Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation

Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PAL1 and PAL2. However, a complete blocking of flavonoid biosynthesis at CHS released PAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols

The Arabidopsis Glucosyltransferase UGT76B1 Conjugates Isoleucic Acid and Modulates Plant Defense and Senescence[C][W][OA]

The orphan small-molecule glycosyltransferase UGT76B1 is a novel player in plant pathogen defense that represses the salicylic acid pathway, yet enhances the jasmonic acid pathway. A nontargeted metabolome approach led to the identification of its substrate, 2-hydroxy-3-methyl-pentanoic acid, which itself was able to induce resistance against Pseudomonas syringae infection

FGF/FGFR2 signaling regulates the generation and correct positioning of Bergmann glia cells in the developing mouse cerebellum.

The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse

Reduced numbers and mispositioning of <i>Tnc</i>⁺ BG cells in the EGL of the <i>Fgfr2</i> cKO CbA.

<p>(<b>A–J</b>) Representative sagittal brightfield views of E16.5 (A–D, n = 4 embryos/genotype), E17.5 (E–H, n = 1 embryo/genotype) and E18.5 (I,J, n = 3 embryos/genotype) control (A,C,E,G,I) and <i>Fgfr2</i> cKO (B,D,F,H,J) cerebella hybridized with radioactive <i>Tnc</i> (A–F,I,J) and <i>Fgfr2</i> (G,H) riboprobes. (C,D) are higher magnifications of the boxed areas in (A,B). (E–H) are higher magnifications of the anterior CbA in adjacent sections from control or mutant embryos. Red arrowheads in (D,F) point at ectopically positioned <i>Tnc</i>⁺ BG cells in the mutant EGL. Note the complete absence of the <i>Fgfr2</i> ISH signal correlating with less <i>Tnc</i>⁺ and intensely Nissl-stained cells in the CbA of the <i>Fgfr2</i> cKO embryo shown in (F,H), although some <i>Fgfr2</i>⁺ cells are detected in the (non-neural) mesenchyme overlying the mutant EGL. Red dotted line in (A,I) delimits the anterior area used for quantification. (<b>K,L</b>) High magnification views of the EGL and PCL on adjacent sections from an E18.5 control (wild-type) embryo, hybridized with a radioactive riboprobe for <i>Fgfr2</i> (red in K) or <i>Tnc</i> (black in L). Black arrowheads point at intensely Nissl-stained cells showing an ISH signal for <i>Fgfr2</i> (K) or <i>Tnc</i> (L). Empty arrowheads point at larger, weakly Nissl-stained cells devoid of <i>Fgfr2</i> (K) or <i>Tnc</i> (L) ISH signals. (<b>M,N</b>) Quantification of <i>Tnc</i>⁺ cells in the anterior CbA (M) and EGL (N) of control (grey bars) and mutant (white bars) embryos at E16.5 and E18.5 (<i>Tnc</i>⁺ cells/µm² (M): E16.5: control, 6.49×10⁻⁴±2.5×10⁻⁵ (n = 4 embryos); <i>Fgfr2</i> cKO, 4.43×10⁻⁴±2.4×10⁻⁵ (n = 4 embryos); E18.5: control, 9.41×10⁻⁴±5.9×10⁻⁵ (n = 3 embryos); <i>Fgfr2</i> cKO, 6.25×10⁻⁴±5.6×10⁻⁵ (n = 3 embryos); <i>Tnc</i>⁺ cells in anterior EGL (N): E16.5: control, 3.00±0.41 (n = 4 embryos); <i>Fgfr2</i> cKO, 17.00±1.29 (n = 4 embryos); E18.5: control, 5.67±0.88 (n = 3 embryos); <i>Fgfr2</i> cKO, 13.33±2.33 (n = 3 embryos); Student's <i>t</i>-test). EGL, external granular layer; PCL, Purkinje cell layer; VZ, cerebellar ventricular zone. Scale bars: 100 µm (B); 50 µm (D,H); 30 µm (L).</p

FGF target gene activation is almost completely abolished in the CbA of <i>Fgfr2</i> cKO embryos.

<p>(<b>A–P</b>) Representative sagittal brightfield views of E16.5 (A–H; n = 5 embryos/genotype) and E18.5 (I-P; n = 4 embryos/genotype) control (A,C,E,G,I,K,M,O) and <i>Fgfr2</i> cKO (B,D,F,H,J,L,N,P) cerebella, hybridized with riboprobes for <i>Etv5</i> (A–D,I–L) and <i>Tnc</i> (E,F,M,N). (C,D) and (K,L) are higher magnifications of the boxed areas in (A,B) and (I,J), respectively. (G,H) and (O,P) are pseudo-colored overlays (<i>Etv5</i> in red, <i>Tnc</i> in green, overlapping expression domains appear in yellow) of the adjacent sections shown in (C–F) and (K–N), respectively. Red arrowheads in (D,F,L,N) point at ectopic <i>Etv5</i>⁺ (D,L) or <i>Tnc</i>⁺ (F,N) cells in the anterior EGL of the mutant embryos. Note that at E18.5, the ectopic <i>Etv5</i>⁺ cells are predominantly located in the outer margin of the EGL, whereas the ectopic <i>Tnc</i>⁺ cells are mostly confined to the inner EGL. EGL, external granular layer; PCL, Purkinje cell layer. Scale bars: 100 µm (B); 50 µm (H).</p

<i>Fgfr2</i> deficiency leads to locomotor deficits in adult mice.

<p>(<b>A–D</b>) Cresyl-violet stained brightfield (A,C) and darkfield (B,D) views of sagittal sections from adult <i>Fgfr2^lox/lox</i> (control, A,B) and <i>Nestin-Cre;Fgfr2^lox/lox</i> (<i>Fgfr2</i> cKO, C,D) cerebella, hybridized with a radioactive <i>Fgfr2 exon 5</i> riboprobe. (<b>E</b>) Western blotting detected the full-length FGFR2 protein (approx. 100 kD) in brain lysates of adult <i>Fgfr2^lox/lox</i> (c, control) and <i>Nestin-Cre;Fgfr2^+/lox</i> (het, heterozygote) but not <i>Nestin-Cre;Fgfr2^lox/lox</i> (hom, homozygote) mice. Hprt is the loading control. (<b>F–J</b>) Behavioral tests revealed an altered horizontal locomotion (maximum velocity (F) and total distance travelled (G)) and unsupported vertical locomotion (latency to first rearing (H) and number of rearings (I) on the board) of male <i>Fgfr2</i> cKO (blue squares; n = 12 males) compared with control (red circles; n = 15 males) mice in the modified hole board paradigm, but no significant differences between both genotypes in the accelerating Rotarod performance (measured by mean latency to fall, J). Values are given in Table S1. I-X, lobuli of the adult cerebellum; ChPl, choroid plexus. Scale bar (C): 500 µm.</p

Strong reduction and ectopic positioning of Sox2⁺/Blbp⁺ BG precursors and cells in the <i>Fgfr2</i> cKO CbA.

<p>(<b>A–J</b>) Representative confocal overviews (A,D,G,I) and close-up views (B,C,E,F,H,J) of the CbA on sagittal sections from control (A,B,C,G,H) and <i>Fgfr2</i> cKO (D,E,F,I,J) embryos at E16.5 (A–F; n = 4 embryos/genotype) and E18.5 (G–J; n = 3 embryos/genotype), immunostained for Sox2 (green) and Blbp (Brain lipid binding protein, red). A marked reduction of Sox2⁺/Blbp⁺ BG precursors/cells was observed in the <i>Fgfr2</i> cKO (D,I) compared to control (A,G) embryos at both stages. A reduction of Sox2⁺ and Blbp⁺ neural progenitors and RG/BG precursors was also apparent in the anterior (left) part of the cerebellar VZ of the mutant embryos at E16.5 (A,D), whereas at E18.5, Sox2⁺ and Blbp⁻ neural progenitor cells appeared to accumulate in the cerebellar VZ of the <i>Fgfr2</i> cKO embryos (G,I). Close-up views (B,C,E,F,H,J) of the boxed areas in (A,D,G,I) revealed an increased number of ectopically positioned Sox2⁺/Blbp⁺ BG cells (white arrowheads) within the EGL of the <i>Fgfr2</i> cKO embryos at E16.5 and E18.5. Only few Blbp⁺ BG fibers reached the pial surface (dashed line in C,F) of the mutant CbA, and these fibers were frequently arranged in a parallel (tangential) rather than perpendicular (radial) manner relative to this surface (white arrows in E,J). EGL, external granular layer; PCL, Purkinje cell layer; VZ, cerebellar ventricular zone. Scale bars: 500 µm (A), 100 µm (B,C).</p