The dicer-like1 Homolog fuzzy tassel Is Required for the Regulation of Meristem Determinacy in the Inflorescence and Vegetative Growth in Maize

Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles.ECU Open Access Publishing Support Fun

Preferential Re-Replication of Drosophila Heterochromatin in the Absence of Geminin

To ensure genomic integrity, the genome must be duplicated exactly once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. Cdt1, also known as Double-parked (Dup) in Drosophila, is a key regulator of the assembly of the pre-replicative complex (pre-RC) and its activity is strictly limited to G1 by multiple mechanisms including Cul4-Ddb1 mediated proteolysis and inhibition by geminin. We assayed the genomic consequences of disregulating the replication licensing mechanisms by RNAi depletion of geminin. We found that not all origins of replication were sensitive to geminin depletion and that heterochromatic sequences were preferentially re-replicated in the absence of licensing mechanisms. The preferential re-activation of heterochromatic origins of replication was unexpected because these are typically the last sequences to be duplicated in a normal cell cycle. We found that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather by the formation of the pre-RC. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin by elevated cyclin A-CDK activity. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1

Re-replication in the Absence of Replication Licensing Mechanisms in Drosophila Melanogaster

<p>To ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. </p><p>DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in <italic> Drosophila Menalogaster </italic>, is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4<super>Ddb1</super> mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase. </p><p>In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.</p><p>In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.</p>Dissertatio

Tissue-specific transcriptomics reveal functional differences in floral development

Abstract Flowers are produced by floral meristems, groups of stem cells that give rise to floral organs. In grasses, including the major cereal crops, flowers (florets) are contained in spikelets, which contain one to many florets, depending on the species. Importantly, not all grass florets are developmentally equivalent, and one or more florets are often sterile or abort in each spikelet. Members of the Andropogoneae tribe, including maize (Zea mays), produce spikelets with two florets; the upper and lower florets are usually dimorphic, and the lower floret is greatly reduced compared to the upper floret. In maize ears, early development appears identical in both florets but the lower floret ultimately aborts. To gain insight into the functional differences between florets with different fates, we used laser capture microdissection coupled with RNA-sequencing to globally examine gene expression in upper and lower floral meristems in maize. Differentially expressed genes were involved in hormone regulation, cell wall, sugar, and energy homeostasis. Furthermore, cell wall modifications and sugar accumulation differed between the upper and lower florets. Finally, we identified a boundary domain between upper and lower florets, which we hypothesize is important for floral meristem activity. We propose a model in which growth is suppressed in the lower floret by limiting sugar availability and upregulating genes involved in growth repression. This growth repression module may also regulate floret fertility in other grasses and potentially be modulated to engineer more productive cereal crops.</jats:p

Tissue-specific transcriptomics reveal functional differences in maize floral development

AbstractFlowers are produced by floral meristems, groups of stem cells that give rise to floral organs. In grasses, including the major cereal crops, flowers (florets) are contained in spikelets, which contain one to many florets, depending on the species. Importantly, not all grass florets are developmentally equivalent, and one or more florets are often sterile or abort in each spikelet. Members of the Andropogoneae tribe, including maize, produce spikelets with two florets; the upper and lower florets are usually dimorphic and the lower floret greatly reduced compared to the upper floret. In maize ears, early development appears identical in both florets but the lower floret ultimately aborts. To gain insight into the functional differences between florets of different fates, we used laser capture microdissection coupled with RNA-seq to globally examine gene expression in upper and lower floral meristems in maize. Differentially expressed genes were involved in hormone regulation, cell wall, sugar and energy homeostasis. Furthermore, cell wall modifications and sugar accumulation differed between the upper and lower florets. Finally, we identified a novel boundary domain between upper and lower florets, which we hypothesize is important for floral meristem activity. We propose a model in which growth is suppressed in the lower floret by limiting sugar availability and upregulating genes involved in growth repression. This growth repression module may also regulate floret fertility in other grasses and potentially be modulated to engineer more productive cereal crops.One sentence summaryFloret-specific differences in cell wall composition and sugar accumulation likely contribute to growth suppression in the lower floret of maize spikelets.</jats:sec

The dicer-like1 Homolog fuzzy tassel Is Required for the Regulation of Meristem Determinacy in the Inflorescence and Vegetative Growth in Maize

Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles.ECU Open Access Publishing Support Fun