealed a downregulated expression of the FtsZ1 gene in ch11 rice, indicating a potential deficiency in chloroplast cell division compared with the wt. This result is consistent with the abnormal chloroplast development and leaf color variation characterizing ch11 rice. The chloroplast abnormalities observed in the ch11 mutant are thus assumed to result in a reduction in Chl b content and a concomitant increase in the Chl a/b. In higher plants, the light-harvesting complex (or antenna complex, LHC) proteins play an important role in the photosynthetic apparatus, which absorbs and regulates the flow of light energy to photosystems I (PSI) and II (PSII) [62,63]. PSII is a multi-protein-pigment complex that contains the PSII core reaction center dimer, LHCII, and minor light-harvesting complexes comprising more than 20 subunits, including PsbR [64,65]. Moreover, LCHII has been reported to be a key factor that regulates the stacking of grana thylakoids in chloroplasts [66][67][68]. Previous studies have found that a reduction in or absence of LHC proteins in Chl-deficient mutants of Arabidopsis thaliana, Hordeum vulgare, and Oryza sativa are associated impairment in stacked grana [26,[69][70][71][72]. Hence, the chloroplast of Chl-deficient mutants is predicted to be characterized by the poorly developed or non-existent stacked grana [69,73]. In the present study, we found that the expression of two PsbR genes was also down-regulated in ch11 rice, by 1.7-and 4.5-fold, respectively, which might be indicative of a reduction in the level of LHCII proteins, and consequently a reduction in photosynthetic capacity. A similar pattern has been detected in Chl b-lacking rice, in which two LHCII family PsbR genes were shown to be markedly down-regulation, leading to a reduced photosynthetic capacity [26]. Moreover, chloroplast FNR proteins include PetH, which plays a role in the final step of the linear flow of electrons, whereby electrons are transferred from ferredoxin to NADP + [74]. In the present study, the PetH gene was observed to be down-regulated in ch11 rice, which might cause a reduction in the transfer of electrons from ferredoxin to NADP + (Table 1). Consequently, it lowers the amounts of NADPH products available for the Calvin reaction, which might lead to the small or non-existent granules in the mutant. These findings indicated that deficiencies in the photosynthetic capacity of the Chl b-deficient ch11 mutant could be related to the down-regulated expression of genes associated with PSII and photosynthetic electron transport. Furthermore, stacked grana in ch11 were found to be thinner than those in the wt, indicating a loss of LCHII.

RNA-Seq Analysis and Chlorophyll-Related Differentially Expressed Genes
Chl a and b play vital roles in light absorption and energy transfer [75,76]. Chl a is the principal pigment in higher plants involved in light-harvesting and primary photochemical reactions, whereas Chl b, which is synthesized from Chl a, functions as an accessory pigment and is only involved in light-harvesting [18,77]. At least 28 genes encode 22 enzymes involved in Chl biosynthesis and degradation in Chl b-lacking rice. Changes in certain DEGs were associated with disorders in Chl metabolism and altered Chl a/b [26]. During the early stages of Chl biosynthesis, glutamyl-tRNA synthetase (GLuRS) regulates the δaminolevulinic acid (ALA), resulting in changes in the expression of GLuRS. Consequently, it could affect the synthesis of early products in the Chl biosynthetic pathway and may contribute to the development of rice plants characterized by a shorter stature and yellow leaves [52,78,79]. Moreover, protochlorophyllide (Pchllide), a key intermediate in the Chl cycle section of the Chl biosynthesis pathway [24,80], is catalyzed by protochlorophyllide reductase (POR), a light-dependent protein present in angiosperms, that reduces the double bond in the D-ring in a stereo-specific manner to yield chlorophyllide (Childe) under light condition [81][82][83]. In the present study, a total of 28 DEGs were detected to associate with Chl biosynthesis and degradation based on KEGG pathway annotation, among which, the GLuRS (Osa_4328818) homolog gene was up-regulated in ch11 rice, whereas Osa_4325167 and Osa_4348464 showed non-significant changes. This study suspects that the expression of GluRS in ch11 may not differ substantially from that in the wt. In addition, no appreciable changes in the expression of GluTR, GSA-AT, ALAD, PBGD, UROS, UROD, CPOX, or PPOX was observed. These observations indicate that the synthesis of products in the early stages of Chl biosynthesis in ch11 rice is essentially unaffected compared to wt rice. These observations contrast with the changes previously observed in Chl blacking rice, in which GluRS was found to be down-regulated leading to an inhibition of the early products of Chl biosynthesis [26]. In the Mg branch of Chl biosynthesis, POR (Osa_4337415) was found to be markedly down-regulated, which might have an effect of disrupting Chlide product synthesis in the Chl cycle. This result indicates that the synthesis of Chl a and Chl b were blocked or significantly reduced in ch11 compared with the wt. Contrastingly, in the Chl cycle, we detected no genes that were significantly differentially expressed in ch11 and wt rice. Furthermore, our detection of Chl b in leaves of the ch11 mutant indicated that Chl b had been normally synthesized from Chl a. Therefore, the enzymes CAO, CHL, NOL, HCAR, and CHLG involved in the Chl cycle had been normally translated and activated. A similar pattern has previously been observed in Chl b-lacking rice, with exception of the NOL gene, which was markedly up-regulated and may thus have promoted a rapid conversion of Chl b to Chl a [26]. In this study, the presence of Chl b in the leaves of ch11 rice may reflect the fact that the expression of NOL in this mutant does not differ significantly from that in wt rice. NOL is unbound to light-harvesting complexes including Chl b, chlorophyllide b, pheophorbide b, and pheophytin b, and has a broad substrate specificity [84]. However, pheophytin b and pheophorbide b were not generated in chloroplast due to the specificity of Mg-dechelatase [85]. Another study stated that pheophorbide b is accumulated during cell death after incubating a plant, whose core antenna complex contained Chl b, in a dark condition [84]. Consequently, the mechanism underlying pheophorbide b remaining in plant cells is still poorly understood. This study suggested that the marked reduction in the Chl content of ch11 can probably be attributed to abnormal development of chloroplasts and a significant reduction in the expression of POR. In addition, a possible explanation for the rising of Chl a/b in ch11 leaves is that Chl b might be degraded by an alternative degradation pathway via pheophorbide b, which causes the absence of Chl b in Chl b-lacking rice [26]. However, the proposed pathway remains unclear, which requires further investigations.

Conclusions
This study reported the photosynthetic properties and transcriptomic profile of a Chl b-deficient mutant rice compared with wt plants. The Chl b-deficient mutant was characterized by a dwarf phenotype along with green leaves, a reduced pigment accumulation, a high Chl a/b, and abnormal chloroplast structure. RNA-Seq analysis revealed that 2870 genes were differentially expressed in ch11. Among these genes, there were 288 TFs related to Chl biosynthesis and degradation, plastid development, cell division, and photosynthetic machinery. This study found the down-regulated expression of POR gene, whereas the GluRS gene was up-regulated in the Chl biosynthesis and degradation pathways. Changes in the expression of genes were related to chloroplast development. An increase in the Chl a/b may be attributed to abnormal chloroplast development and the involvement of an alternative degradation pathway (pheophorbide b pathway). These findings provided insights into the molecular mechanism underlying the Chl a/b and leaf coloration in Chl b-deficient mutant of rice. However, the Chl b degradation in ch11 is still unclear and needs further investigations to clarify.