Cell wall integrity modulates HOOKLESS1 and PHYTOCHROME INTERACTING FACTOR4 expression controlling apical hook formation.

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development is still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and of differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS 1 (HLS1) and PHYTOCHROME INTERACTING FACTOR 4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima and PIF4 and HLS1 expression. Analysis of plants expressing a FRET-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signalling module to control differential cell elongation during hook formation.

Multiple mechanisms behind plant bending.

To survive, plants constantly adapt their body shape to their environment. This often involves remarkably rapid bending of their organs such as stems, leaves and roots. Since plant cells are enclosed by stiff cell walls, they use various strategies for bending their organs, which differ from bending mechanisms of soft animal tissues and involve larger physical forces. Here we attempt to summarize and link different viewpoints on bending mechanisms: genes and signalling, mathematical modelling and biomechanics. We argue that quantifying cell growth and physical forces could open a new level in our understanding of bending and resolve some of its paradoxes.

Endoreplication mediates cell size control via mechanochemical signaling from cell wall.

Endoreplication is an evolutionarily conserved mechanism for increasing nuclear DNA content (ploidy). Ploidy frequently scales with final cell and organ size, suggesting a key role for endoreplication in these processes. However, exceptions exist, and, consequently, the endoreplication-size nexus remains enigmatic. Here, we show that prolonged tissue folding at the apical hook in Arabidopsis requires endoreplication asymmetry under the control of an auxin gradient. We identify a molecular pathway linking endoreplication levels to cell size through cell wall remodeling and stiffness modulation. We find that endoreplication is not only permissive for growth: Endoreplication reduction enhances wall stiffening, actively reducing cell size. The cell wall integrity kinase THESEUS plays a key role in this feedback loop. Our data thus explain the nonlinearity between ploidy levels and size while also providing a molecular mechanism linking mechanochemical signaling with endoreplication-mediated dynamic control of cell growth.

A study of flotation-REST (restricted environmental stimulation therapy) as an insomnia treatment.

Objectives: Flotation-REST is a treatment for deep relaxation, where a person is contained in a stimuli-restricted environment and floats in water with high salt content. The aim was to investigate the effects from flotation-REST on people with insomnia diagnosis, as previous studies of flotation-REST have demonstrated some effects on sleep but have limitations regarding sample selections and sleep measures. Material and Methods: Six participants were recruited through an outpatient psychiatry clinic and posters on a university campus. All participants fulfilled criteria for insomnia diagnosis and four fulfilled criteria for major depressive disorder. Using a single case experimental design, daily changes were investigated on sleep logs regarding sleep onset latency (SOL), wake after sleep onset (WASO), total sleep time (TST), and sleep efficiency over the course of 12 sessions consisting of 45 min of flotation-REST. No other treatments were offered simultaneously. Questionnaire data on insomnia severity (the ISI) and depressive severity (the MADRS) were also collected. Results: Three participants improved on their most salient insomnia symptom (long SOL or WASO), and two improved on sleep efficiency. The improvements were maintained 2 months after treatment. Insomnia severity decreased for three patients, whereas depressive severity decreased for five. No changes in TST were found and two patients did not improve on any sleep measure. The two participants who benefitted the most were students in their 20s. Discussion: The results were mixed. Flotation-REST may be beneficial for young adults with sleep-onset insomnia but more research is warranted.

Plant cell walls as mechanical signaling hubs for morphogenesis.

The instructive role of mechanical cues during morphogenesis is increasingly being recognized in all kingdoms. Patterns of mechanical stress depend on shape, growth and external factors. In plants, the cell wall integrates these three parameters to function as a hub for mechanical feedback. Plant cells are interconnected by cell walls that provide structural integrity and yet are flexible enough to act as both targets and transducers of mechanical cues. Such cues may act locally at the subcellular level or across entire tissues, requiring tight control of both cell-wall composition and cell-cell adhesion. Here we focus on how changes in cell-wall chemistry and mechanics act in communicating diverse cues to direct growth asymmetries required for plant morphogenesis. We explore the role of cellulose microfibrils, microtubule arrays and pectin methylesterification in the transduction of mechanical cues during morphogenesis. Plant hormones can affect the mechanochemical composition of the cell wall and, in turn, the cell wall can modulate hormone signaling pathways, as well as the tissue-level distribution of these hormones. This also leads us to revisit the position of biochemical growth factors, such as plant hormones, acting both upstream and downstream of mechanical signaling. Finally, while the structure of the cell wall is being elucidated with increasing precision, existing data clearly show that the integration of genetic, biochemical and theoretical studies will be essential for a better understanding of the role of the cell wall as a hub for the mechanical control of plant morphogenesis.

Mechanochemical feedback mediates tissue bending required for seedling emergence.

Tissue bending is vital to plant development, as exemplified by apical hook formation during seedling emergence by bending of the hypocotyl. How tissue bending is coordinated during development remains poorly understood, especially in plants where cells are attached via rigid cell walls. Asymmetric distribution of the plant hormone auxin underlies differential cell elongation during apical hook formation. Yet the underlying mechanism remains unclear. Here, we demonstrate spatial correlation between asymmetric auxin distribution, methylesterified homogalacturonan (HG) pectin, and mechanical properties of the epidermal layer of the hypocotyl in Arabidopsis. Genetic and cell biological approaches show that this mechanochemical asymmetry is essential for differential cell elongation. We show that asymmetric auxin distribution underlies differential HG methylesterification, and conversely changes in HG methylesterification impact the auxin response domain. Our results suggest that a positive feedback loop between auxin distribution and HG methylesterification underpins asymmetric cell wall mechanochemical properties to promote tissue bending and seedling emergence.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

Interplay between Cell Wall and Auxin Mediates the Control of Differential Cell Elongation during Apical Hook Development.

Differential growth plays a crucial role during morphogenesis [1-3]. In plants, development occurs within mechanically connected tissues, and local differences in cell expansion lead to deformations at the organ level, such as buckling or bending [4, 5]. During early seedling development, bending of hypocotyl by differential cell elongation results in apical hook structure that protects the shoot apical meristem from being damaged during emergence from the soil [6, 7]. Plant hormones participate in apical hook development, but not how they mechanistically drive differential growth [8]. Here, we present evidence of interplay between hormonal signals and cell wall in auxin-mediated differential cell elongation using apical hook development as an experimental model. Using genetic and cell biological approaches, we show that xyloglucan (a major primary cell wall component) mediates asymmetric mechanical properties of epidermal cells required for hook development. The xxt1 xxt2 mutant, deficient in xyloglucan [9], displays severe defects in differential cell elongation and hook development. Analysis of xxt1 xxt2 mutant reveals a link between cell wall and transcriptional control of auxin transporters PINFORMEDs (PINs) and AUX1 crucial for establishing the auxin response maxima required for preferential repression of elongation of the cells on the inner side of the hook. Genetic evidence identifies auxin response factor ARF2 as a negative regulator acting downstream of xyloglucan-dependent control of hook development and transcriptional control of polar auxin transport. Our results reveal a crucial feedback process between the cell wall and transcriptional control of polar auxin transport, underlying auxin-dependent control of differential cell elongation in plants.

Branching Regulator BRC1 Mediates Photoperiodic Control of Seasonal Growth in Hybrid Aspen.

Cessation of growth as winter approaches is a key adaptive trait for survival of perennial plants, such as long-lived trees native to boreal and temperate regions [1, 2]. The timing of growth cessation in these plants is controlled by photoperiodic cues. As shown recently, perception of growth-repressive short photoperiod (SP) mediated via components of circadian clock results in downregulation of the tree ortholog of Arabidopsis flowering regulator FLOWERING LOCUS T (FT), FT2 [3, 4]. Downregulation of FT2 results in suppression of downstream components LAP1 (orthologous to the Arabidopsis floral meristem identity gene APETALA1) and AIL1 (orthologous to AINTEGUMENTA in Arabidopsis), culminating in induction of growth cessation and bud set [5-7]. Results presented here reveal that, in addition to the CO/FT pathway, a photoperiodically controlled negative feedback loop involving a tree ortholog of Arabidopsis BRANCHED1 (BRC1) (a member of TEOSINTE BRANCHED 1, CYCLOIDEA, PCF family), LAP1, and FT2 participates in regulation of seasonal growth in the model tree hybrid aspen. In growth-promotive long photoperiod, LAP1 suppresses expression of BRC1, but upon perception of growth-repressive SP, downregulation of LAP1 de-represses expression of its downstream target BRC1. BRC1 physically interacts with FT2, and BRC1-FT interaction further reinforces the effect of SP and triggers growth cessation by antagonizing FT action. Accordingly, BRC1 gain and loss of function result in early and retarded growth cessation responses to SP, respectively. Thus, these results reveal a regulatory feedback loop that reinforces responses to SP and induction of seasonal growth cessation.

Long-range mobile signals mediate seasonal control of shoot growth.

In perennial plants, seasonal shifts provide cues that control adaptive growth patterns of the shoot apex. However, where these seasonal cues are sensed and communicated to the shoot apex remains unknown. We demonstrate that systemic signals from leaves play key roles in seasonal control of shoot growth in model tree hybrid aspen. Grafting experiments reveal that the tree ortholog of Arabidopsis flowering time regulator FLOWERING LOCUS T (FT) and the plant hormone gibberellic acid (GA) systemically convey seasonal cues to the shoot apex. GA (unlike FT) also acts locally in shoot apex, downstream of FT in seasonal growth control. At the shoot apex, antagonistic factors-LAP1, a target of FT and the FT antagonist TERMINAL FLOWER 1 (TFL1)-act locally to promote and suppress seasonal growth, respectively. These data reveal seasonal changes perceived in leaves that are communicated to the shoot apex by systemic signals that, in concert with locally acting components, control adaptive growth patterns.

Ethylene Regulates Differential Growth via BIG ARF-GEF-Dependent Post-Golgi Secretory Trafficking in Arabidopsis.

During early seedling development, the shoot apical meristem is protected from damage as the seedling emerges from soil by the formation of apical hook. Hook formation requires differential growth across the epidermis below the meristem in the hypocotyl. The plant hormones ethylene and auxin play key roles during apical hook development by controlling differential growth. We provide genetic and cell biological evidence for the role of ADP-ribosylation factor 1 (ARF1)-GTPase and its effector ARF-guanine-exchange factors (GEFs) of the Brefeldin A-inhibited GEF (BIG) family and GNOM in ethylene- and auxin-mediated control of hook development. We show that ARF-GEF GNOM acts early, whereas BIG ARF-GEFs act at a later stage of apical hook development. We show that the localization of ARF1 and BIG4 at the trans-Golgi network (TGN) depends on ECHIDNA (ECH), a plant homolog of yeast Triacylglycerol lipase (TLG2/SYP4) interacting protein Tgl2-Vesicle Protein 23 (TVP23). BIGs together with ECH and ARF1 mediate the secretion of AUX1 influx carrier to the plasma membrane from the TGN during hook development and defects in BIG or ARF1 result in insensitivity to ethylene. Thus, our data indicate a division of labor within the ARF-GEF family in mediating differential growth with GNOM acting during the formation phase whereas BIGs act during the hook maintenance phase downstream of plant hormone ethylene.

Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains.

The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells.

Promising effects of treatment with flotation-REST (restricted environmental stimulation technique) as an intervention for generalized anxiety disorder (GAD): a randomized controlled pilot trial.

BACKGROUND: During Flotation-REST a person is floating inside a quiet and dark tank, filled with heated salt saturated water. Deep relaxation and beneficial effects on e.g. stress, sleep difficulties, anxiety and depression have been documented in earlier research. Despite that treatments for generalized anxiety disorder (GAD) are effective; it is till the least successfully treated anxiety disorder, indicating that treatment protocols can be enhanced. The use of Flotation-REST as a treatment of GAD has not been researched. The aim of the present study was to conduct an initial evaluation of the effects in a self-diagnosed GAD sample. METHODS: This study was a randomized, parallel group, non-blinded trial with 1:1 allocation ratio to waiting list control group (n = 25) or to a twelve session treatment with flotation-REST (n = 25). Inclusion criteria's were: 18-65 years and GAD (as defined by self-report measures). The primary outcome was GAD-symptomatology, and secondary outcomes were depression, sleep difficulties, emotion regulation difficulties and mindfulness. Assessments were made at three time points (baseline, four weeks in treatment, post-treatment), and at six-month follow-up. The main data analyses were conducted with a two-way MANOVA and additional t-tests. Forty-six participants (treatment, n = 24; control, n = 22) were included in the analyses. RESULTS: A significant Time x Group interaction effect for GAD-symptomatology [F (2,88) = 2.93, p < .001, η p (2) = .062] was found. Further analyses showed that the GAD-symptomatology was significantly reduced for the treatment group (t (23) = 4.47, p < .001), but not for the waiting list control group (t(21) = 0.98, p > .05), when comparing baseline to post-treatment scoring. Regarding clinical significant change, 37 % in the treatment group reached full remission at post-treatment. Significant beneficial effects were also found for sleep difficulties, difficulties in emotional regulation, and depression, while the treatment had ambiguous or non-existent effects on pathological worry and mindfulness. All improved outcome variables at post-treatment, except for depression, were maintained at 6-months follow. No negative effects were found. CONCLUSION: The findings suggest that the method has potential as a complementary treatment alongside existing treatment for GAD. More studies are warranted to further evaluate the treatments efficacy. TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry: ACTRN12613001105730 , Date of registration: 03/10/2013.

Dual role of tree florigen activation complex component FD in photoperiodic growth control and adaptive response pathways.

A complex consisting of evolutionarily conserved FD, flowering locus T (FT) proteins is a regulator of floral transition. Intriguingly, FT orthologs are also implicated in developmental transitions distinct from flowering, such as photoperiodic control of bulbing in onions, potato tuberization, and growth cessation in trees. However, whether an FT-FD complex participates in these transitions and, if so, its mode of action, are unknown. We identified two closely related FD homologs, FD-like 1 (FDL1) and FD-like 2 (FDL2), in the model tree hybrid aspen. Using gain of function and RNAi-suppressed FDL1 and FDL2 transgenic plants, we show that FDL1 and FDL2 have distinct functions and a complex consisting of FT and FDL1 mediates in photoperiodic control of seasonal growth. The downstream target of the FT-FD complex in photoperiodic control of growth is Like AP1 (LAP1), a tree ortholog of the floral meristem identity gene APETALA1. Intriguingly, FDL1 also participates in the transcriptional control of adaptive response and bud maturation pathways, independent of its interaction with FT, presumably via interaction with abscisic acid insensitive 3 (ABI3) transcription factor, a component of abscisic acid (ABA) signaling. Our data reveal that in contrast to its primary role in flowering, FD has dual roles in the photoperiodic control of seasonal growth and stress tolerance in trees. Thus, the functions of FT and FD have diversified during evolution, and FD homologs have acquired roles that are independent of their interaction with FT.

Journey to the cell surface--the central role of the trans-Golgi network in plants.

The secretion of proteins, lipids, and carbohydrates to the cell surface is essential for plant development and adaptation. Secreted substances synthesized at the endoplasmic reticulum pass through the Golgi apparatus and trans-Golgi network (TGN) en route to the plasma membrane via the conventional secretion pathway. The TGN is morphologically and functionally distinct from the Golgi apparatus. The TGN is located at the crossroads of many trafficking pathways and regulates a range of crucial processes including secretion to the cell surface, transport to the vacuole, and the reception of endocytic cargo. This review outlines the TGN's central role in cargo secretion, showing that its behavior is more complex and controlled than the bulk-flow hypothesis suggests. Its formation, structure, and maintenance are discussed along with the formation and release of secretory vesicles.

Methoxetamine (MXE)--a phenomenological study of experiences induced by a "legal high" from the internet.

Methoxetamine (MXE), a ketamine analogue, is one of the new "legal highs" sold on the Internet. The aim of this qualitative study was to provide an initial understanding of what characterizes the experiences induced by MXE. Anonymously written reports (33 persons) on the effects of MXE were collected from public Internet forums and analyzed using the Empirical Phenomenological Psychological Method. The analysis generated 10 themes: (1) preparation, motivation and anticipation; (2) initial effects; (3) malfunction of cognitive processes stabilizing normal state; (4) inner personal processes and learning; (5) emotional processes; (6) altered sensory perception; (7) dissolution and transition; (8) spiritual and transcendental experiences; (9) effects and processes after the experience; (10) re-dosing and addiction. MXE induced a heavily altered state of consciousness. The effects were similar to those induced by classic hallucinogens (such as LSD, psilocybin) and the dissociative ketamine. MXE seemed to have quite a high abuse potential. Beside the positive effects described, negative effects like fear and anxiety were also reported. Acceptance was considered the best coping strategy. Dissolution of identity and body often culminated in spiritual and transcendental experiences. More research is needed on safety issues, how to minimize harm, and the motivation for using legal highs.

ECHIDNA-mediated post-Golgi trafficking of auxin carriers for differential cell elongation.

The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)-mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development.

Repeated Liver Resection for Colorectal Liver Metastases: A Comparison with Primary Liver Resections concerning Perioperative and Long-Term Outcome.

Introduction. 60% of patients operated for colorectal liver metastases (CRLM) will develop recurrent disease and some may be candidates for a repeated liver resection. The study aimed to evaluate differences in intraoperative blood loss and complications comparing the primary and the repeated liver resection for metastases of colorectal cancer (CRC), as well as to evaluate differences in long-time follow-up. Method. 32 patients underwent 34 repeated liver resections due to recurrence of CRLM an studied retrospectively to identify potential differences between the primary and the repeat resections. Results. There was no 30-day postoperative mortality or postoperative hospital deaths. The median blood loss at repeat resection (1850 mL) was significantly (P = 0.014) higher as compared to the primary liver resection (1000 mL). This did not have any effect on the rate of complications, even though increased bleeding in itself was a risk factor for complications. There were no differences in survival at long-term follow-up. Discussion. A repeated liver resection for CRLM was associated with an increased intraoperative bleeding as compared to the first resection. Possible explanations include presence of adhesions, deranged vascular anatomy, more complicated operations and the effects on the liver by chemotherapy following the first liver resection. 30 out of 32 patients had only one reresection of the liver.