Discussion
Cold is a major abiotic stressor to consider when adapting agriculture
to the challenges of climate change, such as avoiding drought or growing
short-cycle crops in northern regions. Seed germination and emergence
are crucial for establishing crops, followed by the vigor and growth of
above- and below-ground parts. Optimal vigor improves nutrient
competition with weeds or after early herbicide treatment, enhances
resistance to pathogens and increases resilience to abiotic stress.
Thus, breeding for vigor is essential to benefit from ES (Houmanatet al. , 2016; Walne and Reddy, 2022).
Assessing vigor usually involves observing above-ground parts, but
assessing the root system is equally important. For instance, a robust
root system that can grow into deeper soil layers allows plants to
withstand more severe drought stress later in the season (Cutforthet al. , 1986; Lamichhane et al. , 2018). Understanding the
influence of chilling on early developmental stages of sunflower is
crucial to identify the components of early cold tolerance, which
increases oil yield in the context of ES.
These field experiments highlight the impacts of ES on early
morphological traits. We observed an 80% decrease in the rate of vigor
growth and a 42% decrease in the rate of height growth. However, in
these ES experiments, low temperatures were correlated with the
daylength and solar radiation intensity. To assess impacts of low
temperatures alone, we developed an indoor experiment that reproduced
the night chilling stress observed and modeled in a French field
environmental network (Mangin et al. , 2017).
Sunflower responded similarly to night chilling (i.e., decrease in leaf
area, hypocotyl diameter, root length and number of secondary roots) as
maize did in other studies (Hussain et al., 2020; Walne and
Reddy, 2022). Low temperatures decreased sunflower development,
resulting in smaller leaves and roots. These phenotypes likely result
from direct effects of low temperatures on plant physiology: cold
prolongs the cell cycle and slows the growth rate, which results in
smaller organs (Rymen et al. , 2007). Low temperatures also
influence the metabolism of auxin, which strongly regulates plant
growth. Rahman (2013) observed that cold stress immobilized the cellular
auxin transport protein and altered the auxin gradient usually
associated with organ formation. This influence could explain the
decrease in root length and increase in root diameter that we observed
for sunflower. A decrease in the root system influences water and
nutrient uptake, which is high near root tips due to increased
expression of nutrient transporters and water channels. Branching is an
effective way to increase the absorptive surface area of the root system
(Dinneny, 2019), which ultimately decreases the yield (Hammer et
al. , 2009). The decrease in leaf area under low temperatures is caused
by lower cell division and cell elongation (Ben-Haj-Salah and Tardieu,
1995), which decreases light interception and thus the yield (Merrien,
1992).
Since early chilling stress decreases oil yield, we explored the
morpho-physiological mechanisms underlying this long-term effect. We
observed no increase in plant height at flowering despite a notable
difference in the number of days required for flowering (+33.2% for
ES). Oil yield increased (80%), as did seed weight and oil content
(52.5% and 16.8%, respectively). Previous studies, such as those of
Tahir et al. (2009), Demir (2019) and Abdelsatar (2020), also
observed an increase in oil content (+7.7%), thousand kernel weight
(+27.2%) and oil yield (+43.2%) with earlier planting dates for
sunflower and wheat. The longer life cycle allowed plants to accumulate
more photo-assimilates and remobilize them to seeds, as suggested by
Evans and Wardlaw (1976) and Giannini et al. (2022) for wheat and
rice. However, the relation between the duration of the vegetative phase
and yield is complex. Some studies observed a positive correlation
between the duration of vegetative growth and yield for sunflower
(Gontcharov and Zaharova, 2008; Abdelsatar, 2020), while other studies
observed a higher yield with a shorter vegetative phase and longer
grain-filling phase for wheat (Sharma, 1992). This complexity is likely
due to interactions among multiple abiotic stressors (e.g., early cold,
late drought) and low temperatures combined with low solar radiation
after ES.
In the present study, UAV and platform phenomics helped distinguish the
impact of night chilling alone on late developmental stage and showed
that it had no influence on leaf area or later growth rates, but did
influence flowering time and thus growth duration, resulting in taller
plants and more biomass. These results agree with those of previous
studies of wheat (Tahir et al., 2009) and sunflower (Alkioet al. , 2003; Demir, 2019). Ferreira and Abreu (2001) observed
similar results for sunflower, with a 29% increase in the number of
days between emergence and flowering with ES compared to normal sowing.
These morphological changes reflect underlying physiological processes
related to cell division, extension, energy pathways and abiotic
signaling. One well-documented physiological process related to cold
resistance is an increase the unsaturation of fatty acids in cell
membranes. We observed similar results, but specifically in hypocotyls
rather than roots or leaves. This hypocotyl response has not been
reported for other plants, but it has been observed for rapeseed
(Brassica napus) , maize and wheat leaves and roots at varying
levels of polyunsaturation (Tasseva et al. , 2004; Makarenkoet al. , 2011; Nejadsadeghi et al. , 2015).
Low temperatures decrease cell membrane fluidity and permeability, which
results in electrolyte leakage and alters internal cell homeostasis
(Barrero-Sicilia et al. , 2017). Destabilization of the
chloroplast membrane has a cascade of negative effects on chlorophyll
content, photosynthetic enzyme activity and the electron-transport chain
(Banerjee and Roychoudhury, 2019), resulting in the production of ROS
until chlorosis symptoms appear (Wise, 1995; Suzuki and Mittler, 2006).
Our results agreed with those of Fabio et al. (2022), given the
10.3% decrease in chlorophyll content and the 9.0% and 7.2% increase
in H2O2 and
O2·-, respectively. Hussain et
al. (2020) observed a 100% and 120% increase in
H2O2 and
O2·-, respectively, in maize leaves.
Zhu et al. (2013) observed similar results for sugarcane
(Saccharum officinarum ) leaves, as did Nejadsadeghi et al.(2015) for wheat leaves. As ROS concentrations increase in organs, the
risk of damaging proteins, DNA and lipids increases (Apel and Hirt,
2004). This negative impact on plant physiology is managed by the ROS
scavenging mechanism, which involves superoxide dismutase, ascorbate
peroxidase, catalase and glutathione peroxidase (Cassia et al. ,
2018). These results demonstrate the complex and interconnected
physiological responses of sunflower to cold stress. Our results
indicate that night chilling decreases the stability of cell membranes,
which leak electrolytes. Disruption of chloroplast membranes is followed
by a decrease in chlorophyll content and production of ROS due to the
decrease in the efficiency of the electron-transport chain, which
results in cell damage (Fig. 10).
To reveal the molecular mechanisms underlying the physiological
responses observed, we explored the transcriptomic responses of leaves,
hypocotyls and roots under chilling stress. The effects of cold stress
were especially pronounced in leaves. Histone genes were the most
abundant group of DEGs in leaves, which is consistent with results of
Kumar and Wigge (2010) for A. thaliana that showed the critical
role of histone H2A.Z in sensing temperature. This histone binds to the
promoters of temperature-responsive genes, which can inhibit the binding
of repressors. It is therefore plausible that an epigenetic mechanism of
stress memory had long-term effects throughout the sunflower life cycle
in our experiments.
Many other DEGs belonged to groups which are potentially linked to
chilling stress, i.e. lipases, germin-like proteins, glycine-rich
proteins, chaperones, heat shock proteins (HSP), and late embryogenesis
abundant proteins (Winfield et al. , 2010; Barrero-Siciliaet al. , 2017). ). Interestingly, we observed no DEGs related to
the ICE-CBF-COR cascade, perhaps due to different sampling strategies.
We sampled tissues after three weeks of exposure to chilling stress,
while these genes are usually found to be differentially expressed after
3-48 h of treatment (Lee et al. , 2005; Song et al. , 2013;
Londo et al. , 2018), which highlights the time-dependent nature
of gene expression in response to chilling.
Analysis of DEGs confirmed the importance of histone and nucleosome
activity, and highlighted other functions potentially related to the
chilling response. This was the case for the ontologies related to
carbohydrate metabolism, whose induction has been identified in peanuts
(Arachis hypogaea ) (Zhang et al. , 2020) and grapevine
(Vitis vinifera ) (Londo et al. , 2018). This was also
observed for ontologies related to ROS metabolism, which have been
described as a chilling stress response in species such as cotton
(Gossypium herbaceum ) (Tang et al. , 2021)
and Chinese cottonwood
(Populus simonii ) (Song et al. ,
2013). The increase in ROS
production, particularly under high light levels, could be associated
with photoinhibition (Banerjee and Roychoudhury, 2019), resulting in the
degradation of chlorophyll, which may explain the decrease in
chlorophyll content in leaves during the cold stress.
The transcriptomic profile of hypocotyls was characterized by two main
processes. The first was lipid metabolism, including lipid unsaturation,
which was represented by two genes and is consistent with the fatty acid
profiles of hypocotyls. Lipid unsaturation in response to chilling
stress is usually observed in leaves (Barrero-Sicilia et al. ,
2017; Zhang et al. , 2020). Our study is the first to identify
this process in hypocotyls. Three other lipid metabolism terms in
response to chilling were alpha-linolenic acid, glycerolipid and
glycerophospholipid metabolisms, all of which are related to membrane
remodeling. To date, these pathways have been described only in leaves,
such as those of peanuts (Zhang et al. , 2020).
The second main process that characterized hypocotyl transcriptomes was
the biosynthesis of cutin, suberin and wax, which was represented by 29
DEGs and confirmed by enrichment and deregulation analyses. The leaf
cuticle is the first line of physical defense against abiotic stresses
(Barrero-Sicilia et al. , 2017), and activation of cutin-related
pathways under chilling stress has been described, for instance in
rubber trees (Hevea brasiliensis ) (Gong et al. , 2018).
However, the role of cutin in plants is usually described in leaves.
In addition to these two main processes, other relevant pathways related
to chilling stress were identified in hypocotyls. First, phenylpropanoid
metabolism, represented by several key DEGs, is homologous to theA. thaliana EARLI1 and phenylalanine ammonia lyase genes,
respectively. The latter is induced by low temperatures in the
hypocotyls of A. thaliana and leaves of rapeseed (Cabane et
al. , 2012) and is related to enhanced lignin synthesis, which increases
the mechanical resistance of cell walls and reduces dehydration.
Overexpression of EARLI1, a lipid transfer protein, reduces
electrolyte leakage during freezing stress, which suggests that it helps
maintain membrane stability (Bubier and Schläppi, 2004).
To better understand the response of sunflower to abiotic stresses, we
adapted the ROS wheel developed for A. thaliana (Willems et
al. , 2016) to the sunflower genome (Badouin et al. , 2017). Doing
so highlighted the relevance of ROS metabolism in hypocotyls, with two
enriched ontologies and six ROS-wheel-related deregulated pathways, and
indicated that hypocotyls and leaves had similar responses to chilling.
Our transcriptomic experiment also revealed a role of the anti-oxidant
vitamin B1 (thiamine) (Subki et al. , 2018). Chilling stress did
not influence the root transcriptome greatly under hydroponic
conditions, but it did deregulate the metabolism of the anti-oxidant
vitamin B2 (riboflavin). Riboflavin has anti-oxidant properties in
several plants, including apples (Malus spp.) (Zha et al. ,
2022). Identification of pathways related to ROS metabolism and
scavenging in the three organs highlighted the major role of these
processes in the cell response to chilling.
We reproduced field conditions in spring in France to characterize
sunflower after three weeks of exposure to moderate cold stress (night
chilling). In this context, we observed no expression of well-known
genes such as those involved in the ICE-CBF-COR cascade, which are
usually induced shortly after exposure to cold stress. Overall, this
shows that the transcriptome is highly plastic and suggests a sequential
action of genes. The literature indicates that during the first few
hours of exposure, the main genes are related to signal transduction and
protein synthesis, indicating a potentially active response strategy. In
contrast, after three weeks, we observed genes associated with long-term
changes, such as those involved in lignin and lipid metabolism, which
alters membrane lipid composition, which may be a passive strategy that
requires less energy to resist stress by structurally modifying plants
and their reactivity through epigenetic changes.
To study long-term impacts of early night chilling, we characterized
sunflower using high-throughput phenotyping platforms combined with
agronomic and biochemical measurements. Our field observations (e.g.,
19% increase in C16:0, 44% decrease in oleic acid, 45% increase in
linoleic acid) agreed with observations of Flagella et al. (2002)
for sunflower, suggesting that the activation of desaturases required in
early stages to maintain membrane fluidity remained in the seeds three
months later. Similarly, the chlorophyll content increased consistently
throughout the life cycle in leaves that had not developed at the time
of stress. This result suggests that molecular memory may be transmitted
through cell division, likely epigenetic changes, such as DNA
methylation, phosphorylation or histone acetylation (Trewavas, 2016),
whose stability is related to memory duration (Villagómez-Arandaet al. , 2022). Although our data did not identify these
epigenetic markers, such as DNA methylation, in later developmental
stages, future studies could explore long-term effects of morphological
modifications induced in early stages or due to epigenetic
modifications.