Context: The photoassimilates and nitrogen (N) allocation in grain crops can be studied via the harvest index (HI) and the N harvest index (NHI). These two variables should be considered in breeding programs seeking more effective use of resources in maize (Zea mays L.) and other grain crops. Molecular biotechnology has emerged as a complement to improve not only maize grain yield but also resource utilization efficiency. A recent study reported a beneficial impact of increasing and extending the expression of a maize MADS-box transcription factor (zmm28) on the NHI. Objective: This research aimed to further identify enhancements of C-N partitioning (HI, NHI) in DP202216 maize hybrids recognizing the main physiological and morphological traits associated to the C-N dynamics via inseason intra-canopy measurements. Methods: Two DP202216 elite hybrids were evaluated with their respective wild-type (WT) controls during two field growing seasons under two N regimes. The N nutrition index (NNI), as crop N status, was quantified to avoid misinterpretations of the results. Results: The DP202216 trait showed, on average, a -6% increase in the HI with a concomitant positive impact of -9% in the NHI across hybrids, N regimes, and seasons. Those increases were consistent at different crop NNI. After accounting for the NNI, it was shown that the increases in both HI and NHI were supported by the accumulation of more N in leaves (mainly in both lower and middle sections of the canopy vertical profile) and more water-soluble carbohydrates (WSC) in stems before flowering, combined with more aggressive postanthesis remobilization. Conclusion: In this study, it was shown an increase in the efficient use of crop resources in DP202216 hybrids with respect to their isogenic WT controls at similar NNI via increments in C and N partitioning (HI and NHI). Implications: This physiological and morphological understanding of C- and N-related traits addressed the challenge of optimizing the effective use of resources in maize breeding programs. To better understand traits by management interactions with yield and yield stability, DP202216 hybrid evaluations across a broad range of commercially relevant N scenarios would be valuable.

As the frequency of intensive storm events increases in the U.S. Corn Belt, reduced stature maize hybrids could provide a new approach to improved climate resilience in maize production. We designed gene-edited D8 (dwarf8, zm-D8) maize germplasm with reduced stature to evaluate morphometric traits and grain yield. Four elite edited zm-D8 maize hybrids and their isogenic standard height hybrid comparators were evaluated over a total of 17 field locations in 2021 and 2022. Across three plant densities (80 K, 100 K and 120 K plants ha(-1)), plant height of the edited zm-D8 reduced stature hybrids was reduced 34 % and ear height was decreased 32 %. In each of the three plant densities, grain yield of the edited zm-D8 reduced stature hybrids was comparable to the isogenic standard height hybrids. Also, edited zm-D8 hybrids had significantly less lodging than the standard hybrids. In a separate study, DP202216 (ZmGos2:zmm28) a transgenic event with increased and extended expression of the zmm28 transcription factor, was crossed with reduced stature edited zm-D8 hybrids. Combining edited zm-D8 with event DP202216 significantly increased hybrid yields over those of edited zm-D8 hybrids under high density (120 K plants ha(-1)). These results support a maize production system where edited zm-D8 reduced stature hybrids, alone or in combination with DP202216, provide improved climate resilience with grain yields competitive to standard height hybrids.

We previously reported that extended and increased expression of the native maize gene zmm28 in maize resulted in transgenic events with greater grain yields under optimum field environments. Here we report additional positive impacts of altered zmm28 expression on improving yield stability of maize in the presence of the key abiotic stresses of water deficit and low nitrogen (N). Transgenic zmm28 elite hybrids were evaluated with their wild type (WT) comparators in multiple Managed Stress Environments (MSE), where a water deficit stress was imposed at flowering (FS) or during grain fill (GFS), or where N application was restricted (LN). Across 5 years of testing (2014-2018, 1600 comparisons, 80 hybrids, 43 locations), the ZmGos2-zmm28 lead event DP202216 increased yield relative to WT an average of 479 kg ha-1 (6.6%), 345 kg ha-1 (3.4%) and 166 kg ha-1 (2.0%) in FS, GFS and LN environments, respectively. Maximum observed yield response across 6-12 transgenic hybrids in a single location x year combination was 1181 kg ha-1 (13.9%), 860 kg ha-1 (9.4%) and 834 kg ha-1 (7.0%), relative to WT, for FS, GFS and LN, respectively. Another independent event, DP382118, provided similar yield improvements in the same environments, confirming overall confidence in the performance of the construct functional gene. In a separate genetic penetration study in 2018, DP202216 increased yield an average of 487 kg ha-1 (4.4%) over a combined set of GFS and FS locations, where 6 SS Bulk F3 populations were crossed with 6 diverse NSS testers (36 hybrids), confirming consistent positive yield responses across diverse germplasm under water-limited conditions. The improved yield stability of multiple elite ZmGos2-zmm28 transgenic hybrids demonstrated over multiple years of abiotic stress exposure indicates this novel transgenic variation can contribute to the sustainable intensification of maize production that will be required to address global yield gaps.

Vegetative storage proteins (VSPs) are known to serve as nitrogen reserves in many dicot plants but remain undiscovered in grasses, most widely grown group of crops globally. We identified and characterized a VSP in maize and demonstrated that its overexpression improved drought tolerance. Nitrogen supplementation selectively induced a mesophyll lipoxygenase (ZmLOX6), which was targeted to chloroplasts by a novel N-terminal transit peptide of 62 amino acids. When ectopically expressed under the control of various tissue-specific promoters, it accumulated to a fivefold higher level upon expression in the mesophyll cells than the wild-type plants. Constitutive expression or targeted expression specifically to the bundle sheath cells increased its accumulation by less than twofold. The overexpressed ZmLOX6 was remobilized from the leaves like other major proteins during grain development. Evaluated in the field over locations and years, transgenic hybrids overexpressing ZmLOX6 in the mesophyll cells significantly outyielded nontransgenic sibs under managed drought stress imposed at flowering. Additional storage of nitrogen as a VSP in maize leaves ameliorated the effect of drought on grain yield.

Biotechnology has emerged as a valuable tool in the development of maize (Zea mays L.) hybrids with enhanced nitrogen (N) use efficiency. Recent work has described the positive effects of an increased and extended expression of the zmm28 transcription factor (Event DP202216) on maize yield productivity. In this study, we expand on the previous findings studying maize N uptake and utilization in DP202216 transgenic hybrids compared to wild-type (WT) controls. Isotope 15N labeling demonstrates that DP202216 hybrids have an improved N uptake during late-vegetative stages (inducing N storage in lower leaves of the canopy) and, thus, N uptake efficiency (N uptake to applied N ratio) relative to WT. Through both greater N harvest index and reproductive N remobilization, transgenic plants were able to achieve better N utilization efficiency (yield to N uptake ratio). Our findings suggest the DP202216 trait could open new avenues for improving N uptake and utilization efficiencies in maize.

Increasing maize grain yield has been a major focus of both plant breeding and genetic engineering to meet the global demand for food, feed, and industrial uses. We report that increasing and extending expression of a maize MADS-box transcription factor gene, zmm28, under the control of a moderate-constitutive maize promoter, results in maize plants with increased plant growth, photosynthesis capacity, and nitrogen utilization. Molecular and biochemical characterization of zmm28 transgenic plants demonstrated that their enhanced agronomic traits are associated with elevated plant carbon assimilation, nitrogen utilization, and plant growth. Overall, these positive attributes are associated with a significant increase in grain yield relative to wild-type controls that is consistent across years, environments, and elite germplasm backgrounds.

Ethylene plays a critical role in many diverse processes in plant development. Recent studies have demonstrated that overexpression of the maize ARGOS8 gene reduces the plant's response to ethylene by decreasing ethylene signaling and enhances grain yield in transgenic maize plants. The objective of this study was to determine the effects of ethylene on the development of nodal roots, which are primarily responsible for root-lodging resistance in maize. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was found to promote the emergence of nodal roots. Transcriptome analysis of nodal tissues revealed that the expression of genes involved in metabolic processes and cell wall biogenesis was upregulated in response to ACC treatment, supporting the notion that ethylene is a positive regulator for the outgrowth of young root primordia. In BSV::ARGOS8 transgenic plants with reduced ethylene sensitivity due to constitutive overexpression of ARGOS8, nodal root emergence was delayed and the promotional effect of ACC on nodal root emergence decreased. Field tests showed that the BSV::ARGOS8 plants had higher root lodging relative to non-transgenic controls. When ARGOS8 expression was controlled by the developmentally regulated promoter FTM1, which conferred ARGOS8 overexpression in adult plants but not in the nodal roots and nodes in juvenile plants, the FTM1::ARGOS8 plants had no significant difference in root lodging compared with the wild type but produced a higher grain yield. These results suggest that ethylene has a role in promoting nodal root emergence and that a delay in nodal root development has a negative effect on root-lodging resistance in maize.

Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions.

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.