Gibberellin and abscisic acid transporters facilitate endodermal suberin formation in Arabidopsis.

The plant hormone gibberellin (GA) regulates multiple developmental processes. It accumulates in the root elongating endodermis, but how it moves into this cell file and the significance of this accumulation are unclear. Here we identify three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) transporters required for GA and abscisic acid (ABA) translocation. We demonstrate that NPF2.14 is a subcellular GA/ABA transporter, presumably the first to be identified in plants, facilitating GA and ABA accumulation in the root endodermis to regulate suberization. Further, NPF2.12 and NPF2.13, closely related proteins, are plasma membrane-localized GA and ABA importers that facilitate shoot-to-root GA12 translocation, regulating endodermal hormone accumulation. This work reveals that GA is required for root suberization and that GA and ABA can act non-antagonistically. We demonstrate how the clade of transporters mediates hormone flow with cell-file-specific vacuolar storage at the phloem unloading zone, and slow release of hormone to induce suberin formation in the maturation zone.

bHLH heterodimer complex variations regulate cell proliferation activity in the meristems of Arabidopsis thaliana.

Root, shoot, and lateral meristems are the main regions of cell proliferation in plants. It has been proposed that meristems might have evolved dedicated transcriptional networks to balance cell proliferation. Here, we show that basic helix-loop-helix (bHLH) transcription factor heterodimers formed by members of the TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) subclades are general regulators of cell proliferation in all meristems. Yet, genetics and expression analyses suggest specific functions of these transcription factors in distinct meristems, possibly due to their expression domains determining heterodimer complex variations within meristems, and to a certain extent to the absence of some of them in a given meristem. Target gene specificity analysis for heterodimer complexes focusing on the LONELY GUY gene targets further suggests differences in transcriptional responses through heterodimer diversification that could allow a common bHLH heterodimer complex module to contribute to cell proliferation control in multiple meristems.

New Onset Cardiac Murmur and Exertional Dyspnea in an Apparently Healthy Child: A Rare Localization of Obstructive Myxoma in the Right Ventricle Outflow Tract without Pulmonary Embolization-A Case Report and Literature Review.

Myxomas are slowly growing benign neoplasms which are rare in children. Up to 80% can be located in the left atrium and generate symptoms such as embolism, cardiac failure, fever and weight loss. Rarely, myxomas can be detected in the right ventricle outflow tract, causing arrhythmias, pulmonary emboli and sudden death. We report the case of a 13-year-old healthy child brought to the Emergency Department (ED) of the Children's Hospital Bambino Gesù, Rome, for recent dyspnea, chest pain on exertion and new onset cardiac murmur. Patient underwent medical examination and echocardiogram with the finding of a rounded and lobulated voluminous mass in the right ventricle outflow tract (RVOT) which caused severe obstruction. The contrast computed tomography (CT) scan confirmed the presence of a heterogeneously enhancing soft-tissue mass occupying the RVOT with no evidence of pulmonary embolization. The mass was surgically excised, and the pathologic examination confirmed our suspicion of myxoma. Our experience suggests that myxoma can have mild clinical symptoms, the presentation may be non-specific, and diagnosis can be a challenge Careful examination and a diagnostic imaging workup, primarily with the transthoracic echocardiogram, are needful to make a rapid differential diagnosis and to better manage surgical treatment and follow-up.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

The Making of Plant Armor: The Periderm.

The periderm acts as armor protecting the plant's inner tissues from biotic and abiotic stress. It forms during the radial thickening of plant organs such as stems and roots and replaces the function of primary protective tissues such as the epidermis and the endodermis. A wound periderm also forms to heal and protect injured tissues. The periderm comprises a meristematic tissue called the phellogen, or cork cambium, and its derivatives: the lignosuberized phellem and the phelloderm. Research on the periderm has mainly focused on the chemical composition of the phellem due to its relevance as a raw material for industrial processes. Today, there is increasing interest in the regulatory network underlying periderm development as a novel breeding trait to improve plant resilience and to sequester CO2. Here, we discuss our current understanding of periderm formation, focusing on aspects of periderm evolution, mechanisms of periderm ontogenesis, regulatory networks underlying phellogen initiation and cork differentiation, and future challenges of periderm research.

ABA homeostasis and long-distance translocation are redundantly regulated by ABCG ABA importers.

The effects of abscisic acid (ABA) on plant growth, development, and response to the environment depend on local ABA concentrations. Here, we show that in Arabidopsis, ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate­binding cassette subfamily G member 17 (ABCG17) and ABCG18 are localized to the plasma membranes of leaf mesophyll and cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance, and transpiration while increasing water use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement and response. The transport mechanism mediated by ABCG17 and ABCG18 allows plants to maintain ABA homeostasis under normal growth conditions.

Two γ-zeins induce the unfolded protein response.

The rapid, massive synthesis of storage proteins that occurs during seed development stresses endoplasmic reticulum (ER) homeostasis, which activates the ER unfolded protein response (UPR). However, how different storage proteins contribute to UPR is not clear. We analyzed vegetative tissues of transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the common bean (Phaseolus vulgaris) soluble vacuolar storage protein PHASEOLIN (PHSL) or maize (Zea mays) prolamins (27-kDa γ-zein or 16-kDa γ-zein) that participate in forming insoluble protein bodies in the ER. We show that 16-kDa γ-zein significantly activates the INOSITOL REQUIRING ENZYME1/BASIC LEUCINE ZIPPER 60 (bZIP60) UPR branch-but not the bZIP28 branch or autophagy-leading to induction of major UPR-controlled genes that encode folding helpers that function inside the ER. Protein blot analysis of IMMUNOGLOBULIN-BINDING PROTEIN (BIP) 1 and 2, BIP3, GLUCOSE REGULATED PROTEIN 94 (GRP94), and ER-localized DNAJ family 3A (ERDJ3A) polypeptides confirmed their higher accumulation in the plant expressing 16-kDa γ-zein. Expression of 27-kDa γ-zein significantly induced only BIP3 and ERDJ3A transcription even though an increase in GRP94 and BIP1/2 polypeptides also occurred in this plant. These results indicate a significant but weaker effect of 27-kDa γ-zein compared to 16-kDa γ-zein, which corresponds with the higher availability of 16-kDa γ-zein for BIP binding, and indicates subtle protein-specific modulations of plant UPR. None of the analyzed genes was significantly induced by PHSL or by a mutated, soluble form of 27-kDa γ-zein that traffics along the secretory pathway. Such variability in UPR induction may have influenced the evolution of storage proteins with different tissue and subcellular localization.

One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study.

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18-24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (101, 103, and 105 CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 101 CFU/g since T0 and Campylobacter from 103 CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Auxin and gibberellin signaling cross-talk promotes hypocotyl xylem expansion and cambium homeostasis.

During secondary growth, the thickening of plant organs, wood (xylem) and bast (phloem) is continuously produced by the vascular cambium. In Arabidopsis hypocotyl and root, we can distinguish two phases of secondary growth based on cell morphology and production rate. The first phase, in which xylem and phloem are equally produced, precedes the xylem expansion phase in which xylem formation is enhanced and xylem fibers differentiate. It is known that gibberellins (GA) trigger this developmental transition via degradation of DELLA proteins and that the cambium master regulator BREVIPEDICELLUS/KNAT1 (BP/KNAT1) and receptor like kinases ERECTA and ERL1 regulate this process downstream of GA. However, our understanding of the regulatory network underlying GA-mediated secondary growth is still limited. Here, we demonstrate that DELLA-mediated xylem expansion in Arabidopsis hypocotyl is mainly achieved through DELLA family members RGA and GAI, which promote cambium senescence. We further show that AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, which physically interact with DELLAs, specifically repress phloem proliferation and induce cambium senescence during the xylem expansion phase. Moreover, the inactivation of BP in arf6 arf8 background revealed an essential role for ARF6 and ARF8 in cambium establishment and maintenance. Overall, our results shed light on a pivotal hormone cross-talk between GA and auxin in the context of plant secondary growth.

Tissue-Autonomous Phenylpropanoid Production Is Essential for Establishment of Root Barriers.

Plants deposit hydrophobic polymers, such as lignin or suberin, in their root cell walls to protect inner tissues and facilitate selective uptake of solutes. Insights into how individual root tissues contribute to polymer formation are important for elucidation of ultrastructure, function, and development of these protective barriers. Although the pathways responsible for production of the barrier constituents are established, our models lack spatiotemporal resolution-especially in roots-thus, the source of monomeric barrier components is not clear. This is mainly due to our restricted ability to manipulate synthesis of the broadly important phenylpropanoid pathway, as mutants in this pathway display lethal or pleiotropic phenotypes. Here, we overcome this challenge by exploiting highly controlled in vivo repression systems. We provide strong evidence that autonomous production of phenylpropanoids is essential for establishment of the endodermal Casparian strip as well as adherence of the suberin matrix to the cell wall of endodermis and cork. Our work highlights that, in roots, the phenylpropanoid pathway is under tight spatiotemporal control and serves distinct roles in barrier formation across tissues and developmental zones. This becomes evident in the late endodermis, where repression of phenylpropanoid production leads to active removal of suberin in pre-suberized cells, indicating that endodermal suberin depositions might embody a steady state between continuous synthesis and degradation.

Pluripotent Pericycle Cells Trigger Different Growth Outputs by Integrating Developmental Cues into Distinct Regulatory Modules.

During post-embryonic development, the pericycle specifies the stem cells that give rise to both lateral roots (LRs) and the periderm, a suberized barrier that protects the plant against biotic and abiotic stresses. Comparable auxin-mediated signaling hubs regulate meristem establishment in many developmental contexts; however, it is unknown how specific outputs are achieved. Using the Arabidopsis root as a model, we show that while LR formation is the main auxin-induced program after de-etiolation, plants with age become competent to form a periderm in response to auxin. The establishment of the vascular cambium acts as the developmental switch required to trigger auxin-mediated periderm initiation. Moreover, distinct auxin signaling components and targets control LR versus periderm formation. Among the periderm-specific-promoting transcription factors, WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNAT1/BREVIPEDICELLUS (BP) stand out as their specific overexpression in the periderm results in an increased number of periderm layers, a trait of agronomical importance in breeding programs targeting stress tolerance. These findings reveal that specificity in pericycle stem cell fate is achieved by the integration of developmental cues into distinct regulatory modules.

The development of the periderm: the final frontier between a plant and its environment.

The periderm acts as the first line of defence for a plant, protecting wood and phloem from abiotic and biotic stresses. During secondary growth, through the increase in girth of plant organs, the periderm replaces the epidermis as the outermost tissue. The phellogen, a bifacial post-embryonic meristem, forms the phelloderm inwards (toward the vasculature) and the suberized phellem outwards (toward the environment). These three tissues are collectively referred to as the periderm. Here, we summarize recent findings on the molecular mechanisms of periderm development by describing periderm formation in connection to the fate of the surrounding tissues, by discussing common regulatory hubs between the vascular cambium and the phellogen, and by highlighting transcription factors (TFs) controlling phellem differentiation.

Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots.

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity1, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development.

Tissue-specific study across the stem reveals the chemistry and transcriptome dynamics of birch bark.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications.

Interrupted versus uninterrupted novel oral anticoagulant peri-implantation of cardiac device: A single-center randomized prospective pilot trial.

BACKGROUND: Many patients requiring cardiac implantable electronic device (CIED) implantation are on long-term oral anticoagulant therapy. While continuation of warfarin has been shown to be safe and reduce bleeding complications compared to interruption of warfarin therapy and heparin bridging, it is not known which novel oral anticoagulants (NOAC) regimen (interrupted vs uninterrupted) is better in this setting. METHODS: One-hundred and one patients were randomized to receive CIED implantation with either interrupted or uninterrupted/continuous NOAC therapy before surgery. No heparin was used in either treatment arm. The primary end-point was the presence of a clinically significant pocket hematoma after CIED implantation. The secondary end-point was a composite of other major bleeding events, device-related infection, thrombotic events, and device-related admission length postdevice implantation. RESULTS: Both treatment groups were equally balanced for baseline variables and concomitant medications. One clinically significant pocket hematoma occurred in the uninterrupted NOAC group and none in the interrupted group (P  =  0.320). There was no difference in other bleeding complications. No thrombotic events were observed in either of the two groups. CONCLUSIONS: Despite the paucity of bleeding events, data from this pilot study suggest that uninterrupted NOAC therapy for CIED implantation appears to be as safe as NOAC interruption and does not increase bleeding complications.

Early prediction of contrast-induced acute kidney injury by a "bedside" assessment of Neutrophil Gelatinase-Associated Lipocalin during elective percutaneous coronary interventions.

Contrast-induced acute kidney injury (CI-AKI) is a serious complication during percutaneous coronary interventions (PCI). Currently, the diagnosis of CI-AKI relies on serum creatinine (SCr) that is however affected by several limitations potentially leading to delayed or missed diagnoses. In this study we examined the diagnostic accuracy of a "bedside" measurement of plasma Neutrophil Gelatinase-Associated Lipocalin (NGAL) in the early detection of CI-AKI in 97 patients undergoing elective PCI. The overall incidence of CI-AKI was 3%. A significant positive correlation was observed between 6-hours NGAL and post-PCI SCr (r = 0.339, p = 0.004) and a significant negative correlation between 6-hours NGAL and post-PCI CrCl (r = -0.303, p = 0.010). In patients with post-PCI SCr increase > 0.24 mg/dl (median SCr absolute increase), delta NGAL 0-6 hours and 6-hours NGAL values were higher compared with patients with SCr elevation below the defined threshold (p = 0.049 and p = 0.056). The ROC analysis showed that a 6 hours NGAL value > 96 ng/ml significantly predicted an absolute SCr increase > 0.24 mg/dl after contrast exposure with sensitivity of 53% and specificity of 74% (AUC 0.819, 95% CI: 0.656 to 0.983, p = 0.005). The use of bedside NGAL assessment may significantly hasten diagnosis and treatment of CI-AKI, with remarkable clinical prognostic consequences.

A molecular framework to study periderm formation in Arabidopsis.

During secondary growth in most eudicots and gymnosperms, the periderm replaces the epidermis as the frontier tissue protecting the vasculature from biotic and abiotic stresses. Despite its importance, the mechanisms underlying periderm establishment and formation are largely unknown. The herbaceous Arabidopsis thaliana undergoes secondary growth, including periderm formation in the root and hypocotyl. Thus, we focused on these two organs to establish a framework to study periderm development in a model organism. We identified a set of characteristic developmental stages describing periderm growth from the first cell division in the pericycle to the shedding of the cortex and epidermis. We highlight that two independent mechanisms are involved in the loosening of the outer tissues as the endodermis undergoes programmed cell death, whereas the epidermis and the cortex are abscised. Moreover, the phellem of Arabidopsis, as in trees, is suberized, lignified and peels off. In addition, putative regulators from oak and potato are also expressed in the Arabidopsis periderm. Collectively, the periderm of Arabidopsis shares many characteristics/features of woody and tuberous periderms, rendering Arabidopsis thaliana an attractive model for cork biology.

Secondary growth as a determinant of plant shape and form.

Plants are the primary producers of biomass on earth. As an almost stereotypic feature, higher plants generate continuously growing bodies mediated by the activity of different groups of stem cells, the meristems. Shoot and root thickening is one of the fundamental growth processes determining form and function of these bodies. Mediated by a group of cylindrical meristems located below organ surfaces, vascular and protective tissues are continuously generated in a highly plastic manner, a competence essential for the survival in an ever changing environment. Acknowledging the fundamental role of this process, which is overall designated as secondary growth, we discuss in this review our current knowledge about the evolution and molecular regulation of the vascular cambium. The cambium is the meristem responsible for the formation of wood and bast, the two types of vascular tissues important for long-distance transport of water and assimilates, respectively. Although regulatory patterns are only beginning to emerge, we show that cambium activity represents a highly rewarding model for studying cell fate decisions, tissue patterning and differentiation, which has experienced an outstanding phylogenetic diversification.

Vascular Morphodynamics During Secondary Growth.

Quantification of vascular morphodynamics during secondary growth has been hampered by the scale of the process. Even in the tiny model plant Arabidopsis thaliana, the xylem can include more than 2000 cells in a single cross section, rendering manual counting impractical. Moreover, due to its deep location, xylem is an inaccessible tissue, limiting live imaging. A novel method to visualize and measure secondary growth progression has been proposed: "the Quantitative Histology" approach. This method is based on a detailed anatomical atlas, and image segmentation coupled with machine learning to automatically extract cell shapes and identify cell type. Here we present a new version of this approach, with a user-friendly interface implemented in the open source software LithoGraphX.