Sequential removal of cation/H+ exchangers reveals their additive role in elemental distribution, calcium depletion and anoxia tolerance.

Multiple Arabidopsis H+ /Cation exchangers (CAXs) participate in high-capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1-4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast-localised H+ /Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants. Leveraging inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP-MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild-type plants grown in Ca-limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+ /Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.

Instead of Calories, Should We Be Counting our Consumption of Exosomes and MicroRNAs?

The specific foods to eat for optimal nutrition remain ill-defined. Studies using plantbased diets or milk suggest that vesicles, termed exosomes, and small RNAs termed microRNAs (miRNAs) are health promoting components in foods. However, numerous studies refute the potential of dietary cross-kingdom communication of exosomes and miRNAs. While research reinforces that plant-based diets and milk are healthy components of a well-rounded diet, the bioavailability and bioactivity of the exosomes and miRNAs present in plant-based diets and milk remain unclear. Further investigations of plant-based diet and milk exosome like particles may open a new era in application of food for overall health enhancement. In addition, the potential biotechnological plantbased diet and milk exosome like particles can aid in cancer treatment.

Generating Reproducing Anoxia Conditions for Plant Phenotyping.

Based on the availability of oxygen, plant growth environment can be normoxic (normal environment), hypoxic (reduced oxygen, <21%), or anoxic (complete depletion of oxygen). Hypoxic/anoxic environment is created when a plant is exposed to stresses such as submergence, flooding, or pathogen attack. Survival of the plants following stress conditions is in part dependent on their ability to overcome the stress induced by anoxia/hypoxia conditions. This shows the need for the development of strategies for understanding the mechanisms involved in plant tolerance to anoxia. Previous studies have employed different methods for establishing an anerobic environment. Here, we describe a simple method for creating anoxic environment using an anaerobic atmosphere generation bag. Anoxic conditions can be maintained in a cylindrical jar, a rectangular box, or a vacuum sealer bag, enabling the screening of a large number of samples. This protocol is particularly useful to screen plant mutants that are tolerant to anoxia. The method is simple, easy, cost-efficient, reproducible, and does not require any sophisticated instruments. Graphic abstract.

Crucial role of Arabidopsis glutaredoxin S17 in heat stress response revealed by transcriptome analysis.

Heat stress can have detrimental effects on plant growth and development. However, the mechanisms by which the plant is able to perceive changes in ambient temperature, transmit this information, and initiate a temperature-induced response are not fully understood. Previously, we showed that heterologous expression of an Arabidopsis thaliana L. monothiol glutaredoxin AtGRXS17 enhances thermotolerance in various crops, while disruption of AtGRXS17 expression caused hypersensitivity to permissive temperature. In this study, we extend our investigation into the effect of AtGRXS17 and heat stress on plant growth and development. Although atgrxs17 plants were found to exhibit a slight decrease in hypocotyl elongation, shoot meristem development, and root growth compared to wild-type when grown at 22°C, these growth phenotypic differences became more pronounced when growth temperatures were raised to 28°C. Transcriptome analysis revealed significant changes in genome-wide gene expression in atgrxs17 plants compared to wild-type under conditions of heat stress. The expression of genes related to heat stress factors, auxin response, cellular communication, and abiotic stress were altered in atgrxs17 plants in response to heat stress. Overall, our findings indicate that AtGRXS17 plays a critical role in controlling the transcriptional regulation of plant heat stress response pathways.

The vacuolar H+/Ca transporter CAX1 participates in submergence and anoxia stress responses.

A plant's oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice (Oryza sativa) cultivation, and submergence tolerance of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters to this process is poorly understood. Four functional cation/proton exchangers (CAX1-4) in Arabidopsis (Arabidopsis thaliana) help regulate Ca homeostasis around the vacuole. Our results demonstrate that cax1 mutants are more tolerant to both anoxic conditions and submergence. Using phenotypic measurements, RNA-sequencing, and proteomic approaches, we identified cax1-mediated anoxia changes that phenocopy changes present in anoxia-tolerant crops: altered metabolic processes, diminished reactive oxygen species production post anoxia, and altered hormone signaling. Comparing wild-type and cax1 expressing genetically encoded Ca indicators demonstrated altered cytosolic Ca signals in cax1 during reoxygenation. Anoxia-induced Ca signals around the plant vacuole are involved in the control of numerous signaling events related to adaptation to low oxygen stress. This work suggests that cax1 anoxia response pathway could be engineered to circumvent the adverse effects of flooding that impair production agriculture.

Redox-engineering enhances maize thermotolerance and grain yield in the field.

Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a sixfold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment.

Crucial Role of Mammalian Glutaredoxin 3 in Cardiac Energy Metabolism in Diet-induced Obese Mice Revealed by Transcriptome Analysis.

Obesity is often associated with metabolic dysregulation and oxidative stress with the latter serving as a possible unifying link between obesity and cardiovascular complications. Glutaredoxins (Grxs) comprise one of the major antioxidant systems in the heart. Although Grx3 has been shown to act as an endogenous negative regulator of cardiac hypertrophy and heart failure, its metabolic impact on cardiac function in diet-induced obese (DIO) mice remains largely unknown. In the present study, analysis of Grx3 expression indicated that Grx3 protein levels, but not mRNA levels, were significantly increased in the hearts of DIO mice. Cardiac-specific Grx3 deletion (Grx3 CKO) mice were viable and grew indistinguishably from their littermates after being fed a high fat diet (HFD) for one month, starting at 2 months of age. After being fed with a HFD for 8 months (starting at 2 months of age); however, Grx3 CKO DIO mice displayed left ventricular systolic dysfunction with a significant decrease in ejection fraction and fractional shortening that was associated with heart failure. ROS production was significantly increased in Grx3 CKO DIO cardiomyocytes compared to control cells. Gene expression analysis revealed a significant decline in the level of transcripts corresponding to genes associated with processes such as fatty acid uptake, mitochondrial fatty acid transport and oxidation, and citrate cycle in Grx3 CKO DIO mice compared to DIO controls. In contrast, an increase in the level of transcripts corresponding to genes associated with glucose uptake and utilization were found in Grx3 CKO DIO mice compared to DIO controls. Taken together, these findings indicate that Grx3 may play a critical role in redox balance and as a metabolic switch in cardiomyocytes contributing to the development and progression of heart failure.

Fusobacterium nucleatum Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation.

Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome.IMPORTANCE Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that F. nucleatum subsp. polymorphum releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals in vitro Using mice harboring a human microbiome, we demonstrate that F. nucleatum can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which F. nucleatum may contribute to intestinal inflammation.

Roles of Regulatory RNAs in Nutritional Control.

Small RNAs (sRNAs), including microRNAs (miRNAs), are noncoding RNA (ncRNA) molecules involved in gene regulation. sRNAs play important roles in development; however, their significance in nutritional control and as metabolic modulators is still emerging. The mechanisms by which diet impacts metabolic genes through miRNAs remain an important area of inquiry. Recent work has established how miRNAs are transported in body fluids often within exosomes, which are small cell-derived vesicles that function in intercellular communication. The abundance of other recently identified ncRNAs and new insights regarding ncRNAs as dietary bioactive compounds could remodel our understanding about how foods impact gene expression. Although controversial, some groups have shown that dietary RNAs from plants and animals (i.e., milk) are functional in consumers. In the future, regulating sRNAs either directly through dietary delivery or indirectly by altered expression of endogenous sRNA may be part of nutritional interventions for regulating metabolism.

Genetically Modified Plants: Nutritious, Sustainable, yet Underrated.

Combating malnutrition is one of the greatest global health challenges. Plant-based foods offer an assortment of nutrients that are essential for adequate nutrition and can promote good health. Unfortunately, the majority of widely consumed crops are deficient in some of these nutrients. Biofortification is the umbrella term for the process by which the nutritional quality of food crops is enhanced. Traditional agricultural breeding approaches for biofortification are time consuming but can enhance the nutritional value of some foods; however, advances in molecular biology are rapidly being exploited to biofortify various crops. Globally, genetically modified organisms are a controversial topic for consumers and governmental agencies, with a vast majority of people apprehensive about the technology. Golden Rice has been genetically modified to contain elevated ß-carotene concentrations and is the bellwether for both the promise and angst of agricultural biotechnology. Although there are numerous other nutritional targets of genetically biofortified crops, here I briefly summarize the work to elevate iron and folate concentrations. In addition, the possibility of using modified foods to affect the gut microbiota is examined. For several decades, plant biotechnology has measured changes in nutrient concentrations; however, the bioavailability of nutrients from many biofortified crops has not been demonstrated.

Gut Bacteria have a novel sweet tooth: ribose sensing and scavenging from fiber.

Dietary fiber is known to influence symbiotic gut microbiota community structure and physiology; however, how and if dietary fiber can induce further exogenous nutrient uptake within gut microbes is ill-defined. Recent findings highlight how during periods of high-fiber consumption, a prevalent gut bacteria senses and scavenges the ubiquitous sugar ribose. This molecular adaptation exemplifies how particular gut microbes have developed a sophisticated system to scavenge nutrients in a diet-dependent manner.

Alteration of iron responsive gene expression in Arabidopsis glutaredoxin S17 loss of function plants with or without iron stress.

Iron (Fe) is a mineral nutrient and a metal cofactor essential for plants. Iron limitation can have detrimental effects on plant growth and development, while excess iron inside plant cells leads to oxidative damage. As a result, plants have evolved complex regulatory networks to respond to fluctuations in cellular iron concentrations. The mechanisms that regulate these responses however, are not fully understood. Heterologous expression of an Arabidopsis thaliana monothiol glutaredoxin S17 (GRXS17) suppresses the over-accumulation of iron in the Saccharomyces cerevisiae Grx3/Grx4 mutant and disruption of GRXS17 causes plant sensitivity to exogenous oxidants and iron deficiency stress. GRXS17 may act as an important regulator in the plant's ability to respond to iron deficiency stress and maintain redox homeostasis. Here, we extend this investigation by analyzing iron-responsive gene expression of the Fer-like iron deficiency-induced transcription factor (FIT) network (FIT, IRT1, FRO1, and FRO2) and the bHLH transcription factor POPEYE (PYE) network (PYE, ZIF1, FRO3, NAS4, and BTS) in GRXS17 KO plants and wildtype controls grown under iron sufficiency and deficiency conditions. Our findings suggest that GRXS17 is required for tolerance to iron deficiency, and plays a negative regulatory role under conditions of iron sufficiency.

Expression of mouse small interfering RNAs in lettuce using artificial microRNA technology.

Artificial miRNA technology enables the generation of siRNAs to regulate the expression of targeted genes. However, the application of siRNAs to alter gene expression is challenging due to their instability and requires a means to efficiently deliver siRNAs into the host. Here, we report that the siRNAs targeted to animal mRNAs can be heterologously expressed and stably produced in lettuce. We have modified rice miRNA precursors to produce siRNAs in lettuce with the potential to target mRNAs of mouse complement 3 (C3) and coagulation factor 7 (CF7). Expression of primary and mature siRNAs in the transgenic lettuce lines was confirmed via Sanger sequencing. Our study demonstrates an applicable tool to alter gene expression in the targeted host and has potential utility in siRNA-based oral therapeutics.

Dietary impact of a plant-derived microRNA on the gut microbiome.

BACKGROUND: Global estimations of 4 billion people living on plant-based diets signify tremendous diversity in plant consumption and their assorted miRNAs, which presents a challenging model to experimentally address how plant-based miRNAs impact the microbiome. Here we establish baseline gut microbiome composition for a mouse model deficient in the specific mammalian miR-146a shown to alter gut microbiomes. We then asses the effect on the gut microbiome when miR-146a-deficient mice are fed a transgenic plant-based diet expressing the murine-derived miR-146a. Mice deficient in miR-146a were maintained either on a baseline diet until 7 weeks of age (day 0) and then fed either vector or miR-146a-expressing plant-based diets for 21 days. The gut microbiomes of mice were examined by comparing the V4 region of 16S rRNA gene sequences of DNA isolated from fecal samples at days 0 (baseline diet) and 21 (vector or miR-146a expressing plant-based diets). RESULTS: Beta-diversity analysis demonstrated that the transition from baseline chow to a plant-based diet resulted in significant longitudinal shifts in microbial community structure attributable to increased fiber intake. Bipartite network analysis suggests that miR-146a-deficient mice fed a plant diet rich in miR-146a have a microbiome population modestly different than mice fed an isogenic control plant diet deficient in miR-146a. CONCLUSION: A mouse diet composed of a transgenic plant expressing a mouse miR-146a may fine tune microbial communities but does not appear to have global effects on microbiome structure and composition.

Cardiac-specific ablation of glutaredoxin 3 leads to cardiac hypertrophy and heart failure.

Growing evidence suggests that redox-sensitive proteins including glutaredoxins (Grxs) can protect cardiac muscle cells from oxidative stress-induced damage. Mammalian Grx3 has been shown to be critical in regulating cellular redox states. However, how Grx3 affects cardiac function by modulating reactive oxygen species (ROS) signaling remains unknown. In this study, we found that the expression of Grx3 in the heart is decreased during aging. To assess the physiological role of Grx3 in the heart, we generated mice in which Grx3 was conditionally deleted in cardiomyocytes (Grx3 conditional knockout (CKO) mice). Grx3 CKO mice were viable and grew indistinguishably from their littermates at young age. No difference in cardiac function was found comparing Grx3 CKO mice and littermate controls at this age. However, by the age of 12 months, Grx3 CKO mice exhibited left ventricular hypertrophy with a significant decrease in ejection fraction and fractional shortening along with a significant increase of ROS production in cardiomyocytes compared to controls. Deletion of Grx3 also impaired Ca2+ handling, caused enhanced sarcoplasmic reticulum (SR) calcium (Ca2+ ) leak, and decreased SR Ca2+ uptake. Furthermore, enhanced ROS production and alteration of Ca2+ handling in cardiomyocytes occurred, prior to cardiac dysfunction in young mice. Therefore, our findings demonstrate that Grx3 is an important factor in regulating cardiac hypertrophy and heart failure by modulating both cellular redox homeostasis and Ca2+ handling in the heart.

Planting the Microbiome.

Plant-derived microRNAs stabilized by species-specific lipid nanoparticles mediate interkingdom communication through bacterial intermediates and impact consumer health. Ingested by distinct gut bacteria, these microRNA-containing particles alter bacterial gene expression to affect host immunity. This three-kingdom interplay provides compelling approaches for health-directed dietary interventions for consumers.

The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H+ gradient to synthesize pyrophosphate.

Energy partitioning and plant growth are mediated in part by a type I H+-pumping pyrophosphatase (H+-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H+-PPase from Arabidopsis also functions in "reverse mode" to synthesize PPi using the transmembrane H+ gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon Pi application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H+-PPase mutant lines (vhp1-1 and fugu5-1) lacking the classical PPi-induced outward currents related to H+ pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H+-PPase. Current amplitudes related to reverse-mode H+ transport depended on the membrane potential, cytosolic Pi concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H+-PPase (AVP1) demonstrated Pi-dependent PPi synthase activity in the presence of a pH gradient. Our work establishes that a plant H+-PPase can operate as a PPi synthase beyond its canonical role in vacuolar acidification and cytosolic PPi scavenging. We propose that the PPi synthase activity of H+-PPase contributes to a cascade of events that energize plant growth.

Intestinal permeability, digestive stability and oral bioavailability of dietary small RNAs.

Impactful dietary RNA delivery requires improving uptake and enhancing digestive stability. In mouse feeding regimes, we have demonstrated that a plant-based ribosomal RNA (rRNA), MIR2911, is more bioavailable than synthetic MIR2911 or canonical microRNAs (miRNAs). Here mutagenesis was used to discern if MIR2911 has a distinctive sequence that aids stability and uptake. Various mutations had modest impacts while one scrambled sequence displayed significantly enhanced digestive stability, serum stability, and bioavailability. To assess if small RNA (sRNA) bioavailability in mice could be improved by increasing gut permeability, various diets, genetic backgrounds and pharmacological methods were surveyed. An intraperitoneal injection of anti-CD3 antibody enhanced gut permeability which correlated with improved uptake of the digestively stable scrambled MIR2911 variant. However, the bioavailability of canonical miRNAs was not enhanced. Similarly, interleukin-10 (IL-10)-deficient mice and mice treated with aspirin displayed enhanced gut permeability that did not enhance uptake of most plant-based sRNAs. This work supports a model where dietary RNAs are vulnerable to digestion and altering gut permeability alone will not impact apparent bioavailability. We suggest that some dietary sRNA may be more digestively stable and methods to broadly increase sRNA uptake requires delivery vehicles to optimize gut and serum stability in the consumer.