Long-term potentiation reconstituted with an artificial TARP/PSD-95 complex

The critical role of AMPA receptor (AMPAR) trafficking in long-term potentiation (LTP) of excitatory synaptic transmission is now well established, but the underlying molecular mechanism is still uncertain. Recent research suggests that PSD-95 captures AMPARs via an interaction with the AMPAR auxiliary subunits-transmembrane AMPAR regulatory proteins (TARPs). To determine if such interaction is a core minimal component of the AMPAR trafficking and LTP mechanism, we engineered artificial binding partners, which individually were biochemically and functionally dead but which, when expressed together, rescue binding and both basal synaptic transmission and LTP. These findings establish the TARP/PSD-95 complex as an essential interaction underlying AMPAR trafficking and LTP

SbCOMT (Bmr12) is involved in the biosynthesis of tricin-lignin in sorghum

Lignin in plant biomass represents a target for engineering strategies towards the development of a sustainable bioeconomy. In addition to the conventional lignin monomers, namely p-coumaryl, coniferyl and sinapyl alcohols, tricin has been shown to be part of the native lignin polymer in certain monocot species. Because tricin is considered to initiate the polymerization of lignin chains, elucidating its biosynthesis and mechanism of export to the cell wall constitute novel challenges for the engineering of bioenergy crops. Late steps of tricin biosynthesis require two methylation reactions involving the pathway intermediate selgin. It has recently been demonstrated in rice and maize that caffeate O-methyltransferase (COMT) involved in the synthesis syringyl (S) lignin units derived from sinapyl alcohol also participates in the synthesis of tricin in planta. In this work, we validate in sorghum (Sorghum bicolor L.) that the O-methyltransferase responsible for the production of S lignin units (SbCOMT / Bmr12) is also involved in the synthesis of lignin-linked tricin. In particular, we show that biomass from the sorghum bmr12 mutant contains lower level of tricin incorporated into lignin, and that SbCOMT can methylate the tricin precursors luteolin and selgin. Our genetic and biochemical data point toward a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units

Expression of a bacterial 3-dehydroshikimate dehydratase (QsuB) reduces lignin and improves biomass saccharification efficiency in switchgrass (Panicum virgatum L.).

BackgroundLignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading.ResultsThe 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass (Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene (pShOMT) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process.ConclusionThis study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants

SbCOMT (Bmr12) is involved in the biosynthesis of tricin-lignin in sorghum.

Lignin in plant biomass represents a target for engineering strategies towards the development of a sustainable bioeconomy. In addition to the conventional lignin monomers, namely p-coumaryl, coniferyl and sinapyl alcohols, tricin has been shown to be part of the native lignin polymer in certain monocot species. Because tricin is considered to initiate the polymerization of lignin chains, elucidating its biosynthesis and mechanism of export to the cell wall constitute novel challenges for the engineering of bioenergy crops. Late steps of tricin biosynthesis require two methylation reactions involving the pathway intermediate selgin. It has recently been demonstrated in rice and maize that caffeate O-methyltransferase (COMT) involved in the synthesis syringyl (S) lignin units derived from sinapyl alcohol also participates in the synthesis of tricin in planta. In this work, we validate in sorghum (Sorghum bicolor L.) that the O-methyltransferase responsible for the production of S lignin units (SbCOMT / Bmr12) is also involved in the synthesis of lignin-linked tricin. In particular, we show that biomass from the sorghum bmr12 mutant contains lower level of tricin incorporated into lignin, and that SbCOMT can methylate the tricin precursors luteolin and selgin. Our genetic and biochemical data point toward a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units

Lignin monomeric composition in wild-type (WT) and <i>bmr12</i> sorghum biomass.

<p>For each genotype, cellulolytic lignin was isolated and analyzed by 2D ¹³C–¹H HSQC NMR spectroscopy. Regions of partial short-range ¹³C–¹H HSQC spectra are shown. Lignin monomer ratios including tricin (T) are provided on the figures. S: syringyl, G: guaiacyl, 5OH-G: 5-hydroxyguaiacyl, H: <i>p</i>-hydroxyphenyl, <i>p</i>CA: <i>p</i>-coumarate, FA: ferulate.</p

Quantification of methanol-soluble luteolin, chrysoeriol, selgin, and tricin extracted from the biomass of wild-type (WT) and <i>bmr12</i> sorghum lines.

<p>Values in <i>bmr12</i> are expressed as a percentage of the values measured in wild-type extracts which correspond to 317 ± 4 µg/g dry weight (DW) for luteolin, 7.8 ± 0.0 µg/g DW for chrysoeriol, 2.0 ± 0.2 µg/g DW for selgin, and 274 ± 3 µg/g DW for tricin. Error bars represent the standard deviation from five experimental replicates. Asterisks indicate significant differences from the wild-type using the unpaired Student’s t-test (*<i>P</i> < 0.05).</p

Amount of tricin in cellulolytic lignin purified from wild-type (WT) and <i>bmr12</i> sorghum lines.

<p>Tricin was released from lignin using the thioacidolysis procedure and subsequently quantified by HPLC-ESI-TOF MS. Error bars represent the standard deviation from three experimental replicates. Asterisks indicate a significant difference from the wild-type using the unpaired Student’s t-test (*<i>P</i> < 0.05).</p

Simplified representation of the lignin and tricin biosynthetic pathways from phenylalanine.

<p>Abbreviations are: Bmr12, Brown midrib12; OMT, <i>O</i>-methyltransferase; SbCOMT, <i>Sorghum bicolor</i> caffeate <i>O</i>-methyltransferase.</p