Plant Quiescence Strategy and Seed Dormancy under Hypoxia.

Plant quiescence and seed dormancy can be triggered by reduced oxygen availability. Under water, oxygen depletion caused by flooding can culminate in a quiescent state, which is a plant strategy for energy preservation and survival. In adult plants, a quiescent state can be activated by sugar starvation, culminating in metabolic depression. In seeds, secondary dormancy can be activated by reduced oxygen availability, which creates an unfavourable state for germination. The physical dormancy of some seeds and buds includes barriers to external conditions, which indirectly results in hypoxia. The molecular processes that support seed dormancy and plant survival through quiescence under hypoxia include the N-degron pathway, which enables the modulation of ethylene responsive factors of group VII and downstream targets. This oxygen- and nitric oxide-dependent mechanism interacts with phytohormone-related pathways to control growth.

Seed bacterial microbiota in post-submergence tolerant and sensitive barley genotypes.

Flooding is a predominant abiotic stress for cultivated plants, including barley. This cereal crop shows a large adaptability to different environmental conditions, suggesting the presence of key traits to tolerate adverse conditions. During germination, genetic variations account for dissimilarities in flooding tolerance. However, differences in the seed microbiota may also contribute to tolerance/sensitivity during seedling establishment. This work investigated differences in microbiome among the grains of barley accessions. Two barley phenotypes were compared, each either tolerant or sensitive to a short submergence period followed by a recovery. The study used a metataxonomic analysis based on 16S ribosomal RNA gene sequencing and subsequent functional prediction. Our results support the hypothesis that bacterial microbiota inhabiting the barley seeds are different between sensitive and tolerant barley accessions, which harbour specific bacterial phyla and families. Finally, bacteria detected in tolerant barley accessions show a peculiar functional enrichment that suggests a possible connection with successful germination and seedling establishment.

Recent progress in understanding the cellular and genetic basis of plant responses to low oxygen holds promise for developing flood-resilient crops.

With recent progress in active research on flooding and hypoxia/anoxia tolerance in native and agricultural crop plants, vast knowledge has been gained on both individual tolerance mechanisms and the general mechanisms of flooding tolerance in plants. Research on carbohydrate consumption, ethanolic and lactic acid fermentation, and their regulation under stress conditions has been accompanied by investigations on aerenchyma development and the emergence of the radial oxygen loss barrier in some plant species under flooded conditions. The discovery of the oxygen-sensing mechanism in plants and unravelling the intricacies of this mechanism have boosted this very international research effort. Recent studies have highlighted the importance of oxygen availability as a signalling component during plant development. The latest developments in determining actual oxygen concentrations using minute probes and molecular sensors in tissues and even within cells have provided new insights into the intracellular effects of flooding. The information amassed during recent years has been used in the breeding of new flood-tolerant crop cultivars. With the wealth of metabolic, anatomical, and genetic information, novel holistic approaches can be used to enhance crop species and their productivity under increasing stress conditions due to climate change and the subsequent changes in the environment.

The inability of barley to germinate after submergence depends on hypoxia-induced secondary dormancy.

Global climate change has dramatically increased flooding events, which have a strong impact on crop production. Barley (Hordeum vulgare) is one of the most important cereals and its cultivation includes a broad range of different environments. We tested the capacity to germinate of a large barley panel after a short period of submergence followed by a period of recovery. We demonstrate that sensitive barley varieties activate underwater secondary dormancy because of a lower permeability to oxygen dissolved in water. In sensitive barley accessions, secondary dormancy is removed by nitric oxide donors. The results of a genome-wide association study uncovered a Laccase gene located in a region of significant marker-trait association that is differently regulated during grain development and plays a key role in this process. Our findings will help breeders to improve the genetics of barley, thereby increasing the capacity of seeds to germinate after a short period of flooding.

Turning earthworms into moonworms: Earthworms colonization of lunar regolith as a bioengineering approach supporting future crop growth in space.

The earthworms beneficial effects on soils may be promising to improve lunar soil fertility, enabling the use of local substrates for space farming. Herein, we investigated the effects of the lunar regolith simulant (LHS-1) at different concentrations in cow manure mixtures on the survival and fitness of Eisenia fetida. During 14 and 60-day experiments, although E. fetida showed an increased mortality with LHS-1 alone, most of the population survived. More numerous tunnels were observed when exposed to the higher concentrations of LHS-1 (poor in nutrients for earthworms). This may be related to an increased mobility for food search. The cocoons production was not affected by different substrate treatments, except for the highest concentration of LHS-1. No effects of different LHS-1 concentrations on the amount of ingested substrate were recorded. This study shows that E. fetida can potentially colonize lunar regolith representing a future valuable biological tool for supporting crops growth on the Moon.

Group VII ethylene response factors, MtERF74 and MtERF75, sustain nitrogen fixation in Medicago truncatula microoxic nodules.

Group VII ethylene response factors (ERF-VII) are plant-specific transcription factors (TFs) known for their role in the activation of hypoxia-responsive genes under low oxygen stress but also in plant endogenous hypoxic niches. However, their function in the microaerophilic nitrogen-fixing nodules of legumes has not yet been investigated. We investigated regulation and the function of the two Medicago truncatula ERF-VII TFs (MtERF74 and MtERF75) in roots and nodules, MtERF74 and MtERF75 in response to hypoxia stress and during the nodulation process using an RNA interference strategy and targeted proteolysis of MtERF75. Knockdown of MtERF74 and MtERF75 partially blocked the induction of hypoxia-responsive genes in roots exposed to hypoxia stress. In addition, a significant reduction in nodulation capacity and nitrogen fixation activity was observed in mature nodules of double knockdown transgenic roots. Overall, the results indicate that MtERF74 and MtERF75 are involved in the induction of MtNR1 and Pgb1.1 expression for efficient Phytogb-nitric oxide respiration in the nodule.

Bacterial Endophytes Contribute to Rice Seedling Establishment Under Submergence.

Flooding events caused by severe rains and poor soil drainage can interfere with plant germination and seedling establishment. Rice is one of the cereal crops that has unique germination strategies under flooding. One of these strategies is based on the fast coleoptile elongation in order to reach the water surface and re-establish the contact with the air. Microorganisms can contribute to plant health via plant growth promoters and provide protection from abiotic stresses. To characterise the community composition of the microbiome in rice germination under submergence, a 16S rRNA gene profiling metagenomic analysis was performed of temperate japonica rice varieties Arborio and Lamone seedlings, which showed contrasting responses in terms of coleoptile length when submerged. This analysis showed a distinct microbiota composition of Arborio seeds under submergence, which are characterised by the development of a long coleoptile. To examine the potential function of microbial communities under submergence, culturable bacteria were isolated, identified and tested for plant growth-promoting activities. A subgroup of isolated bacteria showed the capacity to hydrolyse starch and produce indole-related compounds under hypoxia. Selected bacteria were inoculated in seeds to evaluate their effect on rice under submergence, showing a response that is dependent on the rice genotype. Our findings suggest that endophytic bacteria possess plant growth-promoting activities that can substantially contribute to rice seedling establishment under submergence.

Screening for Abiotic Stress Response in Rice.

Rice (Oryza sativa L.) is the staple food for over half of the world population. However, most rice varieties are severely injured by abiotic stresses, with strong social and economic impacts. Understanding rice responses to stress may guide breeding for more tolerant varieties. However, the lack of consistency in the design of the stress experiments described in the literature limits comparative studies and output assessments. The use of identical setups is the only way to generate comparable data. This chapter comprises three sections, describing the experimental conditions established at the Genomics of Plant Stress (GPlantS) unit of ITQB NOVA to assess the response of rice plants to different abiotic stresses-high salinity, cold, drought, simulated drought, and submergence-and their recovery capacity when intended. All sections include a detailed description of the materials and methodology and useful notes gathered from our team experience. We use seedlings since rice plants at this stage show high sensitivity to abiotic stresses. For the salt, cold, and simulated drought (PEG, polyethylene glycol) stress assays, we grow rice seedlings in a hydroponic system, while for the drought assay, plants are grown in soil and subjected to water withholding. For submergence, we use water-filled Magenta boxes. All setups enable visual score determination and are suitable for sample collection during stress imposition and also recovery. The proposed methodologies are affordable and straightforward to implement in most labs, allowing the discrimination of several rice genotypes at the molecular and phenotypic levels.

Cereal Germination under Low Oxygen: Molecular Processes.

Cereal crops can differ greatly in tolerance to oxygen shortage under germination and seedling establishment. Rice is able to germinate and elongate the coleoptile under submergence and anoxia. This capacity has been attributed to the successful use of starchy reserves through a molecular pathway activated by sugar starvation and low oxygen. This pathway culminates with the expression of α-amylases to provide sugars that fuel the sink organs. On the contrary, barley and wheat are unable to germinate under anoxia. The sensitivity of barley and wheat is likely due to the incapacity to use starch during germination. This review highlights what is currently known about the molecular mechanisms associated with cereal germination and seedling establishment under oxygen shortage with a special focus on barley and rice. Insights into the molecular mechanisms that support rice germination under low oxygen and into those that are associated with barley sensitivity may be of help for genetic improvement programs.

The Oxidative Paradox in Low Oxygen Stress in Plants.

Reactive oxygen species (ROS) are part of aerobic environments, and variations in the availability of oxygen (O2) in the environment can lead to altered ROS levels. In plants, the O2 sensing machinery guides the molecular response to low O2, regulating a subset of genes involved in metabolic adaptations to hypoxia, including proteins involved in ROS homeostasis and acclimation. In addition, nitric oxide (NO) participates in signaling events that modulate the low O2 stress response. In this review, we summarize recent findings that highlight the roles of ROS and NO under environmentally or developmentally defined low O2 conditions. We conclude that ROS and NO are emerging regulators during low O2 signalling and key molecules in plant adaptation to flooding conditions.

Auxin is required for the long coleoptile trait in japonica rice under submergence.

Rice coleoptile elongation under submergence guarantees fast seedling establishment in the field. We investigated the role of auxin in influencing the capacity of rice to produce a long coleoptile under water. In order to explore the complexity of auxin's role in coleoptile elongation, we used gene expression analysis, confocal microscopy of an auxin-responsive fluorescent reporter, gas chromatography coupled to tandem mass spectrometry (GC-MS/MS), and T-DNA insertional mutants of an auxin transport protein. We show that a higher auxin availability in the coleoptile correlates with the final coleoptile length under submergence. We also identified the auxin influx carrier AUX1 as a component influencing this trait under submergence. The coleoptile tip is involved in the final length of rice varieties harbouring a long coleoptile. Our experimental results indicate that auxin biosynthesis and transport underlies the differential elongation between short and long coleoptile-harbouring japonica rice varieties.

Molecular Mechanisms Supporting Rice Germination and Coleoptile Elongation under Low Oxygen.

Rice germinates under submergence by exploiting the starch available in the endosperm and translocating sugars from source to sink organs. The availability of fermentable sugar under water allows germination with the protrusion of the coleoptile, which elongates rapidly and functions as a snorkel toward the air above. Depending on the variety, rice can produce a short or a long coleoptile. Longer length entails the involvement of a functional transport of auxin along the coleoptile. This paper is an overview of rice coleoptiles and the studies undertaken to understand its functioning and role under submergence.

The calcineurin ß-like interacting protein kinase CIPK25 regulates potassium homeostasis under low oxygen in Arabidopsis.

Hypoxic conditions often arise from waterlogging and flooding, affecting several aspects of plant metabolism, including the uptake of nutrients. We identified a member of the CALCINEURIN ß-LIKE INTERACTING PROTEIN KINASE (CIPK) family in Arabidopsis, CIPK25, which is induced in the root endodermis under low-oxygen conditions. A cipk25 mutant exhibited higher sensitivity to anoxia in conditions of potassium limitation, suggesting that this kinase is involved in the regulation of potassium uptake. Interestingly, we found that CIPK25 interacts with AKT1, the major inward rectifying potassium channel in Arabidopsis. Under anoxic conditions, cipk25 mutant seedlings were unable to maintain potassium concentrations at wild-type levels, suggesting that CIPK25 likely plays a role in modulating potassium homeostasis under low-oxygen conditions. In addition, cipk25 and akt1 mutants share similar developmental defects under waterlogging, further supporting an interplay between CIPK25 and AKT1.

Exploring Legume-Rhizobia Symbiotic Models for Waterlogging Tolerance.

Unexpected and increasingly frequent extreme precipitation events result in soil flooding or waterlogging. Legumes have the capacity to establish a symbiotic relationship with endosymbiotic atmospheric dinitrogen-fixing rhizobia, thus contributing to natural nitrogen soil enrichment and reducing the need for chemical fertilization. The impact of waterlogging on nitrogen fixation and legume productivity needs to be considered for crop improvement. This review focuses on the legumes-rhizobia symbiotic models. We aim to summarize the mechanisms underlying symbiosis establishment, nodule development and functioning under waterlogging. The mechanisms of oxygen sensing of the host plant and symbiotic partner are considered in view of recent scientific advances.

Dissection of coleoptile elongation in japonica rice under submergence through integrated genome-wide association mapping and transcriptional analyses.

Rice is unique among cereals for its ability to germinate not only when submerged but also under anoxic conditions. Rice germination under submergence or anoxia is characterized by a longer coleoptile and delay in radicle emergence. A panel of temperate and tropical japonica rice accessions showing a large variability in coleoptile length was used to investigate genetic factors involved in this developmental process. The ability of the Khao Hlan On rice landrace to vigorously germinate when submerged has been previously associated with the presence of the trehalose 6 phosphate phosphatase 7 (TPP7) gene. In this study, we found that, in the presence of TPP7, polymorphisms and transcriptional variations of the gene in coleoptile tissue were not related to differences in the final coleoptile length under submergence. In order to find new chromosomal regions associated with the different ability of rice to elongate the coleoptile under submergence, we used genome-wide association study analysis on a panel of 273 japonica rice accessions. We discovered 11 significant marker-trait associations and identified candidate genes potentially involved in coleoptile length. Candidate gene expression analyses indicated that japonica rice genotypes possess complex genetic elements that control final coleoptile length under low oxygen.

Transcriptome profiling of short-term response to chilling stress in tolerant and sensitive Oryza sativa ssp. Japonica seedlings.

Low temperature is a major factor limiting rice growth and yield, and seedling is one of the developmental stages at which sensitivity to chilling stress is higher. Tolerance to chilling is a complex quantitative trait, so one of the most effective approaches to identify genes and pathways involved is to compare the stress-induced expression changes between tolerant and sensitive genotypes. Phenotypic responses to chilling of 13 Japonica cultivars were evaluated, and Thaibonnet and Volano were selected as sensitive and tolerant genotypes, respectively. To thoroughly profile the short-term response of the two cultivars to chilling, RNA-Seq was performed on Thaibonnet and Volano seedlings after 0 (not stressed), 2, and 10 h at 10 °C. Differential expression analysis revealed that the ICE-DREB1/CBF pathway plays a primary role in chilling tolerance, mainly due to some important transcription factors involved (some of which had never been reported before). Moreover, the expression trends of some genes that were radically different between Thaibonnet and Volano (i.e., calcium-dependent protein kinases OsCDPK21 and OsCDPK23, cytochrome P450 monooxygenase CYP76M8, etc.) suggest their involvement in low temperature tolerance too. Density of differentially expressed genes along rice genome was determined and linked to the position of known QTLs: remarkable co-locations were reported, delivering an overview of genomic regions determinant for low temperature response at seedling stage. Our study contributes to a better understanding of the molecular mechanisms underlying rice response to chilling and provides a solid background for development of low temperature-tolerant germplasm.

New insights into reactive oxygen species and nitric oxide signalling under low oxygen in plants.

Plants produce reactive oxygen species (ROS) when exposed to low oxygen (O2 ). Much experimental evidence has demonstrated the existence of an oxidative burst when there is an O2 shortage. This originates at various subcellular sites. The activation of NADPH oxidase(s), in complex with other proteins, is responsible for ROS production at the plasma membrane. Another source of low O2 -dependent ROS is the mitochondrial electron transport chain, which misfunctions when low O2 limits its activity. Arabidopsis mutants impaired in proteins playing a role in ROS production display an intolerant phenotype to anoxia and submergence, suggesting a role in acclimation to stress. In rice, the presence of the submergence 1A (SUB1A) gene for submergence tolerance is associated with a higher capacity to scavenge ROS. Additionally, the destabilization of group VII ethylene responsive factors, which are involved in the direct O2 sensing mechanism, requires nitric oxide (NO). All this evidence suggests the existence of a ROS and NO - low O2 mechanism interplay which likely includes sensing, anaerobic metabolism and acclimation to stress. In this review, we summarize the most recent findings on this topic, formulating hypotheses on the basis of the latest advances.

A calcineurin B-like protein participates in low oxygen signalling in rice.

Following the identification of the calcineurin B-like interacting protein kinase 15 (CIPK15), which is a regulator of starch degradation, the low O2 signal elicited during rice germination under submergence has been linked to the sugar sensing cascade and calcium (Ca2+) signalling. CIPK proteins are downstream effectors of calcineurin B-like proteins (CBLs), which act as Ca2+ sensors, whose role under low O2 has yet to be established. In the present study we describe CBL4 as a putative CIPK15 partner, transcriptionally activated under low O2 in rice coleoptiles. The transactivation of the rice embryo CBL4 transcript and CBL4 promoter was influenced by the Ca2+ blocker ruthenium red (RR). The bimolecular fluorescence complementation (BiFC) assay associated to fluorescence recovery after photobleaching (FRAP) analysis confirmed that CBL4 interacts with CIPK15. The CBL4-CIPK15 complex is localised in the cytoplasm and the plasma-membrane. Experiments in protoplasts showed a dampening of α-amylase 3 (RAMY3D) expression after CBL4 silencing by artificial miRNA. Our results suggest that under low O2, the Ca2+ sensor CBL4 interacts with CIPK15 to regulate RAMY3D expression in a Ca2+-dependent manner.

Universal stress protein HRU1 mediates ROS homeostasis under anoxia.

Plant survival is greatly impaired when oxygen levels are limiting, such as during flooding or when anatomical constraints limit oxygen diffusion. Oxygen sensing in Arabidopsis thaliana is mediated by Ethylene Responsive Factor (ERF)-VII transcription factors, which control a core set of hypoxia- and anoxia-responsive genes responsible for metabolic acclimation to low-oxygen conditions. Anoxic conditions also induce genes related to reactive oxygen species (ROS). Whether the oxygen-sensing machinery coordinates ROS production under anoxia has remained unclear. Here we show that a low-oxygen-responsive universal stress protein (USP), Hypoxia Responsive Universal Stress Protein 1 (HRU1), is induced by RAP2.12 (Related to Apetala 2.12), an ERF-VII protein, and modulates ROS production in Arabidopsis. We found that HRU1 is strongly induced by submergence, but that this induction is abolished in plants lacking RAP2.12. Mutation of HRU1 through transfer DNA (T-DNA) insertion alters hydrogen peroxide production, and reduces tolerance to submergence and anoxia. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses reveal that HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD, pointing to the existence of a low-oxygen-specific mechanism for the modulation of ROS levels. We propose that HRU1 coordinates oxygen sensing with ROS signalling under anoxic conditions.