A Chlorophyll-Deficient Rice Mutant with Impaired Chlorophyllide Esterification in Chlorophyll Biosynthesis 1

Chlorophyll synthase catalyzes esterification of chlorophyllide to complete the last step of chlorophyll biosynthesis. Although the chlorophyll synthases and the corresponding genes from various organisms have been well characterized, chlorophyll synthase mutants have not yet been reported in higher plants. In this study, a rice ( Oryza Sativa L.) chlorophyll-deficient mutant, yellow-green leaf 1 ( ygl1) , was isolated, which showed yellow-green leaves in young plants with decreased chlorophyll synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the chlorophyll synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with chlorophyll biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of chlorophyll or chlorophyll precursors.


INTRODUCTION
Chlorophyll (Chl) molecules universally exist in photosynthetic organisms. They perform essential processes of harvesting light energy in the antenna systems and driving electron transfer in the reaction centers (Fromme et al., 2003). Their metabolism has been extensively studied in various organisms by both biochemical (Pontoppidan et al., 1994) and genetic approaches (Bollivar et al., 1994a;Nakayashiki et al., 1995;Tanaka et al., 1998). Early enzymatic steps of chlorophyll biosynthesis in converting 5-aminolevulinate acid (ALA) to protoporphyrin IX (Proto IX) are shared with the heme biosynthesis pathway. Many essential data regarding the identity of the associated enzymes were obtained from studies on nonphotosynthetic organisms such as Escherichia coli (Narita et al., 1996). The later steps of chlorophyll biosynthesis are common with bacteriochlorophyll a biosynthesis (Porra, 1997;Suzuki et al., 1997). Directed mutational analysis with a photosynthetic bacterium, Rhodobacter capsulatus, provided abundant information on the genes involved in bacteriochlorophyll biosynthesis (Bollivar et al., 1994b), and homologous genes have been isolated from oxygenic plants (Jensen et al., 1996). With the recent identification of 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), all genes required for chlorophyll biosynthesis have been identified in higher plants (Nagata et al., 2005). Analysis of the complete genome of Arabidopsis thaliana show that there are at least 27 genes encoding 15 enzymes involved in chlorophyll biosynthesis from glutamyl-tRNA to Chl b (Nagata et al., 2005).
We then analyzed the possible phylogenetic relationships between YGL1 and its related proteins from higher plants and cyanobacteria (Fig. 3B). The result indicated that rice YGL1 was more closely related to chlorophyll synthase from monocotyledon plants oat (A. sativa) than to those of other species. Not surprisingly, YGL1 has a phylogenetically much closer relationship to chlorophyll synthases of the higher plant species than to bacteria proteins. In addition, it is interesting to note that bacteriochlorophyll synthases lack a motif  that exists only in the chlorophyll synthase ( Supplementary Fig. 1). Analysis with the transmembrane calculation programs (Nilsson et al., 2002) revealed that the ygl1 mutation site occurred at or close to the end of a transmembrane helix (data not shown).
The identity of ygl1 was subsequently confirmed by genetic complementation experiments (Fig. 4A). The color of leaves, the levels of Chl and the ratio of Chl a:b were all restored to levels of wild-type plants upon transformation with the YGL1 gene ( Fig. 4B and 4C). Therefore, this confirms that observed abnormal phenotypes of the ygl1 mutant plants resulted from mutation of the YGL1 gene.

YGL1 mRNA Expression Level Is Not Affected by the ygl1 Mutation of YGL1
We compared the level of YGL1 transcript in ygl1 mutant and wild-type plants using RT-PCR. Figure 5A showed that YGL1 mRNA was expressed at similar levels in root, leaf sheaths, leaves and young panicles in both the ygl1 mutant and wild-type. We also examined the effect of light and dark growth conditions on the expression of YGL1. No change in transcript levels was observed when ygl1 or wild-type plants were grown under light or dark conditions (Fig. 5B).
Furthermore, no significant differences of YGL1 mRNA levels were observed in the mutant compared to wild-type from early to mature stages (Fig. 5C). These results indicate that the missense mutation of ygl1 does not affect its own mRNA expression.
We next addressed the question of whether the ygl1 mutation affected the transcript of other genes associated with chlorophyll biosynthesis, chloroplast development or photosynthesis.
Analysis of mRNA levels using real-time PCR showed that the expression of genes involved in chlorophyll biosynthesis, such as glutamyl tRNA reductase (HEMA1), was reduced by about 40%, and chlorophyllide a oxygenase (CAO1) and NADPH:protochlorophyllide oxidoreductase

Reduced Activity of Chlorophyll Synthase Results in Accumulation of Intermediates of Chlorophyll Biosynthesis
When angiosperm plants were grown in dark conditions, protochlorophylide (Pchlide) accumulated instead of chlorophyll and plants had an etiolated phenotype (Schoch et al., 1977(Schoch et al., , 1978. Mock and Grimm (1997) characterized transgenic plants with deficiencies in coproporphyrinogen oxidase and uroporphyrinogen decarboxylase, two preceding enzymes in the metabolic pathway of chlorophyll synthesis. These plants accumulated their respective substrates, uroporphyrin(ogen) and coproporphyrin(ogen), in young leaves up to several hundred-fold times the levels in control plants and exhibited necrotic lesions. Since the ygl1 mutant had deficient chlorophyll synthase activity, Chlide was predicted to accumulate in ygl1 mutant plants. Not surprisingly, compared to wild-type plants, ygl1 mutants accumulated higher level of Childe and other intermediates, including ALA (Fig. 8A), Proto IX, Mg-protoporphyrin IX (Mg-Proto IX), and Pchlide, in leaves of seedlings (Fig. 8B). Together these results showed that chlorophyll synthase play a critical role in chlorophyll biosynthesis.

DISCUSSION
Chlorophyll synthase has been the subject of thorough investigation (for reviews, see Willows, 2003；Suzuk, 1997. However, to our knowledge, no corresponding chlorophyll synthase mutant has been found in higher plants. In this study, we have identified and functionally characterized Analysis of YGL1 derived amino acid sequence showed that Pro-198 was in proximity to a motif  specifically found only in chlorophyll synthases, but not in bacteriochlorophyll synthases ( Supplementary Fig. 1), which differ, in part, based on preference of substrates, either Chlide (targeted by chlorophyll synthases) or bacteriochlorphyllide (for bacteriochlorophyll synthases) (Oster et al., 1997a). Therefore, the importance of the essential Pro-198 residue in YGL1 could be attributed to its location in or proximity to the binding site of Chlide. Future studies to pursue this possibility would help further elucidate the substrate specificity and targeting mechanisms between the synthase family members.
In this study, the ygl1 mutant seedlings displayed a yellow-green phenotype and became green with leaf chlorophyll accumulation at the mature stage (Table I). The carotenoid content was significantly lower in the mutant plants compared to wild-type, even in older leaves in which the chlorophyll content was the same as wild-type (Table I). This result might be related to the parallel degradation of pigments and pigment binding proteins of the photosynthetic apparatus (Cunningham FX et al.1998;Papenblock et al., 2000).
Mutation of the YGL1 gene reduced chlorophyll levels, and resulted in a yellow-green phenotype more or less specific to younger plants. Why the ygl1 mutation affects chlorophyll biosynthesis most dramatically in the early developmental stage but is restored in later stages is not yet completely understood. One possible explanation is that there might be other chlorophyll synthase homologs with redundant functional activities in later stages. However, no other rice chlorophyll synthase genes were identified from a survey of rice genome database (International Rice Genome Sequencing Project, 2005). These results were consistent with those of Gaubier et al. (1995) and Schmid et al. (2001), which showed that the chlorophyll synthase sequence represented a single-copy gene in A. thaliana and A. sativa by Southern and Northern blot analysis. Since we did not find significant differences in transcription level of the YGL1 gene at the different development stages, one possibility is that the enzyme is regulated at the translational level. This hypothesis remains to be tested directly.
Moreover, the delayed chloroplast development might lead to a slow accumulation of chlorophyll in ygl1 mutant seedling leaves. Chlorophyll synthase was proposed to localize to the thylakoid membranes where esterification of Chlide a with phytol or earlier alcohol precursors take place (Rüdiger, 1980;Soll and Schultz, 1981;Block et al., 1980). Soll et al. (1983)  that the chlorophyll synthase in chloroplasts is more stable than those from etioplasts. This implied that Chl a biosynthesis catalyzed by chlorophyll synthase was associated with chloroplast development (Biswal et al., 2003;El-Saht, 2000). Therefore, chlorophyll deficiency caused by the ygl1 mutant might be due to delayed formation of thylakoid membranes, and the underdeveloped chloroplast led to the decease of chlorophyll accumulation in ygl1 seedlings stage.
Previous reports indicated that the transcript of the A. thaliana G4 gene was detected only in green or greening tissues, and its expression was not strictly light-dependent, while oat CHLG gene was constitutively expressed (Gaubier et al., 1995;Schmid et al., 2001). Our experiments showed that rice YGL1 was constitutively expressed, which is consistent with trends observed for oat CHLG (Schmid et al., 2001). Another notable observation was the effect of the YGL1 mutation on the mRNA expression of some genes associated with chlorophyll biosynthesis or chloroplast development. Among the genes examined, we found that the expression of most nuclear genes, including cab1R, HEMA1, CAO1 and PORA, were reduced at different levels, whereas the plastid-coded genes, such as psaA, psbA and rbcL, were not significantly influenced in ygl1 seedlings (Fig. 6). This suggests, then, that the expression of the plastid-encoded genes might be regulated at the level of translation rather than transcription.
In the ygl1 mutant, the transcript level of cab1R gene was severely impaired and markedly different from that of cab2R, which was only slightly decreased at the young seedling stage (Fig.   6), indicating that both are differentially regulated (Matsuoka, 1990). Although the expression of the nuclear multi-gene (cab) family was a marker for chloroplast development and tightly Chl a is required for the formation of photosynthetic reaction centers and light-harvesting complexes (LHC), and Chl b is exclusively located in the light-harvesting pigment protein complexes of PS I and PS II. An appropriate ratio of Chl a:b is critical in the regulation of photosynthetic antenna size (Jansson, 1994;Oster et al., 2000;Tatsuru et al., 2003). However, partial block in Chl a biosynthesis caused a decrease of the chlorophyll content and an increase in the ratio of Chl a:b in young leaves of the ygl1 mutant (Falbel et al., 1996a), indicating that the total number of photosystems decreased and light-harvesting antenna complexes might be lower than that of wild-type. Further characterization of the YGL1 gene could provide deeper insight into understanding the relationship between the biosynthesis of Chl a and Chl b, and between the biosynthesis of chlorophyll, carotenoids and proteins, the regulation of photochemical reactions, as well as the assembly of the thylakoid membranes and chloroplast development.

Plant Materials
The rice (Oryza sativa) yellow green leaf mutant (ygl1) was isolated from the indica cultivar Zhenhui249. The ygl1 was crossed with an indica rice cultivar, PA64, to construct the F 2 mapping population. PA64 has a major genetic background of indica and minor gene flows from javanica (Bao et al, 2005).

Genetic Analysis and Marker Development
Genomic DNA was extracted and analyzed for co-segregation using available SSR (McCouch et al., 2002) from F 2 plants. New SSR markers were developed based on the Nipponbare genome sequence information from NCBI database and by searching for simple repeat sequences with the SSRIT program (Temnykh et al., 2001). CAPS markers were developed on comparisons of original or CAPS length by using SNP2CAPS soft (Thiel et al., 2004) between the indica var. The full-length CHLG protein sequences were retrieved from GenBank and used for phylogenetic analysis according to the methods described by Li et al. (2003). The signal peptide was predicted with SignalP version 2.0 (Nielsen et al., 1998). Phylogenetic analysis and Multiple sequence alignment were conducted by using DNAMAN version 6.0 (Lynnon Biosoft). The residue-specific hydropathy index was predicted by using the transmembrane calculation programs (Nilsson et al., 2002).

Complementation of the ygl1 Mutant
As Agrobacterium-mediated transformations are difficult to perform in indica rice, the ygl1 gene was also transferred to Wuyunjing 8 (spp. japonica) by five rounds of backcrosses with Wuyunjing 8 and self crossed for five generations. We obtained an isogenic line with ygl1 allele in japonica genetic background and named it as ygl2, which was used as transforming material.
For complementation of the ygl1 mutation, a full-length cDNA fragment encoding YGL1 was amplified by RT-PCR using the primer 5'-AACTGCAGAGTCTCCAATGGCCACCTC-3' and 5'-GG 14 ACTAGTGCTTTCATCAGTGGCTGGTT-3' from the wild type. The primers incorporated a PstI site at the N-terminal end and a SpeI site at the C-terminal end of the ORF.
PCR products were cloned into the pMD18-T vector (TaKaRa). Then the YGL1 cDNA fragment from wild type was digested with PstI and SpeI and ligated into the PstI and SpeI sites of a binary vector pCUbi1390 (Lu, unpublished data) harboring a hygromycin-resistant gene. The resulting pCUYGL1 plasmid, which contained the YGL1 coding sequence driven by the ubiquitin promoter, was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation, and transformed to japonica rice ygl2 for complementation testing according to a published method (Hiei et al., 1994).

Analysis of RT-PCR and Real-time PCR
Total RNA was extracted from leaves, leaf sheaths, young panicles and roots according to the method described by Wadsworth et al. (1988). cDNA synthesis was performed using 5μg total RNA for each sample. RNA was treated in 1X buffer with 5U of DNase I (MBI Fermentas) added to the reaction and incubated for 30min at 37°C. The reaction was stopped by adding 1μL of 25mM EDTA, followed by 10min incubated at 65°C. For reverse transcription (RT)-PCR, firststrand cDNA was reverse transcribed from total RNA with oligo (dT) and AMV reverse transcriptase (TaKaRa). Amplification of ygl1 and YGL1 cDNA (GenBank accession: EF432576) was carried out with specific primers (forward primer, 5'-CAGTCTCCAATGGCCACCT-3'; reverse primer 5'-TGCTTTCATCAGTGGCTGGT-3'). PCR products were cloned into the HindIII site at the C-terminal end of the ORF. The PCR products were inserted into pMD18-T vectors and sequenced to obtain the correct clones, pMDYGL1 and pMDygl1. The pMDYGL1 and pMDygl1 plasmids were then digested and cloned into the corresponding site of the bacterial expression vector pET28-a(+) (Novagen) to generate pETYGL1 and pETygl1, sequenced to confirm YGL1 and ygl1 sequences, respectively, then introduced into E. coli BL21 for protein expression. Protein expression and recombinant enzyme activity assays was according to the method as described by Schmid et al. (2001).

Pigment and Chlorophyll Precursor Determination
Total chlorophyll and carotenoids were determined with DU 800 UV/Vis Spectrophotometers (Beckman Coulter, Inc.) according to the method of Arnon (1949). Determination of ALA content was based on Richard methods (1975). The precursors, including Proto IX, Mg-Proto IX, Pchlide and Chlide, were assayed as described by Santiago-Ong (2001) and Masuda (2003a). Leaves (approximately 30mg fresh weight) of wild-type and ygl1 mutant were cut and homogenized in 5 mL 9:1 acetone:0.1 m NH4OH, and centrifuged at 3,000g for 10 min. The supernatants were combined and washed successively with an equal volume of hexane three times prior to spectrophotometric analysis. Chlorophyll precursors in the acetone phase were quantified with a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan) using Ex400:Em632 for Protoporphyrin IX, Ex440:Em633 for protochlorophyllide, Ex440:Em672 for chlorophyllide and Ex420:Em595 for Mg-protoporphyrin.

Transmission Electron Microscopy (TEM) analysis
Wild-type and ygl1 mutant leaf samples were harvested from one week and one-month-old plants grown in a greenhouse at medium light intensity (~150μmol photons m -2 s -1 ). Leaf sections were fixed in a solution of 2% glutaraldehyde and further fixed in 1% OsO4. Tissues were stained with uranyl acetate, dehydrated in ethanol, and embedded in Spurr's medium prior to thin sectioning. Samples were stained again and examined with a JEOL (Tokyo) 100 CX electron microscope.  Two BAC contigs (AC144742 and AC136221) cover the ygl1 locus. C, The ygl1 gene was narrowed down to an 11-kb genomic DNA region between the CAPS markers P23 and P8, and co-segregated with P25 and P26.