Drought-related root morphological traits and non-structural carbohydrates in the seedlings of the alien Quercus rubra and the native Quercus robur: possible implication for invasiveness OPEN ACCESS EDITED BY

Abstract

Quercus rubra L. is a moderately shade-tolerant tree species native to eastern North America, readily regenerating since its introduction in the Central and Southern European forests to displace the native pedunculate oak (Quercus robur). Under a scenario of increasing drought, understanding the seedling responses of these two species to water limitation is critical for forest conservation and management. To this aim, morphological, physiological and non-structural carbohydrates analyzes were performed on very-fine and fine roots of Q. robur and Q. rubra seedlings grown under controlled conditions and exposed to two levels of drought before allowing them to recover. Results show significant differences between species for all the investigated traits. The alien Q. rubra showed lower shoot and root biomasses than the native Q. robur, particularly for the thicker fine root 1-2 mm diameter class. However, both species equally invested more biomass in the shoot than the root system (R:S ratio <1). A significant response to drought was observed for the 0.5-1 mm fine root class, with moderate and severe droughted seedlings showing slightly higher biomass than control, particularly in Q. rubra. The overall growth reduction of Q. rubra suggests that the costs associated with the construction and maintenance outweigh the inputs from aboveground, as supported by the lower values of photochemical efficiency (F v /F m), quantum yield (ΦPSII) of PSII and stomatal conductance. In particular, the reduced stomatal conductance assured high midday leaf water potential (i.e., tissue hydration levels) at the expense of growth. The low starch concentration in Q. rubra's very-fine roots correlated positively with the low photochemical efficiency under drought conditions, probably due to the reduction of photosynthate inputs from aboveground. In contrast to the anisohydric behavior reported, these outcomes highlight a rather isohydric behavior for Q. rubra, at least at the seedling stage and in the adopted experimental conditions, making this species highly competitive under the drier condition in the canopy openings during the summer period. Di Iorio A, Caspani AC, Beatrice P and Montagnoli A (2024) Drought-related root morphological traits and non-structural carbohydrates in the seedlings of the alien Quercus rubra and the native Quercus robur: possible implication for invasiveness.

Full text

Frontiers in Forests and Global Change 01 frontiersin.org
Drought-related root
morphological traits and
non-structural carbohydrates in
the seedlings of the alien Quercus
rubra and the native Quercus
robur: possible implication for
invasiveness
AntoninoDi Iorio
1*, AnnaClaudiaCaspani
1,2, PeterBeatrice
1
and AntonioMontagnoli
1
1 Department of Biotechnology and Life Science, University of Insubria, Varese, Italy, 2 Department of
Economics, University of Insubria, Varese, Italy
Quercus rubra L. is a moderately shade-tolerant tree species native to eastern
North America, readily regenerating since its introduction in the Central and
Southern European forests to displace the native pedunculate oak (Quercus
robur). Under a scenario of increasing drought, understanding the seedling
responses of these two species to water limitation is critical for forest conservation
and management. To this aim, morphological, physiological and non-structural
carbohydrates analyzes were performed on very-fine and fine roots of Q. robur
and Q. rubra seedlings grown under controlled conditions and exposed to two
levels of drought before allowing them to recover. Results show significant
dierences between species for all the investigated traits. The alien Q. rubra
showed lower shoot and root biomasses than the native Q. robur, particularly
for the thicker fine root 1–2 mm diameter class. However, both species equally
invested more biomass in the shoot than the root system (R:S ratio <1). A
significant response to drought was observed for the 0.5–1 mm fine root class,
with moderate and severe droughted seedlings showing slightly higher biomass
than control, particularly in Q. rubra. The overall growth reduction of Q. rubra
suggests that the costs associated with the construction and maintenance
outweigh the inputs from aboveground, as supported by the lower values of
photochemical eciency (Fv/Fm), quantum yield (ΦPSII) of PSII and stomatal
conductance. In particular, the reduced stomatal conductance assured high
midday leaf water potential (i.e., tissue hydration levels) at the expense of
growth. The low starch concentration in Q. rubra’s very-fine roots correlated
positively with the low photochemical eciency under drought conditions,
probably due to the reduction of photosynthate inputs from aboveground.
In contrast to the anisohydric behavior reported, these outcomes highlight a
rather isohydric behavior for Q. rubra, at least at the seedling stage and in the
adopted experimental conditions, making this species highly competitive under
the drier condition in the canopy openings during the summer period.
KEYWORDS
fine roots, drought, alien species, non-structural carbohydrates, isohydric, anisohydric
OPEN ACCESS
EDITED BY
Ivika Ostonen,
University of Tartu, Estonia
REVIEWED BY
Jesús Rodríguez-Calcerrada,
Polytechnic University of Madrid, Spain
Joanna Mucha,
Polish Academy of Sciences, Poland
*CORRESPONDENCE
Antonino Di Iorio
antonino.diiorio@uninsubria.it
RECEIVED 04 October 2023
ACCEPTED 16 January 2024
PUBLISHED 31 January 2024
CITATION
Di Iorio A, Caspani AC, Beatrice P and
Montagnoli A (2024) Drought-related root
morphological traits and non-structural
carbohydrates in the seedlings of the alien
Quercus rubra and the native Quercus robur:
possible implication for invasiveness.
Front. For. Glob. Change 7:1307340.
doi: 10.3389/gc.2024.1307340
COPYRIGHT
© 2024 Di Iorio, Caspani, Beatrice and
Montagnoli. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 31 January 2024
DOI 10.3389/gc.2024.1307340

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 02 frontiersin.org
1 Introduction
Quercus rubra L. (red oak) is an economically important tree
species native to eastern North America, well-known for its moderate
shade-tolerance behavior (Sander, 1990). In addition, red oak is the
dominant canopy species in many northern hardwood forests, being
relevant in terms of hydrologic budgets and carbon sequestration
(Cavender-Bares and Bazzaz, 2000).
Although this species is facing diculties in regenerating in
several areas within its native range (Crow, 1992; Abrams, 1996), red
oak has been successfully regenerating since its introduction in
Central European forests estimated at the turn of the 17th and 18th
centuries (Vansteenkiste et al., 2005; Major et al., 2013), and in
Italian forests since the early-19th century (Maniero, 2015).
Concerns are growing over whether its rapid growth in European
forests means this non-native species is becoming an aggressive
invader (Chmura and Sierka, 2005; Vor, 2005; Kuehne etal., 2014),
as dierent studies reported adverse eects on native biodiversity
(Reinhardt et al., 2003; Chmura, 2020; Dimitrova et al., 2022).
Related, an average annual growth of 12m
3
ha
−1
year
−1
was estimated,
outperforming all native oak species, including the indigenous
Quercus robur (English oak, pedunculate oak) (Kiedrzyński etal.,
2011) that reaches only about half of this productivity (Nicolescu
etal., 2020; Kormann etal., 2023).
Several authors highlighted potential recruitment dierences of
red oak when analyzing the native and introduced ranges in favor of
the latter (Steiner etal., 1993; Major etal., 2013). In the years following
the establishment of red oak seedlings, soil moisture content appears
to play a crucial role in their survival and growth (Jacobs etal., 2009),
but several studies and regional reports suggest that Q. rubra has
better resistance to drought than the native Q. robur and Quercus
petraea (sessile oak) (Nicolescu etal., 2020 and references therein). In
the native range, red oak drought adaptation is associated with the
rapid production of large seedlings from large seeds, allowing
seedlings to avoid drought by quickly accessing water in deeper soil
layers (Walters etal., 2023). Similar behavior has also been reported
for Q. robur (Zadworny etal., 2021), whose valid seedling response to
water limitation appears to similarly depend on the root-to-shoot
allometry, the taproot development, and carbon reserve mobilization
for plant functioning under water shortage. In contrast, other evidence
highlights, particularly for red oak seedlings, that large root systems
do not show enhanced drought avoidance during their rst season
aer eld transplant (Jacobs etal., 2009; Walters etal., 2023).
At a ner scale, drought is known to stimulate ne roots (diameter
<2 mm), especially very-ne root (diameter <0.5 mm) growth
(Montagnoli etal., 2012, 2019) and starch accumulation (Domingo
etal., 2023) up to a threshold beyond which growth stops, starch
consumption and even mortality increase (for a review see Brunner
etal., 2015; Di Iorio etal., 2016; Domingo etal., 2023). Sustained dry
soil highlights this pattern also for the ne roots of both Q. robur
(Gieger and omas, 2002; Zadworny et al., 2021) and Q. rubra
(Jacobs etal., 2009; Zadworny etal., 2018). Stimulation of very ne
root growth is oen accompanied by increased specic root length
(SRL, m g
−1
), i.e., the ratio of the length to root mass, which is
proportional to resource acquisition (benet) and construction and
maintenance (cost), respectively (Eissenstat and Yanai, 1997;
Montagnoli etal., 2019). A high SRL value indicates an optimal
benet/cost ratio under drought conditions (Ostonen etal., 2007).
Water shortage and carbon starvation are the leading causes of ne-
roots and other tree compartments’ mortality (Hartmann etal., 2013).
Indeed, even very-ne roots are crucial concerning starch dynamics
since previous studies highlighted that starch and soluble sugars (SS)
were signicantly replenished at the end of the growing season (Nguyen
etal., 1990; Kosola etal., 2002). However, unlike the well-documented
physiological and morphological plasticity, existing studies for these
two species are few and/or of limited scope in terms of temporal
variation of the starch concentration and growth in response to drought.
Drought resistance and mortality of tree species growing under
water-shortage conditions have been widely assessed through the iso/
anisohydric classication (Martínez-Vilalta etal., 2014). In particular,
isohydric species are more prone to carbon starvation, and anisohydric
ones are more vulnerable to hydraulic failure (Garcia-Forner etal.,
2016). Red oak (Yi etal., 2017), pedunculate oak (Gieger and omas,
2002) and sessile oak (Aranda etal., 2000; Klein, 2014; Martínez-
Sancho etal., 2017) are usually considered anisohydric species, i.e.,
able to maintain high transpiration and stomatal conductance and to
track environmental uctuations in water potentials under moderate
drought conditions (Martínez-Vilalta et al., 2014). In their native
range, seedlings of red oak are considered less drought-resistant than
white (Quercus alba) or black oaks (Quercus velutina) (Sander, 1990).
In contrast, in the introduced ranges, red oak seedlings appear more
drought resistant than the native pedunculate and sessile oaks
(Nicolescu etal., 2020, and references therein). erefore, red oak
appears to eciently regenerate under the drier conditions of the
exposed soil surface generated by the regular thinning of managed
European forests. Under a scenario of increasing drought,
understanding the seedling responses of these two species to water
limitation becomes critical, as drought represents an abiotic stress that
in the next future may select the forest tree species composition in
favor of more drought-resistant alien species. Management activities
should promote both the native Q. robur for forest biodiversity
conservation and the alien Q. rubra for its valuable wood properties
and high economic value (Nicolescu etal., 2020).
Based on this evidence, it was hypothesized that Q. rubra seedlings
modulate morpho-physiological traits for better coping with severe
drought stress compared to Q. robur. In particular, for Q. rubra, it was
expected (i) greater biomass production, (ii) higher specic root
length (SRL) and starch accumulation, and (iii) higher PSII yield and
stomatal conductance.
To test this hypothesis, Q. robur and Q. rubra seedlings were
grown under controlled conditions and exposed to progressive soil
drying from moderate to severe before being brought to recover. e
growth and related functional characteristics were evaluated, including
total biomass, leaf chlorophyll uorescence, stomatal conductance,
and water potential for the aboveground portion, ne root length and
biomass by diameter classes and non-structural carbohydrates (starch
and soluble sugars) for the very-ne roots only for the
belowground portion.
2 Materials and methods
2.1 Plant material and growing conditions
Acorns of Q. rubra were collected from a 69 years-old plantation
located within the Parco Regionale delle Groane (Lentate sul Seveso,

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 03 frontiersin.org
Lombardy region, Italy; 45° 41′ 19.748″ N, 9° 8′ 23.308″ E). A similar
amount of Q. robur acorns were provided by the ‘Centro Nazionale
per lo studio e la conservazione della Biodiversità Forestale di Peri’
(VR, Italy). e season preceding the acorn harvest was not aected
by extreme climatic events such as severe droughts, heat waves or
oods. Aer hydration by immersion for 24 h, 12 acorns were sown at
an 8 cm distance from each other in a 3.5 L tray (30 × 23 cm height
10 cm), for a total of ve trays and 60 acorns per species, lled with a
1:1:2 mixture of peat, compost, and sand (pH 6.0–6.8). ese trays
were positioned in a growth chamber under 25/18°C day/night
temperature, 100 μmol m
−2
s
−1
PPFD, 16 h photoperiod, and 45/70%
relative humidity (RH). irty days aer the germination, 96
uniformly sized seedlings (48 per species) were transplanted in larger
pots (24 h × 24 ⌀ cm; volume 9.5 L; one seedling per pot) lled with
the same medium with the addition of 20 g of slow-release fertilizer
(NPK 14-7-14). e plants were grown under 25/18°C day/night
temperature, 350 μmol m
−2
s
−1
PPFD, 16 h photoperiod, and 45/70%
relative humidity (RH) throughout the experiment. All plants, 96in
total, were watered to 70%–80% eld capacity for additional 60 days
until shoot expansion ceased and then were set up for the experimental
design (3 months-old seedlings).
2.2 Drought application and experimental
design
e experiment lasted 44 days, and a sketch of the design is
illustrated in Figure1. A cohort equal to half of the seedlings (24 for
each species) was maintained at eld capacity throughout the
experiment time course (control). Two drought intensities, moderate-
drought stress (MD) and severe-drought stress (SD), were applied
sequentially to the exact second-half cohort of seedlings (24 for each
species). To this aim, volumetric soil water content (SWC, % vol) at
eld capacity was determined by the gravimetric method (Louki and
Al-Omran, 2022). In detail, (i) potting soil was drenched and le to
drain overnight, (ii) the following morning, a wet 200 mL soil sample
was weighed and oven-dried at 105°C to a constant weight, and nally,
(iii) the dried soil sample was weighed to measure the water mass lost.
SWC was calculated by multiplying the gravimetric water value by the
bulk density (0.487 g cm
−3
) and associated with Time Domain
Reectometry (TDR; model ML2x, ΔT Devices, Cambridge, UK)
measurements with 1% error. Successively, SWC was monitored every
3–4 days only by TDR.
Volumetric soil water content decreased over time in drought-
treated plants (Figure2). e MD treatment was xed at ≈35% of the
SWC measured at eld capacity, reached aer 20 days of water
withheld when SWC dropped from 42.93 ± 1.19 and 45.47 ± 0.49% to
13.36 ± 0.89% and 16.84 ± 0.38% for Q. robur and Q. rubra, respectively
(Figure2). e SD treatment, xed at ≈17% of eld capacity, was
reached aer a further 12 days of progressive water withheld (32 days
in total), when water content dropped to 5.54 ± 0.65% and 9.94 ± 0.92%
for Q. robur and Q. rubra, respectively (Figure2). Two events of stress
interruption (i.e., rewatering) were applied on six seedlings per species
on days 20 and 32 by restoring the regular watering regime for 12 days.
Six seedlings per species per treatment were collected at each of the
four sampling points corresponding to day 0 (≈ 62 days from seed
sowing), day 20 (MD and control), day 32 (MD-rewatering, SD, and
control), and day 44 (SD-rewatering and control).
2.3 Morphological measurements
At each sampling point, seedlings were retrieved from the
growth chamber. e root system was carefully washed by hand
FIGURE1
Sketch of schedule and sequential connection between the control, the two drought treatments and the two rewatering treatments. The numbers
indicate the day of the experiment (top) and the age of the seedling (bottom). Arrows indicate the experimental days of the beginning of the rewatering
period (mild and severe drought release).

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 04 frontiersin.org
using a paintbrush aer being freed from the soil to minimize the
loss of ne roots. All lateral roots were separated with scissors from
the main axis and scanned immersed in water at a resolution of 400
dpi with a calibrated atbed scanner coupled to a lighting system for
image acquisition (Epson Expression 10000 XL). e obtained
images were analyzed by WinRhizo Pro V. 2007d (Regent
Instruments Inc. Quebec). A color-coded diameter classes method
was utilized to accurately measure the length, surface area, and
volume of ne roots, according to the protocol described in
Amolikondori et al. (2021). Specically, three sub-classes with
diameters less than 2 mm, d < 0.5 mm, 0.5 < d < 1.0, and
1.0 < d < 2.0 mm were set up, and any roots greater than 2 mm were
excluded. Successively, to evaluate biomass allocation, plants were
divided into shoots, leaves, taproots, and ne roots. Each component
was oven-dried to constant weight at 70°C and then weighed. Finally,
specic root length (SRL, m g−1), dened as the ne root length to
root dry mass ratio, root tissue density (RTD, g cm−3), dened as the
ne root dry mass to root volume ratio, and specic leaf area (SLA,
m
2
kg
−1
), dened as the one-sided leaf surface to dry mass ratio,
were calculated.
2.4 Physiological measurements
e day before each morphology sampling time, leaf
chlorophyll uorescence, leaf stomatal conductance, and leaf water
potentials were measured sequentially on all treated seedlings,
according to the protocol described in Chiatante etal. (2015) for
dierent species of oak seedlings. Chlorophyll uorescence was
measured on two fully expanded leaves per seedling per treatment
with a portable pulse-modulated uorometer (OS1-FL, Opti-
Sciences, Inc. United States). Minimal uorescence (F0) was
measured on dark-adapted leaves during the dark period. e
maximal uorescence yield (F
m
) was recorded aer exposing the
same dark-adapted leaves to a saturating pulse of white light
(800 ms at about 15,000 μmol photons m
−2
s
−1
). e resulting F0
and F
m
values were used to calculate the maximal photochemical
eciency of PSII (Fv/Fm), where Fv is variable uorescence (Fv = Fm
– F0).
e Quantum yield of photosystem II (ΦPSII) was measured on
the same leaves during the light period, i.e., 4 h aer the lights were
switched on. Steady-state uorescence (F
s
) was measured on light-
adapted leaves; the maximal uorescence yield (F
m
′) aer exposing
the same light-adapted leaves to a saturating pulse of white light
(800 ms at about 15,000 μmol photons m−2s−1). Values of Fs and Fms
were used to calculate ΦPSII according to the equation
ΦPSII = (Fms − Fs)/Fms.
Leaf water potential was determined on two dierent fully
expanded leaves per seedling per treatment, 1 h before lights were
switched on (Ψ
pd
) and approximately 4 h later when the temperature
had reached its daily maximum (Ψ
md
), using a pressure chamber
(SKPM 1400, Skye Instruments Ltd., UnitedKingdom).
Stomatal conductance (gs) was measured on the same two leaves
used for midday water potentials and at the same temperature using a
steady-state porometer (PMR 3, PPSystem, MA, UnitedStates).
2.5 Non-structural carbohydrates
measurements
Soluble sugar and starch content of the only very-ne root portion
(d < 0.5 mm) were determined following the method of Landhäusser
etal. (2018). Dried samples were ground in a Wiley Mill to pass
through a 40-mesh screen. A solution of 80% ethanol was used for
extracting soluble sugars (SS), and a spectrophotometric method via
phenol–sulfuric acid assay was applied for their content determination.
Enzymes α-amylase and amyloglucosidase (Sigma-Aldrich, St. Louis,
MO) were used for digesting the starch remaining in the pellet aer
extraction. e enzyme digestion-obtained glucose was determined
spectrophotometrically using glucose oxidase/peroxidase-o-
dianisidine solution and converted to starch equivalent. Sugar and
starch concentrations were calculated as a percent of the sample dry
FIGURE2
Temporal pattern of soil volumetric water content (SWC) over the experimental period for 4 months-old Quercus robur (left column) and Quercus
rubra (right column) seedlings under two drought intensity levels with the respective rewatering events. Continuous (–) and dashed (– – –) lines
indicate watered and drought conditions, respectively; filled and white circles indicate control and droughted plants, respectively; filled square and
triangle indicate the release from drought 12 days from MD and SD stress, respectively. Values are the means of 6 replicates ±1SE. Colored strips
indicate the duration and intensity of the drought treatments: dark-yellow for moderate-drought, red for severe-drought.

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 05 frontiersin.org
weight. Non-structural-carbohydrates (NSC) concentrations are the
sum of starch and soluble sugars.
2.6 Statistical analysis
e experiment was a completely randomized design with
factorial treatments (2 species × 2 watering regimes × 2 drought
duration). e main and interaction eects for the investigated traits
were tested with a three-way ANOVA using the General Linear Model
(SPSS Inc., Chicago, IL). is analysis considered only the control and
drought (MD and SD) treatments of the second and third sampling
points, as the rst sampling was the same for both. Recovery of
rewatered plants was tested against MD and SD treatments only. All
interactions were initially included in the model, the nonsignicant
ones being stepwise excluded. Before the analysis, all above-and
below-ground morpho-physiological and chemical data were tested
with the Kolmogorov–Smirnov test for normality and the Levene test
for the homoscedasticity assumption. In particular, the stomatal
conductance and shoot dry mass met these two requirements aer
being square-root and log transformed, respectively, whereas the
predawn and midday Leaf water potential (Leaf wp), F
v
/F
m
, and ΦPSII
did not. For these latter, the non-parametric two-groups Mann–
Whitney test was carried out on the medians of the main factors. For
each variable, the independent samples student t-test was performed
among treatments within each sampling point and among droughted
and rewatered plants for both MD and SD treatments. All dierences
were considered signicant at p < 0.05.
Principal component analysis (PCA) was performed on all
standardized data to detect the pattern of association between the root
morphological and chemical (Starch and SS) variables and the shoot
physiological variables (soware SYN-TAX 2000, Podani, Budapest,
Hungary).
3 Results
3.1 Morphological traits
In general, both shoot and root biomasses of control plants were
more developed in Q. robur than in Q. rubra throughout the
experiment (time and species main eect p < 0.001, Table 1 and
Figures3A–D). However, the shoot biomass was almost double the
root biomass for both species (Figures3A–D). For Q. robur, shoot and
root biomasses of both MD- and SD-treated plants did not dier from
control (Figures3A,C). Dierently, for Q. rubra, the shoot and root
biomasses were similar to and signicantly higher than those of the
control for MD- and SD-treated plants, respectively (p < 0.05;
Figures3B,D) (S × D interaction p < 0.05, Table1). For both species,
rewatering events (MD-rewatering and SD-rewatering) did not
signicantly change the growth parameters from the previous drought
condition except for root dry mass in Q. robur, which was signicantly
higher (Figure3C).
In terms of the R:S ratio, Q. robur control plants increased
momentarily aer 32 days, with a similar ratio to Q. rubra occurring
12 days later (Figures 3E,F). For both species, droughted plants
showed greater values than the control, highlighting an increase in the
root portion (drought eect p = 0.037, Table1); this ratio decreases
slightly between the second and third (32 days) sampling points. Both
species responded to MD interruption by increasing the R:S ratio,
although not signicantly (Figures3E,F), whereas no dierences were
observed when plants were rewatered aer SD stress.
ese two Quercus species diered signicantly (p < 0.05) for most
of the traits when ne roots were analyzed as one pool <2 mm in
diameter, except for the length (p = 0.336; Table1). If ne roots were
analyzed at the subclass level, signicant dierences between the two
species emerged for the length, with lower and higher values for
0.5–1 mm and 1–2 mm classes in Q. robur than Q. rubra, respectively
(Figures 4A,B). All classes slightly increased their values under
drought conditions.
Fine root (<2 mm) biomass (p = 0.017) and RTD (p = 0.019) were
signicantly higher in Q. robur than in Q. rubra (Table 1 and
Figures5A,B), the opposite for SRL (p = 0.014, Table1 and Figure5C).
In particular, SRL showed a signicant decrease over time in both
species (p < 0.001, Table1 and Figures6A,B), more marked in Q. robur.
A signicant response to drought alone was observed only for the
ne root (<2 mm) biomass trait (p = 0.003, Table1), with MD and SD
slightly higher than control in both species, in particular in Q. rubra
(Figures 6C,D). For both control and drought-treated plants, the
biomass signicantly increased over time (from MD to SD) only in
Q. robur (T × S interaction p = 0.021, Table1 and Figures6C,D).
For both species, rewatering events (MD-rewatering and
SD-rewatering) did not signicantly change the growth parameters
from the previous drought condition except for SRL in Q. robur, which
was signicantly lower (Figure6A).
3.2 Physiological traits
Species signicantly diered for all the investigated physiological
traits except for predawn LWP (Table1). Q. rubra generally showed
mean values lower than Q. robur for the stomatal conductance
(Figure7A), the maximal photochemical eciency (F
v
/F
m
; Figure7B),
and the quantum Yield (Φ; Figure7C) of PSII, the opposite for the
midday LWP (Figure7D).
During the experiment, predawn LWP did not dier between
species (Table 1) and remained higher than midday-LWP
(Figures 8A–D), although drought signicantly decreased both
potentials in both species (Table 1). A signicant drop of LWP to
−2.74 ± 0.22 MPa at predawn (Figure8A) and to −3.40 ± 0.18 MPa at
midday (Figure 8C) occurred only in Q. robur aer 32 days of
progressively soil drying. Leaf water potential was the trait that better
indicated the response to the rewatering, partially increasing aer MD
and signicantly recovering to control values 12 days aer SD
(Figures8A–D). e stomatal conductance was signicantly higher
for well-watered than drought-treated plants in Q. robur (Figure8E),
whereas no dierences emerged in Q. rubra (S × D interaction
p = 0.001, Table1; Figure8F).
3.3 Non-structural carbohydrates
Species significantly differed for the starch rather than the SS
concentrations (Table 1), with the overall mean of starch

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 06 frontiersin.org
concentration significantly higher (p < 0.001) in Q. robur than in
Q. rubra (Figures9A,D). In both species, drought significantly
reduced both starch (p = 0.004) and SS (p = 0.031) concentrations
(Table 1). These differences were of higher magnitude after
32 days of progressive soil drying (T × D interaction p = 0.02,
Table1; Figures9B,C,E,F).
In both species, 12 days of rewatering aer 20 (MD-rewatering)
and 32 (SD-rewatering) days from water withholding signicantly
increased the starch and the SS concentrations from the previous
drought condition (Figures 9B,C,E,F), except for the SS in
MD-rewatering Q. robur plants (Figure9E).
3.4 Multivariate analysis
e most striking dierence among all types of investigated traits
reected by the rst component of PCA (24.37% of total variance) could
beattributed to the dierent responses to SWC changes of the two
species, being all Q. rubra treatments grouped toward negative loadings
in the coincidence of SWC score, the opposite for most of Q. robur
treatments (Figure10). Q. rubra is associated with high LWP and SRL
values, particularly for control and SD-interrupted plants. On the other
hand, Q. robur is associated with growth traits (shoot and root dry mass,
RTD), photochemical eciency, and starch concentration.
FIGURE3
Temporal pattern of shoot (A,B) and root (C,D) dry mass and root-to-shoot ratio (E,F) over the experimental period for 4 months-old Quercus robur
(left column) and Quercus rubra (right column) seedlings under two drought intensity levels with the respective rewatering events. Continuous (–) and
dashed (– – –) lines indicate watered and drought conditions, respectively; filled and white circles indicate control and droughted plants, respectively;
filled square and triangle indicate the recovery from drought 12 days from MD and SD stress, respectively. Values are the means of 6 replicates ±1SE.
Colored strips indicate the duration and intensity of the drought treatments: dark-yellow for moderate-drought, red for severe-drought. If written, a, b,
c and x, y, z indicate significant dierences between dierent sampling points within control and drought treatments, respectively (LSD, p < 0.05); *
indicate significant dierences between control and drought within each sampling point, and ‡ between droughted and rewatered plants for both MD
and SD treatments (student’s t-test, p < 0.05).

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 07 frontiersin.org
e second component explained 18.36% of the total variance. It
divided most of the well-watered (controls and MD-and
SD-rewatering) from the drought-treated plants, particularly for
Q. robur. Moreover, PC2 highlighted the dierent responses to
drought between the morphological structure of ne roots and the
physiology of the shoot, with LWP, stomatal conductance,
TABLE1 p-values of ANOVA (general linear model) for the eects of time (at 20 and 32 days only), species and drought intensity on morphological and
chemical traits for the fine and very-fine roots, respectively, and physiological traits for the leaves of Quercus robur and Quercus rubra.
Time Species Drought T × S T × D S × D T × S × D
df = 1 df = 1 df = 1 df = 1 df = 1 df = 1 df = 1
Plant dry mass
Shoot DM (g) <0.001 <0.001 0.957 — — 0.045 —
Root DM (g) <0.001 <0.001 0.170 0.008 — — —
R:S ratio 0.503 0.142 0.037 — — — —
Fine-root morphological traits
Length 0.988 0.336 0.250 — — — —
DM 0.129 0.017 0.003 0.021 — — —
RTD 0.242 0.019 0.139 — — — —
SRL <0.001 0.014 0.322 — — — —
Leaf physiologic al traits
Leaf Wp predawn <0.001 0.477 0.002 N/A N/A N/A N/A
Leaf Wp midday 0.18 <0.001 0.002 N/A N/A N/A N/A
Stomatal conductance <0.001 0.014 0.002 — — 0.001 —
Fv/Fm0.877 <0.001 0.619 N/A N/A N/A N/A
Yield 0.163 0.019 0.211 N/A N/A N/A N/A
Fine-root chemical traits
Starch <0.001 <0.001 0.004 —0.02 — —
Soluble sugars 0.024 0.466 0.031 — — — —
Non-signicant interaction eects were excluded from the model (—). e non-parametric two-groups Mann–Whitney test was performed for each factor for the predawn and midday Leaf Wp, Fv/Fm and
yield. Boldface p-values are signicant at a probability level of p < 0.05. N/A, not applicable; DM, dry mass; RTD, root tissue density; SRL, specic root length; Wp, water potential.
FIGURE4
Root length for dierent diameter classes of Quercus robur (green) and Quercus rubra (dark red) under well-watered (A) and drought treatments (B).
Values refer to and pool the 20–32 days temporal range; each bar represents the mean of 24 replicates ±1SE. An asterisk (*) indicates a significant
dierence between species within each diameter class (student’s t-test, p < 0.05).

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 08 frontiersin.org
FIGURE6
Temporal pattern of specific root length (A,B) and dry mass (C,D) of very-fine roots over the experimental period for 4 months-old Quercus robur (left
column) and Quercus rubra (right column) seedlings under two drought intensity levels with the respective rewatering events. Continuous (–) and
dashed (– – –) lines indicate watered and drought conditions, respectively; filled and white circles indicate control and droughted plants, respectively;
filled square and triangle indicate the recovery from drought 12 days from MD and SD stress, respectively. Values are the means of 6 replicates ±1SE.
Colored strips indicate the duration and intensity of the drought treatments: dark-yellow for moderate-drought, red for severe-drought. If written, a, b,
c and x, y, z indicate significant dierences between dierent sampling points within control and drought treatments, respectively (LSD, p < 0.05); *
indicate significant dierences between control and drought within each sampling point, and ‡ between droughted and rewatered plants for both MD
and SD treatments (student’s t-test, p < 0.05).
FIGURE5
Fine root (<2 mm) dry mass (A), RTD (B) and SRL (C) for 4 months-old Quercus robur (green) and Quercus rubra (dark red) seedlings. Values refer to the
20–32 days temporal range and pooled well-watered and drought treatments (n = 24). An asterisk (*) indicates a significant dierence between species
(three-way ANOVA main factor, p < 0.05). Vertical boxes represent approximately 50% of the observations (25th to 75th percentiles), and lines extending
from each box are the upper and lower 25% of the distribution. Within each box, the solid horizontal line indicates the median, while the squared filled
dot represents the mean.

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 09 frontiersin.org
photochemical eciency and starch positively correlated with SWC
than RTD and SRL, which are positioned on the other side (Figure10).
Starch and SS concentrations also decreased with the increasing
drought intensity (Figure10).
4 Discussion
Outcomes from this study highlighted that Q. rubra had
signicantly lower biomass than Q. robur independently from the
treatments rejecting the rst (i) hypothesis, although both species
equally invested much more biomass in the shoot than the root.
Moreover, although not measured, based on the literature, the seed
size’s eect on plant growth may beexcluded, as there is no signicant
dierence in average dry weight between the two species (Devetaković
etal., 2019; Woziwoda etal., 2023). e alien Q. rubra responds to
drought dierently from the native Q. robur. In particular, the alien
Q. rubra responded to drought, increasing both shoot and root
biomasses, while the biomass of the native Q. robur remained
unchanged when it underwent drought stress. Moreover, the alien
Q. rubra seemed to respond better to severe drought since this cohort
of plants had similar biomass values to those of the non-stressed
ones. On the other hand, the native Q. robur suered the severe
drought, as demonstrated by the unchanged values of SD-rewatered
plants aer 12 days of water supply.
Consistent with this study, an R:S ratio lower than one was
observed in both species at the sapling stage under dierent canopy
openings (Kuehne etal., 2014). is behavior is the opposite of the
Mediterranean oak species Quercus pubescens (Di Iorio etal., 2011),
Quercus ilex and Quercus suber (Ramírez-Valiente etal., 2018), which
invest much more in the belowground compartment (R:S ratio >1) at
the initial growth stages, even under well-watered conditions. Plants
would allocate more biomass to the roots in their early seedling stages
and direct it to the shoots soon aer. For example, the R:S ratio
signicantly decreases with stem biomass and tree height for forests,
with the accumulation of standing aboveground biomass for shrubs
and grasses (Mokany etal., 2006; Hui etal., 2014). Conversely, the R:S
ratio signicantly increases with elevation and latitude, i.e., under
colder and drier conditions (Qi etal., 2019; Leuschner, 2020). e
optimal partitioning hypothesis (Bloom etal., 1985; Chapin etal.,
1987) suggests that under more stressful conditions, such as those
characterized by low-nutrient or dry soils, greater biomass is allocated
to roots to optimize resource use. Moreover, in both temperate
(omas and Gausling, 2000) and mediterranean (Chiatante etal.,
2006, Chiatante etal., 2015; Di Iorio etal., 2011) oak species, the
predominant process in the short-term response to drought has been
FIGURE7
Stomatal conductance (A), Fv/Fm (B), yield (C), and midday LWP (D) for 4 months-old Quercus robur (green) and Quercus rubra (dark red)
seedlings. Values refer to the 20–32 days temporal range and pooled well-watered and drought treatments (n = 24). An asterisk (*) indicates a
significant difference between species (three-way ANOVA main factor for stomatal conductance, non-parametric Mann–Whitney test for Fv/
Fm, yield, and midday LWP, p < 0.05). Vertical boxes represent approximately 50% of the observations, and lines extending from each box are
the upper and lower 25% of the distribution. Within each box, the solid horizontal line indicates the median, while the squared filled dot
represents the mean.

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 10 frontiersin.org
FIGURE8
Temporal pattern of predawn (A,B) and midday (C,D) LWP and stomatal conductance (E,F) over the experimental period for 4 months-old Quercus
robur (left column) and Quercus rubra (right column) seedlings under two drought intensity levels with the respective rewatering events. Continuous
(–) and dashed (– – –) lines indicate watered and drought conditions, respectively; filled and white circles indicate control and droughted plants,
respectively; filled square and triangle indicate the recovery from drought 12 days from MD and SD stress, respectively. Values are the means of 6
replicates ±1SE. Colored strips indicate the duration and intensity of the drought treatments: dark-yellow for moderate-drought, red for severe-
drought. If written, a, b, c and x, y, z indicate significant dierences between dierent sampling points within control and drought treatments,
respectively (LSD, p < 0.05); * indicate significant dierences between control and drought within each sampling point, and ‡ between droughted and
rewatered plants for both MD and SD treatments (student’s t-test, p < 0.05). Dierences were considered significant at p < 0.05.
found to involve redistribution of biomass toward the ne roots to
increase the soil’s exploitable volume. In this study, the short-term
response of both species to dry conditions mirrors the acclimation
and adaptation pattern of the oak species, with more biomass
allocation to the very-ne and ne roots (Figures6C,D). SRL resulted
higher in Q. rubra than Q. robur independently from the drought
treatments, supporting the rst part of the second hypothesis.
However, over time, the increased ne root biomass was not linked to
a proportional increase in length, resulting in a general decrease of the
specic root length in both species, that is a reduction in resource
acquisition (Eissenstat and Yanai, 1997; Ostonen et al., 2007;
Montagnoli etal., 2019). In particular, in Q. rubra, the magnitude of
the SRL reduction was lower than in Q. robur, regardless of the
drought intensity experienced; this dierence might berelated to the

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 11 frontiersin.org
lower contribution of the heavier 1–2 mm diameter class
(Figures4A,B) in Q. rubra than in Q. robur. Similar behavior was
mirrored at the leaf level, with a higher SLA for Q. rubra
(Supplementary Figure S1).
Therefore, it is possible to speculate that the lower biomass for
the 1–2 mm fine root portion might berelated to the lower inputs
from aboveground, which did not balance construction and
maintenance costs. Indeed, in contrast with the (iii) hypothesis,
photochemical efficiency (F
v
/F
m
), the quantum yield (ΦPSII) of
PSII, and stomatal conductance were consistently lower in
Q. rubra during the experimental period, probably impacting
photosynthesis by reducing the maximum activity of PSII and
limiting the CO
2
availability at the chloroplast level (Cornic,
1994). Moreover, the low root starch concentration, although
limited only to the very-fine class, found in Q. rubra after 32 days
of progressive soil drying does not support the second part of the
(ii) hypothesis and strongly correlates with the low photochemical
efficiency (Figure10), probably being due to the reduction of
photosynthate inputs from aboveground. This hypothesis is
further supported by the significantly low SS concentration in
droughted plants of both species, like the beech seedlings under
similar drought stress conditions (Domingo etal., 2023).
e higher leaf water potential and lower stomatal conductance
of Q. rubra, even under drier conditions, suggests an isohydric
behavior for this species, at least in the seedling stage and in the
adopted experimental conditions. e slightly higher soil water
content measured in Q. rubra pots at the end of the drying period
further supports the reduced stomatal conductance observed. Q. rubra
(Yi etal., 2017) and Q. petraea (Aranda etal., 2000; Klein, 2014;
Martínez-Sancho etal., 2017) are usually considered anisohydric
species, i.e., able to keep their stomata open during drought, allowing
their water potential to decrease while still maintaining C assimilation
rates (Martínez-Vilalta etal., 2014), but with a higher risk of hydraulic
failure. However, the isohydric-anisohydric spectrum should not
beconsidered a xed binary category, as both species have certain
degrees of isohydricity (Martínez-Vilalta etal., 2014; Yi etal., 2017),
FIGURE9
Temporal pattern of starch (B,C) and soluble sugars (E,F) concentrations (% dw) over the experimental period for 4 months-old Quercus robur (left
column) and Quercus rubra (right column) seedlings under two drought intensity levels with the respective rewatering events. Continuous (–) and
dashed (– – –) lines indicate watered and drought conditions, respectively; filled and white circles indicate control and droughted plants, respectively;
filled square and triangle indicate the recovery from drought 12 days from MD and SD stress, respectively. Values are the means of 6 replicates ±1SE.
Colored strips indicate the duration and intensity of the drought treatments: dark-yellow for moderate-drought, red for severe-drought. If written, a, b,
c and x, y, z indicate significant dierences between dierent sampling points within control and drought treatments, respectively (LSD, p < 0.05); *
indicate significant dierences between control and drought within each sampling point, and ‡ between droughted and rewatered plants for both MD
and SD treatments (student’s t-test, p < 0.05). Dierences were considered significant at p < 0.05. On the far-left side, box-plot distribution for the
starch (A) and soluble sugars (B) concentrations; values refer to the 20–32 days temporal range and pooled well-watered and drought treatments
(n = 24) (three-way ANOVA main factor, p < 0.05).

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 12 frontiersin.org
FIGURE10
Principal component analysis (PCA) ordination of the first and second axes showing the relationships between physiological for the leaves, chemical
and morphological traits for very-fine and fine roots, respectively, shoot and root biomass (arrows) in relation to drought stress condition in 4 months-
old Quercus robur and Quercus rubra seedlings. SRL, specific fine root length; length, fine root length; RTD, root tissue density; SS, soluble sugars; pd
LWP, predawn leaf water potential; mid LWP, midday leaf water potential; g, stomatal conductance; yield, quantum yield (Φ); Fv/Fm, maximal
photochemical eciency; and the shoot and root, total shoot and root biomass.
and drought response strategies may change with ontogeny
(Cavender-Bares and Bazzaz, 2000). In fact, red oak seedlings coping
with drought conditions have been observed to reduce the stomata
opening with a consecutive lowering in carbon uptake. Dierently, at
the mature tree level, red oak addresses drought conditions by
reaching deeper soil water layers thanks to a deeper root system
development, sustaining a higher stomatal conductance (Cavender-
Bares and Bazzaz, 2000). e drawback of the isohydric behavior
observed here in Q. rubra was the overall reduced growth. However,
isohydricity may not always beconsidered a carbon limiting factor, for
example, for the evergreen Q. ilex, whose growth performance was
similar to the anisohydric Phyllirea latifolia under extreme drought
(Garcia-Forner etal., 2016).
Both drought releases from MD and SD conditions showed a
marked recovery of the photochemical eciency (F
v
/F
m
) and the
quantum yield (ΦPSII) of PSII to pre-drought values, indicating a
resilient photosynthetic apparatus in these species. Instead, this trend
was not mirrored by the ne roots, whose growth did not recover to
control values, except for a slight increase for Q. robur. Several studies
reported a remarkable resistance of PSII photochemistry to dehydration,
high-temperature and photoinhibition, whether alone or in combination,
in the similar sessile oak (Epron etal., 1992) or cedar (Ladjal etal., 2000)
and the sclerophyll Heteromeles arbutifolia (Valladares and Pearcy, 1997).
e adopted PPFD was relatively lower than that of direct summer
sunlight as the one experienced in a large forest gap (18% of assumed
≈ 2000 μmol m
−2
s
−1
); this light condition could further explain the
reduced growth observed for Q. rubra, supporting its prole as
intermediate shade tolerant and gap specialist.
In conclusion, outcomes from the present study revealed
isohydric behavior rather than specic functional traits as the
character that best explains the competitive performance of Q. rubra
at the seedling stage. e higher resistance to drought under the low
light intensity adopted in this study makes this species highly
competitive under the direr conditions in the canopy openings
during the summer (Amolikondori et al., 2021). However, the
response to the interactive eects of combined stressors may dier
from that elicited when the stresses are imposed singly (Mittler, 2006;
Suseela etal., 2015), so further experiments combining dierent light
and drought intensities are necessary to elucidate better the growth
performance of this alien species in the European habitat.
Data availability statement
e raw data supporting the conclusions of this article will
bemade available by the authors, without undue reservation.

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 13 frontiersin.org
Author contributions
ADI: Conceptualization, Data curation, Formal analysis,
Methodology, Project administration, Supervision, Validation,
Visualization, Writing – original draft, Writing – review &
editing. ACC: Data curation, Formal analysis, Investigation,
Visualization. PB: Formal analysis, Investigation, Supervision.
AM: Conceptualization, Funding acquisition, Methodology,
Project administration, Supervision, Validation, Visualization,
Writing – review & editing, Formal analysis.
Funding
e author(s) declare nancial support was received for the
research, authorship, and/or publication of this article. is work was
supported by the University of Insubria (FAR) and the EC FP7 Project
ZEPHYR-308313.
Acknowledgments
e authors are deeply grateful to Giada Falsetti for her help in the
non-structural carbohydrate analyzes.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
beconstrued as a potential conflict of interest.
e author(s) declared that they were an editorial board member
of Frontiers, at the time of submission. is had no impact on the peer
review process and the nal decision.
Publisher’s note
All claims expressed in this article are solely those of the authors and
do not necessarily represent those of their aliated organizations, or those
of the publisher, the editors and the reviewers. Any product that may be
evaluated in this article, or claim that may be made by its manufacturer,
is not guaranteed or endorsed by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/gc.2024.1307340/
full#supplementary-material
References
Abrams, M. D. (1996). Distribution, historical development and ecophysiological
attributes of oak species in the eastern UnitedStates. Ann. For. Sci. 53, 487–512. doi:
10.1051/FOREST:19960230
Amolikondori, A., Vajari, K. A., Feizian, M., Montagnoli, A., and Di Iorio, A.
(2021). Gap size in Hyrcanian Forest affects the lignin and N concentrations of the
oriental beech (Fagus orientalis Lipsky) fine roots but does not change their
morphological traits in the medium term. Forests 12:137. doi: 10.3390/f12020137
Aranda, I., Gil, L., and Pardos, J. A. (2000). Water relations and gas exchange in Fagus
sylvatica L. and Quercus petraea (Mattuschka) Liebl. in a mixed stand at their southern limit
of distribution in Europe. Trees 146, 344–352. doi: 10.1007/S004680050229
Bloom, A. J., Chapin, F. S., and Mooney, H. A. (1985). Resource limitation in plants-an
economic analogy. Annu. Rev. Ecol. Syst. 16, 363–392. doi: 10.1146/annurev.
es.16.110185.002051
Brunner, I., Herzog, C., Dawes, M. A., Arend, M., and Sperisen, C. (2015). How tree
roots respond to drought. Front. Plant Sci. 6:547. doi: 10.3389/fpls.2015.00547
Cavender-Bares, J., and Bazzaz, F. A. (2000). Changes in drought response strategies
with ontogeny in Quercus rubra: implications for scaling from seedlings to mature trees.
Oecologia 124, 8–18. doi: 10.1007/PL00008865
Chapin, F. S., Bloom, A. J., Field, C. B., and Waring, R. H. (1987). Plant responses to
multiple environmental factors physiological ecology provides tools for studying how
interacting environmental resources control plant growth. Bioscience 37, 49–57. doi:
10.2307/1310177
Chiatante, D., Di Iorio, A., Sciandra, S., Scippa, G. S., and Mazzoleni, S. (2006). Eect
of drought and re on root development in Quercus pubescens Willd. and Fraxinus
ornus L. seedlings. Environ. Exp. Bot. 56, 190–197. doi: 10.1016/j.envexpbot.2005.01.014
Chiatante, D., Tognetti, R., Scippa, G. S., Congiu, T., Baesso, B., Terzaghi, M., et al.
(2015). Interspecic variation in functional traits of oak seedlings (Quercus ilex, Quercus
trojana, Quercus virgiliana) grown under articial drought and re conditions. J. Plant
Res. 128, 595–611. doi: 10.1007/s10265-015-0729-4
Chmura, D. (2020). e spread and role of the invasive alien tree Quercus rubra
(L.) in novel forest ecosystems in central Europe. Forests 11:586. doi: 10.3390/
F11050586
Chmura, D., and Sierka, E. (2005). e occurrence of invasive alien plant species in
selected forest nature reserves in southern Poland as a conservation problem. Nat.
Conserv. 62, 3–11.
Cornic, G. (1994). “Drought stress and high eects on leaf photosynt hesis” in Photoinhibition
of photosynthesis, from molecular mechanisms to the eld. eds. N. R. Baker and J. R. Bowyer
(Oxford: BIOS Scientic Publishers), 297–313.
Crow, T. R. (1992). Population dynamics and growth patterns for a cohort of
northern red oak (Quercus rubra) seedlings. Oecologia 91, 192–200. doi: 10.1007/
BF00317783
Devetaković, J., Nonić, M., Prokić, B., Popović, V., and Šijačić-Nikolić, M. (2019).
Acorn size inuence on the quality of pedunculate oak (Quercus robur L.) one-year old
seedlings. REFORESTA 8, 17–24. doi: 10.21750/REFOR.8.02.72
Di Iorio, A., Giacomuzzi, V., and Chiatante, D. (2016). Acclimation of ne root
respiration to soil warming involves starch deposition in very ne and ne roots: a
case study in Fagus sylvatica saplings. Physiol. Plant. 156, 294–310. doi: 10.1111/
ppl.12363
Di Iorio, A., Montagnoli, A., Scippa, G. S., and Chiatante, D. (2011). Fine root growth
of Quercus pubescens seedlings aer drought stress and re disturbance. Environ. Exp.
Bot. 74, 272–279. doi: 10.1016/j.envexpbot.2011.06.009
Dimitrova, A., Csilléry, K., Klisz, M., Lévesque, M., Heinrichs, S., Cailleret, M., et al.
(2022). Risks, benets, and knowledge gaps of non-native tree species in Europe. Front.
Ecol. Evol. 10:908464. doi: 10.3389/fevo.2022.908464
Domingo, G., Vannini, C., Marsoni, M., Costantini, E., Bracale, M., and Di Iorio, A.
(2023). A multifaceted approach to reveal the very-ne root’s response of Fagus sylvatica
seedlings to dierent drought intensities. Physiol. Plant. 175, e13934–e13915. doi:
10.1111/ppl.13934
Eissenstat, D. M., and Yanai, R. D. (1997). e ecology of root lifespan. Adv. Ecol. Res.
27, 1–60. doi: 10.1016/S0065-2504(08)60005-7
Epron, D., Dreyer, E., and Bréda, N. (1992). Photosynthesis of oak trees [Quercus
petraea (Matt.) Liebl.] during drought under field conditions: diurnal course of net
CO2 assimilation and photochemical efficiency of photosystem II. Plant Cell
Environ. 15, 809–820. doi: 10.1111/J.1365-3040.1992.TB02148.X
Garcia-Forner, N., Biel, C., Savé, R., and Martínez-Vilalta, J. (2016). Isohydric species
are not necessarily more carbon limited than anisohydric species during drought. Tree
Physiol. 37, 441–455. doi: 10.1093/treephys/tpw109
Gieger, T., and omas, F. M. (2002). Eects of defoliation and drought stress on
biomass partitioning and water relations of Quercus robur and Quercus petraea. Basic
Appl. Ecol. 3, 171–181. doi: 10.1078/1439-1791-00091
Hartmann, H., Ziegler, W., and Trumbore, S. (2013). Lethal drought leads to reduction
in nonstructural carbohydrates in Norway spruce tree roots but not in the canopy. Func t.
Ecol. 27, 413–427. doi: 10.1111/1365-2435.12046
Hui, D., Wang, J., Shen, W., Le, X., Ganter, P., and Ren, H. (2014). Near isometric
biomass partitioning in forest ecosystems of China. PLoS One 9:e86550. doi: 10.1371/
journal.pone.0086550

Di Iorio et al. 10.3389/gc.2024.1307340
Frontiers in Forests and Global Change 14 frontiersin.org
Jacobs, D. F., Francis Salifu, K., and Davis, A. S. (2009). Drought susceptibility and
recovery of transplanted Quercus rubra seedlings in relation to root system morphology.
Ann. For. Sci. 66:504. doi: 10.1051/forest/2009029
Kiedrzyński, M., Zielińska, K., and Grzelak, P. (2011). Transformation of Forest
vegetation aer 40 years of protection in the Tomczyce nature reserve (central Poland).
Acta Univ. Lodz., Folia Biol. Oecol. 7, 207–227. doi: 10.2478/V10107-009-0026-X
Klein, T. (2014). e variability of stomatal sensitivity to leaf water potential across
tree species indicates a continuum between isohydric and anisohydric behaviors. Funct.
Ecol. 28, 1313–1320. doi: 10.1111/1365-2435.12289
Kormann, J. M., Liesebach, M., and Liepe, K. J. (2023). Provenances from introduced
stands of northern red oak (Quercus rubra L.) outperform those from the natural
distribution. For. Ecol. Manag. 531:120803. doi: 10.1016/j.foreco.2023.120803
Kosola, K. R., Dickmann, D. I., and Parry, D. (2002). Carbohydrates in individual
poplar ne roots: eects of root age and defoliation. Tree Physiol. 22, 741–746. doi:
10.1093/treephys/22.10.741
Kuehne, C., Nosko, P., Horwath, T., Bauhus, J., and Abrams, M. (2014). A comparative
study of physiological and morphological seedling traits associated with shade tolerance
in introduced red oak (Quercus rubra) and native hardwood tree species in southwestern
Germany. Tree Physiol. 34, 184–193. doi: 10.1093/TREEPHYS/TPT124
Ladjal, M., Epron, D., and Ducrey, M. (2000). Eects of drought preconditioning on
thermotolerance of photosystem II and susceptibility of photosynthesis to heat stress in
cedar seedlings. Tree Physiol. 20, 1235–1241. doi: 10.1093/TREEPHYS/20.18.1235
Landhäusser, S. M., Chow, P. S., Turin Dickman, L., Furze, M. E., Kuhlman, I.,
Schmid, S., et al. (2018). Standardized protocols and procedures can precisely and
accurately quantify non-structural carbohydrates. Tree Physiol. 38, 1764–1778. doi:
10.1093/treephys/tpy118
Leuschner, C. (2020). Drought response of European beech (Fagus sylvatica L.)—a
review. Perspect. Plant Ecol. Evol. Syst. 47:125576. doi: 10.1016/j.ppees.2020.125576
Louki, I. I., and Al-Omran, A. M. (2022). Calibration of soil moisture sensors
(ECH2O-5TE) in hot and saline soils with new empirical equation. Agronomy 13:51. doi:
10.3390/agronomy13010051
Major, K. C., Nosko, P., Kuehne, C., Campbell, D., and Bauhus, J. (2013). Regeneration
dynamics of non-native northern red oak (Quercus rubra L.) p opulations as inuenced
by environmental factors: a case study in managed hardwood forests of southwestern
Germany. For. Ecol. Manag. 291, 144–153. doi: 10.1016/J.FORECO.2012.12.006
Maniero, F. (2015). Cronologia della Flora Esotica Italiana. Leo S. Olschki. Firenze.
Martínez-Sancho, E., Dorado-Liñán, I., Hacke, U. G., Seidel, H., and Menzel, A.
(2017). Contrasting hydraulic architectures of scots pine and sessile oak at their
southernmost distribution limits. Front. Plant Sci. 8:598. doi: 10.3389/fpls.2017.00598
Martínez-Vilalta, J., Poyatos, R., Aguadé, D., Retana, J., and Mencuccini, M. (2014). A
new look at water transport regulation in plants. New Phytol. 204, 105–115. doi: 10.1111/
nph.12912
Mittler, R. (2006). Abiotic stress, the eld environment and stress combination. Trends
Plant Sci. 11, 15–19. doi: 10.1016/j.tplants.2005.11.002
Mokany, K., Raison, R. J., and Prokushkin, A. S. (2006). Critical analysis of root: shoot ratios
in terrestrial biomes. Glob. Chang. Biol. 12, 84–96. doi: 10.1111/j.1365-2486.2005.001043.x
Montagnoli, A., Dumroese, R. K., Terzaghi, M., Onelli, E., Scippa, G. S., and
Chiatante, D. (2019). Seasonality of ne root dynamics and activity of root and shoot
vascular cambium in a Quercus ilex L. forest (Italy). For. Ecol. Manag. 431, 26–34. doi:
10.1016/j.foreco.2018.06.044
Montagnoli, A., Terzaghi, M., Di Iorio, A., Scippa, G. S., and Chiatante, D. (2012).
Fine-root morphological and growth traits in a Turkey-oak stand in relation to seasonal
changes in soil moisture in the southern Ap ennines, Italy. Ecol. Res. 27, 1015–1025. doi:
10.1007/s11284-012-0981-1
Nguyen, P. V., Dickmann, D. I., Pregitzer, K. S., and Hendrick, R. (1990). Late-season
changes in allocation of starch and sugar to shoots, coarse roots, and ne roots in two
hybrid poplar clones. Tree Physiol. 7, 95–105. doi: 10.1093/treephys/7.1-2-3-4.95
Nicolescu, V.-N., Vor, T., Mason, W. L., Bastien, J.-C., Brus, R., Henin, J.-M., et al.
(2020). Ecology and management of northern red oak (Quercus rubra L. syn. Q. borealis
F. Michx.) in Europe: a review. Forestry 93, 481–494. doi: 10.1093/forestry/cpy032
Ostonen, I., Püttsepp, Ü., Biel, C., Alberton, O., Bakker, M. R., Lõhmus, K., et al.
(2007). Specic root length as an indicator of environmental change. Plant Biosyst. 141,
426–442. doi: 10.1080/11263500701626069
Qi, Y., Wei, W., Chen, C., and Chen, L. (2019). Plant root-shoot biomass allocation
over diverse biomes: a global synthesis. Glob. Ecol. Conserv. 18:e00606. doi: 10.1016/j.
gecco.2019.e00606
Ramírez-Valiente, J.-A., Aranda, I., Sanchéz-Gómez, D., Rodríguez-Calcerrada, J.,
Valladares, F., and Robson, T. M. (2018). Increased root investment can explain the
higher survival of seedlings of ‘Mesic’ Quercus suber than “xeric” Quercus ilex in sandy
soils during a summer drought. Tree Physiol. 39, 64–75. doi: 10.1093/treephys/tpy084
Reinhardt, F., Herle, V. M., Bastiansen, B. F., and Streit, B. (2003). Economic impact of
the spread of alien species in Germany. Federal Environmental Agency
(Umweltbundesamt) Berlin.
Sander, I. (1990). “Quercus rubra L.” in Silvics of North America: volume 2. Hardwoods
(Agricultural Handbook). eds. R. M. Burns and B. H. Honkala (U.S. Department of
Agriculture, Forest Service), 727–738. Available at: http://www.na.fs.fed.us/spfo/pubs/
silvics_manual/table_of_contents.htm
Steiner, K. C., Abrams, M. D., and Bowersox, T. W. (1993). “Advance reproduction and
other stand characteristics in Pennsylvania and French stands of northern red oak” in
Proceedings of the 9th Central Hardwood Forest Conference; Gen. Tech. Rep. NC-161. eds.
A. R. Gillespie, G. R. Parker, P. E. Pope and G. Rink (St. Paul, MN: U.S. Department of
Agriculture, Forest Service, North Central Forest Experiment Station), 473–483.
Available at: https://www.fs.usda.gov/treesearch/pubs/15342
Suseela, V., arayil, N., Xing, B., and Dukes, J. S. (2015). Warming and drought
dierentially inuence the production and resorption of elemental and metabolic
nitrogen pools in Quercus rubra. Glob. Chang. Biol. 21, 4177–4195. doi: 10.1111/
gcb.13033
omas, F. M., and Gausling, T. (2000). Morphological and physiological responses
of oak seedlings (Quercus petraea and Q. robur) to moderate drought. Ann. For. Sci. 57,
325–333. doi: 10.1051/forest:2000123
Valladares, F., and Pearcy, R. W. (1997). Interactions between water stress, sun-shade
acclimation, heat tolerance and photoinhibition in the sclerophyll Heteromeles
arbutifolia. Plant Cell Environ. 20, 25–36. doi: 10.1046/j.1365-3040.1997.d01-8.x
Vansteenkiste, D., De Boever, L., and Van Acker, J. (2005). Alternative processing
solutions for Q. rubra (Quercus rubra) from converted forests in Flanders, Belgium.
Proceedings of the COST Action E44 Conference on Broad Spectrum Utilization of
Wood Vienna, Austria).
Vor, T. (2005). Natural regeneration of Quercus rubra L. (red oak) in Germany.
Neobiota 6, 111–123.
Walters, M. B., Kunkle, J. M., Kob e, R. K., and Farinosi, E. J. (2023). Seedling drought
responses governed by root traits, site-soil moisture regimes and overstory competition-
facilitation. For. Ecol. Manag. 544:121159. doi: 10.1016/j.foreco.2023.121159
Woziwoda, B., Gręda, A., and Frelich, L. E. (2023). High acorn diversity of the
introduced Quercus rubra indicates its ability to spread eciently in the new range. Ecol.
Indic . 146:109884. doi: 10.1016/j.ecolind.2023.109884
Yi, K., Dragoni, D., Phillips, R. P., Roman, D. T., and Novick, K. A. (2017). Dynamics
of stem water uptake among isohydric and anisohydric species experiencing a severe
drought. Tree Physiol. 37, 1379–1392. doi: 10.1093/treephys/tpw126
Zadworny, M., Comas, L. H., and Eissenstat, D. M. (2018). Linking ne root
morphology, hydrau lic functioning and shade tolerance of trees. Ann. Bot. 122, 239–250.
doi: 10.1093/aob/mcy054
Zadworny, M., Mucha, J., Jagodziński, A. M., Kościelniak, P., Łakomy, P.,
Modrzejewski, M., et al. (2021). Seedling regeneration techniques aect root systems
and the response of Quercus robur seedlings to water shortages. For. Ecol. Manag.
479:118552. doi: 10.1016/j.foreco.2020.118552