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Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity 1-3 . Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development 1,3,4 . However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.The plant hormone auxin controls essentially all aspects of growth and development, and developmental contexts determine its many unique responses 1,2 . TIR1/AFB F-box proteins perceive auxin and promote the ubiquitination and degradation of Aux/IAA transcriptional repressors. Aux/IAAs inhibit DNA-binding ARF transcription factors through direct interaction, and auxin thus releases ARFs from inhibition 3 . Although this signalling module seems simple, each component is encoded by a large gene family in most land plants, particularly in vascular plants 4,5 , allowing overwhelming combinatorial interaction complexity (Fig. 1a; 6 TIR1/AFBs, 29 Aux/IAAs and 23 ARFs; >4,000 combinations in Arabidopsis thaliana). Given different biochemical properties of family members, sets of response components can trigger unique local responses 1,3 , contributing to the paradoxical functional diversity of the chemically simple auxin hormone. Genetic and functional studies in flowering plants suggest functional interactions and trends in diversification among the many ARFs. ARFs are phylogenetically placed into deeply conserved A/B/C classes [4][5][6] . A-ARFs are considered transcriptional activators, and some B-and C-ARFs are considered repressors 7 . Systems-wide interaction analysis among Arabidopsis Aux/IAAs and ARFs suggests more prominent auxin regulation of A-ARFs than B/C-ARFs 8,9 , and individual A-and B-ARFs in the moss Physcomitrella patens can antagonize through competition for DNA sites 10 . However, there are several counterexamples where A-ARFs directly repress targets 11 , Aux/IAAs interact with B/C-ARFs 8,9 and A-and B-ARFs bind different DNA sequences 12 . Because each gene in a multigene family may have sub-or neofunctionalized during evolution, it is entirely unclear what basic biochemical architecture underlies the auxin response system. Recently, we and others have reconstructed the evolutionary history of auxin response components and found that the irreducible complexity in early-diverging land plants encompasses one TIR1/AFB receptor, one Aux/IAA and three ARFs ...

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37 ؎ 3 Å at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol ؊1 . The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9°C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.

Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene-maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF–DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.