Periodic Reporting for period 1 - HorLumAS (Visualization of plant Hormone dynamics using BioLuminescence during Abiotic Stress)
Periodo di rendicontazione: 2022-11-01 al 2024-10-31
Like in humans, the physiological systems of plants use hormones. Plant hormones govern their development, starting from the formation and maturation of the embryos, the germination of the seed, the seedling growth, and maturation and flowering. The end of a plant’s life is also orchestrated by hormones as a normal procedure. Besides, in their natural habitats, plants are exposed to fluctuations of temperature, light intensity, and water availability, which shape their basic abiotic environment. Their continuous contact and interaction with the living part of their habitats form their biotic environment, consisting of many types of organisms, like bacteria, fungi, viruses, insects and animals, including humans. Plants understand and “decide” how to react to all these interactions, regulating their cellular content, organs’ structure, and ultimately their development. While their development is a relatively anticipated and well-described biological procedure, their environmental responses are rather spontaneous, ad-hoc decisions.
From the moment of perception of an environmental stimulus, plants react at molecular and cellular level. Besides other biochemical compounds, plant hormones are the first, metabolizable effectors on plants’ own physiology, orchestrating their responses. Local hormone biosynthesis, or systematic transport or diffusion alters the overall hormonal homeostasis, signaling the metabolic, transcriptional, or molecular reform of the plant body to alleviate or tolerate stress. For this, the spatiotemporal distribution of hormones in plants is of paramount importance. The methods to determine it are based on biochemical analysis of plant parts, or enzymatic reactions happening within the plant tissues, producing color so we can visualize them. Those methods are destructive to plants’ tissues and do not allow continuous visualization. The less invasive and destructive method is the expression and visualization of fluorescent markers. However, this requires irradiation with strong ultraviolet light, to which long exposure destroys the tissues.
HorLumAS is a research-oriented project addressing this by implementing spontaneously emitted luminescence by plants. In nature, several species of fungi, marine animals, insects, and even some worms, emit luminescence. The biochemical pathway for the autonomous bioluminescence (ABL) of the fungus Neonothopanus nambi was described as a concerted action of 3 enzymes. First, using endogenous precursor molecules, two enzymes synthesize a high-energy biomolecule, termed Fungal Luciferin (FLin), which is oxidized by a specialized enzyme, termed Fungal Luciferase (FLase) with concurrent emission of green light. The idea behind HorLumAS is to create plants and cell suspensions engineered to emit ABL according to the spatiotemporal distribution of plant hormones. First we created transcriptional units for the FLin biosynthetic enzymes and transfer them into the plants, to synthesize FLin throughout their body and lifetime. Then a hormone-regulated transcriptional unit expresses FLase and makes the plant tissues emit ABL wherever and whenever a particular hormone appears.
The objectives of the project are to
- create ABL-emitting model plants of Arabidopsis thaliana and Nicotiana tabacum cell suspensions, Bright Yellow 2 (BY2) cells, and test whether ABL biochemistry affects them,
- standardize imaging methods with both scientific systems and commercial cameras, and script processing and quantification protocols for obtained images,
- create hormone-sensitive ABL reporter plants for auxins, cytokinins, abscisic acid, ethylene, gibberellins, brassinosteroids, salicylic acids, jasmonates, and strigolactones.
- investigate the spatiotemporal distribution of plant hormones when plants experience stressful conditions.
From the moment of perception of an environmental stimulus, plants react at molecular and cellular level. Besides other biochemical compounds, plant hormones are the first, metabolizable effectors on plants’ own physiology, orchestrating their responses. Local hormone biosynthesis, or systematic transport or diffusion alters the overall hormonal homeostasis, signaling the metabolic, transcriptional, or molecular reform of the plant body to alleviate or tolerate stress. For this, the spatiotemporal distribution of hormones in plants is of paramount importance. The methods to determine it are based on biochemical analysis of plant parts, or enzymatic reactions happening within the plant tissues, producing color so we can visualize them. Those methods are destructive to plants’ tissues and do not allow continuous visualization. The less invasive and destructive method is the expression and visualization of fluorescent markers. However, this requires irradiation with strong ultraviolet light, to which long exposure destroys the tissues.
HorLumAS is a research-oriented project addressing this by implementing spontaneously emitted luminescence by plants. In nature, several species of fungi, marine animals, insects, and even some worms, emit luminescence. The biochemical pathway for the autonomous bioluminescence (ABL) of the fungus Neonothopanus nambi was described as a concerted action of 3 enzymes. First, using endogenous precursor molecules, two enzymes synthesize a high-energy biomolecule, termed Fungal Luciferin (FLin), which is oxidized by a specialized enzyme, termed Fungal Luciferase (FLase) with concurrent emission of green light. The idea behind HorLumAS is to create plants and cell suspensions engineered to emit ABL according to the spatiotemporal distribution of plant hormones. First we created transcriptional units for the FLin biosynthetic enzymes and transfer them into the plants, to synthesize FLin throughout their body and lifetime. Then a hormone-regulated transcriptional unit expresses FLase and makes the plant tissues emit ABL wherever and whenever a particular hormone appears.
The objectives of the project are to
- create ABL-emitting model plants of Arabidopsis thaliana and Nicotiana tabacum cell suspensions, Bright Yellow 2 (BY2) cells, and test whether ABL biochemistry affects them,
- standardize imaging methods with both scientific systems and commercial cameras, and script processing and quantification protocols for obtained images,
- create hormone-sensitive ABL reporter plants for auxins, cytokinins, abscisic acid, ethylene, gibberellins, brassinosteroids, salicylic acids, jasmonates, and strigolactones.
- investigate the spatiotemporal distribution of plant hormones when plants experience stressful conditions.
First, the project set the frame for the use of ABL in plants with reliability and reproducibility. We tested whether ABL affects two model plant species, the vascular Arabidopsis thaliana and BY2 cell suspensions. The tests started with plants growing in aseptic, in vitro, normal, or stress conditions, and aimed to capture their ABL while growing in optimal or adverse conditions on simple soil substrates. The first plants with ABL had unaffected development, reproduction, and environmental responses, while their ABL was strong and visible to human eyes. We standardized imaging methods with scientific imaging systems and commercial cameras, and also protocols for processing images and quantification of ABL.
Then we used DR5 synthetic promoter, responsive to the plant hormone auxin. Although the auxin-sensitive ABL was significantly weaker, and, unfortunately not visible to human eyes, Arabidopsis plants glow with particular patterns on their roots, leaf periphery, and other plant parts, denoting where auxin was. The ABL due to auxin’s abundance and distribution was termed Aux-ABL. To ensure that the ABL of these plants truly reports on auxin concentrations and distribution in planta, we challenged the induction of luminescence by exogenously providing natural or synthetic auxins to these plants. We observed and quantified a dose-dependent ABL induction for at least 4 days in vitro and in soil-grown plants, which was stronger than the plants with constitutive ABL. Also, in a novel approach, we used thin layers of BY2 cells, and we also induced DR5-regulated ABL with synthetic and natural auxins.
In parallel experiments, we created Arabidopsis and BY2 lines which constitutively synthesize FLin, which is also sufficient to fuel ABL when FLase was concurrently expressed by strong, constitutive, or hormone-sensitive promoters. We named this plant master line because it is the basis for creating additional lines with ABL. Then we transformed master lines with hormone-sensitive transcriptional units to control the FLase expression.
At this moment, we are testing hormone-sensitive ABL reporter lines for every plant hormone and, we apply hormones exogenously to test their responsiveness. Moreover, we are studying the environmental interactions of these lines with stress conditions and microorganisms.
Then we used DR5 synthetic promoter, responsive to the plant hormone auxin. Although the auxin-sensitive ABL was significantly weaker, and, unfortunately not visible to human eyes, Arabidopsis plants glow with particular patterns on their roots, leaf periphery, and other plant parts, denoting where auxin was. The ABL due to auxin’s abundance and distribution was termed Aux-ABL. To ensure that the ABL of these plants truly reports on auxin concentrations and distribution in planta, we challenged the induction of luminescence by exogenously providing natural or synthetic auxins to these plants. We observed and quantified a dose-dependent ABL induction for at least 4 days in vitro and in soil-grown plants, which was stronger than the plants with constitutive ABL. Also, in a novel approach, we used thin layers of BY2 cells, and we also induced DR5-regulated ABL with synthetic and natural auxins.
In parallel experiments, we created Arabidopsis and BY2 lines which constitutively synthesize FLin, which is also sufficient to fuel ABL when FLase was concurrently expressed by strong, constitutive, or hormone-sensitive promoters. We named this plant master line because it is the basis for creating additional lines with ABL. Then we transformed master lines with hormone-sensitive transcriptional units to control the FLase expression.
At this moment, we are testing hormone-sensitive ABL reporter lines for every plant hormone and, we apply hormones exogenously to test their responsiveness. Moreover, we are studying the environmental interactions of these lines with stress conditions and microorganisms.
The impact of the HorLumAS project cannot be concluded only by the current findings and in the frame of this project. The ease and operability of the ABL imaging methods, analysis, and quantification protocols serve as firm grounds for the observations of plants’ early hormone-sensitive transcriptional outputs.
The produced ABL reporter plants reveal the hormone distributions before stress impacts development. This is important because plants can directly and immediately indicate their stress and that their physiology, and possibly their development or life are imminently changing.
Putting this in perspective, stressed crop plants could warn of their stress so actions can be taken to alleviate it and ensure their lives and production. Studying stress timing can suggest improvements for agricultural practices, propose protocols and treatments for alleviating stress effects, or even prevent forecasted environmental stress.
During scientific actions of dissemination, we could initiate collaboration to study plants’ tropic responses and interactions with microorganisms. We initiated experiments with novel approaches to investigate auxin transport in soil-grown plants in various conditions of light, temperature, and genetic backgrounds.
The produced ABL reporter plants reveal the hormone distributions before stress impacts development. This is important because plants can directly and immediately indicate their stress and that their physiology, and possibly their development or life are imminently changing.
Putting this in perspective, stressed crop plants could warn of their stress so actions can be taken to alleviate it and ensure their lives and production. Studying stress timing can suggest improvements for agricultural practices, propose protocols and treatments for alleviating stress effects, or even prevent forecasted environmental stress.
During scientific actions of dissemination, we could initiate collaboration to study plants’ tropic responses and interactions with microorganisms. We initiated experiments with novel approaches to investigate auxin transport in soil-grown plants in various conditions of light, temperature, and genetic backgrounds.