The ZmbHLH47-ZmSnRK2.9 Module Promotes Drought Tolerance in Maize.

Drought stress globally poses a significant threat to maize (Zea mays L.) productivity and the underlying molecular mechanisms of drought tolerance remain elusive. In this study, we characterized ZmbHLH47, a basic helix-loop-helix (bHLH) transcription factor, as a positive regulator of drought tolerance in maize. ZmbHLH47 expression was notably induced by both drought stress and abscisic acid (ABA). Transgenic plants overexpressing ZmbHLH47 displayed elevated drought tolerance and ABA responsiveness, while the zmbhlh47 mutant exhibited increased drought sensitivity and reduced ABA sensitivity. Mechanistically, it was revealed that ZmbHLH47 could directly bind to the promoter of ZmSnRK2.9 gene, a member of the subgroup III SnRK2 kinases, activating its expression. Furthermore, ZmSnRK2.9-overexpressing plants exhibited enhanced ABA sensitivity and drought tolerance, whereas the zmsnrk2.9 mutant displayed a decreased sensitivity to both. Notably, overexpressing ZmbHLH47 in the zmsnrk2.9 mutant closely resembled the zmsnrk2.9 mutant, indicating the importance of the ZmbHLH47-ZmSnRK2.9 module in ABA response and drought tolerance. These findings provided valuable insights and a potential genetic resource for enhancing the environmental adaptability of maize.

Structural Insights into Polymer-Bounded Lipid Nanodiscs.

Membrane proteins are an essential part of signaling and transport processes and are targeted by multiple drugs. To isolate and investigate them in their native state, polymer-bounded nanodiscs have become valuable tools. In this study, we investigate the lipid model system dimyristoyl-phosphocholine (DMPC) with the nanodisc-forming copolymers styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA). Using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we studied the influence of polymer concentration and temperature on the nanodisc structure. In Tris buffer, the size of nanodiscs formed with SMA is smaller compared to DIBMA at the same polymer ratio. In both cases, the size decreases monotonically with increasing polymer concentration, and this effect is more pronounced when using SMA. Measurements at temperatures (T) between 5 and 30 °C in phosphate buffer showed an incomplete solubilization at high T even at polymer/lipid ratios above that required for complete lipid solubilization. For DIBMA, the nanodiscs developed at lower temperatures are stable and the net repulsion increases, while for SMA, the individual nanodiscs possess smaller sizes and are less affected by T. However, using DLS, one can observe SMA agglomerates at low T. Interestingly, for both polymers, no drastic changes of the observable parameters (radius and bilayer thickness) are seen upon cooling, which would indicate a sharp (first-order) phase transition from liquid-crystalline to gel, but only gradual changes. Hence, we conclude that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer. These results can pave the way toward the development of better protocols for studying membrane proteins stabilized in this type of membrane mimics.

Effective interactions and phase behavior of protein solutions in the presence of hexamine cobalt(III) chloride.

It is well established that deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) exhibit a reentrant condensation (RC) phase behavior in the presence of the trivalent hexamine cobalt(III) cations (Hac) which can be important for their packing and folding. A similar behavior can be observed for negatively charged globular proteins in the presence of trivalent metal cations, such as Y3+ or La3+. This phase behavior is mainly driven by charge inversion upon an increasing salt concentration for a fixed protein concentration (cp). However, as Hac exhibits structural differences compared to other multivalent metal cations, with six ammonia ligands (NH3) covalently bonded to the central cobalt atom, it is not clear that Hac can induce a similar phase behavior for proteins. In this work, we systematically investigate whether negatively charged globular proteins ß-lactoglobulin (BLG), bovine serum albumin (BSA), human serum albumin (HSA) and ovalbumin (OVA) feature Hac-induced RC. Effective protein-protein interactions were investigated by small-angle X-ray scattering. The reduced second virial coefficient (B2/B2HS) was obtained as a function of salt concentration. The virial coefficient analysis performed confirms the reentrant interaction (RI) behavior for BLG without actually inducing RC, given the insufficient strengths of the interactions for the latter to occur. In contrast, the strength of attraction for BSA, HSA and OVA are too weak to show RC. Model free analysis of the inverse intensity [Formula: see text] also supports this finding. Looking at different q-range by employing static (SLS) and dynamic light scattering experiments, the presence of RI behavior can be confirmed. The results are further discussed in view of metal cation binding sites in nucleic acids (DNA and RNA), where Hac induced RC phase behavior.

An alternative approach to the osmotic second virial coefficient of protein solutions and its application to liquid-liquid phase separation.

The osmotic second virial coefficient B2 is an important parameter to describe the interactions and phase behavior of protein solutions, including colloidal systems and macromolecular solutions. Another key parameter to describe the driving force of the nucleation of a new phase is the supersaturation, which is used in the classical nucleation theory framework and is connected with the favorable contribution in the Gibbs free energy in the bulk solution. In this article, we establish a connection between B2 calculated from small angle x-ray scattering (SAXS) data and the values of B2 obtained from supersaturation measurements using thermodynamics considerations. The values of the second virial coefficient calculated employing this method agree with those determined via SAXS in the region near the liquid-liquid phase separation border for human serum albumin and bovine serum albumin. The general relations adopted are shown to be useful for the estimation of the second virial coefficient B2 for globular proteins, in the proximity of the binodal biphasic coexistent region.

Effects of temperature and ionic strength on the microscopic structure and dynamics of egg white gels.

We investigate the thermal gelation of egg white proteins at different temperatures with varying salt concentrations using x-ray photon correlation spectroscopy in the geometry of ultra-small angle x-ray scattering. Temperature-dependent structural investigation suggests a faster network formation with increasing temperature, and the gel adopts a more compact network, which is inconsistent with the conventional understanding of thermal aggregation. The resulting gel network shows a fractal dimension δ, ranging from 1.5 to 2.2. The values of δ display a non-monotonic behavior with increasing amount of salt. The corresponding dynamics in the q range of 0.002-0.1 nm-1 is observable after major change of the gel structure. The extracted relaxation time exhibits a two-step power law growth in dynamics as a function of waiting time. In the first regime, the dynamics is associated with structural growth, whereas the second regime is associated with the aging of the gel, which is directly linked with its compactness, as quantified by the fractal dimension. The gel dynamics is characterized by a compressed exponential relaxation with a ballistic-type of motion. The addition of salt gradually makes the early stage dynamics faster. Both gelation kinetics and microscopic dynamics show that the activation energy barrier in the system systematically decreases with increasing salt concentration.

Molecular Mechanism of Gibberellins in Mesocotyl Elongation Response to Deep-Sowing Stress in Sweet Maize.

Uneven germination is still a common problem in sweet maize planting. The mesocotyl is a key driver for ground-breaking sweet maize, and deep-sowing has a longer mesocotyl. However, the physiological and molecular mechanisms of sweet maize mesocotyl elongation in response to deep-sowing remain unknown. Here we found that sweet maize inbred line Ltx05 could obtain longer mesocotyls in deep soil of 10 cm depth, and that 20 mg/L GA3 was the optimal concentration to promote mesocotyl elongation and seedling emergence. Microstructure observation showed that the longitudinal cell length of mesocotyl at 10 cm sowing depth was significantly longer than that of 1 cm. Transcriptome analysis showed that microtubule process related differentially expressed genes may contribute to the longitudinal cell elongation. The content of GAs in the mesocotyl at 10 cm sowing depth was markedly higher than that of 1 cm. Combining transcriptome data and qRT-PCR at different developmental stages, ZmGA20ox1, ZmGA20ox4 and ZmGA20ox5 were identified as three positive regulation candidate genes during mesocotyl elongation under deep-sowing conditions, and this was further confirmed by the significant elongation of the hypocotyl in heterologous transformation of Arabidopsis thaliana. These results lay a foundation for improving the ability of sweet maize to tolerate deep-sowing stress and improving the breeding of excellent deep-sowing-tolerant germplasms.

Microscopic Dynamics of Liquid-Liquid Phase Separation and Domain Coarsening in a Protein Solution Revealed by X-Ray Photon Correlation Spectroscopy.

While the interplay between liquid-liquid phase separation (LLPS) and glass formation in biological systems is highly relevant for their structure formation and thus function, the exact underlying mechanisms are not well known. The kinetic arrest originates from the slowdown at the molecular level, but how this propagates to the dynamics of microscopic phase domains is not clear. Since with diffusion, viscoelasticity, and hydrodynamics, distinctly different mechanisms are at play, the dynamics needs to be monitored on the relevant time and length scales and compared to theories of phase separation. Using x-ray photon correlation spectroscopy, we determine the LLPS dynamics of a model protein solution upon low temperature quenches and find distinctly different dynamical regimes. We observe that the early stage LLPS is driven by the curvature of the free energy and speeds up upon increasing quench depth. In contrast, the late stage dynamics slows down with increasing quench depth, fingerprinting a nearby glass transition. The dynamics observed shows a ballistic type of motion, implying that viscoelasticity plays an important role during LLPS. We explore possible explanations based on the Cahn-Hilliard theory with nontrivial mobility parameters and find that these can only partially explain our findings.

Kinetics of Network Formation and Heterogeneous Dynamics of an Egg White Gel Revealed by Coherent X-Ray Scattering.

The kinetics of heat-induced gelation and the microscopic dynamics of a hen egg white gel are probed using x-ray photon correlation spectroscopy along with ultrasmall-angle x-ray scattering. The kinetics of structural growth reveals a reaction-limited aggregation process with a gel fractal dimension of ≈2 and an average network mesh size of ca. 400 nm. The dynamics probed at these length scales reveals an exponential growth of the characteristic relaxation times followed by an intriguing steady state in combination with a compressed exponential correlation function and a temporal heterogeneity. The degree of heterogeneity increases with decreasing length scale. We discuss our results in the broader context of experiments and models describing attractive colloidal gels.

Molecular Flexibility of Antibodies Preserved Even in the Dense Phase after Macroscopic Phase Separation.

Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.

Nematic Phase of Plate-like Semicrystalline Block Copolymer Single Crystals in Solution Studied by Small-Angle X-Ray Scattering.

Crystalline block copolymers have been used to prepare plate-like colloidal systems with well-controlled size, shape, and size distribution. The isotropic-to-nematic (I-N) phase transition of the novel plate-like colloidal particle suspensions has been reported previously. In this work, we focus on the characterization of the solution structure of the crystals and the N-phase using small- and ultrasmall-angle X-ray scattering techniques (SAXS/USAXS). The system has polystyrene-block-poly(l-lactide) (PS-b-PLLA) block copolymer single crystals (BCSCs) with different sizes dispersed in p-xylene. These crystals are truncated lozenge in shape and have effective diameters ranging from 550 to 4000 nm with a uniform dry thickness of 18.0 nm. Scattering of the individual crystal in solution can be simplified using a disc model with a core layer of 9-10 nm due to the lower contrast of the tethered PS layer. BCSC suspensions filled in thin quartz capillaries are prepared for monitoring the structural information. SAXS measurements of the isotropic phase show a strong face-to-face correlation, indicating that platelets form small stacked clusters in solutions. The isotropic phase is thus a coexistence of single crystals and the stacked multiple-layered clusters. The face-to-face spacing, d, in the N phases is around 75-90 nm, which increases slightly upon increasing the size of crystals. For a given system, the spacing does not change with increasing concentration under the current experimental conditions. Finally, the possible formation of lamellar domains within the N phase is also discussed due to the lateral attraction of this system. These results demonstrate the importance of the lateral attraction between the polar crystalline PLLA blocks on the formation of the N phase: the BCSCs self-assemble into larger sheets via the lateral attraction, which further enhances the I-N transition.

Bulk Phase Behavior vs Interface Adsorption: Specific Multivalent Cation and Anion Effects on BSA Interactions.

Proteins are ubiquitous and play a critical role in many areas from living organisms to protein microchips. In humans, serum albumin has a prominent role in the foreign body response since it is the first protein which will interact with, e.g., an implant or stent. In this study, we focused on the influence of salts (i.e., different cations (Y3+, La3+) and anions (Cl-, I-) on bovine serum albumin (BSA) in terms of its bulk behavior as well as the role of charges for protein adsorption at the solid-liquid interface in order to understand and control the underlying molecular mechanisms and interactions. This is part of our group's effort to gain a deeper understanding of protein-protein and protein-surface interactions in the presence of multivalent ions. In the bulk, we established two new phase diagrams and found not only multivalent cation-triggered phase transitions, but also a dependence of the protein behavior on the type of anion. The attractive interactions between proteins were observed to increase from Cl- < NO3- < I-, resulting in iodide preventing re-entrant condensation and promoting liquid-liquid phase separation in bulk. Using ellipsometry and a quartz-crystal microbalance with dissipation (QCM-D), we obtained insight into the growth of the protein adsorption layer. Importantly, we found that phase transitions at the substrate can be triggered by certain interface properties, whether they exist in the bulk solution or not. Through the use of a hydrophilic, negatively charged surface (native silica), the direct binding of anions to the interface was prevented. Interestingly, this led to re-entrant adsorption even in the absence of re-entrant condensation in bulk. However, the overall amount of adsorbed protein was enhanced through stronger attractive protein-protein interactions in the presence of iodide salts. These findings illustrate how carefully chosen surface properties and salts can directly steer the binding of anions and cations, which guide protein behavior, thus paving the way for specific/triggered protein-protein, protein-salt, and protein-surface interactions.

Temperature and salt controlled tuning of protein clusters.

The formation of molecular assemblies in protein solutions is of strong interest both from a fundamental viewpoint and for biomedical applications. While ordered and desired protein assemblies are indispensable for some biological functions, undesired protein condensation can induce serious diseases. As a common cofactor, the presence of salt ions is essential for some biological processes involving proteins, and in aqueous suspensions of proteins can also give rise to complex phase diagrams including homogeneous solutions, large aggregates, and dissolution regimes. Here, we systematically study the cluster formation approaching the phase separation in aqueous solutions of the globular protein BSA as a function of temperature (T), the protein concentration (cp) and the concentrations of the trivalent salts YCl3 and LaCl3 (cs). As an important complement to structural, i.e. time-averaged, techniques we employ a dynamical technique that can detect clusters even when they are transient on the order of a few nanoseconds. By employing incoherent neutron spectroscopy, we unambiguously determine the short-time self-diffusion of the protein clusters depending on cp, cs and T. We determine the cluster size in terms of effective hydrodynamic radii as manifested by the cluster center-of-mass diffusion coefficients D. For both salts, we find a simple functional form D(cp, cs, T) in the parameter range explored. The calculated inter-particle attraction strength, determined from the microscopic and short-time diffusive properties of the samples, increases with salt concentration and temperature in the regime investigated and can be linked to the macroscopic behavior of the samples.

Unification of lower and upper critical solution temperature phase behavior of globular protein solutions in the presence of multivalent cations.

In globular protein systems, upper critical solution temperature (UCST) behavior is common, but lower critical solution temperature (LCST) phase transitions are rare. In addition, the temperature sensitivity of such systems is usually difficult to tune. Here we demonstrate that the charge state of globular proteins in aqueous solutions can alter their temperature-dependent phase behavior. We show a universal way to tune the effective protein interactions and induce both UCST and LCST-type transitions in the system using trivalent salts. We provide a phase diagram identifying LCST and UCST regimes as a function of protein and salt concentrations. We further propose a model based on an entropy-driven cation binding mechanism to explain the experimental observations.

Packing and dynamics of a protein solution approaching the jammed state.

The packing of proteins and their collective behavior in crowded media is crucial for the understanding of biological processes. Here we study the structural and dynamical evolution of solutions of the globular protein bovine serum albumin with increasing concentration via drying using small angle X-ray scattering and dynamic light scattering. We probe an evolving correlation peak on the scattering profile, corresponding to the inter-protein distance, ξ, which decreases following a power law of the protein volume fraction, φ. The rate of decrease in ξ becomes faster above a protein concentration of ∼200 mg ml-1 (φ = 0.15). The power law exponent changes from 0.33, which is typical of colloidal or protein solutions, to 0.41. During the entire drying process, we observe the development and the growth of two-step relaxation dynamics with increasing φ as revealed by dynamic light scattering. We find three different regimes of the dependence of ξ as a function of φ. In the dilute regime (φ < 0.22), protein molecules are far apart from each other compared to their size. In this case, the dynamics mainly corresponds to Brownian motion. At an intermediate concentration (0.22 < φ < 0.47), inter-protein distances become comparable to the size of protein molecules, leading to a preferential orientation of the ellipsoidal protein molecules along with a possible deformation. In this regime, the dynamics shows two distinct relaxation times. At a very high concentration (φ > 0.47), the system reaches a jammed state. Subsequently, the secondary relaxation time in this state becomes extremely slow. In this state, the protein molecules have approximately one hydration layer. This study contributes to the understanding of protein molecular packing in crowded environments and the phenomenon of density-driven jamming for soft matter systems.

Evolution of the structure and dynamics of bovine serum albumin induced by thermal denaturation.

Protein denaturation in concentrated solutions consists of the unfolding of the native protein structure, and subsequent cross-linking into clusters or gel networks. While the kinetic evolution of structure has been studied for some cases, the underlying microscopic dynamics of proteins has so far been neglected. However, protein dynamics is essential to understand the specific nature of assembly processes, such as diffusion-limited growth, or vitrification of dense liquids. Here, we present a study on thermal denaturation of concentrated solutions of bovine serum albumin (BSA) in D2O with and without NaCl. Using small-angle scattering, we provide information on structure before, during and after denaturation. Using quasi-elastic neutron scattering, we monitor in real-time the microscopic dynamics and dynamical confinement throughout the entire denaturation process covering protein unfolding and cross-linking. After denaturation, the protein dynamics is slowed down in salty solutions compared to those in pure water, while the stability and dynamics of the native solution appears unaffected by salt. The approach presented here opens opportunities to link microscopic dynamics to emerging structural properties, with implications for assembly processes in soft and biological matter.

Tuning phase transitions of aqueous protein solutions by multivalent cations.

In the presence of trivalent cations, negatively charged globular proteins show a rich phase behaviour including reentrant condensation, crystallisation, clustering and lower critical solution temperature metastable liquid-liquid phase separation (LCST-LLPS). Here, we present a systematic study on how different multivalent cations can be employed to tune the interactions and the associated phase behaviour of proteins. We focus our investigations on the protein bovine serum albumin (BSA) in the presence of HoCl3, LaCl3 and YCl3. Using UV-Vis spectroscopy and small-angle X-ray scattering (SAXS), we find that the interprotein attraction induced by Ho3+ is very strong, while the one induced by La3+ is comparatively weak when comparing the data to BSA-Y3+ systems based on our previous work. Using zeta potential and isothermal titration calorimetry (ITC) measurements, we establish different binding affinities of cations to BSA with Ho3+ having the highest one. We propose that a combination of different cation features such as radius, polarisability and in particular hydration effects determine the protein-protein interaction induced by these cations. Our findings imply that subtle differences in cation properties can be a sensitive tool to fine-tune protein-protein interactions and phase behaviour in solution.

Multivalent-Ion-Activated Protein Adsorption Reflecting Bulk Reentrant Behavior.

Protein adsorption at the solid-liquid interface is an important phenomenon that often can be observed as a first step in biological processes. Despite its inherent importance, still relatively little is known about the underlying microscopic mechanisms. Here, using multivalent ions, we demonstrate the control of the interactions and the corresponding adsorption of net-negatively charged proteins (bovine serum albumin) at a solid-liquid interface. This is demonstrated by ellipsometry and corroborated by neutron reflectivity and quartz-crystal microbalance experiments. We show that the reentrant condensation observed within the rich bulk phase behavior of the system featuring a nonmonotonic dependence of the second virial coefficient on salt concentration c_{s} is reflected in an intriguing way in the protein adsorption d(c_{s}) at the interface. Our findings are successfully described and understood by a model of ion-activated patchy interactions within the framework of the classical density functional theory. In addition to the general challenge of connecting bulk and interface behavior, our work has implications for, inter alia, nucleation at interfaces.

Arrested and temporarily arrested states in a protein-polymer mixture studied by USAXS and VSANS.

We investigate the transition of the phase separation kinetics from a complete to an arrested liquid-liquid phase separation (LLPS) in mixtures of bovine γ-globulin with polyethylene glycol (PEG). The solutions feature LLPS with upper critical solution temperature phase behavior. At higher PEG concentrations or low temperatures, non-equilibrium, gel-like states are found. The kinetics is followed during off-critical quenches by ultra-small angle X-ray scattering (USAXS) and very-small angle neutron scattering (VSANS). For shallow quenches a kinetics consistent with classical spinodal decomposition is found, with the characteristic length (ξ) growing with time as ξ ∼ t1/3. For deep quenches, ξ grows only very slowly with a growth exponent smaller than 0.05 during the observation time, indicating an arrested phase separation. For intermediate quench depths, a novel growth kinetics featuring a three-stage coarsening is observed, with an initial classical coarsening, a subsequent slowdown of the growth, and a later resumption of coarsening approaching again ξ ∼ t1/3. Samples featuring the three-stage coarsening undergo a temporarily arrested state. We hypothesize that, while intermittent coarsening and collapse might contribute to the temporary nature of the arrested state, migration-coalescence of the minority liquid phase through the majority glassy phase may be the main mechanism underlying this kinetics, which is also consistent with earlier simulation results.

Kinetics of liquid-liquid phase separation in protein solutions exhibiting LCST phase behavior studied by time-resolved USAXS and VSANS.

We study the kinetics of the liquid-liquid phase separation (LLPS) and its arrest in protein solutions exhibiting a lower critical solution temperature (LCST) phase behavior using the combination of ultra-small angle X-ray scattering (USAXS) and very-small angle neutron scattering (VSANS). We employ a previously established model system consisting of bovine serum albumin (BSA) solutions with YCl3. We follow the phase transition from sub-second to 104 s upon an off-critical temperature jump. After a temperature jump, the USAXS profiles exhibit a peak that grows in intensity and shifts to lower q values with time. Below 45 °C, the characteristic length scale (ξ) obtained from this scattering peak increases with time with a power of about 1/3 for different sample compositions. This is in good agreement with the theoretical prediction for the intermediate stage of spinodal decomposition where the growth is driven by interface tension. Above 45 °C, ξ follows initially the 1/3 power law growth, then undergoes a significant slowdown, and an arrested state is reached below the denaturation temperature of the protein. This growth kinetics may indicate that the final composition of the protein-rich phase is located close to the high density branch of the LLPS binodal when a kinetically arrested state is reached.

Real-time observation of nonclassical protein crystallization kinetics.

We present a real-time study of protein crystallization of bovine ß-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process.