Transcriptome analysis of nkd1 and nkd2 mutants in W22 maize endosperm
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ABSTRACT: The maize NAKED ENDOSPERM (NKD) genes Nkd1 and Nkd2 encode paralogous INDETERMINATE DOMAIN (IDD) transcription factors (TFs) that have previously been shown to play critical roles in regulating endosperm gene expression and development. However, the function of each individual NKD protein is unclear. To elucidate this, we performed a transcriptome analysis for nkd1-Ds and nkd2-Ds single mutant endosperm. We found that in nkd1-Ds single mutant, nkd2 gene expression is significantly lower than WT, and similar to the nkd2-Ds mutant.
Project description:The transcriptomic analysis of nkd1, nkd2 and o2 homozygous mutant set (including 1 WT, 3 single mutants, 3 double mutants and 1 triple mutant) at 8, 12 and 16 DAP revealed temporal regulatory landscape in endosperm grain-filling stage. The data also showed NKD1, NKD2 and O2 interactively regulate gene network overtime in several processes, such as hormone response, cell wall organization, nutrient storage and endoreduplication, etc.
Project description:Transcription factors (TFs) play an important role in maize endosperm development regulation. The temporal RNA-seq and co-expression network analysis of maize endosperm reveals a hiararchical regulatory network architechture modulating the endosperm development. In the network, NKD1 and NKD2 are central regulators, and GBF1, HSFTF10, NACTF49 and HB115 are secondary regulators downstream of NKD1 and 2. To further test the relationship between these TFs, a DNA affinity purification and sequencing (DAP-seq) assay was performed. The result shows that the assay on NKD1 and NKD2 called 1,951 and 56,855 peaks, and 93 and 1,692 peaks are located within 3 kb distance to transcription starting site (TSS), respectively. And assays on GBF1, HSFTF10, NACTF49 and HB115 called 15,543; 38,147; 7,388 and 936 peaks, respectively.
Project description:Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products and their utilization during germination. However, little is known about the regulatory mechanisms that link cell differentiation processes with those controlling storage product synthesis and deposition, including the activation of zein genes by the maize (Zea mays) bZIP transcription factor Opaque-2 (O2). In this study, we mapped in vivo binding sites of O2 in B73 endosperm, and compared the results with genes differentially expressed in B73 and B73o2. We identified 186 putative direct O2 targets and 1,677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets using real-time RT-PCR experiments revealed at least two distinct modes of O2-mediated gene activation. We also showed that two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2) encoded transcription factors, which can in turn co-activate other O2-network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results provide novel insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function.
Project description:NKD1, 2 and O2 are key transcription factors interactively regulating endosperm development. Their mutation combinations may diversely alter the global gene regulatory landscape. To investigate how interactions between NKD1, 2 and O2 affect endosperm regulatome, which is indicated by accessible regions for regulators on chromatins, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed for 16 DAP endosperms of all 8 genotype combinations (2 biological replicate per genotype). Among 16 sequenced samples, 20,478 to 49,640 (mean: 34,766) peaks were called, and these peaks represent potential chromatin accessible regions. Distribution of the peaks on chromatins varies among samples: 54.35% - 66.73% peaks are located at distal intergenic region (>3kb upstream and >300 bp downstream of a gene) and 6.7% - 12.1% are located at promoter region (within 3 kb flanking transcription starting site), and 4.62% - 8.88% peaks are located within 1kb flanking transcription starting site. Also, in WT background, nkd1 and nkd2 single mutants have overall greater number of opening regions (upregulated peaks) than closing regions (downregulated peaks), whereas nkd1,2 double mutant has similar number of opening and closing regions. In contrast, in o2-background, all nkd1, nkd2 and nkd1,2 mutants have greater number of closing than opening regions. This indicates that o2 may alter differential accessibility of nkd1, nkd2 single or nkd1,2 double mutants.
Project description:Seed size is related to plant evolution and crop yield and is affected by genetic mutations, imprinting, and genome dosage. Imprinting is a widespread epigenetic phenomenon in mammals and flowering plants. ETHYLENE INSENSITIVE2 (EIN2) encodes a membrane protein that links the ethylene perception to transcriptional regulation. Interestingly, during seed development EIN2 is maternally-expressed in Arabidopsis and maize, but the role of EIN2 in seed development is unknown. Here we show that EIN2 is expressed specifically in the endosperm, and the maternal-specific EIN2 expression affects temporal regulation of endosperm cellularization. As a result, seed size increases in the genetic cross using the ein2 mutant as the maternal parent or in the ein2 mutant. The maternal-specific expression of EIN2 in the endosperm is controlled by DNA methylation but not by H3K27me3 or by ethylene and several ethylene pathway genes tested. RNA-seq analysis in the endosperm isolated by laser-capture microdissection show upregulation of many endosperm-expressed genes such as AGAMOUS-LIKEs (AGLs) in the ein2 mutant or when the maternal EIN2 allele is not expressed. EIN2 does not interact with DNA and may act through ETHYLENE INSENSITIVE3 (EIN3), a DNA binding protein present in sporophytic tissues, to activate target genes like AGLs, which in turn mediate temporal regulation of endosperm cellularization and seed size. These results provide mechanistic insights into endosperm and maternal-specific expression of EIN2 on endosperm cellularization and seed development, which could help improve seed production in plants and crops.
Project description:The objective of the current study is to unravel the gene regulatory networks controlled by the nkd genes during maize endosperm developent. We compared wild type (B73) vs. nkd mutant (introgressed into B73 background) transcriptomes in aleurone vs. starchy endosperm cell types captured by laser capture microdissection technology.
Project description:The objective of the current study is to unravel the gene regulatory networks controlled by the nkd genes during maize endosperm development. We compared wild type (B73) vs. nkd mutant (introgressed into B73 background) transcriptomes in aleurone vs. starchy endosperm cell types captured by laser capture microdissection technology. We performed RNA seq analysis of mid-mature (15DAP) endosperm in two cell types [aleurone (A) and starchy endosperm (S)] of wild type B73 (B) and nkd mutant (N) kernels with three independent biological replicates.
Project description:Osteosarcoma (OS) is the most frequent malignant bone tumor with a high propensity for metastases. Through the tissue-specific alteration of p53 status, we have developed a genetically engineered mouse model of localized and metastatic OS. Gene expression analysis revealed naked cuticle homolog 2 (NKD2), a negative regulator of the Wnt signaling pathway, to be significantly downregulated in metastatic OS. An assessment of human OS tumors revealed downregulation of NKD2 in metastatic and recurrent OS. We determined that downregulation was secondary to methylation of the NKD2 promoter region for both mouse and human tumors. Furthermore, in vivo investigations indicate that NKD2 overexpression significantly diminishes OS tumor growth and metastasis and small molecule Wnt inhibitors can decrease OS growth and metastatic potential.