Functions and Significance of Common Physiological and Biochemical Indices in Animals and Plants

Physiological and biochemical activities are fundamental processes that maintain biological structure and function, spanning material metabolism, energy conversion, redox homeostasis, and growth and development. In animals, plants, and microorganisms/cultured cells, environmental changes or genetic differences often manifest as reproducible physiological and biochemical responses that can be captured by quantifiable indices, such as photosynthetic pigments and photochemical efficiency, activities of key enzymes in carbon assimilation and respiratory metabolism, reactive oxygen species (ROS) metabolism and scavenging systems, membrane lipid peroxidation, accumulation of osmolytes, and mineral nutrition and nitrogen assimilation capacity. Systematic measurement and integrated interpretation of multiple indices help identify limiting steps and response pathways, providing a verifiable evidence chain for stress tolerance evaluation, mechanistic studies, and quality analysis.

Keywords: physiological and biochemical indices; photosynthesis; reactive oxygen species; antioxidant system; redox homeostasis; membrane lipid peroxidation; osmotic adjustment; mineral nutrition; nitrogen assimilation

I. Overall Value of Index Systems and Principles for Interpretation

1.1 Scientific Significance of an Index System

(1) Mechanistic localization

Index measurements decompose complex phenotypes into testable key processes, supporting localization judgments for steps such as light energy utilization, carbon assimilation, respiratory energy production, oxidative stress, and membrane damage.

(2) Evidence-chain construction

Multi-index integration reduces nonspecificity associated with any single index and improves causal interpretability and reproducibility.

(3) Comparability and transferability

Under standardized sampling position, developmental stage, and analytical methodology, an index system enables comparisons across treatments, genotypes/lines, and studies, providing a basis for cross-laboratory replication and translation to applications.

1.2 Common Index List

Chlorophyll	Carotenoids	Chlorophyll fluorescence	PEPC (phosphoenolpyruvate carboxylase)
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase)	SOD (superoxide dismutase)	POD (peroxidase; GPX-related)	CAT (catalase)
APX (ascorbate peroxidase)	GR (glutathione reductase)	DHAR (dehydroascorbate reductase)	MDHAR (monodehydroascorbate reductase)
PPO (polyphenol oxidase)	SDH (succinate dehydrogenase)	Glutathione	Ascorbic acid (ascorbate)
H₂O₂ (hydrogen peroxide)	O₂•− (superoxide anion)	Hydroxyl radical (•OH)	Total phenolics
Flavonoids	Reducing power	FRAP (ferric reducing antioxidant power)	ABTS radical scavenging capacity
DPPH radical scavenging capacity	Hydroxyl radical scavenging capacity	Antioxidant defense system	MDA (malondialdehyde)
Proline	Soluble protein	Soluble sugars	Cellulose
Water content	Root activity	Elemental content	NR (nitrate reductase)

II. Photosynthesis- and Carbon Metabolism-Related Indices

2.1 Photosynthetic Pigments and Light Energy Utilization

(1) Chlorophyll

Chlorophyll determines leaf light-harvesting capacity and is associated with chloroplast structural stability. Changes in chlorophyll content can indicate the status of photosynthetic apparatus development, nitrogen nutritional status, and senescence progression, and can serve as an integrated reflection of stress-induced impacts on the photosynthetic system.

(2) Carotenoids

Carotenoids contribute to energy transfer and play essential photoprotective roles. By promoting energy dissipation and suppressing photo-oxidative reactions, they reduce the risk of photosystem damage. Relative changes can help infer photoprotective investment, high-light stress risk, and stability of the pigment system.

2.2 Photochemical Function Characterization

(1) Chlorophyll fluorescence

Chlorophyll fluorescence parameters sensitively report energy partitioning and electron transport efficiency in photosystems. They can detect functional limitation before marked pigment changes occur and help distinguish whether limitation arises mainly from light reactions or from carbon assimilation processes. In stress physiology and recovery assessment, chlorophyll fluorescence provides high temporal resolution and strong diagnostic value.

2.3 Key Enzymes for Carboxylation and Carbon Fixation

(1) PEPC (phosphoenolpyruvate carboxylase)

PEPC catalyzes anaplerotic carboxylation and participates in metabolic regulation. Across biological systems, it is closely associated with carbon-flow partitioning, organic acid metabolism, and coupling with nitrogen metabolism. PEPC activity changes can indicate the direction of metabolic reprogramming and adjustment of carbon assimilation strategies.

(2) Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase)

Rubisco is the core enzyme for CO₂ fixation in the Calvin cycle and is also linked to photorespiration. Its abundance and activity directly determine net assimilation capacity and are highly sensitive to temperature, water status, CO₂, and nitrogen supply. Rubisco is therefore a key basis for judging whether photosynthetic decline is dominated by “dark reaction/carbon fixation limitation.”

III. ROS Metabolism and Antioxidant Defense Systems

3.1 Significance of ROS Indices

(1) H₂O₂

H₂O₂ is both a major indicator of oxidative stress and a central hub in ROS networks. Its accumulation reflects the balance between production and scavenging and is associated with risks of oxidative damage to proteins, nucleic acids, and membrane lipids. Within certain ranges, it also functions in signaling regulation.

(2) O₂•− (superoxide anion)

O₂•− lies upstream in ROS generation pathways. Excess accumulation often indicates enhanced electron leakage or elevated oxidative pressure. Tracking O₂•− helps evaluate oxidative stress initiation intensity and its relationship with subsequent H₂O₂ burden.

(3) Hydroxyl radical (•OH)

Hydroxyl radicals are extremely reactive and can rapidly damage lipids, proteins, and nucleic acids. Measurements related to •OH or •OH scavenging capacity can indicate high-risk oxidative attack environments and are often interpreted together with lipid peroxidation indices to assess damage cascades.

3.2 Key Nodes in Enzymatic Antioxidant Systems

(1) SOD (superoxide dismutase)

SOD converts O₂•− to H₂O₂ and functions as a front-line gatekeeper of ROS detoxification. SOD changes reflect the intensity of response to superoxide pressure, but should be interpreted together with H₂O₂ removal capacity.

(2) CAT (catalase)

CAT decomposes H₂O₂ with high throughput and is a core enzyme controlling H₂O₂ accumulation and lowering oxidative damage risk. When CAT is insufficient and H₂O₂ rises, it often indicates limited detoxification flux and increased downstream damage probability.

(3) POD (peroxidase)/GPX (glutathione peroxidase-like activity)

POD/GPX enzymes use peroxides as substrates for detoxification and are associated with defense responses and certain structural metabolic processes. Their activities can indicate peroxide-removal flux and defense-response intensity.

(4) APX (ascorbate peroxidase)

APX reduces H₂O₂ using ascorbate as an electron donor and is a key component of the ascorbate-dependent detoxification pathway. APX dynamics indicate the efficiency of the ascorbate-based antioxidant barrier, with diagnostic relevance in chloroplasts, cytosol, and other critical compartments.

(5) GR (glutathione reductase)

GR maintains the GSH/GSSG ratio and ensures reducing-power supply, representing a core node of antioxidant regeneration capacity. GR is commonly used to assess whether cellular reducing power can sustain continued detoxification and repair.

(6) DHAR (dehydroascorbate reductase)

DHAR reduces DHA back to ascorbate and regulates the AsA/DHA state, serving as a key step in maintaining an effective ascorbate pool. Upregulation often indicates enhanced regeneration capacity of the ascorbate cycle.

(7) MDHAR (monodehydroascorbate reductase)

MDHAR promotes recycling of MDHA toward ascorbate, reducing the risk of irreversible depletion of the ascorbate pool. Joint interpretation with DHAR and GR enables a more accurate evaluation of overall ascorbate-cycle efficiency.

IV. Non-Enzymatic Antioxidants and Cellular Redox Status

4.1 Glutathione System

(1) Glutathione

GSH, GSSG, and their ratio constitute a key intracellular redox couple, reflecting overall reducing power and antioxidant buffering capacity. When the GSH/GSSG ratio decreases alongside ROS accumulation and insufficient enzymatic detoxification flux, redox homeostasis is typically shifted toward a more oxidized state.

4.2 Ascorbate System

(1) Ascorbate

AsA abundance and redox status reflect antioxidant reserves and cycling efficiency and are tightly coupled to APX-mediated reactions. A decrease in AsA/DHA often indicates depletion of antioxidant pools or restricted regeneration, and should be interpreted together with DHAR, MDHAR, and GR to identify the limiting step.

V. Secondary Metabolism and Browning/Quality-Related Indices

5.1 Phenolic Oxidation Pathway

(1) PPO (polyphenol oxidase)

PPO catalyzes oxidation of phenolics to quinones, leading to browning. PPO activity is used to evaluate browning propensity and the intensity of phenolic oxidation flux. In fruit and vegetable processing, tissue injury, and culture-system browning studies, PPO is a key process indicator.

5.2 Defensive Secondary Metabolites

(1) Total phenolics

Total phenolics reflect the overall level of phenolic compounds and are associated with radical scavenging capacity and allocation to defense-related secondary metabolism. Changes can indicate antioxidant potential and defense strategy adjustment, but interpretation should consider functional differences among phenolic subclasses.

(2) Flavonoids

Flavonoids are important defensive secondary metabolites involved in antioxidant protection and stress adaptation. Their accumulation indicates activation of relevant pathways and is often associated with changes in stress tolerance and certain quality traits.

VI. In Vitro Antioxidant Capacity Assessment Indices

6.1 Total Antioxidant Capacity and Radical Scavenging Capacity

(1) Reducing power

Reducing power reflects electron-donating capacity and is used for relative comparison of antioxidant potential across samples or treatments, serving as a common integrated chemical index.

(2) FRAP

FRAP quantifies total antioxidant capacity via ferric ion reducing power and is suitable for standardized comparative evaluation of biofluids, tissue homogenates, and extracts.

(3) ABTS scavenging capacity

ABTS-based assays evaluate radical scavenging capacity with broad applicability across sample types, enabling horizontal comparisons.

(4) DPPH scavenging capacity

DPPH assays evaluate hydrogen- or electron-donating ability and are commonly used for preliminary screening and relative comparison of antioxidant activity. They primarily reflect chemical scavenging potential and should not be directly equated with in vivo effects.

6.2 Scavenging Capacity Against Highly Reactive Radicals

(1) Hydroxyl radical scavenging capacity

This index evaluates the potential to scavenge highly reactive radicals and is useful for functional component screening and formulation comparison. For mechanistic inference, it should be interpreted together with in vivo ROS levels, enzymatic detoxification flux, and membrane damage indices.

VII. Membrane Damage and Osmotic Adjustment Indices

7.1 Membrane Lipid Peroxidation

(1) MDA (malondialdehyde)

MDA is a widely used indicator of membrane lipid peroxidation and supports evaluation of membrane damage severity and stress tolerance differences. When MDA increases together with ROS accumulation and insufficient antioxidant flux, it strengthens inference of an oxidative damage cascade.

7.2 Osmotic Adjustment and Metabolite Accumulation

(1) Proline

Proline participates in osmotic adjustment and contributes to stabilization of proteins and membranes. Its accumulation indicates the intensity of osmotic adjustment responses and tolerance strategies.

(2) Soluble sugars

Soluble sugars serve as energy sources and carbon skeletons and are also key osmolytes. Their changes reflect carbon partitioning and adaptive regulation and are coupled to stress tolerance and quality formation.

(3) Soluble protein

Soluble protein reflects overall protein metabolism and changes in enzyme systems. It indicates metabolic status and stress impact intensity and also provides a normalization basis for multiple enzyme activity assays.

VIII. Energy Metabolism and Structural Substance Indices

8.1 Key Node in Respiratory Metabolism

(1) SDH (succinate dehydrogenase)

SDH links the tricarboxylic acid cycle to the respiratory electron transport chain. Its activity indicates mitochondrial energy-production flux and energy metabolic status, serving as an important index of respiratory function and energy supply capacity.

8.2 Cell-Wall Structural Substances

(1) Cellulose

Cellulose determines cell-wall mechanical strength and influences tissue mechanical traits. Its content can represent structural construction level and certain quality characteristics, and serves as a foundational index for changes in cell-wall metabolism.

IX. Water Status, Root Function, and Mineral Nutrition Indices

9.1 Water Physiological Status

(1) Water content

Water content reflects tissue water retention and water physiological status, serving as a basic index for evaluating dehydration risk, storage stability, and severity of water stress.

9.2 Root Functional Status

(1) Root activity

Root activity reflects the capacity for water and mineral uptake and metabolic activity in roots. It critically influences nutrient supply to shoots and maintenance of growth, and is an important index of source-side supply capacity.

9.3 Mineral Nutrition and Ionic Homeostasis

(1) Elemental content

Elemental content reflects mineral supply and ionic homeostasis. It affects formation of structural components and influences enzyme activities and signaling processes. Integrated analysis with photosynthetic, osmotic, and oxidative indices improves precision in identifying limiting steps.

X. Key Enzyme Index for Nitrogen Assimilation

10.1 Nitrate reductase

(1) NR (nitrate reductase)

NR is a key rate-limiting step in nitrate assimilation. Its activity reflects the capacity to convert nitrate nitrogen into reduced nitrogen forms and is associated with nitrogen use efficiency and quality formation. Under stress conditions, NR dynamics can also indicate the direction of adjustment in coordination between nitrogen metabolism and carbon metabolism.

XI. Aladdin-Related Products

Indicator	Name	CAS No.	Typical use
H2O2; CAT/APX/POD/GPX activity	Hydrogen peroxide	7722-84-1	Used as a peroxide substrate in catalase and peroxidase assays and for preparing substrate solutions.
SOD activity; O2− related	Nitro blue tetrazolium chloride	298-83-9	Reduced to a colored formazan to report superoxide generation and the inhibitory effect of SOD.
SOD activity; O2− related	Riboflavin	83-88-5	Used to construct a photochemical reaction system that drives NBT reduction for color development.
SOD activity; O2− related	L-Methionine	63-68-3	Used to sustain radical generation/transfer in SOD assay systems and maintain a stable color background.
POD activity	Guaiacol	90-05-1	Used as a POD substrate that forms a colored product under peroxidation conditions for activity calculation.
POD activity; PPO activity	Pyrogallol	87-66-1	Used as a substrate in phenolic oxidation or peroxidation reactions; activity is derived from absorbance change.
Ascorbate content; APX activity	L-Ascorbic acid	50-81-7	Used as the electron donor for APX and as a standard for ascorbate quantification.
Glutathione content; GSH/GSSG status	Glutathione, reduced	70-18-8	Used for glutathione quantification and for preparing GSH-based reaction systems to assess reducing capacity.
Glutathione content; thiol-related	DTNB	69-78-3	Reacts with thiols to yield a yellow product for quantifying thiol groups and GSH-equivalent content.
GR activity; antioxidant regeneration flux	NADPH, reduced	2646-71-1	Used as a hydride donor to drive GR-associated reactions and enable activity/flux readout via absorbance change.
MDA; lipid peroxidation	Thiobarbituric acid	504-17-6	Forms a colored adduct with MDA for TBARS-based estimation of lipid peroxidation level.
MDA; lipid peroxidation	Trichloroacetic acid	76-03-9	Used for extraction and protein precipitation; helps obtain a clear supernatant for TBARS colorimetry.
MDA; lipid peroxidation	Butylated hydroxytoluene	128-37-0	Added during sample handling to suppress further lipid oxidation and reduce processing-induced artifacts.
Proline	Ninhydrin	485-47-2	Produces a colored reaction with proline for spectrophotometric quantification of proline content.
Proline	Sulfosalicylic acid	97-05-2	Used to extract proline and reduce interference from proteins and other macromolecules.
Proline	Acetic acid, glacial	64-19-7	Used to establish the acidic environment required for proline color development and as a solvent component.
Soluble sugars/total sugars	Anthrone	90-44-8	Forms a colored product with carbohydrates under strong-acid conditions for soluble/total sugar determination.
Soluble sugars/total sugars	D-Glucose	50-99-7	Used to build a standard curve for converting absorbance into sugar concentration.
DPPH scavenging capacity	DPPH	1898-66-4	Evaluates radical scavenging via hydrogen/electron donation, quantified by the extent of decolorization.
ABTS scavenging capacity	ABTS	30931-67-0	Uses a stable radical system to quantify scavenging capacity by the decrease in absorbance after reaction with antioxidants.
FRAP total antioxidant capacity	TPTZ	3682-35-7	Forms a colored iron–ligand complex used in FRAP assays to report total antioxidant power via reducing ability.
TEAC reference standard	Trolox	53188-07-1	Used as a reference antioxidant to convert results into Trolox equivalents.
Total phenolics	Gallic acid	149-91-7	Used to construct a standard curve and express total phenolics as gallic acid equivalents.
Flavonoids	Rutin	153-18-4	Used to construct a standard curve and express total flavonoids as rutin equivalents.
Flavonoids	Quercetin	117-39-5	Used to construct a standard curve and express total flavonoids as quercetin equivalents.
Overall ROS load	DCFH-DA	4091-99-0	Converted intracellularly and oxidized by ROS to a fluorescent product for estimating overall ROS burden.

Common physiological and biochemical indices characterize biological status across multiple layers, including light energy utilization, carbon assimilation and energy metabolism, ROS generation and detoxification, membrane damage and osmotic adjustment, secondary metabolism, and nutrient assimilation. Under standardized sampling and consistent analytical conditions, integrated multi-index analysis more accurately localizes limiting steps and response pathways, improves interpretive reliability for stress effects, adaptive mechanisms, and quality changes, and provides a solid basis for reproducibility and translation of research conclusions.

For more related articles, please see below:

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