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Root penetration into the soil is essential for plants to access water and nutrients, as well as to mechanically support aboveground structures. This requires a combination of healthy plant growth, adequate soil mechanical properties, and compatible plant–soil interactions. Despite the current knowledge of the static rheology driving the interactions at the root–soil interface, few theoretical approaches have attempted to describe root penetration with dynamic rheology. In this work, we experimentally showed that radish roots in contact with soil of specific density during a specific growth stage fail to penetrate the soil. To explore the mechanism of root penetration into the soil, we constructed a theoretical model to explore the relevant conditions amenable to root entry into the soil. The theory indicates that dimensionless parameters such as root growth anisotropy, static root–soil competition, and dynamic root–soil competition are important for root penetration. The consequent theoretical expectations were supported by finite element analysis, and a potential mechanism of root penetration into the soil is discussed.

The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting–like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

The Arabidopsis thaliana NAC domain transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) acts as a master regulator of xylem vessel differentiation. To understand the mechanism by which VND7 regulates xylem vessel differentiation, we used a yeast two-hybrid system to screen for proteins that interact with VND7 and identified cDNAs encoding two NAC domain proteins, VND-INTERACTING1 (VNI1) and VNI2. Binding assays demonstrated that VNI2 effectively interacts with VND7 and the VND family proteins, VND1-5, as well as with other NAC domain proteins at lower affinity. VNI2 is expressed in both xylem and phloem cells in roots and inflorescence stems. The expression of VNI2 overlaps with that of VND7 in elongating vessel precursors in roots. VNI2 contains a predicted PEST motif and a C-terminally truncated VNI2 protein, which lacks part of the PEST motif, is more stable than full-length VNI2. Transient reporter assays showed that VNI2 is a transcriptional repressor and can repress the expression of vessel-specific genes regulated by VND7. Expression of C-terminally truncated VNI2 under the control of the VND7 promoter inhibited the normal development of xylem vessels in roots and aerial organs. These data suggest that VNI2 regulates xylem cell specification as a transcriptional repressor that interacts with VND proteins and possibly also with other NAC domain proteins.

Key messageThe homologs of VASCULAR RELATED NAC-DOMAIN in the peat moss Sphagnum palustre were identified and these transcriptional activity as the VNS family was conserved.In angiosperms, xylem vessel element differentiation is governed by the master regulators VASCULAR RELATED NAC-DOMAIN6 (VND6) and VND7, encoding plant-specific NAC transcription factors. Although vessel elements have not been found in bryophytes, differentiation of the water-conducting hydroid cells in the moss Physcomitrella patens is regulated by VND homologs termed VND-NST-SOMBRERO (VNS) genes. VNS genes are conserved in the land plant lineage, but their functions have not been elucidated outside of angiosperms and P. patens. The peat moss Sphagnum palustre, of class Sphagnopsida in the phylum Bryophyta, does not have hydroids and instead uses hyaline cells with thickened, helical-patterned cell walls and pores to store water in the leaves. Here, we performed whole-transcriptome analysis and de novo assembly using next generation sequencing in S. palustre, obtaining sequences for 68,305 genes. Among them, we identified seven VNS-like genes, SpVNS1-A, SpVNS1-B, SpVNS2-A, SpVNS2-B, SpVNS3-A, SpVNS3-B, and SpVNS4-A. Transient expression of these VNS-like genes, with the exception of SpVNS2-A, in Nicotiana benthamiana leaf cells resulted in ectopic thickening of secondary walls. This result suggests that the transcriptional activity observed in other VNS family members is functionally conserved in the VNS homologs of S. palustre.

Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe 3+ -NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H − from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level. Using photosensitive caged-compound for femtosecond crystallography at X-ray free electron lasers would allow the structure determination of reaction intermediates. Here the authors demonstrate the feasibility of this approach with a caged NO-compound and present the initial NO-bound intermediate structure of cytochrome P450 nitric oxide reductase.

Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3′-untranslated region (3′-UTR). Removal of the 3′-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28. Land plants and metazoans are both able to reprogram differentiated cells to stem cells under certain circumstances. Here the authors show that the moss CSP1 protein, which shares conserved domains with the mammalian pluripotent stem cell factor Lin28, promotes reprogramming of leaf cells to apical stem cells.

Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.

Metal-dioxygen adducts, such as metal-superoxo and -peroxo species, are key intermediates often detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. The synthesis and spectroscopic characterization of an end-on nickel( II )-superoxo complex with a 14-membered macrocyclic ligand was reported previously. Here we report the isolation, spectroscopic characterization, and high-resolution crystal structure of a mononuclear side-on nickel( III )-peroxo complex with a 12-membered macrocyclic ligand, [Ni(12-TMC)(O 2 )] + (1) (12-TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane). In contrast to the end-on nickel( II )-superoxo complex, the nickel( III )-peroxo complex is not reactive in electrophilic reactions, but is capable of conducting nucleophilic reactions. The nickel( III )-peroxo complex transfers the bound dioxygen to manganese( II ) complexes, thus affording the corresponding nickel( II ) and manganese( III )-peroxo complexes. Our results demonstrate the significance of supporting ligands in tuning the geometric and electronic structures and reactivities of metal–O 2 intermediates that have been shown to have biological as well as synthetic usefulness in biomimetic reactions. Compounds containing dioxygen bonded to metal atoms – such as metal-peroxo or -superoxo species – are models of enzymes that can activate oxygen. Now a mononuclear side-on nickel(III)-peroxo complex has been made, which is stabilized by a macrocyclic ligand. The complex can transfer the bound dioxygen to another metal, and conduct nucleophilic reactions.