zmm28 transgenic maize increases both N uptake- and N utilization-efficiencies

The study addresses the challenge of improving nitrogen use efficiency (NUE) in maize to meet rising global food demand while reducing the environmental footprint of fertilizers. NUE is framed as comprising nitrogen uptake efficiency (NUpE: total N absorbed as a proportion of N applied) and nitrogen utilization efficiency (NUtE: grain yield per unit of N uptake). Although substantial advances have been made in understanding N uptake, assimilation, and remobilization, validating candidate traits under field conditions remains difficult due to complex genotype-by-environment interactions. Prior work showed that increased and extended expression of the native MADS-box transcription factor zmm28 (event DP202216) improved grain yield across environments. The research question here is whether altered zmm28 expression enhances maize N uptake and N utilization under field conditions and how these effects manifest across growth stages and N supply. The purpose is to mechanistically characterize N dynamics (uptake, allocation, remobilization, and partitioning) in DP202216 versus WT under low and adequate N to inform breeding and biotechnology strategies for NUE.

The authors review NUE concepts, emphasizing division into N uptake and utilization and the influence of root architecture, plant demand, and assimilation/remobilization processes. They note successful laboratory and early-stage efforts to improve NUE via genetic manipulation of N-related transport and signaling genes, including amino acid transporters and regulatory pathways. MADS-box transcription factors are highlighted for roles in nitrate signaling, root development, and vegetative growth, with evidence that upregulating zmm28 can enhance nitrogen assimilation and plant vigor in controlled environments. However, prior to this work, field validation of NUE improvement via overexpression of a single transcription factor in maize had not been reported. The review sets the stage for testing whether zmm28-driven traits translate to field-level improvements in N dynamics and NUE.

Field trials were conducted at the Corteva Agriscience research station in York, Nebraska, USA (40°53′ N, 97°35′ W) during 2019 (irrigated, previous crop soybean) and 2020 (non-irrigated, previous crop wheat) on a silty clay loam (smectitic, mesic Udic Argiustolls). Standard agronomic practices were used. Planting dates: May 14, 2019 (target density 82,000 plants ha⁻¹) and May 1, 2020 (70,000 plants ha⁻¹); row spacing 0.76 m. Two elite hybrids (PH11V8W2Z and P1421) were tested as WT and their corresponding DP202216 transgenic versions. Nitrogen treatments were 0 and 225 kg N ha⁻¹ (N0 and N225) applied two days after planting as 28% UAN. Experimental design was a split-plot with three replicates: whole plots were N supply; subplots were hybrids; plots were eight rows (5 m long, 6 m wide). Short-term 15N multi-stage isotopic labeling quantified N uptake and allocation. In 2019, labeling stages were V17 (≈10 days pre-flowering), R1 (flowering), and R3 (milk). In 2020, V11 was added, with additional samplings at R1, R3, and R6 (physiological maturity). At each labeling, 10.15% 15N Ca(NO3)2 was soil-applied to five-plant microplots at 0.7 g plant⁻¹. Plants were harvested 5 days post-labeling (15 days at R6) and separated into leaves (green blades), stem (stems, sheaths, tassel, husks), and ears (grains + cob when present). Samples were dried to constant weight. Specific leaf nitrogen (SLN, N per unit leaf area) was measured by taking twelve 15.89 mm leaf disks per plant across canopy strata at each sampling. At V11, canopy was divided into upper (three uppermost-expanded leaves) and lower (remaining green leaves). When ears were present, leaves were classified into upper (+2 node and above), middle (ear node ±1), and lower (−2 node and below). Disks were dried, weighed, and analyzed for N. Grain from the four center rows was harvested at maturity for NUE parameters. Laboratory analyses included grinding samples, measuring total N by combustion (FlashEA 1112), and isotopic composition (δ15N) via an elemental analyzer (PyroCube) coupled to IRMS (visION). SLN was calculated as disk N content divided by disk area (1.98 cm²). Tissue N content was calculated as %N × dry mass. 15N uptake rates and allocation fractions were computed from differences in 15N abundance between labeled and non-labeled controls. Indicators such as NUpE (including 15N fertilizer recovery), NUtE (grain yield per unit whole-plant N uptake), N remobilization (balance approach: vegetative N at flowering minus stover N at maturity), and N harvest index (N in grain / whole-plant N at maturity) were derived. Statistics: Bayesian mixed-effects models with population-level effects for N treatment, hybrid, transgene (DP202216 vs WT), and growth stage, and group-level effects for Year (n=2) and Block (n=3). Four MCMC chains, 4000 iterations with 2000 warmup; convergence assessed by trace plots and Gelman–Rubin diagnostics. Medians of posterior distributions reported. Probabilities for differences between DP202216 and WT were derived from posterior pairwise differences and categorized as weak (>65%), moderate (>75%), or strong (>90%) evidence. Year and interaction effects were examined via 90% credible intervals.

• DP202216 increased pre-flowering 15N uptake rates relative to WT, especially up to flowering. At V11 under both N0 and N225, transgenic plants showed higher 15N uptake with >83% probability. Effects diminished in later vegetative stages (V17 to R1) and were not evident during grain filling (R3 and R6).
• DP202216 elevated proportional allocation of 15N to leaves from V11 to R1, and increased specific leaf N (SLN) in lower canopy leaves prior to flowering, particularly under N225: PH11V8W2Z (82% probability) and P1421 (76%). No consistent SLN differences at N0 in upper/middle canopy pre-flowering.
• Ear allocation: From V17 to R6, DP202216 generally allocated more 15N to ears than WT (notably in PH11V8W2Z), though with moderate evidence due to variability.
• Post-flowering N uptake was similar between DP202216 and WT across most conditions; in some N0 conditions (e.g., P1421), DP202216 appeared to maintain growth with reduced N needs.
• N remobilization: DP202216 consistently increased remobilization of N from stover to grain from flowering to maturity versus WT, with probabilities 79–98%, and showed larger improvements under irrigated 2019 conditions.
• N harvest index (NHI): DP202216 had greater NHI under both N0 and N225, with strong evidence (up to 99% at N0 and 96% at N225), averaging an 11% relative increase over WT. Grain N concentration at maturity was maintained similar to WT: 1.26% (N0) and 1.28% (N225).
• 15N fertilizer uptake efficiency (15NUpE) pre-flowering was moderately enhanced in some hybrid × N combinations (e.g., PH11V8W2Z at N0 and P1421 at N225), with a median 6% increase in fertilizer N recovery; otherwise similar to WT. Reproductive-period fertilizer N recovery was similar between genotypes.
• N utilization efficiency (NUtE) was higher in DP202216 versus WT, especially under N0 with >94% probability across experiments. Under N225, improvements tended to be smaller (often <90% probability). Year effects were evident, with lower NUtE in 2019 (median 39 kg kg⁻¹) and higher in 2020 (63 kg kg⁻¹), but DP202216 consistently outperformed WT under N limitation.
• Overall, DP202216 improved NUE via both enhanced pre-flowering N uptake efficiency and improved utilization (greater N remobilization and NHI) without diluting grain N concentration.

The findings demonstrate that earlier and extended expression of the native maize MADS-box transcription factor zmm28 (via a moderately constitutive ZmGos2 promoter) enhances nitrogen dynamics in field-grown maize. DP202216 plants increased N uptake during rapid vegetative growth, likely reflecting elevated source demand and root activity when zmm28 protein in roots peaks (around V9), leading to greater N accumulation and storage in lower canopy leaves. This pre-flowering N reservoir supported stronger reproductive sink establishment (ear size and kernel set) and, after flowering, greater remobilization of N from stover to grain, increasing N harvest index while maintaining grain N concentration. The trait thus improves both components of NUE: pre-flowering NUpE (in some conditions) and NUtE (consistently under N limitation). These mechanisms differ from historical improvements in maize NUE that emphasized post-flowering N uptake; instead, DP202216 leverages vegetative N assimilation and efficient redistribution. The results align with known MADS-box roles in nitrate signaling and root/lateral root development and suggest that modulating zmm28 expression can be a viable strategy to enhance yield stability and sustainability across varying N supplies. The work underscores breeding and biotechnology opportunities focused on intrinsic N uptake capacity and efficient N partitioning to reproductive sinks.

This study provides field-based evidence that altered expression of the zmm28 transcription factor (event DP202216) enhances maize nitrogen use efficiency by: (1) increasing pre-flowering N uptake and storage (notably in lower canopy leaves), and (2) improving post-flowering N utilization via greater N remobilization to the grain and higher N harvest index, without reducing grain N concentration. These coordinated effects improve NUtE, especially under N-limited conditions, and can reduce fertilizer needs or enhance yield under suboptimal N, supporting more sustainable maize production. Future research should dissect the relative contributions of pre- versus post-flowering N to grain filling using isotope tracing across stages, explore root architectural and physiological changes linked to zmm28, evaluate broader germplasm and environments, and assess interactions with fertilization timing and rates to optimize deployment in breeding programs.

• Field validation was limited to two years at a single location with two hybrid backgrounds; broader multi-location and multi-genotype testing is needed for generalization.
• Evidence for some traits (e.g., pre-flowering 15NUpE and ear 15N allocation) was moderate due to variability; not all hybrid × N combinations showed significant differences.
• No late-season N fertilizer applications were included, which may have constrained detection of genotype differences in reproductive N uptake.
• The assumption of no year-by-treatment interaction at V11 and V17 was made; although supported by later-stage consistency, this remains a constraint.
• N remobilization was estimated by the balance approach, which cannot fully resolve complexities of N fluxes (e.g., distinguishing contributions of pre- vs post-flowering N pools).
• Only two discrete N rates (0 and 225 kg N ha⁻¹) were tested, limiting inference across intermediate N supply levels and fertilization strategies.
• Root traits and direct measurements of root N uptake kinetics were not assessed, limiting mechanistic inference about uptake processes.
• Data access is subject to third-party ownership/regulation, and many materials have been destroyed per retention policies, potentially limiting reproducibility assessments.