Exogenous Melatonin Regulates Plant-Disease Interaction by Inducing Maize Resistance and Decreasing the Pathogenicity of Fusarium graminearum.

Plants cannot avoid environmental challenges and are constantly threatened by diverse biotic and abiotic stresses. However, plants have developed a unique immune system to defend themselves against the invasion of various pathogens. Melatonin, N-acetyl-5-methoxytryptamine has positive physiological effects in plants that are involved in disease resistance. The processes underlying melatonin-induced pathogen resistance in plants are still unknown. The current study explores how melatonin regulates the plant-disease interaction in maize. The results showed that 400 µM melatonin strongly reduced the disease lesion on maize stalks by 1.5 cm and corn by 4.0 cm caused by Fusarium graminearum PH-1. Furthermore, after treatment with melatonin, the plant defense enzymes like SOD significantly increased, while POD and APX significantly decreased compared to the control. In addition, melatonin can also improve maize's innate immunity, which is mediated by melatonin treatments through the salicylic acid signaling pathway, and up-regulate the defense-associated expression of PR1, LOX1, OXR, serPIN, and WIPI genes in maize. Melatonin not only inhibits the disease in the maize stalks and corn, but also down-regulates the deoxynivalenol (DON) production-related expression of genes Tri1, Tri4, Tri5, and Tri6 in maize. Overall, this study sheds new light on the mechanisms by which melatonin regulates antioxidant enzymes and defense-related genes involved in plant immunity to effectively suppress plant diseases.

Liquid-Liquid Phase Separation of Peptide/Oligonucleotide Complexes in Crowded Macromolecular Media.

The membraneless organelles (MLOs) and coacervates of oppositely charged polyelectrolytes are both formed by liquid-liquid phase separation. To reveal how the crowded cell interior regulates the MLOs, we chose the coacervates formed by peptide S5 and single-stranded oligonucleotide (ss-oligo) at 1:1 charge ratio and investigated the phase separation processes in polyacrylamide (PAM) and poly(ethylene oxide) (PEO) media at varying concentrations. Results show that the droplet formation unit is the neutral primary complex, instead of individual S5 or ss-oligo. Therefore, the coacervation process can be described by the classic theory of nucleation and growth. The dynamic scaling relationships show that S5/ss-oligo coacervation undergoes in sequence the heterogeneous nucleation, diffusion-limited growth, and Brownian motion coalescence with time. The inert crowders generate multiple effects, including accelerating the growth of droplets, weakening the electrostatic attraction, and slowing down or even trapping the droplets in the crowder network. The overall effect is that both the size and size distribution of the droplets decrease with increasing crowder concentration, and the effect of PEO is stronger than that of PAM. Our study provides a further step toward a deeper understanding of the kinetics of MLOs in crowded living cells.

Protocells with hierarchical structures as regulated by liquid-liquid and liquid-solid phase separations.

The construction of a protocell with dynamic hierarchical structures via spontaneous phase separation sheds light on the mechanisms of life processes. Taking advantage of the transition from the liquid to solid phase, we built composite droplets with PLL/oligo/oligocomp solid particles randomly distributed inside a PLL/oligo liquid coacervate. The circulation and vacuolization under an electric field drive the particles into a fibrous structure and even clusters. A PLL/oligo/oligocomp solid phase can also form on the interface of the PLL/oligo coacervate, turning the droplet into a vesicular structure.

Fission and Internal Fusion of Protocell with Membraneless "Organelles" Formed by Liquid-Liquid Phase Separation.

Construction of protocells with hierarchical structures and living functions is still a great challenge. Growing evidence demonstrates that the membraneless organelles, which facilitate many essential cellular processes, are formed by RNA, protein, and other biopolymers via liquid-liquid phase separation (LLPS). The formation of the protocell in the early days of Earth could follow the same principle. In this work, we develop a novel coacervate-based protocell containing membraneless subcompartments via spontaneous liquid-liquid phase separation by simply mixing single-stranded oligonucleotides (ss-oligo), quaternized dextran (Q-dextran), and poly(l-lysine) (PLL) together. The resulting biphasic droplet, with PLL/ss-oligo phase being the internal subcompartments and Q-dextran/ss-oligo phase as the surrounding medium, is capable of sequestering and partitioning biomolecules into distinct regions. When the droplet is exposed to salt or Dextranase, the surrounding Q-dextran/ss-oligo phase will be gradually dissociated, resulting in the chaotic movement and fusion of internal subcompartments. Besides, the external electric field at a lower strength can drive the biphasic droplet to undergo a deviated circulation concomitant with the fusion of localized subcompartments, while a high-strength electric field can polarize the whole droplet, resulting in the release of daughter droplets in a controllable manner. Our study highlights that liquid-liquid phase separation of biopolymers is a powerful strategy to construct hierarchically structured protocells resembling the morphology and functions of living cells and provides a step toward a better understanding of the transition mechanism from nonliving to living matter under prebiotic conditions.

Dynamic Behavior of Complex Coacervates with Internal Lipid Vesicles under Nonequilibrium Conditions.

During the evolution of life on earth, the emergence of lipid membrane-bounded compartments is one of the most enigmatic events. Endosymbiosis has been hypothesized as one of the solutions. In this work, using a coacervate droplet formed by single-stranded oligonucleotides (ss-oligo) and poly(l-lysine) (PLL) as the protocell model, we monitored the uptake of liposomes of different types and studied the dynamic behavior of the resulting composite droplet under the electric field. The coacervate droplet exhibits affinity for the liposomes of varying charges. However, the permeation of liposome is also controlled by electrostatic interactions. Dominated by electrostatic attraction, the positively charged liposome is retained inside the droplet as growing fibrous structures, while the negatively charged liposome is mainly coated on the droplet surface. Permeation and even distribution occur when the liposome and the droplet carry the same charges, or at least one of them is neutral. As an electric field is applied to trigger repetitive cycles of vacuolization in the ss-oligo/PLL droplet, the fibrous structure formed by the positively charged liposome is basically intact, while a new phase is generated together with uneven mass transport as the negatively charged liposome is internalized. Interestingly, the release of daughter droplets with similar components occurs on the droplet containing neutral liposomes. Our work not only provides a step toward creating protocells with hierarchical structures and biofunctions using a biogenetic material via simple mixing but also sheds light on the possible origin of the lipid structure inside a living organism.

Mass Transport in Coacervate-Based Protocell Coated with Fatty Acid under Nonequilibrium Conditions.

Construction of protocell models from prebiotically plausible components to mimic the basic features or functions of living cells is still a challenge. In this work, we prepare a hybrid protocell model by coating sodium oleate on the coacervate droplet constituted by poly(l-lysine) and oligonucleotide and investigate the transport of different molecules under electric field. Results show that sodium oleate forms a layered viscoelastic membrane on the droplet surface, which is selectively permeable to small, polar molecules, such as oligolysine. As the droplet is stimulated at 10 V cm-1, the oleate membrane slips along the direction of electric field while maintaining its integrity. Most of the molecules are still excluded under such conditions. As repetitive cycles of vacuolization occur at 20 V cm-1, all molecules are internalized and sequestrated in the droplet through their specific pathways except enzyme, which anchors in the oleate membrane and is immune to electric field. Cascade enzymatic reactions are then carried out, and the product generated from the membrane exhibits a time-dependent concentration gradient across the droplet. Our work makes a step toward the nonequilibrium functionalization of synthetic protocells capable of biomimetic operations.

Membrane-mediated transport in a non-equilibrium hybrid protocell based on coacervate droplets and a surfactant.

A hybrid protocell is constructed to investigate the membrane transport of neutral, cationic, and anionic molecules under non-equilibrium conditions. Each model molecule follows a specific pathway to be internalized and generates different distributions in the droplets. This work provides a step towards functionalization of synthetic protocells capable of biomimetic operations.

Electric field-induced circulation and vacuolization regulate enzyme reactions in coacervate-based protocells.

Artificial protocells operating under non-equilibrium conditions offer a new approach to achieve dynamic features with life-like properties. Using coacervate micro-droplets comprising polylysine (PLL) and a short single-stranded oligonucleotide (ss-oligo) as a membrane-free protocell model, we demonstrate that circulation and vacuolization can occur simultaneously inside the droplet in the presence of an electric field. The circulation is driven by electrohydrodynamics and applies specifically to the major components of the protocell (PLL and ss-oligo). Significantly, under low electric fields (E = 10 V cm-1) the circulation regulates the movement of the vacuoles, while high levels of vacuolization produced at higher electric fields can deform or reshape the circulation. By taking advantage of the interplay between vacuolization and circulation, we achieve dynamic localization of an enzyme cascade reaction at specific droplet locations. In addition, the spatial distribution of the enzyme reaction is globalized throughout the droplet by tuning the coupling of the circulation and vacuolization processes. Overall, our work provides a new strategy to create non-equilibrium dynamic behaviors in molecularly crowded membrane-free synthetic protocells.