OsHO2 has a potential regulatory role for tetrapyrrole biosynthesis in rice. 
In higher plants, a major regulatory mechanism for tetrapyrrole metabolism has been attributed to the feedback inhibition of HEMA (glutamyl tRNA reductase) by heme (Beale 1999). HEMA catalyzes the first committed step of tetrapyrrole biosynthesis by generating the immediate precursor (i.e. glutamate 1-semialdehyde) of ALA. Disruption of HO1 or PΦB synthase genes is believed to cause over-accumulation of heme which in turn represses HEMA activities and ALA formation (Tanaka et al. 2011). However, our Osho2 mutants showed decreased levels of heme but increased levels of ALA, indicating that the Chl deficiency was not a consequence of heme-induced HEMA inhibition. In addition, the levels of Proto IX (the last common precursor of Chl and PΦB), Pchlide and Chlide (Chl precursors), and BV (PΦB precursor) were all diminished in the mutant seedlings, presumably reflecting the overall reduction of metabolic flux toward Chl and PΦB production. Interestingly, the Osho2 mutants were found to accumulate elevated levels of Mg-Proto IX, which is the first committed Chl precursor. Meanwhile, the accumulation of transcripts encoding different tetrapyrrole enzymes was consistent with the corresponding metabolite changes. Thus, the expression levels of PPOX (for Proto IX), MgCY (for Pchlide), POR (for Chlide), FeCh (for Heme), and HO1/YGL2 (for BV) were down-regulated while the expression levels of HEMA (for ALA) and CHLI, CHLD, and CHLH (for Mg-Proto IX) were up-regulated in the mutant seedlings. The biosynthesis of different tetrapyrrole-derived biomolecules is under sophisticated controls to meet specific cellular demands and to prevent excessive accumulation of photosensitizing intermediates (Tanaka et al. 2011). For example, chlorophyll biosynthesis is often subject to coordinated transcriptional regulation of genes encoding key enzymes like HEMA1, CHLH, MgMT, MgCy, POR, and CAO  (Waters et al. 2009). Interestingly, the expression of HEMA1 and MgCH genes (CHLI, CHLD, and CHLH) appears to be regulated independently from other tetrapyrrole biosynthesis genes in our Osho2 mutants which accumulated higher levels of ALA and Mg-Proto IX. These unique changes in metabolite and transcript levels suggest the existence of a novel regulatory mechanism, potentially involving OsHO2, for tetrapyrrole biosynthesis, especially during early developmental stages in rice. Instead of binding to heme, the synthetic AtHO2 recombinant protein binds Proto IX with high affinity in vitro, implicating a regulatory role on substrate flow through binding of tetrapyrrole intermediates (Gisk et al. 2010). On the other hand, Arabidopsis GUN4 stimulates MgCH activities by binding to the CHLH subunit as well as the enzyme substrate (Proto IX) and product (Mg-Proto IX) (Adhikari et al. 2011). Meanwhile, OsHO2 may regulate tetrapyrrole biosynthesis through interaction with tetrapyrrole metabolites and/or enzymes inside chloroplasts. Further investigations involving metabolite binding and protein–protein interaction analyses will be necessary to elucidate the biochemical activities of OsHO2.
The ylc2 mutant was identified by screening of a population of rice 60Coγ-irradiated mutants in our laboratory. The chlorosis phenotype was most prominent during the seedling stage (Fig. 2a). In field-grown mutant plants, the mature leaves showed darker green pigmentation but newly emerged leaves were still pale in color (Fig. 2b). Chl contents of WT and ylc2 mutant seedlings (1-week-old) were then measured to characterize the chlorosis mutant phenotype. As shown in Fig. 2c, the contents of Chl a, Chl b, and total Chl in mutant seedlings were 18, 27, and 23 % of those measured in WT Nipponbare plants. The amount of total carotenoids was also reduced by more than 65 % in the ylc2 seedlings. The ultrastructure of chloroplasts was then examined using transmission electron microscopy (TEM). In WT plants, the chloroplasts showed well-developed thylakoid membrane with stacks of grana (Fig. 2d). However, the thylakoid membrane system in the mutant chloroplasts is poorly organized with larger numbers of plastoglobules, indicating that chloroplast development is defective in the ylc2 seedlings.
To elucidate the subcellular localization of the OsHO2 protein, a translational fusion with enhanced yellow fluorescent protein (EYFP) at the C-terminus driven by the CaMV 35S promoter was constructed for Agrobacterium-mediated transient expression in N. benthamiana. As revealed by confocal microscopy analysis, the OsHO2-EYFP fluorescent signals were detected in organelles of mesophyll cells consistent with the sizes and chlorophyll autofluorescence of chloroplasts (Fig. 4a). The sub-organelle location of OsHO2-EYFP was further examined by immunoblot analyses of stromal and membrane fractions from Percoll-purified chloroplasts of infiltrated N. benthamiana leaves. As shown in Fig. 4c, the OsHO2-EYFP fusion protein was only detected in the stromal fraction. Therefore, the above analyses suggested that OsHO2 is a chloroplast protein residing inside the stroma.
Gene expression of OsHO2 in different tissues was first examined in the publicly available Affymetrix GeneChip microarray data. YLC2 expression was detected in almost all tissues of rice with highest levels detected in leaves and shoot apical meristem (Fig. 5a). Levels of OsHO2 expression were then compared in ylc2 mutant and WT seedlings using RT-PCR. Results indicated that the gene expression was not affected by the 8-bp deletion in ylc2 mutant (Fig. 5b). On the other hand, OsHO2 transcript accumulation in WT plants was reduced by approximately 50–60 % in mature leaves (third leaf) of 12-week-old plants in comparison to newly emerged leaves or young seedlings (Fig. 5c), further suggesting that the rice gene plays a more important role during early development.
BLAST search in the rice genome revealed that Os03g27770 is a single-copy gene with an ORF of 993 bp. The coding region contains 4 exons (3 introns) and encodes a protein of 330 amino acids with a molecular weight of approximately 36.5 kDa. The 8-bp deletion in ylc2 generated a frame-shift mutation, resulting in premature translational termination. Consequently, the ylc2 mutant protein is identical to the WT sequence up to the 244th position, followed by a short stretch of 15 amino acid residues (Fig. S2). The WT protein harbors a predicted N-terminal chloroplast-targeted sequence of 47 amino acids (www.cbs.dtu.dk/service/TargetP). The rice protein is annotated as heme oxygenase 2 (HO2) which contains a heme oxygenase (HO) domain (Pfam 01126) in the second half of the protein (176th–325th residues). Pairwise 3D structural alignment  (Gelly et al. 2011) demonstrated the tight superimposition of the mutant protein with OsHO2 except for the absence of three C-terminal α-helices (Fig. S3) which may be important for OsHO2 functions. The HO signature sequence which includes the His residue required for protein stability  (Matera et al. 1997) is conserved in OsHO2 (His-246). The His residue involved in HO-ascorbic acid binding  (Matera et al. 1997) can also be identified (His-254). However, the His residue essential for heme binding in HO is replaced by Arg-102 in OsHO2. Phylogeny analysis of plant HO homologous sequences clearly revealed the separation of the HO1 and HO2 subfamilies (Fig. S4). OsHO2 is clustered with two highly conserved sequences from maize and sorghum in the HO2 family. These cereal sequences share more than 75 % identity but are only weakly homologous to the dicot HO2 sequences with less than 50 % identity. By contrast, the HO1 sequences are highly homologous between monocot and dicot species. For example, OsHO1 shares 70 % sequence identity to the Arabidopsis HO1.
Labs working on this gene
1. State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China 2. School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China 3. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 311121, China 4. Department of Biology, Concordia University, Montreal, QC, H4B 1R6, Canada
- Qingzhu Li, Fu-Yuan Zhu, Xiaoli Gao, Yi Sun, Sujuan Li, Yuezhi Tao, Clive Lo, Hongjia Liu (2014) Young Leaf Chlorosis 2 encodes the stroma-localized heme oxygenase 2 which is required for normal tetrapyrrole biosynthesis in rice. Planta 10.1007/s00425-014-2116-0
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