Evaluation of metabolomics profiles of grain from maize hybrids derived from near-isogenic GM positive and negative segregant inbreds demonstrates that observed differences cannot be attributed unequivocally to the GM trait.

INTRODUCTION: Past studies on plant metabolomes have highlighted the influence of growing environments and varietal differences in variation of levels of metabolites yet there remains continued interest in evaluating the effect of genetic modification (GM). OBJECTIVES: Here we test the hypothesis that metabolomics differences in grain from maize hybrids derived from a series of GM (NK603, herbicide tolerance) inbreds and corresponding negative segregants can arise from residual genetic variation associated with backcrossing and that the effect of insertion of the GM trait is negligible. METHODS: Four NK603-positive and negative segregant inbred males were crossed with two different females (testers). The resultant hybrids, as well as conventional comparator hybrids, were then grown at three replicated field sites in Illinois, Minnesota, and Nebraska during the 2013 season. Metabolomics data acquisition using gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) allowed the measurement of 367 unique metabolite features in harvested grain, of which 153 were identified with small molecule standards. Multivariate analyses of these data included multi-block principal component analysis and ANOVA-simultaneous component analysis. Univariate analyses of all 153 identified metabolites was conducted based on significance testing (α = 0.05), effect size evaluation (assessing magnitudes of differences), and variance component analysis. RESULTS: Results demonstrated that the largest effects on metabolomic variation were associated with different growing locations and the female tester. They further demonstrated that differences observed between GM and non-GM comparators, even in stringent tests utilizing near-isogenic positive and negative segregants, can simply reflect minor genomic differences associated with conventional back-crossing practices. CONCLUSION: The effect of GM on metabolomics variation was determined to be negligible and supports that there is no scientific rationale for prioritizing GM as a source of variation.

Physiological responses related to increased grain yield under drought in the first biotechnology-derived drought-tolerant maize.

Maize (Zea mays ssp. mays L.) is highly susceptible to drought stress. This work focused on whole-plant physiological mechanisms by which a biotechnology-derived maize event expressing bacterial cold shock protein B (CspB), MON 87460, increased grain yield under drought. Plants of MON 87460 and a conventional control (hereafter 'control') were tested in the field under well-watered (WW) and water-limited (WL) treatments imposed during mid-vegetative to mid-reproductive stages during 2009-2011. Across years, average grain yield increased by 6% in MON 87460 compared with control under WL conditions. This was associated with higher soil water content at 0.5 m depth during the treatment phase, increased ear growth, decreased leaf area, leaf dry weight and sap flow rate during silking, increased kernel number and harvest index in MON 87460 than the control. No consistent differences were observed under WW conditions. This indicates that MON 87460 acclimated better under WL conditions than the control by lowering leaf growth which decreased water use during silking, thereby eliciting lower stress under WL conditions. These physiological responses in MON 87460 under WL conditions resulted in increased ear growth during silking, which subsequently increased the kernel number, harvest index and grain yield compared to the control.

Comparative analysis of maize (Zea mays) crop performance: natural variation, incremental improvements and economic impacts.

Grain yield from maize hybrids continues to improve through advances in breeding and biotechnology. Despite genetic improvements to hybrid maize, grain yield from distinct maize hybrids is expected to vary across growing locations due to numerous environmental factors. In this study, we examine across-location variation in grain yield among maize hybrids in three case studies. The three case studies examine hybrid improvement through breeding, introduction of an insect protection trait or introduction of a transcription factor trait associated with increased yield. In all cases, grain yield from each hybrid population had a Gaussian distribution. Across-location distributions of grain yield from each hybrid partially overlapped. The hybrid with a higher mean grain yield typically outperformed its comparator at most, but not all, of the growing locations (a 'win rate'). These results suggest that a broad set of environmental factors similarly impacts grain yields from both conventional- and biotechnology-derived maize hybrids and that grain yields among two or more hybrids should be compared with consideration given to both mean yield performance and the frequency of locations at which each hybrid 'wins' against its comparators. From an economic standpoint, growers recognize the value of genetically improved maize hybrids that outperform comparators in the majority of locations. Grower adoption of improved maize hybrids drives increases in average U.S. maize grain yields and contributes significant value to the economy.

Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants.

Zebrafish (Danio rerio) have a number of strengths as a host model for infection, including genetic tractability, a vertebrate immune system similar to that of mammals, ease and scale of laboratory handling, which allows analysis with reasonable throughput, and transparency, which facilitates visualization of the infection. With these advantages in mind, we examined whether zebrafish could be used to study Pseudomonas aeruginosa pathogenesis and found that infection of zebrafish embryos with live P. aeruginosa (PA14 or PAO1) by microinjection results in embryonic death, unlike infection with Escherichia coli or heat-killed P. aeruginosa, which has no effect. Similar to studies with mice, P. aeruginosa mutants deficient in type three secretion (pscD) or quorum sensing (lasR and mvfR) are attenuated in zebrafish embryos infected at 50 h postfertilization (hpf), a developmental stage when both macrophages and neutrophils are present. In contrast, embryos infected at 28 hpf, when only macrophages are initially present, succumb to lethal challenge with far fewer P. aeruginosa cells than those required for embryos infected at 50 hpf, are susceptible to infection with lasR and pscD deletion mutants, and are moderately resistant to infection with an mvfR mutant. Finally, we show that we can control the outcome of infection through the use of morpholinos, which allow us to shift immune cell numbers, or small molecules (antibiotics), which rescue embryos from lethal challenge. Thus, zebrafish are a novel host model that is well suited for studying the interactions among individual pathogenic functions of P. aeruginosa, the role of individual components of host immune defense, and small-molecule modulators of infection.

The bacteriophage lambda Q antiterminator protein contacts the beta-flap domain of RNA polymerase.

The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (alpha(2)beta beta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between sigma and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of sigma and the flap domain of the beta-subunit (beta-flap). The nascent RNA-mediated destabilization of the sigma region 4/beta-flap interaction is required for the bacteriophage lambda Q antiterminator protein (lambdaQ) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that lambdaQ establishes direct contact with the beta-flap during the engagement process, thus competing with sigma(70) region 4 for access to the beta-flap. We also show that lambdaQ's affinity for the beta-flap is calibrated to ensure that lambdaQ activity is restricted to the lambda late promoter P(R'). Specifically, we find that strengthening the lambdaQ/beta-flap interaction allows lambdaQ to bypass the requirement for specific cis-acting sequence elements, a lambdaQ-DNA binding site and a RNAP pause-inducing element, that normally ensure lambdaQ is recruited exclusively to transcription complexes associated with P(R'). Our findings demonstrate that the beta-flap can serve as a direct target for regulators of elongation.

A sigma-core interaction of the RNA polymerase holoenzyme that enhances promoter escape.

The sigma subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation and can also participate in downstream events. Several functionally important intersubunit interactions between Escherichia coli sigma(70) and the core enzyme (alpha(2)betabeta'omega) have been defined. These include an interaction between conserved region 2 of sigma(70) (sigma(2)) and the coiled-coil domain of beta' (beta' coiled-coil) that is required for sequence-specific interaction between sigma(2) and the DNA during both promoter open complex formation and sigma(70)-dependent early elongation pausing. Here, we describe a previously uncharacterized interaction between a region of sigma(70) adjacent to sigma(2) called the nonconserved region (sigma(70) NCR) and a region in the N-terminal portion of beta' that appears to functionally antagonize the sigma(2)/beta' coiled-coil interaction. Specifically, we show that the sigma(70) NCR/beta' interaction facilitates promoter escape and hinders early elongation pausing, in contrast to the sigma(2)/beta' coiled-coil interaction, which has opposite effects. We also demonstrate that removal of the sigma(70) NCR results in a severe growth defect; we suggest that its importance for growth may reflect its role in promoter escape.