A lil3 chlp double mutant with exclusive accumulation of geranylgeranyl chlorophyll displays a lethal phenotype in rice

Background Phytyl residues are the common side chains of chlorophyll (Chl) and tocopherols. Geranylgeranyl reductase (GGR), which is encoded by CHLP gene, is responsible for phytyl biosynthesis. The light-harvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis. Results In this study, we isolated a yellow-green leaf mutant, 637ys, in rice (Oryza sativa). The mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains and displayed a yellow-green leaf phenotype through the whole growth period. The development of chloroplasts was suppressed, and the major agronomic traits, especially No. of productive panicles per plant and of spikelets per panicle, dramatically decreased in 637ys. Besides, the mutant exhibited to be sensitive to light intensity and deficiency of tocopherols without obvious alteration in tocotrienols in leaves and grains. Map-based cloning and complementation experiment demonstrated that a point mutation on the OsLIL3 gene accounted for the mutant phenotype of 637ys. OsLIL3 is mainly expressed in green tissues, and its encoded protein is targeted to the chloroplast. Furthermore, the 637ys 502ys (lil3 chlp) double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage. Conclusions We identified the OsLIL3 gene through a map-based cloning approach. Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.


Isolation and characterization of the 637ys mutant
In a previous study, we isolated the yellow-green leaf mutant 502ys from japonica cultivar Nipponbare (NP), which accumulated the Chls with unsaturated side chains, and was resulted from a point mutation causing an amino acid substitution G206S in OsCHLP (LOC_Os02g51080) gene [15]. Here, we obtained a new mutant 637ys from japonica cultivar ZH11 via EMS mutagenesis, which accumulated majority of Chls with unsaturated side chains as well as a small amount of Chl phy (about 10% of Chl phy in the wild type) (Additional file 1: Figure S1). The 637ys mutant displayed a yellow-green leaf phenotype through the whole growth period and grew at a very slow rate. The young leaves from leaf sheaths stayed green in 637ys, but rapidly turned yellow in several days (Fig. 1). Despite 19 days delay to heading compared to wild type ZH11, 637ys showed dramatic declines in major agronomic traits. For instance, while the wild type plants had an average on 7.2 of productive panicles, 637ys plants had only one to at most three, decreasing by 84.7%. The other agronomic traits, plant height, panicle length, No. of spikelets per panicle, seed setting rate, and 1000-grain weight declined by 38.6%, 28.2%, 80.7%, 42.7%, and 33.8% correspondingly (Table 1). To quantify the mutant phenotype of 637ys, we determined the contents of photosynthetic pigments in the 637ys and ZH11 plants at both seedling and heading stages. The amount of total Chl, Chl a, Chl b, and Caro in 637ys remarkably decreased by 50.6% to 58.2%, 47.4% to 57.3%, 63.4% to 61.4%, and 24.0% to 53.4%, respectively, compared to those in wild type (Fig. 2). These results suggested that the yellow-green leaf phenotype was due to its decreased level of photosynthetic pigments.
To explore if the reduced contents of photosynthetic pigments affect the development of chloroplasts in 637ys, we investigated the ultrastructure of chloroplasts under transmission electron microscopy. A number of grana stacks consisting of well-developed grana lamellae connected by stroma lamellae were present in wild type chloroplasts ( Fig.   3a, b). However, the chloroplasts were swollen in 637ys. Even if some grana stacks existed, the grana lamellae were less densely spaced than those in wild type and changed into disarray arrangement. Furthermore, stroma density decreased and osmiophilic globules occurred in the stroma in 637ys (Fig. 3c, d). These results revealed that the development of chloroplast was suppressed in the 637ys mutant.

Sensitivity of 637ys mutant to temperature and light intensity
To investigate if the mutant phenotype was dependent upon temperature, the 637ys and wild type plants grown in the growth chamber were treated by two different temperature conditions (constant 23 °C and 30 °C). As a consequence, the 637ys mutant grown under different temperature conditions exhibited indistinguishable leaf-color phenotype (Additional file 2: Figure S2 a1, a2). Its Chl contents significantly reduced, compared to wild type, but there was no obvious difference between low temperature and high temperature, which was similar to those in its wild type ( Fig. 4; Additional file 3: Table S1; Additional file 4: Table S2). These data suggested that the phenotype of 637ys was independent upon temperature.
All mutants accumulating the Chls with unsaturated side chains displayed sensitivity to light intensity [14,30]. Correspondingly, the phenotype of 637ys mutant under low light (80 μmol m -2 s -1 ) and high light (300 μmol m -2 s -1 ) was also investigated. The mutant displayed yellow-green leaf phenotype under high light condition (Additional file 2: Figure   S2 a1-b2). Meanwhile, its Chl contents significantly declined, compared to that under low light condition, while the Chl contents in wild type remained relatively stable ( Fig. 4; Additional file 5: Table S3). These data suggested that the phenotype of 637ys depended on light intensity.

Analysis of vitamin E in leaves and grains
Tocopherols and tocotrienols constitute vitamin E. The phytyl-PP forms the side chains of both Chl phy and tocopherols, and the GGPP forms the side chains of Chl GG and tocotrienols [4,5]. Because of the accumulation of Chls with unsaturated side chains in 637ys mutant, to investigate whether the composition of vitamin E was affected, we analyzed the tocopherol and tocotrienol compositions in leaves and grains in 637ys and its wild type ZH11 by HPLC. In leaves, HPLC profiles of vitamin E showed that α-tocopherol was abundant in wild type, while the elution peak of α-tocopherol in 637ys was much lower than that in wild type and significantly decreased by 89.2% (peak 1 in Fig. 5b, c; Fig. 5f).
At the same time, a small amount of γ-tocopherol was detected in the wild type leaves (peak 2 in Fig. 5b), but not in the 637ys mutant. It is noteworthy that a minor peak (peak 7 in Fig. 5c), whose retention time was 0.5 min fewer than the peak of γ-tocopherol in wild type, was detected in the 637ys mutant. We speculated that the minor peak in the 637ys was likely to be an isomer of γ-tocopherol [4]. In addition, tocotrienols in leaves were almost undetectable either in wild type or 637ys mutant (Fig. 5b, c). In grains, HPLC analysis showed that the 637ys mutant had few of α-tocopherol or γ-tocopherol declining by 90.9% and 89.7% respectively (peaks 1 and 2 in Fig. 5d, e; Fig. 5f), but considerable level of tocotrienols comparable to ZH11 (peaks 4, 5 and 6 in Fig. 5d, e; Fig. 5f; Additional file 6: Figure S3) [4,14]. These results indicated that tocopherols were deficient, but the accumulation of tocotrienols was not affected in 637ys.

Map-based cloning of the 637ys mutant gene
We crossed 637ys with 502ys ( chlp) mutant, and the resulting F 1 plants all displayed normal green phenotype, which indicated that 637ys and 502ys mutant genes are not allelic. In order to genetically analyze the 637ys mutant, we crossed 637ys with its wild type ZH11 and normal green indica cultivar G46B. All resulting F 1 plants exhibited a normal green phenotype. Leaf-color phenotypes of the F 2 populations segregated with a ratio of 3:1 (χ 2 <χ 2 0.05 =3.84, P>0.05), suggesting that a single recessive gene was responsible for the yellow-green leaf phenotype of 637ys.
Next, the F 2 population from the cross between 637ys and G46B was constructed for mapping. Preliminary mapping results suggested that the 637ys locus was linked with the SSR marker RM6641 on the short arm of Chromosome 2, and then we used 2 SSR markers and 3 InDel markers (Additional file 7: Table S4) to locate 637ys in a 334-kb region between SSR markers RM110 and RM7033 with 0.2 and 3.7 cM respectively (Fig 6a, b).
Within this region, we have further developed a total of 8 InDel and SSR markers which however showed no polymorphism between 637ys and G46B.
Although there are 62 putative genes within the 334-kb region according to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml), the genetic distance between the 637ys locus and RM110 is much shorter than that between 637ys and RM7033, which suggested that the 637ys locus should be close to RM110 (Fig. 6b, c). mutant and its wild type, and the results revealed that a G-to-A substitution occurred in position 368 of the gene (Fig. 6d). Furthermore, we sequenced cDNA of LOC_Os02g03330 in 637ys mutant and ZH11. Sequence alignment showed that the substitution occurred at the first nucleotide of the first intron in the DNA sequence of LOC_Os02g03330, and consequently caused 10 bp-intron sequence insertion in cDNA sequence of this gene in 637ys mutant (Fig. 6e). Thereby the reading frame shift in LOC_Os02g03330 resulted in premature translation of its encoded protein in 637ys (Additional file 8: Figure S4a).
Therefore, LOC_Os02g03330 was considered as the candidate gene of 637ys mutant, and designated as OsLIL3.
Searching in the rice genome database revealed that OsLIL3 is a single copy gene.
Alignment of sequenced DNA and cDNA showed OsLIL3 has three exons and two introns and its full length of genomic sequence and cDNA are 2384 bp and 753 bp, respectively.
The protein encoded by OsLIL3 comprises 250 of amino acids, which has a molecular weight of 27.6 kDa. The OsLIL3 contains a predicted chloroplast transit peptide of 44 amino acids at N-terminus (Additional file 8: Figure S4) Figure S5). According to multiple alignment of OsLIL3 and its homologues in different species, OsLIL3 has a high similarity to its homologues in monocotyledonous plants, barley (Hordeum vulgare), and maize (Zea mays) and dicotyledonous plants, cucumber (Cucumis sativus) and tobacco (Nicotiana tabacum), with 72%, 72%, 70% and 63% respectively. Phylogenetic analysis revealed that OsLIL3 is more closely related to its homologues from barley and maize than those from other species ( Fig. 7).

Complementation of the 637ys mutant
To confirm that the mutation of OsLIL3 caused the yellow-green leaf phenotype in 637ys mutant, we performed a complementation assay. The construct pC2300-OsLIL3 carrying OsLIL3 driven by rice Actin1 promoter was generated by inserting full length cDNA of and tocopherols in 637ys, from which we conclude that the mutant phenotype of 637ys was due to the single base pair mutation in the OsLIL3 gene.

Subcellular localization of OsLIL3 protein
OsLIL3 was predicted to contain a chloroplast transit peptide with 44 amino acid residues at its N-terminus by using TargetP and ChloroP (Additional file 8: Figure S4) [31,32]. In order to prove this prediction, we generated constructs expressing OsLIL3-green fluorescent protein (GFP) fusion protein, pCAMBIA2300-35s-OsLIL3-GFP, transformed rice protoplasts with the final construct and pCAMBIA2300-35s-GFP (as control) respectively, and observed transformed protoplasts under a laser-scanning confocal microscopy. In accordance with what was predicted by TargetP and ChloroP, the green fluorescence of OsLIL3-GFP fusion protein overlapped with the red autofluorescence of Chl in the chloroplasts, while GFP itself was expressed all over the whole cell (Fig. 9). These data provide strong evidence that OsLIL3 is chloroplast targeted.

Expression pattern of the OsLIL3 gene
To investigate where the OsLIL3 gene was expressed, we analyzed its level of transcripts in different tissues by qRT-PCR, including roots and leaf blades at seedling stage, stems, leaf blades, leaf sheaths, and young panicles of the wild type at both seedling stage and booting stage. The results demonstrated that the OsLIL3 was differentially expressed in different tissues. Specifically, leaf blades ranked the first, with leaf sheaths and young panicles followed, while stems and roots had relatively low levels of transcripts (Fig. 10).
The results indicated that OsLIL3 was mainly expressed in green tissues.

Expression analysis of genes at seedling stage for photosynthesis and Chl synthesis
Since the Chl compositions changed in the 637ys mutant, to investigate whether expressions of the genes associated with photosynthesis and Chl synthesis were affected, we examined transcript levels of 19 related genes and OsLIL3 at seedling stage in 637ys. 502ys double mutant plants were retained in the soil. Unlike 502ys or 637ys (Fig. 13b, c), only Chl GG a and Chl GG b were exclusively accumulated, and none of Chl DHGG , Chl THGG , or Chl phy was detectable in the double mutants by HPLC (Fig. 13d). To investigate the chloroplast development in the double mutants, we also observed the ultrastructure of chloroplasts under transmission electron microscopy. Compared to 637ys and 502ys (Fig.   3) [15], few of well-developed grana stacks existed in the double mutant (Additional file 10: Figure S6). Unfortunately, the double mutants all died at the three-leaf stage (Fig. 12).
In addition, we also investigated the 637ys 502ys double mutant grown in a growth chamber under low light at constant 23 °C, and obtained similar results of exclusive accumulation of Chl GG and lethal phenotype at the three-leaf stage (Additional file 11: Figure S7). These results suggested that the complete absence of Chl phy or only the presence of Chl GG in the double mutant could be fatal to rice seedling. predicted to contain 78 of wild-type plus 11 of new amino acids (Additional file 8: Figure   S4a). The 637ys mutant displayed a yellow-green leaf phenotype with a very slow growth rate and arrested chloroplast development. Correspondingly, the 637ys mutant accumulated about 10% of Chl phy in leaves, and 11.8% and 9.1% of α-tocopherol in leaves and grains respectively, equivalent to those of the wild type (Additional file 1: Figure S1; Fig. 5c, e, f). These data suggested that the function of the been confirmed to interact with OsGGR as well [41]. In the present study, the 502ys mutant was more comprehensively investigated to do a comparison analysis with 637ys.

Discussion
637ys showed similar characteristics with 502ys, including sensitivity to temperature and light intensity ( Fig. 4; Additional file 12: Figure S8), tocopherol and tocotrienol compositions in both leaves and grains ( Fig. 5; Additional file 13: Figure S9), and alteration in expression of 18 genes involved in photosynthesis and Chl synthesis ( Fig. 11; Additional file 14: Figure S10). Moreover, OsLIL3 and OsCHLP genes displayed a consistent expression pattern ( Fig. 10; Additional file 15: Figure S11). Together with the lack of substantial Chl phy and tocopherols in 637ys, these data suggested that OsLIL3 is of Consequently, it died at the three-leaf stage not only under natural sunlight (Figs 12, 13), but also under the low-light condition in the growth chamber (Additional file 11: Figure   S7). These data suggested that Chl phy is critical to plant growth and development, and the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.

Conclusions
In this study, we identified the OsLIL3 gene through a map-based cloning approach.
Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.

Plant materials
The plant materials used in this study were originally from our lab. The yellow-green mutants, 637ys and 502ys, were obtained from commonly used japonica cultivar Zhonghua

Transmission electron microscopy analysis
The fully expanded leaves of the wild-type ZH11 and the 637ys mutant were harvested from seedlings grown in the field under natural planting season at the three-leaf stage.
The second leaf of the 637ys 502ys double mutant was harvested when the third leaf started to emerge from the sheath. The treatment on leaf sections and observation of chloroplast ultrastructure followed the method described by Wang et al [38]. were quantified by using α-, γ-, and δ-tocopherol standards (Sigma).

Marker development
The information of SSR markers was acquired from Gramene database

Sequence analysis
The DNA and amino acid sequences of OsLIL3 and its homologues were acquired from

Complementation analysis of the 637ys mutant
For the complementation assay, the full-length cDNA sequence (753 bp) of OsLIL3 (LOC_Os02g03330) was amplified from the cDNA of wild-type ZH11 by using the primer set 5′-GGGTCTAGAATGGCCATGGCGACCTCC-3′ and 5′-CTCTGCAGCTATTTCTTGGGCTGAGAAG-3′, containing an Xba I site and a Pst I site, respectively. After treating with enzymes Xba I and Pst I, the amplified fragment was inserted into the binary vector pCAMBIA2300 to produce construct PC2300-OsLIL3 driven by the Actin 1 promoter. The pC2300-OsLIL3 some field experiments. CL and PW analyzed the experimental data and drafted the manuscript. PW and XD designed the experiments, supervised the study and revised the manuscript. All authors read and approved the manuscript. temperature treatments, in mg g fresh weight -1 Additional file 5: Table S3. Comparison of pigment contents in leaves of the 637ys and 502ys mutants and their wild-type ZH11 and Nipponbare between two different lightintensity treatments, in mg g fresh weight -1 Additional file 6: Figure S3. The peak area of tocotrienols in grains of ZH11 (WT) and 637ys. α-T3, γ-T3 and δ-T3 represent α-tocotrienol, γ-tocotrienol and δ-tocotrienol, respectively.
Additional file 7: Table S4. Insertion/deletion (InDel) markers used for mapping of the 637ys locus Additional file 8: Figure S4. Sequence alignment of OsLIL3 and its homologues.
Identical residues are boxed in black, and similar residues (≥75% identical) are and 502ys. α-T, α-tocopherol; γ-T, γ-tocopherol. The tocopherol standards were prepared as described in Figure 5. α-T3, γ-T3 and δ-T3 represent α-tocotrienol, γ-tocotrienol and δtocotrienol, respectively. Peaks 1 and 2 represent α-tocopherol and γ-tocopherol; Peak 3 is δ-tocopherol which does not exist in rice and was used as control. Peaks 4, 5 and 6 represent α-tocotrienol, γ-tocotrienol and δ-tocotrienol, respectively. Peak 7 might be the isomer of γ-tocopherol. Error bars represent standard errors of three independent biological replicates. Asterisks indicate statistically significant differences compared with the wild-type at P < 0.01.  Error bars represent standard deviations of three independent biological replicates. Asterisks indicate statistically significant differences (with Student's t test) compared with the wild-type at P < 0.01.  Data are shown as mean ± SD. Error bars represent standard deviations of three independent biological replicates. Asterisks indicate statistically significant differences (with Student's t test) compared with the wild-type at P < 0.01.       Expression analysis of genes involved in photosynthesis and Chl biosynthesis between the 637ys mutant and its wild-type ZH11. Actin 1 was amplified as an internal reference. The expression level of each gene in wild types was set to 1.0, and those in 637ys mutant were calculated accordingly. Error bars represent standard errors of three independent biological replicates. The asterisk indicates statistically significant differences (with Student's t test) compared with the wildtype at P < 0.05.  Chl composition analysis of the wild-type, the 637ys mutant, the 502ys mutant and the 637ys 502ys double mutant (DM). The elution profiles of Chls in wild-type ZH11 and Nipponbare (a), 637ys (b), 502ys (c) and DM (d) were detected at 660 nm by using HPLC. Peaks 2, 3, 4, and 5 represent Chlphy a, ChlTHGG a, ChlDHGG a, and ChlGG a, respectively. Peaks 1, 6 and 7 represent Chlphy b, ChlDHGG b,