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BRZ-INSENSITIVE-LONG 1 (BIL1)/BRASSINAZOLE-RESISTANT 1 (BZR1) and its homologues are plant-specific transcription factors that convert the signalling of the phytohormones brassinosteroids (BRs) to transcriptional responses, thus controlling various physiological processes in plants. Although BIL1/BZR1 upregulates some BR-responsive genes and downregulates others, the molecular mechanism underlying the dual roles of BIL1/BZR1 is still poorly understood. Here we show that BR-responsive transcriptional repression by BIL1/BZR1 requires the tight binding of BIL1/BZR1 alone to the 10 bp elements of DNA fragments containing the known 6 bp core-binding motifs at the centre. Furthermore, biochemical and structural evidence demonstrates that the selectivity for two nucleobases flanking the core motifs is realized by the DNA shape readout of BIL1/BZR1 without direct recognition of the nucleobases. These results elucidate the molecular and structural basis of transcriptional repression by BIL1/BZR1 and contribute to further understanding of the dual roles of BIL1/BZR1 in BR-responsive gene regulation.

In plants, vascular stem cells located in the cambium continuously undergo self-renewal and differentiation during secondary growth. Recent advancements in cell sorting techniques have enabled access to the transcriptional regulatory framework of cambial cells. However, mechanisms underlying the robust control of vascular stem cells remain unclear. Here, we identified a new cambium-related regulatory module through co-expression network analysis using multiple transcriptome datasets obtained from an ectopic vascular cell transdifferentiation system using Arabidopsis cotyledons, Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL). The cambium gene list included a gene encoding the transcription factor BES1/BZR1 Homolog 3 (BEH3), whose homolog BES1 negatively affects vascular stem cell maintenance. Interestingly, null beh3 mutant alleles showed a large variation in their vascular size, indicating that BEH3 functions as a stabilizer of vascular stem cells. Genetic analysis revealed that BEH3 and BES1 perform opposite functions in the regulation of vascular stem cells and the differentiation of vascular cells in the context of the VISUAL system. At the biochemical level, BEH3 showed weak transcriptional repressor activity and functioned antagonistically to other BES/BZR members by competing for binding to the brassinosteroid response element. Furthermore, mathematical modeling suggested that the competitive relationship between BES/BZR homologs leads to the robust regulation of vascular stem cells.

Leguminous plants produce nodules for nitrogen fixation; however, nodule production incurs an energy cost. Therefore, as an adaptive strategy, leguminous plants halt root nodule development when sufficient amounts of nitrogen nutrients, such as nitrate, are present in the environment. Although legume NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors have recently been identified, understanding how nodulation is controlled by nitrate, a fundamental question for nitrate-mediated transcriptional regulation of symbiotic genes, remains elusive. Here, we show that two Lotus japonicus NLPs, NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1)/LjNLP4 and NRSYM2/LjNLP1, have overlapping functions in the nitrate-induced control of nodulation and act as master regulators for nitrate-dependent gene expression. We further identify candidate target genes of LjNLP4 by combining transcriptome analysis with a DNA affinity purification-seq approach. We then demonstrate that LjNLP4 and LjNIN, a key nodulation-specific regulator and paralog of LjNLP4, have different DNA-binding specificities. Moreover, LjNLP4–LjNIN dimerization underlies LjNLP4-mediated bifunctional transcriptional regulation. These data provide a basic principle for how nitrate controls nodulation through positive and negative regulation of symbiotic genes.

Application of high concentrations of sodium ascorbate suppresses necrosis caused by the expression of recombinant proteins in Nicotiana benthamiana, resulting in an increase in protein accumulation.

Transient protein expression in plant cells is less time consuming than the production of whole transgenic plants. For transient expression, agroinfiltration is a simple and effective method to deliver transgenes into plant cells. After an Agrobacterium infection, recombinant proteins can be produced in plant cells from 3 to 10days. To increase protein yield, a deconstructed viral vector has been used. This chapter provides a detailed description of the transient expression of recombinant proteins in a well-developed host strain of Nicotiana benthamiana. This study also describes the necessary steps for the extraction of soluble proteins from agroinfiltrated leaves.

Legumes have adaptive mechanisms that regulate nodulation in response to the amount of nitrogen in the soil. In Lotus japonicus, two NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors, LjNLP4 and LjNLP1, play pivotal roles in the negative regulation of nodulation by controlling the expression of symbiotic genes in high nitrate conditions. Despite an improved understanding of the molecular basis for regulating nodulation, how nitrate plays a role in the signaling pathway to negatively regulate this process is largely unknown. Here, we show that nitrate transport via NITRATE TRANSPORTER 2.1 (LjNRT2.1) is a key step in the NLP signaling pathway to control nodulation. A mutation in the LjNRT2.1 gene attenuates the nitrate-induced control of nodulation. LjNLP1 is necessary and sufficient to induce LjNRT2.1 expression, thereby regulating nitrate uptake/transport. Our data suggest that LjNRT2.1-mediated nitrate uptake/transport is required for LjNLP4 nuclear localization and induction/repression of symbiotic genes. We further show that LjNIN, a positive regulator of nodulation, counteracts the LjNLP1-dependent induction of LjNRT2.1 expression, which is linked to a reduction in nitrate uptake. These findings suggest a plant strategy in which nitrogen acquisition switches from obtaining nitrogen from the soil to symbiotic nitrogen fixation.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)—the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

The maltose-binding protein (MBP) fusion tag is one of the most commonly utilized crystallization chaperones for proteins of interest. Recently, this MBP-mediated crystallization technique was adapted to Arabidopsis thaliana (At) BRZ-INSENSITIVE-LONG (BIL1)/BRASSINAZOLE-RESISTANT (BZR1), a member of the plant-specific BZR TFs, and revealed the first structure of AtBIL1/BZR1 in complex with target DNA. However, it is unclear how the fused MBP affects the structural features of the AtBIL1/BZR1-DNA complex. In the present study, we highlight the potential utility of the MBP crystallization chaperone by comparing it with the crystallization of unfused AtBIL1/BZR1 in complex with DNA. Furthermore, we assessed the validity of the MBP-fused AtBIL1/BZR1-DNA structure by performing detailed dissection of crystal packings and molecular dynamics (MD) simulations with the removal of the MBP chaperone. Our MD simulations define the structural basis underlying the AtBIL1/BZR1-DNA assembly and DNA binding specificity by AtBIL1/BZR1. The methodology employed in this study, the combination of MBP-mediated crystallization and MD simulation, demonstrates promising capabilities in deciphering the protein-DNA recognition code.

During secondary growth in plants, vascular stem cells located in the cambium continuously undergo self-renewal and differentiation throughout the lifetime. Recent cell-sorting technique enables to uncover transcriptional regulatory framework for cambial cells. However, the mechanisms underlying the robust control of vascular stem cells have not been understood yet. By coexpression network analysis using multiple transcriptome datasets of an ectopic vascular cell transdifferentiation system using Arabidopsis cotyledons, VISUAL, we newly identified a cambium-specific gene module from an alternative approach. The cambium gene list included a transcription factor BES1/BZR1 homolog 3 (BEH3), whose homolog BES1 is known to control vascular stem cell maintenance negatively. Interestingly, the vascular size of the beh3 mutants showed a large variation, implying the role of BEH3 as a stabilizer. BEH3 almost lost the transcriptional repressor activity and functioned antagonistically with other BES/BZR members via competitive binding to the same motif BRRE. Indeed, mathematical modeling suggests that the competitive relationship among BES/BZRs leads to the robust regulation of vascular stem cells.

Transient protein expression in plant cells is less time consuming than the production of whole transgenic plants. For transient expression, agroinfiltration is a simple and effective method to deliver transgenes into plant cells. After an Agrobacterium infection, recombinant proteins can be produced in plant cells from 3 to 10 days. To increase protein yield, a deconstructed viral vector has been used. This chapter provides a detailed description of the transient expression of recombinant proteins in a well-developed host strain of Nicotiana benthamiana. This study also describes the necessary steps for the extraction of soluble proteins from agroinfiltrated leaves.

Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N38-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N38-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined., Author summary Expression of genes is tightly regulated by transcription factors that physically interact with DNA. Transcription factors recognize specific DNA sequences and selectively regulate the expression of target genes through the specific interactions. We previously found that a group of plant-specific transcription factors, NIGT1 family proteins, recognize two different types of DNA sequences to modulate the expression of a number of genes associated with uptake of nitrogen and phosphorus or related metabolism in Arabidopsis. However, it was unknown how NIGT1 family proteins recognize different types of sequences. In this study, we reveal that dimerization of NIGT1 family proteins is essential to the dual specificity of NIGT1 family proteins and therefore loss of the dimerization ability alters specificity and affinity of NIGT1 family proteins to target DNA sequences. We further suggest that the dimerization of NIGT1 family proteins is an evolutionarily conserved feature to properly respond to changes in nutrient availability. These findings would advance our knowledge on the mode of DNA recognition by transcription factors and have further implications for structural basis on the diverse patterns of the interaction between DNA and transcription factors.

BRZ-INSENSITIVE-LONG HYPOCOTYL 1 (BIL1)/BRASSINAZOLE-RESISTANT 1 (BZR1) is a master transcription factor of brassinosteroid (BR) signalling. The varieties of nucleobase recognition of the NN-BRRE-core motif (NNCGTG), one of variant G-box motifs, distinguish BIL1/BZR1 from basic helix-loop-helix transcription factors, underlying the specific regulation of BR-responsive genes. Here, we show the non-canonical bHLH dimer formation of BIL1/BZR1 to optimize the interaction network with DNA and the orientation of a key residue for NN-BRRE-core motif recognition. The authors report the non-canonical crystal structure of the DNA binding domain from BIL1/BZR1, a transcription factor involved in brassinosteroid signalling, in complex with its target DNA fragment.

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest. We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/ . REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.