Stress responses studying techniques in plants

stress response study techniques

1.   Stress Responses Preeti Sharma Ph.D. Scholar 2023BS11D Department of Biochemistry, COB&H, CCSHAU, Hisar

2. Thermal Reflectance Thermal reflectance refers to the ability of a material to reflect thermal radiation, typically in the infrared part of the electromagnetic spectrum. Thermal reflectance is typically measured as a percentage of the incident thermal radiation that is reflected by the material . Higher values indicate greater reflectivity, meaning more thermal radiation is being reflected rather than absorbed by the material. Remote sensing technologies, such as thermal imaging from satellites or drones, are commonly used to capture thermal reflectance data. This data is then processed using various algorithms to derive useful information. Additionally, ground-based thermal cameras can also be employed for more localized assessments.

3. Thermal characters as a measure of water status Principle:  Plants with good water content tend to have higher emissivity (ability to radiate heat) and lower reflectance in the thermal infrared (TIR) region compared to water-stressed plants. Reasoning:  Water has a high heat capacity, meaning it takes more energy to heat it up and it releases heat more slowly. So, well-hydrated plants with more water content will feel cooler and radiate more heat in the TIR compared to dry plants that will feel warmer and reflect more thermal radiation. Monitoring:  By measuring the TIR reflectance of plants, we can indirectly estimate their water stress levels. As water stress increases, the TIR reflectance increases. This data can be collected from satellites or drones for large-scale monitoring of agricultural fields or natural ecosystems.

4. Thermal characters as a measure of root characteristics Dense root systems can increase soil moisture content in the shallow layers, influencing the overall thermal signature of the plant-soil system. Applications:  By analyzing thermal data alongside other information like vegetation indices, root depth or density can be inferred based on observed variations in thermal reflectance. Thermal reflectance offers a non-invasive way to assess plant health indirectly. While thermal data is valuable, other factors like soil type, weather conditions, and plant species can influence the results.

5. Oxidative stress induction Oxidative stress induction refers to the process by which cells experience an imbalance between the production of reactive oxygen species (ROS) and the ability of antioxidant systems to detoxify these reactive intermediates or repair the resulting damage. This imbalance can lead to oxidative damage to various cellular components, including lipids, proteins, and DNA, ultimately impacting cell function and viability. Reactive Oxygen Species (ROS) : These are highly reactive molecules containing oxygen. They include free radicals like superoxide anion (O2•-) and hydroxyl radical (OH•), as well as non-radicals like hydrogen peroxide (H₂O₂). ROS are normal byproducts of cellular metabolism, but excess ROS can damage cells and contribute to various diseases.  

6. ROS Structure and formation The process begins with molecular oxygen (O2), which has two unpaired electrons in the outer orbital. Thus, it can acquire an electron to form the superoxide anion (O2 -), which is a radical (i.e., with an unpaired electron). Superoxide dismutase (SOD) converts superoxide into hydrogen peroxide (H2O2) and oxygen. Catalase can further break down H2O2 into water and oxygen. In the presence of transition metals, the Haber-Weiss reaction generates hydroxyl radicals (OH˙) and hydroxyl ions (OH -) from superoxide and H2O2. The Fenton reaction also produces hydroxyl radicals and hydroxyl ions by reducing H2O2 in the presence of iron ions. O: Oxygen atom; H: Hydrogen atom; H2O: water molecule; O2˙: superoxide anion; H2O2: Hydrogen Peroxide; OH˙: Hydroxyl Radical; OH-: Hydroxyl Ion; NO: Nitric Oxide; Fe 2+ : iron in reduced state; Fe 3+ : iron in oxidized state; SOD: superoxide dismutase.

7. Oxidative stress-induced lipid peroxidation Lipid peroxidation is a specific consequence of oxidative stress that involves the oxidative degradation of lipids, particularly polyunsaturated fatty acids (PUFAs), within cell membranes. This process generates lipid peroxides and other reactive aldehydes, which can further damage cellular structures and disrupt membrane integrity.

8. Asses sing the response to oxidative stress-induced lipid peroxidation Assessment of Antioxidant Enzyme Activity : Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase ( GPx ), and glutathione reductase (GR) are key antioxidant enzymes that help mitigate oxidative stress. Changes in the activity levels of these enzymes can indicate the cellular response to oxidative stress. Measurement of Lipid Peroxidation Products : Thiobarbituric acid reactive substances (TBARS) assay: This assay measures the levels of malondialdehyde (MDA), a byproduct of lipid peroxidation, through its reaction with thiobarbituric acid (TBA). Lipid peroxide assays: Various methods exist to directly quantify lipid peroxides, such as the ferric thiocyanate assay or the FOX assay (ferrous oxidation-xylenol orange assay).

9. Asses sing the response to oxidative stress-induced lipid peroxidation Evaluation of Cellular Viability and Damage : Cell viability assays (e.g., MTT assay, propidium iodide staining) can assess the impact of oxidative stress on cell survival. Morphological changes, such as membrane blebbing or cytoplasmic vacuolization, observed under microscopy, may indicate cellular damage.

10. Asses sing the response to oxidative stress-induced lipid peroxidation Assessment of Lipid Membrane Integrity : Fluorescent probes like DPPP (diphenyl-1-pyrenylphosphine) or C11-BODIPY can be used to assess lipid peroxidation-induced membrane damage. These probes selectively bind to oxidized lipids, emitting fluorescence signals that can be quantified spectrofluorometrically . Gene Expression Analysis : Quantitative real-time PCR (qPCR) can be employed to analyze the expression levels of genes involved in antioxidant defense , lipid metabolism, and stress response pathways.

11. ROS Quantification Dihydroethidium (DHE) Assay : DHE is a fluorescent dye that selectively reacts with superoxide anions (O2^-) to form ethidium, which emits red fluorescence. The intensity of fluorescence is proportional to the level of superoxide. Dichlorodihydrofluorescein Diacetate (DCFH-DA) Assay : DCFH-DA is a non-fluorescent probe that is oxidized by various ROS to form the fluorescent compound, 2',7'-dichlorofluorescein (DCF). The fluorescence intensity correlates with the amount of ROS present. Amplex Red Assay : This assay utilizes the oxidation of non-fluorescent Amplex Red to fluorescent resorufin by hydrogen peroxide (H2O2) in the presence of horseradish peroxidase (HRP). The fluorescence intensity is proportional to the concentration of H2O2. Chemiluminescence Assay : Chemiluminescent probes like luminol or lucigenin can detect ROS generation by measuring the intensity of light emitted during the reaction with ROS.

12. Reactive Nitrogen Species (RNS) RNS is similar to ROS, but contain nitrogen. Examples include nitric oxide (NO•) and peroxynitrite (ONOO⁻). RNS play roles in cell signaling and inflammation, but excess RNS can also be harmful. RNS Quantification: Diaminofluorescein-2 Diacetate (DAF-2DA) Assay: DAF-2DA is a non-fluorescent probe that reacts specifically with nitric oxide (NO) to form the fluorescent compound, DAF-2T, whose fluorescence intensity correlates with NO levels. Griess Assay: This assay measures nitrite (NO2^-), a stable breakdown product of NO, by its reaction with Griess reagents to form a colored azo compound. Nitrate (NO3^-) can be converted to nitrite using nitrate reductase for total NOx measurement. Electron Paramagnetic Resonance (EPR): EPR spectroscopy can directly detect and quantify free radicals, including NO, by their interaction with paramagnetic probes such as spin traps or spin labels.

13. Reactive Carbonyl Species (RCS) and their Quantification RCS are α,β-unsaturated aldehydes and ketones derived from various sources in plants. These are a group of highly reactive molecules. They are formed through the breakdown of: Lipids  by reactive oxygen species (ROS) or lipoxygenase enzymes. Sugars  and  amino acids  during metabolic processes. RCS act as a  signaling molecule , transmitting information about stress conditions like drought, wounding, or pathogen attack. They covalently modify proteins, altering their activity and function. This modification can be beneficial at low levels, triggering defense mechanisms. At high levels, however, RCS can damage proteins and other biomolecules, leading to cell death.

14. Quantifying RCS in Plants Since RCS are formed from various sources, interpreting their levels requires considering the specific stress or condition being studied. Quantifying RCS alongside ROS can provide a more complete picture of cellular oxidative stress. By understanding RCS levels and their role in signaling pathways, insights into plant responses to stress an be gained and strategies to improve plant resilience can be developed. Derivatization methods: These methods convert RCS into more stable derivatives that can be easily detected. DNPH (2,4-dinitrophenylhydrazine) derivatization:  Forms colored hydrazones with RCS, allowing for spectrophotometric measurement. Hydrophilic derivatization reagents:  Convert RCS into water-soluble compounds facilitating separation and analysis techniques like HPLC (High-Performance Liquid Chromatography). Oximation methods: These methods utilize specific reagents that react with the carbonyl group of RCS to form oximes. Hydroxylamine trapping:  Followed by detection using various methods like HPLC or mass spectrometry (MS).

15. Fluorescence to assess the stress response When a molecule absorbs light of a specific wavelength, it gets excited to a higher energy state. When it relaxes back to its ground state, it releases energy, often as light of a longer wavelength. This emitted light is called fluorescence. Certain molecules in cells, particularly chlorophyll (plants) or biomolecules like protein and aromatic amino acids (all organisms), naturally fluoresce. By measuring chlorophyll fluorescence, researchers can assess the efficiency of photosynthetic electron transport. A decrease in efficiency often indicates stress. Monitoring changes in protein fluorescence can indicate alterations in protein structure or function due to stress. Scientists can introduce specific fluorescent probes into cells. These probes bind to target molecules or react with specific products of stress pathways, allowing for targeted monitoring of stress responses. Non-invasive  (can be performed on living organisms without harming them), highly sensitive  and can be automated for large-scale studies. Expensive equipments like fluorescence microscopes and spectrometers are required. Fluorescence signals can sometimes be influenced by factors other than stress reducing specificity of the technique.

16. Tissue Localization of ROS and RNS Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules involved in various cellular processes. However, their excess can damage tissues. Accurately pinpointing their location within tissues is crucial for understanding their roles in health and disease. This can be achieved by qualitative staining and fluorescence-based methods. Qualitative Staining:   Simple, inexpensive, and readily available but limited specificity (may stain for multiple ROS/RNS or unrelated molecules) and difficulty in quantification. Some common Qualitative Stains for ROS include: Diaminobenzidine (DAB):  Reacts with H₂O₂ (a ROS) to form a brown precipitate, marking sites of ROS production. Nitroblue tetrazolium (NBT):  Reduced by superoxide radicals (another ROS) to form a blueformazan precipitate, indicating superoxide presence. Some Common Qualitative Stains for RNS are: Griess reagent:  Reacts with nitrite (a breakdown product of nitric oxide, an RNS) to form a colored complex, revealing nitric oxide production sites.

17. Tissue Localization of ROS and RNS Fluorescence-Based Methods: High sensitivity and improved specificity through probe selection, but generally more expensive and require specialized equipment (fluorescence microscopes). Common Fluorescence Probes for ROS: DCFH-DA:  A cell-permeable dye that is cleaved by intracellular esterases into DCFH, which reacts with various ROS to emit green fluorescence. MitoSOX Red:  Specifically targets mitochondria, a major site of ROS production, and emits red fluorescence upon oxidation by ROS. Common Fluorescence Probes for RNS: DAF-2 DA:  A cell-permeable dye that reacts with nitric oxide to emit blue fluorescence, indicating nitric oxide production. Dihydrorhodamine (DHR):  Oxidized by various reactive species, including some RNS, to emit red fluorescence. For initial investigations or screening:  Qualitative stains offer a quick and easy approach. For more specific localization and potential quantification:  Fluorescence methods are preferred.

18. Water use efficiency Water use efficiency (WUE) is a crucial metric in plant science, indicating how well a plant utilizes water for growth and productivity. We can quantify WUE at different scales and employ surrogate measures when direct measurement is challenging. Water use efficiency is typically defined as the ratio of biomass produced (or yield) to the amount of water consumed. It can be expressed in various units such as grams of biomass per liter of water (g/L), kilograms of biomass per cubic meter of water (kg/m³), or similar measures. Factors affecting WUE : Genetics: D ifference in varieties Environmental conditions : Temperature, humidity, light intensity, and CO2 concentration Soil characteristics : Soil type, structure, and moisture content can affect a plant's ability to uptake water efficiently. Management practices : Irrigation techniques, fertilization, and other agricultural practices

19. WUE Quantification Leaf Level: This is the most fundamental measurement. It focuses on gas exchange processes within leaves. Formula:  WUE (intrinsic) = Net CO₂ assimilation (A) / Stomatal conductance ( gs ) A: Measured with a gas exchange system, representing the rate of CO₂ fixation by photosynthesis. gs : Represents the ease with which stomata open and close, affecting water vapor loss. Methods:  Portable gas exchange systems allow for non-destructive, in-field measurements. Plant Level: Here, WUE considers water used by the entire plant for biomass production. Formula:  WUE (plant) = Total dry biomass / Total water transpired Dry biomass: Measured by harvesting and drying plant material. Transpiration: Can be estimated through gravimetric methods (weighing pots) or using specialized chambers. Methods:  This approach is more laborious but provides a holistic picture of water use by the whole plant.

20. Surrogates for WUE Direct measurement of WUE, especially at the plant level, can be time-consuming or impractical. Some commonly used surrogates are as follows: Carbon isotope discrimination (Δ¹³C):  Plants with higher WUE discriminate more against the heavier ¹³C isotope during CO₂ fixation. Measuring Δ¹³C in leaf tissue can be a good indicator of WUE, especially for long-term water use strategies. Leaf traits:  Certain leaf characteristics, such as smaller size, thicker cuticle, and pubescence (hairs), are associated with higher WUE as they reduce water loss through transpiration. Studying these traits can provide insights into water use strategies. Remote sensing data:  Spectral reflectance data captured by satellites or drones can be used to estimate canopy water content or vegetation indices indirectly linked to WUE. For detailed physiological studies:  Leaf-level WUE with gas exchange measurements provides precise data. For screening large populations:  Surrogates like Δ¹³C or leaf traits may be more efficient. For large-scale field studies:  Remote sensing data can offer valuable insights into water use patterns over vast areas.