Mutation of the <i>Light-Induced Yellow Leaf 1</i> Gene, Which Encodes a Geranylgeranyl Reductase, Affects Chlorophyll Biosynthesis and Light Sensitivity in Rice

<div><p>Chlorophylls (Chls) are crucial for capturing light energy for photosynthesis. Although several genes responsible for Chl biosynthesis were characterized in rice (<i>Oryza sativa</i>), the genetic properties of the hydrogenating enzyme involved in the final step of Chl synthesis remain unknown. In this study, we characterized a rice <i>light-induced yellow leaf 1-1</i> (<i>lyl1-1</i>) mutant that is hypersensitive to high-light and defective in the Chl synthesis. Light-shading experiment suggested that the yellowing of <i>lyl1-1</i> is light-induced. Map-based cloning of <i>LYL1</i> revealed that it encodes a geranylgeranyl reductase. The mutation of <i>LYL1</i> led to the majority of Chl molecules are conjugated with an unsaturated geranylgeraniol side chain. <i>LYL1</i> is the firstly defined gene involved in the reduction step from Chl-geranylgeranylated (Chl_GG) and geranylgeranyl pyrophosphate (GGPP) to Chl-phytol (Chl_Phy) and phytyl pyrophosphate (PPP) in rice. <i>LYL1</i> can be induced by light and suppressed by darkness which is consistent with its potential biological functions. Additionally, the <i>lyl1-1</i> mutant suffered from severe photooxidative damage and displayed a drastic reduction in the levels of α-tocopherol and photosynthetic proteins. We concluded that <i>LYL1</i> also plays an important role in response to high-light in rice.</p></div

A working model of branched pathway starting from GGPP and Chlide to Chl_phy and α-tocopherol in rice.

<p>Geranylgeranyl reductase, encoded by the <i>LYL1</i>, generates PPP and Chl_phy using GGPP and Chlide as substrates. PPP is then directed into the tocopherol-synthesizing pathway.</p

Effects of different light density on the lipid peroxidation and ROS levels.

<p>The changes in MDA content (A), H₂O₂ content (B) and HO· level (C) between ZH11 and <i>lyl1-1</i> plants under low-light (LL, 100 µmol photonm m⁻² s⁻¹) and high-light (HL, 400 µmol photon m⁻² s⁻¹) conditions. Data presented are mean ±SD. NS =  No significant, ** Significant at the 0.01 level.</p

Expression analysis of rice <i>LYL1</i> gene.

<p>(A) Transcript levels of <i>LYL1</i> relative to <i>OsActin</i> in various tissues detected by quantitative real-time PCR. (B) Time-course of <i>LYL1</i> expression in response to light and dark conditions. About 3-week-old seedlings under dark, low-light (LL, 100 µmol photonm m⁻² s⁻¹) and high-light conditions (HL, 400 µmol photon m⁻² s⁻¹) at 27°C were used for expression analysis. T1, 12 hours of dark treatment; T2–T4, 3, 6 and 9 hours after the initiation of light treatment; T5–T8, 3, 6 and 9 hours after dark exposure.</p

HPLC analysis of the accumulation of Chl derivatives.

<p>The fluorescence intensity at 650(A) HPLC chromatograms of extracts from leaves of ZH11 (top) and <i>lyl1-1</i> (bottom). (B) HPLC chromatograms of extracts from leaves of Nipp (top) and <i>lyl1-2</i> (bottom). Peak 1, Chl <i>b</i>_GG; peak 2, Chl <i>b</i>_DHGG; peak 3, Chl <i>b</i>_THGG; peak 4, Chl <i>b</i>_phy; peak 5, Chl <i>a</i>_GG; peak 6, Chl <i>a</i>_DHGG; peak 7, Chl <i>a</i>_THGG; and peak 8, Chl <i>a</i>_phy. (C) The contents of Chl <i>a</i> derivatives in ZH11 and <i>lyl1-1</i>. (D) The contents of Chl <i>a</i> derivatives in Nipp and <i>lyl1-2</i>. (E) The contents of Chl <i>b</i> derivatives in ZH11 and <i>lyl1-1</i>. (F) The contents of Chl <i>b</i> derivatives in Nipp and <i>lyl1-2</i>. The extracts were isolated from the first, second and third leaves of ZH11, <i>lyl1-1</i>, Nipp and <i>lyl1-2</i> plants grown under natural conditions (high light). Data presented are mean ±SD. ND =  Not Detected. The Chl <i>b</i>_GG of ZH11 and Chl intermediates in Nipp were not detected. ** Significant at the 0.01 level.</p

HPLC analysis of tocopherols and tocotrienols.

<p>(A) HPLC chromatograms of reference standards. Peak 1, δ-tocotrienol; Peak 2, γ-tocotrienol; Peak 3, α-tocotrienol; Peak 4, δ-tocopherol; Peak 5, γ-tocopherol; Peak 6, α-tocopherol. (B) HPLC chromatograms of extracts from the leaves of ZH11 (top) and <i>lyl1-1</i> (bottom), respectively. (C) The contents of α-tocopherol in ZH11 and <i>lyl1-1</i> plants. (D) HPLC chromatograms of extracts from the leaves of Nipp (top) and <i>lyl1-2</i> (bottom), respectively. (E) The contents of α-tocopherol in Nipp and <i>lyl1-2</i> plants. The tocopherols were extracted from the first, second and third leaves of ZH11, <i>lyl1-1</i>, Nipp and <i>lyl1-2</i> plants grown under natural conditions (high light). Data presented are mean ±SD. ** Significant at the 0.01 level.</p

Phenotype of the rice <i>lyl1-1</i> mutant.

<p>(A) wild type ZH11 (left) and <i>lyl1-1</i> mutant (right) at the seeding stage. (B) ZH11 plants at tillering stage. (C) The <i>lyl1-1</i> plants at tillering stage. (D–G), The electron microscopic analysis of ZH11 and <i>lyl1-1</i> leaves. (D) and (E), The mesophyll cells of ZH11 and <i>lyl1-1</i> mutant. Bar  = 1 µm. (F) and (G), The chloroplasts of ZH11 and <i>lyl1-1</i> mutant. g, grana stack; p, plastoglobule; s, starch granule; sl, stroma lamellae. Bar  = 2 µm.</p

Map-based cloning of <i>LYL1</i>.

<p>(A) Rough mapping of the <i>LYL1</i> locus. <i>LYL1</i> is located between two markers, W243 and W226, on the long arm of chromosome 2. (B) Fine mapping of the <i>LYL1</i> locus. The <i>LYL1</i> gene is limited in a 33-kb genomic DNA region between markers W235 and W246, and it co-segregates with marker W247. Six candidate genes are located within this region in the Nipponbare genome, according to the TIGR Rice Genome Annotation Database. LOC_Os02g51080 is the candidate for <i>LYL1</i>. (C) <i>LYL1</i> gene structure at the genomic level. Three exons and the mutation positions are indicated. (D) Confirmation of the splice variation by analysis of the PCR amplicon size in the <i>lyl1-1</i> mutant and 22 rice cultivars. Lane 1, ZH11; lane 2, <i>lyl1-1</i>; lane 3-24, normal rice varieties, Zhongxian 3037, Tianegu, Wuxiangjing 3, Wuyunjing 8, Wuxiangjing 9, 9915, Nantehao, Wu 2661, Nanjing 46, Guandong 194, Nippobare, Guangluai 4, Balillar, Wuyunjing 7, 9516, 3015, Dular, 9311, Wujing 5, Miyang 23, Zhengdao 88 and Fengsizhan. (E) Phenotype of a 3-week-old RNAi transgenic plant. (F) Phenotype of a 2-month-old RNAi plant. (G) Comparison of Chl content between wild type Nipponbare and RNAi line. (H) Examination of <i>LYL1</i> expression level in the RNAi line by RT-PCR. <i>OsActin</i> was amplified as a control. (I) PCR and RT-PCR identification of the Tos17 insertion mutant <i>lyl1-2</i>. D1F was a primer derived from the Tos17 region. The D1R, D2F and D2R primers were derived from the genes examined. For RT-PCR analysis, <i>OsActin</i> was amplified as a control. (J) Phenotype of a 3-week-old <i>lyl1-2</i> mutant. (K) Comparison of Chl content between Nipp and <i>lyl1-2</i>. Data presented are mean ±SD. ** Significant at the 0.01 level.</p

Phylogenetic tree of green plant <i>LYL1</i> proteins.

<p>The numbers above the branches show bootstrap values for maximum likelihood and distance analysis, respectively. Asterisks indicate values lower than 50%. The locus name for each gene was from the database of Phyzotome (v9.0). A species acronym is included before the gene name: At, <i>Arabidopsis thaliana</i>; Bd, <i>Brachypodium distachyon</i>; Cr, <i>Chlamydomonas reinhardtii</i>; Gm, <i>Glycine max</i>; Mt, <i>Medicago truncatula</i>; Mg, <i>Mimulus guttatus</i>; Os, <i>Oryza sativa</i>; Pp, <i>Physcomitrella patens</i>; RC, <i>Ricinus communis</i>; Sb, <i>Sorghum bicolor</i>; Si, <i>Setaria italica</i>; Sm, <i>Selaginella moellendorffii</i>; Vc, <i>Volvox carteri</i>; Zm, <i>Zea mays</i>.</p

Effects of light intensity on leaf Chl content of ZH11 and <i>lyl1-1</i>.

<p>(A) Comparison of the total Chl content of ZH11 and <i>lyl1-1</i> plants exposed to different light intensities. (B) Comparison of the Chl <i>a</i> and Chl <i>b</i> contents of ZH11 and <i>lyl1-1</i> plants exposed to different light intensities. (C) Phenotype of ZH11 and <i>lyl1-1</i> leaves grown under high-light conditions for various periods of time (0, 6 and 12 days). Plants grown under low-light (LL, 100 µmol photonm m⁻² s⁻¹) at a 12 h photoperiod at 27°C for 30 days were transferred to high-light conditions (HL, 400 µmol photon m⁻² s⁻¹) at 27°C. The leaves were detached from the illuminated plants and photographed in water. (D) Light-shading experiment. Plants were initially grown under low-light conditions. A black integument covered in the leaf center to block out light before transfer to high-light conditions (up). The same leaves are shown on day 6 after high-light stress (down). The parts between the two dotted lines were covered by black integuments.</p