520YS , Encoding an Ankyrin Repeat Protein, is Involved in Chloroplast Development in Rice


 BackgroundThe ankyrin repeat (ANK) proteins are widely distributed in organisms ranging from viruses to plants, which play key roles in plastid differentiation, embryogenesis, chloroplast biogenesis and so on. However, only a few ANK genes have been identified in rice.ResultsIn this study, we isolated a yellow-green leaf mutant, 520ys, from japonica rice cultivar Nipponbare through ethyl methane sulfonate mutagenesis. The mutant exhibited a yellow-green leaf phenotype throughout the life cycle, arrested development of chloroplasts, reduced levels of photosynthetic pigments, and accumulated reactive oxide species. Map-based cloning suggested that the candidate gene was LOC_Os07g33660, which encodes an expressed protein containing one ankyrin repeat and showing sequence similarity with the Arabidopsis LTD/GDC1 (At1g50900). Transgenic complementation experiment confirmed that LOC_Os07g33660 is the causal gene for the mutant type of 520ys. 520YS (LOC_Os07g33660) is mainly expressed in green tissues and its encoded protein is targeted to the chloroplast. In 520ys mutant, expression levels of four light-harvesting chlorophyll a/b-binding protein translocation-related genes and eight photosynthesis-related genes were significantly down-regulated.ConclusionWe characterized a novel ANK gene, 520YS, which plays a key role in chloroplast development in rice.


Abstract Background
The ankyrin repeat (ANK) proteins are widely distributed in organisms ranging from viruses to plants, which play key roles in plastid differentiation, embryogenesis, chloroplast biogenesis and so on. However, only a few ANK genes have been identi ed in rice.

Results
In this study, we isolated a yellow-green leaf mutant, 520ys, from japonica rice cultivar Nipponbare through ethyl methane sulfonate mutagenesis. The mutant exhibited a yellow-green leaf phenotype throughout the life cycle, arrested development of chloroplasts, reduced levels of photosynthetic pigments, and accumulated reactive oxide species. Map-based cloning suggested that the candidate gene was LOC_Os07g33660, which encodes an expressed protein containing one ankyrin repeat and showing sequence similarity with the Arabidopsis LTD/GDC1 (At1g50900). Transgenic complementation experiment con rmed that LOC_Os07g33660 is the causal gene for the mutant type of 520ys. 520YS (LOC_Os07g33660) is mainly expressed in green tissues and its encoded protein is targeted to the chloroplast. In 520ys mutant, expression levels of four light-harvesting chlorophyll a/b-binding protein translocation-related genes and eight photosynthesis-related genes were signi cantly down-regulated.

Conclusion
We characterized a novel ANK gene, 520YS, which plays a key role in chloroplast development in rice.

Background
Ankyrin repeat (ANK) is one of the most common protein domains and is widely distributed in organisms ranging from viruses to plants (Sedgwick and Smerdon 1999). The primary structure of ANK consists of an approximately 33-residue motif that is repeated in tandem to build speci c secondary and tertiary structures (Mosavi et al. 2002). This protein domain was rst identi ed in the two yeast cell-cycle regulators Swi6 and Cdc10 and in the Drosophila signalling protein Notch (Breeden and Nasmyth 1987a, b). In Arabidopsis thaliana, 105 ANK proteins have been discovered and classi ed into 12 subgroups (Becerra et al. 2004). In rice (Oryza sativa) and tomato (Solanum lycopersicum), 175 and 130 ANK genes have been identi ed and classi ed into 10 and 13 subfamilies respectively (Huang et al. 2009;Yuan et al. 2013). Recently, 87, 85 and 96 ANK genes have been reported in C. baccatum, C. annuum and C. chinense genomes, respectively (Lopez-Ortiz et al. 2020).
ANK proteins usually function in a number of important biological processes in plants, such as plastid differentiation, chloroplast biogenesis, embryogenesis, leaf morphogenesis, pollen germination and pollen tube growth, and so on (Zhang et al. 1992;Albert et al. 1999;Ha et al. 2004;Garcion et al. 2006;Huang et al. 2006;Bae et al. 2008). Arabidopsis AKR was the rst ANK protein reported in higher plants, whose loss of function blocks chloroplast differentiation and causes a chlorotic phenotype (Zhang et al. 1992). BOP1 encodes a BTB/POZ domain protein with ankyrin repeats, and is required for leaf morphogenesis (Ha et al. 2004). LIANK, a Lily ANK gene, is indispensable for pollen germination and pollen tube growth (Huang et al. 2006). LTD encodes an ANK protein, which is involved in both facilitating light-harvesting chlorophyll a/b-binding proteins (LHCP) translocation across the inner envelope and subsequently delivering them to the chloroplast signal recognition particle (cpSRP) pathway in Arabidopsis chloroplasts (Ouyang et al. 2011).
In addition, ANK proteins play key roles in the response to biotic and abiotic stresses. Arabidopsis ANH protein AKR2 was identi ed as an interacting partner of GF14λ (G-box factor 14-3-3 homolog), and plays a role in both disease resistance and antioxidation metabolism. Antisense AKR2 in plants exhibits the necrosis phenotype (Yan et al. 2002). AKR2A (AKR2) was further found to function as a molecular chaperone for APX3 (ascorbate peroxidase3), a major H 2 O 2 -degrading enzyme, by forming an AKR2A-APX3 protein complex (Shen et al. 2010). Rice XB3 (XA21-binding protein3) containing an ANK domain interacs with XA21 and is required for full accumulation of the XA21 protein and for Xa21-mediated resistance to Xanthomonas oryzae (Wang et al. 2006). OsBIANK1 gene, encoding a protein containing a typical ANK domain, may be involved in regulation of disease resistance response in rice (Zhang et al.

2010).
So far, only a few ANK genes have been characterized in rice, such as XB3, OsBIANK1, OsNPR1/OsNH1 and OsPIANK1, which are all involved in disease resistance (Wang et al. 2006;Huang et al. 2009;Zhang et al. 2010;Sugano et al. 2010;Mou et al. 2013). In this study, we characterized a novel ANK gene, 520YS (LOC_Os07g33660), by using a yellow-green leaf mutant 520ys in rice. 520YS encoded an expressed protein containing one ankyrin repeat. Transgenic complementation experiment con rmed that LOC_Os07g33660 is responsible for mutant phenotype of 520ys. Furthermore, we examined expression changes of the 12 genes associated with LHCP translocation and photosynthesis in 520ys mutant and its wild type. Our data suggested that the ANK gene 520YS plays an important role in chloroplast development in rice.

Characterization of the 520ys mutant
The 520ys mutant exhibited yellow-green leaf phenotype throughout development (Fig. 1). Its plants grew slower, and days to heading were delayed. At maturity, its number of productive panicles per plant, seedsetting rate and 1000-grain weight were signi cantly decreased by 20.0%, 10.9% and 5.4% respectively, and the plant height decreased by 3.6% (Table 1). However, its panicle length and number of spikelets per panicle were not affected signi cantly.
To characterize effect of the yellow-green phenotype of 520ys on its photosynthetic pigments, we measured the pigment levels in the leaves of the mutant and its wild-type plants. At the seedling stage, contents of total chlorophyll (Chl), Chl a, Chl b, and carotenoids in the mutant were signi cantly reduced by 57.9%, 52.5%, 79.5% and 29.3% respectively, relative to those in the wild type. At the heading stage, contents of these pigments further decreased by 63.7%, 59.5%, 82.9% and 59.2% respectively ( Table 2).
The results suggested that the mutant phenotype of 520ys resulted from remarkable reduction of photosynthetic pigment levels.
Subsequently, we also observed the ultrastructure of chloroplasts in 520ys and its wild type at seedling stage by transmission electron microscopy. As shown in Fig. 2, thylakoid membrane structures were obvious, and grana stacks were dense and ordered in the wild-type chloroplasts. By contrast, in the 520ys chloroplasts, thylakoid membrane structures were disordered, and no obvious grana stack was observed.
Besides, many vesicular structures occurred in the mutant chloroplasts. The data suggested that the mutantion in 520ys arrested the development of rice chloroplasts.

Temperature sensitivity of the 520ys mutant
To investigate whether the yellow-green leaf phenotype of 520ys was dependent on temperature, the 520ys mutant and its wild type were grown in two different temperature conditions (23 ℃ and 30 ℃) in the growth chamber, respectively. As shown in Fig. 3, 520ys displayed similar yellow-green leaf phenotypes under different temperature conditions. On the other hand, pigment analysis showed that contents of total Chls, Chl a, Chl b and carotenoids in 520ys were signi cantly reduced by 70.4%, 66.4%, 89.4%, and 55.0% under 23℃ condition, and reduced by 63.7%, 59.5%, 82.9%, and 59.2% under 30℃ condition (Table 3), respectively, compared with those in the wild type. The results indicated that the leafcolor phenotype of 520ys was insensitive to temperature.

ROS accumulation in 520ys leaves
Besides variegation leaves, pale green leaves could also accumulate excessive ROS (Sakuraba et al. 2013). To investigate whether 520ys had ROS accumulation, we examined the levels of hydrogen peroxide (H 2 O 2 ) and superoxide anion radicals (O 2 -) in leaves of 520ys seedling by staining with DAB and NBT, respectively. As a result, the brown spots shown by staining with DAB were not obvious on leaves of both 520ys and wild type (Fig. 4). However, there were many blue spots shown by staining with NBT on the 520ys leaf, by contrast, only few blue spots on the wild type leaf, suggesting that the accumulation level of superoxide anion radicals was signi cantly higher in 520ys than that in wild type.

Map-based cloning of 520ys gene
For genetic analysis of the mutant, 520ys was crossed with the wild-type Nipponbare. All F 1 plants displayed normal green leaf phenotype. The F 2 population was separated into two groups, the normal green leaf plants and the yellow-green leaf plants, with a ratio of 3:1 (χ 2 = 0.55 χ 2 0.05 = 3.84, P 0.05; Table S1). The result suggested that the mutant phenotype of 520ys is controlled by a single recessive nuclear gene.
Subsequently, 520ys was crossed with normal green indica cv Minghui 63, and the F 2 population was used to mapping of the 520YS locus. With the proportionally spaced SSR markers which scattered on rice chromosomes, 520ys gene was preliminarily mapped on the long arm of chromosome 7 between SSR markers RM11 and RM336 (Fig. 5a). For ne mapping, we developed four polymorphic insertion/deletion (InDel) markers (Table 4), and analyzed 832 recessive individuals with a yellow-green leaf phenotype from the F 2 population. Finally, the 520ys locus was narrowed down to a 168-kb region between InDel maker X3 and SSR marker RM21725, at genetic distances of 0.6 and 0.4 cM (Fig. 5b), respectively The 168-kb region harbors 17 putative genes according to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu, Fig. 5c). So we sequenced these genes in the 520ys mutant and its wild type, and found that 520ys only contains a single-base G-to-A substitution at position 1158 in LOC_Os07g33660 (Fig. 5d). Next, we sequenced cDNA of LOC_Os07g33660 gene and con rmed the substitution at codon 415 in its coding sequence, which resulted in an amino acid change from Ala139 to Thr in the encoded protein. This gene encodes an expressed protein, which is predicated to contains a chloroplast transit peptide of 28 amino acid residues at its N terminus (http://www. cbs.dtu.dk/services/TargetP/) and one ankyrin repeat between residues 114 and 146 (Fig. 6a). Furthermore, the mutated amino acid residue is located on the ankyrin repeat and highly conserved in the 520ys mutant (Fig. 6a, Mosavi et al. 2002). Therefore, LOC_Os07g33660 was identi ed as the candidate gene of 520ys mutant, and de ned as 520YS gene.
The sequencing results of DNA and cDNA indicated that LOC_Os07g33660 has two exons and one intron, and its full length of genomic sequence and cDNA are 1696 bp and 519 bp, respectively. LOC_Os07g33660 encodes an 172-amino acid protein, with a molecular mass of approximately 18 kD. BLASTP search in the Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org) showed that LOC_Os07g33660 has high similarity to AT1G50900 (GDC1/LTD, 68% identities), which is essential for grana formation in Arabidopsis. Multiple amino acid sequence alignment revealed that this protein has similarity to its homologs from monocotyledonous plants such as Setaria italica, Sorghum bicolor, Zea mays, Hordeum vulgare, with identities of 80.1%, 77.1%, 77.4%, and 73.6%, and dicotyledonous plants such as Gossypium mustelinum, Arachis hypogaea, Glycine max, Momordica charantia, with identities of 64.1%, 63.9%, 62.3% and 61.7%, respectively. Moreover, phylogenetic analysis suggested that LOC_Os07g33660 is more closely related to the proteins from monocotyledon plants than those of other species (Fig. 6b).

Complementation analysis
To con rm that LOC_Os07g33660 was causal gene for the yellow-green phenotype of 520ys, we performed a complementation assay. The expression vector pCAMBIA2300-520YS containing the rice Actin1 promoter and the full-length cDNA sequence of wild-type LOC_Os07g33660 gene was transformed into 520ys mutant mediated by Agrobacterium, and 11 independent transgenic lines were obtained. Among these, nine lines were PCR-positive (Fig. 7c) and showed normal green phenotype (Fig. 7a, b), while other two lines were PCR-negative and showed yellow-green phenotype. Meanwhile, plant growth and photosynthetic pigment contents of the nine PCR-positive lines were restored nearly to those of wild type (Fig. 7a, b, d). Therefore, the yellow-green leaf phenotype of 520ys was caused by the mutation in the 520YS (LOC_Os07g33660) gene.
520YS protein was localized to the chloroplast TargetP analysis (http://www.cbs.dtu.dk/services/TargetP/) showed that 520YS protein contains a chloroplast transit peptide of 28 amino acid residues at its N terminus (Fig. 6a). To verify the actual intracellular localization, we constructed the expression vector pCAMBIA2300-520YS-GFP, carrying the encoding sequence of 520YS along with the GFP reporter gene under control of the 35S promoter. The resulting plasmid pCAMBIA2300-520YS-GFP and the empty eGFP vector were transformed into rice protoplasts (as negative control), respectively. As shown in Fig. 8a, the green uorescent signals of 520YS-eGFP fusion proteins were clearly co-localized with chlorophyll auto uorescence in the chloroplasts, while GFP itself was expressed all over the whole cell (Fig. 8b). The results indicated that the 520YS protein is localized to the chloroplast.
Expression analysis of 520YS and the 12 genes associated with LHCP-translocation and photosynthesis To investigate the spatiotemporal expression pattern of 520YS gene, we analyzed its level of transcripts in different tissues of the wild type by qRT-PCR at the seedling stage and the booting stage. The result showed that 520YS was expressed in root, leaves, stem, leaf sheaths and young panicle (Fig. 9a). However, expression levels of 520YS had diversity in different tissues. More speci cally, leaf blades had the highest expression at both stages, followed by leaf sheaths and young panicles, stems and roots had lower expression levels at the booting stage.
In addition, we examined transcriptional changes of the 12 related genes. Among them, four genes are involved in LHCP translocation in the chloroplast (Ouyang et al. 2011), including Tic110 and Tic40 (encoded translocon at the inner envelop membrane of chloro plast), and cpSRP43 and cpSRP54 (encoded chloroplast signal recognition particle). The other eight genes are associated with photosynthesis (Matsuoka et al. 1990;Kyozuka et al. 1993;Cui et al. 2011), including CAB1R, CAB2R, Lhcb2.1 and Lhcb3 (encoded light harvesting Chl a/b binding proteins of PSII), psaA and psbA (encoded reaction center subunits of PSI and PSII), rbcL and rbcS (encoded Rubisco large subunit and small subunit respectively). qRT-PCR analysis revealed that expression levels of the detected genes were all down-regulated signi cantly. Among them, except for rbcS, other genes very remarkably down-regulated (Fig. 9b). The results suggested that the mutation of 520ys gene inhibits the transcription levels of the genes associated LHCP translocation and photosynthesis.

Discussion
Rice genome contains many ANK genes. However, only a few ANK genes have been characterized, and they are all involved in disease resistance (Huang et al. 2009;Wang et al. 2006;Zhang et al. 2010;Sugano et al. 2010;Mou et al. 2013). In this study, we isolated a mutant, 520ys, in rice. The mutant displayed yellow-green leaves and arrested chloroplast development. Map-based cloning revealed that the candidate gene 520YS (LOC_Os07g33660) encoded an expressed protein containing one ankyrin repeat. A single-base G-to-A substitution occurred at its coding sequence in the 520ys mutant, resulting in an amino acid change in its encoded protein. Furthermore, the yellow-green leaf phenotype of 520ys was rescued by complementation with the wild-type 520YS gene, con rming that 520YS was responsible for the mutant phenotype. Therefore, we successfully identi ed a novel ANK gene involved in chloroplast development in rice.
520YS has sequence similarity with Arabidopsis At1g50900 gene. In the T-DNA insertion mutants of At1g50900, the ltd mutant showed yellow leaves, retarded growth, and impaired grana stacking in its chloroplasts. ltd seedlings only could grow on sucrose-supplemented medium and did not develop fertile ow ers (Ouyang et al. 2011). The gdc1 mutant could survive for approximately 5 to 6 weeks with a pale green phenotype on soil. It grew much slower and ceased at the vegetative growth stage before bolting. In the knockout line of gdc1-3, only stromal thylakoids were observed in the chloroplasts, and they could not stack together to form appressed grana (Cui et al. 2011). Sequencing of the TAIL-PCR products suggested that T-DNA insertion sites were located near/in ankyrin repeat in both ltd and gdc1-3 mutants. In the present study, a single amino acid of 520YS changed from Ala139 to Thr in the 520ys mutant, which was also located in the ankyrin repeat, and furthermore, this amino acid residue was highly conserved (Fig 6a; Mosavi et al. 2002). 520ys mutant exhibited a yellow-green leaf phenotype throughout development, grew slower, reduced photosynthetic pigment contents, and no grana stacking in the chloroplasts. The results suggested that the ankyrin repeat in 520YS plays an important role in grana formation and chloroplast development in rice, which is similar with LTD/GDC1 in Arabidopsis. In addition, the mutant phenotype of 520ys was less severe than those of Arabidopsis ltd and gdc mutants, implying that the single nucleotide substitution could not completely abolish the gene function in 520ys.
In the ltd mutant of Arabidopsis, immunoblot analysis showed that LHCPs, including Lhcb1, Lhcb2, Lhca1 and Lhca2, were signi cantly decreased, compared with those in wild type, but Tic110, Tic40, cpSRP43 and cpSRP54 accumulated rather normally or were increased (Ouyang et al. 2011). In the gdc1-3 mutant, Arabidopsis protein Lhcb1 and Lhcb2 levels were reduced, but the transcription levels of them were not different (Cui et al. 2011). In this study, the transcript levels of photosynthesis-and LHCP translocation-associated genes, including Tic40, Tic110, cpSRP43, cpSRP54, CAB1R, CAB2R, Lhcb2.1, Lhcb3, PsaA, PsbA, rbcL and rbcS, were all down-regulated signi cantly in the 520ys mutant. The expression differences of these genes between the above Arabidopsis and rice mutants may be caused by different functional damage of the proteins, which might result in the damage of the thylakoid membrane structures to different extent in ltd, gdc1-3 and 520ys mutants.

Conclusions
520YS encodes an expressed protein containing one ankyrin repeat, and change of a single amino acid in the ankyrin repeat resulted in yellow-green leaf phenotype and arrested chloroplast development in 520ys mutant, suggested that the ankyrin repeat play a key role in rice grow and development.

Plant materials
The yellow-green leaf mutant, 520ys, was isolated from japonica cultivar Nipponbare, through ethyl methane sulfonate (EMS) mutagenesis. For genetic analysis and gene mapping of 520ys, the F 2 populations were generated by crossing the 520ys mutant with its wild-type Nipponbare and normal green indica cultivar Minghui 63, respectively. Rice materials were grown in the paddy eld during the normal rice growing seasons in experimental farms of Sichuan Agricultural University, Chengdu City, China (Wang et al. 2010).

Photosynthetic pigment measurement
Pigments were extracted from 0.2 g fresh rice leaves with 80% acetone in darkness for 48 h at 4°C. The contents of chlorophyll (Chl) and Carotenoid (Caro) were measured with UV-1700UV-visible spectrophotometer (shimadzu) at 663nm, 646nm and 470nm, and were calculated according to the method of Lichtenthaler and Wellburn (1983).

Transmission electron microscopy analysis
The fully expanding leaves of 520ys mutant and its wild-type Nipponbare were harvested at the seedling stage, respectively. Leaf sections were xed, dehydrated, embedded, sectioned, and stained according to the previous method (Wang et al. 2010). Finally, the ultrastructure of mesophyll cells and chloroplasts was observed using H-600IV transmission electron microscope (Hitachi).

Complementation of the 520ys mutant
To create the complementation construct for transgenic plants, the wild type full-length cDNA sequence of 520YS (LOC_Os07g33660) was ampli ed by RT-PCR, using the primers 5'-GATCTAGAATGGCATCCATCCCGTGCAC-3' and 5'-AGCCTGCAGTCAGGCGGCCAAGGTGGCG-3'. The primers incorporated XbaI site at the 5'-end and a PstI site at the 3'-end of the ORF. The PCR products were digested with XbaI and PstI and inserted into the corresponding site of the binary vector pCAMBIA2300. The pCAMBIA2300-520YS plasmid under the control of the rice Actin1 promoter was introduced into the 520ys mutant by Agrobacterium tumefaciens-mediated transformation. The transgenic plants were detected with the primers 5'-ATGGCATCCATCCCGTGC -3' and 5'-GCGATCATAGGCGTCTCG-3', which were located on the rice 520YS gene and pCAMBIA2300 vector, respectively.

RT-PCR analysis
Total RNA from both wild-type and 520ys plants were isolated using the TRIzol kit (CWBIO), and cDNA was made using the rst strand cDNA synthesis kit (TOYOBO) following the manufacturer's instructions. Real-time PCR was carried out using the CFX96 real-time PCR system (Bio-Rad) with the SYBR Premix Ex 55 o C for 30 s. For each sample, qRT-PCR was performed with three technical replicates on each of three biological replicates, and the rice actin 1 gene was used for normalization as an internal control. All qRT-PCR primers are listed in Table S2.

Subcellular localization of GFP proteins
The full length cDNA sequence encoding 520YS protein was ampli ed from the wild-type Nipponbare by RT-PCR with the primers 5'-GAGGTACCATGGCATCCATCCCGTGCAC-3' and 5'-AGCTCTAGACGAGGCGGCCAAGGTGGCG-3'. The primers incorporated a KpnI site at the 5'-end and an XbaI site at the 3'-end of the ORF. The PCR products were inserted into vector pCAMBIA2300-35S-eGFP to generate construct pCAMBIA2300-35S-520YS-eGFP. Next, the plasmid pCAMBIA2300-35S-520YS-eGFP and empty vector pCAMBIA2300-35S-eGFP were introduced into normal rice protoplasts, respectively, according to previously described procedures (Zhang et al. 2011). GFP uorescence was observed using a laser-scanning confocal microscope after being incubated overnight in the dark (NikonA1).
Declarations Sugano S, Jiang CJ, Miyazawa SI, Masumoto C, Yazawa K, Hayashi N, Shimono M, Nakayama A, Miyao M, Takatsuji H (2010) Figure 1 Phenotypic comparison of the 520ys mutant and its wild-type Nipponbare (WT). a Seedlings at the seedling stage. b Plants at the grain lling stage.

Figure 2
Ultrastructure of chloroplasts of 520ys and its wild-type at the seedling stage. a and c Chloroplasts in the wild-type leaves. b and d Chloroplast in the mutant 520ys leaves. Bars = l μm.

Figure 3
Phenotypic characterization of the 520ys mutant and its wild type (WT) grown in the growth chamber under different temperature conditions. a and b Seedlings grown for two-week under 12 h light/12 h darkness at constant 23°C and 30°C, respectively.

Figure 4
Superoxide anion radicals (O2-) and hydrogen peroxide (H2O2) in 520ys leaves. The accumulation of superoxide anion radicals was visualized by staining with NBT, and no obvious accumulation of hydrogen peroxide was visualized by staining with DAB.

Figure 5
Map-based cloning of the 520YS locus. a The 520ys locus was preliminarily located on the long arm of chromosome 7 (Chr.7) between SSR markers RM11 and RM336. b The 520ys locus was narrowed to a genomic region between InDel marker X3 and SSR marker RM21725. c 17 putative ORFs have been annotated in this 168-kb region. d Candidate gene LOC_Os07g33660 consists of two exons and one intron. The mutation site (G1158A) is indicated by the arrow head.

Figure 6
Sequence analysis of 520YS. a Sequence alignment of 520YS and its homologues. Identical residues were boxed in dark blue, similar residues (≥75% identical) were highlighted in gray. The cleavage site for the putative chloroplast-targeting sequence is marked by a black arrowhead. The ankyrin repeat (ANK) is indicated with the overline.The Ala-139 to Thr change in the 520ys mutant is indicated by an inverted triangle. b Phylogenetic analysis of 520YS and its homologues. The rooted tree using percentage identities is based on a multiple sequence alignment generated with the program DNAMAN. Scale represents percentage substitution per site. Gen Bank accession numbers for the respective protein sequences are as follows: Oryza sativa (520YS, LOC_Os07g33660, 4343402); Sorghum bicolor (XP_002460789.1); Zea mays (NP_001144531.1); Hordeum vulgare (KAE8808993.1); Arabidopsis thaliana (At1g50900); Gossypium mustelinum (TYI96610.1); Arachis hypogaea (XP_025693353.1); Glycine max (NP_001236074.2); Momordica charantia (XP_022150983.1).

Figure 7
Complementation of the 520ys mutant by the wild-type gene. a and b Phenotypes of wild type (WT), 520ys and PCR-positive transgenic plants (520ys-C)) at the seedling stage and at the grain-lling stage, respectively. c Identi cation of transgenic plants by PCR. M, D5000 marker; 1, pC2300-520YS plasmid (positive control); 2, 520ys (negative control); 3-5, positive transgenic plants. d Pigment contents in leaves of wild type (WT), 520ys and 520ys-C at seedlings stage. * Signi cantly different at P = 0.01.  Expression analysis of 520YS. a The expression pattern of 520YS in root (R), leaf (L), stem (S), leaf sheath (LS), and young panicle (P) of the wild-type plants at the seedling stage and the booting stage, respectively. b Expression analysis of the LHCP translocation and photosynthesis associated genes by real-time PCR. Total RNA was extracted from 4-week-old plants. The expression level of each gene in wild types was set to 1.0, and those in 520ys mutant were calculated accordingly. Actin 1 was amplified as a