# Source paper archive

- Source title: Genetic Variation in Photosynthetic Maintenance and Post-Chill Recovery for Early Season Maize Plantings
- Author(s): Tyler J McCubbin, Emma N Leary, Donald Coon, Jacob D Washburn
- Source landing URL: https://www.biorxiv.org/content/10.64898/2026.10.30.763178v1
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[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

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## **Genetic Variation in Photosynthetic Maintenance and Post-Chill** **Recovery for Early Season Maize Plantings**


McCubbin, Tyler J [1,3*] [†] ; Leary, Emma N [2,3*] ; Coon, Donald [1,3] ; Washburn, Jacob D [ 1,3 ]


1 USDA-ARS, Plant Genetics Research Unit, Columbia, MO 65211
2 Division of Plant Science and Technology, University of Missouri, Columbia, MO 65211
3 Interdisciplinary Plant Group, University of Missouri, Columbia, MO 65211
         - These authors contributed equally to the work

         - Correspondence: tyler.mccubbin@usda.gov


**Abstract**


Earlier maize planting could extend the growing season and improve synchronization of crop
nitrogen demand with biogenically available soil nitrogen in spring, but seedling photosynthesis
and development are strongly constrained by chilling. Eleven genetically diverse maize inbred
lines were evaluated under a spring-like controlled environment regime containing five days of
chilling stress followed by a recovery period, and across four early-spring field environments.
Gas exchange, chlorophyll fluorescence, chlorophyll status, immature-leaf growth, vegetative
stage, biomass, and visible cold injury were quantified to characterize physiological and
developmental responses during chilling and recovery. Chilling substantially reduced net CO₂
assimilation and rapidly decreased ΦPSII, while stomatal conductance responded slower.
Genotypic differences in assimilation and ΦPSII were compressed during prolonged chilling but
reemerged during recovery. Chilling also slowed immature-leaf elongation and largely arrested
vegetative developmental progression; both resumed rapidly after warming, and advanced
recovery stage development was associated with a greater fraction of mature leaf biomass.
Assimilation and ΦPSII were most tightly associated during recovery and remained positively
associated across field environments, although the strength of this relationship depended strongly
on environment. Field expression of genotype effects was highly environment dependent, but
broad-sense heritability was moderate to high for a variety of measured photosynthetic and
physiological traits. Compared with the benchmark chilling-tolerant C 4 grass _Miscanthus,_ the
maize panel exhibited similar photosynthetic performance, underscoring the accession
dependence of this comparative benchmark. Taken together, these results identify photosynthetic
maintenance and recovery as separable components of early-season cold performance and
characterize physiologically informative and heritable traits associated with chilling stress

resilience.


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


**Introduction**


C 4 photosynthesis supports some of the world's most productive crops, including maize
( _Zea mays_ L.), sorghum ( _Sorghum bicolor_ ), and sugarcane ( _Saccharum officinarum_ ). C 4
lineages exhibit considerable biochemical and evolutionary diversity (Washburn _et al._, 2015,
2021), and provide particularly high photosynthetic efficiency under conditions of high light and
warm temperature. However, because of the system’s tropical ancestry advantages are less
pronounced at low temperature maize production in temperate regions (Ehleringer and
Björkman, 1977; Zhu _et al._, 2008; Long and Spence, 2013). Maize growth and photosynthesis
are strongly inhibited by suboptimal temperature, with chilling injury occurring as temperatures
approach or fall below approximately 10°C (Greaves, 1996; Long and Spence, 2013). This cold
sensitivity limits the extent to which maize can exploit the early spring growing season. The
Circular Economy that Reimagines Corn Agriculture (CERCA) initiative has proposed that
increasing seedling cold tolerance could enable earlier planting, extend the annual period of C 4
carbon assimilation, and better synchronize maize growth and nitrogen demand with the spring
period of natural soil nitrogen availability (Ojeda-Rivera _et al._, 2025). Accordingly, development
of maize capable of maintaining growth and photosynthetic function under early-season chilling
conditions represents an important component of the proposed CERCA ideotype.


Photosynthesis is among the most chilling-sensitive physiological processes in maize
(Sowiński _et al._, 2005; Hund _et al._, 2008). Following the transition to autotrophic growth,
continued seedling development depends on the capacity to capture energy from light and
maintain carbon assimilation despite the limitations imposed by suboptimal temperatures to
chloroplast development, C 4 metabolism, and the photosynthetic electron transport system
(Greaves, 1996; Long and Spence, 2013). Chilling decreases the abundance or activity of
important photosynthetic enzymes, including pyruvate phosphate dikinase (PPDK),
phosphoenolpyruvate carboxylase (PEPC), and RubisCO, while simultaneously increasing
susceptibility of the photosynthetic apparatus to excess excitation energy (Naidu _et al._, 2003;
Long and Spence, 2013). These effects are particularly important when low temperature occurs
under high irradiance, when limitations to carbon metabolism can increase excitation pressure on
photosystem II (PSII) and contribute to photoinhibition (Pimentel _et al._, 2005; Grzybowski _et al._,
2019). Chlorophyll fluorescence therefore provides a complementary measure of photosynthetic
function to gas exchange measurements. In particular, the operating quantum efficiency of
photosystem II (ΦPSII) reflects the proportion of absorbed light being used in PSII
photochemistry under light-adapted conditions and provides a measure of the capacity for
photosynthetic electron transport (Murchie and Lawson, 2013). Maintenance of carbon
assimilation during chilling may therefore depend not on a single physiological process, but on
coordination among carbon fixation, PSII photochemistry, stomatal behavior, and protection or
recovery of the photosynthetic apparatus. Moreover, these photosynthetic traits also exhibit
useful genetic variation. Chlorophyll fluorescence has long been proposed as a selection tool for


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


cold tolerance of photosynthesis in maize (Fracheboud _et al._, 1999), and more recent work has
identified genetic variation affecting PSII efficiency during early growth under cold conditions
(Urzinger _et al._, 2025).


The strong chilling sensitivity of maize appears to be an avoidable consequence of C 4
photosynthesis. Several species within the genus _Miscanthus_, a member of the Andropogoneae
tribe and relatively close to maize and other major C 4 grasses, are notable for their ability to
remain photosynthetically productive at low temperature (Beale and Long, 1995; Naidu _et al._,
2003; Dohleman _et al._, 2009; Long and Spence, 2013). Comparisons between maize and
_Miscanthus × giganteus_ have shown substantially greater maintenance of CO 2 assimilation in
_Miscanthus_ during chilling. Whereas maize grown at low temperature can exhibit reductions in
photosynthesis approaching 80%, _M. × giganteus_ maintains a much greater proportion of its
warm-grown photosynthetic capacity (Naidu _et al._, 2003). This response has been associated
with maintenance of C 4 -cycle and Calvin-cycle enzymes, including PPDK and RubisCO, as well
as rapid repair of PSII and increased photoprotective capacity (Naidu _et al._, 2003; Long and
Spence, 2013; Spence _et al._, 2014). These observations demonstrate that sustained C 4
photosynthesis at low temperatures is physiologically possible and provides a useful comparative
model for improvement of maize. However, chilling tolerance also varies within _Miscanthus_, and
much of the mechanistic understanding of cold-tolerant C 4 photosynthesis has been derived from
a limited number of exceptionally tolerant genotypes (Głowacka _et al._, 2015; Ojeda-Rivera _et al._,
2025). Thus, _Miscanthus_ provides an informative benchmark for chilling-tolerant C 4
photosynthesis but may not necessarily define a uniform species-level response against which
maize can be compared (Głowacka _et al._, 2014).


Following domestication from teosinte in southwestern Mexico, maize diversified
through farmer selection and adaptation as it spread throughout the Americas and subsequently
into temperate production regions worldwide (Vigouroux _et al._, 2008; Hufford _et al._, 2012; FlintGarcia, 2013; Larkins, 2017). This history generated extensive phenotypic and genetic diversity
among landraces and modern germplasm, including adaptation to high elevations, northern
latitudes, and cooler growing environments. Highland maize and Northern European germplasm
provide prominent examples of adaptation to cooler environments (Eagles and Lothrop, 1994;
Rodríguez _et al._, 2010), and genetic variation for seedling cold tolerance has been identified in
diverse maize panels (Revilla Temiño _et al._, 2014; Yi _et al._, 2021). Multiple loci and natural
variants associated with low-temperature responses have subsequently been identified (Revilla
Temiño _et al._, 2014; Zeng _et al._, 2021; Jiang _et al._, 2022), demonstrating that cold tolerance is
amenable to genetic improvement.


Despite this progress, the physiological architecture underlying genetic variation in maize
chilling tolerance remains partially resolved. Many studies have focused on germination,
emergence, survival, or individual photosynthetic traits, while fewer have examined how carbon
assimilation, PSII photochemistry, stomatal behavior, chlorophyll status, and growth respond
together across genetically diverse germplasm during both chilling and subsequent recovery.


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


Recovery is particularly important because genetic differences that are not apparent during the
period of maximal stress may be expressed after favorable temperatures return. For example,
several European Flint genotypes exhibit strongly reduced leaf growth during chilling but have
been shown to rapidly resume growth after returning to a warm temperature; this response was
described as a physiological "standby mode" (Riva-Roveda _et al._, 2016). However, this and
many other detailed physiological studies have evaluated relatively small sets of maize
genotypes, often under light environments substantially below the 1,000-2,000 µmol m [-2] sec [-1]
encountered during clear spring days in the U.S. Corn Belt. Despite the genetic variation of cold
tolerance being well established, it remains unclear how much capacity for maintenance of
photosynthesis under chilling and high irradiance that maize germplasm possesses. In addition,
the physiological processes that govern that capacity exhibit genetic variation that can be
exploited through breeding.


The present study addressed the question: What capacity does maize germplasm have for
maintenance of photosynthesis during chilling stress, and what physiological processes are
essential to chilling and recovery? A genetically diverse panel of 11 maize inbred lines was
evaluated under controlled chilling and recovery conditions designed to approximate
physiologically relevant early-season cold stress while maintaining comparatively high
irradiance. Gas exchange, PSII photochemistry, chlorophyll status, immature-leaf growth,
developmental progression and biomass accumulation were quantified to characterize variation
in photosynthetic performance and its relationship to whole plant growth and recovery from
chilling stress. _Miscanthus_ accessions were included as comparative C 4 germplasm representing
a lineage in which exceptional chilling tolerance of photosynthesis has previously been
documented. The controlled-environment analyses were complemented by field experiments
testing whether physiological and growth responses translated across naturally variable earlyseason cold environments. Finally, broad-sense heritability was estimated for component
photosynthetic and growth traits to assess the extent of genetic control and their potential utility
as breeding targets in the development of an earlier-planted, cold-tolerant maize ideotype.
Together, these experiments link photosynthetic maintenance and recovery with vegetative
development, field performance, and the trait architecture relevant to development of the coldtolerant CERCA ideotype.


**Materials and Methods**


**Plant material and controlled-environment growth**


Eleven maize inbred lines were selected to span broad germplasm and heterotic
backgrounds: 3IIH6 (Iodent); B73 and LH244 (Stiff Stalk); B97, PHK76, and PHN46 (Non-Stiff
Stalk); CML247, CML322, and NC358 (Tropical); Il14H (Sweet Corn); and F2 (European
Flint). Seed was obtained through the USDA National Plant Germplasm System. The panel
included public and ex-PVP material and was used consistently across controlled-environment
and field analyses. Two _Miscanthus_ accessions, Pi417947 and a _Miscanthus × giganteus_ hybrid,


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


were obtained from the USDA GRIN stock center and from Gilbert H. Wild and Son, LLC

(Reeds, MO, USA).


Seeds were sown in 3-inch-deep, 36-cell Star Plug trays containing a 3:1 mixture of ProMix BX (Premier Tech LTD., Rivière-du-Loup, QC, Canada) and Turface MVP (Profile
Products, Buffalo Grove, IL, USA). After 10-14 d, three to five seedlings per genotype were
transplanted into 1.72-gallon (6.5 l) nursery pots (A.M.A. Horticulture, BM600, Kingsville, ON,
Canada) filled with Pro-Mix BX amended with 2 Tsp (10.2 ml) of a blend consisting of one part
Osmocote Plus (ICL Growing Solutions, Geldermalsen, Netherlands) 15-9-12 slow-release
fertilizer, one part Plant Doctor Tomato Maker (Organic Laboratories, Inc., Stuart, FL, USA) 42-6 cal mag, 1/12 part Sprint 330 (BASF, Ludwigshafen am Rhein, Germany), and 1/100 part
ferrous sulfate (Diamond Brand, Cary, NC, USA). Pots were wrapped with R-3 reflective
insulation, the soil surface was covered with an insulated disk, and pots were placed on R-10
rigid foam board on top of a short bench.


Plants were grown in Conviron walk-in chambers (Controlled Environments Limited,
Winnipeg, MB, Canada). The prechill and recovery program was designed to approximate earlyseason Corn Belt conditions associated with substantially earlier planting. Early May, when
approximately half of the Illinois maize crop is typically planted (Irwin and Good, 2012), was
used as an agronomic reference for the warm-stage environment. A 22/16 °C day/night regime
was selected for prechill and recovery, with the overnight temperature chosen to remain above
temperatures that commonly impair normal maize development (Miedema, 1982; Riva-Roveda
_et al._, 2016). The intervening chilling treatment used 12/10 °C day/night temperatures, near the
lower thermal boundary for maize vegetative growth (Ojeda-Rivera _et al._, 2025). Plants
experienced 5 days (D) of prechill, 5 D of chilling, and 5 D of recovery under a 14-h photoperiod
with photosynthetic photon flux density ramped from 300 to a peak of 1,200 μmol m⁻² s⁻¹, for a
daily light integral of 51.7 mol photons m⁻² day⁻¹. Relative humidity was programmed to 60%
during Prechill, 40% during Chilling, and 60% during Recovery stages. To prevent the
occurrence of thermal gradients, two horizontal fans were suspended from the ceiling 1m above
canopy height, while one fan was placed on the opposite side of the chamber on the floor,
circulating air under the table. Air at canopy level and soil temperatures were verified using
EasyLog EL-SIE-2+ loggers (Lascar Electronics, Erie, PA, USA) and CR1000X dataloggers
(Campbell Scientific, Logan, UT, USA), respectively.


**Sampling design and physiological measurements**


Measurements were organized around the transition into and out of chilling; the
controlled environment experiment was replicated four times. Gas exchange and light-adapted
fluorescence were collected at the end (D5) of the Prechill stage, at the beginning and end (D1
and D5) of the Chilling and Recovery stages. Growth and SPAD measurements were collected
at D1 and D5 of each stage, allowing growth to be estimated directly within each five-day
interval, and vegetative stage was recorded at the D5 endpoint of each stage. The youngest


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


actively growing leaf was reassessed at the beginning of each stage; consequently, immature-leaf
growth is interpreted as a within-stage D1-to-D5 response rather than as a continuous trajectory
of the same organ across all three stages.


Gas exchange and chlorophyll fluorescence were measured with an LI-6800 Portable
Photosynthesis System equipped with a Multiphase Flash Fluorometer and 2-cm² chamber (LICOR Environmental, Lincoln, NE, USA). Measurements were made on the youngest fully
expanded mature leaf. Chamber irradiance was set to 1,200 μmol m⁻² s⁻¹ for light-adapted
measurements, from which net CO₂ assimilation, stomatal conductance, ΦPSII, and light-adapted
Fv′/Fm′ were obtained. Leaf temperature in the LI-6800 chamber was controlled to match the
stage environment (22 °C during prechill and recovery; 12 °C during chilling). Fluorescence
nomenclature and interpretation followed established practice (Murchie and Lawson, 2013).
Dedicated dark-adapted fluorescence was measured on the same relevant leaf after at least 1 h of
dark adaptation. For paired same-leaf measurements, NPQ was calculated from dedicated darkadapted Fm and the corresponding light-adapted Fm′ as (Fm − Fm′)/Fm′.


For morphology, the mature source leaf and the youngest actively growing immature leaf
were measured for length and width. Mature-leaf relative chlorophyll content was measured with
a Konica Minolta SPAD-502DL meter (Konica Minolta, Tokyo, Japan); five readings distributed
across the illuminated leaf surface were averaged for each leaf. Developmental stage was
assigned from the number of fully mature leaves; a leaf was considered mature only when the
ligule was fully formed (Hanway, 1966). At final harvest, plant material was separated into
immature leaves, mature leaves, stem, and roots and dried at 65 °C for at least 4 d before dry
mass determination.


**Controlled-environment measurements and statistical analysis**


Quality control for LI-6800 measurements was adjudicated on a trait-specific basis so
that a technical problem with one measurement channel did not automatically invalidate other
measurements from the same plant and timepoint. Inclusion criteria for gas-exchange and
simultaneous light-adapted fluorescence measurements were based on whether leaf temperature
and measurement conditions matched the intended experimental stage; instrument identity was
retained as quality-control metadata. Technically valid negative assimilation values were
retained, whereas negative stomatal-conductance values and failed all-zero fluorescence
measurements were treated as invalid. Dark-adapted fluorescence was evaluated independently
of the gas-exchange temperature filter removing any need for quality control selection.


Longitudinal analyses of assimilation, stomatal conductance, light-adapted F V ′/F M ′, and
ΦPSII used mixed models containing genotype × timepoint and experimental-run × timepoint
effects with repeated measurements on individual plants. Timepoint-specific residual variances
were allowed; ΦPSII was analyzed on the logit scale and displayed on the response scale,
whereas the other primary physiology traits were analyzed on their observed scales. Genotype
estimated marginal means and Tukey-adjusted comparisons were used for stage-specific


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


statistical inference. Statistical model selection for all photosynthesis traits is described in
Supplementary Table S1. Vegetative stage at Prechill D5, Chilling D5, and Recovery D5 was
analyzed as a repeated numeric response with genotype × timepoint and experimental-rep ×
timepoint effects and a plant random intercept. Mature-leaf SPAD was analyzed separately using
the controlled-environment experiment with complete six-timepoint coverage, with D5 analyses
across additional experiment replicates used as sensitivity checks. Dark-adapted F V /F M was
analyzed cross-sectionally at D5. Relationships among assimilation, ΦPSII, dark-adapted Fv/Fm,
growth, SPAD, developmental stage, and endpoint biomass allocation were evaluated using
linear models with experimental run included where supported. Biomass responses were
analyzed at the individual-plant level, and allocation was evaluated using both tissue ratios and
allometric comparisons among biomass components. All statistical analysis for controlled
environment and field experiments was performed using R language programming (R Core
Team, 2026).


**Early-spring field experiments**


The maize panel was evaluated in four early-spring field environments over 2025 and
2026 at two locations: Location A refers to the University of Missouri Rollins Bottoms
Experiment Station (38.92 N, -92.35W; Haymond silt loam soil) while Location B refers to the
University of Missouri Genetics Farm (38.90 N, -92.27W, Mexico silt loam soil). V3 seedlings
were transplanted into three replicate blocks at each site, with five plants per genotype per block
targeted at establishment. A cold sensitive Dekalb commercial hybrid (P1213AM;
LibertyLink®/Roundup Ready® 2) was included as a field phenotyping reference but was
excluded from the genomic and heritability analysis. Plants were germinated and grown before
transplant in the same way as described above for the controlled environment experiments. Upon
transplant, they were exposed to ambient field conditions for 5 d. Air temperature, 4-inch soil
temperature, PPFD, relative humidity, and precipitation were recorded for each environment
(https://agebb.missouri.edu/). Visible cold injury was scored daily and summarized as the
fraction of plants within each genotype × block that remained free of visible injury.


Immature-leaf length and mature-leaf SPAD were measured immediately before
transplanting and again after the five-day field exposure. Immature-leaf growth was calculated
over this interval, and mature-leaf SPAD at the endpoint was also expressed relative to the
pretransplant value. Net CO₂ assimilation, stomatal conductance, ΦPSII, and light-adapted
F V ′/F M ′ were collected at the field endpoint. When multiple plants were measured within a
genotype × environment × block, plant subsamples were averaged to a single block-level
experimental unit before statistical analysis.


**Field statistical analysis**


Genotype effects were evaluated separately within each field environment using mixed
models with genotype as a fixed effect and block as a random effect. Tukey-adjusted genotype
comparisons were used when the omnibus genotype effect was significant. A pooled analysis


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


across all four environments evaluated genotype × year × location effects with block nested
within an environment. Trait-relationship figures used the same block-level data nested within
environment. The assimilation–ΦPSII relationship was summarized across all environments by
linear regression; assimilation–immature-leaf growth and stomatal conductance–SPAD
relationships were evaluated as complementary field associations.


**Genomic marker validation and population structure**


Marker calls shared between the maize HapMap and Genomes to Fields (G2F) resources
were evaluated for cross-source concordance. Seven highly concordant accessions selected from
79 accessions represented in both resources were used as trusted controls. Shared SNPs were
assigned stability tiers from full-panel discordance and leave-one-accession-out validation; the
highest-confidence Platinum tier required complete concordance across the trusted set and
validation in every leave-one-out analysis. The final panel contained 1,310 SNPs distributed
across all 10 maize chromosomes. These markers were used to construct the realized genomic
relationship matrix for the 11 study lines and to summarize population structure by principalcomponent analysis. The workflow and raw data are hosted electronically.


**Broad-sense heritability across field environments**


Broad-sense heritability was estimated from the field block means for the 11 maize
inbred lines across four environments. The multi-environment phenotype model included
environment as a fixed effect and genotype, genotype × environment, and block-withinenvironment as random effects. This phenotype-only best linear unbiased predictor (BLUP)
model, which assumes independent genotype effects, was treated as the primary analysis. A
complementary genomic BLUP (GBLUP) model replaced the identity covariance among
genotype effects with the realized genomic relationship matrix from the SNP panel. Entry-mean
H², Cullis H², and the mean pairwise difference reliability (mean ΔH²) were calculated for the
phenotype model as described (Schmidt _et al._, 2019 _b_, _a_ ); genomic Cullis and mean ΔH² estimates
accounted for the realized genetic variance of each genotype pair. Stomatal conductance was
excluded _a priori_ because block-level coverage was insufficient for reliable multi-environment

variance estimation.


**Results**


**A modified growth chamber system imposed repeatable spring-like chilling events**


A repeatable combination of high light intensity and low temperatures was considered
critical for testing hypotheses about early-season photosynthesis in the U.S. Cornbelt region.
However, achieving these conditions in growth chambers is technically challenging due to
factors including heat generation from high intensity lamps, thermal gradients, higher heat
exchange of potting conditions compared to field soils, and the increasing difficulty of humidity
control as temperatures decrease. Considerable time and experimentation were invested to
develop systems for buffering these issues and monitoring protocols to ensure conditions were


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


maintained. Supplementary Fig. S1 demonstrates the success of the growth chamber
modifications and configurations in maintaining desired diurnal temperatures (both soil and air),
soil moisture, and humidity levels during each phase of the experiment and across experiment
repetitions. The resulting schema therefore imposed a defined transient cold event within an
otherwise spring-like growth regime, without imposing frost damage. Of the 165 plants grown
across the trials 96% were measured at all timepoints in Prechill, Chilling, and Recovery stages.


**Chilling rapidly inhibits carbon assimilation and PSII photochemistry**


Net CO₂ assimilation, stomatal conductance, ΦPSII, and light-adapted F V ′/F M ′ were
followed from prechill through chilling and recovery to determine how maize photosynthesis
responds to sustained chilling (Fig. 1). Mean assimilation across the entire maize panel declined
from approximately 8.4 μmol CO₂ m⁻² s⁻¹ at Prechill D5 to 3.3 at Chilling D1 and 1.8 at Chilling
D5, corresponding to an approximately 78% reduction over the chilling period. Stomatal
conductance increased slightly at Chilling D1 before returning near the prechill value by Chilling
D5. In contrast, ΦPSII fell sharply from approximately 0.163 before chilling to 0.055 at Chilling
D1, increasing slightly to 0.090 by D5 of the Chilling period. Light-adapted F V ′/F M ′ declined
more gradually from approximately 0.259 before chilling to 0.201 by Chilling D5. Thus, the
rapid loss of carbon assimilation at the onset of chilling coincided more closely with reduced
PSII operating efficiency than with stomatal closure.


Photosynthetic recovery began immediately after the return to spring-like temperatures in
the recovery stage. Assimilation increased to approximately 5.2 μmol CO₂ m⁻² s⁻¹ at Recovery
D1 and 12.6 by Recovery D5, while stomatal conductance rose to approximately 0.087 mol H₂O
m⁻² s⁻¹. Recovery of fluorescence traits followed a different trajectory: light-adapted Fv′/Fm′
reached its lowest mean value at Recovery D1 before returning near the prechill value by
Recovery D5, and ΦPSII recovered more gradually to approximately 0.134. Carbon gain
therefore recovered rapidly after chilling even while PSII fluorescence parameters continued to
readjust during the early recovery period.


Two _Miscanthus_ accessions were included as physiological comparisons for C 4 chilling
performance in three growth chamber experiments (Supplementary Fig. S4). During both
chilling timepoints, neither accession maintained higher assimilation than the maize panel, and
ΦPSII converged to similarly low values across maize and _Miscanthus_ . Differences re-emerged
during recovery, but the direction and magnitude of those differences were not consistent across
the two accessions. These data show that the expected superior maintenance of chilling
photosynthesis was not uniformly expressed in the _Miscanthus_ material evaluated here.


Photosynthetic trajectories varied among maize genotypes, with significant genotype ×
timepoint interactions for assimilation, stomatal conductance, light-adapted F V ′/F M ′, and ΦPSII.
Stage-end estimates illustrate the strongest feature of these interactions: assimilation was distinct
among genotypes before chilling, but no pairwise differences remained at Chilling D5.
Substantial differentiation reappeared by Recovery D5 (Fig. 2A). ΦPSII followed the same


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pattern of a chilling compression, but genotype-specific responses were only seen during
recovery (Fig. 2B).


Several genotypes exhibited distinct stage-dependent responses. LH244 exhibited the
highest assimilation before chilling, whereas B97 displayed the strongest photosynthetic
recovery. At Recovery D5, B97 had the highest assimilation (17.3 μmol CO₂ m⁻² s⁻¹), stomatal
conductance (0.124 mol H₂O m⁻² s⁻¹), light-adapted F V ′/F M ′ (0.291), and ΦPSII (0.195) among
the maize genotypes. Prolonged chilling therefore compressed expression of genetic differences
in photosynthetic performance, while return to favorable temperature revealed substantial genetic
variation in recovery capacity.


**Coordination between PSII operating efficiency and carbon assimilation changes through**
**chilling and recovery**


Because carbon assimilation depends on continued conversion of absorbed light into
biochemical energy, the relationship between assimilation and ΦPSII was evaluated at the D5
endpoint of each stage with differences among controlled-environment experiment reps
considered. Assimilation and ΦPSII were moderately associated before chilling and strongly
associated during recovery., Conversely, the relationship during chilling was weak and the
observed range of both traits was compressed (Fig. 3). For all genotypes, a steep positive
association was generally evident during recovery, while chilling values fell within a narrow
range of both low-assimilation and low-ΦPSII (Supplementary Fig. S5). These data show that
operating PSII efficiency tracked carbon assimilation most closely during recovery, when
variation in both traits expanded beyond the narrow variability observed in the chilling stage.


Dark-adapted F V /F M provided a complementary measure of PSII status. Mean darkadapted F V /F M declined from approximately 0.72 before chilling to 0.50 at Chilling D5 and
recovered to approximately 0.73 by Recovery D5 (Supplementary Fig. S6). Genotypic
differences were detectable for Prechill D5 but were not resolved at Chilling D5 or Recovery D5.
Nonphotochemical quenching (NPQ) showed no detectable genotypic differentiation at the D5
endpoint of any experimental stage, while mean NPQ was greater during chilling and recovery
than before chilling (Supplementary Fig. S7A). Relationships of NPQ with assimilation rate and
mature-leaf SPAD were stage dependent, with no clear resolvable relationships (Supplementary
Fig. S7B,C). Thus, variation in operating PSII efficiency was more closely aligned with variation
in carbon assimilation than were dark-adapted maximum PSII efficiency or NPQ.


**Chilling restricts immature-leaf growth and vegetative developmental progression**


The same actively growing immature leaf was marked at D1 and measured again at D5
within each stage to determine whether inhibition of photosynthetic function was accompanied
by reduced vegetative growth and development. Vegetative stage was also recorded from fully
collared leaves at the D5 endpoint of each stage (Fig. 4A). Mean rate of immature-leaf
elongation declined from 22.5 mm d [-1] at Prechill to 14.8 mm d [-1] during Chilling and recovered to


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158% of the Prechill amount by Recovery D5 (35.6 mm d [-1] ). Genotypes differed in growth
before chilling, whereas genotype differences were not detected during the chilling or recovery
intervals. Chilling therefore restricted expansion of developing leaf tissue while compressing
detectable genetic variation in this short-term growth response.


Developmental progression showed a similar response; mean vegetative stage increased
only from V2.86 to V3.12 during the five-day chilling interval but reached V3.92 after five days
of recovery. Most plants failed to advance a complete vegetative stage during chilling, whereas
most developed by at least one stage during recovery. Genotype-specific trajectories differed
across the treatment, with the strongest separation evident by Recovery D5 (Fig. 4B). Thus,
chilling delayed both expansion of the actively growing leaf and the emergence of successive
fully mature leaves, whereas recovery restored both elongation and developmental progression.


Genotypic differences were evident for immature leaf, root, and shoot biomass, whereas
no statistically significant differences were observed among genotypes for mature leaf biomass.
Partitioning between above- and belowground biomass diverged among genotypes, with the
shoot:root ratio ranging from approximately 1.13 in PHK76 to 1.88 in F2 (Supplementary Fig.
S8). Vegetative stage at Recovery D5 was positively associated with the fraction of leaf biomass
present as mature tissue (Fig. 4C). Thus, developmental progression during recovery was
reflected as a higher fraction of mature leaf biomass than developing tissue at the conclusion of
the experiment.


For the majority of genotypes assessed, mature-leaf SPAD increased across the prechill
period, declined during chilling, and recovered after restoration of warm conditions
(Supplementary Fig. S9A). SPAD trajectories differed among genotypes through most of the sixtimepoint sequence, although genotype differences were no longer detected at Recovery D5.
Chlorophyll status therefore retained a substantial genotype component while following a pattern
distinct from the rapid changes in carbon assimilation and ΦPSII. Mature-leaf SPAD was
positively associated with stomatal conductance at the D5 endpoint of Prechill, Chilling, and
Recovery, although substantial variation remained within each stage (Supplementary Fig. S9B).

**Carbon assimilation remains coupled to ΦPSII in the early-spring field experiments, but**
**the strength of association is environment-dependent**


The same maize panel was evaluated in a natural experiment consisting of four earlyspring field environments spanning two locations and two years. Air and soil temperature, PPFD,
humidity, and precipitation differed substantially among the four five-day exposure periods, but
approximately reflect the environment imposed in the controlled environment experiments with
more extreme overnight low temperatures for three of the four experiments (Fig. 5A). The
increased chill resulted in distinct patterns of cold injury which could be visually scored (Fig.
5B). Injury developed most rapidly and extensively in the 2025 Location A environment,
whereas cold resistance remained comparatively high across most genotypes in 2026 Location A;


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the two Location B environments produced intermediate but genotype-dependent injury
trajectories (Fig. 5C). The field experiments therefore extended the controlled-environment
analysis into naturally variable combinations of low temperature, high irradiance, humidity, and
precipitation.


**Field expression of genotypic variation is strongly environment dependent**


Morphometric and physiological traits fluctuated markedly among field environments,
and the expression of genotype effects was not uniform across sites or years (Fig. 6).
Assimilation differed significantly among genotypes in both Location A environments but not in
either Location B environment. In contrast, light-adapted F V ′/F M ′, immature-leaf growth, and
relative mature-leaf SPAD showed significant genotype differences in both Location B
environments but not in the corresponding Location A environments. Genotype rankings also
shifted among environments, and no single genotype was consistently superior across traits
although the commercial check variety (Dekalb Hybrid) was frequently among the top
performers in all environments in which it was evaluated.


Across all four field environments, assimilation increased with ΦPSII. A moderate

positive correlation was observed among the pooled block-level dataset (Fig. 7). The distribution
was strongly structured by environment, however, and environment-specific regressions ranged
from little apparent relationship in 2025 Location A to much tighter positive relationships in the
2026 sites. Thus, ΦPSII remained positively associated with carbon assimilation across the
combined multi-year multi-environment experiment, but the degree of association depended
strongly on the environment in which plants were measured.


Additional field relationships were consistent with the broader physiological response.
Greater endpoint assimilation was positively associated with greater immature-leaf growth
accumulated over the same five-day exposure (Supplementary Fig. S10). By contrast, stomatal
conductance showed no clear positive association with mature-leaf SPAD at the field endpoint
(Supplementary Fig. S11). Together with the controlled-environment results, these field
observations support maintenance of carbon assimilation and PSII operating efficiency as more
closely coordinated components of early-season performance than chlorophyll status and

stomatal conductance alone.


**Field traits differ substantially in broad-sense heritability**


The cross-source validated panel of 1,310 SNPs was distributed across all 10 maize
chromosomes and revealed substantial variation in realized genomic relatedness among the 11
inbred lines (Supplementary Fig. S2). Principal-component analysis separated the most
genetically distinct genotypes and recapitulated broad germplasm relationships, with PC1 and
PC2 accounting for 36.2% and 14.2% of marker variation, respectively (Supplementary Fig. S3).


Broad-sense heritability estimated across the four field environments differed markedly
among traits (Table 1). In the primary phenotype-BLUP analysis, entry-mean H² was highest for


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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mature-leaf SPAD both before field exposure (0.73) and after chilling exposure (0.86). ΦPSII
showed moderate to high heritability (0.55). Assimilation was moderately heritable (0.45),
whereas immature-leaf growth was lower (0.32) and light-adapted F V ′/F M ′ showed comparatively
little heritable variation (0.22). The complementary GBLUP estimates reinforced the high
heritability of SPAD and the low heritability of light-adapted F V ′/F M ′, while estimates for the
remaining growth and photosynthetic traits shifted in magnitude after genomic relatedness was
incorporated.


Taken together, the controlled-environment and field experiments identify a common
dynamic response: chilling rapidly restricts carbon assimilation, PSII operating efficiency, leaf
elongation and developmental progression, with prolonged stress compressing the expression of
genetic differences. Both photosynthetic function and development are restored when favorable
temperatures return, revealing substantial variation in the extent and rate of recovery among
maize genotypes. The persistence of an assimilation–ΦPSII relationship under controlled and
field conditions, together with measurable field heritability for both traits, identifies
photosynthetic maintenance and recovery as experimentally tractable components of the coldtolerant early-planted maize ideotype. The V-stage and biomass allocation results further show
that recovery includes renewed maturation of leaf tissue, linking leaf-level physiological
recovery with whole-plant developmental progression.


**Discussion**


**Maize chilling resilience is defined by both photosynthetic maintenance and recovery**


The capacity of maize to experience a temporary reduction of photosynthetic capacity
during chilling followed by its quick return with favorable temperatures represents
complementary components of early-season cold resiliency. This contrasts with the concept of
cold tolerance, which describes the ability of maize to maintain photosynthetic capacity
throughout the chill stress. The results shown here depict resiliency over tolerance through
declines of assimilation, ΦPSII, and stomatal conductance upon exposure to chilling, that
rebounded to baseline level or greater when the chill was lifted. The drops seen were consistent
with the established sensitivity of maize photosynthesis to temperatures below its optimum and
indicate that the initial limitation to carbon gain was not attributable solely to stomatal closure
(Long _et al._, 1983; Garzon _et al._, 2020). Convergence of physiology under severe limitation
should not necessarily be interpreted as an absence of genetic variation for chilling resilience.
Rather, prolonged exposure may drive diverse genotypes toward a common low-function state in
which differences in physiological capacity become difficult to resolve, a model which has been
previously postulated (Riva-Roveda _et al._, 2016).


The photosynthetic resiliency of maize to chilling is a complex phenotype encompassing
multiple physiological processes and potential breeding targets (Burnett and Kromdijk, 2022).
That concept was seen here where genotypes differed in the magnitude of recovery,
demonstrating that restoration of photosynthetic function is itself a variable trait. These


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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differences are biologically important because episodic chilling in agricultural environments is
followed by periods in which plants must rapidly resume carbon gain and development.
Moreover, chilling stress is known to sensitize the photosynthetic apparatus to light intensity
(Nie _et al._, 1995; Szalai _et al._, 1996) and an early spring recovery period is likely to be
accompanied by high incident irradiance, as evidenced by the field experiments reported here.


Photosynthetic recovery can occur at different rates among processes; classical studies of
chilling-dependent photoinhibition in maize demonstrated that components of carbon
assimilation and photochemical efficiency need not recover synchronously (Ortiz-Lopez _et al._,
1990; Fryer _et al._, 1995). Chilling injury is often characterized as tolerance by measurements
made during or immediately following stress, whereas these results follow the recovery process
of light-adapted fluorescence, dark-adapted F V /F M, and nonphotochemical energy dissipation
immediately after the stress is lifted as well as five days later. The collective responses of those
photosynthetic parameters show that the reduction and subsequent recovery of carbon
assimilation are governed through coordinated changes across carbon metabolism,
photochemistry, photoprotection, and ultimately photosystem function. This is consistent with
previous work concluding that chilling sensitivity is not monocausal (Foyer _et al._, 2002; Long
and Spence, 2013).


**Sustained chilling and high light disrupt coordination of carbon assimilation and**
**photochemistry**


In many controlled-environment studies of maize, chilling has been imposed with low to
moderate irradiances (150–300 µmol m⁻² s⁻¹), in some cases specifically to limit photooxidative
damage during cold exposure (Capell and Dörffling, 1993; Irigoyen _et al._, 1996; Szalai _et al._,
1997, 2018; Aroca _et al._, 2001, 2003; Kościelniak and Biesaga-Kościelniak, 2006). The use of
sustained high irradiance (1,200 μmol m⁻² s⁻¹) comparable to field conditions captures an
important physiological challenge relevant to clear spring days in the U.S. Corn Belt. That is,
leaves must intercept substantial solar radiation while cool air and soil temperatures constrain the
ability to use that energy.


The differing speed and magnitude of change in the response to chilling across the maize
panel demonstrates that chilling did not affect photosynthesis through a single regulatory step,
instead pointing towards nonstomatal limitation and a concurrent restriction of photochemical
energy utilization. In general, operating PSII efficiency remained closely related to carbon
assimilation even as absolute photosynthetic rates changed markedly among temperature stages
and genotypes. Changes in NPQ were consistent with those of photoprotective energy dissipation
as the capacity to use absorbed light declined. Reductions in dark-adapted F V /F M after sustained
chilling provide evidence that prolonged stress extended beyond the rapidly reversible regulation
of operating efficiency to persistent changes in maximum PSII efficiency. These responses are
consistent with the broader understanding of maize chilling injury as a consequence of imbalance
among light capture, electron transport, carbon metabolism, antioxidant capacity, and repair of


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


the photosynthetic apparatus (Fryer _et al._, 1995, 1998; Aroca _et al._, 2001; Foyer _et al._, 2002).
Genotypic differences following the return to favorable temperatures may reflect both the
severity of accumulated impairment and the capacity to restore functional coordination among
carbon metabolism and photochemistry.


The present experiments cannot independently partition the effects of low temperature,
irradiance, and their interaction because irradiance was not manipulated factorially during
chilling. Instead, exposing maize to sustained chilling while maintaining substantial light
availability created an integrated physiological environment relevant to the conditions
encountered by seedlings planted earlier into temperate production systems. Future factorial
experiments varying both temperature and irradiance could determine which genotypes are
particularly sensitive to excess excitation pressure during chilling and whether genotypic
differences in recovery originate primarily from metabolic limitation, accumulated
photoinhibition, or their interaction.


The acclimation of maize to suboptimal temperatures has been well described in the
literature; such acclimation processes have shown to contribute to subsequent tolerance of more
extreme chilling. Leaves developed at 15 °C exhibited greater antioxidant capacity and
photoprotective energy dissipation and sustained less photooxidative damage during successive
exposure to 5 °C and high irradiance than leaves developed at 25 °C (Leipner _et al._, 1997), while
growth at moderately low temperature induces broader adjustments in photosynthetic
metabolism and antioxidant status (Kingston‐Smith _et al._, 1999). Thus, the transition from
favorable conditions to sustained chilling imposed here represents only one form of cold stress
that may present in an early-spring field environment. Seedlings may experience gradual cooling,
repeated cool nights, disproportionate chilling of the root zone, or shorter cold events
interspersed with favorable conditions, each of which may alter the balance among hydraulic,
metabolic, and photoprotective responses (Melkonian _et al._, 2004; Riva-Roveda _et al._, 2016).
Consequently, genotypic performance under the present regime should be interpreted as
tolerance to sustained chilling under high light rather than as a complete representation of lowtemperature resilience. Importantly, however, field experiments indicated that the controlledenvironment treatment captured physiologically relevant cold responses, with genotypic patterns
observed in the growth chamber broadly translating to field performance despite the greater
stochasticity and factors which are difficult to reproduce experimentally.


The C 4 species _Miscanthus × giganteus_ is known for its ability to productively function
at temperature ranges below that of modern maize (Głowacka _et al._, 2014; Bilska-Kos _et al._,
2018). The _Miscanthus_ accessions included here did not outperform maize across all measured
traits, emphasizing that cold resilience is not represented by a universally superior value for any
single fluorescence or gas exchange parameter. Nevertheless, the broader _Miscanthus_
comparison remains an important physiological benchmark: productive C 4 photosynthesis under
cool conditions is biologically achievable, and improvement of maize will likely require
coordinated changes across multiple components of photosynthetic function.


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**Genetic variation in recovery translates across whole-plant and field responses**


Variation in photosynthetic recovery was accompanied by differences in growth and
whole-plant performance, indicating that the consequences of chilling extended beyond
instantaneous leaf-level carbon exchange. Immature-leaf growth was strongly affected by
chilling, consistent with temperature-dependent limitations of cell division and expansion (BenHaj-Salah and Tardieu, 1995; Rymen _et al._, 2007; Louarn _et al._, 2010), and growth resumed to
differing degrees following return to favorable temperatures (Lainé _et al._, 2024; Ojeda-Rivera _et_
_al._, 2025). Likewise, the restoration of assimilation in mature leaves does not guarantee the
rapid restoration of whole-plant growth that is needed to overcome the stress induced limitation
of new leaf area production and expansion.


Recovery of both leaf growth and development are critical to the chilling resiliency of
maize to ensure minimal loss of future photosynthetic area. Renewed developmental progression
can increase future carbon capture even when individual leaves do not fully return to their
prechill physiological state. The combined gas-exchange and growth responses observed here
therefore support a broader definition of chilling recovery that includes the restoration of
physiological function and resumption of developmental activity, both of which impact biomass
accumulation. Differences in biomass accumulation reflect the cumulative consequences of
carbon acquisition, respiration, developmental rate, and allocation over time and therefore cannot
be assigned to any single photosynthetic trait.


Chlorophyll related traits provide an additional, although more indirect, dimension of
leaf-level responses to chilling and high light. SPAD exhibited substantial genetic repeatability
and may therefore provide a comparatively tractable phenotype for large-scale screening.
However, chlorophyll retention should not be interpreted as equivalent to maintenance of
photosynthetic function. Leaves can retain chlorophyll while experiencing substantial reductions
in carbon assimilation or operating photochemical efficiency, particularly during short-duration
stress (Ting _et al._, 1991; Andrews _et al._, 1995). This creates an important practical distinction for
breeding programs: traits that are highly heritable and inexpensive to measure are not necessarily
the traits most closely aligned with the physiological process of interest. Additionally, the most
mechanistically direct measurements may be difficult to deploy at scale.


**Physiological cold resilience** **provides a route toward earlier-planted maize**


The agronomic importance of traits described here becomes clearest when chilling
resilience is considered within the context of the maize planting window. Earlier planting has
contributed to historical increases in maize productivity in the central United States by increasing
the effective growing season and facilitating the use of germplasm adapted for a longer duration
growing season. Across 12 central U.S. states from 1979 to 2005, it was estimated that trends
toward earlier planting for half of the states accounted for 19–53% of state-level yield increases
(Kucharik, 2008). Additional calendar days afforded by earlier planting only contribute to
productivity if plants can develop photosynthetically active leaf area and maintain growth under


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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the chill stresses encountered during that period (Bollero _et al._, 1996). Thus, genotypes capable
of maintaining greater photosynthetic activity during moderate chilling, limiting accumulated
photochemical impairment under high light, and rapidly restoring carbon gain and leaf
development following stress are needed to make early-season plantings successful.


The results presented here should not be taken as a counterfactual to existing crop
modeling assumptions; these experiments do not directly establish how much grain yield would
be gained by improving assimilation, ΦPSII, or photosynthetic recovery during chilling. Instead,
the present results identify genetic variation in physiological processes that could contribute to
improving grain yield of early-season maize plantings. This is particularly relevant to the
CERCA cold-tolerant maize ideotype, in which early-season cold resilience is an enabling trait
rather than an endpoint (Ojeda-Rivera _et al._, 2025). Photosynthetic cold resilience directly links
the seedling stress phenotypes to broader agronomic objectives: maintenance of carbon
assimilation during chilling to preserve immediate carbon gain; maintenance of ΦPSII and
effective photoprotection to help sustain productive use of intercepted radiation; rapid recovery
of carbon acquisition following transient stress; and quick restoration of leaf development to
increasing photosynthetic photon capture.


In total, these results demonstrate substantial standing variation in the capacity of maize
to maintain and recover photosynthetic function and growth following chilling under high
irradiance. Carbon assimilation, operating PSII efficiency, photoprotection, maximum PSII
efficiency, leaf development, and whole-plant growth respond on different timescales and differ
in their degrees of genetic variation. This complexity argues against defining chilling resilience
by a single endpoint or trait, and it provides multiple physiological routes for crop improvement.
Of these, maintenance and recovery of carbon assimilation and ΦPSII provide a particularly
direct connection between the leaf-level response to chilling and the ability of an early-planted
crop to productively use captured solar radiation. Describing the heritable genetic variation of
photosynthetic maintenance, photochemical regulation, recovery, and development provides a
route toward that objective. In this way, the physiological variation identified here establishes a
foundation for developing maize capable of converting an earlier planting window into greater
seasonal carbon assimilation, more effective resource acquisition, and ultimately a more
productive and resource efficient crop.


**Supplementary Data**


Fig. S1. Description of controlled environment conditions


Fig. S2. Genome-wide distribution of SNP markers and resulting genomic relationship matrix


Fig. S3. Population structure of the 11 inbred maize panel


Fig. S4. Photosynthetic trajectories of maize panel and two _Miscanthus_ accessions across chilling
and recovery


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Fig. S5. CO 2 assimilation as a function of Φ PSII at D5 of each experimental stage for all

genotypes


Fig. S6. Dark adapted F V /F M measurements


Fig. S7. Chlorophyll content as a function of NPQ at D5 of each experimental stage


Fig. S8. Tissue dry weights by component and biomass ratios


Fig. S9. Chlorophyll content trajectory by genotype and chlorophyll content as a function of
stomatal conductance at D5 of each experimental stage


Fig. S10. Immature-leaf growth as a function of CO 2 assimilation rate at the end of field
experiments


Fig. S11. Chlorophyll content as a function of stomatal conductance at the end of field
experiments


Supplementary Table 1. Model selection criteria


**Acknowledgements**


We thank Harper LaFond (USDA-ARS) and Amy Litherland (University of Missouri) for their
assistance with logistics and data collection for all experiments. We thank Ray Wright (USDAARS) and Chris Browne (University of Missouri) for their aid with the field experiments.


**Author Contributions**


TJM, ENL, JDW: Conceptualization; TJM, ENL, JDW: methodology; TJM, ENL: formal
analysis; TJM, ENL: investigation; JDW: resources; TJM and ENL: writing—original draft;
TJM, ENL, DC, JDW: writing—review & editing; TJM and JDW: data curation; TJM:
visualization; JDW: funding acquisition.


**Funding**


This research was supported by the United States Department of Agriculture (USDA)
Agricultural Research Service (USDA-ARS), including funding from projects 8062-21000-052005-T. TJM and DC were supported by ORISE postdoctoral fellowships, ENL was supported by
an ORISE predoctoral fellowship.


**Data Availability**


The primary data supporting this study were not made publicly available at the time of
publication. The data that support the findings of this study are available from the corresponding
author upon request


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**Supplementary Figure S1.** Environmental conditions imposed during controlled-environment
experiments. (A) Air and soil temperature across the 15-day experimental sequence comprising
five days of prechilling, five days of chilling, and five days of recovery. (B) Relative humidity and
soil water potential (Ψ W ) over the same experimental sequence. The shaded interval denotes
the chilling treatment. Environmental records were compiled from four experiments conducted
under the common treatment regime; lines denote mean values and shaded ribbons display

95% confidence interval.


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#### 10 0 -10 -20



Origin class


Ex-PVP


Public


Heterotic group


European Flint


Iodent


Non-Stiff Stalk


Stiff Stalk


Sweet Corn


Tropical
















|Il14H L<br>CML322<br>F2<br>B73|PHK76<br>H244<br>PHN46|
|---|---|
|B97<br>CML247<br>NC358|3IIH6|


#### -10 0 10 20
## PC1 (36.2%)

**Supplementary Figure S3** . Population structure of the maize inbred panel based on the
validated marker set. Principal component analysis of the 11 maize inbred lines using
the same Platinum marker matrix used to construct the genomic relationship matrix.
PC1 and PC2 account for 36.2% and 14.2% of marker variation, respectively. Point
colors identifies germplasm/heterotic group, while the outer symbols denote origin
(Public or Ex-PVP).


# **A** **B** **C** **D**



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16


12


8



4


0.125


0.100


0.075


0.050


0.20


0.15


0.10


0.05


0.30


0.25


0.20


0.15



|Col1|Col2|Col3|Col4|a|Col6|
|---|---|---|---|---|---|
||b|||||
|||~~d~~||c||
||||e|||


Chilling

D5


|Col1|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
|||||||
|||||||
||c|b<br>|c<br>|||
|||||||



Prechill

D5

|Col1|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
|||||b||
||||c<br>|c||
|||d||||



Prechill

D5

|Col1|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
||a|a||a||
||||b|||
|||||c||
|||||||



Prechill

D5



Prechill

D5



Chilling

D1



Recovery

D5


Recovery

D5


Recovery

D5


Recovery

D5





Chilling

D5


Chilling

D5


Chilling

D5



Recovery

D1


Recovery

D1


Recovery

D1


Recovery

D1



Chilling

D1


Chilling

D1


Chilling

D1



**Figure 1.** Photosynthetic physiology of the maize diversity panel across prechilling, chilling, and recovery. (A) Net CO₂ assimilation, (B)
stomatal conductance, (C) photosystem II quantum yield (ΦPSII), and (D) light-adapted F V ′/F M ′ at the indicated experimental timepoints.
The shaded interval denotes the five-day chilling treatment. Points represent the mean of model-adjusted genotype estimated marginal
means (EMMs) at each timepoint, and error bars represent the SE among the 11 genotype EMMs. Statistical analyses included 703, 678,
529, and 539 valid plant-time observations for assimilation, stomatal conductance, ΦPSII, and light-adapted Fv′/Fm′, respectively.
Different letters indicate Tukey-adjusted differences among timepoints at P < 0.05. Longitudinal inference used mixed models containing
genotype × timepoint and experiment × timepoint effects, a plant random intercept, and timepoint-specific residual variances; ΦPSII was
modeled on the log scale, untransformed values displayed.


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|a|Col2|Col3|Col4|Col5|a|
|---|---|---|---|---|---|
|ab|~~N~~<br>b|~~S.~~|N.|S.|b<br>ab|
|||N|.S.|||








|N|S|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
|~~.~~|~~.~~|||a||
|||||||
||N.|S.<br>N|.S.<br>N.|~~a~~<br><br>S.|b<br>|
|||||||





**Supplementary Figure S4.** Photosynthetic physiology of maize and two _Miscanthus_
accessions across prechilling, chilling, and recovery. (A) Net CO₂ assimilation and (B) ΦPSII
are shown for the pooled maize panel, _Miscanthus_ accession Pi417947, and _Miscanthus ×_
_giganteus_ at five experimental timepoints. The shaded interval denotes the chilling
treatment. Multiple instrumental measurements from the same plant and timepoint were
averaged before analysis, and each trait was restricted to experiments providing complete
five-timepoint support for both maize and _Miscanthus_ . Points are model-estimated marginal
means ± SE. N= 56 maize plants and 7-10 miscanthus replicates. Letter denote comparison
of the three groups within a timepoint; groups not sharing a letter differed at Tukey-adjusted P
< 0.05. Statistical inference was made using repeated measures models which included
group × timepoint and experiment × timepoint effects, as well as a genotype effect.


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made












































|Prechill|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|a|a|bc<br>a|bc<br>a|bc<br>a|b<br>a|b<br>a|b<br>a|bc~~b~~|bc~~b~~|bc~~b~~|~~c b~~|~~c b~~|~~c b~~|~~ca~~|~~ca~~|~~ca~~|~~bc~~|~~bc~~|~~bc~~|a|a|a|bc<br>a|bc<br>a|bc<br>a|a|a|a|b<br>~~b~~|b<br>~~b~~|b<br>~~b~~|~~c~~|~~c~~|
||||||||||||||||||c|c|c|||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||


|Chilling|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|
|---|---|---|---|---|---|---|---|---|---|---|---|
||||||N.|S.||||||
|||||||||||||
|||||||||||||
|||||||||||||
|||||||||||||


|Recovery|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
||||||a|a|a|||||||||||||||||||||||||||
|||a|a|a|b|b|b||||a|a|a|bc|bc|bc|a|a|a|bc|bc|bc|a|a|a|ba|ba|ba|bc<br>~~b~~|bc<br>~~b~~|bc<br>~~b~~|~~c~~|~~c~~|
|||a|a|a|b|b||||||||||||||||||||||||||||
|||a|a|||||||||||||||||||||||||||||||
|~~b~~|~~b~~|~~c~~|~~c~~||||||b|b|c|c|c|c|c|c||||b|b|b|c|c||||||||||
|~~b~~|~~b~~|~~c~~|~~c~~||||||b|b|c|c||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||














|Prechill|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||N.|N.|N.|S.|S.|S.|||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||


|Chilling|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||N.|N.|S.|S.||||||||||
||||||||||||||||||||||||
||||||||||||||||||||||||
||||||||||||||||||||||||
||||||||||||||||||||||||
||||||||||||||||||||||||


|Recovery|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
||||||||||||||||||||||||||||||
|||a|a|a|b a|b a|b a|||~~a~~|~~a~~|~~b~~|~~b~~|~~b~~|~~a~~|~~a~~|~~b~~|~~b~~|~~a~~|~~a~~|~~ba~~|~~ba~~|~~ba~~|~~bc~~a|~~bc~~a|~~bc~~a|b|b|
|a|a|bc|bc|bc|||||b|c|c||||||b|b|c<br>|c<br>|||||||||
||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||



**Figure 2.** Genotypic variation in photosynthetic traits at the end of each experimental stage. Genotypespecific EMMs for (A) net CO₂ assimilation and (B) ΦPSII at Prechill D5, Chilling D5, and Recovery D5 for the
11 maize inbred lines. Error bars represent 95% confidence intervals around the EMMs; ΦPSII estimates
are displayed on the response scale following analysis on the logit scale. The longitudinal datasets
contained 703 valid plant-time observations for assimilation and 529 for ΦPSII. Within each stage,
genotypes not sharing a letter differed at Tukey-adjusted _P_ < 0.05. EMMs and contrasts were obtained from
longitudinal mixed models containing genotype × timepoint and experiment × timepoint effects, a plant
random intercept, and timepoint-specific residual variances.


[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


**Prechill** **Chilling** **Recovery**









20


10


0

|Prechill|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||R²<br>P <|= 0.57<br> 0.001|
||||||
||||||
||||||


|Chilling|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||R²<br>P <|= 0.47<br> 0.001|
||||||
||||||
||||||


|Recovery|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
||||R²<br>P <|R²<br>P <|= 0.79<br> 0.001|
||||R²<br>P <|R²<br>P <|= 0.79<br> 0.001|
|||||||
|||||||
|||||||



0.0 0.25 0.50 0.75 0.0 0.25 0.50 0.75 0.0 0.25 0.50 0.75
#### PSII quantum yield (ΦPSII)


**Figure 3.** Relationship between net CO₂ assimilation and ΦPSII at the end of prechilling, chilling, and
recovery. Net CO₂ assimilation is plotted against ΦPSII for individual maize plants at Prechill D5, Chilling
D5, and Recovery D5, with points colored by genotype. Black lines show the linear relationship within each
stage, with shaded 95% confidence intervals. Panel annotations report the R2 for ΦPSII after adjustment
for Experiment and the corresponding P-value. Analyses included _N_ = 59, 88 and 99 observations for
Prechill, Chilling, and Recovery, respectively (N = 246 total).


20


10


0


20


10


0


20


10


0



[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


**3IIH6** **B73** **B97** **NC358**

|3IIH6|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|B73|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|B97|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|NC358|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||



**CML247** **CML322** **F2** **LH244**


|CML247|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|CML322|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|F2|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|LH244|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||



**PHK76** **PHN46** **Il14H**

|PHK76|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|PHN46|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||


|Il14H|Col2|Col3|Col4|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||



0.0 0.25 0.50 0.75 0.0 0.25 0.50 0.75 0.0 0.25 0.50 0.75

#### ΦPSII


Prechill Chilling Recovery



0.0 0.25 0.50 0.75



**Supplementary Figure S5.** Effect of genotype on the relationship between net CO ₂ assimilation
and ΦPSII across experimental stages **.** Net CO ₂ assimilation is plotted against ΦPSII for each of
the 11 maize genotypes at Prechill D5, Chilling D5, and Recovery D5. Points represent paired
individual biological observations, and fitted lines show genotype and stage specific leastsquares. _N_ = 114 Prechill, 111 Chilling, and 128 Recovery observations ( _N_ = 353 total). Fitted
lines are descriptive genotype × stage regressions.


# **A**

#### **B**



[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


0.8



|Col1|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
|a|b||||a|
|||||||
||||c|||
|||||||


Chilling

D5



Recovery

D5



0.7


0.6


0.5



Prechill

D5



Chilling

D1



Recovery

D1



**Prechill D5** **Chilling D5** **Recovery D5**


N.S. N.S.







0.75


0.50


0.25


0.00


|PrechillD5|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|a|a|a|a|a|a|a|a|b<br>a|b<br>a||||a<br>|a<br>|a<br>|||||||a|a|a|b a|b a|b a|b a|b a|b a|b|b|
||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||
||||||||||||||||||||||||||||||||||


|ChillingD5|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||N.|N.|N.|S.|S.|S.|||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||


|Recovery D5|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||N.|N.|N.|S.|S.|S.|||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||



**Supplementary Figure S6** . Dark-adapted Fv/Fm across the chilling treatment and among
maize genotypes. (A) Model-adjusted dark-adapted Fv/Fm across the course of the
experiment; the shaded interval denotes the chilling treatment. Points are estimated
marginal means with associated SE shown with error bars. (B) Dark-adapted Fv/Fm for each
of the 11 maize genotypes at Prechill D5, Chilling D5, and Recovery D5. Bars/points represent
genotype EMMs with associated 95% confidence intervals shown by error bars. N= 67. In (A),
different letters indicate significant differences among timepoints; in (B), letters compare
genotypes within each stage, with groups not sharing a letter differing at Tukey-adjusted _P_ <
0.05; N.S. indicates _P_ ≥ 0.05.


### **A**



2.2 ~~5~~


2.0 ~~0~~


1.7 ~~5~~


1.5 ~~0~~



[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)
### (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made B


20


10


0

|g<br>NC N<br>-|D40 Interna tio nal<br>.|p y<br>lice ns e<br>.|p p p p|y<br>.|Col6|
|---|---|---|---|---|---|
|||||||
|||||||
|||||||



0 2 4 6 8

#### Nonphotochemical quenching (NPQ)


|Col1|y<br>N|p<br>S a vai lable u<br>. .|nd|
|---|---|---|---|
|||||
|||||
|||||
|||||


### **C**



Prechill Chilling Recovery



50


40


30


0 1 2 3
#### Nonphotochemical quenching (NPQ)


**Supplementary Figure S7.** Nonphotochemical quenching across experimental stages and its relationships
with carbon assimilation and mature-leaf chlorophyll status. (A) Nonphotochemical quenching (NPQ) at Prechill
D5, Chilling D5, and Recovery D5. Points and error bars represent estimated marginal means and 95%
confidence intervals from stage-specific models; the shaded interval denotes the chilling stage. (B)
Relationship between net CO₂ assimilation and NPQ at the D5 endpoint of each experimental stage. (C)
Relationship between mature-leaf SPAD and NPQ at the same stage endpoints. In (B) and (C), points represent
individual plants with matched measurements, lines show stage-specific linear relationships. N = 51, 36, and
26 NPQ observations at Prechill D5, Chilling D5, and Recovery D5, respectively.


[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made


























|Prechill|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||
|||||||||||||||||a|a|||||
|||a|b<br>a|b<br>b|a|a|b<br>a|b<br>a|b<br>|b<br>|b<br>~~a~~|b<br>~~a~~|~~b~~|~~b~~|~~b~~|a|a|b<br>~~a~~|b<br>~~a~~|~~b~~|~~b~~|
|||||||||||||||||||||||
|||||||||||||||||||||||
|||||||||||||||||||||||


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||~~N.~~|~~N.~~|~~S.~~||
|||||||||||||||
|||||||||||||||
|||||||||||||||
|||||||||||||||
|||||||||||||||


### **B**





|Col1|Timepointeffect:P<<br>Genotype×Tim epoint: P <<br>N=1 5 8plants|0001<br>0.001<br>.|d|
|---|---|---|---|
|||||
||||c<br>~~ac~~|
||b<br><br>ab<br>|b<br>abc<br>|ab<br><br>b|
|||||


Recovery

D5


### **C**



3 4 5 6
#### V-stage at Recovery D5



5


4


3





90 ~~0~~


60 ~~0~~


30 ~~0~~


0









Prechill
D5



Chilling

D5



**Figure 4.** Vegetative development and biomass allocation during chilling and recovery. A) Immature-leaf
growth rate for the 11 maize genotypes across all experiment stages, calculated from repeated
measurements of the marked immature leaf within each stage. (B) D5 vegetative stage across Prechill,
Chilling, and Recovery. (C) Mature-leaf biomass fraction is plotted against Recovery-D5 V-stage using
matched endpoint biomass observations; the line shows the experiment-adjusted linear trend, shaded
region shows 95% CI. N = 54, 55 and 51 for prechill, chilling and recovery stages in (A); 158, and 191
for B and C, respectively. Within a stage or biomass trait, genotypes not sharing a letter differed at
Tukey-adjusted P < 0.05.


### **A** **C** **A**


### bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. B The copyright holder for this preprint

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 I](http://creativecommons.org/licenses/by-nc-nd/4.0/) ~~nternational license.~~


### **B**



40 ~~[0](http://creativecommons.org/licenses/by-nc-nd/4.0/)~~










|(|Col2|w c|Col4|Col5|wasn<br>a|Col7|Col8|otcert<br>b|Col10|Col11|e y|Col13|Col14|peer<br>a|Col16|Col17|revew<br>b|Col19|Col20|) st<br>ava|Col22|Col23|eaut<br>i lable|Col25|Col26|or un<br>under<br>a|Col28|Col29|er w<br>a C, C -<br>a|Col31|Col32|o a<br>B Y<br>-<br>b|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|(|(|wc|wc|wc|a<br> was n|a<br> was n|a<br> was n|b<br>  t cert|b<br>  t cert|b<br>  t cert|e|e|e|~~a~~<br>     peer|~~a~~<br>     peer|~~a~~<br>     peer|~~b~~<br>      revew|~~b~~<br>      revew|~~b~~<br>      revew|ava<br>      ) s t|ava<br>      ) s t|ava<br>      ) s t|ilable<br>         aut|ilable<br>         aut||||||||||
|b|b||||b|b|b||||b<br>|b<br>|b<br>|b<br>|b<br>|b<br>||||b<br>|b<br>|b<br>|b|b|||||b<br>|b<br>|b<br>|||
|b|b||||b|b||||||||||||||||||||||||||||
|b||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||


|x<br>ern|va ce<br>a tio na|Col3|Col4|nseto<br>llice ns|Col6|Col7|sp<br>e<br>.|Col9|Col10|ayt e|Col12|Col13|prepr|Col15|Col16|nt np|Col18|Col19|erpetu|Col21|Col22|ty t s<br>.|Col24|ma e|Col26|Col27|Col28|Col29|Col30|Col31|Col32|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
||||||||||||||~~N.~~|~~N.~~|~~N.~~|~~S.~~|~~S.~~|~~S.~~||||||||||||||
|||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||


### **D** **B**



20 ~~0~~


10 ~~0~~


0


40 ~~0~~


30 ~~0~~


20 ~~0~~

















30 ~~0~~


20 ~~0~~


100


0


60 ~~0~~


40 ~~0~~


20 ~~0~~


0


2


1


0















10 ~~0~~


0

|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||~~a~~|~~a~~|~~a~~|~~b~~<br>|~~b~~<br>|~~b~~<br>|a<br>a|a<br>a|a<br>a|b<br>a|b<br>a|b<br>a|b<br>|b<br>|b<br>|a|a|a|~~a~~|~~a~~|~~a~~|~~b~~<br>|~~b~~<br>|~~b~~<br>|a<br>a|a<br>a|a<br>a|b<br>a|b<br>a|b<br>a|b|b|
|b|b|||||||||||||||||||b|b|b||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||


### **E F**


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|a|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||b|b|b|ab|ab|ab|cdeab|cdeab|cdeab|cde<br>b|cde<br>b|cde<br>b|cd<br>b|cd<br>b|cd<br>b|cd<br>b|cd<br>b|cd<br>b|c<br>|c<br>|d|d|d|e<br>b|e<br>b|e<br>b|c|c|
|||||b|b|b|ab|ab|ab|cdeab|cdeab|cdeab|cde<br>b|cde<br>b|cde<br>b|cd<br>b|cd<br>b|cd<br>b|cd<br>b|cd<br>b|cd<br>b|c<br>||||||||||
|c|e<br>|e<br>|e<br>|||||||||||||||||||||||||||||
|c||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||



1.2


0.9


0.6


0.3


0.0






|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|Col25|Col26|Col27|Col28|Col29|Col30|Col31|Col32|Col33|Col34|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||P = 0|P = 0|P = 0|.07|.07|.07|||||||N.|N.|N.|S.|S.|S.|||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||
|||||||||||||||||||||||||||||||||||


|Col1|Col2|Col3|a|Col5|bc|Col7|Col8|Col9|Col10|Col11|a|Col13|Col14|Col15|Col16|Col17|Col18|a|Col20|b|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|b|c<br>a|c<br>a|bc<br>|bc<br>|a|a|bc<br>b|bc<br>b|c<br>b|c<br>b|c|c|b|b|c<br>b|c<br>b|c<br>|c|c||
|b|c<br>a|c<br>a|bc<br>|bc<br>|a|a|bc<br>b|bc<br>b|c<br>b|c<br>b|c||||||||||
||||||||||||||||||||||
||||||||||||||||||||||



**Supplementary Figure S8.** Dry weights of (A) immature leaves, (B) mature leaves, (C) roots, and (D) shoots
(mature leaves, immature leaves, and stem tissue), E) Immature:mature leaf biomass ratio. F) Shoot:root
biomass ratio measured at harvest. Values are genotype EMMs with 95% CI displayed as error bars; biomass
ratios were analyzed on the log scale and are displayed as ordinary biomass ratios. N= 195


### **A**

40


30


20



|Col1|a|a|a|a|N|S|
|---|---|---|---|---|---|---|
|a|ab<br><br>b<br>|~~b~~<br><br>~~b~~<br>~~cde~~<br>abc~~d~~<br>bcd~~e~~<br>ab<br>abc|ab<br>bc|abc<br>ab|||
|cd<br>ab<br>bcd<br>abc<br>abcd||de<br>e|c<br>c|bc<br>c|||
|d|||||||


Prechill

D1



[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)





Chilling

D5



Recovery

D5







Prechill

D5





Recovery

D1



Chilling

D1



B73 CML247 Il14H PHN46 3IIH6 NC358 F2 PHK76 B97 CML322 LH244

### **B**


5 ~~0~~


4 ~~0~~


3 ~~0~~


2 ~~0~~


1 ~~0~~

|Col1|Col2|Prechill|Chilling Recovery|Col5|
|---|---|---|---|---|
||||||
||||||
||||||
||||||
||||||



0.0 0.1 0.2 0.3
#### Stomatal Conductance (mol H 2 O m ⁻ ² s ⁻ ¹)


**Supplementary Figure S9.** A) Genotypic variation in mature-leaf chlorophyll content across prechilling,
chilling, and recovery. Mature-leaf relative chlorophyll content (SPAD) is shown for each maize genotype at
Prechill D1 and D5, Chilling D1 and D5, and Recovery D1 and D5. The shaded interval denotes the chilling
treatment. Points are genotype EMMs and error bars represent SE. N = 66 plants. Within each timepoint,
genotypes not sharing a letter differed at Tukey-adjusted P < 0.05; N.S. indicates that the genotype effect
was not significant P _<_ 0.05. B) Mature-leaf SPAD is plotted against stomatal conductance (g sw ) at the D5
endpoint of Prechill, Chilling, and Recovery. Points represent individual plants with matched SPAD and gasexchange measurements. Lines show the stage-specific linear relationships; shaded bands indicate 95%
confidence intervals. N = 153, 117, and 148 matched observations for Prechill, Chilling, and Recovery,
respectively. Stage-specific linear models included g sw and replicate experiment as fixed effects; g sw was
significantly associated with SPAD within each stage (P<0.05).


## **A** **B**



























Air Temp 4-inch Soil Temp PPFD Humidity Precipitation





3IIH6


Nc358

Cml247

Cml322







Il14H

LH244


Phk76

Phn46

Dekalb Hybrid



**Figure 5.** Environmental conditions and visible cold injury during four early-spring field experiments. A) Air temperature, 4-inch soil

|Col1|Col2|Col3|Col4|B|Col6|
|---|---|---|---|---|---|
|||||||
|||||||
|||||||
|||||||
|||||||
|||||** B**|** B**|
|||||||
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|||||D5|D5|

temperature, PPFD, relative humidity, and precipitation during the 2025 and 2026 experiments at Locations A and B. B) Representative
plant from the 2025 Rollins ; the red box identifies damaged leaf tissue. C) Resistance to cold injury, expressed as the fraction of plants
within each genotype that had not developed visible cold injury on each day following transplanting. Lines show block-averaged genotype
values for the four field environments. The experimental design comprised three replicate blocks per environment, with five plants per
genotype per block.


bioRxiv preprint **2025 Location A** [doi: https://doi.org/10.64898/2026.10.30.763178](https://doi.org/10.64898/2026.10.30.763178) **2025 Location B** ; this version posted November 5, 2026. **2026 Location A** The copyright holder for this preprint **2026 Location B**

(which 20 ~~was not certified by peer review) is~~ t ~~he author/funder, who has grante~~ d ~~bioRxiv a license to display the p~~ r ~~eprint in perpetuity. It is made~~
available under a N.S. [CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) b N.S.



20



**2025 Location A** [doi: https://doi.org/10.64898/2026.10.30.763178](https://doi.org/10.64898/2026.10.30.763178) **2025 Location B** ; this version posted November 5, 2026. **2026 Location A** The copyright holder for this preprint **2026 Location B**

~~was not certified by peer review) is~~ t ~~he author/funder, who has grante~~ d ~~bioRxiv a license to display the p~~ r ~~eprint in perpetuity. It is made~~
available under a N.S. [CC-BY-NC-ND 4.0 International license](http://creativecommons.org/licenses/by-nc-nd/4.0/) b N.S.





15


10


5


0


0.5


0.4


0.3


0.2


0.1


0.0


12


10


8


6


4


2


0


100%


80%


60%






















|a|s|no|tc|erti|fie|db|yp|eer|re|vie|w)|is|
|---|---|---|---|---|---|---|---|---|---|---|---|---|
||||||||||||||
||||||||||||||
||~~a~~|~~b~~<br>~~a~~|~~a~~|~~a~~|~~a~~|~~b~~|~~a~~|~~a~~|~~ba~~|~~b~~|~~b~~||


|heaut<br>ai labl|hor/fund<br>eunder|er<br>N<br>a .|C,S w<br>C<br>.|hohas<br>B YNC<br>- -|grante<br>ND4<br>-|
|---|---|---|---|---|---|
|||||||
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|b<br>I|io<br>nt|Col3|Rxi<br>ern|Col5|va<br>a ti|Col7|lic<br>on|Col9|en<br>all|se<br>ice|Col12|to<br>ns|Col14|dis<br>e<br>.|Col16|pl<br>a|Col18|ay<br>b|Col20|the|Col22|b|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|a|b<br>a|b<br>a|b<br>a|b<br>a|b|b||||a|a|b<br>a|b<br>a|b|b||||||||
|a|b<br>a|b<br>a|b<br>a|b<br>a|b|b||||a|a|b<br>a|b<br>a|b|b||||||||
|a|b<br>a||||||||||||||||||||||
||||||a|a|b|b|a|b|b|||a|a|b|||~~a~~|~~b~~|||
||||||||||||||||||||||||
||||||||||||||||||||||||


|e|prin|Col3|ti|Col5|np|Col7|er|Col9|pet<br>N<br>.|Col11|uSit|Col13|y I<br>.<br>.|Col15|tis|Col17|m|Col19|ad|Col21|e|Col23|Col24|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||||
|||||||||||||||||||||||||
|||||||||||||||||||||||||




















|Col1|Col2|Col3|Col4|Col5|Col6|Col7|N<br>.|Col9|S<br>.|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||
|||||||||||||||||||||
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|Col1|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
|||||||
|||||||
|a<br>|~~b~~ab|~~a~~|~~b~~a|bab a|ab|
|b|~~a~~|||||


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|N<br>.|Col11|S<br>.|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||||
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|||||||||||||||||||||||||
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|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||||
||a|a|b<br>|b<br>|a|a|b<br>a|b<br>a|b<br>~~a~~|b<br>~~a~~|~~b~~<br>|~~b~~<br>|a<br>~~ba~~|a<br>~~ba~~|~~b~~<br>|~~b~~<br>|a~~a~~|a~~a~~|~~b~~|~~b~~|a~~a~~|a~~a~~|~~b~~|
||a|a|b<br>|||||||||||||||||||||
|||||||||||||||||||||||||
|||||||||||||||||||||||||






















|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
||||||~~N.~~|~~N.~~|~~S.~~|~~S.~~||||||||
|||||||||||||||||
|||||||||||||||||
|||||||||||||||||


|Col1|Col2|Col3|Col4|Col5|Col6|
|---|---|---|---|---|---|
|ab<br>~~ab~~|~~a~~|||~~b~~<br>||
|||||~~ab~~||
||~~b~~<br>~~ab~~|bab|bab|~~b~~<br>|abb|
|||||||


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
||||||||~~N.~~|~~S.~~|~~S.~~||||||||
||||||||||||||||||
||||||||||||||||||
||||||||||||||||||


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||||
|a|b|b|||a|a|||a<br>|a<br>|~~a~~<br>b<br>|~~a~~<br>b<br>|b|b|~~a~~<br>b<br>|~~a~~<br>b<br>|||a<br>~~b~~|a<br>~~b~~|b<br>|b<br>||
|a|b|b|||a|a|||a<br>|a<br>|~~a~~<br>b<br>|~~a~~<br>b<br>|b|||||||||||
|||c|d<br>~~a~~<br>c<br>~~d~~|d<br>~~a~~<br>c<br>~~d~~|c<br>d|c<br>d|c|c|d<br><br>c<br>d|d<br><br>c<br>d||||||d|d|d|c|c||||
|||||||||||||||||||||||||

























40%


20%


0%

|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|N<br>.|Col11|S<br>.|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||
|||||||||||||||||||||||
|||||||||||||||||||||||
|||||||||||||||||||||||


|a|a|a a|Col4|a a<br>a|Col6|
|---|---|---|---|---|---|
|~~b~~<br>c a<br>b<br>~~d ~~|~~d~~<br>ac<br>~~b~~cd|bc~~d~~<br>bcd<br>|bc~~d~~<br>bcd<br>|~~b~~cd<br>~~b~~<br>c<br>bcd|~~b~~<br>d<br>~~c~~|
|||||||
|||||||
|||||||


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|N<br>.|Col11|S<br>.|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|||||||||||||||||||||||||
|||||||||||||||||||||||||
|||||||||||||||||||||||||
|||||||||||||||||||||||||


|Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19|Col20|Col21|Col22|Col23|Col24|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
||~~ab~~|~~ab~~|~~c~~|~~c~~|~~ab~~|~~ab~~|~~c~~|~~c~~|a<br>~~b~~|a<br>~~b~~|c|c|a|a|b|b|||~~b~~|~~b~~|~~c~~<br>a<br>b<br>~~c~~|~~c~~<br>a<br>b<br>~~c~~||
|a|b|b|a|a|b|b|~~a~~|~~a~~|c|c|||||a|a|ba|ba|b|||||
|a|b|b|a|a|b|b|~~a~~|||||||||||||||||
|a|b|b|a|a|b|b|~~a~~|||||||||||||||||
|||||||||||||||||||||||||
|||||||||||||||||||||||||



**Figure 6.** Morphometric and physiological variation among maize genotypes in four early-spring field environments. Columns
show the 2025 and 2026 experiments at Locations A and B. Rows show net CO₂ assimilation at five days after transplanting,
light-adapted Fv′/Fm′ measured on the same sampling date, immature-leaf growth over the five-day field exposure, and
mature-leaf relative chlorophyll content (SPAD) at day 5 expressed relative to the pretransplant value. Values are means of
block-level experimental units and error bars represent SE across replicate blocks. Experiments contained three replicate
blocks, with five plants per genotype per block targeted; plant subsamples within a block were summarized to a single blocklevel value before statistical analysis. Different letters indicate Tukey-adjusted genotype comparisons at P < 0.05 within an
environment; N.S. indicates a nonsignificant genotype effect. Individual-environment analyses used genotype as a fixed effect

and block as a random effect.


[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)



20





15


10


5


0

|Col1|Col2|Col3|R² 0<br>=|48<br>.|
|---|---|---|---|---|
||||||
||||||
||||||
||||||
||||||



0.0 0.1 0.2 0.3

#### PSII quantum yield (ΦPSII) 2025 Location A 2025 Location B 2026 Location A 2026 Location B


**Figure 7.** Relationship between net CO ₂ assimilation and ΦPSII across four field
environments. Net CO ₂ assimilation is plotted against ΦPSII for block-level observations
collected from the 2025 and 2026 field experiments at Locations A and B. Points are colored
by field environment. The black line and shaded region show the pooled ordinary leastsquares fit and its 95% confidence interval across all environments. The pooled relationship
had an R2 = 0.48; N =112. The plotted regression is a descriptive ordinary least-squares fit
to the block-level observations.


[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)









**Supplementary Figure S10.** Relationship between net CO₂ assimilation and immature-leaf
growth during field exposure. Immature-leaf growth over the five-day field interval is plotted
against net CO₂ assimilation measured at five days after transplanting. Points represent blocklevel observations from the four field environments and are colored by genotype. The black line
and shaded region show the pooled ordinary least-squares fit and 95% confidence interval. N =
135. The plotted line is an ordinary least-squares regression across the available block-level

field observations.


[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)









**Supplementary Figure S11** . Relationship between stomatal conductance and mature-leaf
chlorophyll content during field exposure **.** Mature-leaf SPAD measured at the end of the field
exposure is plotted against stomatal conductance measured at the same endpoint. Points
represent block-level observations from the four field environments and are colored by
genotype. The black line and shaded region show the pooled ordinary least-squares fit and
95% confidence interval. N = 112. The displayed relationship is an ordinary least-squares
regression across the available block-level field observations.


[bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763178; this version posted November 5, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.10.30.763178)

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


**Phenotype BLUP** **GBLUP**



_Mean Δ_
_Cullis H²_
_H²_ _H²_



_H²_



_Entry-_
_Trait_
_mean H²_



_Cullis_


_H²_



_Mean Δ_



Mature leaf

SPAD
(prechill)


Mature leaf
SPAD (after
chill)


Immature
leaf growth


Assimilation

rate



0.73 0.73 0.73 0.83 0.78


0.86 0.86 0.86 0.92 0.89


0.32 0.32 0.32 0.51 0.44


0.45 0.45 0.45 0.45 0.38



ΦPSII 0.55 0.51 0.51 0.40 0.33



F’ V /F’ M
(light
adapted)



0.22 0.20 0.20 0.00 0.00



Phenotype BLUP is the primary broad-sense heritability analysis;
GBLUP is complementary. Δ H² is the difference-based reliability
estimate.


**Table 1.** Broad-sense heritability of maize morphological and photosynthetic traits across field
environments. Heritability estimates were obtained from multi-environment mixed models fitted to
genotype × environment × block means from 11 maize inbred lines evaluated across four field
environments. Phenotype BLUP estimates treat genotype effects as independent and identically distributed
and represent the primary phenotypic heritability analysis, whereas GBLUP estimates incorporate realized
genomic relationships among the inbred panel. For the phenotype BLUP model, entry-mean H2 was
calculated from estimated genotype, genotype × environment, and residual variance components for the
four-environment, three-block design. Cullis H2 is a prediction-error-variance-based measure of the
reliability of pairwise genotype differences, and mean ΔH2 is the mean of the corresponding pair-specific
reliability estimates. For GBLUP, Cullis H2 and mean ΔH2 were calculated using the genomic variance
expected for each pairwise genotype difference to account for unequal genomic relatedness among lines.