Functions of maize genes encoding pyruvate orthophosphate dikinase in developing maize endosperm and their enzymatic properties

The research described herein, focuses on investigating the role of pyruvate phosphate dikinase (PPDK) and factors influencing its activity in maize starchy endosperm (SE) tissue. PPDK reversibly converts phosphoenolpyruvate (PEP), pyrophosphate (PPi), and AMP, to pyruvate, phosphate (Pi), and ATP. PPDK is found in microorganisms and plant tissues with diverse functions. PPDK is abundant in maize SE during grain fill, but little is known about its role. Hypotheses were tested using transposon-insertion alleles affecting individual genes and by eliminating all PPDK expression using RNAi. PPDK\u27s C4 metabolism function in mesophyll was proven to be essential in maize. RNAi transgenes completely eliminated PPDK protein and enzyme activity from developing SE. An opaque kernel phenotype resulted, indicative of changes in storage compound organization. Metabolite analyses indicated PPDK acts in the pyruvate-forming direction but is not necessary for synthesis of sufficient ATP to generate normal storage compound abundance. PPDK activity affects energy charge, i.e., ATP/(ADP + AMP) ratio, and through this is proposed to regulate central metabolic fluxes. PPDK activity in SE decreases during prolonged growth in high temperature, coincident with kernel weight decrease compared to normal. Recombinant PPDK was used to test whether this effect is inherent to the enzyme. When incubated without substrates, PPDK maintained full activity up to 35oC, but was completely inactivated within 5 min at 45oC. Pre-incubation with AMP prevented PPDK from inactivation at 45oC. The data suggest PPDK activity in vivo can be affected by availability of AMP, and confirm that PPDK is a particularly temperature-labile enzyme that could contribute to reduced plant performance at elevated temperatures. Recombinant PPDK isoforms were also used in protein-protein interaction tests that demonstrated formation of hetermultimeric forms including both the PPDK1 and PPDK2 polypeptides in addition to homomultimers of each isoform individually. Technical capacities developed to perform these experiments were distributed to the research community. These include a new series of vectors designed for SE-specific, high-level expression of transgenes through the course of grain fill, which were proven highly effective for plant transformation and transgene expression. New targeted metabolomics methods were developed to detect PEP, pyruvate, and PPi in total SE extracts using GC-MS

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression

A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores

Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome‐inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti‐insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV‐mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV‐expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize

Vitamin D supplementation and breast cancer prevention : a systematic review and meta-analysis of randomized clinical trials

In recent years, the scientific evidence linking vitamin D status or supplementation to breast cancer has grown notably. To investigate the role of vitamin D supplementation on breast cancer incidence, we conducted a systematic review and meta-analysis of randomized controlled trials comparing vitamin D with placebo or no treatment. We used OVID to search MEDLINE (R), EMBASE and CENTRAL until April 2012. We screened the reference lists of included studies and used the “Related Article” feature in PubMed to identify additional articles. No language restrictions were applied. Two reviewers independently extracted data on methodological quality, participants, intervention, comparison and outcomes. Risk Ratios and 95% Confident Intervals for breast cancer were pooled using a random-effects model. Heterogeneity was assessed using the I2 test. In sensitivity analysis, we assessed the impact of vitamin D dosage and mode of administration on treatment effects. Only two randomized controlled trials fulfilled the pre-set inclusion criteria. The pooled analysis included 5372 postmenopausal women. Overall, Risk Ratios and 95% Confident Intervals were 1.11 and 0.74–1.68. We found no evidence of heterogeneity. Neither vitamin D dosage nor mode of administration significantly affected breast cancer risk. However, treatment efficacy was somewhat greater when vitamin D was administered at the highest dosage and in combination with calcium (Risk Ratio 0.58, 95% Confident Interval 0.23–1.47 and Risk Ratio 0.93, 95% Confident Interval 0.54–1.60, respectively). In conclusions, vitamin D use seems not to be associated with a reduced risk of breast cancer development in postmenopausal women. However, the available evidence is still limited and inadequate to draw firm conclusions. Study protocol code: FARM8L2B5L

Resilient emotionality and molecular compensation in mice lacking the oligodendrocyte-specific gene Cnp1

Altered oligodendrocyte structure and function is implicated in major psychiatric illnesses, including low cell number and reduced oligodendrocyte-specific gene expression in major depressive disorder (MDD). These features are also observed in the unpredictable chronic mild stress (UCMS) rodent model of the illness, suggesting that they are consequential to environmental precipitants; however, whether oligodendrocyte changes contribute causally to low emotionality is unknown. Focusing on 2′-3′-cyclic nucleotide 3′-phosphodiesterase (Cnp1), a crucial component of axoglial communication dysregulated in the amygdala of MDD subjects and UCMS-exposed mice, we show that altered oligodendrocyte integrity can have an unexpected functional role in affect regulation. Mice lacking Cnp1 (knockout, KO) displayed decreased anxiety- and depressive-like symptoms (i.e., low emotionality) compared with wild-type animals, a phenotypic difference that increased with age (3–9 months). This phenotype was accompanied by increased motor activity, but was evident before neurodegenerative-associated motor coordination deficits (⩽9–12 months). Notably, Cnp1KO mice were less vulnerable to developing a depressive-like syndrome after either UCMS or chronic corticosterone exposure. Cnp1KO mice also displayed reduced fear expression during extinction, despite normal amygdala c-Fos induction after acute stress, together implicating dysfunction of an amygdala-related neural network, and consistent with proposed mechanisms for stress resiliency. However, the Cnp1KO behavioral phenotype was also accompanied by massive upregulation of oligodendrocyte- and immune-related genes in the basolateral amygdala, suggesting an attempt at functional compensation. Together, we demonstrate that the lack of oligodendrocyte-specific Cnp1 leads to resilient emotionality. However, combined with substantial molecular changes and late-onset neurodegeneration, these results suggest the low Cnp1 seen in MDD may cause unsustainable and maladaptive molecular compensations contributing to the disease pathophysiology

Injury rates and injury risk factors among federal bureau of investigation new agent trainees

<p>Abstract</p> <p>Background</p> <p>A one-year prospective examination of injury rates and injury risk factors was conducted in Federal Bureau of Investigation (FBI) new agent training.</p> <p>Methods</p> <p>Injury incidents were obtained from medical records and injury compensation forms. Potential injury risk factors were acquired from a lifestyle questionnaire and existing data at the FBI Academy.</p> <p>Results</p> <p>A total of 426 men and 105 women participated in the project. Thirty-five percent of men and 42% of women experienced one or more injuries during training. The injury incidence rate was 2.5 and 3.2 injuries/1,000 person-days for men and women, respectively (risk ratio (women/men) = 1.3, 95% confidence interval = 0.9-1.7). The activities most commonly associated with injuries (% of total) were defensive tactics training (58%), physical fitness training (20%), physical fitness testing (5%), and firearms training (3%). Among the men, higher injury risk was associated with older age, slower 300-meter sprint time, slower 1.5-mile run time, lower total points on the physical fitness test (PFT), lower self-rated physical activity, lower frequency of aerobic exercise, a prior upper or lower limb injury, and prior foot or knee pain that limited activity. Among the women higher injury risk was associated with slower 300-meter sprint time, slower 1.5-mile run time, lower total points on the PFT, and prior back pain that limited activity.</p> <p>Conclusion</p> <p>The results of this investigation supported those of a previous retrospective investigation emphasizing that lower fitness and self-reported pain limiting activity were associated with higher injury risk among FBI new agents.</p

Functions of maize genes encoding pyruvate orthophosphate dikinase in developing maize endosperm and their enzymatic properties

The research described herein, focuses on investigating the role of pyruvate phosphate dikinase (PPDK) and factors influencing its activity in maize starchy endosperm (SE) tissue. PPDK reversibly converts phosphoenolpyruvate (PEP), pyrophosphate (PPi), and AMP, to pyruvate, phosphate (Pi), and ATP. PPDK is found in microorganisms and plant tissues with diverse functions. PPDK is abundant in maize SE during grain fill, but little is known about its role. Hypotheses were tested using transposon-insertion alleles affecting individual genes and by eliminating all PPDK expression using RNAi. PPDK's C4 metabolism function in mesophyll was proven to be essential in maize. RNAi transgenes completely eliminated PPDK protein and enzyme activity from developing SE. An opaque kernel phenotype resulted, indicative of changes in storage compound organization. Metabolite analyses indicated PPDK acts in the pyruvate-forming direction but is not necessary for synthesis of sufficient ATP to generate normal storage compound abundance. PPDK activity affects energy charge, i.e., ATP/(ADP + AMP) ratio, and through this is proposed to regulate central metabolic fluxes. PPDK activity in SE decreases during prolonged growth in high temperature, coincident with kernel weight decrease compared to normal. Recombinant PPDK was used to test whether this effect is inherent to the enzyme. When incubated without substrates, PPDK maintained full activity up to 35oC, but was completely inactivated within 5 min at 45oC. Pre-incubation with AMP prevented PPDK from inactivation at 45oC. The data suggest PPDK activity in vivo can be affected by availability of AMP, and confirm that PPDK is a particularly temperature-labile enzyme that could contribute to reduced plant performance at elevated temperatures. Recombinant PPDK isoforms were also used in protein-protein interaction tests that demonstrated formation of hetermultimeric forms including both the PPDK1 and PPDK2 polypeptides in addition to homomultimers of each isoform individually. Technical capacities developed to perform these experiments were distributed to the research community. These include a new series of vectors designed for SE-specific, high-level expression of transgenes through the course of grain fill, which were proven highly effective for plant transformation and transgene expression. New targeted metabolomics methods were developed to detect PEP, pyruvate, and PPi in total SE extracts using GC-MS.</p

Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing

Viral vectors are being engineered to deliver CRISPR/Cas9 components systemically in plants to induce somatic or heritable site-specific mutations. It is hypothesized that RNA mobility signals facilitate entry of viruses or single guide RNAs (sgRNAs) into the shoot apical meristem where germline mutations can occur. Our objective was to understand the impact of RNA mobility signals on virus-induced somatic and germline gene editing in Nicotiana benthamiana and Zea mays. Previously, we showed that foxtail mosaic virus (FoMV) expressing sgRNA induced somatic mutations in N. benthamiana and Z. mays expressing Cas9. Here, we fused RNA mobility signals to sgRNAs targeting the genes encoding either N. benthamiana phytoene desaturase (PDS) or Z. mays high affinity potassium transporter 1 (HKT1). Addition of Arabidopsis thaliana Flowering Locus T (AtFT) and A. thaliana tRNA-Isoleucine (AttRNAIle) did not improve FoMV-induced somatic editing, and neither were sufficient to facilitate germline mutations in N. benthamiana. Maize FT homologs, Centroradialus 16 (ZCN16) and ZCN19, as well as AttRNAIle were found to aid somatic editing in maize but did not enable sgRNAs delivered by FoMV to induce germline mutations. Additional viral guide RNA delivery systems were assessed for somatic and germline mutations in N. benthamiana with the intention of gaining a better understanding of the specificity of mobile signal-facilitated germline editing. Potato virus X (PVX), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV) were included in this comparative study, and all three of these viruses delivering sgRNA were able to induce somatic and germline mutations. Unexpectedly, PVX, a potexvirus closely related to FoMV, expressing sgRNA alone induced biallelic edited progeny, indicating that mobility signals are dispensable in virus-induced germline editing. These results show that PVX, BSMV, and TRV expressing sgRNA all have an innate ability to induce mutations in the germline. Our results indicate that mobility signals alone may not be sufficient to enable virus-based delivery of sgRNAs using the viruses, FoMV, PVX, BSMV, and TRV into cell types that result in germline mutations.This article is published as Beernink BM, Lappe RR, Bredow M and Whitham SA (2022) Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing. Front. Genome Ed. 4:925088. DOI: 10.3389/fgeed.2022.925088. Copyright 2022 Beernink, Lappe, Bredow and Whitham. Attribution 4.0 International (CC BY 4.0). Posted with permission