Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice

Abstract

Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice.

Figure 1.

Phenotypic analysis of the wild type (WT) and oshub1 and oshub2 mutants. A, Comparison of the percentage seed set of the wild type (left), oshub1-1 (middle), and oshub1-2 (right). B, Comparison of the percentage seed set of the wild type (left) and oshub2 (right). Arrowheads in A and B indicate sterile spikelets. C, Seed set of wild-type and oshub1-1 (1-1), oshub1-2 (1-2), and oshub2 (2) mutant plants. Seed set was calculated as the proportion of fertile spikelets to all spikelets for each plant at maturity. Data are means ± sd (n = 20). D and E, Comparison of anther phenotype before anthesis (D) and I₂-KI-stained pollens (E) of the wild type and oshub1 mutants. F, The number of sterile and fertile pollen grains per anther as determined by I₂-KI staining of wild-type, oshub1-1 (1-1), oshub1-2 (1-2), and oshub2 (2) materials. Data are means ± sd of total pollen grains (n = 5). G, Comparison of aniline blue-stained pollen grains on stigmas for the wild type and oshub1 mutants. Arrows indicate the pollen grains with pollen tubes. Student’s t test was used to analyze significant differences between the wild type and mutants (*P < 0.05; and **P < 0.01). Bars = 5 cm (A and B), 2 mm (D), 50 µm (E), and 200 µm (G).

Figure 2.

Comparison of male gametogenesis in wild-type and oshub1 plants. A to D and M to P show the wild type. E to H, I to L, and Q to T show the oshub1-1, oshub1-2, and oshub1-1 mutant, respectively. E, Epidermis; En, endothecium; ML, middle layer; T, tapetum; Msp, microspore; MP, mature pollen; AMsp, aborted microspore. A, E, and I, Cross section of anthers at stage 9. B, F, and J, Cross section of anthers at stage 10. C, G, and K, Cross section of anthers at stage 11. D, H, and L, Anthers at stage 13. M and Q, N and R, O and S, and P and T show microspores at stage 8b, stage 10, stage 11, and stage 12, respectively. N to P and R to T are DAPI stained to show nuclei. M and Q are karbol fuchsin stained to show tetrad. Bars = 100 µm (A–L) and 10 µm (M–T).

Figure 3.

Scanning electron microscopy and transmission electron microscopy of wild-type (WT) and oshub1 anthers. A and B, Comparison of anthers before anthesis (A) and mature pollen grains (B) derived from the wild type and oshub1 mutants. C, Higher magnification of pollen grains from wild-type and oshub1 mutant plants. D to G, The cross section of wild-type anthers at stage 10 showing anther wall layers (D), mitochondria (E) and nucleus (F) in tapetal layer, and microspore (G). H to K, The cross section of oshub1-1 anthers at stage 10 showing anther wall layers (H), mitochondria (I) and nucleus (J) in tapetal layer, and microspore (K). Mitochondria is marked by the white arrowhead in E and I. E, Epidermis; En, endothecium; ER, endoplasmic reticulum; Ex, exine; Ml, middle layer; Msp, microspore; Mt, mitochondria; Nu, nucleus; T, tapetum; Ub, ubisch body. Bars = 1 mm (A), 50 µm (B), 20 µm (C), 5 µm (D, H, G, and K), and 0.5 µm (E, F, I, and J).

Figure 4.

Global histone modification in the wild type (WT) and oshub mutants. A and B, Analysis of H2B monoubiquitination in the wild type and mutants using an anti-H2Bub1 antibody. H2B was used as a loading control. C, Detection of H3 methylation and H3 and H4 acetylation on a genome-wide scale in the wild type and oshub1 mutants. D, Analysis of H3K4 di- and trimethylation in the wild type and oshub2 mutant. H3 was used as loading control in C and D.

Figure 5.

Self-interaction and pairwise interaction of OsHUB1 and OsHUB2. A, Physical interactions between OsHUB1 and OsHUB2 in the yeast two-hybrid assay. pACT2 is the pray vector (AD), and pAS2 is the bait vector (BD). Yeast stains cotransformed with OsHUB1 and OsHUB2, OsHUB1 and OsHUB1, and OsHUB2 and OsHUB2 were assayed for growth on selective medium (-Leu, -Trp, -His, and -Ade; left) and β-galactosidase activity (right). Self-interaction of HUB1 (Cao et al., 2008) was used as a positive control (pAD-HUB1 + pBD-HUB1). Cotransformations of pAD-OsHUB1 with pBD, pBD-OsHUB1 with pAD, pAD-OsHUB2 with BD, and pBD-OsHUB2 with pAD are included as negative controls. B, Bimolecular fluorescence complementation assay showing the (self-)interaction of OsHUB1 and OsHUB2 in vivo. OsHUB1 and OsHUB2 were transiently coexpressed or were coexpressed with the vector alone in Nicotiana benthamiana leaf cells. Bars = 50 µm. SD, Synthetically defined medium; X-Gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside acid; YFP, yellow fluorescent protein.

Figure 6.

Expression pattern of OsHUB1 and OsHUB2. A and B, qRT-PCR analysis of expression profiles for OsHUB1 and OsHUB2. S6 to S8 and S9 and S10 represent anther development at stages 6 to 8 and stages 9 and 10, respectively. ACTIN1 was used as internal control. Error bars indicate sd of three independent samples. YL, Young leaf; YR, young root; ML, mature leaf; MS, mature sheath; N, node; C, culm; P, panicle. C to H, Histochemical analysis of OsHUB1 expression in a germinating seed (C), young root (D), young leaf (E), mature leaf (F), leaf sheath (G), spikelet at various stages (H), and mature pollen grains (I) from transgenic plants expressing GUS driven by the OsHUB1 promoter. S6 to S13 refer to stages 6 to 13. J and K, GUS staining in a transverse section of a stage-8 (J) and a stage-10 (K) anther locule. Arrowheads in K show the GUS signal. Bars = 2 mm (C–H) and 20 µm (I–K).

Figure 7.

Biological process and molecular function by GO analysis of genes that were up-regulated and down-regulated at least 2-fold in oshub1-1. A, Biological process indicated by GO analysis. B, Molecular function indicated by GO analysis. Hydrolase activity (1) refers to hydrolase activity acting on acid anhydrides in phosphorus-containing anhydrides. Hydrolase activity (2) refers to hydrolase activity acting on acid anhydrides. Oxidoreductase activity (1) refers to oxidoreductase activity acting on single donors with incorporation of molecular oxygen. Oxidoreductase activity (2) refers to oxidoreductase activity acting on NAD(P)H.

Figure 8.

qRT-PCR expression analysis of genes reported to be involved in rice anther development in wild-type (WT) and oshub1 plants. S6 to S8 and S9 and S10 represent stages 6 to 8 and stages 9 and 10 of anther development, respectively. For RNA extraction, anthers were used. ACTIN1 was used as the internal control. Each data point is the average of three biological repeats, and error bars indicate sd.

Figure 9.

Evaluation of H2B monoubiquitination and H3K4 dimethylation levels in UDT1, OsC4, and OsCP1 chromatin. A, The structure of UDT1. B and C, ChIP-qPCR analysis to determine the level of uH2B (B) and H3K4me2 (C) in UDT1 chromatin. D, The structure of OsC4. E and F, ChIP-qPCR analysis to determine the level of the uH2B (E) and H3K4me2 (F) in OsC4 chromatin. G, The structure of OsCP1. H and I, ChIP-qPCR analysis to determine the level of uH2B (H) and H3K4me2 (I) in OsCP1 chromatin. The start codon ATG and stop codon TGA (or TAA or TAG) are indicated. +1 indicates the translation initiation point. Boxes and lines represent exons and introns, respectively. P1 to P3 represent regions covered by the primers used to assess the level of uH2B and H3K4me2 by qPCR following ChIP. Data were normalized to the input chromatin, the P1 region in the wild type (WT) was set to be 1 in B, C, E, F, and H, and the P2 region in the wild type was set to be 1 in I. This experiment was repeated three times with independent samples. Data are means ± se. ND, Not detected.

Figure 10.

A proposed model for H2B monoubiquitination mediated by OsHUB1 and OsHUB2 during anther development in rice. In this model, OsHUB1 and OsHUB2 form a heterotetramer and recruit E2s to UDT1 and OsCP1 chromatin, leading to transfer of an ubiquitin molecule to H2B possibly at Lys-143. H2Bub1 formation enhances H3K4me2, together promoting transcription initiation. The fine-tuning of gene expression of UDT1/OsCP1 is important for late anther development of rice.