Far-red light enrichment affects gene expression and architecture as well as growth and photosynthesis in rice.

Plants use light as a resource and signal. Photons within the 400-700 nm waveband are considered photosynthetically active. Far-red photons (FR, 700-800 nm) are used by plants to detect nearby vegetation and elicit the shade avoidance syndrome. In addition, FR photons have also been shown to contribute to photosynthesis, but knowledge about these dual effects remains scarce. Here, we study shoot-architectural and photosynthetic responses to supplemental FR light during the photoperiod in several rice varieties. We observed that FR enrichment only mildly affected the rice transcriptome and shoot architecture as compared to established model species, whereas leaf formation, tillering and biomass accumulation were clearly promoted. Consistent with this growth promotion, we found that CO2-fixation in supplemental FR was strongly enhanced, especially in plants acclimated to FR-enriched conditions as compared to control conditions. This growth promotion dominates the effects of FR photons on shoot development and architecture. When substituting FR enrichment with an end-of-day FR pulse, this prevented photosynthesis-promoting effects and elicited shade avoidance responses. We conclude that FR photons can have a dual role, where effects depend on the environmental context: in addition to being an environmental signal, they are also a potent source of harvestable energy.

A low-cost open-source imaging platform reveals spatiotemporal insight into leaf elongation and movement.

Plant organs move throughout the diurnal cycle, changing leaf and petiole positions to balance light capture, leaf temperature, and water loss under dynamic environmental conditions. Upward movement of the petiole, called hyponasty, is one of several traits of the shade avoidance syndrome (SAS). SAS traits are elicited upon perception of vegetation shade signals such as far-red light (FR) and improve light capture in dense vegetation. Monitoring plant movement at a high temporal resolution allows studying functionality and molecular regulation of hyponasty. However, high temporal resolution imaging solutions are often very expensive, making this unavailable to many researchers. Here, we present a modular and low-cost imaging setup, based on small Raspberry Pi computers that can track leaf movements and elongation growth with high temporal resolution. We also developed an open-source, semiautomated image analysis pipeline. Using this setup, we followed responses to FR enrichment, light intensity, and their interactions. Tracking both elongation and the angle of the petiole, lamina, and entire leaf in Arabidopsis (Arabidopsis thaliana) revealed insight into R:FR sensitivities of leaf growth and movement dynamics and the interactions of R:FR with background light intensity. The detailed imaging options of this system allowed us to identify spatially separate bending points for petiole and lamina positioning of the leaf.

Differential leaf flooding resilience in Arabidopsis thaliana is controlled by ethylene signaling-activated and age-dependent phosphorylation of ORESARA1.

The phytohormone ethylene is a major regulator of plant adaptive responses to flooding. In flooded plant tissues, ethylene quickly increases to high concentrations owing to its low solubility and diffusion rates in water. Ethylene accumulation in submerged plant tissues makes it a reliable cue for triggering flood acclimation responses, including metabolic adjustments to cope with flood-induced hypoxia. However, persistent ethylene accumulation also accelerates leaf senescence. Stress-induced senescence hampers photosynthetic capacity and stress recovery. In submerged Arabidopsis, senescence follows a strict age-dependent pattern starting with the older leaves. Although mechanisms underlying ethylene-mediated senescence have been uncovered, it is unclear how submerged plants avoid indiscriminate breakdown of leaves despite high systemic ethylene accumulation. We demonstrate that although submergence triggers leaf-age-independent activation of ethylene signaling via EIN3 in Arabidopsis, senescence is initiated only in old leaves. EIN3 stabilization also leads to overall transcript and protein accumulation of the senescence-promoting transcription factor ORESARA1 (ORE1) in both old and young leaves during submergence. However, leaf-age-dependent senescence can be explained by ORE1 protein activation via phosphorylation specifically in old leaves, independent of the previously identified age-dependent control of ORE1 via miR164. A systematic analysis of the roles of the major flooding stress cues and signaling pathways shows that only the combination of ethylene and darkness is sufficient to mimic submergence-induced senescence involving ORE1 accumulation and phosphorylation. Hypoxia, most often associated with flooding stress in plants, appears to have no role in these processes. Our results reveal a mechanism by which plants regulate the speed and pattern of senescence during environmental stresses such as flooding. Age-dependent ORE1 activity ensures that older, expendable leaves are dismantled first, thus prolonging the life of younger leaves and meristematic tissues that are vital to whole-plant survival.

Plant responses to limited aeration: Advances and future challenges.

Limited aeration that is caused by tissue geometry, diffusion barriers, high elevation, or a flooding event poses major challenges to plants and is often, but not exclusively, associated with low oxygen. These processes span a broad interest in the research community ranging from whole plant and crop responses, post-harvest physiology, plant morphology and anatomy, fermentative metabolism, plant developmental processes, oxygen sensing by ERF-VIIs, gene expression profiles, the gaseous hormone ethylene, and O2 dynamics at cellular resolution. The International Society for Plant Anaerobiosis (ISPA) gathers researchers from all over the world contributing to understand the causes, responses, and consequences of limited aeration in plants. During the 14th ISPA meeting, major research progress was related to the evolution of O2 sensing mechanisms and the intricate network that balances low O2 signaling. Here, the work moved beyond flooding stress and emphasized novel underexplored roles of low O2 and limited aeration in altitude adaptation, fruit development and storage, and the vegetative development of growth apices. Regarding tolerance towards flooding, the meeting stressed the relevance and regulation of developmental plasticity, aerenchyma, and barrier formation to improve internal aeration. Additional newly explored flood tolerance traits concerned resource balance, senescence, and the exploration of natural genetic variation for novel tolerance loci. In this report, we summarize and synthesize the major progress and future challenges for low O2 and aeration research presented at the conference.

Ethylene augments root hypoxia tolerance via growth cessation and reactive oxygen species amelioration.

Flooded plants experience impaired gas diffusion underwater, leading to oxygen deprivation (hypoxia). The volatile plant hormone ethylene is rapidly trapped in submerged plant cells and is instrumental for enhanced hypoxia acclimation. However, the precise mechanisms underpinning ethylene-enhanced hypoxia survival remain unclear. We studied the effect of ethylene pretreatment on hypoxia survival of Arabidopsis (Arabidopsis thaliana) primary root tips. Both hypoxia itself and re-oxygenation following hypoxia are highly damaging to root tip cells, and ethylene pretreatments reduced this damage. Ethylene pretreatment alone altered the abundance of transcripts and proteins involved in hypoxia responses, root growth, translation, and reactive oxygen species (ROS) homeostasis. Through imaging and manipulating ROS abundance in planta, we demonstrated that ethylene limited excessive ROS formation during hypoxia and subsequent re-oxygenation and improved oxidative stress survival in a PHYTOGLOBIN1-dependent manner. In addition, we showed that root growth cessation via ethylene and auxin occurred rapidly and that this quiescence behavior contributed to enhanced hypoxia tolerance. Collectively, our results show that the early flooding signal ethylene modulates a variety of processes that all contribute to hypoxia survival.

Nocturnal gibberellin biosynthesis is carbon dependent and adjusts leaf expansion rates to variable conditions.

Optimal plant growth performance requires that the presence and action of growth signals, such as gibberellins (GAs), are coordinated with the availability of photo-assimilates. Here, we studied the links between GA biosynthesis and carbon availability, and the subsequent effects on growth. We established that carbon availability, light and dark cues, and the circadian clock ensure the timing and magnitude of GA biosynthesis and that disruption of these factors results in reduced GA levels and expression of downstream genes. Carbon-dependent nighttime induction of gibberellin 3-beta-dioxygenase 1 (GA3ox1) was severely hampered when preceded by reduced daytime light availability, leading specifically to reduced bioactive GA4 levels, and coinciding with a decline in leaf expansion rate during the night. We attributed this decline in leaf expansion mostly to reduced photo-assimilates. However, plants in which GA limitation was alleviated had significantly improved leaf expansion, demonstrating the relevance of GAs in growth control under varying carbon availability. Carbon-dependent expression of upstream GA biosynthesis genes (Kaurene synthase and gibberellin 20 oxidase 1, GA20ox1) was not translated into metabolite changes within this short timeframe. We propose a model in which the extent of nighttime biosynthesis of bioactive GA4 by GA3ox1 is determined by nighttime consumption of starch reserves, thus providing day-to-day adjustments of GA responses.

Age-Dependent Abiotic Stress Resilience in Plants.

Developmental age is a strong determinant of stress responses in plants. Differential susceptibility to various environmental stresses is widely observed at both the organ and whole-plant level. While it is clear that age determines stress susceptibility, the causes, regulatory mechanisms, and functions are only now beginning to emerge. Compared with concepts on age-related biotic stress resilience, advancements in the abiotic stress field are relatively limited. In this review, we focus on current knowledge of ontogenic resistance to abiotic stresses, highlighting examples at the organ (leaf) and plant level, preceded by an overview of the relevant concepts in plant aging. We also discuss age-related abiotic stress resilience mechanisms, speculate on their functional relevance, and outline outstanding questions.

Shape shifting by amphibious plants in dynamic hydrological niches.

Amphibious plants thrive in areas with fluctuating water levels, partly as a result of their capacity to make specialized leaves when submerged or emerged. The tailor-made leaves improve gas exchange underwater or prevent aerial desiccation. Aquatic leaves are thin with narrow or dissected forms, thin cuticles and fewer stomata. These traits can combine with carbon-concentrating mechanisms and various inorganic carbon utilization strategies. Signalling networks underlying this plasticity include conserved players like abscisic acid and ethylene, but closer inspection reveals greater variation in regulatory behaviours. Moreover, it seems that amphibious leaf development overrides and reverses conserved signalling pathways of their terrestrial counterparts. The diversity of physiology and signalling makes plant amphibians particularly attractive for gaining insights into the evolution of signalling and crop improvement.

Keeping the shoot above water - submergence triggers antithetical growth responses in stems and petioles of watercress (Nasturtium officinale).

The molecular mechanisms controlling underwater elongation are based extensively on studies on internode elongation in the monocot rice (Oryza sativa) and petiole elongation in Rumex rosette species. Here, we characterize underwater growth in the dicot Nasturtium officinale (watercress), a wild species of the Brassicaceae family, in which submergence enhances stem elongation and suppresses petiole growth. We used a genome-wide transcriptome analysis to identify the molecular mechanisms underlying the observed antithetical growth responses. Though submergence caused a substantial reconfiguration of the petiole and stem transcriptome, only little qualitative differences were observed between both tissues. A core submergence response included hormonal regulation and metabolic readjustment for energy conservation, whereas tissue-specific responses were associated with defense, photosynthesis, and cell wall polysaccharides. Transcriptomic and physiological characterization suggested that the established ethylene, abscisic acid (ABA), and GA growth regulatory module for underwater elongation could not fully explain underwater growth in watercress. Petiole growth suppression is likely attributed to a cell cycle arrest. Underwater stem elongation is driven by an early decline in ABA and is not primarily mediated by ethylene or GA. An enhanced stem elongation observed in the night period was not linked to hypoxia and suggests an involvement of circadian regulation.

Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress.

Timely perception of adverse environmental changes is critical for survival. Dynamic changes in gases are important cues for plants to sense environmental perturbations, such as submergence. In Arabidopsis thaliana, changes in oxygen and nitric oxide (NO) control the stability of ERFVII transcription factors. ERFVII proteolysis is regulated by the N-degron pathway and mediates adaptation to flooding-induced hypoxia. However, how plants detect and transduce early submergence signals remains elusive. Here we show that plants can rapidly detect submergence through passive ethylene entrapment and use this signal to pre-adapt to impending hypoxia. Ethylene can enhance ERFVII stability prior to hypoxia by increasing the NO-scavenger PHYTOGLOBIN1. This ethylene-mediated NO depletion and consequent ERFVII accumulation pre-adapts plants to survive subsequent hypoxia. Our results reveal the biological link between three gaseous signals for the regulation of flooding survival and identifies key regulatory targets for early stress perception that could be pivotal for developing flood-tolerant crops.

A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

Signal Dynamics and Interactions during Flooding Stress.

Flooding is detrimental for nearly all higher plants, including crops. The compound stress elicited by slow gas exchange and low light levels under water is responsible for both a carbon and an energy crisis ultimately leading to plant death. The endogenous concentrations of four gaseous compounds, oxygen, carbon dioxide, ethylene, and nitric oxide, change during the submergence of plant organs in water. These gases play a pivotal role in signal transduction cascades, leading to adaptive processes such as metabolic adjustments and anatomical features. Of these gases, ethylene is seen as the most consistent, pervasive, and reliable signal of early flooding stress, most likely in tight interaction with the other gases. The production of reactive oxygen species (ROS) in plant cells during flooding and directly after subsidence, during which the plant is confronted with high light and oxygen levels, is characteristic for this abiotic stress. Low, well-controlled levels of ROS are essential for adaptive signaling pathways, in interaction with the other gaseous flooding signals. On the other hand, excessive uncontrolled bursts of ROS can be highly damaging for plants. Therefore, a fine-tuned balance is important, with a major role for ROS production and scavenging. Our understanding of the temporal dynamics of the four gases and ROS is basal, whereas it is likely that they form a signature readout of prevailing flooding conditions and subsequent adaptive responses.

Functional Balancing of the Hypoxia Regulators RAP2.12 and HRA1 Takes Place in vivo in Arabidopsis thaliana Plants.

Plants are known to respond to variations in cellular oxygen availability and distribution by quickly adapting the transcription rate of a number of genes, generally associated to improved energy usage pathways, oxygen homeostasis and protection from harmful products of anaerobic metabolism. In terrestrial plants, such coordinated gene expression program is promoted by a conserved subfamily of ethylene responsive transcription factors called ERF-VII, which act as master activators of hypoxic gene transcription. Their abundance is directly regulated by oxygen through a mechanism of targeted proteolysis present under aerobic conditions, which is triggered by ERF-VII protein oxidation. Beside this, in Arabidopsis thaliana, the activity of the ERF-VII factor RAP2.12 has been shown to be restrained and made transient by the hypoxia-inducible transcription factor HRA1. This feedback mechanism has been proposed to modulate ERF-VII activity in the plant under fluctuating hypoxia, thereby enhancing the flexibility of the response. So far, functional balancing between RAP2.12 and HRA1 has been assessed in isolated leaf protoplasts, resulting in an inverse relationship between HRA1 amount and activation of RAP2.12 target promoters. In the present work, we showed that HRA1 is effective in balancing RAP2.12 activity in whole arabidopsis plants. Examination of a segregating population, generated from RAP2.12 and HRA1 over-expressing plants, led to the first quantitative proof that, over a range of either transgene expression levels, HRA1 counteracts the phenotypic and transcriptional effects of RAP2.12. This report supports the occurrence of fine-tuned regulation of the hypoxic response under physiological growth conditions.

Molecular Profiles of Contrasting Shade Response Strategies in Wild Plants: Differential Control of Immunity and Shoot Elongation.

Plants growing at high densities elongate their shoots to reach for light, a response known as the shade avoidance syndrome (SAS). Phytochrome-mediated detection of far-red light reflection from neighboring plants activates growth-promoting molecular pathways leading to SAS However, it is unknown how plants that complete their life cycle in the forest understory and are shade tolerant prevent SAS when exposed to shade. Here, we show how two wild Geranium species from different native light environments regulate contrasting responses to light quality cues. A comparative RNA sequencing approach unveiled the molecular underpinnings of their contrasting growth responses to far-red light enrichment. It also identified differential phytochrome control of plant immunity genes and confirmed that far-red enrichment indeed contrastingly affects resistance against Botrytis cinerea between the two species. Furthermore, we identify a number of candidate regulators of differential shade avoidance. Three of these, the receptor-like kinases FERONIA and THESEUS1 and the non-DNA binding bHLH protein KIDARI, are functionally validated in Arabidopsis thaliana through gene knockout and/or overexpression studies. We propose that these components may be associated with either showing or not showing shade avoidance responses.

Variation in Arabidopsis flooding responses identifies numerous putative "tolerance genes".

Plant survival in flooded environments requires a combinatory response to multiple stress conditions such as limited light availability, reduced gas exchange and nutrient uptake. The ability to fine-tune the molecular response at the transcriptional and/or post-transcriptional level that can eventually lead to metabolic and anatomical adjustments are the underlying requirements to confer tolerance. Previously, we compared the transcriptomic adjustment of submergence tolerant, intolerant accessions and identified a core conserved and genotype-specific response to flooding stress, identifying numerous 'putative' tolerance genes. Here, we performed genome wide association analyses on 81 natural Arabidopsis accessions that identified 30 additional SNP markers associated with flooding tolerance. We argue that, given the many genes associated with flooding tolerance in Arabidopsis, improving resistance to submergence requires numerous genetic changes.

Plant responses to flooding stress.

Most plant species cannot survive prolonged submergence or soil waterlogging. Crops are particularly intolerant to the lack of oxygen arising from submergence. Rice can instead germinate and grow even if submerged. The molecular basis for rice tolerance was recently unveiled and will contribute to the development of better rice varieties, well adapted to flooding. The oxygen sensing mechanism was also recently discovered. This system likely operates in all plant species and relies on the oxygen-dependent destabilization of the group VII ethylene response factors (ERFVIIs), a cluster of ethylene responsive transcription factors. An homeostatic mechanism that controls gene expression in plants subjected to hypoxia prevents excessive activation of the anaerobic metabolism that could be detrimental to surviving the stress.

Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress.

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress.

Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit.

The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions.

Group VII ethylene response factor diversification and regulation in four species from flood-prone environments.

Flooding events negatively affect plant performance and survival. Flooding gradients thereby determine the dynamics in vegetation composition and species abundance. In adaptation to flooding, the group VII Ethylene Response Factor genes (ERF-VIIs) play pivotal roles in rice and Arabidopsis through regulation of anaerobic gene expression and antithetical survival strategies. We investigated if ERF-VIIs have a similar role in mediating survival strategies in eudicot species from flood-prone environments. Here, we studied the evolutionary origin and regulation of ERF-VII transcript abundance and the physiological responses in species from two genera of divergent taxonomic lineages (Rumex and Rorippa). Synteny analysis revealed that angiosperm ERF-VIIs arose from two ancestral loci and that subsequent diversification and duplication led to the present ERF-VII variation. We propose that subtle variation in the regulation of ERF-VII transcript abundance could explain variation in tolerance among Rorippa species. In Rumex, the main difference in flood tolerance correlated with the genetic variation in ERF-VII genes. Large transcriptional differences were found by comparing the two genera: darkness and dark submergence-induced Rumex ERF-VIIs, whereas HRE2 expression was increased in submerged Rorippa roots. We conclude that the involvement of ERF-VIIs in flooding tolerance developed in a phylogenetic-dependent manner, with subtle variations within taxonomic clades.

Two Rumex species from contrasting hydrological niches regulate flooding tolerance through distinct mechanisms.

Global climate change has increased flooding events, which affect both natural vegetation dynamics and crop productivity. The flooded environment is lethal for most plant species because it restricts gas exchange and induces an energy and carbon crisis. Flooding survival strategies have been studied in Oryza sativa, a cultivated monocot. However, our understanding of plant adaptation to natural flood-prone environments remains scant, even though wild plants represent a valuable resource of tolerance mechanisms that could be used to generate stress-tolerant crops. Here we identify mechanisms that mediate the distinct flooding survival strategies of two related wild dicot species: Rumex palustris and Rumex acetosa. Whole transcriptome sequencing and metabolite profiling reveal flooding-induced metabolic reprogramming specific to R. acetosa. By contrast, R. palustris uses the early flooding signal ethylene to increase survival by regulating shade avoidance and photomorphogenesis genes to outgrow submergence and by priming submerged plants for future low oxygen stress. These results provide molecular resolution of flooding survival strategies of two species occupying distinct hydrological niches. Learning how these contrasting flood adaptive strategies evolved in nature will be instrumental for the development of stress-tolerant crop varieties that deliver enhanced yields in a changing climate.