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Abstract: Most calmodulin (CaM) in apo and Ca2+-bound states show a dumb-bell-like structure, involving the N- and C-terminal domains, connected with a flexible linker. However, Ca2+-bound yeast calmodulin (yCaM) takes on a unique globular structure; the target-binding site of this protein is autoinhibited. We applied NMR relaxation dispersion experiments to yCaM in the Ca2+-bound state. The amide 15N and 1HN relaxation dispersion profiles indicated the presence of conformational dynamics for specific residues at the interface between the N- and C-terminal domains. We conclude that these conformational dynamics were derived from the mobility of the C-terminal domain. [Copyright &y& Elsevier]

N-Acetyl-γ-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-γ-glutamyl phosphate to N-acetylglutamate-γ-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His)6] extension. OsAGPR(Ala50–Pro366) was purified and crystals were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P61, with unit-cell parameters a = 86.11, c = 316.3 Å. [ABSTRACT FROM AUTHOR]

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP+ oxidoreductase (FNR). The redox centers in the complex ? the 2Fe?2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR ? are in close proximity; the shortest distance is 6.0 Å. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR. [ABSTRACT FROM AUTHOR]

Studies the hydrogen, nitrogen and carbon backbone and side-chain assignments of the rice phytochrome B PAS1 domain and backbone assignments of the PAS1-PAS2 domain. Regulation of photomorphogenic events ranging from seed germination and deetiolation, to the induction of flowering; Absorption of red light, phytochromes translocate from the cytoplasm to the nucleus; Interactions with transcription factors.

Conferring drought resistant traits to crops is one of the major aims of current breeding programs in response to global climate changes. We previously showed that exogenous application of acetic acid to roots of various plants could induce increased survivability under subsequent drought stress conditions, but details of the metabolism of exogenously applied acetic acid, and the nature of signals induced by its application, have not been unveiled. In this study, we show that rice rapidly induces jasmonate signaling upon application of acetic acid, resulting in physiological changes similar to those seen under drought. The major metabolite of the exogenously applied acetic acid in xylem sap was determined as glutamine—a common and abundant component of xylem sap—indicating that acetic acid is not the direct agent inducing the observed physiological responses in shoots. Expression of drought-responsive genes in shoot under subsequent drought conditions was attenuated by acetic acid treatment. These data suggest that acetic acid activates root-to-shoot jasmonate signals that partially overlap with those induced by drought, thereby conferring an acclimated state on shoots prior to subsequent drought. [ABSTRACT FROM AUTHOR]

• We developed an RNP-mediated CRISPR/Cas9 system in the basidiomycete, Pseudozyma antarctica. • Addition of RNP improved both transformation and gene targeting efficiencies. • Highly efficient gene targeting was successful at two loci in the P. antarctica genome. The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate- co -adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, Pa ADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms. [ABSTRACT FROM AUTHOR]

Abstract Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5′-triphosphates. Highlights • Tomato mosaic virus replication-associated protein 130 K, which contains the methyltransferase and helicase domains, was expressed using the baculovirus-silkworm expression system. • Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution. • 130K protein hydrolyzed nucleoside 5'-triphosphate. [ABSTRACT FROM AUTHOR]

Amylomaltase (AM) catalyzes inter- and intra-molecular transglycosylation reactions of glucan to yield linear and cyclic oligosaccharide products. The functional roles of the conserved histidine at position 461 in the active site of AM from Corynebacterium glutamicum ( Cg AM) was investigated. H461 A/S/D/R/W were constructed, their catalytic properties were compared to the wild-type (WT). A significant decrease in transglucosylation activities was observed, especially in H461A mutant, while hydrolysis activity was barely affected. The transglucosylation factor of the H461A- Cg AM was decreased by 8.6 folds. WT preferred maltotriose (G3) as substrate for disproportionation reaction, but all H461 mutants showed higher preference for maltose (G2). Using G3 substrate, k ca t / K m values of H461 mutated Cg AMs were 40–64 folds lower, while the K m values were twice higher than those of WT. All mutants could not produce large-ring cyclodextrin (LR-CD) product. The heat capacity profile indicated that WT had higher thermal stability than H461A. The X-ray structure of WT showed two H-bonds between H461 and heptasaccharide analog at subsite +1, while no such bonding was observed from the model structure of H461A. The importance of H461 on substrate binding with Cg AM was evidenced. We are the first to mutate an active site histidine in AM to explore its function. [ABSTRACT FROM AUTHOR]

Tm-1, the protein product of Tm-1, a semidominant resistance gene of tomato, inhibits tomato mosaic virus (ToMV) replication by binding to ToMV replication proteins. Previous studies suggested the importance of the Tm-1 N-terminal region for its inhibitory activity; however, it has not been determined if the N-terminal region is sufficient for inhibition. Furthermore, the three-dimensional structure of Tm-1 has not been determined. In this study, an N-terminal fragment of Tm-1 (residues 1–431) as a fusion protein containing an upstream maltose-binding protein was expressed in E. coli Rosetta (DE3) cells at 30°C and then purified. The solubility of the fusion protein was greater when the cells were cultured at 30°C than when cultured at lower or higher temperatures. The purified N-terminal Tm-1 fragment from which the maltose-binding protein tag had been removed has inhibitory activity against ToMV RNA replication. [ABSTRACT FROM AUTHOR]

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA -responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions. [ABSTRACT FROM AUTHOR]

Many seed storage proteins, including monomeric 2S albumin and polymeric prolamin, contain conserved sequences in three separate regions, termed A, B, and C, which contain the consensus motifs LxxC, CCxQL, and PxxC, respectively. Protein-sorting mechanisms in rice (Oryza sativa) endosperm were studied with a green fluorescent protein (GFP) fused to different segments of rice α-globulin, a monomeric, ABC-containing storage protein. The whole ABC region together with GFP was efficiently transported to protein storage vacuoles (type II protein bodies [PB-II]) in the endosperm cells and sequestered in the matrix that surrounds the crystalloids. Peptide Gin-23 to Ser-43 in the A region was sufficient to guide GFP to PB-II. However, GFP fused with the AB or B region accumulated in prolamin protein bodies. Substitution mutations in the CCxQL motif in the B region significantly altered protein localization in the endosperm cells. Furthermore, protein extracts containing these substituted proteins had increased amounts of the endoplasmic reticulum (ER) chaperons BiP (for binding protein), protein disulfide isomerase, and calnexin as a part of protein complexes that were insoluble in a detergent buffer. These results suggest that the ER chaperons and disulfide bonds formed at the dicysteine residues in CCxQL play critical roles in sorting fused proteins in the endosperm cells. [ABSTRACT FROM AUTHOR]

Rice is the staple food for over half the world's population. Genes associated with rice yield include THOUSAND GRAIN WEIGHT 6 (TGW6), which negatively regulates the number of endosperm cells as well as grain weight. The 1-bp deletion allele of tgw6 cloned from the Indian landrace rice cultivar Kasalath, which has lost function, enhances both grain size and yield. TGW6 has been utilized as a target for breeding and genome editing to increase the yield of rice. In the present study, we describe an improved heterologous expression system of TGW6 in Escherichia coli to enable purification of the recombinant protein. The best expression was achieved using codon optimized TGW6 with a 30 amino acid truncation at the N-terminus (Δ30TGW6) in the Rosetta-gami 2(DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Δ30TGW6. Crystals of Δ30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted X-rays to at least 2.6 Å resolution. Herein, we established an efficient procedure for the production and purification of TGW6 in sufficient quantities for structural and functional studies. Detailed information concerning the molecular mechanism of TGW6 will enable the design of more efficient ways to control the activity of the enzyme. • Efficient expression/purification procedures for TGW6 which inhibits the rice yields. • Codon optimization and N-terminus truncation improved the yield of recombinant TGW6. • The stability of TGW6 was greatly influenced by the calcium ion. • The purified TGW6 was crystallized and diffracted X-rays at 2.6 Å resolution. [ABSTRACT FROM AUTHOR]

Cassia nomame , an annual herb belonging to the Leguminosae family, is traditionally consumed as tea in the form of dried whole herbs or beans. To deepen our understanding of the chemical constituents of C. nomame, we isolated 16 compounds, categorised as flavone derivatives ( 1 - 4 ), including a novel compound nomameflavone A ( 4 ), a chalcone derivative ( 5 ), an aurone derivative ( 6 ), a chromone derivative ( 7 ), a juglone derivative ( 8 ), anthraquinone derivatives ( 9 - 11 ), phenolic compounds ( 12 - 14 ), and sterol derivatives ( 15 , 16 ). The structure of each compound was elucidated using NMR and MS. Compounds 2 , 6 , 7 , 8 , 12 , 13 , and 14 were successfully isolated from C. nomame for the first time. Furthermore, compounds 1 , 3 , 5 , and 6 exhibited DPPH radical scavenging activities, and the bioactivity of 3 was discovered. The elucidation of the diverse chemical constituents of C. nomame underscores its potential functional ingredients.

Background and Aims: HCO3- can be a major carbon resource for photosynthesis in underwater environments. Here we investigate the underlying mechanism of uptake and membrane transport of HCO3- in submerged leaves of Hygrophila difformis, a heterophyllous amphibious plant. To characterize these mechanisms, we evaluated the sensitivity of underwater photosynthesis to an external carbonic anhydrase (CA) inhibitor and an anion exchanger protein inhibitor, and we attempted to identify components of the mechanism of HCO3- utilization., Methods: We evaluated the effects of the external CA inhibitor and anion exchanger protein inhibitor on the NaHCO3 response of photosynthetic O2 evolution in submerged leaves of H. difformis. Furthermore, we performed a comparative transcriptomic analysis between terrestrial and submerged leaves., Key Results: Photosynthesis in the submerged leaves was decreased by both the external CA inhibitor and anion exchanger protein inhibitor, but no additive effect was observed. Among upregulated genes in submerged leaves, two α-CAs, Hdα-CA1 and Hdα-CA2, and one β-carbonic anhydrase, Hdβ-CA1, were detected. Based on their putative amino acid sequences, the α-CAs are predicted to be localized in the apoplastic region. Recombinant Hdα-CA1 and Hdβ-CA1 showed dominant CO2 hydration activity over HCO3- dehydration activity., Conclusions: We propose that the use of HCO3- for photosynthesis in submerged leaves of H. difformis is driven by the cooperation between an external CA, Hdα-CA1, and an unidentified HCO3- transporter., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance ( 19 F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed β-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19 F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis., (© 2024. The Author(s).)

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yanagawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fragment-based screening using 19 F NMR ( 19 F-FS) is an efficient method for exploring seed and lead compounds for drug discovery. Here, we demonstrate the utility and merits of using 19 F-FS for methionine γ-lyase-binding fragments, together with a 19 F NMR-based competition and mutation assay, as well as enzymatic and in silico methods. 19 F NMR-based assays provided useful information on binding between 19 F-FS hit fragments and target proteins. Although the 19 F-FS and enzymatic assay were weakly correlated, they show that the 19 F-FS hit fragments contained compounds with inhibitory activity. Furthermore, we found that in silico calculations partially account for the differences in activity levels between the 19 F-FS hits as per NMR analysis. A comprehensive approach combining the 19 F-FS and other methods not only identified fragment hits but also distinguished structural differences in chemical groups with diverse activity levels.

Solid-state cross-polarization magic-angle spinning carbon-13 nuclear magnetic resonance ( 13 C CP/MAS NMR) spectroscopy is used to analyze starch derived from plants including wheat, maize, and potato, but few reports have described its application to rice starch. Here, we combined 13 C CP/MAS NMR with deconvolution and subtraction methods to analyze rice lines including mutants that are deficient in at least one enzyme involved in amylose and/or amylopectin biosynthesis. We found that differences in the content of ordered structures between rice lines could be evaluated using C1 signal deconvolution and subtraction. The content of the V-type ordered structure increased with increasing amylose content. Furthermore, starch derived from a starch synthase (SS) IIIa/starch branching enzyme (BE) IIb-deficient mutant formed B- and V-type ordered structures and significantly more nonordered structures than the other rice lines. These data indicate that 13 C CP/MAS NMR analysis is useful for discriminating the genetic backgrounds of starch derived from different rice cultivars., Competing Interests: The authors declare no competing financial interest.

19 F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19 F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19 F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19 F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A recently introduced (13)C polarization technique based on the nuclear Overhauser effect in rotating solid (nuclear Overhauser polarization-magic-angle spinning, NOP-MAS) (Takegoshi, K.; Terao, T. J. Chem. Phys. 2002, 117, 1700-1707) is applied to uniformly (13)C, (15)N-labeled proteins. NOP enhancement factors per scan of 1.5 approximately 2.0 are obtained, while that by cross polarization (CP) is less than 1.0. We show that uniform enhancement of all (13)C signals by CP is difficult to attain, while it is easily achieved by NOP, thus enabling quantitative comparison of signal intensities. NOP is easy to carry out under fast MAS and works well even for somewhat mobile molecules, for which CP does not work. Moreover, in labeled protein samples containing nonlabeled additives, NOP can eliminate the latter signals. For these features, NOP is superior to CP in many uniformly (13)C labeled proteins.

The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors.