Non-transgenic Research Articles

Overexpression of ZmPCK2, a phosphoenolpyruvate carboxykinase gene from maize confers enhanced tolerance to water deficit stress in rice

Water deficit is one of the major abiotic stresses that limit plant growth and global crop yields. Phosphoenolpyruvate carboxykinase (PCK) plays important roles in regulating plant growth and development, but its role in water-deficit stress remains elusive. In this study, we found that overexpression of ZmPCK2 significantly enhanced the water-deficit tolerance of transgenic rice. The expression level of ZmPCK2 was strongly induced by PEG and ABA treatments. Overexpression of ZmPCK2 in rice increased stomatal closure and water saving by regulating malate metabolism under water-deficit conditions. Moreover, the expression of ZmPCK2 in rice up-regulated ABA biosynthesis and responsive genes under water-deficit stress, and ZmPCK2 transgenic rice showed hypersensitive to exogenous ABA at germination stage, suggesting that ZmPCK2 may be involved in ABA signalling pathway. Under water-deficit stress, the ZmPCK2 transgenic rice showed higher antioxidant enzyme activities and lower accumulation of reactive oxygen species (ROS) compared with non-transgenic (NT) plants, resulting in less oxidative damage. Taken together, we suggest that ZmPCK2 plays multiple roles in response to water-deficit stress by enhancing ABA signalling pathway, regulating malate metabolism, promoting stomatal closure and further activating the ROS-scavenging system.

Rice microRNA171f/SCL6 module enhances drought tolerance by regulation of flavonoid biosynthesis genes.

Plants have evolved sophisticated defense systems to enhance drought tolerance. These include the microRNA (miRNA) group of small noncoding RNAs that act as post‐transcriptional regulators; however, details of the mechanisms by which they confer drought tolerance are not well understood. Here, we show that osa‐MIR171f, a member of osa‐MIR171 gene family, is mainly expressed in response to drought stress and regulates the transcript levels of SCARECROW‐LIKE6‐I (SCL6‐I) and SCL6‐II in rice ( Oryza sativa ). The SCL6 genes are known to be involved in shoot branching and flag leaf morphology. Osa‐MIR171f‐overexpressing (osa‐MIR171f‐OE) transgenic plants showed reduced drought symptoms compared with non‐transgenic (NT) control plants under both field drought and polyethylene glycol (PEG)‐mediated dehydration stress conditions. Transcriptome analysis of osa‐MIR171f‐OE plants and osa‐mir171f‐knockout (K/O) lines generated by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) revealed that osa‐mature‐miR171a‐f (osa‐miR171) regulates the expression of flavonoid biosynthesis genes, consequently leading to drought tolerance. This upregulation in the osa‐MIR171f‐OE plants, which did not occur in NT control plants, was observed under both normal and drought conditions. Our findings indicate that osa‐miR171 plays a role in drought tolerance by regulating SCL6‐I and SCL6‐II transcript levels.

Transcriptional activation of rice CINNAMOYL-CoA REDUCTASE 10 by OsNAC5, contributes to drought tolerance by modulating lignin accumulation in roots.

SummaryDrought is a common abiotic stress for terrestrial plants and often affects crop development and yield. Recent studies have suggested that lignin plays a crucial role in plant drought tolerance; however, the underlying molecular mechanisms are still largely unknown. Here, we report that the rice (Oryza sativa) gene CINNAMOYL‐CoA REDUCTASE 10 (OsCCR10) is directly activated by the OsNAC5 transcription factor, which mediates drought tolerance through regulating lignin accumulation. CCR is the first committed enzyme in the monolignol synthesis pathway, and the expression of 26 CCR genes was observed to be induced in rice roots under drought. Subcellular localisation assays revealed that OsCCR10 is a catalytically active enzyme that is localised in the cytoplasm. The OsCCR10 transcript levels were found to increase in response to abiotic stresses, such as drought, high salinity, and abscisic acid (ABA), and transcripts were detected in roots at all developmental stages. In vitro enzyme activity and in vivo lignin composition assay suggested that OsCCR10 is involved in H‐ and G‐lignin biosynthesis. Transgenic rice plants overexpressing OsCCR10 showed improved drought tolerance at the vegetative stages of growth, as well as higher photosynthetic efficiency, lower water loss rates, and higher lignin content in roots compared to non‐transgenic (NT) controls. In contrast, CRISPR/Cas9‐mediated OsCCR10 knock‐out mutants exhibited reduced lignin accumulation in roots and less drought tolerance. Notably, transgenic rice plants with root‐preferential overexpression of OsCCR10 exhibited higher grain yield than NT controls plants under field drought conditions, indicating that lignin biosynthesis mediated by OsCCR10 contributes to drought tolerance.

Overexpression of Orange Gene (OsOr-R115H) Enhances Heat Tolerance and Defense-Related Gene Expression in Rice (Oryza sativa L.).

In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.

Mitochondrial Fusion Suppresses Tau Pathology-Induced Neurodegeneration and Cognitive Decline.

Abnormalities of mitochondrial fission and fusion, dynamic processes known to be essential for various aspects of mitochondrial function, have repeatedly been reported to be altered in Alzheimer's disease (AD). Neurofibrillary tangles are known as a hallmark feature of AD and are commonly considered a likely cause of neurodegeneration in this devastating disease. To understand the pathological role of mitochondrial dynamics in the context of tauopathy. The widely used P301S transgenic mice of tauopathy (P301S mice) were crossed with transgenic TMFN mice with the forced expression of Mfn2 specifically in neurons to obtain double transgenic P301S/TMFN mice. Brain tissues from 11-month-old non-transgenic (NTG), TMFN, P301S, and P301S/TMFN mice were analyzed by electron microscopy, confocal microscopy, immunoblot, histological staining, and immunostaining for mitochondria, tau pathology, and tau pathology-induced neurodegeneration and gliosis. The cognitive function was assessed by the Barnes maze. P301S mice exhibited mitochondrial fragmentation and a consistent decrease in Mfn2 compared to age-matched NTG mice. When P301S mice were crossed with TMFN mice (P301S/TMFN mice), neuronal loss, as well as mitochondria fragmentation were significantly attenuated. Greatly alleviated tau hyperphosphorylation, filamentous aggregates, and thioflavin-S positive tangles were also noted in P301S/TMFN mice. Furthermore, P301S/TMFN mice showed marked suppression of neuroinflammation and improved cognitive performance in contrast to P301S mice. These in vivo findings suggest that promoted mitochondrial fusion suppresses toxic tau accumulation and associated neurodegeneration, which may protect against the progression of AD and related tauopathies.

Overexpression of IbFAD8 Enhances the Low-Temperature Storage Ability and Alpha-Linolenic Acid Content of Sweetpotato Tuberous Roots.

Sweetpotato is an emerging food crop that ensures food and nutrition security in the face of climate change. Alpha-linoleic acid (ALA) is one of the key factors affecting plant stress tolerance and is also an essential nutrient in humans. In plants, fatty acid desaturase 8 (FAD8) synthesizes ALA from linoleic acid (LA). Previously, we identified the cold-induced IbFAD8 gene from RNA-seq of sweetpotato tuberous roots stored at low-temperature. In this study, we investigated the effect of IbFAD8 on the low-temperature storage ability and ALA content of the tuberous roots of sweetpotato. Transgenic sweetpotato plants overexpressing IbFAD8 (TF plants) exhibited increased cold and drought stress tolerance and enhanced heat stress susceptibility compared with non-transgenic (NT) plants. The ALA content of the tuberous roots of TF plants (0.19 g/100 g DW) was ca. 3.8-fold higher than that of NT plants (0.05 g/100 g DW), resulting in 8–9-fold increase in the ALA/LA ratio in TF plants. Furthermore, tuberous roots of TF plants showed better low-temperature storage ability compared with NT plants. These results indicate that IbFAD8 is a valuable candidate gene for increasing the ALA content, environmental stress tolerance, and low-temperature storage ability of sweetpotato tuberous roots via molecular breeding.

Transcriptome analysis reveals differing response and tolerance mechanism of EPSPS and GAT genes among transgenic soybeans.

Glyphosate is a broad-spectrum, non-selective systemic herbicide. Introduction of glyphosate tolerance genes such as EPSPS or detoxification genes such as GAT can confer glyphosate tolerance on plants. Our previous study revealed that co-expression of EPSPS and GAT genes conferred higher glyphosate tolerance without "yellow flashing". However, the plant response to glyphosate at the transcriptional level was not investigated. To investigate the glyphosate tolerance mechanism, RNA-seq was conducted using four soybean genotypes, including two non-transgenic (NT) soybeans, ZH10 and MD12, and two GM soybeans, HJ698 and ZH10-6. Differentially expressed genes (DEGs) were identified in these soybeans before and after glyphosate treatment. Similar response to glyphosate in the two NT soybeans and the different effects of glyphosate on the two GM soybeans were identified. As treatment time was prolonged, the expression level of some DEGs involved in shikimate biosynthetic pathway and herbicide targeted cross-pathways was increased or declined continuously in NT soybeans, and altered slightly in HJ698. However, the expression level of some DEGs was altered in ZH10-6 at 12hpt, while similar expression level of some DEGs involved in shikimate biosynthetic pathway and herbicide targeted cross-pathways was observed in ZH10-6 at 0hpt and 72hpt. These observations likely explain the higher glyphosate tolerance in ZH10-6 than in HJ698 and NT soybeans. These results suggested that GAT and EPSPS genes together play a crucial role in response to glyphosate, the GAT gene may work at the early stage of glyphosate exposure, whereas the EPSPS gene may be activated after the uptake of glyphosate by plants. These findings will provide valuable insight for the molecular basis underlying glyphosate tolerance or glyphosate detoxication.

Gene Transmission, Growth, and Exogeneous Growth Hormone Expression of G2 Transgenic Betta Fish (Betta imbellis)

Highlight ResearchThe F2 of GH-transgenic B. imbellis was successfully producedThe transgene inheritance by the F2 fish was more than 90%The growth and body size of transgenic fish was significantly higher than controlF2 fish reached a larger body size in a shorter period compared to the F1 AbstractIn our previous research, we had successfully produced G0 and G1 Pangasianodon hypophthalmus growth hormone (PhGH) transgenic B. imbellis, native ornamental betta from Indonesia, which its giant-sized variant has valuable price for the breeders. The G0 and G1 transgenic (TG) fish showed higher growth rate and body size compared to the non-transgenic (NT) fish. The study was aimed to produce and evaluate the consistencies of transgene transmission and expression in G2 generation. The growth rate and body size between TG and NT fish was also compared. The G2 generation was produced using crosses between TG and NT G1 fish: ♂TG × ♀TG, ♂TG × ♀NT, ♂NT × ♀TG, and ♂NT ×♀ NT. Fish were reared for 12 weeks, and transgene detection was performed using the polymerase chain reaction method (PCR) on isolated DNA from the caudal fin clips. The endogenous and exogenous GH expression analysis was conducted using the quantitative real-time PCR (qPCR) method. The results showed that the inheritance of the GH transgene by the G2 fish was more than 90% in all transgenic crosses. Endogenous GH was expressed at the same levels in the brain of TG and NT fish, but the exogenous GH expression was highly detected only in the TG fish. The G2 transgenic fish had a higher specific growth rate, up to 31%, compared to the control. The body length of TG crosses were 23−35% higher and had 111−135% higher body weight compared to NT fish. These results showed a promising approached in mass-producing stable lines of giant-sized betta using the GH-transgenic technology.

Overexpression of Ginkgo BBX25 enhances salt tolerance in Transgenic Populus

B-box (BBX) genes play important roles in plant growth, light morphogenesis, and environmental stress responses. Ginkgo (Ginkgo biloba L.) is known as a living fossil species that has a strong ability to adapt to environmental changes and tolerate harsh conditions. In this study, we chose this species to investigate the function of the GbBBX25 gene. We isolated the BBX gene from ginkgo and named it GbBBX25; this gene consists of an 819 bp open reading frame (ORF) that encodes 273 amino acids with two B-box domains but no CCT domain. GbBBX25 was localized in only the nucleus. The expression of GbBBX25 transcripts was observed in the leaves and was significantly enhanced under salt stress conditions. To further verify its function, we overexpressed the GbBBX25 gene in Populus davidiana × Populus bolleana and found that the transgenic Populus had greater soluble sugar levels and higher peroxidase (POD) activity in response to salt stress than nontransgenic (NT) Populus. Five genes related to salt stress were induced in transgenic plants with significantly higher expression levels than those in NT plants. This finding suggests that GbBBX25 improves the salt adaptation abilities of transgenic Populus and provides a scientific basis for related research.

Transgenic japonica rice expressing the cry1C gene is resistant to striped stem borers in Northeast China

Rice production and quality are seriously affected by the lepidopteran pest, striped stem borer (SSB), in Northeast China. In this study, a synthetic cry1C gene encoding Bacillus thuringiensis (Bt) δ-endotoxin, which is toxic to lepidopteran pest, was transformed into a japonica rice variety (Jigeng 88) in Northeast China by Agrobacterium-mediated transformation. Through molecular detection and the Basta resistance germination assay, a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1C. Finally, four cry1C-transgenic lines (JL16, JL23, JL41, and JL42) were selected by evaluation of the Cry1C protein level, insect-resistance and agronomic traits. The cry1C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic (NT) control plants. T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1C gene was inserted into the intergenic region of chromosome 11, indicating that its insertion may not interfere with the genes near insertion site. In summary, this study developed four cry1C-transgenic japonica rice lines with high insect resistance and high yield. They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.

Abstract P417: Cardiomyopathy-associated Variant In Troponin T Tail Domain Promotes Disruption Of Both Frank-starling Mechanism And Cardiac Myofilament Performance

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) tail region has been linked to familial hypertrophic cardiomyopathy (HCM), arrhythmia, and sudden cardiac death. It has been reported that inotropic stimulation with high extracellular Ca 2+ or isoproterenol led to diastolic dysfunction in both isolated and in vivo HcTnT-I79N mice hearts. Although HcTnT-I79N effects are acknowledged to be dependent on the inotropic state of the cardiac muscle, little is known about how this pathogenic variant affects the Frank-Starling law of the heart. To further investigate the functional and structural consequences of this deadly variant in a stretch-dependent manner, cardiac tissues were harvested from non-transgenic (NTg) control mice and transgenic mice bearing HcTnT-I79N. Left ventricular papillary muscle bundles were permeabilized and mounted for mechanical measurements. Sarcomere length (SL 1.9, 2.1 or 2.3 μm) was set at pCa 8 using HeNe laser diffraction and then Ca 2+ -dependence of isometric force, sinusoidal stiffness (SS, 0.2% PTP length oscillation) and rate of tension redevelopment ( k TR ) were measured. We observed that HcTnT-I79N tissue exhibited increased myofilament Ca 2+ -sensitivity of force, increased SS, slower k TR at all levels of Ca 2+ -activation, and diminished length-dependent activation (LDA). Small-angle X-ray diffraction revealed that HcTnT-I79N permeabilized cardiac muscles exhibit smaller myofilament lattice spacing at longer SLs (2.1 μm and 2.3 μm) compared to NTg. Using 3% Dextran T500 to osmotically compress the myofilament lattice (SL 2.1 μm), HcTnT-I79N showed no change in myofilament lattice spacing and little change in contractile indices associated with LDA. Interestingly, upon osmotic compression, HcTnT-I79N displayed a decrease in disordered relaxed state (DRX, ON state) of myosin and an increase in super-relaxed state (SRX, OFF state) of myosin. We conclude that altered cardiac myofilament performance, lack of responsiveness to osmotic compression, and reduced LDA observed with HcTnT-I79N are partially due to a combination of smaller myofilament lattice and disturbed ON and OFF states of myosin.

Abstract P492: Putative Protein Biomarkers For Reductive Stress Cardiomyopathy

Background: Nuclear factor erythroid 2-related factor 2 (NRF2) signaling is vital for redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2) exhibit reductive stress (RS). Here in, we identified novel protein signatures reacting to RS-induced cardiomyopathy. Methods: Tandem Mass Tag (TMT) proteomic analysis was performed in the heart tissues of Ca-Nrf2-transgenic (TG-low & TG-high) and non-transgenic (NTg) mice at 6 months of age (N= 4/group). Differentially expressed proteins (DEPs) were then identified using Scaffold. Validated the key DEPs using immunoblotting. PANTHER and STRING analysis were used to identify potential targets and their interactions. Results: A total of 1105 proteins were extracted from 24369 spectra. Of note, 226 and 261 proteins were differentially expressed in TG-L and TG-H vs. NTg hearts indicating a unique proteome signature for RS. Heat map analysis revealed a clear distinction between the TG-L and TG-H due to the dose-dependent effects of transgene/RS. Majority of the DEPs that are significantly altered in RS mice found to involve in stress related pathways such as antioxidants, NADPH, protein quality control (PQC), etc. Interestingly, some of these proteins were redox modified at their cysteine residues under chronic RS setting. Conclusions: TMT based proteomic analyses revealed unique proteome signatures for RS. The cysteine modifications in multiple proteins likely to cause pathological alterations via impaired PQC mechanisms. Molecular studies related to RS-mediated redox modifications in structural and functional cardiac proteome are underway.

Overexpression of 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) increases abiotic stress tolerance in transgenic sweetpotato plants

Tocopherols are lipid-soluble compounds regarded as vitamin E compounds and they function as antioxidants in scavenging lipid peroxyl radicals and quenching reactive oxygen species (ROS). In our previous studies, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants including 4-hydroxyphenylpyruvate dioxygenase (IbHPPD). HPPD is the first regulatory enzyme in vitamin E biosynthesis and serves to catalyze in the first steps α-tocopherol and plastoquinone biosynthesis by converting 4-hydroxyphenylpyruvate (HPP) to homogentisic acid (HGA). In this study, we generated transgenic sweetpotato plants overexpressing IbHPPD under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as HP plants) via Agrobacterium-mediated transformation to understand the function of IbHPPD in sweetpotato. Three transgenic lines (HP3, HP14 and HP15) with high transcript levels of IbHPPD were selected for further characterization. Compared with non-transgenic (NT) plants, HP plants exhibited enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses. In addition, HP plants showed increased tolerance to the herbicide sulcotrione, which is involved in the inhibition of the HPPD. Interestingly, after stress treatments, HP plants also showed higher abscisic acid (ABA) contents than NT plants. Under dehydrated condition, HP plants displayed an elevated α-tocopherol content to 19–27% in leaves compared with NT plants. These results indicate that increased abiotic stress tolerance in HP plants is related to inducing enhancement of α-tocopherol and ABA contents.

Preserved Thermal Pain in 3xTg-AD Mice With Increased Sensory-Discriminative Pain Sensitivity in Females but Affective-Emotional Dimension in Males as Early Sex-Specific AD-Phenotype Biomarkers.

The increase of the aging population, where quite chronic comorbid conditions are associated with pain, draws growing interest across its investigation and the underlying nociceptive mechanisms. Burn injuries associated problems might be of relevance in the older adult’s daily life, but in people with dementia, exposure to high temperatures and heat sources poses a significantly increased risk of burns. In this brief report, the hind paws and tail pain withdrawal reflexes and the emotional responses to thermal nociception in 3xTg-AD mice were characterized for the first time in the plantar test and compared to their non-transgenic (NTg) counterparts. We studied a cohort of male and female 3xTg-AD mice at asymptomatic (2 months), early (6 months), middle (9 months), and advanced (12 and 15 months) stages of the disease and as compared to sex- and age-matched NTg control mice with normal aging. At 20 and 40W intensities, the sensorial-discriminative thresholds eliciting the withdrawal responses were preserved from asymptomatic to advanced stages of the disease compared to NTg counterparts. Moreover, 3xTg-AD females consistently showed a greater sensory-discriminative sensitivity already at premorbid ages, whereas increased emotionality was shown in males. False-negative results were found in “blind to sex and age” analysis, warning about the need to study sexes independently. The current results and previous report in cold thermal stimulation provide two paradigms unveiling sex-specific early AD-phenotype nociceptive biomarkers to study the mechanistic underpinnings of sex-, age- and AD-disease-dependent thermal pain sensitivity.

Effects of a low-dose IL-2 treatment in HLA-B27 transgenic rat model of spondyloarthritis

Introduction/AimHLA-B27/human β2m transgenic rats (B27-rats) develop an inflammatory disorder resembling spondyloarthritis (SpA) with dysregulated IL-10/IL-17 production by regulatory T cells (Treg). Treg plays a major role in controlling pathogenic inflammatory processes. Interleukin 2 (IL-2), a cytokine which promotes Treg cell survival and function, may thus have therapeutic efficacy in SpA. Here, we tested this hypothesis using a low dose of IL-2 treatment in B27-rat.Material and methodsB27-rats aged 4 weeks (before disease onset) and nontransgenic (NTG) littermates were administered intraperitoneally recombinant human IL-2 (Sanofi®; 2,000IU/injection) or PBS, 3 days per week during 6 weeks. Assessment of treatment effect was performed, based on clinical (weight, diarrhea, arthritis), histological (proximal and distal colon, caecum, ileum and tarsal/ankle joint) scores, and frequency of Treg in the spleen and lymph nodes (LN).ResultsIL-2 administration had no effect on weight gain, either in B27- or NTG-rats. Over the 6 weeks of treatment, the clinical disease score worsened similarly in both IL-2-treated and control groups of B27-rats. The macroscopic and histological evaluation of gut and joints showed marked inflammation in B27-rats; however, no change related to IL-2 treatment was observed. In the B27-rats, the percentage of Treg was moderately increased after IL-2 treatment in the spleen, but neither in mesenteric nor peripheral LN in those rats.ConclusionOur data demonstrate that a low dose of IL-2 administered before disease onset was moderately effective for boosting Treg but failed to prevent SpA development in B27-rat.

Enhanced aluminum tolerance in sugarcane: evaluation of SbMATE overexpression and genome-wide identification of ALMTs in Saccharum spp.

BackgroundA major limiting factor for plant growth is the aluminum (Al) toxicity in acidic soils, especially in tropical regions. The exclusion of Al from the root apex through root exudation of organic acids such as malate and citrate is one of the most ubiquitous tolerance mechanisms in the plant kingdom. Two families of anion channels that confer Al tolerance are well described in the literature, ALMT and MATE family.ResultsIn this study, sugarcane plants constitutively overexpressing the Sorghum bicolor MATE gene (SbMATE) showed improved tolerance to Al when compared to non-transgenic (NT) plants, characterized by sustained root growth and exclusion of aluminum from the root apex based on the result obtained with hematoxylin staining. In addition, genome-wide analysis of the recently released sugarcane genome identified 11 ALMT genes and molecular studies showed potential new targets for aluminum tolerance.ConclusionsOur results indicate that the transgenic plants overexpressing the Sorghum bicolor MATE has an improved tolerance to Al. The expression profile of ALMT genes revels potential candidate genes to be used has an alternative for agricultural expansion in Brazil and other areas with aluminum toxicity in poor and acid soils.

Tuberous roots of transgenic sweetpotato overexpressing IbCAD1 have enhanced low-temperature storage phenotypes

Lignin is associated with cell wall rigidity, water and solute transport, and resistance to diverse stresses in plants. Lignin consists of polymerized monolignols (p-coumaryl, coniferyl, and sinapyl alcohols), which are synthesized by cinnamyl alcohol dehydrogenase (CAD) in the phenylpropanoid pathway. We previously investigated cold-induced IbCAD1 expression by transcriptome profiling of cold-stored tuberous roots of sweetpotato (Ipomoea batatas [L.] Lam). In this study, we confirmed that IbCAD1 expression levels depended on the sweetpotato root type and were strongly induced by several abiotic stresses. We generated transgenic sweetpotato plants overexpressing IbCAD1 (TC plants) to investigate CAD1 physiological functions in sweetpotato. TC plants displayed lower root weights and lower ratios of tuberous roots to pencil roots than non-transgenic (NT) plants. The lignin contents in tuberous roots of NT and TC plants differed slightly, but these differences were not significant. By contrast, monolignol levels and syringyl (S)/guaiacyl (G) ratios were higher in TC plants than NT plants, primarily owing to syringyl unit accumulation. Tuberous roots of TC plants displayed enhanced low-temperature (4 °C) storage with lower malondialdehyde and H2O2 contents than NT plants. We propose that high monolignol levels in TC tuberous roots served as substrates for increased peroxidase activity, thereby enhancing antioxidation capacity against cold stress–induced reactive oxygen species. Increased monolignol contents and/or increased S/G ratios might contribute to pathogen-induced stress tolerance as a secondary chilling-damage response in sweetpotato. These results provide novel information about CAD1 function in cold stress tolerance and root formation mechanisms in sweetpotato.

Chronic HDAC6 Activation Induces Atrial Fibrillation Through Atrial Electrical and Structural Remodeling in Transgenic Mice.

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.

Macrophage polarization in obesity induced inflammation: The effect of HSP70

Abstract Obesity is a chronic inflammatory disease that plagues our nation’s healthcare system. Tissue macrophages and recruited monocytes are implicated in the systemic release of pro-inflammatory cytokines that foster disease progression. Diet-induced obesity (DIO) is prevented in heat shock protein 70 over-expressing (HSP70 transgenic; TG) mice. While there are several mechanisms responsible for this protective response, prevention of macrophage polarization to a pro-inflammatory phenotype (M1) may be one contributory mechanism that warrants further investigation. Using a TG mouse line with selective over-expression of HSP70 in villin-expressing epithelial cells, male TG and non-transgenic (NTG) littermates were fed a low fat (LF; 10 kcal% fat) or high fat (HF; 60 kcal% fat) diet for 13 weeks (Research Diets, Inc). Peritoneal cavity macrophages were harvested from these mice and analyzed by flow cytometry (Beckman Coulter Cytoflex) using the following markers: total (F4/80 APC), M1 (CD38 PE), and M2 (CD301 APC/Fire 750) macrophages. TG mice demonstrated increased F4/80+high macrophages and more F4/80+high M2 cells in comparison to their NTG littermates (p=0.051). These results demonstrate HSP70 over-expression in vivo increases the population of macrophages, particularly anti-inflammatory M2 macrophages, in the peritoneal cavity. Preferential M2 phenotype could be a significant contributory factor to HSP70-mediated protection from DIO.

Social Nesting, Animal Welfare, and Disease Monitoring.

Simple SummaryMost standardized tools to evaluate welfare and disease progression in animals assess the individuals, while social behaviors are scarcely monitored, despite being useful to detecting acute illness and chronic and mental health problems. The main reason is that social behavior is complex and time-consuming. We are currently using the nests built by animals living together, a species-typical behavior naturally occurring in standard housing conditions, to monitor them. Here, we provide an example of its use to evaluate social deficits and the long-term effects of a neonatal tactile-proprioceptive sensorial treatment from postnatal day 1 to 21, in male and female adult mice modeling Alzheimer’s disease compared to mice with normal aging. Social nesting was worse in the mutants, mostly in males, since the number of days needed to build a perfect nest was longer or unsuccessful in a three-day test. Early life intervention was successful. Social nesting, easily included in housing routines, can be a useful tool to assess animal welfare, monitor disease progress, and evaluate potential risk factors and effects of preventive/therapeutical strategies. Other advantages, such as being a noninvasive, painless, simple, short, and low-cost, rend social nesting feasible to be implemented in most animal department settings.The assessment of welfare and disease progression in animal models is critical. Most tools rely on evaluating individual subjects, whereas social behaviors, also sensitive to acute illness, chronic diseases, or mental health, are scarcely monitored because they are complex and time-consuming. We propose the evaluation of social nesting, a species-typical behavior naturally occurring in standard housing conditions, for such behavioral monitoring. We provide an example of its use to evaluate social deficits and the long-term effects of neonatal tactile-proprioceptive sensorial stimulation from postnatal day 1 to 21, in male and female adult 3xTg-AD mice for Alzheimer’s disease compared to sex- and age-matched non-transgenic (NTg) counterparts with normal aging. Social nesting was sensitive to genotype (worse in 3xTg-AD mice), sex (worse in males), profile, and treatment (distinct time to observe the maximum score and incidence of the perfect nest). Since social nesting can be easily included in housing routines, this neuroethological approach can be useful for animal welfare, monitoring the disease’s progress, and evaluating potential risk factors and effects of preventive/therapeutical strategies. Finally, the noninvasive, painless, simple, short time, and low-cost features of this home-cage monitoring are advantages that make social nesting feasible to be successfully implemented in most animal department settings.