CHR729 Is a CHD3 Protein That Controls Seedling Development in Rice

<div><p>CHD3 is one of the chromatin-remodeling factors that contribute to controlling the expression of genes associated with plant development. Loss-of-function mutants display morphological and growth defects. However, the molecular mechanisms underlying CHD3 regulation of plant development remain unclear. In this study, a rice CHD3 protein, CHR729, was identified. The corresponding mutant line (<i>t483</i>) exhibited late seed germination, low germination rate, dwarfism, low tiller number, root growth inhibition, adaxial albino leaves, and short and narrow leaves. <i>CHR729</i> encoded a nuclear protein and was expressed in almost all organs. RNA-sequencing analysis showed that several plant hormone-related genes were up- or down-regulated in <i>t483</i> compared to wild type. In particular, expression of the gibberellin synthetase gibberellin 20 oxidase 4 gene was elevated in the mutant. Endogenous gibberellin assays demonstrated that the content of bioactive GA₃ was reduced in <i>t483</i> compared to wild type. Moreover, the seedling dwarfism, late seed germination, and short root length phenotypes of <i>t483</i> were partially rescued by treatment with exogenous GA₃. These results suggest that the rice CHD3 protein CHR729 plays an important role in many aspects of seedling development and controls this development via the gibberellin pathway.</p></div

Expression analysis of <i>CHR729</i> and subcellular localization of the encoding protein.

<p>A, qRT-PCR analysis of <i>CHR729</i> expression in WT roots (R), culms (C), leaves (L), leaf sheaths (Ls), and young 3 cm inflorescence (In). Dissected anthers (An), pistils (Pi), lemmas (Le) and paleas (Pa) at inflorescence stage 9 were also analyzed. Values are means ±SD of three replicates. B–D, <i>CaMV35S</i>:CHR729-GFP fusion protein localization in rice protoplast. B, Subcellular localization of CHR729-GFP fusion protein. C, Subcellular localization of MADS3-mCherry fusion protein (nuclear marker). D, Merged image of (B) and (C) in bright field. Scale bars: 10 μm (B–D).</p

Seed germination of WT and <i>t483</i>.

<p>A, Germination of WT and <i>t483</i> seeds after 3 days. B–E, Seedlings of WT and <i>t483</i> at 8 days post-germination. F, Seed germination rate. Values are means ±SD of three independent experiments. Significance of differences between WT and <i>t483</i> was determined by Student’s <i>t-</i>test (**<i>P</i><0.01). Scale bars: 2 cm (A–D); 1 cm (E).</p

Factors Affecting the Radiosensitivity of Hexaploid Wheat to γ-Irradiation: Radiosensitivity of Hexaploid Wheat (<i>Triticum aestivum</i> L.)

<div><p>Understanding the radiosensitivity of plants, an important factor in crop mutation breeding programs, requires a thorough investigation of the factors that contribute to this trait. In this study, we used the highly radiosensitive wheat (<i>Triticum aestivum</i> L.) variety HY1 and J411, a γ-irradiation-insensitive control, which were screened from a natural population, to examine the factors affecting radiosensitivity, including free radical content and total antioxidant capacity, as well as the expression of <i>TaKu70</i> and <i>TaKu80</i> (DNA repair-related genes) as measured by real-time PCR. We also investigated the alternative splicing of this gene in the wild-type wheat ecotype by sequence analysis. Free radical contents and total antioxidant capacity significantly increased upon exposure of HY1 wheat to γ-irradiation in a dose-dependent manner. By contrast, in J411, the free radical contents exhibited a similar trend, but the total antioxidant capacity exhibited a downward trend upon increasing γ-irradiation. Additionally, we detected dose-dependent increases in <i>TaKu70</i> and <i>TaKu80</i> expression levels in γ-irradiated HY1, while in J411, <i>TaKu70</i> expression levels increased, followed by a decline. We also detected alternative splicing of <i>TaKu70</i> mRNA, namely, intron retention, in HY1 but not in J411. Our findings indicate that γ-irradiation induces oxidative stress and DNA damage in hexaploid wheat, resulting in growth retardation of seedlings, and they suggest that <i>TaKu70</i> may play a causal role in radiosensitivity in HY1. Further studies are required to exploit these factors to improve radiosensitivity in other wheat varieties.</p></div

Map-based cloning and confirmation of the <i>CHR729</i> gene.

<p>A, Map of the genomic region containing the <i>t483</i> mutant locus of interest. Numerals below the corresponding markers indicate the number of recombinants identified among F₂ plants with the mutant phenotype mutant. The mutated gene was located in a 27 kb region between markers IN27 and IN35. Three ORFs were predicted in the mapped region. Sequencing analysis revealed that an A to T substitution in the fifth exon of the ORF3 resulted in a stop codon in <i>t483</i>. B, Phenotypes of control and typical T₂ transgenic knockdown plants (RNAi-3) at the heading stage. C, Germination of control and RNAi-3 seeds at 3 day. D, Control and RNAi-3 seedlings 8 days post-germination. E, Two-week-old seedlings. F, Expression analysis of <i>CHR729</i> in leaves of control and RNAi-3 by qRT-PCR. G, Seed germination rates in control and RNAi-3 plants. H, Chlorophyll contents in control and RNAi-3 plants. Chlorophyll was extracted from above-ground parts of plants shown in (E). Values are means ±SD (n = 3, **<i>P</i><0.01). Scale bars: 25 cm (B); 2 cm (C, D); 5 cm (E).</p

Phenotypic comparisons of the <i>t483</i> mutant and wild-type Nipponbare (Beijing).

<p>Data are presented as means ± SE. Flag leaf widths were measured through the middle region of leaves at the mature stage. Significance of differences between WT and <i>t483</i> was detected using Student’s <i>t</i>-test (n = 20).</p><p>Phenotypic comparisons of the <i>t483</i> mutant and wild-type Nipponbare (Beijing).</p

Phenotypic characteristics of WT and <i>t483</i>.

<p>A, Plants at the tillering stage after removal of soil. B, Mature plant stage. C, Three uppermost internodes from the main tiller. D, Panicles of WT and <i>t483</i>. E, Adaxial side of leaf segments. F, Leaf chlorophyll contents in WT and <i>t483</i>. Values are means ±SD (n = 3) (**<i>P</i><0.01). G–H, Ultrastructure of chloroplasts in adaxial mesophyll cells of WT (G) and <i>t483</i> (H). I–J, Thylakoid lamellar structure of WT (I) and <i>t483</i> (J). <i>C</i> chloroplast, <i>Thy</i> thylakoid lamellar. Scale bars: 10 cm (A, C); 25 cm (B); 5 cm (D); 1cm (E); 0.5 μm (G, H); 0.2 μm (I, J).</p

Phenotypic comparisons of control and homozygous T₂ transgenic plants of RNAi-3 line (Sanya).

<p>Data are presented as means ± SE. Significance of differences between control and RNAi-3 was detected using Student’s <i>t</i>-test (n = 5).</p><p>Phenotypic comparisons of control and homozygous T₂ transgenic plants of RNAi-3 line (Sanya).</p

Effect of gamma irradiation on free radical levels.

<p>The X-axis represents the treatment dosage, including 0 Gy, 100 Gy, 150 Gy and 250 Gy. The Y-axis represents the free radical levels. Dark gray bars indicate free radical contents in HY1, light gray bars represent free radical contents in the J411 variety. Significant differences between treatment groups and the control groups in the HY1and J411 variety were analyzed by spass 16.0 (P < 0.05).</p

Effect of gamma irradiation on DNA repair-related genes <i>TaKu70</i> and <i>TaKu80 and plant phenotypes</i>.

<p>(A, B) Photographs of HY1 and J411 plants under different dosages of γ-irradiation. Seedling height and root length decreased significantly with increasing gamma irradiation dosage more quickly in HY1 than in J411. (C, D) Histogram analysis of variation rate of root length and seedling height in HY1 and J411. The X-axis represents the treatment dosage, including 0 Gy, 100 Gy, 150 Gy and 250 Gy. The Y-axis represents the variation rate of root length and seedling in HY1 and J411. Significant differences were analyzed by spass 16.0 (P < 0.05) (E, F)The X-axis represents the treatment dosage, including 0 Gy, 100 Gy, 150 Gy and 250 Gy. The Y-axis represents <i>Taku70</i> gene expression level. Dark gray bars indicate <i>Taku70</i> and <i>Taku80</i> gene expression values in HY1, and light gray bars indicate those in J411. Significant differences between treatment groups and the control groups in the HY1and J411 variety were analyzed by spass 16.0 (P < 0.05).</p