Hypoxia-activated fluorescent probes as markers of oxygen levels in plant cells and tissues.

Low oxygen signalling in plants is important in development and stress responses. Measurement of oxygen levels in plant cells and tissues is hampered by a lack of chemical tools with which to reliably detect and quantify endogenous oxygen availability. We have exploited hypoxia-activated fluorescent probes to visualise low oxygen (hypoxia) in plant cells and tissues.

Tools to understand hypoxia responses in plant tissues.

Our understanding of how low oxygen (O2) conditions arise in plant tissues and how they shape specific responses has seen major advancement in recent years. Important drivers have been (1) the discovery of the molecular machinery that underpins plant O2 sensing; and (2) a growing set of dedicated tools to define experimental conditions and assess plant responses with increasing accuracy and resolution. While some of those tools, such as the Clark-type O2 electrode, were established decades ago, recent customization has set entirely new standards and enabled novel research avenues in plant hypoxia research.

Flooding stress and responses to hypoxia in plants.

In recent years, research on flooding stress and hypoxic responses in plants has gathered increasing attention due to climate change and the important role of O2 in metabolism and signalling. This Collection of Functional Plant Biology on 'Flooding stress and responses to hypoxia in plants' presents key contributions aimed at progressing our current understanding on how plants respond to low-O2 conditions, flooding stress and a combination of stresses commonly found in flooded areas. The Collection emphasises the characterisation of diverse plant responses across different developmental stages, from seed germination to fully developed plants, and under different water stress conditions ranging from waterlogging to complete submergence, or simply low-O2 conditions resulting from limited O2 diffusivity in bulky tissues.

Mechanodetection of neighbor plants elicits adaptive leaf movements through calcium dynamics.

Plants detect their neighbors via various cues, including reflected light and touching of leaf tips, which elicit upward leaf movement (hyponasty). It is currently unknown how touch is sensed and how the signal is transferred from the leaf tip to the petiole base that drives hyponasty. Here, we show that touch-induced hyponasty involves a signal transduction pathway that is distinct from light-mediated hyponasty.

Target of rapamycin signaling couples energy to oxygen sensing to modulate hypoxic gene expression in .

Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs).

Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes.

N-terminal cysteine oxidases (NCOs) use molecular oxygen to oxidise the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway. To expand the characterisation of the plant family of NCOs (plant cysteine oxidases [PCOs]), we performed a phylogenetic analysis across different taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups.

Exogenous miRNAs induce post-transcriptional gene silencing in plants.

Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi.

In Vivo Imaging of Plant Oxygen Levels.

Oxygen is essential for multicellular aerobic life due to its central role in energy metabolism. The availability of oxygen can drop below the level to sustain oxidative phosphorylation when plants are flooded, posing a severe threat to survival. However, under non-stressful conditions, the internal oxygen concentration of most plant tissue is not in equilibrium with the environment, which is attributed to cellular respiration and diffusion constrains imposed by O2 barriers and bulky tissue.

An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue.

Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead.

Jasmonate Signalling Contributes to Primary Root Inhibition Upon Oxygen Deficiency in .

Plants, including most crops, are intolerant to waterlogging, a stressful condition that limits the oxygen available for roots, thereby inhibiting their growth and functionality. Whether root growth inhibition represents a preventive measure to save energy or is rather a consequence of reduced metabolic rates has yet to be elucidated. In the present study, we gathered evidence for hypoxic repression of root meristem regulators that leads to root growth inhibition.

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.

Botrytis cinerea induces local hypoxia in Arabidopsis leaves.

Low oxygen availability often is associated with soil waterlogging or submergence, but may occur also as hypoxic niches in otherwise aerobic tissues. Experimental evidence assigns a role in Botrytis cinerea resistance to a group of oxygen-unstable Ethylene Response Factors (ERF-VII). Given that infection by B.

Molecular oxygen as a signaling component in plant development.

While traditionally hypoxia has been studied as a detrimental component of flooding stress, the last decade has flourished with studies reporting the involvement of molecular oxygen availability in plant developmental processes. Moreover, proliferating and undifferentiated cells from different plant tissues were found to reside in endogenously generated hypoxic niches. Thus, stress-associated acute hypoxia may be distinguished from constitutively generated chronic hypoxia.

A Ratiometric Sensor Based on Plant N-Terminal Degrons Able to Report Oxygen Dynamics in Saccharomyces cerevisiae.

The ability to perceive oxygen levels is crucial to many organisms because it allows discerning environments compatible with aerobic or anaerobic metabolism, as well as enabling rapid switch between these two energy strategies. Organisms from different taxa dedicate distinct mechanisms to associate oxygen fluctuations with biological responses. Following from this observation, we speculated that orthogonal oxygen sensing devices can be created by transfer of essential modules from one species to another in which they are not conserved.

An apical hypoxic niche sets the pace of shoot meristem activity.

Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism. Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation. Here we demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves.

Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation.

infection of wounded plant tissues causes the formation of crown gall tumors. Upon infection, genes encoded on the tumor inducing plasmid are integrated in the plant genome to induce the biosynthesis of auxin and cytokinin, leading to uncontrolled cell division. Additional sequences present on the bacterial T-DNA encode for opine biosynthesis genes, which induce the production of opines that act as a unique carbon and nitrogen source for .

Oxygen Sensing and Integrative Stress Signaling in Plants.

Integration of multiple cellular signals provides new opportunities in understanding oxygen sensing and response mechanisms in plants.

Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation.

Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway.

In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins.

A PLETHORA-auxin transcription module controls cell division plane rotation through MAP65 and CLASP.

Despite their pivotal role in plant development, control mechanisms for oriented cell divisions have remained elusive. Here, we describe how a precisely regulated cell division orientation switch in an Arabidopsis stem cell is controlled by upstream patterning factors. We show that the stem cell regulatory PLETHORA transcription factors induce division plane reorientation by local activation of auxin signaling, culminating in enhanced expression of the microtubule-associated MAP65 proteins.

Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization.

The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now.

Hypoxia responsive gene expression is mediated by various subsets of transcription factors and miRNAs that are determined by the actual oxygen availability.

• Reduced oxygen availability is not only associated with flooding, but occurs also during growth and development. It is largely unknown how hypoxia is perceived and what signaling cascade is involved in activating adaptive responses. • We analysed the expression of over 1900 transcription factors (TFs) and 180 microRNA primary transcripts (pri-miRNAs) in Arabidopsis roots exposed to different hypoxic conditions by means of quantitative PCR.