The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply.

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Whereas several transcriptional regulators of nitrate signaling have been identified, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN7 (NLP7), additional regulators are expected to fine-tune this pivotal physiological response. Here, we characterized Arabidopsis NLP2 as a top-tier transcriptional regulator of the early nitrate response gene regulatory network. NLP2 interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, DNA-binding motif, and some target genes with NLP7. Genetic, genomic, and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multitiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

Interplay between NIN-LIKE PROTEINs 6 and 7 in nitrate signaling.

NLP7 (NIN-LIKE-PROTEIN 7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its homolog, NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared with single mutants, the nlp6nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of the PNR showed that NLP6 and NLP7 govern ∼50% of nitrate-induced genes, with cluster analysis highlighting 2 distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high- and low-nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in high ammonium conditions. Growth phenotypes and transcriptome data revealed that NLP6 and NLP7 are completely functionally redundant and may act as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that differ depending on nitrogen sources and gene clusters.

Controlled Ring Opening of a Tetracyclic Tetraphosphane with Twofold Metallocene Bridging.

A direct route to a doubly ferrocene bridged tetracyclic tetraphosphane 1 was developed via reductive coupling of Fe(CpPCl2)2 (2 a), where a chlorine terminated linear P4-compound 3 could be identified as an intermediate. Selective P-P bond activation was further achieved by reacting 1 with elemental selenium or [Cp*Al]4, where regioselective insertion of Se or Al atoms resulted in ferrocenylene bridged [P4Se] (4) or [P4Al] (7) moieties. Compound 7 can be transformed to a hydrogen terminated linear P4 species, 8, with protic solvents. Methylation of compound 1 with MeOTf, proceeds via intermediate formation of monomethylated species 5, which gradually produced Me2-terminated dicationic 6, again containing a linear P4-unit. Besides spectroscopic characterization, the structural details of compounds 1, 4, 6, and 8 could be determined by SC-XRD. Moreover, DFT calculations were used to rationalize the reactivity of 1, derived compounds and intermediates. As a key feature, 1 undergoes ring opening polymerization to a linear polyphosphane 9.

Iron(II) Complexes of P3 -Chain Ligands: Structural Diversity.

Iron(II) complexes containing ligands with a R2 P-P-PR2 unit were synthesized by metathesis reactions. With R=tBu, a mixture of two isomers is formed; in one of them, the terminal phosphorus binds to the Fe center (ylidic structure), while in the other one, the central P atom is linked to Fe. Starting from differently functionalized parent triphosphanes and corresponding functionalized Fe complexes, the ratio of isomers does not change. The outcome of the reaction and therefore the binding modes of the triphosphane ligands in the resulting compounds can be influenced by the size of the substituents. In the case of R=iPr a chelate complex is formed (both terminal P atoms are linked to the Fe center). Applying the mixed-substituted triphosphane, the ylidic structure of the resulting complex is preferred. The new compounds were characterized by NMR spectroscopy in solution and single-crystal X-ray diffraction in solid-state. The synthetic work was supported by DFT calculations.

Gradual Donor Stabilization of a Transient Ferrocene Bridged Bisphosphanyl Phosphenium Cation.

A transient phosphenium cation embedded into a [3]ferrocenophane scaffold was formed via chloride abstraction. The cation has been trapped with phosphane, carbene, and silylene donors resulting in stable adducts or bond activation of the ferrocenophane bridge. In the absence of donors, dimerization of the phosphenium cation to the corresponding dication is observed or P-C bond activation with migration of a substituent leading to a putative phosphoniodiphosphene. Using 1,3-di-tert-butylimidazol-2-silylene as the donor, further reaction of the initially formed chlorosilane leads to activation of a P-P bond of the ferrocenophane scaffold with ring expansion of the ansa-bridge. The donor formation and bonding situation are investigated by density functional theory calculations as well as experimental methods (e.g., NMR spectroscopy and X-ray crystallography).

Reversible Redox Chemistry of Anionic Imidazole-2-thione-Fused 1,4-Dihydro-1,4-diphosphinines.

Anionic 1,4-dihydro-1,4-diphosphinines were synthesized from tricyclic 1,4-diphosphinines and isolated as blue powdery salts M[2a-2c]. Reaction of solutions of these monoanions with iodomethane led to P-methylated compounds 3a-3c. An oxidation/reduction cycle was examined, starting from solutions of K[2a] via P-P coupled product 4a and back to K[2a], and the recyclability and redox chemistry of this cycle were confirmed by experimental and simulated cyclic voltammetry analysis, which is proposed as a potential 2-electron cathode for rechargeable cells. TD-DFT studies were used to examine species that might be involved in the process.

Chalcogen-Transfer Rearrangement: Exploring Inter- versus Intramolecular P-P Bond Activation.

Invited for the cover of this issue are Rudolf Pietschnig and co-workers at the University of Kassel and Zsolt Kelemen at Budapest University of Technology and Economics. The image depicts Selene, the goddess of the moon in ancient Greek mythology, and the cycle of chalcogen-transfer rearrangement in the starry sky over the hills of the mountainous region of the northern shore of Lake Balaton (Mts. Badacsony and Gulács) in Hungary. Read the full text of the article at 10.1002/chem.202002481.

Phosphetes via Transition Metal Free Ring Closure - Taking the Proper Turn at a Thermodynamic Crossing.

A transition metal free route to phosphetes featuring an exocyclic alkene unit is presented. In this approach phosphanides are added to a variety of diynes generating phosphaallylic intermediates which depending on the reaction conditions transform either to phosphetes or the corresponding phospholes. Investigation of the reaction mechanism by combined quantum chemical and experimental means identifies phosphole formation as thermodynamically controlled reaction path, whereas kinetic control furnishes the corresponding phosphetes. Structural and luminescence properties of the rare class of phosphetes are explored, as well as for selected key intermediates.

Chalcogen-Transfer Rearrangement: Exploring Inter- versus Intramolecular P-P Bond Activation.

tert-Butyl-substituted diphospha[2]ferrocenophane has been used as a stereochemically confined diphosphane to explore the addition of O, S, Se and Te. Although the diphosphanylchalcogane has been obtained for tellurium, all other chalcogens give diphosphane monochalcogenides. The latter transform via chalcogen-transfer rearrangement to the corresponding diphosphanylchalcoganes upon heating. The kinetics of this rearrangement has been followed with NMR spectroscopy supported by DFT calculations. Intermediates during rearrangement point to a disproportionation/synproportionation mechanism for the S and Se derivatives. Cyclic voltammetry together with DFT studies indicate ferrocene-centred oxidation for most of the compounds presented.

Basicity-Tuned Reactivity: diaza-[1,2]-Wittig versus diaza-[1,3]-Wittig Rearrangements of 3,4-Dihydro-2H-1,2,3-benzothiadiazine 1,1-Dioxides.

The base-induced (t-BuOK) rearrangement reactions of 3,4-dihydro-2H-1,2,3-benzothiadiazine 1,1-dioxides result in a ring opening along the N-N bond, followed by ring closure with the formation of new C-N bonds. The position of the newly formed C-N bond can selectively be tuned by the amount of the base, providing access to new, pharmacologically interesting ring systems with high yield. While with 2 equiv of t-BuOK 1,2-benzisothiazoles can be obtained in a diaza-[1,2]-Wittig reaction, with 6 equiv of the base 1,2-benzothiazine 1,1-dioxides can be prepared in most cases as the main product, in a diaza-[1,3]-Wittig reaction. DFT calculations and detailed NMR studies clarified the mechanism, with a mono- or dianionic key intermediate, depending on the amount of the reactant base. Also, the role of an enamide intermediate formed during the workup of the highly basic (6 equiv of base) reaction was clarified. The substrate scope of the reaction was also explored in detail.

Toward a 1,4-Diphosphinine-Based Molecular CPS-Ternary Compound.

Synthesis of the tricyclic 1,3-dithiole-2-thione-derived 1,4-dihydro-1,4-diphosphinine is presented using a base-induced ring formation protocol and chloro(diethylamino)(1,3-dithiole-2-thion-4-yl)phosphane as the starting point. P-oxidation reactions of dihydrodiphosphinine by chalcogens led to bis(P-oxide), bis(P-sulfide), or bis(P-selenide), respectively; all tricyclic compounds were obtained as cis/trans mixtures. 1,4-Dihydro-1,4-diphosphinine was converted into 1,4-dichloro-1,4-dihydro-1,4-diphosphinine. This compound is almost insoluble in organic solvents, furnished selectively the trans-bis(amino) derivative upon a 2-fold P-substitution reaction with the weak nucleophile potassium bis(trimethylsilyl)amide, and reacted also with alcohols ROH (R = nBu, iPr, tBu) to give cis/trans mixtures of the corresponding bis(alkoxy) derivatives. Furthermore, the dichloro derivative could be reduced to a 1,4-diphosphinine using PnBu3, but, unfortunately, the stubbornly insoluble product could be neither purified nor crystallized. Despite this, we achieved a thermal [4 + 2] cycloaddition reaction of this first CPS-ternary compound with diethylacetylene dicarboxylate to obtain the corresponding diphosphabarrelene, thus providing indirect evidence for the aromatic tricyclic diphosphinine. Detailed density functional theory studies on the formation of 1,4-diphosphinine provided insights into formation pathways as well as NMR, IR, and UV/vis data.

2-(Dimethylamino)phosphinine: A Phosphorus-Containing Aniline Derivative.

The yet unknown 2-amino-substituted λ3 ,σ2 -phosphinines are phosphorus-containing aniline derivatives. Calculations show that the strong interaction of the π-donating NR2  group with the aromatic system results in a high π-density at the phosphorus atom. We could now synthesize 2-N(CH3 )2 -functionalized phosphinines, starting from a 3-N(CH3 )2 -substituted 2-pyrone and (CH3 )3 Si-C≡P. Their reaction with CuBr⋅S(CH3 )2 affords CuI  complexes with the first example of a neutral phosphinine acting as a rare bridging µ2 -P-4e donor-ligand between two CuI  centers. Our experimental and theoretical investigations show that 2-aminophosphinines are missing links in the series of known 2-donor-functionalized phosphinines.

Metallacarboranes as Photoredox Catalysts in Water.

Metallacarboranes with the shape of the Greek letter θ, such as [Co(C2 B9 H11 )2 ]- , were tested, for the first time, as efficient photoredox catalysts in the oxidation of aromatic and aliphatic alcohols in water. Their efficiency is linked to their high solubility in water, their high oxidizing power (Co4+/3+ ), and their absence of fluorescence on excitation, among others. In most of the studied examples, using a catalyst load of 0.4 mol % gave high yields of 90-95 % with selectivity greater than 99 %. By reducing the catalyst load to 0.01 mol %, quantitative conversion of reactants to products was achieved, in some cases with greater than 99 % yield, high catalyst efficiency reaching a turnover number of 10 000, and a higher yield with a 45 times lower concentration of catalyst. The metallacarboranes can be recovered easily by precipitation on addition of [NMe4 ]Cl. A pathway for the photoredox-catalyzed oxidation of alcohols is proposed.

Stretching the P-C Bond. Variations on Carbenes and Phosphanes.

The stability and the structure of adducts formed between four substituted phosphanes (PX3, X:H, F, Cl, and NMe2) and 11 different carbenes have been investigated by DFT calculations. In most cases, the structure of the adducts depends strongly on the stability of the carbene itself, exhibiting a linear correlation with the increasing dissociation energy of the adduct. Carbenes of low stability form phosphorus ylides (F), which can be described as phosphane → carbene adducts supported with some back-bonding. The most stable carbenes, which have high energy lone pair, do not form stable F-type structures but carbene → phosphane adducts (E-type structure), utilizing the low-lying lowest unoccupied molecular orbital (LUMO) of the phosphane (with electronegative substituents), benefiting also from the carbene-pnictogen interaction. Especially noteworthy is the case of PCl3, which has an extremely low energy LUMO in its T-shaped form. Although this PCl3 structure is a transition state of rather high energy, the large stabilization energy of the complex makes this carbene-phosphane adduct stable. Most interestingly, in case of carbenes with medium stability both F- and E-type structures could be optimized, giving rise to bond-stretch isomerism. Likewise, for phosphorus ylides (F), the stability of the adducts G formed from carbenes with hypovalent phosphorus (PX-phosphinidene) is in a linear relationship with the stabilization of the carbene. Adducts of carbenes with hypervalent phosphorus (PX5) are the most stable when X is electronegative, and the carbene is highly nucleophilic.

m-Carborane as a Novel Core for Periphery-Decorated Macromolecules.

Closo m-C2B10H12 can perform as a novel core of globular periphery-decorated macromolecules. To do this, a new class of di and tetrabranched m-carborane derivatives has been synthesized by a judicious choice of the synthetic procedure, starting with 9,10-I2-1,7-closo-C2B10H10. The 2a-NPA (sum of the natural charges of the two bonded atoms) value for a bond, which is defined as the sum of the NPA charges of the two bonded atoms, matches the order of electrophilic reaction at the different cluster bonds of the icosahedral o-and m- carboranes that lead to the formation of B-I bonds. As for m-carborane, most of the 2a-NPA values of B-H vertexes are positive, and their functionalization is more challenging. The synthesis and full characterization of dibranched 9,10-R2-1,7-closo-carborane (R = CH2CHCH2, HO(CH2)3, Cl(CH2)3, TsO(CH2)3, C6H5COO(CH2)3, C6H5COO(CH2)3, N3(CH2)3, CH3CHCH, and C6H5C2N3(CH2)3) compounds as well as the tetrabranched 9,10-R2-1,7-R2-closo-C2B10H8 (R = CH2CHCH2, HO(CH2)3) are presented. The X-ray diffraction of 9,10-(HO(CH2)3)2-1,7-closo-C2B10H10 and 9,10-(CH3CHCH)2-1,7-closo-C2B10H10, as well as their Hirshfeld surface analysis and decomposed fingerprint plots, are described. These new reported tetrabranched m-carborane derivatives provide a sort of novel core for the synthesis of 3D radially grown periphery-decorated macromolecules that are different to the 2D radially grown core of the tetrabranched o-carborane framework.

Regulation of FUSCA3 Expression During Seed Development in Arabidopsis.

FUSCA3 (FUS3) is a master regulator of seed development important in establishing and maintaining embryonic identity whose expression is tightly regulated at genetic and epigenetic levels. Despite this prominent role, the control of FUS3 expression remains poorly understood. Promoter and functional complementation analyses provided insight into the regulation of FUS3. W-boxes present in the promoter proximal to the start of transcription are recognized by WRKY type-1 factors which are necessary for the activation of FUS3 expression. The RY motif, the binding site of B3 factors, is important for the activation of FUS3 in the embryo proper but not in the suspensor. The loss of a negative regulatory sequence (NRS) leads to preferential expression of FUS3 in the vasculature of vegetative tissues. Since the NRS includes the RY motif, mechanisms of activation and repression target adjacent or overlapping regions. These findings discriminate the regulation of FUS3 from that of LEAFY COTYLEDON2 by the control exerted by WRKY factors and by the presence of the RY motif, yet also confirm conservation of certain regulatory elements, thereby implicating potential regulation by BASIC PENTACYSTEINE (BPC) factors and POLYCOMB REPRESSIVE COMPLEX2 (PRC2).

Are the Accompanying Cations of Doping Anions Influential in Conducting Organic Polymers? The Case of the Popular PEDOT.

Conducting organic polymers (COPs) are made of a conjugated polymer backbone supporting a certain degree of oxidation. These positive charges are compensated by the doping anions that are introduced into the polymer synthesis along with their accompanying cations. In this work, the influence of these cations on the stoichiometry and physicochemical properties of the resulting COPs have been investigated, something that has previously been overlooked, but, as here proven, is highly relevant. As the doping anion, metallacarborane [Co(C2 B9 H11 )2 ]- was chosen, which acts as a thistle. This anion binds to the accompanying cation with a distinct strength. If the binding strength is weak, the doping anion is more prone to compensate the positive charge of the polymer, and the opposite is also true. Thus, the ability of the doping anion to compensate the positive charges of the polymer can be tuned, and this determines the stoichiometry of the polymer. As the polymer, PEDOT was studied, whereas Cs+ , Na+ , K+ , Li+ , and H+ as cations. Notably, with the [Co(C2 B9 H11 )2 ]- anions, these cations are grouped into two sets, Cs+ and H+ in one and Na+ , K+ , and Li+ in the second, according to the stoichiometry of the COPs: 2:1 EDOT/[Co(C2 B9 H11 )2 ]- for Cs+ and H+ , and 3:1 EDOT/[Co(C2 B9 H11 )2 ]- for Na+ , K+ , and Li+ . The distinct stoichiometries are manifested in the physicochemical properties of the COPs, namely in the electrochemical response, electronic conductivity, ionic conductivity, and capacitance.

A Stabilized Bisphosphanylsilylene and Its Heavier Congeners.

A bisphosphanylsilylene with [3]ferrocenophane backbone and its heavier analogues are described in the form of donor adducts. These heterocarbenes can be formed by dehydrochlorination (Si) or cycloreversion (Sn, Pb) using NHC (N-heterocyclic carbene) tetramethylimidazol-2-ylidene. The structures of the bisphosphanyl-silylene, -stannylene and -plumbylene NHC adducts are presented, and the bonding and stability of these compounds were elucidated using DFT calculations. Reactivity studies confirm the stability of the silylene adduct, where the electrophilic character of the silylene center is comparable to a borane.

Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa.

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta-12-desaturase (FAD2) genes was achieved by CRISPR-Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss-of-function mutants revealed the importance of delta-12-desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.

Deciphering the Molecular Mechanisms Underpinning the Transcriptional Control of Gene Expression by Master Transcriptional Regulators in Arabidopsis Seed.

In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors.