Translational Insights into Abiotic Interactions: From Arabidopsis to Crop Plants

Adrienne H.K. Roeder¹*, Yiting Shi², Shuhua Yang², Mohamad Abbas³, Rashmi

Sasidharan³, Marcelo J. Yanovsky⁴, Jorge José Casal⁴,⁵, Sandrine Ruffel⁶, Nicolaus von

Wirén⁷, Sarah M. Assmann⁸, Noah A. Kinscherf⁸, ⁹, Arkadipta Bakshi¹⁰, Burcu

Alptekin¹⁰,¹¹, Simon Gilroy¹⁰, Malleshaiah SharathKumar¹², Salomé Prat¹³ and Cristiana

T. Argueso¹⁴*

#Authors listed in the order in which their vignettes appear

* Authors for correspondence: A.H.K.R (ahr75@cornell.edu) and C.T.A

(cris.argueso@colostate.edu)

¹School of Integrative Plant Science, Section of Plant Biology and Weill Institute for Cell
and Molecular Biology, Cornell University, Ithaca, NY 14853 USA

²State Key Laboratory of Plant Environmental Resilience, Frontiers Science Center for
Molecular Design Breeding, College of Biological Sciences, China Agricultural
University, Beijing, China

³Plant Stress Resilience, Institute of Environmental Biology, Utrecht University, The
Netherlands

⁴Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires,
Consejo Nacional de Investigaciones Científicas y Técnicas, 1405 Buenos Aires,
Argentina

⁵Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura,
Facultad de Agronomía, Universidad de Buenos Aires, Consejo Nacional de
Investigaciones Científicas y Técnicas, 1417 Buenos Aires, Argentina

⁶Institute for Plant Sciences of Montpellier, Univ. Montpellier, CNRS, INRAE, Montpellier,
France

⁷Molecular Plant Nutrition, Department of Physiology & Cell Biology, Leibniz Institute of
Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben,
Germany

⁸Department of Biology, Penn State University, 208 Mueller Laboratory, University Park,
PA, 16802, USA

⁹Huck Institutes Intercollege Graduate Degree Program in Plant Biology

¹⁰Department of Botany, University of Wisconsin-Madison, Birge Hall, 430 Lincoln Drive,
Madison, WI 53706

¹¹Department of Bacteriology, 5345 Microbial Sciences Building, 1550 Linden Drive,
Madison, WI 53706

¹²Horticulture and Product Physiology, Department of Plant Sciences, Wageningen
University and Research, Wageningen 6700 AA, the Netherlands

¹³Centre for Research in Agricultural Genomics (CRAG), Barcelona, 08193 Spain

© The Author(s) 2025. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals permissions@oup.com.
1 <sup>14</sup>Department of Agricultural Biology, Colorado State University, Fort Collins, CO 80523

2 USA

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4 **Short Title:**

5 Arabidopsis abiotic research translation

6

7 The authors responsible for distribution of materials integral to the findings presented in

8 this article in accordance with the policy described in the Instructions for Authors

9 (https://academic.oup.com/plcell/pages/General-Instructions) are: Adrienne H.K. Roeder

10 (ahr75@cornell.edu) and Cristiana T. Argueso (cris.argueso@colostate.edu)

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12 **Abstract:**

13 Understanding crop plants responses to abiotic stress is increasingly important in this
14 changing climate. We asked experts how discoveries in *Arabidopsis thaliana* have
15 translated into advancements in abiotic crop stress resilience. The theme is that core
16 regulatory networks identified in Arabidopsis are conserved in crops, but the molecular
17 regulation varies among species. For cold tolerance, the regulatory framework is
18 conserved, but MAP Kinase signaling promotes degradation of the INDUCER OF
19 DREB1 EXPRESSION (ICE) transcription factor in Arabidopsis but inhibits it in rice. For
20 hypoxia, manipulation of the oxygen sensing Arg/N-degron pathway discovered in
21 Arabidopsis has improved waterlogging and flood tolerance in barley, maize, wheat, and
22 soybean. For light signaling, overexpression of PHYTOCHROME B reduces shade
23 avoidance, improving yield under dense planting in potato, soybean, and maize. In rice,
24 understanding of nitrogen responsiveness, uptake, and transport in Arabidopsis has
25 inspired engineering of the NRT1 nitrate transceptor to increase yield. Overexpressing
26 Arabidopsis genes in crops confers drought tolerance, although none have been
27 commercialized. Growing plants in space generates a complex array of stresses, and
28 Arabidopsis experiments in the space station prepare for future development of robust
29 crops as integral components of the life support systems. For environmental regulation
30 of flowering time, the role of the GIGANTEA (GI) - CONTANS (CO) - FLOWERING
31 LOCUS T (FT) module elucidated in Arabidopsis is largely conserved in crop plants,
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although additional regulators modify short day responsiveness in rice, soybean,

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chrysanthemum, and potato.

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Introduction

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Abiotic stress is a major contributor to crop yield loss, with factors such as extreme temperatures, changes in light, drought prevalence and nutrient deficiencies challenging the ability of plants to thrive (reviewed in work cited by Eckhardt et al., 2023).

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Because plants are often ill-equipped to cope with stress conditions outside those they have evolved to endure, the growing unpredictability of climate patterns exacerbates the negative impact of these stresses on agricultural productivity (Ray et al., 2019). To address this, breeding plants that are more resilient to abiotic stress is crucial for securing food production (Cooper and Messina, 2023). However, engineering plants for abiotic resilience, through genome editing and/or synthetic biology efforts, requires a comprehensive understanding of the genetic pathways that govern these stress responses.

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Studies on plant adaptation to extreme environments, including space—where agricultural production may be essential in the future—have greatly benefited from the ease of cultivation and advanced genetic tools provided by the plant model species *Arabidopsis thaliana* (Yashencko et al., 2024, in this Focus Issue), hereafter *Arabidopsis*. Even though different plant species have evolved in distinct environments, withstanding different abiotic stresses, work on Arabidopsis has been instrumental in uncovering the genes and molecular networks involved in abiotic stress signaling and adaptation, many of which are conserved in crops. While challenges translating what has been learned in Arabidopsis into crops do exist (highlighted in Uauy et al., 2025, also in this Focus Issue), this review explores how some of the insights gained from Arabidopsis have informed the development of crop plants with increased abiotic stress tolerance, in relation to cold stress, hypoxia, light signaling, drought, nitrogen use efficiency, growth in space and flowering under different daylengths, also highlighting future avenues for enhanced crop productivity under rapid environmental change.

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1 Cold/Freezing Tolerance

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3 (Written by Yiting Shi and Shuhua Yang)

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As sessile organisms, plants constantly face temperature fluctuations, with cold stress posing significant threats to different developmental stages such as germination, seedling growth, and reproduction. The increasing frequency of extreme low-temperature events due to climate change exacerbates crop vulnerability, threatening agricultural productivity. Understanding the molecular mechanisms underlying plant cold responses is essential for developing resilient crop varieties.

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Plants have evolved sophisticated biochemical and molecular defense mechanisms to cope with cold stress. Temperate plants employ cold acclimation, a physiological and biochemical adjustment triggered by exposure to sub-lethal low temperatures, as a primary survival strategy (Thomashow, 1999). Arabidopsis has been instrumental in elucidating this process, particularly through the characterization of the DREB1/CBF-COR regulatory signaling pathway (Thomashow, 1999; Shi et al., 2018). The discovery of DEHYDRATION RESPONSIVE ELEMENT (DRE)-BINDING 1/C-REPEAT BINDING FACTOR (DREB1/CBF) transcription factor in the 1990s marked a major breakthrough in understanding cold stress responses (Jaglo-Ottosen et al., 1998; Liu et al., 1998) (Figure 1A). *DREB1* genes are rapidly induced by cold stress, encoding transcription factors that regulate COLD-RESPONSIVE (COR) genes to mitigate cold-induced damage (Thomashow, 1999; Song et al., 2021). *DREB1* expression is tightly regulated by transcription factors, including the INDUCER OF DREB1 EXPRESSION (ICE), CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR (CAMTA), NIN-LIKE PROTEIN 7 (NLP7), and MYB15 (Ding et al., 2024). Plant hormone signaling also modulate *DREB1*: brassinosteroid and strigolactone enhance its expression, whereas the role of ethylene appears to be species-dependent (Ding et al., 2024). Additionally, *DREB1* expression is governed by light signaling and circadian clock (Kidokoro et al., 2022; Ding et al., 2024). This intricate regulatory network presents both challenges and opportunities, making the DREB1 pathway a potential target for genetic manipulation to optimize cold tolerance in crops.
Plants perceive cold signals via specialized cold sensors, triggering signal transduction pathways mediated by secondary messengers (Figure 1B). Rice CHILLING TOLERANCE DIVERGENCE 1 (COLD1) acts as a cold sensor that enhances cold tolerance by activating Ca²⁺ signaling (Ma et al., 2015). In Arabidopsis, cold stress induces the activation of calcium-permeable transporters and channels, such as ANNEXINS and CYCLIC NUCLEOTIDE-GATED CHANNELS (CNGCs), generating calcium signals (Liu et al., 2020; Peng et al., 2024). Downstream of these signals, calcium-related protein kinases, including CDPKs and CIPKs, have been reported to play a role in regulating cold tolerance in plants (Ding et al., 2022, 2024). Moreover, a recent study revealed that COLD6 mediates cold responses through 2',3'-cAMP signaling in rice (Luo et al., 2024). Nonetheless, the full repertoire of cold sensors and the underlying molecular mechanisms require further investigation.

Cold stress responses involve both positive and negative regulatory kinases that fine-tune cold tolerance. OPEN STOMATA 1 (OST1), a key regulator in the abscisic acid (ABA) signaling pathway (Yamaguchi-Shinozaki and Shinozaki, 2006), is activated by cold stress in an ABA-independent manner and phosphorylates its targets, including ICE1 and the E3 ligases PUB25/26, increasing their stability and/or activity, which in turn enhances *DREB1* expression and freezing tolerance (Ding et al., 2024). In contrast, COLD-RESPONSIVE PROTEIN KINASE 1 (CRPK1) and MPK3/6 negatively regulate freezing tolerance by destabilizing DREB1 and ICE1 proteins, respectively (Liu et al., 2017a; Chen et al., 2021). These findings reveal a complex and dynamic regulatory network to modulate plant cold stress responses.

Arabidopsis cold signaling research provides a basis for identifying homologous genes in diverse crop species. For example, the functions of CBF/DREB1 homologs have been characterized in many crops, such as rice, wheat, maize, barley and rye (Nie et al., 2022). In rice, the OST1 homolog OsSAPK8 phosphorylates and activates OsCNGC9 (Wang et al., 2021a). These findings highlight the value of comparative genomics in elucidating evolutionarily conserved components of cold responses. However, emerging evidence also points functional divergence of homologous genes among species. For instance, while MPK3 promotes ICE1 protein degradation in Arabidopsis, its rice homolog instead enhances the stability of OsICE1 through
1 phosphorylation (Zhang et al., 2017a). Such differences highlight the need for species-
2 specific strategies when translating insights from Arabidopsis to agriculturally crops.

3 Extensive studies on natural genetic variation has revealed key alleles associated
4 with cold tolerance in crops. In rice, polymorphisms in COLD1, COLD6, COLD11, COG1
5 (Chilling-tolerance in Gengdao/japonica rice 1), qLTG3-1 (Low-temperature
germinability on chromosome 3), LTG1,and bZIP73 confer the divergence in seedling-
stage cold tolerance among different varieties (Ma et al., 2015; Liu et al., 2018a; Li et
8 al., 2023; Xia et al., 2023; Luo et al., 2024), while natural variants of COLD
9 TOLERANCE AT BOOTING STAGE (CTB4a), and CTB5 enhance rice cold tolerance
during the booting stage (Zhang et al., 2017b; Guo et al., 2025). In maize, natural
alleles of ZmICE1, ZmHSF21, ZmTIP4;3, and ZmRR1 have been identified to improve
11 cold tolerance among maize inbred lines (Zeng et al., 2021; Jiang et al., 2022; Yang et
12 al., 2023; Gao et al., 2024). A natural variant of bZIP68 from teosinte, the wild ancestor
13 of maize, also contributes to enhanced maize cold tolerance (Li et al., 2022a). These
14 findings highlight the value of both cultivated and wild germplasm for identifying superior
15 alleles for improving cold resilience in crops.
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17 Population genetics studies have revealed evidence of cold-driven adaptive
18 evolution in plants. Natural variation in CBF2 in Arabidopsis correlates with local
19 adaptation across different ecotypes, while copy number variations at the HvCBF locus
20 in barley are similarly linked to cold tolerance (Gehan et al., 2015; Francia et al., 2016).
21 In wheat, variations in TaVRN1 and TaHY5-like genes play crucial roles in
22 environmental adaptation (Kippes et al., 2018; Guo et al., 2020a). In rice, the selection
23 of favorable alleles in genes such as natural variation of COLD1, COLD6, HAN1,
24 COG1, CTB4a, CTB5, and bZIP73 has facilitated the adaptation of japonica rice to high-
25 latitude or high-altitude regions (Ma et al., 2015; Zhang et al., 2017b; Liu et al., 2018a;
26 Mao et al., 2019; Xia et al., 2023; Luo et al., 2024; Guo et al., 2025). A recent
27 breakthrough study in maize uncovered that a natural variant of the COOL1 (COLD-
RESPONSIVE OPERATION LOCUS 1) gene contributes to enhanced cold tolerance
29 and high-latitude adaptation (Zeng et al., 2025). These evolutionary and population
30 genomic insights offer promising directions for breeding cold-resilient crop varieties.
While key components of cold signaling network show remarkable evolutionary conservation across plant species (Nie et al., 2022), their direct application in crop improvement often faces challenges due to growth-defense trade-offs (Zhang et al., 2020). To address these challenges, several strategies can be employed: 1) Utilizing tissue-specific or stress-inducible promoters to achieve precise spatiotemporal regulation of key genes; 2) Identifying key regulatory elements or amino acid residues within the cold regulatory networks as genome-editing targets to fine-tune activity; and 3) Exploring natural variations to improve cold tolerance without sacrificing growth or yield (Figure 1C). Recent advances in artificial intelligence and genome editing offer powerful tools to accelerate the development of climate-smart crop varieties capable of thriving in increasingly unpredictable climates.

## Hypoxia

(Written by Mohamad Abbas and Rashmi Sasidharan)

Plant tissues experience hypoxia or sub-ambient oxygen conditions, when oxygen demand exceeds availability. Hypoxic conditions imposed by abiotic factors include flooding events, winter ice encasement, and soil compaction (Figure 2). Reduced oxygen availability impairs oxidative phosphorylation-mediated ATP generation and negatively impacts cellular energy status. Hypoxia can also occur naturally during development, in densely packed tissues with high respiratory demand such as seeds, pollen, meristems, and tubers, and are vital signals shaping developmental programs (Figure 2). Whether in the developmental or environmental stress context, plants need to sense prevailing oxygen conditions and react appropriately to adjust developmental programs or trigger stress-acclimation/adaptive mechanisms. Arabidopsis research has been instrumental in progressing our understanding of how plants perceive, transduce, and respond to low oxygen signals in environmental and developmental settings.

The negative consequences of abiotic environment-induced hypoxia and how plants tolerate it have been extensively studied in the context of flooding. Submerged plant tissues experience impaired gas exchange due to diminished gas diffusion rates in
water (Figure 2). Historically, research in this area focused on tolerant species, investigating flood adaptive traits such as aerenchyma or adventitious roots. Studies in Arabidopsis were few but intensified following the sequencing of its genome. Facilitated by a genome sequence, facile transient and stable transformation, microarray technology, community resources/tools, and the relative ease of genetic investigations, these early studies provided the first detailed insights into hypoxia-activated signaling and identified regulatory components (Sedbrook et al., 1996; Peng et al., 2001; Hunt et al., 2002; Shiao et al., 2002; Baxter-Burrell et al., 2002; Tournaire-Roux et al., 2003; Gonzali et al., 2005; Liu et al., 2005; Mustroph et al., 2009). Despite this rich body of work implicating roles for transients in calcium, reactive oxygen species, and ATP, evidence of a direct oxygen-sensing mechanism in plants proved elusive. In 2011, two independent studies identified an Arg/N-degron pathway mediating oxygen sensing in Arabidopsis (Licausi et al., 2011; Gibbs et al., 2011). This involved oxygen-dependent targeted proteolysis of Group VII ETHYLENE RESPONSE FACTORs (ERFVIIIs) transcription factors mediating hypoxia-responsive gene expression. Both studies stemmed from intriguing observations of altered hypoxia responses in Arabidopsis mutants and transgenics. Arabidopsis has five ERFVIIIs all of which are substrates of the N-degron pathway. In the presence of oxygen, oxidation of a conserved N-terminal cysteine residue marks these ERFVIIIs for recognition and degradation by the N-degron pathway machinery leading to proteasomal degradation (Figure 2A).

The discovery of this molecular mechanism of oxygen sensing spurred extensive follow-up studies in Arabidopsis and crops. There was further characterization of the N-degron pathway components and greater scrutiny of the molecular features, regulation, and functions of the ERFVIIIs (e.g. Gasch et al., 2016; Weits et al., 2014). The rice ERFVII SUBMERGENCE1 SUB1A-1 confers tolerance to complete submergence by restricting growth and limiting energy expenditure (the so-called quiescence survival strategy), and facilitating post-submergence recovery (Emerick and Ronald, 2019). Despite possessing a similar N-terminal degron sequence and function in triggering hypoxia responses and mediating submergence tolerance, SUB1A was found not to be an N-degron substrate *in vitro* (Gibbs et al., 2011). Subsequent studies have shed light on regulatory interactions between ERFVIIIs and rice hypoxia sensing and comparisons
1 of the flooding-induced quiescence between rice and Arabidopsis (Lin et al., 2019;
2 Reynoso et al., 2019).
3 Importantly, the Arabidopsis studies revealed the N-degron pathway, controlling
4 ERFVII stability, to be an attractive target for altering flood tolerance. The oxygen-
5 sensing PLANT CYSTEINE OXIDASEs (PCOs), catalyzing the N-terminal cysteine
6 oxidation of ERFVIIs (Figure 2A), are particularly promising in this respect. Efforts
7 towards unraveling the biochemical features of these enzymes could be instrumental for
8 conditional manipulation of PCO activity via engineering of their oxygen sensitivity or
9 substrate specificity (Taylor-Kearney et al., 2022). The manipulation of N-degron E3
10 ligase PROTEOLYSIS 6 (PRT6) expression in waterlogging sensitive barley,
11 significantly improved plant performance and yields (Mendiondo et al., 2016). ERFVIIs
12 have been successfully characterized and targeted to improve flood tolerance in other
13 crops such as maize, wheat, and soybean (Loreti and Perata, 2023). Refined
14 knowledge of ERFVII target binding sites could be useful in promoter editing to tune the
15 expression of key regulators and enzymes (Gasch et al., 2016; Lee et al., 2019;
16 Reynoso et al., 2019).

17 Recent Arabidopsis work has established how flood sensing and signaling
18 encompasses a complex network involving interactions between oxygen, hormones
19 (Hartman et al., 2019);, redox (Sasidharan et al., 2021), energy (Kunkowska et al.,
20 2023), and nutrients (Shahzad et al., 2016) sensing pathways. Mechanisms unraveling
21 hypoxia-mediated modulation of root architecture (Shukla et al., 2019; Eysholdt-Derzsó
22 et al., 2024) and post-hypoxic injury (Yeung et al., 2018) have provided important
23 knowledge on flood resilient traits. Vital post-submergence recovery mechanisms
24 shared by rice and Arabidopsis underscore further possibilities for translation.
25 Arabidopsis hypoxia and flooding-induced molecular responses remain a key
26 comparative benchmark for multi-species comparisons assessing how variation in
27 hypoxia tolerance is determined at the molecular level (Reynoso et al., 2019; van Veen
28 et al., 2024).

29 Plants do not always perceive hypoxia as stressful but as a developmental cue.
30 Hypoxia occurring due to soil compaction impacts developmental processes such as
31 seed germination, seedling establishment, and the transition to photomorphogenesis via
1 the N-degron components PRT6 and ARGININE-TRNA PROTEIN TRANSFERASE 1
2 and 2 (ATE1,2) (ATEs) (Figure 2) (Holman et al., 2009; Gibbs et al., 2011; Abbas et al.,
3 2015). PRT6 and the ERFVIIIs constitute a molecular mechanism responsible for
4 adaptation to altitudes, where the partial pressure of oxygen naturally changes based
5 on elevation (Abbas et al., 2022) (Figure 2). Knowledge of the role of chronic hypoxia as
6 a developmental regulator has greatly benefitted from Arabidopsis
7 research. Arabidopsis shoot apical meristems maintain 3-6 % oxygen (Figure 2), which
8 is essential for the N-degron substrates VERNALIZATION2 (VRN2) and LITTLE
9 ZIPPER 2 (ZPR2) to modulate flowering and meristematic activity (Gibbs et al., 2018;
10 Weits et al., 2019). Hypoxic niches of advanced lateral root primordia (LRP) allow
11 stabilization of ERFVIIIs and their binding to AUXIN RESPONSE FACTOR 7 (ARF7).
12 This inhibits LRP gene expression, mainly LOB DOMAIN-CONTAINING PROTEIN 16
13 and 18 (LBD16,18), and contributes to the optimal spacing between LRs and root
14 branching (Shukla et. al., 2019).

15 The discovery of the molecular sensing mechanism whereby plants sense oxygen
16 and in parallel nitric oxide (NO) was a game changer. Oxygen-requiring pathways in
17 plants are diverse and oxygen is involved in many enzymatically catalyzed redox
18 reactions. In theory, any enzyme that facilitates redox reactions could potentially
19 function as an oxygen sensor, especially when oxygen levels drop below the enzyme's
20 affinity constant for its substrate. Thus, while the PCO branch of the N-degron pathway
21 is the current main oxygen sensor in plants, the discovery of new oxygen sensors will
22 not be surprising.
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23 Our understanding of the molecular mechanism of oxygen sensing is relatively new
24 (<15 years ago). The rich knowledge base established since retains Arabidopsis's
25 status as an important model to address new questions and existing knowledge gaps.
26 This includes determining the site of NO action, possible substrates of the PCO branch
27 of the N-degron pathway, and identification of, as yet, unknown oxygen sensors. How
28 plants differentiate between developmental and environment-induced hypoxia remains
29 an intriguing question. Understanding these mechanisms and their engineering can aid
30 the improvement of crop resilience to hypoxia-related challenges, including yield loss
31 from floods and post-harvest preservation. Arabidopsis still boasts an impressive
1 toolbox in molecular biology and genetics. Combined with the publicly accessible 1001

2 Genomes collection it remains a vital model for further comprehension of hypoxia stress

3 and resilience and will be needed to ensure that more success stories like that of SUB1

4 rice will emerge.

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6 **Light signaling**

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8 (Written by Marcelo J. Yanovsky and Jorge José Casal)

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10 Plant tolerance to high population densities (crowding) and plant cycle duration are
11 crucial agronomic traits that significantly impact crop resource utilization and stress
tolerance. Crowding intensifies mutual shading within the canopy, prompting
12 architectural adaptations in response to changes in light quality and quantity. These
13 adaptations directly modulate crowding tolerance and ultimately determine the optimal
14 planting density (Figure 3). Sowing date and location along the latitudinal axis influence
15 the photoperiod experienced by the crop, which, depending on plant photoperiod
16 sensitivity, dictates flowering time, the length of the plant cycle, and consequently, the
17 optimal sowing window (Figure 3). This section demonstrates how studying Arabidopsis
18 mutants with altered seedling phenotypes under varying light and dark conditions has
19 provided valuable insights into the genetic underpinnings of these critical crop
20 responses.
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22 The reduced red/far-red light ratios and UVA-blue irradiance caused by light
absorption and reflection from green leaves (canopy light cues) respectively diminish
23 the activities of phytochrome B (PHYB) (Reed et al., 1993) and cryptochrome 1 (CRY1)
24 (Ahmad and Cashmore, 1993), the primary photoreceptors governing plant responses
25 to shade (Casal and Fankhauser, 2023). Accurately simulating the profound impact of
26 dense canopies on these light cues without compromising photosynthetic light
27 availability is extremely challenging. The identification and functional characterization of
28 PHYB and CRY1 genes in Arabidopsis facilitated the finding of mutants in various crop
29 species, revealing a deeper-than-expected influence of photomorphogenesis. For
30 instance, ZmphyB and Zmcry1b mutants in maize exhibit reduced leaf expansion in field
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conditions, suggesting that the decreased leaf area per plant in high-density crops is a
sensory response rather than solely a consequence of resource competition (Wies et
al., 2019; Chen et al., 2024). Studies in Arabidopsis have also provided a valuable
model system to understand the molecular mechanisms by which canopy shade
regulates important agronomic traits. For example, low blue light reduces the protective
effect of GmCRY1 on DELLA proteins in soybean, and this enhances the expression of
the GmWRKY100 transcription factor, ultimately leading to basal leaf senescence (Li et
al., 2024a).

Arabidopsis mutants revealed that, contrary to PHYB, phytochrome A (PHYA) activity
is maximal under far-red light (Quail et al., 1995) and phyA mutants fail to survive under
deep shade, rich in far-red light (Yanovsky et al., 1995). In maize, high PHYA activity in
the shade, promotes the degradation of the transcription factor RAVL1 that enhances
the expression of LEAF ANGLE ARCHITECTURE OF SMART CANOPY 1 (LAC1) (Tian
et al., 2024). The maize lac mutant displays leaf angles that increase tolerance to high
population densities, supporting a crucial role of PHYA in plant adjustment to crowded
environments (Tian et al., 2024).

The notion that plants may overreact to canopy light cues, potentially sacrificing
yield, has persisted for years. Consequently, attenuating shade-avoidance responses in
crops could be advantageous. Ectopic expression of the Arabidopsis PHYB gene, which
maintains higher levels of active PHYB and reduces shade sensitivity, has successfully
increased potato tuber yield under high-density planting (Boccalandro et al., 2003).
Similarly, soybean and maize overexpressing GmCRY1b and ZmCRY1b, respectively,
demonstrated improved lodging resistance and yield under dense conditions (Lyu et al.,
2021; Chen et al., 2024).

However, reducing plant's sensitivity to neighbor cues has caveats. For instance,
ectopic expression of PHYB hinders vegetative growth and light foraging capacity in
potato (Boccalandro et al., 2003). Tomato plants overexpressing S/CRY1a exhibited
increased carotenoid content per fruit but showed a dwarf and bushy phenotype and a
significant reduction in overall yield (Liu et al., 2018b). Noteworthy, fruit-specific
expression of a constitutively active tomato PHYB2^Y252H, was enough to enhance
carotenoids, tocopherols, flavonoids, and ascorbate, highlighting a promising tool for
1 fruit fortification (Alves et al., 2020) that could avoid the negative impact of whole plant
2 overexpression on vegetative growth.

3 Plants can anticipate seasonal changes by sensing day length. This ability comes
4 from a combination of photoreceptors, which distinguish light from dark periods, and the
5 circadian clock that tracks the passage of time. Research in Arabidopsis has been
6 instrumental to identify the photoreceptors that perceive daylength in crops. For
7 instance, phytochrome C (PHYC) mediates natural variation in flowering time responses
8 of Arabidopsis (Liu et al., 2011), and plays a major role in the long-day induction of
9 flowering in wheat (Chen et al., 2014).

10 Furthermore, genetic variants of genes that operate at the interphase of light and
11 clock signaling have allowed crops grown at different latitudes to adjust their growth and
12 development for optimal yield (Maeda and Nakamichi, 2022). For instance, the major
13 locus determining photoperiodic sensitivity in cereals, *Photoperiod-1* (*Ppd-1*), encodes
14 orthologues of Arabidopsis *PSEUDO-RESPONSE REGULATOR 7* (*PRR7*). Similarly,
15 the *E2* locus controlling photoperiodic sensitivity in soybean encodes a homolog of
16 *Arabidopsis GIGANTEA* (*GI*). Furthermore, a deletion in the *NIGHT LIGHT-INDUCIBLE
17 AND CLOCK-REGULATED 2* (*LNK2*) gene uncovered in Arabidopsis was involved in
18 the adaptation of tomato from growing in the equator to its cultivation in northern Europe
19 (Maeda and Nakamichi, 2022). Variations in the *EARLY FLOWERING 3* (*ELF3*) gene
20 have been linked to domestication and breeding practices that maximize crop
21 productivity at different latitudes in soybean, wheat, barley, rice, and other species (Lu
22 et al., 2017; Wittern et al., 2023; Zakhrabekova et al., 2012; Faure et al., 2012; Maeda
23 and Nakamichi, 2022). All the genes mentioned above are transcriptional regulators that
24 function within the complex circadian network and also play a role in regulating light
25 signaling.

26 For reasons of space, we only focus on ELF3. In *Arabidopsis*, the *elf3* mutation
27 disrupts flowering time and circadian rhythms, as well as light inhibition of hypocotyl
28 growth (Zagotta et al., 1996; Hicks et al., 1996). ELF3 functions as a scaffold protein
29 that interacts with other clock-regulated proteins to form the evening complex (EC)
30 (Nusinow et al., 2011), which acts by repressing the expression of hundreds of genes,
31 including the clock genes *PRR9*, *PRR7*, and *GI*. In addition, ELF3 interacts with PHYB
1 (Liu et al., 2001) and other light signaling proteins to modulate light control of the
2 circadian clock and plant growth and development in both EC-dependent and EC-
3 independent manners. This suggests that ELF3 acts as a bridge between light signaling
4 and the circadian clock, potentially affecting a plant's response to day length
5 (photoperiodism) through either pathway. For more details on the mechanisms involving
6 ELF3, see the section on "Flowering Response to Rising Temperatures."

7 Soybeans are short-day plants originally cultivated in the temperate regions of
8 China. Until a few decades ago, their cultivation in more equatorial regions was not
9 feasible due to very low yields associated with extremely early flowering. Genetic
10 variants at the *J* locus, which encodes an *ELF3* orthologue, enabled soybean plants to
11 flower much later in Brazil (Lu et al., 2017), allowing this country to become the largest
12 producer of soybeans in the world. In wheat, a long-day plant cultivated in temperate
13 zones, genetic variants associated with orthologues of *ELF3* lead to early flowering,
14 allowing the crop to elude the hot and dry summers of the Mediterranean region. In
15 Japanese Wheat varieties, the early flowering resulting from mutations in *ELF3*
16 orthologues helps anticipate the rainy season, avoiding pre-harvest sprouting and
17 reducing the risk of fungal infections.

18 In crop species, ELF3 variations can affect photoperiodism by altering the clock, as
19 seen in barley (Faure et al., 2012; Zakhrabekova et al., 2012). However, studies in
20 soybean with altered ELF3 function revealed no significant changes in daily gene
21 expression patterns, suggesting the circadian clock remained largely unaffected (Yue et
22 al., 2021). Similarly, while mutations in all four ELF3 homologs of wheat disrupt
23 circadian rhythms, variations in individual genes still influence flowering time without
24 affecting the clock (Wittem et al., 2023). These findings suggest that natural variations
25 in ELF3 likely influence flowering time and maturity in crops primarily through its role in
26 light signaling, rather than through its role in the circadian clock.

27

28 Nitrogen Use Efficiency

29

30 (Written by Sandrine Ruffel and Nicolaus von Wirén)

31
Nitrogen (N), representing 1-6% of the dry matter, is one of the most limiting elements for plant growth. Often, massive use of fertilizers constitutes the main strategy to avoid any impact of N limitation on yield. Since at high fertilizer doses plants absorb less than 50% of applied N, a large amount of reactive N forms is lost, leading to water pollution and greenhouse gas emissions with consequences on climate change (Gong et al., 2024). Reducing environmental impacts of fertilizers requires optimizing agricultural practices but also Nitrogen Use Efficiency (NUE; defined as grain yield or biomass relative to the amount of N applied) in crops. The most common and straightforward approach to improve NUE is genetic selection and classical breeding of N-efficient crop cultivars (Hawkesford and Griffiths, 2019). Alternatively, genetic improvement of crops may profit from our advanced understanding of N responsiveness, uptake, transport, assimilation and reallocation in the model plant Arabidopsis, in which a cascade of inter-linked metabolic and developmental processes provides already a deep and comprehensive picture of the N-related regulatory network (Shanks et al., 2024).

In Arabidopsis, the perception of nitrate (NO3-), the main inorganic form used in most agrosystems, has been shown to rely on the NO3- transceptor NITRATE TRANSPORTER1.1/NRT1.PTR FAMILY6.3/CHLORATE RESISTANT1 (NRT1.1/NPF6.3/CHL1) and on the NODULE INCEPTION-like protein (NLP) transcription factor family (mainly NLP7 and NLP2,4,5,6,8,9) (Ho et al., 2009; Liu et al., 2017b; Liu et al., 2022a). Interestingly, different ways of targeting NRT1s have been shown to improve NUE in rice. Based on AtNRT1.1 homology, three orthologs have been identified in rice genome: OsNRT1.1A, OsNRT1.1B and OsNRT1.1C (Plett et al., 2010). Initially, genetic variability between indica and japonica rice at the OsNRT1.1B locus, causing higher NO3- influx, was found to account on average for up to 10% higher grain yield and NUE (Hu et al., 2015). Then, the elite allele of indica OsNRT1.1B was introduced in japonica by gene replacement using an innovative CRISPR/Cas9 method based on homology-directed DNA repair (Li et al., 2018). Here, the previous characterization of AtNRT1.1 as transceptor at the plasma membrane (Ho et al., 2009) has been pivotal to define OsNRT1.1B as the functional ortholog and to decipher its broader impact on NO3- uptake, translocation and signaling (Hu et al., 2015). Although
1 OsNRT1.1A localizes to the tonoplast, shows ammonium-inducible expression and
2 shares even higher homology with AtNRT1.1 than OsNRT1.1B (Wang et al., 2020), it
3 also stimulates the expression of genes involved in N metabolism and utilization.

4 Indeed, overexpression of OsNRT1.1A in rice increases grain yield, NUE and reduces
5 the time required to reach maturation due to its impact on N assimilation and flowering
6 (Wang et al., 2018).

7 Combining fundamental understanding of NO3- transport and remobilization in
8 Arabidopsis provided alternative strategies to improve NUE in crops. On basis of the
9 low-affinity NO3- transporter AtNRT1.2/NPF4.6, involved in NO3- reallocation from old to
10 young leaves, Chen et al. (2020) constructed a chimeric and hyperactive NO3-
11 transporter, named NC4N. To do so, the four transmembrane domains 2 to 5 were
12 replaced by those of AtNRT1.1, increasing the uptake level by 12-fold magnitude
13 compared to NRT1.2. To target the activity of this hyperactive low-affinity transporter to
14 source organs, NC4N was fused to the AtNRT1.7/NPF2.13 promoter, conferring high
15 expression in source leaves. Expressing this transgene in the Arabidopsis *nrt1.7* mutant
16 enhanced NO3- remobilization from leaves, and ectopic expression in wild-type rice
17 improved plant growth, NUE and grain yield by 8-11% in field experiments (Chen et al.,
18 2020). This approach illustrates how successful translational approaches including re-
19 designed structure-function aspects can profit from fundamental Arabidopsis
20 knowledge.

21 Manipulating NO3- reduction, N assimilation or N remobilization during leaf
22 senescence and reproductive stage is also considered as a valuable strategy to
23 improve NUE in crops. Our current understanding of the function of individual
24 isoenzymes or organic N transporters in this N metabolism is based on a large number
25 of in-depth studies in Arabidopsis (Coruzzi, 2003; Liu et al., 2022b; Havé et al., 2017).
26 These have inspired transgenic approaches, in which overexpression of individual N
27 metabolism genes led to better growth on low N or higher NUE. For example, the
28 overexpression of the *GLUTAMINE-DEPENDENT ASPARAGINE SYNTHASE1*
29 (*OsASN1*), encoding an asparagine synthetase, increases rice yield under N-limiting
30 conditions (Lee et al., 2020a). Likewise, overexpressing initial steps in ammonium
31 assimilation are a promising strategy. The concurrent activation of the *AMMONIUM*
1 TRANSPORTER1;2 (OsAMT1;2), an NH₄⁺ transporter, and the FERREDOXIN-DEPENDENT GLUTAMATE SYNTHASE1 (OsGOGAT1), a glutamate synthase, led to higher grain yield under N-limiting conditions in rice (Lee et al., 2020b). The understanding of autophagy processes, involved into the degradation of unnecessary constituents in lytic compartments, also largely benefited from studies in model organisms. In particular, the role of autophagy and ATG8 (AUTOPHAGY-RELATED8), a protein involved in the autophagosome functioning, for nutrient recycling and organic N remobilization from leaves to seeds has been shown in Arabidopsis (Guiboileau et al., 2012). In rice, under normal conditions, the number of tillers, the N content in grains, as well as the yield, are increased with the overexpression of OsATG8a (Yu et al., 2019).

11 Successful improvement of crop yield can also be achieved by targeting central regulators, such as transcription factors, involved in the coordination of the different steps of N-use (Kasemsap and Bloom, 2022). Arabidopsis studies once again can play a significant role in the search for NUE improvement. Based on the central role of NLPs in NO₃⁻ perception in Arabidopsis, analyses on rice homologs have been undertaken to assess their role in NUE. The overexpression of OsNLP3 and OsNLP4, the two closest homologs to AtNLP7 in rice, led to yield and NUE increases from 10 to 30% under moderate or sufficient N levels, and transcriptome analysis showed that they indeed coordinate the regulation of genes related to N-signaling and assimilation (Wu et al., 2021; Zhang et al., 2022a). Supervised machine-learning algorithm combined to gene network analysis on a large set of genomic data in Arabidopsis was another approach to identify candidate coordinators of nutrition and growth. This way, the scaffold protein BT2 has been predicted to be a central node in a network of genes related to N-use. Experimentally, the agronomical NUE (i.e., seed amount per plant / quantity of N applied) was found increased in the Arabidopsis *bt1/bt2* double mutant as well as in a rice Osbt mutant, specifically in N-limited conditions. In both species, the NUE improvement in mutants is likely due to a higher NO₃⁻ transport level depending on the NRT2 transporter family, defining these BTB family members as repressors of the N-use (Araus et al., 2016). More recently, a study presented an evolutionarily informed machine learning approach that uses conserved transcriptome responses to predict phenotypic outcomes, specifically nitrogen use efficiency, across species like
1 Arabidopsis and maize. By reducing feature dimensionality with biologically relevant
2 genes, the approach enhances predictive power and reveals in maize the role of the
3 NFYA3 transcription factor in NUE under low N conditions (Cheng et al. 2021).

4 In Arabidopsis, the transcription factor TGA1 has been identified as a hub in a N-
5 dependent network displaying a unique and interesting property to control plant growth
6 in response to N dose (Swift et al., 2020). Evaluating the role of its homologs for NUE in
7 crops provides an exciting perspective. Instead of being only a resource to identify
8 candidate genes for NUE improvement, Arabidopsis has also been used for its simplicity
to delineate the role of a gene isolated from crops. This was the case for the
9 transcription factor Dof1. Initially, this regulator has been isolated and studied in maize
10 (ZmDof1) in which it coordinates the expression of some genes involved in carbon
11 metabolism. In particular, it regulates the synthesis of oxaloacetate, a precursor for the
12 synthesis of 2-oxoglutarate which is the carbon-skeleton required for N assimilation
13 (Yanagisawa, 2000). To test the hypothesis that by overexpressing DOF, the activity of
14 the nitrogen metabolism should be stimulated to stay coordinated with carbon
15 metabolism, Arabidopsis transgenic plants expressing ZmDOF1 have been produced
16 and analyzed. In addition to the expected increase of the phosphoenolpyruvate
17 carboxylase activity, the N assimilation was found enhanced as well as a better growth
18 in low nitrogen conditions (Yanagisawa et al; 2004). Later, the same approach has been
19 performed in rice, giving similar results (Kurai et al., 2011).
20

21 Eventually, optimizing the root system architecture potentially belongs to targets that
22 may improve the use of nutrient acquisition and yield (Den Herder et al., 2010). A
23 tremendous and essential work has and still is being done to delineate the architectural
24 phenotypes improving the capture of nitrogen, as well as water and other nutrients,
25 studying crops in soil (Lynch et al., 2024). Even in this context, a better knowledge and
26 an optimization of phenotypic traits could be considered with model systems combined
27 to genetic and structure-function approaches. Indeed, determining genetic networks
28 underlying root structure characteristics would reveal possible tight coordination,
29 favorable or unfavorable, with metabolism. It is noteworthy that among the genes
30 presented above, some of them control root development. AtNRT1.1 is a perfect
31 example of a central node for NO3- acquisition and root development (Krouk et al.,
1 2010). Eventually, an extensive research on the signaling pathways controlling root development in response to N availability in Arabidopsis identified allelic variation in several molecular targets (Jia et al., 2022). Exploiting allelic variation in genes for beneficial root traits identified in Arabidopsis for crop improvement by genome editing provides another promising avenue for translational research (Figure 4).

6

7 Drought

8

(Written by Sarah M. Assmann and Noah A. Kinscherf)

10

11 Plants respond to drought in complex ways. In drought escape, plants achieve a
12 measure of reproductive success through acceleration of the life cycle. In drought
13 tolerance, plants withstand desiccation, but this is often with a penalty on seed
14 production. For crop improvement, optimal approaches are likely to capitalize on
15 drought avoidance, defined here as morphological, anatomical, or physiological
16 changes in response to water limitation that allow plant water status and thus yield to be
17 minimally impacted. Morphological changes include altered root and aboveground
18 architecture; anatomical changes include reduced stomatal density and changes in cell
19 wall properties that favor turgor maintenance; and physiological changes include
20 osmotic adjustment, as well as stomatal closure and consequent reduction of
21 transpirational water loss.

22 As for many aspects of plant biology, research on Arabidopsis has led the field in the
23 identification of genes involved in drought response. As reviewed in Wang et al.
24 (2021b), more than a dozen genes have been identified in Arabidopsis that confer
25 drought tolerance when their orthologs are overexpressed in a crop species -- yet none
26 of these has been commercialized. In fact, transgenes commercialized for performance
27 under drought are rare; they include a bacterial RNA chaperone, CspB, that is utilized in
28 Bayer's DroughtGard maize (Castiglioni et al., 2008; Nemali et al., 2015), and a
29 sunflower homeodomain-leucine zipper transcription factor gene, *HaHD4*, with its
30 stress-responsive promoter, favorably evaluated in soybean (Ribichich et al., 2020) and
31 commercialized in wheat (Gupta et al., 2024). While the stress-protective attributes of
heterologous expression of these two genes were evaluated in Arabidopsis (Castiglioni et al., 2008; Dezar et al., 2005), the paucity of commercial varieties developed based on leads from this model species (Nuccio et al., 2018) raises the question of its role in translational biology for improved crop performance under drought.

One possible explanation for this situation relates to the types of gene candidates identified from Arabidopsis: 1) transcription factors, especially those responsive to the desiccation-signaling hormone, ABA; 2) genes involved in metabolic responses to drought, such as those that regulate synthesis of compatible solutes, and; 3) genes involved in protein synthesis, modification, or turnover. It is possible that overexpression of these types of genes is, paradoxically, too protective, conferring survival under drought at the expense of yield, and/or that their overexpression leads to yield drag under non-water-limited conditions. One example in support of this hypothesis is a study by Yamaguchi-Shinozaki and colleagues (Kudo et al., 2019). They observed that overexpression in Arabidopsis of *DREB1A/CBF3*, a transcription factor that promotes drought and freezing tolerance, also decreased growth. The growth phenotype was partially alleviated, without compromising drought tolerance, upon co-expression of a GA<sub>5</sub>, rate limiting enzyme in GA biosynthesis.

Further challenges to translation from Arabidopsis to crops are that the genetic makeup of Arabidopsis and crop plants differs (Inze and Nelissen, 2022); this is particularly true over larger phylogenetic distances such as those between Arabidopsis and grain crops. Moreover, the conditions used to uncover drought-related genes in Arabidopsis may not always sufficiently mimic the conditions that crops actually experience in the field (Langstroff et al., 2022).

However, the above observations scarcely mean that Arabidopsis research is irrelevant to crop improvement. First, Arabidopsis historically has been the *sine qua non* system for research into plant developmental biology and its genetic underpinnings. As argued herein, our resultant knowledge of plant development has been under-exploited toward improving crop performance under drought. Second, when candidate QTLs or genes are identified directly in a crop species, fundamental knowledge of gene function, most often garnered from Arabidopsis, is vital in prioritizing leads. Third, Arabidopsis is a hotbed of methods development, with such methods often subsequently deployed in
1 crop species. Each of these topics is touched on below in the context of plant
2 development and drought response (Figure 5).

3 Consider the path that a water molecule takes through the plant. Water that is taken
4 up by the root moves symplastically across the endodermis (Ranathunge et al., 2011),
5 transits the vasculature of the shoot xylem, enters shoot tissue across the living cells of
6 the xylem parenchyma, hydrates mesophyll tissue, and ultimately is lost to the
7 atmosphere through stomata via the transpiration stream. At each step along the way,
8 plant development influences water movement. Starting with roots, it has been argued
9 that under drought a "steep, cheap, and deep" root architecture (Lynch, 2018) is
10 favorable: fewer longer roots (Voothuluru et al., 2024) with enhanced aerenchyma
11 production, accompanied by lateral roots that grow at a more acute angle and are
12 differentially produced at deeper locations. This architecture is proposed to reflect a
13 strengthening of the gravitropic response (Rellán-Álvarez et al., 2015; Feng et al., 2016)
14 and has been characterized in Arabidopsis through the development of specialized
15 imaging rhizotrons (Rellán-Álvarez et al., 2015). Hydrotropism, or preferential root
16 growth toward water independent of gravity, has been studied primarily in Arabidopsis,
17 with four regulatory mutants, *nhr1*, *miz1*, *miz2*, and *ahr1* identified (reviewed in Feng et
18 al., 2016) that arguably could be targets for biotechnology. Additionally, hydropatterning,
19 whereby lateral root development is promoted by local water availability (Bao et al.,
20 2014), is a favorable trait under drought, and genetic control of lateral root formation has
21 been elucidated largely in Arabidopsis (Banda et al., 2019).

22 Classically, the Casparian strip had been described as a suberized ring around
23 endodermal cells that restricts transit of water and solutes to the xylem to a symplastic
24 route. It was research in Arabidopsis that determined that the Casparian strip is
25 fundamentally a lignified zone, albeit endodermal cell surfaces are also heavily
26 suberized (Naseer et al., 2012). Observation of drought-induced increases in
27 endodermal suberization in rice have been interpreted as a mechanism to retain water
28 in the xylem when soil water potential decreases (Henry et al., 2012). Many of the
29 developmental genes governing formation of the endodermis (Cui et al., 2007), as well
30 as multiple other root cell types (Cederholm et al., 2012), were first identified in
31 Arabidopsis by the seminal work of Philip Benfey and colleagues. Exemplifying how
1 functional annotations from Arabidopsis inform studies in crops, Zaman et al. (2024)
2 built on this knowledge to assign putative functions to a number of candidate loci
3 associated (via GWAS and TWAS) with root morphology traits in spring wheat.

4 Xylem hydraulic efficiency, defined as hydraulic conductance for a given
5 pressure/tension gradient, and xylem safety, defined as lack of susceptibility to
6 embolism formation and spread (Gleason et al. 2016), has been studied primarily in tree
7 species (Torres-Ruiz et al., 2024). However, studies on Arabidopsis accessions and the
8 Arabidopsis woody *soc1 ful* double mutant have importantly revealed that the extent of
9 xylem wall lignification and the thickness of pit membranes within the pits that connect
10 xylem elements are positively correlated with improved xylem safety (Thonglim et al.,
11 2021, 2023), providing potential anatomical and gene targets for manipulation. Related
12 to this, one GWAS study in Arabidopsis identified a transcription factor, XYLEM NAC
13 DOMAIN 1 (XND1), that negatively regulates both xylem vessel formation and drought
14 tolerance (defined as vegetative biomass production) (Tang et al., 2018).

15 In addition, novel aspects of hydraulic control by the vascular parenchyma, through
16 which water transits the plasma membrane via water-conducting channels named
17 aquaporins, have been studied extensively in Arabidopsis (Maurel et al., 2015). Simple
18 overexpression of aquaporins can have either positive or negative effects on drought
19 response (reviewed in Tenorio Berrío et al., 2022); this makes sense given that
20 aquaporins can facilitate both water entry into the root xylem and water loss to the
21 substomatal cavity. Manipulation of aquaporin activity, as elucidated in Arabidopsis
22 (Torne-Srivastava et al., 2024), rather than aquaporin expression levels *per se*, may
23 hold particular promise for improved drought resilience.

24 Most of the water loss from mature leaves occurs through stomata. Dynamic
25 stomatal closure in response to signals engendered by soil drought and high VPD
26 (sometimes referred to as "atmospheric drought") is central to protection of plant water
27 potential and hydraulic safety. Because stomatal closure in response to changes in light
28 intensity or CO2 concentration is slow as compared to the rapid effects of these
29 parameters on photosynthesis, there can be a period when the ratio of assimilation to
30 transpiration (water use efficiency; WUE) is reduced. Shortening this period by
31 manipulating stomatal kinetics could theoretically improve water conservation. Although
studies in Arabidopsis have approached stomatal kinetics in many ways (Nguyen et al., 2023), the translation of these strategies into crop species is still lacking. However, there are some promising leads. For example, overexpression in wheat of the wheat ortholog (TaNHX2) of an Arabidopsis vacuolar cation/H⁺ antiporter (NHX)-type exchanger (AtNHX2) that mediates K⁺ accumulation in the guard cell vacuole (Andrés et al., 2014) resulted in smaller stomatal apertures and improved drought tolerance, as assessed by plant survival rates and foliar fresh weight (Li et al., 2024b). Several groups are also assessing optogenetic approaches that physically link guard cell ion transporters, such as K⁺ channels and H⁺-ATPases, to light-responsive proteins to alter stomatal kinetics in Arabidopsis (Nguyen et al., 2023; Horaruang et al., 2024), with potential for translation into crop species.

The density of stomata on a leaf surface can be a critical influence on transpiration and thus water use efficiency (Doheny-Adams et al., 2012; Bertolino et al., 2019). Indeed, Ferguson et al. (2024) note that "optimal" stomatal density will likely become progressively lower as atmospheric concentrations of CO₂ continue to rise. Stomatal development is one area wherein manipulation of developmental genes first identified in Arabidopsis, while not always guaranteed to succeed (Schuler et al., 2018), has shown promise in crop species.

The EPIDERMAL PATTERNING FACTOR (EPF) family of genes first identified in Arabidopsis encodes secretory peptides involved in the regulation of stomatal spacing and density (Hara et al., 2007). This family is composed of *EPF* genes, which are negative regulators of stomatal development, and *EPF*-like (*EPFL*) genes, which include positive regulators of stomatal development (Karavolias et al., 2023). Multiple studies in Arabidopsis found that overexpressing *EPF* genes or knocking out *EPFL* genes decreased stomatal density (Hara et al., 2007; Hunt et al., 2010; Doheny-Adams et al., 2012). These results have been translated into grain crops, with overexpression of *EPF1* orthologs *OsEPF1* in rice (Caine et al., 2019) and *HvEPF1* in barley (Hughes et al., 2017) achieving reduced stomatal density and improved performance under drought: at ~50% reduction in stomatal density in rice, there was no penalty on either assimilation or yield at light intensities below 1000 µmol/m²/s. Furthermore, CRISPR/Cas9-mediated knock-out in rice of a paralog of *OsEPFL9*, *OsEPFL10*,
1 exhibited a moderate decrease in stomatal density and improved water conservation,
2 without reducing photosynthesis or impacting thermoregulation under field-relevant light
3 conditions (Karavolias et al., 2023).

4 It has been predicted that due to rising atmospheric levels of CO2, concentrations of
5 intracellular CO2 in C4 plants may exceed those required to maximize photosynthesis,
6 theoretically permitting decreased stomatal conductance without negatively impacting
7 carbon gain (Leakey et al., 2019). In sorghum, a C4 crop, constitutive expression of an
8 *EPF* gene fusion, Sb*EPF2syn*, containing domains from Sb*EPF2* and Sb*EPF9*, the
9 sorghum homologs of At*EPF2* and At*EPF9*, reduced stomatal density and stomatal
10 conductance without penalty to photosynthesis. However, while WUE in the Sb*EPF2syn*
11 lines was improved relative to wild-type, pleiotropic defects in development of flowers
12 and panicles resulted in reduced seed yield (Ferguson et al., 2024).

13 Similarly to *EPF* genes, in Arabidopsis the subtilase STOMATAL DENSITY AND
14 DISTRIBUTION 1 (AtSDD1) regulates stomatal patterning, decreasing stomatal density
15 when overexpressed (Von Groll et al., 2002). Overexpression in cultivated tomato,
16 *Solanum lycopersicum*, of *SchSDD1*, an *AtSDD1* ortholog from the stress-tolerant wild
17 tomato *Solanum chilense*, reduced stomatal density and improved water retention in
18 excised leaves (Morales-Navarro et al., 2018). Together with the results from *OsEPF10*
19 knockout in rice (Karavolias et al., 2023), these studies support the value of identifying
20 critical genes regulating stomatal density and patterning in Arabidopsis so that
21 researchers can take advantage of orthologs and paralogs in crop species and their wild
22 relatives.

23 Early on in Arabidopsis research, thermal imaging was used to measure the cooling
24 effect of transpiration on leaf temperature as a proxy for stomatal conductance (Plessis
25 et al., 2011). Scaling up this approach and combining it with rapid stomatal density
26 phenotyping via optical tomography interpreted by machine-learning (Ferguson et al.,
27 2021), allowed Leakey and colleagues to identify QTLs related to these traits in *Setaria
28 italica* x *Setaria viridae* recombinant inbred lines grown under well-watered vs. water-
29 limited conditions (Prakash et al., 2021). Similar high-throughput field phenotyping,
30 coupled with GWAS and TWAS, has identified candidate genes in sorghum that include
31 orthologs of Arabidopsis genes known for their roles in stomatal behavior or
1 development (Ferguson et al., 2021; Pignon et al., 2021). Such results highlight the
2 importance of Arabidopsis research in both technique development and biological
3 understanding. Another example of this dual role is provided by identification of ABA
4 receptors in Arabidopsis, followed by development of receptor agonists as
5 antitranspirants (e.g. Okamoto et al., 2013; Vaidya et al., 2019; Roeder et al., 2023),
6 heralding a new type of agrochemical that could be used to improve drought resilience.

7 In both model and crop species, a growing CRISPR toolkit, enabling editing of both
8 gene regulatory regions and ORFs (Soyk et al., 2020; Li et al., 2024c), offers new
9 approaches to dissect and manipulate quantitative traits such as drought avoidance.
10 Inducible and reversible systems of gene regulation arising from such approaches could
11 foster crop productivity under drought in the absence of a yield penalty. Moreover, single
12 cell RNA-seq in Arabidopsis, including studies of drought effects (Illouz-Eliaz et al.,
13 2023), is providing new insights into exactly which cell types to target. Although the path
14 that water takes through the plant is a continuum, the ability to target specific aspects of
15 that continuum via these and other advanced molecular approaches foreshadows a new
16 type of precision agriculture, holding promise that crops will flourish despite the growing
17 threat of drought engendered by climate change.
18

19 Plant stress responses to space
20

21 (Written by Arkadipta Bakshi, Burcu Alptekin, and Simon Gilroy)

22
23 The Earth is teeming with life and millions of years of evolution have equipped
24 organisms with a vast array of adaptations to our planet's many and varied
25 environments. However, biology's voyage into space began less than 80 years ago. If
26 all of life's history on the Earth were compressed to a single year, the longest human
27 mission in space would have lasted about 10 milliseconds. How then does biology fare
28 in these truly novel and alien conditions? Some of the stresses found in spaceflight are
29 identical to those on Earth. Drought is still drought, even for a plant grown on the
30 International Space Station. The sensing and response systems that help plants survive
31 these kinds of challenges in a terrestrial setting are equally relevant in space. However,
there are a suite of factors that impact plant physiology and development that are unique to spaceflight and where biology's evolutionary history may be of little help (Figure 6).

Why study these effects of space on plants? One of the goals of space plant biologists is to develop robust crops as integral components of the life support systems that can sustain a crew on long-duration missions. Although these plants are grown in the highly engineered, closed environment of a spacecraft, many of their most desirable features will be familiar to plant breeders on Earth. Alongside selecting for plants that can thrive despite the unique stressors found in spaceflight, space agriculture targets the development of crops with reduced stature, high yield and harvest index, strong resilience to biotic and abiotic stress and critically, plants that the crew enjoy eating. A compact, high yielding tomato plant that grows robustly in space is of little value if the crew members just don't like how it tastes. Arabidopsis has proven a critical model in these studies, not least because its small size and rapid generation time fit well with the often-significant physical constraints of a spaceflight mission. However, space also provides a unique laboratory to conduct experiments that are simply impossible to perform on Earth. How does gravity shape plant growth and development on Earth? A direct approach to answering this question is to remove gravity and ask what happens. The insights that are being drawn from how spaceflight impacts Arabidopsis physiology and development are expanding our basic understanding of plant biology and offering opportunities to translate this knowledge into practical applications on Earth, especially in the context of closed environment agriculture (Mortimer and Gilliham, 2022).

Without the pervasive 1 x gravity of Earth, plants show disrupted morphogenesis, although they do still grow and develop robustly, being capable of germinating, flowering and setting seeds. For example, Arabidopsis grown in spaceflight develops a relatively normal looking rosette and root system but the roots exhibit randomized growth directions and an enhanced skewing that is likely related in part to disruption of the cytoskeleton (e.g., Califar et al., 2020; Nakashima et al., 2014; Millar et al., 2011). In addition, lacking the internal signals to develop robust support tissues that are normally generated by their own weight on Earth, space grown Arabidopsis shows evidence of cell wall remodeling in its transcriptomic, proteomic and glycomic profiles (e.g.,
1 Nakashima et al., 2023; Johnson et al., 2017; Kruse et al., 2020; Choi et al., 2019).
2 These changes likely reflect a shift away from depositing the strengthening polymers
3 needed at 1 x gravity, offering an insight into how manipulating mechanical signals
4 might be used to tailor cell wall characteristics to enhance, for example,
5 biomanufacturing or biofuel feedstocks.

6 In addition to these direct effects on the plant's biology, the microgravity environment
7 profoundly alters the physical world that plants inhabit in spaceflight. Absent gravity's
8 pervasive effects, liquid movements become dominated by forces such as surface
9 tension and capillarity (Jones and Or, 1999). Under these conditions, water tends to
10 adhere to, and then creep over, hydrophilic surfaces such as the plant's root and shoot
11 systems. This process can result in a flooding-like stress for the plants even at watering
12 levels determined to be optimal for growth on Earth. This effect is compounded by the
13 altered behavior of gasses in space where density-related phenomena, such as
14 buoyancy-driven convection, are absent (Lappa, 2004). On Earth, convection leads to a
15 constant mixing of the atmosphere around the plant and its loss allows for the formation
16 of gradients of gasses that would normally be rapidly dissipated. This physical effect
17 means that steep, stable oxygen depletion zones can form around rapidly respiring
18 tissues (Liao et al., 2004). Indeed, many transcriptomic studies of Arabidopsis growing
19 in spaceflight have revealed an underlying molecular signature of hypoxic stress (Barker
20 et al., 2023). *Fusarium oxysporum* was found as an opportunistic pathogen that
eventually killed several Zinia plants grown on the International Space Station, with the
22 cause of the infection thought to be related to suppression of the plants' defenses due to
23 flooding and hypoxic stress (Schuerger et al., 2021). Crewed spacecraft are far from
24 sterile environments, offering ample opportunities for such plant-microbe interactions.
25 For example, the International Space Station has been continuously inhabited for 25
26 years and not surprisingly has a rich and diverse microbiome, including a wide range of
27 plant pathogens (Urbaniak et al., 2022).

28 The effect of altered gravity is just one of many new challenges biology encounters
29 once it moves into space. The Earth's magnetic field and atmosphere shields our
30 biology from the high levels of ionizing radiation, such as from Galactic Cosmic Rays
31 and Solar Particle Events, that are an inherent factor for life in space. Most of our
knowledge on how plants respond to ionizing radiation comes from radioactively contaminated sites such as Chernobyl and Fukushima, and a few studies of simulated ionizing radiation with heavy ions (Mousseau and Møller, 2020). This research suggests plants have a much higher radiation tolerance than animals. While the lethal dose of ionizing radiation for humans is only 5 Gy, plants can easily survive up to 50 Gy (Gudkov et al., 2019). Although the precise reasons behind this higher tolerance remain to be discovered elements such as plasticity in development, innate regenerative capabilities, lack of mobile, proliferative cells and the distributed, modular nature of plant structures and functions likely all contribute to this remarkable resilience. However, despite this radiation resistance, plants are not immune to radiation damage, with exposure to simulated Galactic Cosmic Rays and Solar Particle Events leading to developmental abnormalities and widespread changes in patterns of gene expression, particularly those related to DNA repair and glucosinolate metabolism (Dixit et al., 2023; Zhang et al., 2022b). Molecular mechanisms leading to these physiological changes have yet to be intensively explored, although it is notable that oxidative damage is one key effect of radiation exposure, likely accounting for 70-80% of radiation-related DNA damage (Gudkov et al., 2019). Of note in this context, induction of molecular markers of oxidative stress is one of the most commonly reported characteristics of plants growing in the spaceflight environment (e.g., Choi et al., 2019; Barker et al., 2023; Sugimoto et al., 2014; Johnson et al., 2017; Kwon et al., 2015; Zupanska et al., 2013). The upregulation of telomerase activity seen in Arabidopsis grown on orbit has even been proposed as a response to protect the genome from oxidative damage (Barcenilla et al., 2023). Researchers have attempted to leverage the unique combination of radiation exposure and other spaceflight-related stresses to develop new agricultural characteristics in plants grown in space. Indeed, Chinese scientists have reported using a combination of spacecraft and high-altitude balloons to develop ~200 new crop varieties (Mohanta et al., 2021).

There are a host of other effects reported for Arabidopsis growing in spaceflight from the induction of novel alternative splicing (Beisel et al., 2019) to shifts in the patterns of cellulose synthase production (Olanrewaju et al., 2023). Determining the chain of cause and effect for these responses is a key element of current research. This knowledge is
crucial to help develop potential biology- and engineering-based mitigation strategies that can be applied to the broad range of plants targeted for future space agriculture (Johnson et al., 2021). To give just one example, the extensive molecular dissection of spaceflight response spearheaded by work in Arabidopsis has led to the realization that spaceflight induces significantly increased levels of oxidative stress. This understanding has then helped target growing crops high in antioxidants, such as the Outredgeous cultivar of lettuce, microgreens and even the cultivation of the carotenoid-rich duck weeds as strategies to help protect both the plant and the crew that consumes them.

With all these stresses and challenges impacting on organisms in space, you might easily conclude that plants could not survive in the spaceflight environment. However, Arabidopsis can complete a full lifecycle from seed to seed in space (reviewed in Medina et al., 2022), a finding that has paved the way for experiments with crop plants. Indeed, the crew of the International Space Station have grown and eaten their own lettuce and pepper harvests. Plants are remarkable in their developmental plasticity and physiological adaptability and rather than looking to science fiction writers for clues as to the green extraterrestrials we may meet in the future, simply looking to our gardens might provide a much better view.

## Flowering response to rising temperature

(Written by Malleshaiah Sharath-Kumar and Salomé Prat)

The circadian clock synchronizes flowering with seasonal changes in day-length and temperature, ensuring reproductive transitions occur at optimal time of year. Seasonal and daily variations in both environmental cues drove the evolution of shared molecular mechanisms for sensing these input signals, as exemplified by the phytochromes. Red light absorption induces a conformational change of these light receptors leading to the active Pfr form, while elevated temperatures accelerate the reversal of nuclear phyB Pfr to its inactive Pr form, oppositely affecting light signaling (reviewed in Casal et al., 2024). As such, rising global temperature due to climate change significantly affects
1 flowering timing in several plants, decreasing crop productivity and shrinking habitats for
2 wild species.

3 Day-length follows a predictable seasonal pattern, unlike temperature, which
4 fluctuates with weather, cloud cover, and regional climate. Hence, the timing of flowering
5 strongly relies on the day-length (a process known as photoperiodism), categorizing
6 plants as long-day plants (LDPs), short-day plants (SDPs), or day-neutral plants in
7 which flowering is independent of day-length (Gendron and Staiger, 2023). Temperate
8 crops are typically LDPs flowering in spring, when light hours exceed a critical
9 threshold, ensuring seed production before autumn frosts. Near the equator, changes in
10 day-length are modest throughout the year, with seasons marked by dry summers and
11 moist autumns. Tropical and subtropical crops are predominantly SDPs, initiating
12 flowering when the day-length is shorter than a critical threshold, close to 12 hours. This
13 response is suppressed when long nights are interrupted by a brief exposure to light
14 (night-break, NB), indicating that these plants primarily measure the length of the night.

15 As global temperatures rise, understanding how this cue affects flowering time is
16 crucial for selecting crop cultivars that better cope with climate change. Warm
17 temperatures induce early flowering in LDPs as an adaptive strategy to escape heat
18 stress. On the opposite, elevated temperatures suppress *FT* expression and delay
19 flowering in SDPs such as soybean or chrysanthemum (Tang et al., 2023; Nakano et al.,
20 2013), while inhibiting tuber formation in potato (Hancock et al., 2014; Koch et al., 2024)
21 via a yet poorly understood mechanism. Homologs of CONSTANS (CO) play a
22 conserved role in activating *FT* gene expression in these short-day crops. However,
23 additional negative regulators such as the soybean *E1 B3* domain factor (Lin et al.,
24 2021; Fang et al., 2024), or the chrysanthemum *CsAFT* and potato *StSP5G* anti-
25 florigens play a central role in suppressing *FT* expression or its inducing activity in non-
26 inductive long days (Kloostermann et al., 2013; Higuchi et al., 2013; Abelenda et al.,
27 2016). This section summarizes the day-length pathways controlling *FT* gene
28 expression in these SDPs, drawing on lessons learned from Arabidopsis. We also
29 discuss how warm temperatures during the night hours distinctly affect *FT* expression,
30 making these SDPs particularly susceptible to climate change.

31
# 1 The photoperiodic flowering model of Arabidopsis

Genetic studies in Arabidopsis were pivotal to identification of a central regulatory module driving flowering in long-days (LDs) (Wang et al., 2024). The B-box CCT domain CONSTANS (CO) factor has a central role in this pathway by activating expression of the *FLOWERING LOCUS-T* (*FT*) gene in leaves. In LD conditions, CO transcription is elevated in the afternoon, and the light stabilized CO protein goes on to activate *FT* expression (reviewed in Song et al., 2015; Takagi et al., 2023). In short days (SDs), CO is expressed at night and the protein is degraded by CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which prevents *FT* upregulation (Jang et al., 2008). GIGANTEA (GI) links the circadian clock and photoperiodic flowering by regulating *FT* expression through both CO-dependent and CO-independent mechanisms (Mizoguchi et al., 2005; Sawa et al., 2007). In the CO-dependent pathway, GI interacts with FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) in blue light, and destabilizes the CO repressor CYCLING DOF FACTOR 1 (CDF1), therefore promoting CO-mediated *FT* activation (Sawa et al., 2007; Takagi et al., 2023). *FT* is expressed in leaf phloem companion cells and the protein transported through sieve elements to the shoot apical meristem (SAM) (Corbesier et al., 2007). In the SAM, *FT* interacts with the bZIP transcription factor FLOWERING LOCUS D (FD) and 14-3-3 proteins to form a florigen activation complex (FAC) that triggers flowering transition (Taoka et al., 2011; Abe et al., 2005). Similar ternary complexes induce potato tuber formation, and bud set in perennial trees (Teo et al. 2017; Parmentier-Line and Coleman, 2016), showing that these *FT* complexes are highly conserved in angiosperms, while regulating other developmental processes beyond flowering (Figure 7).

# 25 Elevated temperature control of flowering time in Arabidopsis

In Arabidopsis, flowering time is accelerated by warm temperatures, and a similar response is observed when temperatures are elevated in the day or during nighttime. Mild temperatures promote a faster elongation of the young seedlings' hypocotyl and the upward positioning of leaves, in a characteristic response known as thermomorphogenesis. This adaptive response is aimed at cooling the leaves and protecting the meristem from the warm soil and is found to in part rely on an effect on
1 the circadian clock (Kerbler and Wigge, 2023). The effects of warm temperature differ
2 from those of heat stress that causes irreversible plant damage, whereas these
3 responses are governed by distinct signaling pathways (reviewed in Sato et al., 2024).

4 The light receptor phytochromes act as major temperature sensors. Elevated
5 temperatures promote the faster reversion of nuclear phyB Pfr into the non-active Pr
6 form, causing inactivation of these photoreceptors and the nuclear accumulation of
7 PHYTOCHROME INTERACTING FACTORS (PIFs) (Casal et al., 2024). *FT* transcript
8 levels are elevated at 28°C, due to the removal of histone H2A.Z from the *FT* promoter,
9 which enables PIF4 mediated activation of the *FT* gene (Kumar and Wigge, 2010;
10 Kumar et al., 2012). Other proteins were also revealed to act as temperature sensors,
11 including the evening complex (EC) protein EARLY FLOWERING 3 (ELF3), which at
12 warm temperatures undergoes phase separation through a prion-like domain (Jung et
13 al., 2020). ELF3 forms condensates that disrupt EC function and alleviate *PIF4*
14 repression, which indirectly promotes activation of *FT* (Nusinow et al., 2011; Jung et al.,
15 2020). While elevated temperatures accelerate flowering in Arabidopsis, a flowering
16 delay is observed in oilseed rape (*Brassica napus*) underscoring that this response
17 varies among Brassicaceae species. Analyses of H2A.Z occupancy revealed H2A.Z
18 histone levels not to be increased in the *BnaFTA2* locus at 28°C (Abelenda et al., 2023).
19 Likewise, *BnARP6*-RNAi plants, silenced for the *ACTIN-RELATED PROTEIN6* (*ARP6*),
20 a component of the H2A.Z-incorporating SWR1 complex, showed little H2A.Z
21 accumulation but still delayed flowering in response to elevated temperatures, indicating
22 that *BnaFTA2* is repressed via additional mechanism (Abelenda et al., 2023).
23 Temperature indeed affects function of the circadian clock by inactivating the
24 Arabidopsis evening complex (EC), hence suppressing EC feedback inhibition of genes
25 like *PRR9*, *PRR7*, *GI*, and *LUX*, in addition to leading to de-repressed expression of
26 *PIF4* (Mizuno et al., 2014). Therefore, thermal flowering induction may result from
27 temperature effects on the EC, *GI* and on other upstream *FT* regulators, which offers
28 multiple possible strategies to improve climate resilience of this model plant as well as
29 other LD crops.

30

31

Translating Insights from LD Arabidopsis to SD Crops
Studies in Arabidopsis accentuate a central role of the GI-CO-FT module in flowering control. The main function of this central module is largely conserved in crops, including SDPs. Throughout natural evolution and domestication several additional regulators contributed to modifying LD activation of this module into a SD response. These changes relied on LD activation of negative regulators or antiflorigenic homologs, SDs thus being required to release flowering inhibition by these repressors. Core clock regulators such as Gl and the EC play also a central role in flowering time regulation in SDPs but defects in the central clock genes lead in these plants to an opposite flowering phenotype as compared to Arabidopsis (Andrade et al., 2022; Lu et al., 2017; Bu et al. 2021). Unlike Arabidopsis, flowering is inhibited in SDPs by a NB or short exposure to light in the middle of the night. Red light is the most effective for NB interruption, while its inhibitory effects are reversed by an additional pulse of FR-light, implicating phytochromes in this response. NB pulses as short as 10 min suppress flowering in most SDPs, while they do not affect entrainment of the clock (Ishikawa et al., 2005). Likewise, NB treatments suppress flowering only when given around 8-10 h after dusk, and an identical gating is observed in non-24h light/dark cycles, indicating that this inhibition relies on a clock-regulated factor whose phase expression is set at dusk (Hayama et al., 2007). NB effects were found in chrysanthemum to activate the CsAFT anti-florigen at night (Higuchi et al., 2013), emphasizing that insights obtained in Arabidopsis are always not directly transferable to SD crops.

21

## The photoperiodic flowering model of SD crops

A significant progress has been made in understanding the rice, soybean, chrysanthemum, or potato day-length pathways. Rice is a facultative SD plant. The CO homolog Heading date 1 (Hd1) has a central role in SD flowering and activation of the FT homolog Hd3a. OsGI enhances OsHd1 expression (Hayama et al., 2003) while identification of the Ehd1 locus revealed that this pathway is more complex than in Arabidopsis. The OsGI-Hd1-Hd3a/RFT1 segment of the pathway has in SD a similar function as the Arabidopsis GI-CO-FT module, while OsHd1 acts in LDs as a repressor of OsEhd1 and OsHd3a/RFT1 on heterodimerization with OsGhd7 and OsDHT8 (reviewed by Vicentini et al., 2023). OsHd1 abundance is not affected by light (Yang et
al., 2015), while diurnal sensing depends on OsGhd7, that is upregulated in LDs and the protein stabilized by the OsPHYA and OsPHYB phytochromes. These photoreceptors interfere with OsGI-mediated degradation of OsGhd7, linking photoperiod sensing with the circadian clock (Zheng et al., 2019).

In soybean, flowering is regulated by the *GmPHYA* (*E3*, *E4*)-*E1-FT* pathway. Key loci, include *E1–E4*, *J* (*GmELF3*), and *GmFT2a/FT5a*, where E1-E4 delay flowering in LDs (Dong et al., 2023; Xia et al., 2012). The B3 domain E1 factor suppresses *GmFT2a* and *GmFT5a* expression, while *GmPHYA2* and *GmPHYA3* serve as the primary photoreceptors of the GmphyA-LUX-E1-FT module (Lin et al., 2024; Zhao et al., 2022). In potato, StSP6A functions as FT in Arabidopsis, regulating in this crop tuber formation. In LDs, StSP6A is suppressed by the anti-florigen StSP5G, whose expression is induced by StCOL1 in a phyB-dependent manner. Under SDs, StCDF1 represses StCOL1 and suppresses StSP5G, enabling StSP6A activation, whereas stabilizing alleles of StCDF1 were critical for potato adaptation to higher latitudes (Abelenda et al., 2016; Kloosterman et al., 2013). In chrysanthemum SD flowering is promoted by *CsFTL3* (florigen), while the anti-florigen *CsAFT* inhibits flowering under LDs, and in response to a NB. *CsphyB* senses the NB, while *CsGI* extends the photosensitive phase for NB responsive *CsAFT* induction and flowering inhibition (Higuchi et al., 2013; Oda et al., 2020). Hence, the roles of GI, CO-like factors, and florigen/anti-florigen pairs are conserved in soybean, chrysanthemum and potato, while *Ehd1* (rice), *E1* (soybean), and *StCDF1* (potato) were specifically recruited to a SD-dependent control of these pathways.

23

24

## High temperature stress on SD crops

SD plants, such as soybean, chrysanthemum, and potato, are significantly affected by elevated temperatures at night, which strongly suppress flowering and tuberization. Inhibition of flowering or tuberization correlates with suppressed expression of the *GmFT2a/5a*, *CsFTL3*, and *StSP6A* inducing signals. Interestingly, response of the *FT* florigen and antiflorigen genes is not identical to that observed on interruption of long nights by a NB, suggesting that warm temperatures impair activity of a different regulator in the pathway than the NB. Light and temperature input signals are both

31
1 integrated by the circadian clock and thus it is possible that high temperatures
2 desynchronize the endogenous diel, leading to defective regulation of the *FT* florigen
3 genes.

4 In rice, key loci such as *Hd1*, *PRR37*, *Ghd8*, and *Hd6* had a critical role in spreading
5 its growth habitat and adapting flowering time to new geographical latitudes (Takahashi
6 et al., 2001; Guo et al., 2020b). *OsHd1* acts as a stronger LD repressor at cooler
7 temperatures, while sensitivity to temperature is reduced in *hd1* mutants (Luan et al.,
8 2009; Nagalla et al., 2021). Similarly, flowering suppression by *OsGhd7* is enhanced
9 under cooler conditions, while *OsPRR37* was found to repress flowering at low
10 temperatures, but promoting heading when temperature is over a critical temperature.
11 Indeed, phyB enhances *OsGhd7* transcription and stabilizes this repressor, thereby
12 enhancing *OsHd1* repressive function in LDs. This control is more relevant in cool
13 conditions, whereas warmth induced phyB inactivation decreases *OsGhd7* abundance,
14 reversing *OsGhd7*-*OsHd1* repression of *OsEhd1*, *OsHd3a*, and *OsRFT1*. *OsGhd7*
15 transcript levels are also reduced in warm conditions in a phyB independent manner,
16 which leads to activation of *OsRFT1* and flowering induction (Nagalla et al., 2021).

17 In soybean moderate increase in temperature (30°C) promote flowering, while
18 flowering transition is delayed at 35°C. *GmFT2a* and *GmFT5a* are up-regulated at 30°C
19 independently of E1, in contrast to the flowering delay at 35°C where suppression of
20 *GmFT2a* and *GmFT5a* is mediated through the phyA-E1 photoperiodic pathway (Tang
21 et al., 2023). The molecular mechanisms underlying this control remain however
22 obscure.

23 In chrysanthemum, elevated temperatures (~30°C) suppress flowering in SDs due to
24 the inhibition of *CsFTL3* in leaves (Nakano et al., 2013). Warm temperatures impair
25 *CsFTL3* expression and affect its downstream activation cascade. Heat sensitivity is
26 higher during nighttime and particularly during the second half of the night (ZT 12–16),
27 consistently with this response relying on an output of the circadian clock (Nakano et al.,
28 2015; 2020). phyB was also established to sense NB treatments, while a circadian
29 clock-dependent gating mechanism defines a photosensitive NB phase that coincides
30 with midnight (Higuchi et al., 2013). Overexpression of *GI* extends the critical dark
1 length (CDL) for flowering and lengthens this photosensitive phase, hence suggesting
2 that GI plays a pivotal role in gating CsAFT activation (Oda et al., 2020).

3
4 **Conclusions**
5
6 Temperature acts as a positive or negative signal for flowering, depending on the LD
7 or SD-photoperiodic behavior of plants. The day length flowering regulators that
8 integrate this environmental cue remain still unknown. Future research will thus be
9 crucial to gain a molecular understanding of the mechanisms involved at perceiving
10 warm temperatures at night. Revealing which actors mediate this poorly understood
11 process will help to explain the opposing effects of temperature in flowering time
12 regulation in LD and SD plants, while pave the way to the design of novel strategies
13 addressed to improving climate change resilience of SD crops.

14 Acknowledgements

15 We apologize to authors whose work could not be cited due to space constraints. MA
16 and RS also grateful to Julia Bailey-Serres and Pierdomenico Perata for their valuable
17 feedback. The authors gratefully acknowledge the funding support for their research
18 groups: Research in the Roeder lab is supported by grants NIH NIGMS R01
19 GM134037, NSF MCB-2203275, NSF EF-2222434, and NSF DBI-2320251. Research
20 in the Yang laboratory is funded by the National Natural Science Foundation of China
21 (32230005), and Pinduoduo-China Agricultural University Research Fund
22 (PC2023A02004). Research in the Sasidharan laboratory is supported by Utrecht
23 University and Netherlands Organization for Scientific Research (NWO)
24 (OCENW.M20.197) to RS. Research in the Yanovsky and Casal laboratories is
25 supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT,
26 Argentina, grants PICT-2019-04450 to MJY and PICT-2021-I-A-01070 to JJC) and
27 University of Buenos Aires (grant 20020170100505BA to JJC). Research in the Ruffel
28 laboratory is supported by the Agence Nationale de la Recherche (ANR) with the
29 FatRoot and ModLSys projects (ANR-21-CE13-0019 and ANR-23-CE45-0018) and by
30 the DIGIT-BIO MetaProgram. Research in the Assmann laboratory is funded by the
31 National Institute of General Medical Sciences of the National Institutes of Health under
award number 1 R35GM153492-01 to SMA. Research in the Gilroy laboratory is supported by NASA Space Biology: 80NSSC19K0126, 80NSSC21K0577, 80NSSC23K0380 and 80NSSC24K0705. Potato research conducted in the Prat laboratory lab has been supported by the ADAPT project, funded by the European Union's Horizon 2020 Research and Innovation program under grant agreement No. 862858. Chrysanthemum research led by the Sharath laboratory has been funded by Deliflor Chrysant BV. Research in the Argueso lab is supported by NSF MCB-1818111.

## Author Contributions:

All authors contributed to the writing, reviewing and editing of the article.

## Data Availability:

No new data were generated or analysed in support of this research.

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**Figure Legends:**

42

<caption>**Figure 1. Genetic regulation of cold tolerance in Arabidopsis and major crops. (A)**</caption>

43

44 Overview of the current understanding of the DREB1 regulatory network in Arabidopsis.
1 Both positive and negative regulators that modulate *DREB1* expression and/or protein
2 stability through various mechanisms are depicted. (B) Key genetic components
3 identified in various crops, including homologous genes, natural variations, and loci
4 associated with cold adaptation. These elements represent promising targets for genetic
5 manipulation aimed at mitigating the trade-offs between yield and stress tolerance in the
6 development of cold-tolerant crop varieties.
7

8 **Figure 2.** Arabidopsis research has yielded groundbreaking insights into plant
9 low-oxygen sensing, signaling and response in both developmental and
10 environmental contexts. (A) Schematic representation of the PLANT CYSTEINE
11 OXIDASE (PCO) branch of the N-degron pathway and its current known substrates.
12 MetAP, methionine amino-peptidase; ATE, arginyl transferase; PRT6, proteolysis 6;
13 <sup>ox</sup>Cys, oxidized cysteine. The oxygen and possible nitric oxide (NO) positions in the
14 pathway are shown. Oxygen is used by PCOs to oxidize amino-terminal Cys of the
15 substrates. (B) The different aspects of hypoxia. Arabidopsis studies have been
16 instrumental in our current understanding of how plants deal with environment-induced
17 and developmental hypoxia. Stressful hypoxia (right side panel) occurs in the roots of
18 waterlogged plants or shoots of plants during complete submergence in turbid flood
19 waters. Impaired gas diffusion in water also causes a rapid rise in ethylene in flooded
20 tissues. Oxygen and ethylene sensing and signaling pathways are part of a complex
21 molecular network essential for plant responses to abiotic-stress-induced hypoxia. Non-
22 stressful hypoxia (left side panel) corresponds to hypoxic niches that affect plant
23 development. Soil oxygen content can vary from 3 – 15% depending on soil type, depth,
24 and compactness, whereas shoot apical meristem (SAMs) maintain 3 – 6 % O₂.
25 Flowering plants grow on an altitudinal range from sea level (pO₂ 21%) up to 6000
26 meters above sea level (pO₂ 10%).

28 **Figure 3.** Plant responses to light cues modified by crop management practices
29 significantly influence key agronomic traits and Arabidopsis research has been
30 instrumental in identifying the crucial genetic players involved. In dense stands,
31 shade perceived by photoreceptors (PHYB, CRY1, PHYA) triggers architectural
changes that modulate crowding tolerance. The photoperiod, determined by location (latitude) and sowing date, influences flowering time and cycle length, depending on genes involved in light signaling, in some cases interacting with the circadian clock (PHYC, PRR7, GI, LNK2, ELF3). These changes in crowding tolerance and cycle length ultimately feedback to determine the optimal population density, sowing dates, and suitable locations for crop cultivation. The light signaling genes identified in Arabidopsis are highlighted in white boxes.

**Figure 4. Deciphering nitrogen (N) nutrition and signaling in Arabidopsis contributes to improve yield and Nitrogen Use Efficiency (NUE) in crops.**

The main N sources in plant nutrition are the two inorganic forms nitrate (NO3⁻) and ammonium (NH4⁺). In Arabidopsis, molecular mechanisms associated to their perception, uptake, transport, remobilization and assimilation have been deciphered. In rice, it is mainly the overexpression of homolog genes that led to increase yield and NUE. Regulators, such as transcription factors, are also promising targets as potential coordinators of the different steps of N journey in plants. Making root systems more efficient at capturing nutrients by optimizing the architecture is another interesting avenue.

**Figure 5. Approaches and targets toward improved drought resilience.**

Popular techniques for identifying and assessing candidate genes include genome-wide association studies (GWAS), transcriptome-wide association studies (TWAS), Inducible and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-targeted gene regulation, chemical genetics, and single-cell RNA sequencing. Advanced techniques for assessing drought response phenotypes include hyperspectral and thermal imaging and imaging rhizotrons. Gene targets for enhancing drought tolerance and/or avoidance in Arabidopsis are associated with xylem anatomy, stomatal kinetics and/or density, stress signaling pathways, and root architecture. Candidates indicated with OE (overexpression) or KO (knockout) were shown to confer improved drought phenotypes after that manipulation. Of the gene targets in this figure, only *CspB* from *Bacillus subtilis* and *HaHD4* from sunflower have been incorporated into commercial
1. crop lines; however, several candidate genes that regulate stomatal behavior or reduce
2. stomatal density upon manipulation in Arabidopsis have produced favorable drought-
3. related phenotypes upon manipulation of a related gene in crop species.

4. 
5. **Figure 6. The unique stresses of living in space.** Spaceflight imposes a series of
6. novel factors that biology must adapt to. These include the biological effects of reduced
7. gravity (microgravity) on physiology and development (such as altered gravity-
8. responsive growth) and microgravity's physical effects on the growth environment (such
9. as loss of convection and increased impact of the adhesion of water) and increased
10. radiation exposure. The space vehicle itself imposes other challenges such as
11. constraints on physical space for plant growth, and often an elevated CO₂ level (that
12. can reach several thousands of ppm from crew respiration) along with a potentially
13. complex environment of volatiles arising from outgassing from the plastics and polymers
14. of the vehicle's construction.
15.

16. **Figure 7. Overview of flowering regulation in Arabidopsis (LD model plant) and its**
17. parallels in SD crops: Soybean (*Glycine max*), Chrysanthemum (*Chrysanthemum seticuspe*), and the tuberization pathway in Potato (*Solanum tuberosum*). In
18. Arabidopsis, central flowering regulators include the circadian clock and the interplay of
19. florigens and antiflorigens to regulate flowering. Under long-day (LD) photoperiods, the
20. CONSTANS (CO) transcription factor activates *FLOWERING LOCUS T* (*FT*)
21. expression. The *FT* protein moves from the leaves to the shoot apex, where it forms the
22. Flowering Activation Complex (FAC) to initiate flowering. These regulators are
23. conserved in short-day (SD) crops, where they play critical roles in photoperiodic
24. flowering, temperature-responsive flowering, and tuberization pathways. SD plants have
25. evolved additional negative or antiflorigenic regulators (*GmFT1a/4a* in soybean, *CsAFT*
26. in chrysanthemum, and *StSP5G* in potato) that suppress flowering and tuberization
27. signals under long-day (LD), night-break (NB) conditions, or warm temperature.
28.
29.
30
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