Reassess the t Test: Interact with All Your Data via ANOVA.

Plant biology is rapidly entering an era where we have the ability to conduct intricate studies that investigate how a plant interacts with the entirety of its environment. This requires complex, large studies to measure how plant genotypes simultaneously interact with a diverse array of environmental stimuli. Successful interpretation of the results from these studies requires us to transition away from the traditional standard of conducting an array of pairwise t tests toward more general linear modeling structures, such as those provided by the extendable ANOVA framework. In this Perspective, we present arguments for making this transition and illustrate how it will help to avoid incorrect conclusions in factorial interaction studies (genotype × genotype, genotype × treatment, and treatment × treatment, or higher levels of interaction) that are becoming more prevalent in this new era of plant biology.

The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination.

All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in an msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild-type Ler background, MSL8I711S suppressed pollen germination, similar to wild-type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer.

Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine.

OBJECTIVE: The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene-related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. BACKGROUND: Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate-buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. METHODS: Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP. RESULTS: Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP-related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP-related genes were upregulated by both IS and PBS applications. CONCLUSIONS: This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation.

United in diversity: mechanosensitive ion channels in plants.

Mechanosensitive (MS) ion channels are a common mechanism for perceiving and responding to mechanical force. This class of mechanoreceptors is capable of transducing membrane tension directly into ion flux. In plant systems, MS ion channels have been proposed to play a wide array of roles, from the perception of touch and gravity to the osmotic homeostasis of intracellular organelles. Three families of plant MS ion channels have been identified: the MscS-like (MSL), Mid1-complementing activity (MCA), and two-pore potassium (TPK) families. Channels from these families vary widely in structure and function, localize to multiple cellular compartments, and conduct chloride, calcium, and/or potassium ions. However, they are still likely to represent only a fraction of the MS ion channel diversity in plant systems.

Mechanosensitive channel MSL8 regulates osmotic forces during pollen hydration and germination.

Pollen grains undergo dramatic changes in cellular water potential as they deliver the male germ line to female gametes, and it has been proposed that mechanosensitive ion channels may sense the resulting mechanical stress. Here, we identify and characterize MscS-like 8 (MSL8), a pollen-specific, membrane tension-gated ion channel required for pollen to survive the hypoosmotic shock of rehydration and for full male fertility. MSL8 negatively regulates pollen germination but is required for cellular integrity during germination and tube growth. MSL8 thus senses and responds to changes in membrane tension associated with pollen hydration and germination. These data further suggest that homologs of bacterial MscS have been repurposed in eukaryotes to function as mechanosensors in multiple developmental and environmental contexts.

Impaired expression of neuroprotective molecules in the HIF-1alpha pathway following traumatic brain injury in aged mice.

Elderly traumatic brain injury (TBI) patients have higher rates of mortality and worse functional outcome than non-elderly TBI patients. The mechanisms involved in poor outcomes in the elderly are not well understood. Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a basic helix-loop-helix transcription factor that modulates expression of key genes involved in neuroprotection. In this study, we studied the expression of HIF-1alpha and its target survival genes, heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), and erythropoietin (EPO) in the brains of adult versus aged mice following controlled cortical impact (CCI) injury. Adult (5-6 months) and aged (23-24 months) C57Bl/6 mice were injured using a CCI device. At 72 h post-injury, mice were sacrificed and the injured cortex was used for mRNA and protein analysis using real-time reverse transcription--polymerase chain reaction (RT-PCR) and Western blotting protocols. Following injury, HIF-1alpha, HO-1, and VEGF showed upregulation in both the young and aged mice, but in the aged animals the increase in HIF-1alpha and VEGF in response to injury was much lower than in the adult injured animals. EPO was upregulated in the adult injured brain, but not in the aged injured brain. These results support the hypothesis that reduced expression of genes in the HIF-1alpha neuroprotective pathway in aging may contribute to poor prognosis in the elderly following TBI.