Non-transgenic Research Articles

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds

BackgroundDeoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated.ResultsThe accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn’t in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a ‘Three Faucets and Cisterns Model’ about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a “Faucet”, the biosynthetic capacity of intermediate metabolites as a “Cistern”, and the carotenoid accumulations as the content of “Cistern”.ConclusionOur study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the “Three Faucets and Cisterns model” presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

INVESTIGATING MOTOR LEARNING IN A LRRK2 MOUSE MODEL OF PARKINSON’S DISEASE

While most cases of Parkinson’s disease are idiopathic, meaning of unknown origin, in 10% of cases, a genetic link has been identified. Of these, the most common is the G2019S mutation of the leucine‐rich repeating kinase 2 (LRRK2) gene. Investigation into this mutation could prove useful not only for those with LRRK2 Parkinson’s, but for idiopathic Parkinson’s as well. Genetic mouse models of this mutation are important for translational research, but despite the increasingly large number of studies on these animals, few have identified a reliable phenotype. In this study, we measured the average latency to fall during a rotarod‐based motor‐learning task conducted twice a week for two weeks with transgenic (Tg) and non‐transgenic (NTg) mice 6–8 months old. During the second week, half the animals were given a LRRK2 inhibitor, MLi‐2. Although we detected no effect of the drug, results have been mixed for overall motor performance with a trend towards LRRK2 Tg animals performing worse on the task. In this study, we examine whether LRRK2 Tg mice perform similarly to NTg mice for within‐day learning, between‐day learning, and overall latency to fall measures.Support or Funding InformationFunding from Michael J. Fox Foundation.

Modifications of Sarcoplasmic Reticulum Function Prevent Progression of Sarcomere-Linked Hypertrophic Cardiomyopathy Despite a Persistent Increase in Myofilament Calcium Response.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in different genes mainly encoding myofilament proteins and therefore called a “disease of the sarcomere.” Despite the discovery of sarcomere protein mutations linked to HCM almost 30 years ago, the cellular mechanisms responsible for the development of this disease are not completely understood and likely vary among different mutations. Moreover, despite many efforts to develop effective treatments for HCM, these have largely been unsuccessful, and more studies are needed to better understand the cellular mechanisms of the disease. In experiments reported here, we investigated a mouse model expressing the mutant cTnT-R92Q, which is linked to HCM and induces an increase in myofilament Ca2+ sensitivity and diastolic dysfunction. We found that early correction of the diastolic dysfunction by phospholamban knockout (PLNKO) was able to prevent the development of the HCM phenotype in troponin T (TnT)-R92Q transgenic (TG) mice. Four groups of mice in FVB/N background were generated and used for the experiments: (1) non-transgenic (NTG)/PLN mice, which express wild-type TnT and normal level of PLN; (2) NTG/PLNKO mice, which express wild-type TnT and no PLN; (3) TG/PLN mice, which express TnT-R92Q and normal level of PLN; (4) TG/PLNKO mice, which express TnT-R92Q and no PLN. Cardiac function was determined using both standard echocardiographic parameters and speckle tracking strain measurements. We found that both atrial morphology and diastolic function were altered in TG/PLN mice but normal in TG/PLNKO mice. Histological analysis showed a disarray of myocytes and increased collagen deposition only in TG/PLN hearts. We also observed increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation only in TG/PLN hearts but not in TG/PLNKO hearts. The rescue of the HCM phenotype was not associated with differences in myofilament Ca2+ sensitivity between TG/PLN and TG/PLNKO mice. Moreover, compared to standard systolic echo parameters, such as ejection fraction (EF), speckle strain measurements provided a more sensitive approach to detect early systolic dysfunction in TG/PLN mice. In summary, our results indicate that targeting diastolic dysfunction through altering Ca2+ fluxes with no change in myofilament response to Ca2+ was able to prevent the development of the HCM phenotype and should be considered as a potential additional treatment for HCM patients.

Saline–alkaline resistance analysis of rice overexpressing the CsCYP1A gene of alkaline Chlorella

AbstractPlant cyclophilin (CYP) is related to chloroplast photoprotection, redox and other growth and developmental regulatory processes and responds to stress and improves tolerance to saline–alkali stress. Besides, it exerts peptidyl-prolyl cis/trans isomerase activity to participate in protein synthesis and folding. In this study, Northern blot was used to analyse the CsCYP1A gene (a CYP gene isolated from a Chlorella species) (accession number: KY207381) of tolerant Chlorella vulgaris in soda saline–alkali soil. The result showed that the expression of the CsCYP1A gene was induced by NaHCO3, NaCl and sorbitol. Additionally, Agrobacterium tumefaciens was used to infect the callus of rice (Oryza sativa var. Longjing11) for genetic transformation. Molecular detection confirmed that transgenic seedlings overexpressing CsCYP1A were obtained by hygromycin selection. Treatment with NaCl and NaHCO3 at the five-leaf stage was performed in the seedlings, and the results showed that there were significant differences between the CsCYP1A overexpressed rice lines and non-transgenic (NT) Longjing11 in terms of plant height, fresh weight, chlorophyll content, malondialdehyde content and ascorbate peroxidase activity. The CsCYP1A overexpression rice lines had higher tolerance to NaCl and NaHCO3 than NT. The current findings indicate that CsCYP1A can enhance the tolerance of rice to saline–alkali stress possibly through its involvement in reactive oxygen scavenging pathways.

Loci associated with variation in gene expression and growth in juvenile salmon are influenced by the presence of a growth hormone transgene

BackgroundGrowth regulation is a complex process influenced by genetic and environmental factors. We examined differences between growth hormone (GH) transgenic (T) and non-transgenic (NT) coho salmon to elucidate whether the same loci were involved in controlling body size and gene expression phenotypes, and to assess whether physiological transformations occurring from GH transgenesis were under the influence of alternative pathways. The following genomic techniques were used to explore differences between size classes within and between transgenotypes (T vs. NT): RNA-Seq/Differentially Expressed Gene (DEG) analysis, quantitative PCR (qPCR) and OpenArray analysis, Genotyping-by-Sequencing, and Genome-Wide Association Study (GWAS).ResultsDEGs identified in comparisons between the large and small tails of the size distributions of T and NT salmon (NTLarge, NTSmall, TLarge and TSmall) spanned a broad range of biological processes, indicating wide-spread influence of the transgene on gene expression. Overexpression of growth hormone led to differences in regulatory loci between transgenotypes and size classes. Expression levels were significantly greater in T fish at 16 of 31 loci and in NT fish for 10 loci. Eleven genes exhibited different mRNA levels when the interaction of size and transgenotype was considered (IGF1, IGFBP1, GH, C3–4, FAS, FAD6, GLUT1, G6PASE1, GOGAT, MID1IP1). In the GWAS, 649 unique SNPs were significantly associated with at least one study trait, with most SNPs associated with one of the following traits: C3_4, ELA1, GLK, IGF1, IGFBP1, IGFII, or LEPTIN. Only 1 phenotype-associated SNP was found in common between T and NT fish, and there were no SNPs in common between transgenotypes when size was considered.ConclusionsMultiple regulatory loci affecting gene expression were shared between fast-growing and slow-growing fish within T or NT groups, but no such regulatory loci were found to be shared between NT and T groups. These data reveal how GH overexpression affects the regulatory responses of the genome resulting in differences in growth, physiological pathways, and gene expression in T fish compared with the wild type. Understanding the complexity of regulatory gene interactions to generate phenotypes has importance in multiple fields ranging from applications in selective breeding to quantifying influences on evolutionary processes.

Alteration of Carotenoid Metabolic Machinery by β-Carotene Biofortification in Rice Grains

To increase nutritional values as dietary sources of provitamin A and health-promoting antioxidants in rice grains, carotenoids have been biofortified in a Golden Ricelike variety, the stPAC (stPsy-2A-stCrtI) rice. In both of non-transgenic (NT) and stPAC seeds, total chlorophylls and carotenoids were gradually decreased during seed development while de novo biosynthesized carotenoids being comprised mainly of β-carotene, lutein and zeaxanthin were accumulated from early stage of 10 DAF and peaked at 20 DAF in stPAC seeds. The de novo production of carotenoids coincided with the high levels of transgene expression driven by the rice globulin gene promoter. Interestingly, expression levels of endogenous carotenoid metabolic pathway genes were the highest at 30 DAF in NT seeds whereas they were generally down-regulated in stPAC seeds, suggesting a negative feedback control mechanism of carotenoid metabolism by enhanced carotenoid production. The transgenic protein levels in stPAC seeds were not changed much during seed storage for up to 5 years, while carotenoid contents in the seeds were decreasing after 1 year of storage. The decrease in carotenoid contents was restored when the transgenic plants re-grown, supporting the reliability of transgenic pathways for carotenoid biofortification in rice grains. Thus, our results showed that transgene-driven biofortification of carotenoids was made and maintained over several transgenic generations with a possible negative feed-back control of endogenous carotenoid metabolism during seed development.

Treatment with JQ1, a BET bromodomain inhibitor, is selectively detrimental to R6/2 Huntington's disease mice.

Transcriptional and epigenetic alterations occur early in Huntington's disease (HD), and treatment with epigenetic modulators is beneficial in several HD animal models. The drug JQ1, which inhibits histone acetyl-lysine reader bromodomains, has shown promise for multiple cancers and neurodegenerative disease. We tested whether JQ1 could improve behavioral phenotypes in the R6/2 mouse model of HD and modulate HD-associated changes in transcription and epigenomics. R6/2 and non-transgenic (NT) mice were treated with JQ1 daily from 5 to 11weeks of age and behavioral phenotypes evaluated over this period. Following the trial, cortex and striatum were isolated and subjected to mRNA-seq and ChIP-seq for the histone marks H3K4me3 and H3K27ac. Initially, JQ1 enhanced motor performance in NT mice. In R6/2 mice, however, JQ1 had no effect on rotarod or grip strength but exacerbated weight loss and worsened performance on the pole test. JQ1-induced gene expression changes in NT mice were distinct from those in R6/2 and primarily involved protein translation and bioenergetics pathways. Dysregulation of HD-related pathways in striatum was exacerbated by JQ1 in R6/2 mice, but not in NTs, and JQ1 caused a corresponding increase in the formation of a mutant huntingtin protein-dependent high molecular weight species associated with pathogenesis. This study suggests that drugs predicted to be beneficial based on their mode of action and effects in wild-type or in other neurodegenerative disease models may have an altered impact in the HD context. These observations have important implications in the development of epigenetic modulators as therapies for HD.

Overexpression of the Arabidopsis and winter squash superoxide dismutase genes enhances chilling tolerance via ABA-sensitive transcriptional regulation in transgenic Arabidopsis

ABSTRACTThe winter squash (Cucurbita moschata, Cm) superoxide dismutase (SOD) CmSOD gene and Arabidopsis thaliana (At)SOD gene were transferred under a ubiquitin promoter into Arabidopsis via Agrobacterium tumefaciens. The expression and amount of SOD and the SOD activities in the AtSOD and CmSOD transgenic lines were significantly higher than those of non-transgenic (NT) plants exposed to 23 or 4°C treatment for 6 ~ 192-h periods. Furthermore, expressions of the cold-inducible gene (AtCBF2) and desiccation-responsible transcription factors (AtRD29A/B) were also activated in all transgenic lines compared to NT plants after chilling treatments. Compared to NT plants under chilling stress, superoxide (•O2−) accumulation was significantly lower, and chlorophyll (Chl) contents were significantly higher in all transgenic lines with higher SOD activity. Moreover, Arabidopsis seedlings overexpressing AtSOD and CmSOD also displayed greater resistance to chilling and less oxidative injury than NT plants under chilled conditions, indicating that the overexpression of AtSOD and CmSOD in Arabidopsis enhanced chilling tolerance by eliminating •O2−. The expression of AtRD29A was strongly up-regulated only in AtSOD transgenic plants treated with abscisic acid (ABA), while it was repressed in other transgenic plants, indicating ABA-sensitive AtCBF2 and AtRD29A/B transcriptional regulation signaling pathways in transgenic Arabidopsis under chilling conditions.

Enhancement of Iron Acquisition in Rice by the Mugineic Acid Synthase Gene With Ferric Iron Reductase Gene and OsIRO2 Confers Tolerance in Submerged and Nonsubmerged Calcareous Soils.

Iron (Fe) is an essential micronutrient for plants. Plants encounter Fe deficiency when grown in calcareous soil with low Fe availability, leading to reduced crop yield and agricultural problem. Rice acquires Fe from the soil via Strategy I–related system (ferrous ion uptake by OsIRT1) and Strategy II system (ferric ion uptake by chelation). However, rice plants have a weak ability in Fe(III) reduction and phytosiderophore secretion. We previously produced an Fe deficiency–tolerant rice harboring OsIRT1 promoter-refre1/372 (for higher Fe(III) reductase ability) and a 35S promoter-OsIRO2 (for higher phytosiderophore secretion). In this study, we produced a new Fe deficiency–tolerant rice by the additional introduction of a barley IDS3 genome fragment with refre1/372 and OsIRO2 (named as IRI lines) for further enhancement in Strategy II phytosiderophore productivity and better growth performance in various environments. Our results show that an enhanced tolerance was observed in OsIRO2 introduced line at the early growth stage, refre1/372 introduced line in the late stage, and RI line in all stages among five types of cultivation method. Moreover, we demonstrated that new IRI rice lines exhibited enhanced tolerance to Fe deficiency compared to nontransgenic (NT) rice and rice lines harboring the overexpressing OsIRO2 or the IDS3 fragment under submerged calcareous soil. The yields of IRI lines were ninefold higher than the NT line. Furthermore, under Fe-limited nonsubmerged calcareous soil condition (a new cultivation condition), IRI lines also conferred enhanced tolerance than NT, lines introducing only the OsIRT1 promoter-refre1/372 or overexpressing OsIRO2, and lines harboring both. Our results demonstrate that further enhancement of the Strategy II Fe uptake system by the mugineic acid synthase gene in addition to Fe uptake by enhanced ferric Fe reduction and phytosiderophore production in rice contributes Fe deficiency tolerance and broaden its utility in calcareous soil cultivation under paddy or nonpaddy field conditions.

Transgenic sweetpotato plants overexpressing tocopherol cyclase display enhanced α-tocopherol content and abiotic stress tolerance

Oxidative stress caused by reactive oxygen species (ROS) under various environmental stresses significantly reduces plant productivity. Tocopherols (collectively known as vitamin E) are a group of lipophilic antioxidants that protect cellular components against oxidative stress. Previously, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including tocopherol cyclase (IbTC). In this study, we generated transgenic sweetpotato plants overexpressing IbTC under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as TC plants) via Agrobacterium-mediated transformation to understand the function of IbTC in sweetpotato. Three transgenic lines (TC2, TC9, and TC11) with high transcript levels of IbTC were selected for further characterization. High performance liquid chromatography (HPLC) analysis revealed that α-tocopherol was the most predominant form of tocopherol in sweetpotato tissues. The content of α-tocopherol was 1.6–3.3-fold higher in TC leaves than in non-transgenic (NT) leaves. No significant difference was observed in the tocopherol content of storage roots between TC and NT plants. Additionally, compared with NT plants, TC plants showed enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses, and showed consistently higher levels of photosystem II activity and chlorophyll content, indicating abiotic stress tolerance. These results suggest IbTC as a strong candidate gene for the development of sweetpotato cultivars with increased α-tocopherol levels and enhanced abiotic stress tolerance.

Global Metabolic Shifts in Age and Alzheimer's Disease Mouse Brains Pivot at NAD+/NADH Redox Sites.

Age and Alzheimer’s disease (AD) share some common features such as cognitive impairments, memory loss, metabolic disturbances, bioenergetic deficits, and inflammation. Yet little is known on how systematic shifts in metabolic networks depend on age and AD. In this work, we investigated the global metabolomic alterations in non-transgenic (NTg) and triple-transgenic (3xTg-AD) mouse brain hippocampus as a function of age by using untargeted Ultrahigh Performance Liquid Chromatography-tandem Mass Spectroscopy (UPLC-MS/MS). We observed common metabolic patterns with aging in both NTg and 3xTg-AD brains involved in energy-generating pathways, fatty acids oxidation, glutamate, and sphingolipid metabolism. We found age-related downregulation of metabolites from reactions in glycolysis that consumed ATP and in the TCA cycle, especially at NAD+/NADH-dependent redox sites, where age- and AD-associated limitations in the free NADH may alter reactions. Conversely, metabolites increased in glycolytic reactions in which ATP is produced. With age, inputs to the TCA cycle were increased including fatty acid β-oxidation and glutamine. Overall age- and AD-related changes were > 2-fold when comparing the declines of upstream metabolites of NAD+/NADH-dependent reactions to the increases of downstream metabolites (p = 10-5, n = 8 redox reactions). Inflammatory metabolites such as ceramides and sphingosine-1-phosphate also increased with age. Age-related decreases in glutamate, GABA, and sphingolipid were seen which worsened with AD genetic load in 3xTg-AD brains, possibly contributing to synaptic, learning- and memory-related deficits. The data support the novel hypothesis that age- and AD-associated metabolic shifts respond to NAD(P)+/NAD(P)H redox-dependent reactions, which may contribute to decreased energetic capacity.

Abstract 334: Cardiac Myosin Binding Protein C Phosphorylation Regulates Calcium Homeostasis

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is heavily phosphorylated to regulate normal cardiac function under basal conditions. However, its phosphorylation level is significantly decreased in patients with heart failure and atrial fibrillation. Furthermore, decreased cMyBP-C phosphorylation causes reduced myofilament contractility and decreased calcium sensitivity. The impact of such decreases in cMyBP-C phosphorylation and myofilament calcium sensitivity on the overall calcium handling and potential induction of arrhythmogenesis is not known. Objective: To determine the necessity and sufficiency of cMyBP-C phosphorylation to regulate calcium cycling and contractility at the isolated cardiomyocyte level. Methods and Results: Contractile properties and calcium kinetics were measured in intact cardiomyocytes isolated from 3-month-old, mixed sex, mice expressing phospho-ablated (S273A/S282A/S302A) cMyBP-C (AAA) or phospho-mimetic (S273D/S282D/S302D) cMyBP-C (DDD) and nontransgenic (NTG) control mice. AAA cells displayed a significant decrease in fractional shortening compared to NTG and DDD cells (9.4% vs 12.8% in NTG, and 12.3% in DDD, p<0.0001). Similarly, AAA myocytes demonstrated abnormal calcium kinetics with increased diastolic calcium levels (20%, p<0.001 vs . NTG and DDD) and prolonged decay time of the calcium transient (26%, p<0.01 vs NTG and DDD) when compared with NTG and DDD myocytes. Caffeine-induced calcium release in AAA myocytes indicated no change in SR calcium content, while sodium-calcium exchanger function, assessed as the time constant (τau) of calcium decline, was increased (89%, p<0.01 vs NTG). However, these depressive effects in AAA myocytes were relieved by isoproterenol (100 nmol/L) stimulation. Furthermore, stress conditions (2 Hz + ISO) increased after-contractions in AAA cardiomyocytes (60% in AAA vs 13% in NTG, p<0.001 and 15% in DDD, p<0.001). In addition, preliminary data indicate increased susceptibility of the AAA mice to arrhythmias under stress conditions in vivo. Conclusion: Dephosphorylation of cMyBP-C is sufficient to reduce sarcomere contractility and impair calcium cycling resulting in spontaneous after-contractions and arrhythmias under stress conditions.

Reversibility of Age-related Oxidized Free NADH Redox States in Alzheimer\u2019s Disease Neurons by Imposed External Cys/CySS Redox Shifts

Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer’s disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer’s neurons.

Highly Dynamic Changes in the Activity and Regulation of Macroautophagy in Hearts Subjected to Increased Proteotoxic Stress

Macroautophagy (referred to as autophagy hereafter) plays an important role in the quality control of cellular proteins and organelles. Transcription Factor EB (TFEB) globally activates the expression of genes in the autophagic-lysosomal pathway (ALP) to replenish lysosomes and ALP machineries. We previously reported that myocardial TFEB signaling was impaired in advanced cardiac proteinopathy; however, myocardial ALP status and TFEB activity at earlier stages of cardiac proteinopathy remain uncharacterized. Here a stable line of CryABR120G transgenic (R120G) and non-transgenic (NTG) littermate mice with cardiomyocyte-restricted overexpression of CryABR120G were used at 1, 3, and 6 months of age. At 1 month when no cardiac phenotypes other than aberrant protein aggregation are discernible, R120G mice displayed a 5-fold increase in myocardial LC3-II flux. Interestingly, the LC3-II flux increase co-existed with increases in mTOR complex 1 (mTORC1) activities as well as cytoplasmic, but not nuclear, TFEB proteins. This increase in cytoplasmic TFEB proteins occurred without any discernible alteration in TFEB activity as reflected by unchanged mRNA levels of representative TFEB target genes (Mcoln1, M6pr, Sqstm1, Vps18, and Uvrag). At 3 months of age when hypertrophy and diastolic malfunction start to develop, the LC3-II flux remained significantly increased but to a lesser degree (2-fold) than at 1 month. The LC3-II flux increase was associated with decreased mTORC1 activities and with increased nuclear TFEB proteins and TFEB activities. At 6 months of age when congestive heart failure is apparent in R120G mice, both LC3-II flux and TFEB activities were severely suppressed, while mTORC1 activity increased. We conclude that changes in both autophagy and TFEB signaling are highly dynamic during the progression of cardiac proteinopathy. Increases in autophagy occur before increases in TFEB activities but both increase in the compensatory stage of cardiac proteinopathy. Once congestive heart failure develops, both autophagy and TFEB signaling become impaired. Our results suggest that TFEB signaling is regulated by both mTORC1-dependent and -independent mechanisms in hearts subjected to increased proteotoxic stress. For therapeutic exploration, it will be important to test the effect of TFEB stimulation at the early, intermediate, and late stages of cardiac proteinopathy.

Comparative Proteomics of Phytase-transgenic Maize Seeds Indicates Environmental Influence is More Important than that of Gene Insertion

Proteomic differences were compared between phytase-transgenic (PT) maize seeds and nontransgenic (NT) maize seeds through two-dimensional electrophoresis (2-DE) with mass spectrometry (MS). When maize was grown under field conditions, 30 differentially accumulated proteins (DAPs) were successfully identified in PT seeds (PT/NT). Clusters of Orthologous Groups (COG) functional classification of these proteins showed that the largest group was associated with posttranslational modifications. To investigate the effects of environmental factors, we further compared the seed protein profiles of the same maize planted in a greenhouse or under field conditions. There were 76 DAPs between the greenhouse- and field-grown NT maize seeds and 77 DAPs between the greenhouse- and field-grown PT maize seeds However, under the same planting conditions, there were only 43 DAPs (planted in the greenhouse) or 37 DAPs (planted in the field) between PT and NT maize seeds. The results revealed that DAPs caused by environmental factors were more common than those caused by the insertion of exogenous genes, indicating that the environment has much more important effects on the seed protein profiles. Our maize seed proteomics results also indicated that the occurrence of unintended effects is not specific to genetically modified crops (GMCs); instead, such effects often occur in traditionally bred plants. Our data may be beneficial for biosafety assessments of GMCs at the protein profile level in the future.

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60–80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

Enhanced Keap1-Nrf2 signaling protects the myocardium from isoproterenol-induced pathological remodeling in mice

Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/Nrf2) is an inducible transcription factor that is essential for maintenance of redox signaling in response to stress. This suggests that if Nrf2 expression response could be enhanced for a defined physiological pro-oxidant stress then it would be protective. This has important implications for the therapeutic manipulation of the Keap1/Nrf2 signaling pathway which is now gaining a lot of attention. We tested this hypothesis through the generation of Nrf2 transgene expression mouse model with and without isoproterenol-induced cardiac stress. Cardiac-specific mouse Nrf2 transgenic (mNrf2-TG) and non-transgenic (NTG) mice were subjected to isoproterenol (ISO) treatment and assessed for myocardial structure, function (echocardiography and electrocardiography), and glutathione redox state. Myocardial infarction and fibrosis along with increased inflammation leading to myocardial dysfunction was noted in NTG mice exposed to ISO, while mNrf2-TG hearts were resistant to the ISO insult. Preservation of myocardial structure and function in the mNrf2-TG mice was associated with the enhanced Nrf2 expression displayed in these hearts with an increased basal and post-treatment expression of redox modulatory genes and an overall enhanced antioxidant status. Of note, myocardium of ISO-treated TG mice displayed significantly increased stabilization of the KEAP1-NRF2 complex and enhanced release of NRF2 to the nucleus resulting in overall decreased pro-oxidant markers. Taken together, we suggest that a basal enhanced Nrf2 expression in mouse heart results in maintenance of redox homeostasis and counteracts ISO-induced oxidative stress, and suppresses pathological remodeling. These data suggest that an alternative therapeutic approach to enhance the efficacy of the Keap1-Nrf2 system is to stimulate basal expression of Nrf2.

Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy.

A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson's trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E': 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Biodegradation Profiles of Proanthocyanidin-Accumulating Alfalfa Plants Coexpressing Lc- bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes.

The utilization of the nutrient potential of alfalfa ( Medicago sativa L.) cannot be maximized because of its rapidly degradable protein content in the rumen, leading to waste and various digestive disorders. This might be alleviated if protein-binding proanthocyanidins are present in aerial parts of alfalfa forage in adequate amounts. The Lc (bHLH) and C1 (MYB) genes of maize are transcription factors known to be collectively involved in the regulation of anthocyanin biosynthetic pathways. The objective of this study was to investigate the effect of Lc and C1 gene transformations on the proanthocyanidin content, nutrient composition, and degradation characteristics of proteins and carbohydrates by comparing the transgenic alfalfa with its parental nontransgenic (NT) alfalfa and commercial AC-Grazeland cultivar. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Both Lc-single and LcC1-double transgenic alfalfa accumulated both monomeric and polymeric proanthocyanidins with total proanthocyanidins ranging from ca. 460 to 770 μg/g of DM. The C1-transgenic alfalfa did not accumulate proanthocyanidins similar to NT alfalfa. The C1 gene increased the NPN content significantly only in C1-single and Lc1C1-double transgenic alfalfa. The LcC1 combination seemed to have a synergic effect on reducing sugar in alfalfa. In contrast, the Lc gene appears to have a negative effect on starch content. The C1 gene tended to lower the PB3 content irrespective of the presence of the Lc gene. Although the cotransformation of Lc and C1 increased the total N/CHO ratio compared to Lc single gene transformation, the total N/CHO ratio of transgenic alfalfa was not significantly different from NT. In conclusion, Lc-bHLH single and LcC1 double gene transformation resulted in the accumulation of proanthocyanidins and affected the chemical profiles in alfalfa, which altered ruminal degradation patterns and impacted the nutrient availability of alfalfa in ruminant livestock systems.

Chronic Reductive Stress Impairs Endoplasmic Reticulum Function and Cause Proteotoxic Cardiac Disease

BackgroundHeart failure (HF) is a growing cause of global human morbidity and mortality and elevated ROS are generalized as drivers of HF pathology. However, antioxidant treatments have failed to offer sufficient therapeutic benefits; the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic reductive stress (cRS) induces endoplasmic reticulum (ER) dysfunction, which leads to proteotoxicity and pathological cardiac remodeling.MethodsTransgenic mice expressing cardiac specific, constitutively active Nrf2 (caNrf2‐TG) and their non‐transgenic (NTG) littermates at the age of 3 and ~6 months (n=6–8/group) were used to determine ER redox states and protein aggregation levels. Diastolic function was measured by echocardiography.ResultscRS substantially increased ER glutathione levels and suppressed protein oxidation (DMPO‐adducts) in TG vs. NTG mouse hearts. The co‐localization of KDEL (ER‐marker) with GSH or DMPO, showed by immunofluorescent imaging, indicates a hyper‐reductive redox milieu in the ER. Immunoblotting revealed a dose‐dependent induction of ER‐proteins (CHOP, GRP78, GRP94 and PDI) in TG hearts at 3‐months‐old. However, at 6‐months of age, this response was significantly repressed along with impaired protein folding response in TG mice, evidencing that progressive cRS is associated with a transition from adaptive ER stress‐response to ER dysfunction. Notably, electron micrograph and Proteostat® staining showed a dose‐ and age‐dependent augmentation of misfolded/aggregated proteins in TG hearts; these aggregates were highly ubiquitinated as demonstrated. Patterns of Mitral valve movement showed that ≥6 month‐old TG mice developed overt LV diastolic dysfunction as evidenced by increased mitral inflow velocity (MV E/A ratio).ConclusionER dysfunction and proteotoxic insults triggered pathological cardiac remodeling and diastolic dysfunction in the TG hearts experiencing cRS. The heterogeneity of redox profiles in HF subjects demands further investigation into the potential impact of RS in cardiovascular pathology. Research focusing on correlations between redox stress intensity, redox imbalance and disease progression may offer insights into redox‐based therapies.Support or Funding InformationThis study was supported by funding from NHLBI (HL118067), NIA (AG042860), the AHA (BGIA 0865015F), University of Utah Center for Aging Pilot grant (2009), the Division of Cardiovascular Medicine/Department of Medicine, University of Utah and the start‐up funds (for NSR) by Department of Pathology, the University of Alabama at Birmingham, AL and UABAMC21 grant by the University of Alabama at Birmingham, AL.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.