The structure of B-ARR reveals the molecular basis of transcriptional activation by cytokinin.

The phytohormone cytokinin has various roles in plant development, including meristem maintenance, vascular differentiation, leaf senescence, and regeneration. Prior investigations have revealed that cytokinin acts via a phosphorelay similar to the two-component system by which bacteria sense and respond to external stimuli. The eventual targets of this phosphorelay are type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs), containing the conserved N-terminal receiver domain (RD), middle DNA binding domain (DBD), and C-terminal transactivation domain. While it has been established for two decades that the phosphoryl transfer from a specific histidyl residue in ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEINS (AHPs) to an aspartyl residue in the RD of B-ARRs results in a rapid transcriptional response to cytokinin, the underlying molecular basis remains unclear. In this work, we determine the crystal structures of the RD-DBD of ARR1 (ARR1RD-DBD) as well as the ARR1DBD-DNA complex from Arabidopsis. Analyses of the ARR1DBD-DNA complex have revealed the structural basis for sequence-specific recognition of the GAT trinucleotide by ARR1. In particular, comparing the ARR1RD-DBD and ARR1DBD-DNA structures reveals that unphosphorylated ARR1RD-DBD exists in a closed conformation with extensive contacts between the RD and DBD. In vitro and vivo functional assays have further suggested that phosphorylation of the RD weakens its interaction with DBD, subsequently permits the DNA binding capacity of DBD, and promotes the transcriptional activity of ARR1. Our findings thus provide mechanistic insights into phosphorelay activation of gene transcription in response to cytokinin.

Cryo-EM structure and molecular mechanism of abscisic acid transporter ABCG25.

Abscisic acid (ABA) is one of the plant hormones that regulate various physiological processes, including stomatal closure, seed germination and development. ABA is synthesized mainly in vascular tissues and transported to distal sites to exert its physiological functions. Many ABA transporters have been identified, however, the molecular mechanism of ABA transport remains elusive. Here we report the cryogenic electron microscopy structure of the Arabidopsis thaliana adenosine triphosphate-binding cassette G subfamily ABA exporter ABCG25 (AtABCG25) in inward-facing apo conformation, ABA-bound pre-translocation conformation and outward-facing occluded conformation. Structural and biochemical analyses reveal that the ABA bound with ABCG25 adopts a similar configuration as that in ABA receptors and that the ABA-specific binding is dictated by residues from transmembrane helices TM1, TM2 and TM5a of each protomer at the transmembrane domain interface. Comparison of different conformational structures reveals conformational changes, especially those of transmembrane helices and residues constituting the substrate translocation pathway during the cross-membrane transport process. Based on the structural data, a 'gate-flipper' translocation model of ABCG25-mediated ABA cross-membrane transport is proposed. Our structural data on AtABCG25 provide new clues to the physiological study of ABA and shed light on its potential applications in plants and agriculture.

The oligomeric structures of plant cryptochromes.

Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.

Publisher Correction: The oligomeric structures of plant cryptochromes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Kinetically Controllable Hydrogen Generation at Low Temperatures by the Alcoholysis of CaMg2 -Based Materials in Tailored Solutions.

The alcoholysis of CaMg2 -based materials for hydrogen generation is reported. Compared to hydrolysis in water, hydrogen supply from alcoholysis shows an excellent potential for outdoor applications, which not only bypasses the formation of passivation layers deposited on the surface of particles but also breaks the temperature bottleneck in which hydrolysis occurs over 0 °C. To remove the troublesome freezing issue of the water solution system in low-temperature conditions, here, instead of pure methanol, methanol/water and methanol/ethanol solutions are applied to react with CaMg2 alloy (CM2 ) and its hydrides (H-CM2 ) for hydrogen generation. Compared with pure water and ethanol, the reaction of CaMg2 -based materials with methanol possesses much faster reaction kinetics and gives a considerable hydrogen yield. CM2 can generate 858 mL H 2 g-1 within only 3 min at room temperature as it reacts vigorously with methanol, as opposed to a low hydrogen yield with ethanol and water (395 and 224 mL H 2 g-1 within 180 min, respectively) under the same conditions. Even at -20 °C, there is still over 600 mL H 2 g-1 released at a conversion rate of 70.7 % within 100 min for methanolysis, which shows its prominent advantage for hydrogen production, especially in winter or subzero areas. Interestingly, the methanolysis byproducts can transform into metal hydroxides and methanol in the reaction with water, and the methanol may be separated and reused as an intermediate. Moreover, the hydrogen behavior of CaMg2 methanolysis can be well controlled by tailoring the components of the solutions to deliver a promising hydrogen supply system for the hydrogen economy.

Two-dimensional triphenylene cored hole-transporting materials for efficient perovskite solar cells.

Two organic hole-transporting materials comprising a two-dimensional triphenylene core and methoxyl-arylamine terminal units are developed and applied in perovskite solar cells. Enhanced photovoltaic and stability performance are obtained using TPH-T compared with those of spiro-OMeTAD.

Structural and Biochemical Insights Into Two BAHD Acyltransferases (AtSHT and AtSDT) Involved in Phenolamide Biosynthesis.

Phenolamides represent one of the largest classes of plant-specialized secondary metabolites and function in diverse physiological processes, including defense responses and development. The biosynthesis of phenolamides requires the BAHD-family acyltransferases, which transfer acyl-groups from different acyl-donors specifically to amines, the acyl-group acceptors. However, the mechanisms of substrate specificity and multisite-acylation of the BAHD-family acyltransferases remain poorly understood. In this study, we provide a structural and biochemical analysis of AtSHT and AtSDT, two representative BAHD-family members that catalyze the multisite acylation of spermidine but show different product profiles. By determining the structures of AtSHT and AtSDT and using structure-based mutagenesis, we identified the residues important for substrate recognition in AtSHT and AtSDT and hypothesized that the acyl acceptor spermidine might adopt a free-rotating conformation in AtSHT, which can undergo mono-, di-, or tri-acylation; while the spermidine molecule in AtSDT might adopt a linear conformation, which only allows mono- or di-acylation to take place. In addition, through sequence similarity network (SSN) and structural modeling analysis, we successfully predicted and verified the functions of two uncharacterized Arabidopsis BAHD acyltransferases, OAO95042.1 and NP_190301.2, which use putrescine as the main acyl-acceptor. Our work provides not only an excellent starting point for understanding multisite acylation in BAHD-family enzymes, but also a feasible methodology for predicting possible acyl acceptor specificity of uncharacterized BAHD-family acyltransferases.

Structural basis of rifampin inactivation by rifampin phosphotransferase.

Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.

Fabrication of superhydrophobic/superoleophilic cotton for application in the field of water/oil separation.

Cotton with superhydrophobic and superoleophilic properties had been successfully fabricated for application in the field of oil/water separation by the combination of SiO2 nanoparticles on cotton fiber surface and subsequent octadecyltrichlorosilane modification. The as-prepared cotton could be used to selectively absorb various common oils and organic solvents up to above 50 times of its own weight while repelling water completely. The absorbed oils were easily collected by a simple vacuum filtration, and the recovered cotton could be reused for several cycles while still keeping high absorption capacity. Moreover, the as-prepared cotton was simply spun into cloth, which not only could be tailored to the water-repellent clothing but also could be used in the oil/water separation filter system. The results presented in this work might provide a simple, low-cost and environment friendly approach for application in the field of water/oil separation.

Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli.

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu(2+), either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu(2+) associated proenzymes. Cu(2+) associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu(2+) showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu(2+) than rPPO2 did. Surprisingly, however, high concentration of Cu(2+) (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2