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Journal of Genetics and Genomics 38 (2011) 29e37 www.jgenetgenomics.org

RLIN1, encoding a putative coproporphyrinogen III oxidase, is involved in lesion initiation in rice

Changhui Sun a,b,d,1, Linchuan Liu b,c,1, Jiuyou Tang b, Aihong Lin b,c, Fantao Zhang b,d, Jun Fang b, Genfa Zhang a,*, Chengcai Chu a,b,c,*

a Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Science, Beijing Normal University, Beijing 100875, China b The State Key Laboratory of Plant Genomics and National Plant Gene Research Center (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, China c Graduate School of the Chinese Academy of Sciences, Yuquan Road, Beijing 100039, China d Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China

Received 11 October 2010; revised 17 October 2010; accepted 20 October 2010

Abstract

Lesion mimic is necrotic lesions on plant leaf or stem in the absence of pathogenic infection, and its exact biological mechanism is varied. By a large-scale screening of our T-DNA mutant population, we identified a mutant rice lesion initiation 1 (rlin1), which was controlled by a single nuclear recessive gene. Map-based cloning revealed that RLIN1 encoded a putative coproporphyrinogen III oxidase in biosynthesis pathway. Sequencing results showed that a G to T substitution occurred in the second exon of RLIN1 and led to a missense mutation from Asp to Tyr. Ectopic expression of RLIN1 could rescue rlin1 lesion mimic phenotype. Histochemical analysis demonstrated that lesion formation in rlin1 was light-dependent accompanied by reactive oxygen species accumulated. These results suggest that tetrapyrrole participates in lesion formation in rice.

Keywords: Rice; Map-based cloning; Lesion mimic; Coproporphyrinogen III oxidase; Tetrapyrrole

1. Introduction pathway is very complex and fine regulated (Mock and Grimm, 1997; Grimm, 1998). In higher plant, there are four Tetrapyrrole has been studied over several decades as well- kinds of : , , , and known photosensitizers (Tanaka and Tanaka, 2007; Hirashima phytochromobilin (Tanaka and Tanaka, 2007). All the classes et al., 2009). It has been found that tetrapyrroles play impor- of tetrapyrroles share the same pathway from glutamate to tant roles in many biological processes such as light harvest- III. Subsequently, uroporphyrinogen III is ing, photophosphorylation, removal of reactive oxygen converted to protoporphyrin IX in three steps catalyzed by species, oxygen transport etc. (Grimm, 1998; Ishikawa et al., three distinct enzymes respectively: uroporphyrinogen decar- 2001). It has been shown that the tetrapyrrole biosynthesis boxylase (UROD), coproporphyrinogen III oxidase (CPOX) and protoporpyrinogen IX oxidase (PPOX), all these three Abbreviations: rlin1, rice lesion initiation 1; UROD, uroporphyrinogen steps are shared by chlorophyll, heme, and phytochromobilin decarboxylase; CPOX, coproporphyrinogen III oxidase; PPOX, proto- synthesis (Tanaka and Tanaka, 2007), the other remaining porpyrinogen IX oxidase. steps are categorized into the siroheme branch (Tanaka and * Corresponding authors. Tanaka, 2007)(Fig. 1). E-mail addresses: [email protected] (G. Zhang), [email protected] (C. Chu). Previous reports have demonstrated that lesion mimic can be 1 These authors contributed equally to this work. induced by several intermediate molecules of tetrapyrrole 1673-8527/$ - see front matter Copyright Ó 2011, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. doi:10.1016/j.jcg.2010.12.001 30 C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37

Here, we identified a lesion initiation mutant rlin1, which presented a lesion mimic phenotype, affected by a single recessive nuclear gene in rice. RLIN1 gene was mapped on the long arm of chromosome 4 between two markers STS3 and STS4 in an 85 kb region, among the 13 ORFs in this region, LOC_Os04g52130 encoding a putative CPOX, substituted one base of G to T in the second exon, causing a missense muta- tion from Asp to Tyr. The lesion phenotype of rlin1 could be rescued by overexpression of LOC_Os04g52130. Taken together, we show that RLIN1 which may be a tetrapyrrole biosynthesis gene is involved in lesion formation in rice.

2. Materials and methods

2.1. Rice materials

The spontaneous lesion formation mutant rlin1 was first identified from a T-DNA insertion population which was generated in our laboratory (Ma et al., 2009), but the pheno- type of rlin1 was not co-segregated with T-DNA, to this end, the rlin1 line without T-DNA insertion was isolated for progenies for further analysis. F1 and F2 population from a cross between rlin1 and Minghui 63 (Oryza sativa ssp. Fig. 1. Tetrapyrrole biosynthesis pathway. The four classes of tetrapyrroles, indica) were used to determine whether rlin1 was controlled chlorophyll, heme, phytochromobilin, and siroheme are shown in bold. CPOX by single nuclear recessive gene. This F2 population was also represents coproporphyrinogen III oxidase; PPOX indicates protoporpyrinogen used to map the gene RLIN1. All the plants were grown in IX oxidase; UROD indicates uroporphyrinogen decarboxylase. a paddy field at the Changping experimental station of Insti- tute of Genetics and Developmental Biology in Beijing, China. metabolism. So far, a number of genes responsible for lesion initiation involved in tetrapyrrole metabolism have been iso- 2.2. DNA extraction and PCR lated in higher plants (Hirashima et al., 2009). Repressing the expression levels of UROD, CPOX, and PPOX can lead to Genomic DNA was extracted from leaves following the a light-dependent lesion formation in tobacco and Arabidopsis protocol described by Edwards et al. (1991). The PCR mixture m m m respectively (Kruse et al., 1995; Mock and Grimm, 1997; Mock was mixed with 1 L DNA (10 ng/ L), 2 L10 buffer, m m m m et al., 1997, 1998, 1999; Molina et al., 1999; Ishikawa et al., 0.4 L primers (10 mol/ L), 0.4 L dNTP (10 mmol/L), m m m 2001). The CPOX-deficiency mutant lin2 shows a lesion 0.3 L Taq (5 U/ L), and 15.9 L ddH2O. PCR for mapping was performed as the following: pre-denaturation at 95 C for formation phenotype on both leaves and siliques in Arabidopsis (Ishikawa et al., 2001). Les22, encoding a UROD, appears to 5 min followed by 31 cycles of denaturation at 95 C for 30 s; annealing at 50e55 C for 30 s; extension at 72 C for 40 s; participate in natural porphyria in maize (Hu et al., 1998). Besides UROD, CPOX and PPOX, some other molecules of with a final extension at 72 C for 20 min. The PCR products tetrapyrrole metabolism are also involved in lesion formation. were separated on 4.0% agarose gels, stained with ethidium Reduction of the plastidic ferrochelatase by antisense RNA bromide and photographed. expression leads to leaf necrosis in transgenic tobacco plants (Papenbrock et al., 2001). Accumulated can 2.3. Molecular markers cause light-independent cell death along with chlorophyll breakdown (Hirashima et al., 2009); Lls1 encoding a pheo- In our lab, there were totally 290 SSR (simple sequence phorbide a oxygenase involves in lesion initiation in maize repeats) and STS (sequence-tagged site markers) markers (Yang et al., 2004). In Arabidopsis, two chlorophyll break- distributed on 12 chromosomes of rice. Among them, we down related genes, Accelerated cell death 1 (Acd1) and obtained 119 SSR and STS markers showing polymorphisms Accelerated cell death 2 (Acd2) induce lesions when they are between Zhonghua 11 and Minghui 63 for linkage analysis. absent, respectively (Mach et al., 2001; Tanaka et al., 2003; SSR were obtained in microsatellite sequences database Yang et al., 2004; Ishikawa, 2005). Taken together, all these (http://www.gramene.org/microsat). The new STS markers results suggest that lesion formation and tetrapyrrole biosyn- were designed based on the different DNA sequences between thesis are closely related, but the biological mechanism of Nipponbare (Oryza sativa ssp. japonica) and 9311 (O. sativa lesion formation which is caused by the process of tetrapyrrole ssp. indica)(Tong et al., 2009). All primers were designed metabolism is still unknown. using Primer Premier 5.0 (http://www.premierbiosoft.com). C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37 31

2.4. Mapping and linkage map TritonX-100, pH 3.8) at 28 C for 8 h. After that, all the seedlings were cleared in boiling ethanol (95%) at 28 C for Two DNA pools were constructed from F2 mapping pop- 20 min, then destained in ethanol (95%) overnight at 28 Cto ulation; one was mixed with 22 individuals with the lesion bleach the chlorophyll before photographing. mimic phenotype of rlin1, and the other contained 22 individ- uals with wild-type phenotype. The first DNA pool was used to 2.9. RNA extraction and quantitative RT-PCR screen the markers linkage to the gene RLIN1, the second DNA pool was used to confirm the results. All the markers covering Total RNA was extracted and quantitative RT-PCR was per- the 12 chromosomes of rice were screened in Zhonghua 11 and formed with Bio-Rad CFX96 Real-time System following the Minghui 63, and then additional 836 individuals with rlin1 protocol as described previously (Yang et al., 2009). The rice phenotype were used for fine mapping. The linkage map was Actin gene was used as an internal control in quantitative RT-PCR constructed based on the segregation data. analysis, the primer pairs were 50-ACATCGCCCTGGACTAT- GACCA-30 and 50-GTCGTACTCAGCCTTGGCAAT-30,the 2.5. Gene annotation and sequences analysis primers for quantitative RT-PCR analysis of RLIN1 expression were 50-ATCATACACCTGAAGAGGGAACT-30 and 50-AGG- The 85 kb region was analyzed in the rice genome automated AAAGCTAACAAACGATTGGA-30. annotation database (http://www.tigr.org). All the possible target ORFs were amplified using KOD polymerase (Toyobo 3. Results company, Japan) and purified using DNA gel extraction kit. Then, all the PCR fragments were sequenced by Invitrogen 3.1. Identified lesion initiation mutants Biotechnology Company of China. Sequencing results were assembled using Lasergene (http://www.dnastar.com/) and To assess the molecular mechanism of lesion formation in aligned using Vector NTI Suite 8 (http://www.invitrogen.com/). rice, a large-scale screening was carried out, and an rlin1 mutant line was obtained from our T-DNA population (Ma 2.6. Vector construction and transformation et al., 2009). Two weeks after seeding, the mutant rlin1 had already begun to develop lesions on the young leaves and leaf The following primers 50-CCCGGGATGGCGCCCTCTCT- sheaths shortly after full leaf expansion in both Beijing (N: CCTCAC-30 and 50-TCTAGATCAGAGATCGAGCCATTC- 39.92) and Hainan (N: 18.48). The lesions initiated as small CT-30 were used for RT-PCR to isolate the full-length cDNA of spots or stripes, then spreaded through the main vein of leaves LOC_Os04g52130.1. These primers contained the restriction or the middle part of the leaves. At the same time, more severe enzyme sites Xma I and Xba I at their 50 end for subsequent lesions were formed on the leaf sheaths. In the reproductive cloning. The PCR product was first cloned into pMD18-T vector growth phase, lesions were also found on the spikes and and then introduced into the binary plasmid pXQAct (a deriva- spermoderms. Besides these, rlin1 showed slightly reduced tive of pCAMBIA2300 carrying the rice Actin1 promoter and the plant height and later flowering phenotype compared to the OCS terminator with a multicloning site in between), yielding wild-type (Fig. 2). the pActin1:RLIN1 construct. The construct was then introduced into Agrobacterium tumefaciens strain AGL1, and transgenic 3.2. Genetic analysis rice plants were generated by method as Liu et al. (2007). The mutant rlin1 was crossed with Minghui 63, F1 plants 2.7. Light and dark treatments showed a wild-type phenotype (without lesion mimic), and in the F2 population plants, segregation ratio of wild-type Both the wild-type Zhonghua 11 and the mutant rlin1 were phenotype plants to lesion formation phenotype plants was seeded in 1/2 Murashige and Skoog medium in the same growth about 2.88:1 (Table 1), suggesting the lesion mimic phenotype chamber for 7 days, then divided into two groups, one was at of rlin1 was controlled by a single nuclear recessive gene. 30 C 14 h light/24 C 10 h dark condition, the other was at 24 C continuous dark condition. All of the plants had grown for 3.3. Preliminary gene mapping 2 weeks after seeding. In order to map the RLIN1 gene, map-based cloning was 2.8. Histochemical analysis carried out by using F2 population obtained from a cross of rlin1 and Minghui 63. By screening 290 pairs of SSR and STS DAB (diamino benzidine) staining was performed markers in our lab, we found 119 pairs of markers exhibited following the modified protocol described previously polymorphisms between rlin1 and Minghui 63, and then these (Thordal-Christensen et al., 1997). The wild-type Zhonghua markers were used for analysis of the linkage relationship with 11 and the mutant rlin1 were seeding in growth chamber at the the RLIN1 gene. The DNA pool mixed by 22 lesion initiation photoperiod of 12 h light at 30 C and 12 h dark at 24 C for 7 mutant individuals from F2 population was used for linkage days, then the entire seedlings of Zhonghua 11 and rlin1 were relationship analysis. STS1 and STS9 on chromosome 4 showed placed in DAB staining solution (1 mg/mL DAB, 0.01% a brightness electrophoretic band of rlin1 along with very weak 32 C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37

Fig. 2. Phenotypes of mutant rlin1. A: plants of wild-type (light) and rlin1 (right). B: leaf sheath of wild-type (right) and rlin1 (left). C: leaves of wild-type (right) and rlin1 (left). D: panicles of wild-type (right) and rlin1 (left).

electrophoretic band of Minghui 63 polymorphism, suggesting Table 1 STS1 and STS9 might be linkage with RLIN1. Furthermore, the Segregation ratio of F2 populations. 22 F2 individuals DNA polymorphism segregation data c2 < Cross NP LP Segregation ratio 0.05 3.84 P confirmed the conclusion. In a word, RLIN1 located between the rlin1/Minghui 63 2410 836 2.88 1.00 P < 0.05 two markers, STS1 and STS9, and the genetic distances were NP: normal phenotype plants. LP: lesion mimic phenotype plants. 16.2 cM and 9.2 cM, respectively (Fig. 3).

Fig. 3. Map-based cloning of RLIN1. A: primary mapping of RLIN1 between the marker STS1 and STS9. B and C: fine mapping of RLIN1 between STS3 and STS4. D: BAC contigs spanning the RLIN1 locus. E: structures of LOC_Os04g52130.1 (down) and LOC_Os04g52130.2 (up). Black boxes indicate exons, white boxes indicate UTRs, and black lines indicate introns, AAA-SR indicates the most likely cleavage site of the signal peptide between 21 aa and 22 aa (http://www. cbs.dtu.dk/services/SignalP/). F: the mutation in rlin1. C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37 33

Table 2 Markers used in fine mapping of RLIN1. Markers Forward (50 / 30) Reverse (50 / 30) Loci (bp) STS1 TTTGGATACACGTGAAAAGC GAAGGACTTGTGATGATGGA 27209352 STS2 GGGTTTTGGAAGGGAAGAAG TACCCCTAGCATAGGCCTCC 30144526 STS3 CAATAATGCGAGGACAAAAT CTGATTTGGTAGCGTGTGT 30719669 STS4 CCAGCTCCCTAAACATTCCA CTCCTTTTGAACAATGGATG 30804224 STS5 AGCCTTGGATTGTGCATT TGAATGACAAGCAGCGACAC 30832954 STS6 TTAGCCTTATCATCCCAGAA GCTGACACGTACGATACTCC 30966527 STS7 TCAAGGCTACGGAGTAGTTC CCGTACTATGTCGTCGATCT 31458587 STS8 CCATCGGTGGTTACGTATAG TATGAAGTCGGTGTACATGC 32377554 STS9 GGCAGTCTCGTTGGAGTC AGCCAAGGTAGATAGGGTTC 32897074

Fig. 4. Phylogenic analysis and sequence alignment of CPOX proteins in different species. A: phylogenetic tree analysis using Clustal X and MEGA4. Accession numbers of the protein sequences were as follows: Oryza sativa (RLIN1, Q7XPL2.2); Zea mays (NP_001183946.1); Hordeum vulgare (Q42840.1); Vitis vinifera (CAN76809.1); Glycine max (P35055.1); Nicotiana tobacum (Q42946.1); Arabidopsis thaliana (LIN2, NP_171847.4); Ricinus communis (XP_002518396.1); Picea sitchensis (ABR16358.1); Isochrysis galbana (ABA55505.1); Homo sapiens (BAA04033.1); Musmus culus (BAA03840.1). B: protein sequences alignment using Clustal X and GenDoc. Shades of black and gray indicate the conserved amino acids, white letter with black background indicates 100% identity, white letter with gray background indicates 80% identity and black letter with gray background indicates 60% identity. The gray arrow indicates the mutation site of rlin1. Protein accessions were used as described in (A). Nt, Nicotiana tobacum; At, Arabidopsis thaliana; Gm, Glycine max; Os, Oryza sativa; Zm, Zea mays; Hv, Hordeum vulgare. 34 C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37

3.4. Fine mapping and candidate gene analysis

In the region between STS1 and STS9, 31 pairs of new STS markers were developed for fine mapping. Seven pairs of them showed polymorphisms between rlin1 and Minghui 63, and were used for fine mapping (Table 2). According to the genetic linkage analysis, the RLIN1 gene was finally narrowed down between two STS markers, STS2 and STS5, with the genetic distances of 6.7 cM and 1.0 cM, respectively. RLIN1 was finally mapped to a 1.5 cM region between STS3 and STS4, with the genetic distances of 1.0 cM and 0.5 cM, respectively. The physical distance was about 85 kb with 13 candidate genes (Fig. 3). DNA sequencing results revealed that the 622nd base G of the CPOX gene LOC_Os04g52130 changed to T, and caused a missense mutation from Asp to Tyr in rlin1 mutant (Fig. 3), suggesting that LOC_Os04g52130 was most likely the candidate gene of RLIN1. This LOC_Os04g52130 protein was further analyzed by subcellular localization prediction program ChloroP 1.1 Fig. 5. Analysis of LOC_Os04g52130.1 overexpressed plants. A: vector (http://www.cbs.dtu.dk/services/ChloroP)(Chou and Shen, construction. R-Border, the right border; L-Border, the left border; 2008). We found that the LOC_Os04g52130 included P35S:CaMV35S promoter; NPTII: neomycin phosphotransferase gene; PAc- a 21-amino acid putative chloroplast transit peptide, implying tin1, rice Actin1 promoter; OCS: OCS terminator. B: transgenic plants that LOC_Os04g52130 might be localized in the chloroplast phenotype. While rlin1 shows lesion mimic phenotype, rlin1-C shows wild- type phenotype. WT: Wild-type (left); rlin1: rlin1 mutant (middle); rlin1- (Fig. 3). C: LOC_Os04g52130.1 overexpressed plant (right). CPOX proteins were widely existed in living organisms, and they could be found in both lower forms of life such as 3.7. The light-dependent phenotype of rlin1 unicellular marine microalga Isochrysis galbana and higher living organisms like Homo sapiens, suggesting the impor- To check whether the lesion initiation caused by deficiency tance of CPOX in evolution (Fig. 4A). Furthermore, alignment of the putative CPOX was a light-dependent phenotype in of RLIN1 with its homologs showed that the CPOX proteins rlin1, we seeded both wild-type and rlin1 under light and dark were highly conserved in different species, especially in the conditions for 2 weeks, rlin1 showed lesion formation under C-terminal region (Fig. 4B). light condition but not under dark condition, the wild-type did not show lesion formation in both conditions. The results 3.5. Complementation analysis suggested that the lesion mimic was a light-dependent phenotype in rlin1 (Fig. 7). To make sure that the defect of LOC_Os04g52130 was H2O2 is a major reactive oxygen species (ROS) produced responsible for rlin1, we constructed LOC_Os04g52130.1 during programmed cell death (PCD) like lesion formation, overexpressed vector using Actin1 promoter and transformed it and DAB staining could confirm the degree and the regions of to rlin1. About 20 individual transgenic plants were obtained, and all of them showed a wild-type phenotype without lesion mimic phenotype, demonstrating that LOC_Os04g52130 was the candidate gene of RLIN1 (Fig. 5).

3.6. Expression pattern of RLIN1

The expressions of RLIN1 were examined in different organs by quantitative real-time RT-PCR, and it showed RLIN1 was expressed in all the organs tested, and the expression level was much higher in roots than in the other organs, suggested RLIN1 had important functions in roots. This was corresponded to the report that the tetrapyrroles such as heme might be present in all organs (Boese et al., 1991). Besides roots, RLIN1 expression levels decreased in the order of leaf sheaths, leaves, culms, with the lowest expression in the panicles, this expression pattern was consistent with the lesion mimic phenotype of rlin1 (Fig. 6). Fig. 6. Expression patterns of RLIN1 in different organs. C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37 35

Fig. 7. Light-dependent analyses of rlin1. A: rlin1 mutant (right) and wild-type (left) grown under light condition; red arrows show the lesion mimic in rlin1. B: rlin1 mutant (right) and wild-type (left) grown under dark.

H2O2 accumulation. H2O2 can be observed as a reddish-brown and maize, indicating that tetrapyrroles and their derivatives are coloration by DAB. After DAB staining, we found that the involved in lesion formation in plants (Mock and Grimm, 1997; mutant rlin1 had higher background compared with the wild- Mock et al., 1997, 1998, 1999; Hu et al., 1998; Molina et al., type, no matter of the regions with or without the lesion mimic 1999; Ishikawa et al., 2001; Mach et al., 2001; Papenbrock (Fig. 8). These results indicate that the cell death may be et al., 2001; Tanaka et al., 2003; Yang et al., 2004; Ishikawa, associated with the burst of ROS in rlin1. 2005; Hirashima et al., 2009). It is known that 15 enzymes encoded by 27 genes in Arabidopsis are involved in tetrapyrrole 4. Discussion biosynthesis (Nagata et al., 2005). However, only a few genes have been identified in rice, and none of them is involved in Lesion mimic mutants can autonomously present necrotic lesion initiation (Jung et al., 2003; Lee et al., 2005; Zhang et al., lesions in the absence of any pathogenic infection (Matin et al., 2006; Wu et al., 2007; Wang et al., 2010). 2010). In 1995, Kruse first found that a defective in tetrapyrrole Here, we identified a new lesion initiation mutant rlin1, and metabolism caused development of lesions in transgenic the RLIN1 gene was finally delimited in an 85 kb region on the tobacco with antisense mRNA of CPOX. Since then, quite long arm of chromosome 4. In this region, we speculated a number of genes had been identified in Arabidopsis, tobacco, LOC_Os04g52130 was the candidate gene for lesion mimic

Fig. 8. DAB staining of rlin1 and wild-type. 36 C. Sun et al. / Journal of Genetics and Genomics 38 (2011) 29e37 phenotype. Firstly, by aligning sequences of CPOX in different rlin1 but not the wild-type could be stained by DAB, indicated species, we found that the Asp which was changed to Tyr is that rlin1 contained much more H2O2 than the wild-type, and quite conserved in many plant species, this mutation might the cell lesions in rlin1 were resulted from ROS accumulation. cause function defect in CPOX in rice (Fig. 4B). Secondly, the Furthermore, it was found that the defect of PPOX led to a cell putative CPOX is a single copy gene in rice, indicates that the death response which could trigger resistance to Peronospora pathway from coproporphyrinogen III to protoporphyrinogen parasitica infection in Arabidopsis (Molina et al., 1999). In IX may rely on LOC_Os04g52130 (Tanaka and Tanaka, 2007). our following work, it will be interesting to determine whether Thirdly, it has been shown that the defect of CPOX could also rlin1 plants can be resistant to some pathogen infection of rice. lead to lesion mimic phenotype in tobacco and Arabidopsis. Defects of CPOX could cause different phenotypes in Last, ectopic expression experiment showed that constitutive Arabidopsis and rice, the mutant lin2 exhibited pale green expression of LOC_Os04g52130 could rescue lesion mimic leaves phenotype besides lesion mimic in Arabidopsis phenotype of rlin1. In conclusion, LOC_Os04g52130 is the (Ishikawa et al., 2001), but rlin1 only showed a lesion candidate gene of RLIN1. formation phenotype in our study, suggesting that the func- In the lesion formation mutants as described above, the tions of CPOX might be partly different in monocotyledonous function of UROD, CPOX, PPOX, plastidic ferrochelatase and plants and dicotyledonous plants. uroporphyrinogen III synthase are partly deficient rather than In summary, this is the first demonstration about the rela- knockout (Ayliffe et al., 2009). Tetrapyrrole metabolism genes tionship between tetrapyrrole biosynthesis and lesion forma- involved in lesion mimics are likely to cause lethal if tion in rice. Our study declares that the necrotic phenotype in completely deleted. Contrast to the wild-type, there is only an rlin1 which is caused by partial suppression of RLIN1, is light- amino acid mutation from Asp to Tyr in rlin1, which may dependent and may be related to ROS accumulated. Moreover, cause partial deficiency in CPOX. Therefore, the necrotic study of RLIN1 will help us to further understand the bio- phenotype of rlin1 may be caused by reduced enzyme activity logical mechanism of lesion formation affected by tetrapyrrole rather than completely loss-of-function. biosynthesis in plants. In our study, we found that the formation of lesions initi- ated shortly after leaves fully expansion, this phenomenon was Acknowledgements also observed in lin2, Les22 and transgenic plants of antisense suppression of coproporphyrinogen oxidase and uroporphyri- This work was supported by grants from the Ministry of nogen decarboxylase genes in tobacco (Kruse et al., 1995; Science and Technology of China (No. 2009CB118506) and Mock and Grimm, 1997; Hu et al., 1998; Ishikawa et al., the National Natural Science Foundation of China (Nos. 2001). It has been supposed that the phenomenon was corre- 30825029 and 30621001). We thank Prof. Xiaojian Deng and lated with peak flux through the chlorophyll biosynthetic Pingrong Wang from Sichuan Agricultural University for their pathway, and this hypothesis could also explain why fully critical readings of the manuscript in this work. expanded mature leaves of dark-grown plants did not show lesion mimic phenotype when grown under light condition (Hu et al., 1998; Ishikawa et al., 2001; Ayliffe et al., 2009). References It has been reported that the surplus of tetrapyrrole inter- mediate molecules could lead to a series of reactions involved in Ayliffe, M.A., Agostino, A., Clarke, B.C., Furbank, R., von Caemmerer, S., ROS and finally caused a lesion mimic phenotype. In the Pryor, A.J., 2009. Suppression of the barley uroporphyrinogen III synthase beginning, redundant energy accumulated from light by tetra- gene by a Ds activation tagging element generates developmental photo- sensitivity. 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