Mice lacking myotubularin-related protein 14 show accelerated high-fat diet-induced lipid accumulation and inflammation

The phosphoinositide phosphatase, myotubularin-related protein 14 (MTMR14), has been reported to play an important role in the regulation of muscle performance, autophagy, and aging in mice. We previously showed that MTMR14-knockout (KO) mice gain weight earlier than their wild-type (WT) littermates even on a normal chow diet (NCD), suggesting that this gene might also be involved in regulating metabolism. In the present study, we evaluated the effect of MTMR14 deficiency on high-fat diet (HFD)-induced obesity, lipid accumulation, metabolic disorders, and inflammation in WT and MTMR14-KO mice fed with NCD or HFD. To this end, MTMR14-KO mice fed with HFD showed significantly increased body weight, blood glucose levels, serum triglyceride (TG) levels, and total cholesterol (TC) levels as compared to their age-matched WT control. Additionally, lipid accumulation also increased in the KO mice. Simultaneously, the expression of metabolism-associated genes (Glut4, adiponectin, and leptin) was different in the liver, muscle, and fatty tissue of MTMR14-KO mice fed with HFD. More importantly, the expression of several inflammation-associated genes (TNF-α, IL-6, IL-1β, and MCP-1) dramatically increased in the liver, muscle, and fatty tissue of MTMR14-KO mice relative to control. Taken together, these results suggest that MTMR14 deficiency accelerates HFD-induced metabolic dysfunction and inflammation. Furthermore, the results showed that exacerbated metabolic dysfunction and inflammation may be regulated via the PI3K/Akt and ERK signaling pathways.     

Taurine regulation of short term synaptic plasticity in fragile X mice

Background

Fragile X Syndrome is the most common known genetic cause of autism. The Fmr1-KO mouse, lacks the fragile X mental retardation protein (FMRP), and is used as a model of the syndrome. The core behavioral deficits of autism may be conceptualized either as excessive adherence to patterns as seen in repetitive actions and aberrant language, or as insensitivity to subtle but socially important changes in patterns. The hippocampus receives information from the entorhinal cortex and plays a crucial role in the processing of patterned information. To gain more insight into the physiological function of FMRP and the neuronal mechanisms underlying fragile X syndrome, we examined the electrophysiological response of the hippocampus to pair pulse stimulation as a measure of patterned information processing and how it is affected in the Fmr1-KO mouse.

Methods

In this study, we used paired-pulse stimulation of the afferent perforant path and recorded from the CA1 region of the hippocampus. Two-month-old FVB/NJ male mice and age-matched Fmr1-KO mice were used in this study. Hippocampal slices were prepared, equilibrated in artificial cerebrospinal fluid (aCSF), and excitatory post synaptic potentials (EPSPs) measured by stimulating the perforant path of the dentate gyrus (DG) while recording from the molecular layer of CA1. Stimulation occurred by setting current and pulse width to evoke a fixed percentage of maximal EPSP amplitude. This stimulation paradigm allowed us to examine the processing capabilities of the hippocampus as a function of increasing interstimulus intervals (ISI) and how taurine, a GABA_A receptor agonist, affects such information processing.

Results

We found that hippocampal slices from wild type (WT) showed pair-pulse facilitation at ISI of 100-300 ms whereas slices from Fmr1-KO brains showed a consistent pair-pulse depression at a comparable ISI. Addition of 10 μM taurine to WT slices resulted in a drastic decrease of the peak response to the second stimulus, resulting in an initial depression at 100 ms ISI followed by potentiation at higher ISI (150 ms and above). In the presence of taurine, the amplitude of the second response remained significantly lower than in its absence. Fmr1-KO mice however, were completely insensitive to taurine application and pair-pulse stimulation always resulted in a depression of the response to the second stimulus.

Conclusions

Previously we reported that Fmr1-KO mice have reduced beta subunits of the GABA_A receptors. We also showed as well as others that taurine acts as an agonist or a modulator for GABA_A receptors. Therefore, the insensitivity of Fmr1-KO slices to taurine application could be due to the reduced binding sites on the GABA_A receptors in the Fmr1-KO mice.

     

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Key message

Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis.

Abstract

Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two β subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1–CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

     

Gene expression and localization of a β-1,3-glucanase of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Phytohormone abscisic acid (ABA) inhibits root nodule formation of leguminous plants. LjGlu1, a β-1,3-glucanase gene of Lotus japonicus, has been identified as an ABA responsive gene. RNA interference of LjGlu1 increased nodule number. This suggests that LjGlu1 is involved in the regulation of nodule formation. Host legumes control nodule number by autoregulation of nodulation (AON), in which the presence of existing root nodules inhibits further nodulation. For further characterization of LjGlu1, we focused on the expression of LjGlu1 in relation to AON. In a split-root system, LjGlu1 expression peaked when AON was fully induced. Hairy roots transformed with LjCLE-RS1, a gene that induces AON, were generated. Expression of LjGlu1 was greater in the transgenic roots than in untransformed roots. LjGlu1 was not induced in a hypernodulating mutant inoculated with Mesorhizobium loti. These results suggest that the expression of LjGlu1 is involved in the system of AON. However, neither hypernodulation nor enlarged nodulation zone was observed on the transgenic hairy roots carrying LjGlu1-RNAi, suggesting that LjGlu1 is not a key player of AON. Recombinant LjGlu1 showed endo-β-1,3-glucanase activity. LjGlu1-mOrange fusion protein suggested that LjGlu1 associated with M. loti on the root hairs. Exogenous β-1,3-glucanase inhibited infection thread formation by both the wild type and the mutant, and nodule numbers were reduced. These results suggest that LjGlu1 is expressed in response to M. loti infection and functions outside root tissues, resulting in the inhibition of infection.     

The role of pre-symbiotic auxin signaling in ectendomycorrhiza formation between the desert truffle <Emphasis Type="Italic">Terfezia boudieri</Emphasis> and <Emphasis Type="Italic">Helianthemum sessiliflorum</Emphasis>

The ectendomycorrhizal fungus Terfezia boudieri is known to secrete auxin. While some of the effects of fungal auxin on the plant root system have been described, a comprehensive understanding is still lacking. A dual culture system to study pre mycorrhizal signal exchange revealed previously unrecognized root–fungus interaction mediated by the fungal auxin. The secreted fungal auxin induced negative taproot gravitropism, attenuated taproot growth rate, and inhibited initial host development. Auxin also induced expression of Arabidopsis carriers AUX1 and PIN1, both of which are involved in the gravitropic response. Exogenous application of auxin led to a root phenotype, which fully mimicked that induced by ectomycorrhizal fungi. Co-cultivation of Arabidopsis auxin receptor mutants tir1-1, tir1-1 afb2-3, tir1-1 afb1-3 afb2-3, and tir1-1 afb2-3 afb3-4 with Terfezia confirmed that auxin induces the observed root phenotype. The finding that auxin both induces taproot deviation from the gravity axis and coordinates growth rate is new. We propose a model in which the fungal auxin induces horizontal root development, as well as the coordination of growth rates between partners, along with the known auxin effect on lateral root induction that increases the availability of accessible sites for colonization at the soil plane of fungal spore abundance. Thus, the newly observed responses described here of the root to Terfezia contribute to a successful encounter between symbionts.     

Molecular cloning,expression, and subcellular localization of a PAL gene from Citrus reticulata under iron deficiency

Phenylalanine ammonia lyase (PAL) is a specific branch point enzyme of primary and secondary metabolism. The Citrus reticulata Blanco PAL gene was cloned and designated as CrPAL1. The cDNA sequence of CrPAL1 was 2 166 bp, encoding 721 amino acid residues. Sequence alignment indicates that CrPAL1 shared a high identity with PAL genes found in other plants. Both the dominant and catalytic active sites of CrPAL1 were similar to PAL proteins observed in Petroselinum crispum. Phylogenetic tree analysis indicates that CrPAL1 was more closely related to PALs in Citrus clementina × C. reticulata and Poncirus trifoliata than to those from other plants. Subcellular localization reveals that CrPAL1-green fluorescent protein fusion protein was specifically localized in the plasma membrane. Activity of PAL as well as CrPAL1 expression increased under Fe deficiency. A similar result was noted for total phenolic content. The root exudates of C. reticulata strongly promoted reutilization of apoplastic Fe in roots. Furthermore, Fe was more desorbed from the cell wall under Fe deficiency than in sufficient Fe supply.     

Distribution of manganese and other biometals in <Emphasis Type="Italic">flatiron</Emphasis> mice

Flatiron (ffe) mice display features of “ferroportin disease” or Type IV hereditary hemochromatosis. While it is known that both Fe and Mn metabolism are impaired in flatiron mice, the effects of ferroportin (Fpn) deficiency on physiological distribution of these and other biometals is unknown. We hypothesized that Fe, Mn, Zn and/or Cu distribution would be altered in ffe/+ compared to wild-type (+/+) mice. ICP-MS analysis showed that Mn, Zn and Cu levels were significantly reduced in femurs from ffe/+ mice. Bone deposits reflect metal accumulation, therefore these data indicate that Mn, Zn and Cu metabolism are affected by Fpn deficiency. The observations that muscle Cu, lung Mn, and kidney Cu and Zn levels were reduced in ffe/+ mice support the idea that metal metabolism is impaired. While all four biometals appeared to accumulate in brains of flatiron mice, significant gender effects were observed for Mn and Zn levels in male ffe/+ mice. Metals were higher in olfactory bulbs of ffe/+ mice regardless of gender. To further study brain metal distribution, ⁵⁴MnCl₂ was administered by intravenous injection and total brain ⁵⁴Mn was measured over time. At 72 h, ⁵⁴Mn was significantly greater in brains of ffe/+ mice compared to +/+ mice while blood ⁵⁴Mn was cleared to the same levels by 24 h. Taken together, these results indicate that Fpn deficiency decreases Mn trafficking out of the brain, alters body Fe, Mn, Zn and Cu levels, and promotes metal accumulation in olfactory bulbs.     

Involvement of polar auxin transport in the inhibition of Arabidopsis seedling growth induced by Stenotrophomonas maltophilia

A wide range of microorganisms found in the rhizhosphere are able to regulate plant growth and development, but little is known about the mechanism by which epiphytic microbes inhibit plant growth. Here, an epiphytic bacteria Stenotrophomonas maltophilia, named as LZMBW216, were isolated and identified from the potato (Solanum tuberosum L. cv. Da Xi Yang) leaf surface. They could decrease primary root elongation and lateral root numbers in Arabidopsis seedlings. The inhibitory effects of LZMBW216 on plant growth were not due to a reduced indole-3-acetic acid (IAA) content, as exogenously applied IAA did not recover the inhibition. Furthermore, LZMBW216 did not affect the expression of DR5::GUS and CycB1;1::GUS. However, we found that LZMBW216 exhibited little effect on the primary root elongation in the pin2 mutant and on the lateral root numbers in the aux1-7 mutant. Moreover, LZMBW216 decreased expressions of AUX1 and PIN2 proteins. Together, these results suggest that root system architecture alterations caused by LZMBW216 may involve polar auxin transport.     

BMAL1 deficiency promotes skeletal mandibular hypoplasia via OPG downregulation

Objectives

Skeletal mandibular hypoplasia (SMH), a common type of developmental deformities, results in impaired aesthetics of facial profile, occlusal dysfunction and poor life quality. In this study, BMAL1 deficiency leads to SMH formation, and we aim to investigate the mechanism by which BMAL1 deficiency induces SMH.

Materials and methods

Circadian rhythm‐disordered mouse models were constructed by placing animals in a jet lag schedule of 6‐h light advance every 7 days for 4 or 8 weeks. The OPG expression was evaluated by histomorphometry, immunohistochemistry and western blot analysis. The mechanism by which BMAL1 affects OPG expression was investigated by chromatin immunoprecipitation and luciferase reporter assays. The phenotypes caused by BMAL1 knockout can be rescued by exogenous supplementation with OPG.

Results

We demonstrate that the expressions of BMAL1 and OPG decreased in SMH patients. Circadian rhythm‐disordered mice and Bmal1^?/? mice exhibited decreased expression of OPG, reduced bone mass and bone size of mandibles. Our results revealed that BMAL1 bound directly to the Opg promoter and upregulated its expression, thus inhibiting osteoclast differentiation. BMAL1 deficiency increased osteoclast differentiation by downregulating OPG expression. In vitro, the enhancement effect of osteoclast differentiation caused by BMAL1 knockdown was significantly reversed by exogenous supplementation with OPG. Importantly, bone loss caused by BMAL1 knockout can be partially reversed by injecting OPG Intraperitoneally.

Conclusions

These results indicate that the circadian clock plays a critical role in the growth and development of mandible by regulating OPG expression, and present a potential therapeutic strategy to prevent SMH.

     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.     

A strong root-specific expression system for stable transgene expression in bread wheat

Key message

A strong, stable and root-specific expression system was developed from a rice root-specific GLYCINE - RICH PROTEIN 7 promoter for use as an enabling technology for genetic manipulation of wheat root traits.

Abstract

Root systems play an important role in wheat productivity. Genetic manipulation of wheat root traits often requires a root-specific or root-predominant expression system as an essential enabling technology. In this study, we investigated promoters from rice root-specific or root-predominant expressed genes for development of a root expression system in bread wheat. Transient expression analysis using a GREEN FLUORESCENT PROTEIN (GFP) reporter gene driven by rice promoters identified six promoters that were strongly expressed in wheat roots. Extensive organ specificity analysis of three rice promoters in transgenic wheat revealed that the promoter of rice GLYCINE-RICH PROTEIN 7 (OsGRP7) gene conferred a root-specific expression pattern in wheat. Strong GFP fluorescence in the seminal and branch roots of wheat expressing GFP reporter driven by the OsGRP7 promoter was detected in epidermal, cortical and endodermal cells in mature parts of the root. The GFP reporter driven by the promoter of rice METALLOTHIONEIN-LIKE PROTEIN 1 (OsMTL1) gene was mainly expressed in the roots with essentially no expression in the leaf, stem or seed. However, it was also expressed in floral organs including glume, lemma, palea and awn. In contrast, strong expression of rice RCg2 promoter-driven GFP was found in many tissues. The GFP expression driven by these three rice promoters was stable in transgenic wheat plants through three generations (T1–T3) examined. These data suggest that the OsGRP7 promoter can provide a strong, stable and root-specific expression system for use as an enabling technology for genetic manipulation of wheat root traits.