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

Biological effect of lncRGMB-AS1 downregulation on cell proliferation in lung adenocarcinoma in vivo.

<p>(A, C) In mice injected with A549 cells, the luciferase signal was lower in the si-RNA nude mice than in the control groups at 2, 3, and 4 weeks, and the signal intensity became even lower in the si-RNA group with time (<i>P</i> < 0.05). (B, D) In mice injected with SPC-A-1 cells, the luciferase signal was lower in the si-RNA nude mice than in the control groups at 2, 3, and 4 weeks, and the signal intensity also became lower in the si-RNA group with time (<i>P</i> < 0.05). (E, F) Western blot analysis of Ki67 and RGMB expression in xenograft tissues from the three groups of nude mice. Compared with the control groups, Ki67 was downregulated and RGMB was upregulated in the si-RNA group (<i>P</i> < 0.05). si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; NC: cells that were transfected with negative control oligonucleotides, and Blank: cells that were not transfected. *Indicated statistical significance (<i>P</i> < 0.05).</p

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

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

Biological effect of lncRGMB-AS1 downregulation on cell cycle progression in A549 and SPC-A-1 cells.

<p>(A and B) Flow cytometric analysis showed a marked increase in the percentage of cells in the G0/G1 phase and a reduction in the percentage of cells in the S phase in the si-lncRNA group in A549 cells (<i>P</i><0.05), without significant changes in the NC and Blank groups (<i>P</i> > 0.05). (C and D) Flow cytometric analysis of SPC-A-1 cells showed a marked increase in the percentage of G0/G1 phase cells and a decrease the percentage of S phase cells in the si-lncRNA group (<i>P</i><0.05), without significant changes in the NC and Blank groups (<i>P</i> > 0.05). si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; NC: cells that were transfected with negative control oligonucleotides, and Blank: cells that were not transfected.</p

Potential mechanism of lncRGMB-AS1 in lung adenocarcinoma.

<p>(A) Exon1 and Exon2 of RGMB were amplified from human genomic DNA and inserted into the pEGFP-N3 vector with Hind III and BamHI to construct the recombination plasmids pEGFP-N3-Exon1 and pEGFP-N3-Exon2, respectively. The third exon of lncRNA RGMB-AS1 was also amplified from human genomic DNA and inserted into the pSilencer 3.1 vector with BamHI and Hind III to construct the recombination plasmid pSilencer-RGMB-AS1. (B) The fluorescence intensity of cells co-transfected with pEGFP-N3-Exon1 and pSilencer-RGMB-AS1 did not differ from that of cells transfected with pEGFP-N3-Exon1 only or cells co-transfected with pEGFP-N3-Exon1 and pSilencer 3.1 (<i>P</i> > 0.05). (C) The fluorescence intensity of cells co-transfected with pEGFP-N3-Exon2 and pSilencer-RGMB-AS1 was weaker than that of cells transfected with pEGFP-N3-Exon2 only or cells co-transfected with pEGFP-N3-Exon2 and pSilencer 3.1 (<i>P</i> < 0.05). The fluorescence intensity of the pEGFP-N3-Exon1 group was used as a control. *Indicates statistical significance (<i>P</i> < 0.05).</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

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