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, 29 (10), 2661-2675

The Maize Imprinted Gene Floury3 Encodes a PLATZ Protein Required for tRNA and 5S rRNA Transcription Through Interaction With RNA Polymerase III

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The Maize Imprinted Gene Floury3 Encodes a PLATZ Protein Required for tRNA and 5S rRNA Transcription Through Interaction With RNA Polymerase III

Qi Li et al. Plant Cell.

Abstract

Maize (Zea mays) floury3 (fl3) is a classic semidominant negative mutant that exhibits severe defects in the endosperm but fl3 plants otherwise appear normal. We cloned the fl3 gene and determined that it encodes a PLATZ (plant AT-rich sequence and zinc binding) protein. The mutation in fl3 resulted in an Asn-to-His replacement in the conserved PLATZ domain, creating a dominant allele. Fl3 is specifically expressed in starchy endosperm cells and regulated by genomic imprinting, which leads to the suppressed expression of fl3 when transmitted through the male, perhaps as a consequence the semidominant behavior. Yeast two-hybrid screening and bimolecular luciferase complementation experiments revealed that FL3 interacts with the RNA polymerase III subunit 53 (RPC53) and transcription factor class C 1 (TFC1), two critical factors of the RNA polymerase III (RNAPIII) transcription complex. In the fl3 endosperm, the levels of many tRNAs and 5S rRNA that are transcribed by RNAPIII are significantly reduced, suggesting that the incorrectly folded fl3 protein may impair the function of RNAPIII. The transcriptome is dramatically altered in fl3 mutants, in which the downregulated genes are primarily enriched in pathways related to translation, ribosome, misfolded protein responses, and nutrient reservoir activity. Collectively, these changes may lead to defects in endosperm development and storage reserve filling in fl3 seeds.

Figures

Figure 1.
Figure 1.
Kernel Phenotypes and Scanning Electron Microscopy of Starch Granules in WT, WT × fl3, fl3 × WT, and fl3. (A) Ear phenotypes of WT, WT × fl3, fl3 × WT, and fl3. Bar = 5 cm. (B) Kernel phenotypes of WT, WT × fl3, fl3 × WT, and fl3. Bar = 2 mm. (C) Scanning electron microscopy of starch granules in the areas, as indicated in (B). Bars = 10 µm.
Figure 2.
Figure 2.
Storage Carbohydrates and Proteins in the Wild Type and fl3. (A) The 100-kernel weight of wild type and fl3. (B) The content of starch in wild type and fl3. (C) The content of soluble sugars in wild type and fl3. (D) The contents of proteins in wild type and fl3. Error bars in (A) to (D) represent the sd of five independent replicates. *P < 0.05 and **P < 0.01. (E) SDS-PAGE analysis of zein in developing endosperms of wild type and fl3. D, days after pollination. The size of each band is indicated by numbers besides it. M, protein markers from top to bottom correspond to 25, 20, and 15 kD.
Figure 3.
Figure 3.
Light and Transmission Electron Microscopy of Immature Endosperms at Different Developmental Stages the Wild Type and fl3. (A) to (E) The wild type. (F) to (J) fl3. (A) and (F) Light microscopy of 6-DAP endosperms of the wild type (A) and fl3 (F). Bars = 100 µm. (B) and (G) Light microscopy of 10-DAP endosperms of the wild type (B) and fl3 (G). Bars = 2 mm. (C) and (H) Light microscopy of 18-DAP endosperms of the wild type (C) and fl3 (H). Bars = 50 µm. (D) and (I) Transmission electron microscopy of 18-DAP endosperms of the wild type (D) and fl3 (I). Bars = 1 µm. (E) and (J) Transmission electron microscopy of the magnified mitochondria in the wild type (E) and fl3 (J). Bars = 200 nm. En, endosperm; asterisk, embryo; SG, starch granule; PB, protein body; CW, cell wall; RER, rough endoplasmic reticulum; M, mitochondria.
Figure 4.
Figure 4.
Map-Based Cloning of fl3. (A) Fine-mapping of fl3 using the BC1F1 populations of (B73 × fl3) × B73 and (Mo17 × fl3) × Mo17 with 2800 and 6000 individuals, respectively. The numbers under each bar indicate the number of recombinants between fl3 and the molecular marker. The red dot indicates the centromere. Between two markers SNP5194 and SNP7666, 19 genes were annotated. Gene IDs in gray indicate that they are not expressed in the endosperm. Gene IDs in black indicate that they are expressed in the endosperm. The gene ID in red indicates that it is specifically expressed in the endosperm. (B) A diagram showing the GRMZM2G006585 gene structure and the A/C substitution in the second exon in fl3. (C) A diagram showing the GRMZM2G006585 protein structure and the Asn to His replacement in the PLATZ domain in fl3.
Figure 5.
Figure 5.
Transgenic Confirmation of fl3. (A) Ear phenotypes of the transgenic maize expressing fl3. Bar = 5 cm. Upper left, ear phenotypes. The floury and normal seeds on the self-pollinated cobs are indicated by arrows and asterisks; lower left, statistical analysis of the kernel phenotype from two independent transgenic maize lines; right, PCR analysis of transgenic maize kernels. Forty-eight randomly selected kernels from two transgenic T0 lines were phenotyped and analyzed by PCR. The upper line indicates the phenotype of each kernel and the lower line indicates the PCR amplification of the corresponding kernel. w, the wild type; f, the floury phenotype. (B) Reciprocal crosses of the wild type and fl3-OE6/+. The floury and normal seeds are indicated by arrows and asterisks. (C) Rescue of fl3 with the Fl3-RNAi. In the cross of fl3 × Fl3-RNAi/+, the progeny that inherited the RNAi were restored to a normal phenotype as indicated by asterisks, whereas those without the RNAi remained floury as indicated by arrows.
Figure 6.
Figure 6.
The Expression Pattern of Fl3. (A) Quantitative RT-PCR analysis of Fl3 in root, leaf, stalk, tassel, embryo, and developing endosperms from 3 to 32 DAP. All expression levels are normalized to Actin. Three replicates for each sample were made and are illustrated as ±sd. (B) to (E) RNA in situ hybridization of Fl3 at 6 (B), 8 (C), 12 (D), and 16 DAP (E). Longitudinal sections of wild-type kernels from 6 to 16 DAP were hybridized with an antisense RNA probe of Fl3. The positive signals (blue violet) of Fl3 were only observed in the starchy endosperm. (F) and (G) RNA in situ hybridization of Fl3 at 12 DAP showing no expression in the aleurone and a few outmost starchy endosperm cell layers (F) and BETL (G). (H) No signal was seen when a 12-DAP kernel section was hybridized with a sense probe. NU, nucellus; En, endosperm; asterisk, embryo; SE, starchy endosperm; SA, subaleurone ; A, aleurone. Bars = 1 mm in (B) to (E) and (H) and 100 μm in (F) and (G).
Figure 7.
Figure 7.
Gene Imprinting of Fl3. (A) Allelic expression analysis of Fl3 and fl3 in their reciprocal crosses. (B) Allelic expression analysis of Fl3-A619 and Fl3-Fangyin in their reciprocal crosses. (C) Diagram showing the polymorphisms between the Fl3-A619 and Fl3-Fangyin promoters. W168 and W169 are a pair of primers designed to amplify the allelic promoters of Fl3-A619 and Fl3-Fangyin that comprise four SNPs and a ClaI site. (D) Differential DNA methylation in the Fl3 promoters of two parents in the endosperm. ClaI is a DNA methylation-sensitive restriction enzyme. Without ClaI digestion, the Fl3 promoter sequences amplified from the female and male alleles were ∼2:1, consistent with their gene dosage contribution in the endosperm. With ClaI digestion, the sequences were more amplified from the male allele, which has a higher DNA methylation in its promoter.
Figure 8.
Figure 8.
FL3 Interacts with RPC53 and TFC1. (A) Yeast two-hybrid assay showing that FL3 and RPC53/TFC1 interacts. (B) BiLC showing that FL3 and RPC53/TFC1 interacts. Fluorescence signal intensities represent their interaction activities. (C) A diagram showing different deletions in FL3 designed for protein-protein interaction analysis. (D) Yeast two-hybrid assay showing that the PLATZ domain is required for protein-protein interaction with RPC53. The full-length, N terminus, PLATZ domain region, and C terminus of FL3 were tested for their interaction with RPC53. In (A) and (D), the yeast cultures with four different dilutions (100, 10−1, 10−2, and 10−3) were grown on the SD medium lacking Trp, Leu, His, and adenine (left panel) and lacking Trp and Leu (right panel, as control), respectively. AD, activating domain; BD, binding domain.
Figure 9.
Figure 9.
Quantitative RT-PCR Analysis of 17 tRNAs and 5S rRNA Genes in 12-DAP Endosperms of the Wild Type and fl3. All expression levels are normalized to Actin. Three replicates for each sample were made and are illustrated as ±sd.
Figure 10.
Figure 10.
Global Transcriptome Analysis Based on RNA-Seq Data in 10-DAP Endosperms of the Wild Type and fl3. (A) Principal component analysis. (B) The numbers of differentially expressed genes in the fl3 endosperm compared with the wild type. (C) GO classification for genes with downregulated expression in the fl3 endosperm compared with the wild type.
Figure 11.
Figure 11.
A Proposed Model for FL3/fl3 Controlling the Storage Filling in the Maize Endosperm.

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