Regulation of aleurone cell fate determinants in Zea mays

In grass species, the endosperm is a major source of food, feed and industrial raw materials. In maize, the aleurone is the peripheral cell layer of the endosperm that functions primarily as a digestive tissue. The aluerone layer of the endosperm is an excellent system to study cell differentiation and fate specification. Aleurone identity is not fixed and positional cues are continuously required during development to specify and maintain aleurone cell fate in the peripheral layer. A putative genetic hierarchy is proposed based on the collections of aleurone mutants. Cell fate mutants define early factors and aluerone differentiation mutants define factors late in the aleurone development. CRINKLY4 (CR4), a plant receptor-kinase defines an early step in aleurone cell fate and VIVIPAROUS1 (VP1), a B3 domain -containing transcription factor that regulates seed maturation in maize. The intermediate steps between CR4 mediated cell fate specification and Vp1 transcriptions are unknown.CSN5 subunit of the COP9Siganalosome (CSN) interacts with the cytoplasmic domain of Arabidopsis CR4 (ACR4).The kinase domain of ACR4 was shown to bind and phosphorylate the CSN5 subunits in-vitro. In-vivo FRET experiments confirmed that ACR4 was in close proximity to CSN5 within the context of a plant cell. Curcumin treatment (COP9 signalosome inhibitor) of transgenic plants resulted in the accumulation of ACR4-GFP. ACR4-GFP accumulated in a csn5 double mutant suggesting the role of the CSN complex in the turnover of ACR4 receptor. To better understand the transcriptional regulation of the Vp1 and identify upstream candidates in the seed maturation and dormancy process, transfactors that bind to the CE1L element of Vp1 was identified. CE1LBP1, a novel zinc binding protein present in all angiosperms appears to be single copy in maize. Electrophoretic mobility shift assays (EMSAs) with recombinant CE1LBP1 protein and supershift EMSAs using maize embryos nuclear proteins with CE1LBP1 specific antibodies confirmed binding. This study enhances our understanding of cell fate specification and maturation processes that operate in the aleurone cells during cereal grain development

Correlation between a loss of auxin signaling and a loss of proliferation in maize antipodal cells

The plant life cycle alternates between two genetically active generations: the diploid sporophyte and the haploid gametophyte. In angiosperms the gametophytes are sexually dimorphic and consist of only a few cells. The female gametophyte, or embryo sac, is comprised of four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In some species the antipodal cells are indistinct and fail to proliferate, so many aspects of antipodal cell function and development have been unclear. In maize and many other grasses, the antipodal cells proliferate to produce a highly distinct cluster at the chalazal end of the embryo sac that persists at the apex of the endosperm after fertilization. The antipodal cells are a site of auxin accumulation in the maize embryo sac. Analysis of different families of genes involved in auxin biosynthesis, distribution, and signaling for expression in the embryo sac demonstrates that all steps are expressed within the embryo sac. In contrast to auxin signaling, cytokinin signaling is absent in the embryo sac and instead occurs adjacent to but outside of the antipodal cells. Mutant analysis shows a correlation between a loss of auxin signaling and a loss of proliferation of the antipodal cells. The leaf polarity mutant Laxmidrib1 causes a lack of antipodal cell proliferation coupled with a loss of DR5 and PIN1a expression in the antipodal cells

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND: Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS: By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans- genetic factors. CONCLUSIONS: We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors. CONCLUSIONS We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping

Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes

BACKGROUND: Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life cycle is that gene expression is required in the haploid gametophytes. Consequently, many mutant phenotypes are expressed in this phase. RESULTS: We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. Transposon-related transcripts are present in high levels in both gametophytes, suggesting a connection between gamete production and transposon expression in maize not previously identified in any female gametophytes. Two classes of small signaling proteins and several transcription factor gene families are enriched in gametophyte transcriptomes. Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2 indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than subgenome 2 genes with other expression patterns. Analysis of available insertion mutant collections shows a statistically significant deficit in insertions in gametophyte-expressed genes. CONCLUSIONS: This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies maize gametophyte functions, gametophyte expression of transposon-related sequences, and unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed genes is supporting evidence for their function in the gametophytes. Expression patterns of extant, duplicated maize genes reveals that selective pressures based on male gametophytic function have likely had a disproportionate effect on plant genomes

Maize RNA PolIV affects the expression of genes with nearby TE insertions and has a genome-wide repressive impact on transcription

Abstract Background RNA-directed DNA methylation (RdDM) is a plant-specific epigenetic process that relies on the RNA polymerase IV (Pol IV) for the production of 24 nucleotide small interfering RNAs (siRNA) that guide the cytosine methylation and silencing of genes and transposons. Zea mays RPD1/RMR6 gene encodes the largest subunit of Pol IV and is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs) and transcriptional regulation of specific alleles. Results In this study we applied a total RNA-Seq approach to compare the B73 and rpd1/rmr6 leaf transcriptomes. Although previous studies indicated that loss of siRNAs production in RdDM mutants provokes a strong loss of CHH DNA methylation but not massive gene or TEs transcriptional activation in both Arabidopsis and maize, our total RNA-Seq analysis of rpd1/rmr6 transcriptome reveals that loss of Pol IV activity causes a global increase in the transcribed fraction of the maize genome. Our results point to the genes with nearby TE insertions as being the most strongly affected by Pol IV-mediated gene silencing. TEs modulation of nearby gene expression is linked to alternative methylation profiles on gene flanking regions, and these profiles are strictly dependent on specific characteristics of the TE member inserted. Although Pol IV is essential for the biogenesis of siRNAs, the genes with associated siRNA loci are less affected by the pol IV mutation. Conclusions This deep and integrated analysis of gene expression, TEs distribution, smallRNA targeting and DNA methylation levels, reveals that loss of Pol IV activity globally affects genome regulation, pointing at TEs as modulator of nearby gene expression and indicating the existence of multiple level epigenetic silencing mechanisms. Our results also suggest a predominant role of the Pol IV-mediated RdDM pathway in genome dominance regulation, and subgenome stability and evolution in maize

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND: Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS: By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors. CONCLUSIONS: We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping

Regulation of aleurone cell fate determinants in Zea mays

In grass species, the endosperm is a major source of food, feed and industrial raw materials. In maize, the aleurone is the peripheral cell layer of the endosperm that functions primarily as a digestive tissue. The aluerone layer of the endosperm is an excellent system to study cell differentiation and fate specification. Aleurone identity is not fixed and positional cues are continuously required during development to specify and maintain aleurone cell fate in the peripheral layer. A putative genetic hierarchy is proposed based on the collections of aleurone mutants. Cell fate mutants define early factors and aluerone differentiation mutants define factors late in the aleurone development. CRINKLY4 (CR4), a plant receptor-kinase defines an early step in aleurone cell fate and VIVIPAROUS1 (VP1), a B3 domain -containing transcription factor that regulates seed maturation in maize. The intermediate steps between CR4 mediated cell fate specification and Vp1 transcriptions are unknown.CSN5 subunit of the COP9Siganalosome (CSN) interacts with the cytoplasmic domain of Arabidopsis CR4 (ACR4).The kinase domain of ACR4 was shown to bind and phosphorylate the CSN5 subunits in-vitro. In-vivo FRET experiments confirmed that ACR4 was in close proximity to CSN5 within the context of a plant cell. Curcumin treatment (COP9 signalosome inhibitor) of transgenic plants resulted in the accumulation of ACR4-GFP. ACR4-GFP accumulated in a csn5 double mutant suggesting the role of the CSN complex in the turnover of ACR4 receptor. To better understand the transcriptional regulation of the Vp1 and identify upstream candidates in the seed maturation and dormancy process, transfactors that bind to the CE1L element of Vp1 was identified. CE1LBP1, a novel zinc binding protein present in all angiosperms appears to be single copy in maize. Electrophoretic mobility shift assays (EMSAs) with recombinant CE1LBP1 protein and supershift EMSAs using maize embryos nuclear proteins with CE1LBP1 specific antibodies confirmed binding. This study enhances our understanding of cell fate specification and maturation processes that operate in the aleurone cells during cereal grain development.</p