Introduction

Chlorophyll (Chl) is a green pigment found in plants and cyanobacteria, which absorbs sunlight and uses its energy to synthesize carbohydrates from CO2 and water. Chl molecules are specifically around photosystems that are embedded in the thylakoid membranes of chloroplasts (Fromme et al. 2003). Heme is a vital component required for both photosynthesis and respiration (Terry and Kendrick 1996). HO1 converts heme to BV-IXα with the release of iron (Fe2+) and carbon monoxide (CO) (Terry et al. 1993, 1995; Weller et al. 1996). Disruption of HO1 may indirectly affect chlorophyll biosynthesis, because both heme and chlorophyll branches share same substrates from 5-aminolevulinic acid to protoporphyrin IX (Weller et al. 1996). Chl and heme are two major tetrapyrroles of photosynthetic organisms in nature, and their metabolism has been extensively studied in various organisms such as cyanobacteria and Arabidopsis thaliana (von Wettstein et al. 1995; Grimm 1998). Analysis of the complete genome of Arabidopsis reveals that there are at least 27 genes encoding 15 enzymes involved in Chl biosynthesis (Nagata et al. 2005).

Rice (Oryza sativa) is a staple food crop for more than half of the world’s population. Chl biosynthesis in rice plays a vital role to rice production. Chl-deficient mutants generally affect leaf colors, and are ideal genetic materials to investigate molecular mechanisms that regulate Chl biosynthesis and chloroplast development. More than ninety rice mutants were identified associated with leaf colors in a rice science database, for example, albino, chlorina, stripe (st), virescent (v) and zebra according to their phenotypes (http://www.shigen.nig.ac.jp/rice/oryzabaseV4/). Some of the genes underlying the mutations have been cloned. The V1 mutant of rice develops chlorotic leaves under low-temperature condition and the V1 gene encodes a chloroplast-localized protein NUS1, which promotes the establishment of the plastid genetic system during early chloroplast development in rice (Kusumi et al. 1997, 2011). The magnesium chelatase is a key enzyme catalyzing the chelation of Mg2+ into proto IX to produce Mg-Proto IX, and it comprises three subunits (ChlH, ChlD and ChlI) (Jung et al. 2003; Davison et al. 2005). Mutations of the Chlorina-1 and Chlorina-9 genes encoding ChlD and ChlI subunits, respectively, lead to deficiency in Chl content and cause the incomplete development of chloroplasts (Zhang et al. 2006). The Yellow-Green Leaf1 (YGL1) gene catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis, and a missense mutation of which results in yellow-green leaves in young plants with decreased Chl synthesis and delayed chloroplast development (Wu et al. 2007). The Young Seedling Albino (YSA) encodes a protein with pentatricopeptide repeat and its mutation causes albino leaves before three-leaf stage in rice (Su et al. 2012). The Young Leaf Chlorosis1 (YLC1) gene encodes a protein of unknown function belonging to the DUF3353 superfamily, disruption of which leads to a chlorosis phenotype at the seedling stage (Zhou et al. 2013). These above-mentioned genes provided useful information in uncovering Chl synthesis in rice.

In this study, we isolated a pigment-deficient mutant, which was induced by 60Co γ-rays. The mutant exhibited a yellow-green leaf phenotype which was similar to yellow-green leaf1 (ygl1) (Wu et al. 2007), and therefore was named ygl2. The ygl2 mutant showed a reduction in Chl content and an obvious increase in the Chl a/b ratio. By map-based cloning, ygl2 locus was isolated, and it encodes a plastid heme oxygenase (HO1). Genetic complementary experiments indicated YGL2 could completely restore ygl2 mutant phenotype. Our results provided the direct evidences that YGL2 could play an important role to Chl biosynthesis in rice.

Materials and methods

Plant materials and growth conditions

The rice ygl2 mutant was isolated from Gang 46B (indica type) by 60Co radiation mutagenesis. All plants were grown in the experimental farms of the Institute of Crop Science in Beijing (China) during the natural growing season. For temperature treatments, the seedlings were grown in a growth chamber with 12/12 h (light/dark) at a constant air temperature of 23 °C (C23) or 30 °C (C30).

Measurement of Chl and carotenoid (Car) contents

Chl and Car content were determined as the study described previously (Su et al. 2012). Equal weight of the second top leaf tissues was cut and suspended in 95 % ethanol for 48 h under dark conditions, and then centrifuged at 3,000g for 30 min to remove residual plant debris. The supernatants of these samples were analyzed with a SpectraMax Plus384 spectrophotometer (Molecular Devices LLC, Sunnyvale, CA), and the concentrations of Chls and Cars were measured at 665, 649, and 470 nm and determined using the method of Lichtenthaler (1987).

Transmission electron microscopy

To investigate the status of chloroplast development, leaf samples grown in growth chambers were harvested from 2-week-old days seedlings after germination. Leaf sections were fixed in a solution of 2 % glutaraldehyde and further fixed in 1 % OsO4 overnight at 4 °C. Thereafter, they were stained with uranyl acetate before dehydrating through an ethanol series, and then embedded in Spurr’s medium prior to thin sectioning. Samples were stained again and viewed with a Hitachi H-7650 transmission electron microscope.

Map-based cloning of ygl2

For map-based cloning of ygl2 gene, a mapping population was constructed by a cross between the ygl2 mutant (indica) and Nipponbare (japonica). Individual plants showing yellow leaf phenotype in the F2 progeny were selected for a genetic linkage analysis. Molecular markers distributed throughout the rice genome (McCouch et al. 2002a, b) were chosen for preliminary mapping. New InDel (insertion and deletion) markers for fine mapping were developed based on the DNA sequences of indica and japonica whole genomes (Yu et al. 2002; International Rice Genome Sequencing Project 2005), and primer sequences were listed in Supplemental Table 1.

Complementation test and RNA interference

For functional complementation of the rice ygl2 mutant, a full-length cDNA fragment encoding YGL2 was amplified by RT-PCR with primers HO1-CF/HO1-CR, and then inserted into empty binary vector pCUbi1390 through the Clontech In-Fusion® PCR cloning system (TaKaRa). The resulting pUBI-YGL2 plasmid, which contained the YGL2 coding sequence driven by the ubiquitin promoter and a hygromycin resistant marker, was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. Because YGL2 mutant was originated from an indica variety, and its calli were validated to produce difficultly, a near isogenic line with ygl2 allele in a japonica genetic background was obtained from ygl2/Nipponbare F3 population using marker assistant selection. This line was further used as transforming receptor.

For RNAi experiments, the RNAi vector pCUbi1390-ΔFAD 2 (Stoutjesdijk et al. 2002; Zhou et al. 2013) was fused with a 328 bp specific coding region of YGL2, which was amplified with primer pairs RNAi-SF/RNAi-SR and RNAi-AF/RNAi-AR (Supplemental Table 1). The resulting construct (pUbi-RNAi-YGL2) was transformed into Agrobacterium tumefaciens as described above. The empty vector pCUbi1390 was used as a control. Kitaake, an easily regeneratable japonica variety, was chosen as transforming receptor. The protocol of Agrobacterium tumefaciens-mediated transformation in rice followed Hiei’s previous study (Hiei et al. 1994).

RT-PCR, qRT-PCR and histochemical assays

Total RNA was extracted from tissues of the wild type and ygl2 mutant, respectively, using an RNA Prep Pure Plant kit (Tiangen Co. Beijing, China), and treated with DNase I following the manufacturer’s instruction. The total cDNA was reverse transcribed using a SuperScript II kit (TaKaRa). The qRT-PCR analysis was performed using the ABI7500 Fast Real-Time PCR System with SYBR Premix Ex Taq kit (TaKaRa), and rice ubiquitin gene was chosen as endogenous control. Reactions containing SYBR premix were carried out in final volumes of 25 μl containing 0.2 μmol of the appropriate primers (Supplemental Table 1) and 1× SYBR green PCR master mix (PE Applied Biosystems). The 2−ΔΔCT method was used to calculate relative levels of gene expression (Schmittgen and Livak 2008). The primers for the YGL2, Chl biosynthesis and photosynthesis associated genes were given in Supplemental Table 1.

Histochemical β-glucuronidase (GUS) staining was performed according to methods described previously (Dai et al. 1996). Freshly collected samples from transgenic plants expressing the promoter-GUS fusion were put in the staining solution under vacuum-infiltrated for 30 min, and followed by incubation in darkness at 37 °C overnight. After staining, the samples were completely rinsed with 70 % ethanol and then photographed. Staining solution contained 50 mM sodium phosphate, 1 mM 5-bromo-4-chloro- 3-indolyl-b-D-glucuronide, 10 mM EDTA, 1 mM potassium ferrocyanide and 0.1 % [v/v] Triton X-100, pH 7.0.

Subcellular localization

The full-length YGL2 open reading frame (ORF) was amplified with primer pair GFP-F/GFP-R (see Supplemental Table 1) and ligated into binary vector AHLG under the control of the rice ACTIN promoter (Delaroche et al. 2010). Then the recombinant plasmid was introduced into Kitaake by Agrobacterium tumefaciens-mediated transformation. To exam the subcellular localization precisely, we isolated leaf protoplasts from those positive transgenic plants. Fluorescence of GFP was observed by a confocal laser scanning microscope (Leica TCS SP5).

Determination of Chl precursors

Determination of ALA content was based on Richard’s (1975) methods. The precursors of Chl synthesis pathway, including Proto IX, Mg-Proto IX, Pchlide, and Chlide, were assayed as described by Santiago-Ong et al. (2001) and Masuda et al. (2003). Leaves (0.2 g fresh weight) of wild type and ygl2 mutant were cut and homogenized in 5 mL 9:1 acetone:0.1 mNH4OH, and centrifuged at 3,000g for 10 min. The supernatants were combined and washed three times successively with an equal volume of hexane prior to spectrophotometric analysis. Chl precursors in the acetone phase were quantified with a Hitachi F-4600 fluorescence spectrophotometer using Ex400:Em632 for Proto IX, Ex440:Em633 for Pchlide, Ex440:Em672 for Chlide, and Ex420:Em595 for Mg-Proto.

Statistical analysis of the results

The data for comparison included at least three samples. T test was carried out using Microsoft Excel 2007 at significance level of 0.001, 0.01 or 0.05.

Results

The ygl2 mutant shows reduced Chl content and less number of thylakoid lamellar in chloroplast

The ygl2 mutant exhibited a yellow-green leaf phenotype when leaves emerged after germination. At three-leaf stage, the yellowish phenotype was the most obvious (Fig. 1a). Thereafter, from tiller initiating to heading stage, yellowish leaves gradually turned green and approached almost as normal as wild type did (Fig. 1b, c). In addition, the ygl2 mutant showed a significant earlier flowering phenotype (near 20 days, p < 0.001) than that of wild type. Plant height is significantly lower than wild type throughout whole developmental stages (Supplemental Table 2) (t test, p < 0.05).

Fig. 1
figure 1

Phenotypic characterization of wild type (WT) and the rice ygl2 mutant. a Leaf color of two-week-old WT and ygl2 seedlings. b Leaf color of 6-week-old WT and ygl2 seedlings. c Plants of WT and ygl2 at the flowering stage. d, e Overview of chloroplast of wild type (d) and ygl2 mutant (e) from 2-week-old plants. f, g Ultrastructures of chloroplasts in the mesophyll cells of WT (d) and ygl2 mutant (e) plants from 2-week-old plants. C chloroplast, Thy thylakoid lamellar. Bars 0.5 μm (d, e), 0.2 μm (f, g)

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Pigments contents at different developmental stages were examined and the results were showed in Table 1. At three-leaf stage, the ygl2 mutant had a reduction of 37.7 % for Chl and 50 % for Car levels as compared to the wild type. During tillering stage, the percentages of Chl and Car reduction were narrowed to 19.4 and 24.5 % of normal levels, respectively. Compared to wild type, ygl2 mutant showed higher Chl a/b ratio at three-leaf stage (t test, p < 0.05). The reduction of Chl contents and increase of Chl a/b ratio corresponded perfectly to yellowish scores of leaf colors.

Table 1 Pigments contents in leaves of wild type and ygl2 mutant, in mg g−1 fresh weight
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To determine if ygl2 mutation affects chloroplast development and morphology, we compared the ultrastructures of chloroplasts between the ygl2 mutant and wild type plants at three-leaf stage using transmission electron microscopy. The number of thylakoid lamellar in chloroplasts of the ygl2 was significantly less than that of wild type (Supplemental Fig. 1), whereas the structure of the thylakoid lamellar seemed normal and similar to that of wild type (Fig. 1d–g).

The YGL2 gene encodes a heme oxygenase on chromosome 6

Map-based cloning was used to isolate ygl2 mutant gene. Initially, 190 individuals from the ygl2× Nipponbare F2 population were used for genetic analysis. The phenotypic separation ratio fitted with 3:1 (150 normal: 40 yellow-green, χ 2 = 1.37 < χ 20.05,1   = 3.84), suggesting the ygl2 mutant phenotype was controlled by a single recessive gene. To map ygl2 locus, 30 F2 ygl2-like individuals were utilized. The ygl2 gene was mapped to an interval between two InDel markers In9 and In11 on the long arm of chromosome 6 (Fig. 2a). 502 segregants showed mutant phenotype and were used for fine mapping; more markers were developed by comparing the difference of genomic sequences between indica and japonica whole genome. The ygl2 locus was finally narrowed to a region of 66.8-kb between two InDel markers, In 44 and In 39 (Fig. 2a). Within the targeting region, there were eight recognizable open reading frames that were identified by Rice Genome Research Program (http://rgp.dna.affrc.go.jp/). Among these candidate genes, the sixth ORF (Os06g40080) encodes a heme oxygenase (HO1). Because a mutation of the HO1 homolog in pea could cause pale foliage (Weller et al. 1996), we speculated that Os06g40080 was likely the candidate gene underlying the ygl2 phenotype.

Fig. 2
figure 2

Map-based cloning of the ygl2 gene. a Fine mapping of ygl2 locus. The ygl2 was first mapped to a region between the markers In 9 and In 11 on the long arm of rice chromosome (Chr) 6 with 30 recessive individuals. Then it was narrowed to a 66.8 kb region between the In 44 and In 39 using 502 recessive individuals from F2 population. Eight open reading frames (ORFs) were predicted in the mapped region. b YGL2 gene structure modeling. Black boxes indicate the coding sequence, gray boxes the 5′- and 3′-UTRs, and lines between boxes introns. ATG and TGA represent the start and stop codons, respectively. The red letters represent the 7-kb insertion which was almost removed as the first intron in the mature transcript of ygl2, and black letters denote wild type sequences. The black arrows represent splicing sites. c Identification of the genomic DNA fragment between wild type and ygl2. M molecular weight marker

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To define the mutation(s) in ygl2, we sequenced genomic sequence of Os06g40080 in ygl2, and found a fragment of 7-kb insertion in the first exon of Os06g40080 (Fig. 2b, c). Based on exon/intron splicing rules (Black 2003), the 7-kb insertion was almost removed as the first intron (Fig. 2b). By sequencing RT-PCR product, a 46-bp insertion and 19-bp nucleotide deletion from codon 344 to 363 were detected in the mature transcript of ygl2 (Supplemental Fig. 2a), leading to fifteen amino acids insertion and six amino acids deletion in putative protein (Supplemental Fig. 2b).

To confirm the identity of YGL2/Os06g40080, we performed a genetic complementation by expressing Os06g40080 of wild type in the selected ygl2 recessive homozygote of F3 plant line. In the transgenic plants, all the characters including color of leaves (Fig. 3a), levels of Chl (Fig. 3b), and ratio of Chl a/b (Fig. 3c) were restored to the levels of wild type. In addition, we also constructed an RNAi vector of YGL2 and transformed it into japonica variety Kitaake. A total of 12 positive transgenic plants displayed the similar phenotype as that in ygl2 mutant (Fig. 3a). The expression levels of YGL2 in leaves were consistent with the corresponding phenotypes in overexpression and RNAi plants (Supplemental Fig. 3). Taken together, we concluded that the abnormal phenotype of ygl2 mutant resulted from the mutation of YGL2 gene.

Fig. 3
figure 3

Functional complementation and RNAi knock-down analysis of YGL2. a Leaf color of a representative wild type (WT), ygl2 mutant, a representative transgenic plant (ygl2/YGL2), control (Kitaake) and RNAi line, respectively. b Total chlorophyll (Chl) levels of the plants shown in (a). Chl was extracted from the second leaf of three-leaf-old plants. c Chl a/b ratio calculated from b. Values are the mean ± SD (n = 3)

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YGL2 protein belongs to a highly conserved protein family and localized in chloroplast

Searching the rice genome database revealed that YGL2 is a single-copy gene, and its coding sequence contains 870 nucleotides encoding a protein with 289 amino acids. Based on cDNA sequence analysis, the YGL2 protein has a calculated molecular mass of 31.9 KD with a putative transit peptide at its N-terminus. Research in the national center for biotechnology information (NCBI) (http://www.ncbi.nlm.nih.gov/) revealed that both the wild type and mutant proteins contain a heme binding pocket and a heme ligand (Fig. 4). The HO1 encoded by YGL2 has 67.8–97.8 % predicted amino acid sequence identity with other orthologs in higher plants (Supplemental Fig. 4 and Supplemental Fig. 5), suggesting it is a family member of highly conserved proteins. The mutation site in ygl2 was resided in a relatively conserved region of YGL2 (Supplemental Fig. 4). In addition, according to modeling of swissmodel (http://swissmodel.expasy.org/workspace), secondary and 3D structure of mutant ygl2 protein were compared with wild type. The results showed that ygl2 increased one additional β strand in amino acid (AA) 105–110 but missed three a-helix in AA 45–52, 60–72, and 101–104, leading to disappearance of one loop and enlargement of another loop in tertiary structure (Fig. 4b).

Fig. 4
figure 4

Structure comparison of the predicted protein between YLG2 and ygl2. a The alignment of complete amino acid sequence of the YGL2 and its mutant ygl2. The heme binding pocket and heme ligand sites are marked by solid and empty circles, respectively. Identical residues are demonstrated in shade. Bars indicate the positions of helices within the WT and ygl2 protein. Arrows indicate the positions of β strands within the WT and ygl2 protein. b Docking structure of YGL2 and heme. The model was constructed according to the swissmodel (http://swissmodel.expasy.org/workspace). The ball-and-stick model structure indicates a heme molecular was embedded in both proteins of WT and ygl2. The difference of 3D-structure is showed by arrows

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To determine the subcellular localization of the YGL2 protein, the construct YGL2-GFP driven by rice Actin promoter was applied to transform rice plants. Confocal microscopy analysis of YGL2-GFP localization showed that GFP fluorescence overlapped with Chl auto fluorescence in isolated leaf protoplasts (Fig. 5), suggesting YGL2 is localized in chloroplast. To test whether the mutation in ygl2 affected the subcellular localization, we also performed a transient expression analysis of the ygl2-GFP fusion protein. Most of ygl2 mutant proteins were localized in chloroplast (Supplemental Fig. 6), indicating the ygl2 mutation did not affect protein anchoring.

Fig. 5
figure 5

Subcellular localization of YGL2. a Fluorescence of YGL2 mostly located in the chloroplast of rice protoplasts. b Chlorophyll auto fluorescence. c Bright field. d Merged image of a, b and c. Bar indicates 10 μm

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YGL2 is constitutively expressed in various tissues and the ygl2 mutation down-regulates its mRNA expression level

The qRT-PCR analysis was performed to study the expression profile of YGL2. As shown in Fig. 6a, the expression of YGL2 was abundantly in flag leaf and penultimate leaf but relatively lower in root, culm, leaf sheath and young panicle. Compared with wild type, the expression level of YGL2 was significantly decreased in various organs in ygl2 mutant (p < 0.05, Fig. 6a), suggesting that the 7-kb insertion in the first exon obviously reduced the expression of YGL2 in mutant. To verify qRT-PCR results, we further used transgenic plants carrying the YGL2 promoter-GUS fusion construct to detect the YGL2 endogenous expression level. GUS signals were detected in root, culm, leaf, leaf sheath and young panicle (Fig. 6b–e), and their relative strengths were consistent with expression pattern from qRT-PCR analysis.

Fig. 6
figure 6

Expression analysis of YGL2 by qRT-PCR. a Expression patterns of YGL2 in root (R), culms (C), flag leaves (FL), the penultimate leaves (PL), leaf sheaths (LS), young panicles (YP) of wild type and ygl2 mutant. b–e YGL2 expression patterns of various tissues as revealed by GUS assay of pYGL2-GUS transgenic plants before heading. b Culms and roots. c Flag leaves. d Leaf sheaths. e Young panicles. f Expression level of OsHO1 in the leaves of 3-week-old seedlings and tillering stage of plants, respectively. g Expression level of OsHO2 in the leaves of 3-week-old seedlings and tillering stage of plants, respectively. Ubiquitin gene was amplified as a control. Values are mean ± SD (n = 3)

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A paralogue of OsHO1 (named OsHO2) was also analyzed. The results showed that in wild type, the transcripts of both OsHO1 and OsHO2 at seedling stage were much higher than those at tillering stage. While in ygl2, the tendency was reversed (Fig. 6f, g).

YGL2 is temperature sensitive and affects expression of other genes associated with Chl biosynthesis and photosynthesis

To research whether the phenotype of ygl2 was affected by temperature, we treated ygl2 and wild type seedlings at two different temperatures. The yellowish color of ygl2 became more obvious and the difference of Chl content between ygl2 and wild type was even more significant at low temperature (23 °C) than high temperature (30 °C) (Fig. 7a, b). From 30 to 23 °C, the leaf color of wild type showed no obvious change, though their Chl levels had a reduction of 15.6 %. In contrast, significant color changes were observed in the ygl2 leaves, and their Chl levels had a reduction of 45.6 % (Fig. 7c), indicating ygl2 a temperature sensitive mutant. In wild type, YGL2 expression levels at 23 °C were 6.6-fold higher than those at 30 °C treatment. While in mutant, YGL2 exhibited only modest up-regulation (2.7-fold increase) at 23 °C, as compared with those at 30 °C (Fig. 7d).

Fig. 7
figure 7

Quantitative PCR (qPCR) expression analysis of YGL2 under temperature treatments and the genes associated with Chl biosynthesis and photosynthesis in WT and ygl2 at 23 °C(C23) or 30 °C(C30), respectively. a Leaf phenotypes of wild type and ygl2 mutant in 2-weeks-old seedlings grown at C30, 12 h light/12 h darkness. b Leaf phenotypes of wild type and ygl2 mutant in 2-weeks-old seedlings grown at C23, 12 h light/12 h darkness. c Chlorophyll a/b contents at C30 and C23. d qPCR analysis of YGL2 transcript levels in wild type and ygl2 mutant at C30 and C23. Total RNA was isolated from leaves. e qPCR analysis of genes associated with Chl biosynthesis and photosynthesis in WT and ygl2 at C30. f qPCR analysis of the genes associated with Chl biosynthesis and photosynthesis in WT and ygl2 at C23. Total RNA was extracted from leaves of 2-week-old plants at C30 and 3-week-old at C23. Ubiquitin gene was amplified as a control. Values are the mean ± SD (n = 3) (*p < 0.05)

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The expression of ten genes associated with Chl biosynthesis (CHLD, CHLI, CHLH, YGL1, PORA, and CAO1) and photosynthesis (Cab1, Cab2, PsaA and PsbA) was analyzed to investigate their possible regulations by YGL2. At 30 °C, all tested genes in ygl2 were significantly up-regulated as compared to wild type. Among them, Cab1 showed the most obvious increase. Along with temperature decreased, most detecting genes in ygl2 showed down-regulated trends except CHLH (Fig. 7e, f). The results indicated that YGL2 gene played an important role in the regulation network of Chl biosynthesis and photosynthesis.

Reduced expression of YGL2 results in decreased intermediates of Chl biosynthesis

HO1 encoded by YGL2 catalyzes the degradation of heme to form biliverdin IXα, a precursor of phytochrome (Cornejo et al. 1992; Weller et al. 1996). It was reported that heme accumulation could feedback inhibit Chl synthesis (Terry and Kendrick 1999). To detect whether the reduction of Chl resulted from the feedback inhibition of heme accumulation, we tested key intermediates in Chl biosynthesis by absorption spectroscopy. The results showed that ygl2 had significantly decreased levels of protochlorophyllide (Pchlide) and Chlide as compared with wild type (p < 0.05) (Fig. 8). In contrast, no significant difference was found between mutant and wild type in other intermediates of Chl biosynthesis including ALA, proto IX and Mg- proto IX.

Fig. 8
figure 8

Analysis of chlorophyll (Chl) intermediates in wild type and ygl1 mutant. Chl intermediates were measured in second leaf from 2-week-old wild type and ygl2 mutant. a Levels of 5-aminolevulinate (ALA). FW fresh weight. b Contents of Proto IX, Mg-proto IX, Pchlide, and Chlide. Values are the mean ± SD (n = 3) (*p < 0.05)

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Discussion

Heme is a vital component of the electron transport chains required for photosynthesis and degraded in the plastid through HO (Terry and Kendrick 1996). To date, genes encoding HOs have been isolated from a wide variety of organisms including animals, higher plants, red algae, cyanobacteria, and pathogenic bacteria (Ortiz de Montellano and Wilks 2001; Terry et al. 2002; Balestrasse et al. 2008). Structural analysis of HO revealed that it is a single compact domain consisting of mostly α-helices (Liu and Hu 2011). In higher plants, heme oxygenases are involved in the pathway leading to phytochrome chromophore metabolism, which was thought to be important in photoresponse, protection against oxidative stress and tissue injury, and lateral root formation (Terry et al. 1993, 2002; Ortiz de Montellano and Wilks 2001; Quail 2002a, b; Xie et al. 2012; Chen et al. 2013).

In this study, we described a rice mutant with yellow-green leaf color, ygl2. Map-based cloning is a powerful tool to isolate mutant gene(s) and has been widely used in various plants (Jander et al. 2002). Through fine mapping, an ORF encoding a HO was preliminary chosen from several candidates for further verification. Mutants with yellowish leaves caused by defects of HOs had been identified in other species. The phytochrome chromophore-deficient 1 (pcd1) mutant of pea demonstrated pale yellow-green foliage and elongated internodes, and is caused by HO1 deficiency (Weller et al. 1996; Linley et al. 2006). The hy1 mutants in Arabidopsis showed partially etiolated morphology and three independent alleles of hy1 all carried nucleotide substitutions/deletions within transcript region of AtHO1 (Muramoto et al. 1999; Davis et al. 1999). The yellow-green-2 (yg2) mutants of tomato had reduced Chl and anthocyanin, which resulted in a pale-green or yellow looking plants (Koornneef et al. 1985). Through high pressure liquid chromatography-based assay, it proposed HO dysfunction was responsible for yg2 mutant phenotype (Terry and Kendrick 1996, 1999). Together these studies provided a strong hint that ygl2 is probably encoded by a HO, whose mutation (s) as other ones in higher plants affects Chl synthesis and leaf color.

Sequence analysis indicated a 7-kb fragment was inserted in YGL2 from ygl2. Previous studies showed that YGL2/OsHO1 is a phytochrome synthesis factor, SE5, to control flowering in rice (Izawa et al. 2000; Andres et al. 2009). However, in se5 the revertible yellow-green phenotype showing in ygl2 was not focused on (Izawa et al. 2000; Andres et al. 2009). Besides the lesion of leaf color, ygl2 also showed decreased plant height and early flowering (Supplemental Table 2), which is phenotypically similar to se5 as a photochrome-deficient mutant (Andres et al. 2009). These results suggest YGL2 has a pleiotropic effect to plant development. Here we addressed the Chl biosynthesis and chloroplast structure regulated by YGL2.

Chl contents (including Chl a and b) are required for the formation of photosynthetic reaction centers and light-harvesting complexes, whereas an appropriate ratio of Chl a and b is critical in the regulation of photosynthetic antenna size (Oster et al. 2000). In the ygl2, a decrease of Chl contents and an increase of Chl a/b ratio were observed, indicating both photosystems and light-harvesting antenna complexes might be impaired, just like another rice Chl-deficient mutant (ygl1, Wu et al. 2007). Granal stacks of the ygl2 mutant appeared less dense (Fig. 1e, g) and decreased granal membranes as compared to wild type (Fig. 1d, f). It has long been established that mutations leading to defects in chlorophyll biosynthesis have a profound effect on the structure of plastids (Von Wettstein et al. 1995). The reduction of Chl synthesis and abnormal Chl a/b may disrupt the thylakoid ultrastructure in the mutant, which result in the reduction of filling of light-harvesting complexes in the thylakoid membrane. A Chl-deficient rice mutant has been reported to impair plastid development (Wu et al. 2007).

Transient expression indicated YGL2 from wild type and the mutant was both located in chloroplasts, suggesting the transit peptide of YGL2 in ygl2 mutant is functional and sufficient to transport proteins to plastids. The mutation was resided in a relatively conservative region of YGL2, which affected the higher structure of putative protein (Fig. 4), thus might impair enzyme activity. We tried to purify YGL2 proteins in vitro through Escherichia coli and compare their differences of enzyme activities between mutant and wild type, but were unsuccessful. Further studies maybe conducted to reveal the effect of the ygl2 mutation on enzyme activity.

Our results showed that the YGL2 was constitutively expressed in a variety of tissues, which is consistent with the trends observed for Arabidopsis HY1 (Davis et al. 1999). The mutation of YGL2 directly caused the reduction of its expressions in various tissues, suggesting YGL2 is regulated at transcript levels. The phenotypic effect of ygl2 is much more severe in seedlings, but ygl2 mutant is relatively healthy and recovered to wild type in the later stages of plant development. The presence of OsHO2, which is supposed to be functionally similar to YGL2 in enzymatic reaction(s) and abundantly expressed later than YGL2, is expected to have responsibility in plant later development.

Both Chl and heme are synthesized by photosynthetic organisms (Grimm 1998). The biosynthesis pathway for Chl is common to holophytochrome synthesis from 5-aminolevulinic acid to protoporphyrin IX (Terry et al. 1995). It has been reported in other Chl-deficient mutants, the expressions of Chl biosynthesis and photosynthesis related genes were lower than those in wild type (Wu et al. 2007; Chai et al. 2011; Zhou et al. 2013). In contrast, these genes were up-regulated in ygl2 mutant at normal air temperature. A simple explanation is, despite the ygl2 mutation on phytochrome synthesis pathway, Chl biosynthesis and photosynthesis pathway are relatively functional. Therefore, plants can remedially up-regulate some genes related with the synthesis of Chl and photosynthesis and struggle to survive. In addition, negative feedback effect of free heme on ALA synthesis has been proposed (Chereskin et al. 1982; Terry and Kendrick 1999) and ALA synthesis is rate limiting for Chl synthesis in tomato (Beale and Weinstein 1991). The inability of YGL2/OsHO1 caused by ygl2 mutation should lead to, more or less, heme accumulation, which then blocks Chl synthesis by feedback inhibition.

Our results showed ygl2 had significantly decreased levels of protochlorophyllide (Pchlide) and Chlide, but no effects to other intermediates of Chl biosynthesis, such as Mg-proto IX and proto IX. These results are a little bit difference to those in a tomato HO mutant (yg2), which showed reduced effects of yg2 to synthesize not only Pchlide but also Mg-proto IX (Terry and Kendrick 1999). This suggests YGL2 has a function slightly divergent from its counterpart in tomato.

In conclusion, the results presented here indicate that YGL2 in rice encodes a heme oxygenase required for Chl biosynthesis. Molecular cloning of YGL2 may provide a new prospect to analyze regulation of Chl biosynthesis pathway during rice developmental process.