Two Discrete cis Elements Control the Abaxial Side–Specific Expression of the FILAMENTOUS FLOWER Gene in Arabidopsis

Our previous studies showed that a member of the YABBY gene family, FILAMENTOUS FLOWER (FIL), plays a role in specifying the abaxial side tissues in the development of lateral organs such as cotyledons, leaves, young flower buds, and flower organs. We examined the expression pattern of FIL and found a temporal change of expression domains in the developmental process of the floral meristem. We also examined the cis control regions by constructing a series of transgenic plants that carry green fluorescent protein under the control of the FIL promoter with several types of deletions, base changes, and tandem repeats and showed that the unique expression pattern is dependent on at least two cis-acting elements in the 5′ regulatory region. One element proximal to the FIL gene would be responsible for the expression of both the abaxial and adaxial sides, and the other element of the 12-bp sequence would work to repress expression on the adaxial side

Enhancement of Neprilysin Activity by Natural Polyphenolic Compounds and Their Derivatives in Cultured Neuroglioma Cells

The onset of Alzheimer’s disease (AD) is characterized by accumulation of amyloid β peptide (Aβ) in the brain. Neprilysin (NEP) is one of the major Aβ-degrading enzymes. Given findings that NEP expression in the brain declines from the early stage of AD before apparent neuronal losses are observed, enhancement of NEP activity and expression may be a preventive and therapeutic strategy relevant to disease onset. We screened for compounds that could enhance the activity and expression of NEP using a polyphenol library previously constructed by our research group and investigated the structure–activity relationships of the identified polyphenols. We found that amentoflavone, apigenin, kaempferol, and chrysin enhanced the activity and expression of NEP, suggesting that chemical structures involving a double bond between positions 2 and 3 in the C ring of flavones are important for NEP enhancement, while catechol or pyrogallol structures, except for the galloyl group of catechins, abolished these effects. Moreover, natural compounds, such as quercetin, were not effective per se, but were changed to effective compounds by adding a lipophilic moiety. Using our study findings, we propose improvements for dietary habits with experimental evidence, and provide a basis for the development of novel small molecules as disease-modifying drugs for AD

Histone Deacetylases and ASYMMETRIC LEAVES2 Are Involved in the Establishment of Polarity in Leaves of Arabidopsis

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs

Pattern dynamics in adaxial-abaxial specific gene expression are modulated by a plastid retrograde signal during Arabidopsis thaliana leaf development.

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes

The <i>enf2</i> mutant shows slow FMB shifting and an abaxialized leaf phenotype.

<p>(A–E) Confocal images of transverse sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression in the wild-type (A) and <i>enf2</i> (B–E) leaf primordia. The arrowheads indicate the provascular cells. (C–E), A series of sections from a leaf of approximately 300 µm in height. The approximate heights of the observation plane from the leaf base are indicated. The comparable WT data are <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003655#pgen-1003655-g002" target="_blank">Figure 2D–F</a>. (F) <i>FIL</i>-expression area sizes (%, y-axis) at different stages (grouped by section area sizes, x-axis) of the wild-type and <i>enf2</i> leaf primordia. Bars indicate standard errors. n.s., not significantly different; *, significantly different (<i>p</i><0.05, t-test) between the wild type and <i>enf2</i>. (G, H) Seedlings of the wild type and <i>enf2</i>. (I, J) Scanning electron microscope images of leaf sections from the wild type and <i>enf2</i>. Scale bars represent 50 µm (A–E, I, J) and 1 mm (G, H). WT, wild type.</p

The boundary between the <i>FIL</i>-expression and miR165/166-free domains shifts during leaf development.

<p>(A–F) Confocal images of longitudinal (A–C) and transverse (D–F) sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression patterns at different stages: 50-µm-long (A), 200-µm-long (B) and 300-µm-long (C–F). Lower schematic illustrations represent each boxed region in (A–C). (D–F), A series of sections from a leaf of approximately 300 µm in height. The approximate heights of the observation plane from the leaf base are indicated in (D–F). Arrowheads indicate the distal (A–C) and marginal (D–F) tip cells. (G–R) Confocal (G, Q, R) and stereoscopic (H–P) images showing VENUS expression patterns (yellow and yellow-green) of the <i>FILpro:CRE-GR 35Spro:loxP-Ter-loxP-VENUS</i> system in the third leaves of 12-day-old plants. The timing of DEX treatment for CRE/loxP recombination is indicated at the bottom left of each panel. The confocal imaging planes are a transverse section of a shoot apex (G) and third leaves (Q, R). The red color represents chlorophyll fluorescence. “+” marks the meristem center in all figures. Scale bars represent 50 µm (A–G), 1 mm (H–P) and 100 µm (Q, R). ad, adaxial side; ab, abaxial side.</p