Author Correction: Acute and chronic hypoxia differentially predispose lungs for metastases.

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Acute and chronic hypoxia differentially predispose lungs for metastases.

Oscillations in oxygen levels affect malignant cell growth, survival, and metastasis, but also somatic cell behaviour. In this work, we studied the effect of the differential expression of the two primary hypoxia inducible transcription factor isoforms, HIF-1α and HIF-2α, and pulmonary hypoxia to investigate how the hypoxia response of the vascular endothelium remodels the lung pre-metastatic niche. Molecular responses to acute versus chronic tissue hypoxia have been proposed to involve dynamic HIF stabilization, but the downstream consequences and the extent to which differential lengths of exposure to hypoxia can affect HIF-isoform activation and secondary organ pre-disposition for metastasis is unknown. We used primary pulmonary endothelial cells and mouse models with pulmonary endothelium-specific deletion of HIF-1α or HIF-2α, to characterise their roles in vascular integrity, inflammation and metastatic take after acute and chronic hypoxia. We found that acute hypoxic response results in increased lung metastatic tumours, caused by HIF-1α-dependent endothelial cell death and increased microvascular permeability, in turn facilitating extravasation. This is potentiated by the recruitment and retention of specific myeloid cells that further support a pro-metastatic environment. We also found that chronic hypoxia delays tumour growth to levels similar to those seen in normoxia, and in a HIF-2α-specific fashion, correlating with increased endothelial cell viability and vascular integrity. Deletion of endothelial HIF-2α rendered the lung environment more vulnerable to tumour cell seeding and growth. These results demonstrate that the nature of the hypoxic challenge strongly influences the nature of the endothelial cell response, and affects critical parameters of the pulmonary microenvironment, significantly impacting metastatic burden. Additionally, this work establishes endothelial cells as important players in lung remodelling and metastatic progression.

Wheat root length and not branching is altered in the presence of neighbours, including blackgrass.

The effect of neighbouring plants on crop root system architecture may directly interfere with water and nutrient acquisition, yet this important and interesting aspect of competition remains poorly understood. Here, the effect of the weed blackgrass (Alopecurus myosuroides Huds.) on wheat (Triticum aestivum L.) roots was tested, since a low density of this species (25 plants m-2) can lead to a 10% decrease in wheat yield and herbicide resistance is problematic. We used a simplified growth system based on gelled medium, to grow wheat alongside a neighbour, either another wheat plant, a blackgrass or Brachypodium dystachion individual (a model grass). A detailed analysis of wheat seminal root system architecture showed that the presence of a neighbour principally affected the root length, rather than number or diameter under a high nutrient regime. In particular, the length of first order lateral roots decreased significantly in the presence of blackgrass and Brachypodium. However, this effect was not noted when wheat plants were grown in low nutrient conditions. This suggests that wheat may be less sensitive to the presence of blackgrass when grown in low nutrient conditions. In addition, nutrient availability to the neighbour did not modulate the neighbour effect on wheat root architecture.

HIF2α-arginase axis is essential for the development of pulmonary hypertension.

Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodeling. The hypoxia-inducible transcription factors (HIFs) HIF-1α and HIF-2α are known to contribute to the process of hypoxic pulmonary vascular remodeling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2α is a critical regulator of hypoxia-induced pulmonary arterial hypertension. The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2α deletion. The RVSP of mice lacking HIF-2α in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic WT mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1α exposed to hypoxia. Endothelial HIF-2α deletion also protected mice from hypoxia remodeling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2α, likewise attenuated many of the pathophysiological symptoms associated with hypoxic pulmonary hypertension. We propose a mechanism whereby chronic hypoxia enhances HIF-2α stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2α in regulating the pulmonary vascular response to hypoxia.

Salinity-induced calcium signaling and root adaptation in Arabidopsis require the calcium regulatory protein annexin1.

Salinity (NaCl) stress impairs plant growth and inflicts severe crop losses. In roots, increasing extracellular NaCl causes Ca²âº influx to elevate cytosolic free Ca²âº ([Ca²âº](cyt)) as a second messenger for adaptive signaling. Amplification of the signal involves plasma membrane reduced nicotinamide adenine dinucleotide phosphate oxidase activation, with the resultant reactive oxygen species triggering Ca²âº influx. The genetic identities of the Ca²âº-permeable channels involved in generating the [Ca²âº](cyt) signal are unknown. Potential candidates in the model plant Arabidopsis (Arabidopsis thaliana) include annexin1 (AtANN1). Here, luminescent detection of [Ca²âº](cyt) showed that AtANN1 responds to high extracellular NaCl by mediating reactive oxygen species-activated Ca²âº influx across the plasma membrane of root epidermal protoplasts. Electrophysiological analysis revealed that root epidermal plasma membrane Ca²âº influx currents activated by NaCl are absent from the Atann1 loss-of-function mutant. Both adaptive signaling and salt-responsive production of secondary roots are impaired in the loss-of-function mutant, thus identifying AtANN1 as a key component of root cell adaptation to salinity.

Cytosolic Ca(2+) determinations in studying plant responses to salinity and oxidative stress.

Salinity and oxidative stress can transiently elevate cytosolic free Ca(2+) ([Ca(2+)](cyt)) of plant cells. The [Ca(2+)](cyt) increase may be part of a signaling cascade or cell death, depending on cell type, the magnitude and the duration of stress exposure. Several approaches for determining [Ca(2+)](cyt) responses are available to plant biologists, but some require highly specialized equipment. Here we describe protocols for using aequorin as a standard [Ca(2+)](cyt) reporter, with output detected with a plate-reader luminometer.

Receptor-like activity evoked by extracellular ADP in Arabidopsis root epidermal plasma membrane.

Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca(2+) influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca(2+) in root epidermal protoplasts. An inward Ca(2+)-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca(2+) efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

Targeting of pollen tubes to ovules is dependent on nitric oxide (NO) signaling.

The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to a great extent, unknown. Previous studies in vitro showed that nitric oxide (NO) works as a negative chemotropic cue for pollen tube growth in lily (Lilium longiflorum). Furthermore, Arabidopsis thaliana Atnos1 mutant plants, which show defective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen-pistil communication, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnos1 plants shows that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF-2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the micropyle. The pollen-pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1 and two Nitrate Reductases (nr1 and nr2), which collectively are thought to trigger a putative NO signaling pathway. Semi-vivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca(2+) signaling. A role of NO in modulating Ca(2+) signaling was further substantiated by direct imaging the cytosolic free Ca(2+) concentration during NO-induced re-orientation, where two peaks of Ca(2+) occur-one during the slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide evidence for the participation of NO signaling events during pollen-pistil interaction. Of special relevance, NO seems to directly affect the targeting of pollen tubes to the ovule's micropyle by modulating the action of its diffusible factors.