Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C or C photosynthesis. As part of a multinational effort to introduce C traits into rice to boost crop yield, candidate regulators of C leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C -like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C plants

An efficient transformation method for genome editing of elite bread wheat cultivars

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far

Water exchange reaction of a manganese catalase mimic: oxygen-17 NMR relaxometry study on (aqua)manganese(III) in a salen scaffold and its reactions in a mildly basic medium

The kinetics of water exchange for trans-[MnIII(salen)(OH2)2]+ has been investigated by the 17O NMR line broadening technique in 17OH2 enriched aqueous medium. The rate and activation parameters for the exchange reaction, trans-[MnIII(salen)(OH2)2]+ + 17OH2 → trans-[MnIII(salen)(OH2)(17OH2)]+ + H2O are: k298ex/s−1 = 3.8 × 106, ΔH≠/kJ mol−1 = 18.3 ± 5.9, ΔS≠/J K−1 mol−1 = −57.6 ± 20.2, which support the associative interchange mechanism (Ia). The kinetic parameters for the corresponding reaction of trans-[MnIII(salen)(OH2)(OH)] are but tentative (k298ex/s−1 = 3.5 × 107, ΔH≠/kJ mol−1 = 54.1 ± 4.3, ΔS≠/J K−1 mol−1 = +80.9 ± 17.0) due to its dimerization, and thermal instability of the dimer in a basic medium (pH ∼ 10). The kinetic parameters for the dimerisation of the (aqua)(hydroxo) complex are: k2981/dm−3 mol−1 s−1 = 1.7 ± 0.4, ΔH≠/kJ mol−1 = 52 ± 4, ΔS≠/J K−1 mol−1 = −64 ± 14; the reaction is reversible with a virtually temperature insensitive equilibrium constant, K298eq./dm3 mol−1 = 1.5 ± 0.3 × 105. The negative value of the activation entropy is also consistent with an associative interchange mechanism (Ia) for the dimerisation reaction. The reaction mixture is EPR silent in X-band mode thus indicating that the +3 oxidation state of Mn is preserved. The dimer (OH2)MnIII(salen)(μ-OH)–MnIII(salen)(OH) decays to hydrolysed product(s) obeying first order kinetics with k2982/s−1 = (0.8 ± 0.1) × 10−5. The relevance of water exchange kinetics of [MnIII(salen)(OH2)(OH2/OH)]+/0 with catalase and SOD (superoxide dismutase) activities of [MnIII(salen)] has been discussed

Ligand substitution and electron transfer reactions of trans-(diaqua)(salen)manganese(iii) with oxalate: an experimental and computational study

The trans-Mn^III(salen)(OH₂)₂⁺ undergoes reversible aqua ligand substitution by HOX⁻ (H₂salen = N,N′-bis(salicylidene)ethane-1,2-diamine; HOX⁻ = ⁻O–COCO₂H) with k₁/dm³ mol⁻¹ s⁻¹ (k₋₁/s⁻¹) = 11.8 ± 0.7 (0.255 ± 0.02), ΔH≠/kJ mol⁻¹ = 54.6 ± 0.8 (64.2 ± 6.7), ΔS≠/J K⁻¹ mol⁻¹ = −41.2 ± 2.6 (−40.8 ± 22.7) at 25.0 °C and I = 0.3 mol dm⁻³. The low values of the activation enthalpy and nearly the same and negative values of the activation entropy are ascribed to an associative transition state for this interchange process (I_a mechanism). The redox reaction that follows involves several paths and the products are Mn^II and CO₂ identified by ESR spectroscopy and conventional test, respectively. The rate retardation by acrylamide monomer with no perceptible polymerization during the course of the redox reaction supports the involvement of the radical intermediate, C₂O₄−˙ (= CO₂ + CO₂−˙) which succeeds in reducing Mn^III species much faster than the dimerisation of its congener, CO₂^−˙ in keeping with the stoichiometry, |[ΔMn^III]/Δ[OX]| = 2. The trans-[Mn^III(salen)(OH₂)(HOX) and its conjugate base, trans-Mn^III(salen)(OH₂)(OX)− are virtually inert to intramolecular reduction of the Mn^III centre by the bound oxalate species but undergo facile electron transfer by H₂OX, HOX⁻ and very slowly by OX²⁻ following the reactivity sequence, k_H₂OX > kHOX [triple gtr-than] kOX and featuring second order kinetics. The rate retardation by the anionic micelles of SDS (sodium dodecyl sulfate) and rate enhancement by N3− provide supportive evidence in favor of the proposed mechanistic pathways. The structure optimization of trans-Mn^III(salen)(OH₂)(HOX) (A), trans-Mn^III(salen)(HOX)₂⁻ (B), trans-Mn^III(salen)(OH₂)(OX)⁻ (C), trans-Mn^III(salen)(OH₂)(H₂OX)⁺ (E1), and trans-Mn^III(salen)(HOX)(H₂OX) (E2) {all high spin Mn^III(d⁴)} by Density Functional Theory (DFT) reveals that the structural trans-effect of the unidentately bonded OX²⁻ in C is the strongest and Mn^III assumes five coordination with the H2O molecule (displaced from the Mn^III centre), hydrogen bonded to the phenoxide oxygen moiety. The computational study highlights different modes of H-bonding in structures A–E. The activation parameters for the redox reactions, A + HOX⁻ and A + H₂OX, ΔH≠/kJ mol⁻¹ (ΔS≠/J K⁻¹ mol⁻¹): 42.5 ± 6.2, (−106 ± 20) and 71.7 ± 7.7 (+12 ± 25), respectively, are indicative of different degrees of ordering and reorganization of bonds as expected in the case of a proton coupled electron transfer (PCET) process