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, 17 (1), 178-187

Chromatin Interacting Factor OsVIL2 Increases Biomass and Rice Grain Yield

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Chromatin Interacting Factor OsVIL2 Increases Biomass and Rice Grain Yield

Jungil Yang et al. Plant Biotechnol J.

Abstract

Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3-LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA-sequencing analyses revealed that 1958 genes were up-regulated and 2096 genes were down-regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.

Keywords: OsCKX2; OsVIL2; biomass; chromatin interacting factor; grain yield; rice.

Figures

Figure 1
Figure 1
Phenotypes of WT and osvil2 mutants. (a) Comparison of panicles among WT, osvil2‐1, and osvil2‐2. Scale bar = 5 cm. (b) Number of primary branches on main panicle. (c) Number of secondary branches on main panicle. (d) Number of grains from main panicle. Error bars show standard deviations; = 10. Statistical significance is indicated by * (< 0.01) and ** (< 0.001).
Figure 2
Figure 2
Morphological comparison between wild‐type (WT) and OsVIL2‐overexpression (OX) plants #1 and #2. (a) Phenotypes at seed‐ripening stage. Scale bar = 10 cm. (b) Lengths of panicles and internodes at seed‐ripening stage. (c) Diameter of each internode in major culm. (d) Comparison of dry weights among WT and OsVIL2OX plants at heading stage. Error bars indicate standard deviations; = 5 or more. Statistical significance is indicated by ** (< 0.001).
Figure 3
Figure 3
Longitudinal and cross sections of internode. (a) Longitudinal sections of culm from first internode of WT and OsVIL2OX #1 at heading stage. Scale bar: 100 μm. Comparison of cell length (b) and cell number (c) at about 0.5 cm upper part from the division region of the first internode of WT and OsVIL2OX #1 at heading stage. Error bars indicate standard deviations from four individual sections. Statistical significance is indicated by ** (< 0.001). (d) Cross sections of culm from first internode of WT and OsVIL2OX #1 plants. Scale bar = 200 μm. (e) Large vascular bundle from first internode. Arrows indicate xylem parenchyma cells (XP), sieve tubes (S) and companion cells (C). Scale bar = 500 μm.
Figure 4
Figure 4
Panicle phenotypes. (a) Comparison between WT and two OsVIL2 OX transgenic lines. Scale bar = 5 cm. (b) Number of primary branches on main panicle. (c) Number of secondary branches on main panicle. (d) Number of grains from main panicle. Error bars show standard deviations; = 10. Statistical significance is indicated by * (< 0.01) and ** (< 0.001).
Figure 5
Figure 5
OsCKX2 transcription and cytokinin concentrations in OsVIL2OX, mutant and WT plants. (a) Transcript levels in first internodes from WT and OsVIL2OX #1 were measured by quantitative real‐time PCR. Y‐axis, relative transcript level of OsCKX2 compared with that of Ubi. Error bars indicate standard deviations; n = 3 or more. (b) Cytokinin levels in first internodes of WT and OsVIL2OX #1. iPRPs, N6‐(Δ2‐isopentenyl) adenine ribotides; tZRPs, trans‐zeatin ribotides; iPR, N6‐(Δ2‐isopentenyl) adenine riboside; tZ, trans‐zeatin; tZR, trans‐zeatin riboside; iP, N6‐(Δ2‐isopentenyl) adenine. Error bars indicate standard deviations; = 3 or more. Statistical significance is indicated by ** (< 0.001).
Figure 6
Figure 6
Chromatin immunoprecipitation assay of OsCKX2 chromatin with OsVIL2‐Myc. (a) Genomic structure of OsCKX2 and OsLP. Tested regions are numbered. (b) ChIP analysis of OsVIL2 enrichment on OsCKX2 chromatin. OsVIL2‐Myc epitope‐tagged transgenic lines were used to detect enrichment. Actin chromatin served as control. Samples from OsVIL2‐Myc plants are indicated in red, while those from control plants expressing only Myc are in blue. (c) ChIP analysis of OsVIL2 enrichment on OsLP1 chromatin. OsVIL2‐Myc epitope‐tagged transgenic lines were used to detect enrichment. Actin chromatin served as control. Samples from OsVIL2‐Myc plants are indicated in red, while those from control plants expressing only Myc are in blue. (d) Analysis of H3K27me3 level on OsCKX2 chromatin in WT (blue) and OsVIL2OX #1 (red) using antibodies against H3K27me3. Actin chromatin served as control. Error bars show standard deviations; = 3.
Figure 7
Figure 7
Analysis of interaction between OsVIL2 and OsEMF2b by co‐immunoprecipitation assay. (a) Schematic representation of OsVIL2 protein. Scale bar = 100 a.a. (b) Co‐immunoprecipitation assay between OsEMF2b and 3 domains of OsVIL2. Total proteins were extracted from Oc‐cell protoplasts after transient expression of OsVIL2_PHD‐Myc, OsVIL2_FNIII‐Myc, OsVIL2_VID‐Myc and OsEMF2b‐HA. Extracts were immuno‐precipitated with anti‐HA antibodies and interaction signals were detected using anti‐Myc antibody after SDSPAGE. Inputs, total protein extracts before immunoprecipitation; IP, elutes from agarose beads after immunoprecipitation. The entire experiment was conducted three times.

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