Arabidopsis GLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III

Abstract

Precise control of gene expression is critical for embryo development in both animals and plants. We report that Arabidopsis thaliana GLUTAMINE-RICH PROTEIN23 (GRP23) is a pentatricopeptide repeat (PPR) protein that functions as a potential regulator of gene expression during early embryogenesis in Arabidopsis. Loss-of-function mutations of GRP23 caused the arrest of early embryo development. The vast majority of the mutant embryos arrested before the 16-cell dermatogen stage, and none of the grp23 embryos reached the heart stage. In addition, 19% of the mutant embryos displayed aberrant cell division patterns. GRP23 encodes a polypeptide with a Leu zipper domain, nine PPRs at the N terminus, and a Gln-rich C-terminal domain with an unusual WQQ repeat. GRP23 is a nuclear protein that physically interacts with RNA polymerase II subunit III in both yeast and plant cells. GRP23 is expressed in developing embryos up to the heart stage, as revealed by beta-glucuronidase reporter gene expression and RNA in situ hybridization. Together, our data suggest that GRP23, by interaction with RNA polymerase II, likely functions as a transcriptional regulator essential for early embryogenesis in Arabidopsis.

Figure 1.

Mutant Embryos Were Arrested before the Globular Stage and Displayed Aberrant Cell Division Patterns. (A) A wild-type silique showing a full seed set. (B) A heterozygous set09078 silique with approximately one-quarter of the embryos lethal (red arrows). (C) Whole-mounted, cleared seeds from siliques of heterozygous set09078 plants. The same silique contains a mutant embryo arrested at the early globular stage (right) compared with a normal embryo developed at the heart stage (left). (D) to (Q) Terminal phenotypes of set09078 embryos. (D) to (G) Wild-type embryos at the 1-cell zygote (D), 16-cell dermatogen (E), triangular (F), and late heart (G) stages. (H) to (Q) Homozygous set09078 embryos arrested from the 1-cell to the 16-cell stage. (H) A mutant embryo with a misplaced cell wall of the apical cell at the one-cell stage. (I) and (J) Mutant embryos with elongated and enlarged embryo proper at the two-cell (I) or four-cell (J) stage. (K) A six-cell embryo with an asymmetric division pattern. (L) and (M) Abnormal eight-cell embryos in the mutant. (N) An elongated 16-cell embryo. (O) An eight-cell embryo with misplaced cell division planes. (P) A six-cell embryo with an aberrant division pattern. (Q) A 16-cell embryo with asynchronous cell division. ac, apical cell; bc, basal cell; Ep, embryo proper; Su, suspensor. Bars = 20 μm.

Figure 2.

Molecular Characterization of GRP23 Protein. (A) Diagram of the insertion positions of Ds and T-DNAs in GRP23. The hatched box indicates the predicted open reading frame of the GRP23 gene. Ds insertion caused a 9-bp nucleotide duplication in set09078. The nucleotide numbers are consistent with those in BAC clone F14N23. (B) Predicted GRP23 amino acid sequences. The insertion positions of Ds in set09078 and T-DNA in SALK_128329 and SALK_074740 are indicated by arrowheads. The basic region and Leu zipper motif are shown in boldface italic and underlined, respectively. The shaded amino acids representing the nine PPR motifs and the three overlapping TPR motifs are underlined. Boxed columns show the conserved Trp (W) and Gln (Q) residues in the WQQ repeats. Q amino acids are highlighted in boldface. (C) Scheme of the GRP23 protein domains. The bZIP domain and nine PPR repeats are indicated. The white open boxes represent the WQQ motifs in the Gln-rich region.

Figure 3.

Alignment of the GRP23 Protein with Its Homologs from Cucumis melo, Oryza sativa (cv japonica), and Arabidopsis thaliana. Identical amino acids are shown with white letters in black boxes, and similar amino acids are shown with shaded boxes.

Figure 4.

GRP23-YFP Is Localized to the Nucleus in the Root Cells of Transgenic Plants. (A) Confocal image of a transgenic root cell under the YFP channel showing GRP23-YFP localization in yellow. (B) Confocal image of the same cell with transmitted light. (C) Merged image of (A) and (B) showing GRP23-YFP localization in the nucleus (arrow). (D) Confocal section of a transgenic root cell. DNA fluorescence (DAPI) is shown in red. (E) The same cell as in (D) showing GRP23-YFP fluorescence in green. (F) Merged image of (D) and (E) showing colocalization of GRP23-YFP with DAPI in the nucleus. Bars = 5 μm.

Figure 5.

Expression Patterns of the GRP23 and RBP36B Genes. (A) Tissue-specific expression of GRP23 and RBP36B using RT-PCR analysis. RT-PCR was performed on total RNAs from different tissues, including roots (Rt), stems (St), leaves (Lf), inflorescences (Fr), siliques (Se), and seedlings (Sd), with (+) or without (−) reverse transcriptase (R.T.) as indicated. After 35 cycles, the resulting products were stained with ethidium bromide and analyzed by gel electrophoresis. ACTIN2 RNA was used as an internal template control. (B) and (C) Expression profiles of GRP23 (B) and RBP36B (C) in various organs. The y axis represents the expression level. Data used in this analysis were retrieved from the public GENEVESTIGATOR microarray data set (https://www.genevestigator.ethz.ch; Zimmermann et al., 2004).

Figure 6.

Temporal and Spatial Patterns of GRP23 Gene Expression. (A) to (J) Histochemical assays for the expression pattern of the P_GRP-GUS transgene revealed by the P_GRP23-GUS reporter. (A) Young seeds in a silique expressing GUS. Bar = 1 mm. (B) Micrograph showing GUS activity specifically detected in anthers and young ovules during different flower development stages. Bar = 1 mm. (C) A stage 12 flower showing GUS activity in pollen grains (arrowhead) and embryo sacs at about the one- to two-nucleate stage (arrow). Bar = 100 μm. (D) GUS staining of a 7-d-old seedling showing GRP23 expression in the shoot apex, leaf primordium, lateral root primordia, and root meristem (inset). Bar = 1 mm. (E) GUS staining detected in an early two-nucleate embryo sac (arrow). (F) A mature embryo sac expressing GUS. (G) A zygote expressing GUS. (H) Approximately 12 h after fertilization, GUS staining is detected in the apical cell, basal cell, and endosperm cells. (I) Micrograph showing GUS activity in the embryo proper at the globular stage. (J) Micrograph showing GUS activity in the embryo proper and weak GUS activity in the endosperm cells at the heart stage. (K) to (P) RNA in situ hybridization confirmed the GRP23 expression during embryogenesis. (K) to (O) Antisense probe hybridization to longitudinal sections of seeds at different developmental stages. (K) Nomarski micrograph showing signal in the embryo proper and suspensor cell at the two- to four-cell stage. (L) and (M) Nomarski micrographs showing strong signal in the embryo proper, suspensor, and endosperm cells at the eight-cell stage. (N) Nomarski micrograph showing signal in the endosperm cells at the globular stage. (O) Nomarski micrograph showing strong signal in the embryo proper at the late heart stage. (P) No signal was detected using sense probe hybridization to seed sections with embryo development at the globular stage. Ac, apical cell; An, anther; Bc, basal cell; En, endosperm; Ep, embryo proper; Su, suspensor; Zy, zygote. Bars = 60 μm ([E] to [J]) and 25 μm ([K] to [P]).

Figure 7.

GRP23 Interacts with RBP36B in Both Yeast and Onion Cells. (A) Scheme of GRP23 representing the nine PPR motifs as nine open boxes and the Gln-rich domain as a hatched bar. GRP23 deletion constructs containing either the PPR domain or the WQQ repeat domain are shown below. Numbers indicate the first and last amino acids of the peptides. (B) GRP23 interacts with RBP36B in yeast cells. Yeast cells were cotransformed with pGBKT7 and pAD-RBP36B (sector 1), pBD-GRP23 and pGAD-GH (sector 2), and pBD-GRP23 and pAD-RBP36B (sector 3). Transformants were streaked on a plate containing synthetic dropout selection medium that lacked Trp, Leu, and His supplemented with 5 mM 3-AT (SD/-Trp-Leu-His + 5 mM 3-AT). (C) The WQQ repeat–containing Gln-rich domain of GRP23 interacts with RBP36B in yeast. Yeast cells were cotransformed with pGBKT7 and pAD-RBP36B (sector 1), pBD-WQQ and pGAD-GH (sector 2), and pBD-WQQ and pAD-RBP36B (sector 3). Transformants were streaked on a SD/-Trp-Leu-His + 5 mM 3-AT plate. (D) GRP23 deletions containing only its PPR domain failed to interact with RBP36B in yeast. Yeast cells were cotransformed with pGBKT7 and pAD-RBP36B (sector 1), pBD-PPR and pGAD-GH (sector 2), and pBD-PPR and pAD-RBP36B (sector 3). Transformants were streaked on a SD/-Trp-Leu-His + 5 mM 3-AT plate. (E) β-Galactosidase activities by o-nitrophenyl-β-d-galactopyranosidase assays of yeast cells cotransformed with pGBKT7 and pGAD-GH (bar 1), pBD-GRP23 and pGAD-GH (bar 2), pBD-PPR and pGAD-GH (bar 3), pBD-WQQ and pGAD-GH (bar 4), pBD-GRP23 and pAD-RBP36B (bar 5), pBD-WQQ and pAD-RBP36B (bar 6), and pBD-PPR and pAD-RBP36B (bar 7). These assays were repeated three times. Mean ± SD values are shown. mU, milliunits. (F) BiFC visualization of GRP23 interaction with RBP36B in nuclei of onion cells (arrows). Left, YFP fluorescence image of onion epidermal cells cotransfected with GRP23-YFP^N and RBP36B-YFP^C; middle, bright-field image of the onion cells; right, merged image showing YFP florescence in nucleus. Bars = 5 μm.