INTRODUCTION:

Plants encounter a multitude of diverse abiotic and biotic stresses in nature. Abiotic stress enhances reactive oxygen species (ROS) which leads to the upregulation of methylglyoxal¹. Methylglyoxal is known to be a cytotoxic metabolite when upregulated in excess serves as a key molecule involving in stress response generated during environmental stress². Stress responses of plants restrict them from distribution in a region, change the growth and development also retards the productivity³.

Multiple stress inducible glyoxalase upregulated followed by desiccation stress⁴. To tolerate and survive the harsh environmental stress plants have many protective machineries which include non-enzymatic antioxidants like anthocyanin, carotenoids, flavonoids, phenolics ascorbate and reduced glutathione which helps to remove the ROS generated during abiotic stress⁵. As a response to the oxidative stress plants produce elevated amount of Methylglyoxal (MG) which is a cytotoxic α ketoaldehyde, end product of glycolysis. Glyoxalase and methylglyoxal serve as a potential biomarker for stress response⁶. To detoxify the MG, plants have a ubiquitous enzyme glyoxalase system, which consists of enzyme Gly I and Gly II which plays a crucial role in the pathway of biotransformation of MG into D – lactate involving GSH (fig .3) but in bacteria another enzyme Gly III is involved in the process without GSH⁷.

The well-studied angiosperm resurrection plant reported so far is from the family Linderniaceae found to be Craterostigma plantagineum Hochst and Lindernia brevidens Skan. Craterostigma plantagineum Hochst is one of the model angiosperm resurrection plant⁸. Majority of Lindernia sp. are desiccation sensitive such as Lindernia rotundata (Pilg.) Eb. Fisch, L. subracemosa De Wild. The family Linderniaceae also contains desiccation tolerant Lindernia brevidens Skan and Craterostigma plantagineum Hochst. Linderniaceae is a family of flowering plants in the order Lamiales comprising 13 genera and 195 species around the world⁹. Quantitative analysis of expression and promoter activity pcC13-62 promoters from the resurrection plant: Craterostigma plantagineum; desiccation tolerant: L. brevidens and together with desiccation sensitive L. subracemosa revealed lower level of DRP gene pcC13-62 accumulation which involves a DRE motif that is absent in desiccation sensitive plant. Also, the pcC13-62 promoters could also be activated in Arabidopsis thaliana plant which are transformed¹⁰.

There are huge reports of stress tolerance by the contribution of glyoxalase system in the higher plants. Many studies presented the quantitative data on the level of MG produced under stress condition as a signal of plant stress response¹¹. The enzymatic antioxidants which boost up against from the effects of oxidative stress like glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX), glutathione peroxidase (GPX).¹²

Fig. 1: Methylglyoxal Detoxification Pathway

This figure illustrates the formation of methylglyoxal (MG) from glycolysis and the Calvin cycle. Under stress, MG is detoxified by the glyoxalase system, involving Glyoxalase I (Gly I), Glyoxalase II (Gly II), and Glyoxalase III (Gly III), converting it to D-lactic acid with reduced glutathione (GSH).

Fig. 2: Plants collected for the study

This figure shows a) Torenia crustacea (L) Cham. and schltdl, b) Bonnaya ciliata (Colsm.) Spreng.c) Lindernia hyssopioides (L.) Haines, and d) Bonnaya antipoda (L.) Alston

Fig.3: Study area Madayi

MATERIAL AND METHODS:

Plant Material Collection:

Mature leaves from Torenia crustacea (L) Cham. & Schltdl., Bonnaya ciliata (Colsm.) Spreng., Lindernia hyssopioides (L.) Haines, and Bonnaya antipoda (L) Druce were gathered from Madayipara fields in Kerala (fig. 3). The species were authenticated, and voucher specimens were deposited at the Madras Herbarium for future reference.

DNA Extraction and Quantification:

Genomic DNA was isolated using the CTAB method, yielding high-quality DNA (fig.4 ). Tissue was frozen and ground, then digested in CTAB buffer, and contaminants were removed through phenol-chloroform extraction. DNA concentration and purity were optimized for PCR, revealing 10 ng DNA per reaction as the optimal concentration.¹³

Agarose Gel Electrophoresis:

Amplified DNA was visualized on 1.5% agarose gel, stained with ethidium bromide, and photographed under UV light. This step confirmed the presence of expected bands, indicating successful amplification.¹⁴

PCR Amplification of Glyoxalase I:

PCR amplification targeted partial Glyoxalase I sequences, using primers based on sequences from related species. Various DNA concentrations (5–50 ng) and primer concentrations (10 nM to 1 µM) were tested. The best amplification was achieved with 250 nM primers at an annealing temperature of 45°C, minimizing non-specific bands.

RESULTS AND DISCUSSION:

Glyoxalase I Gene Amplification and Optimization

Optimized PCR conditions, particularly the 10 ng DNA concentration and 45°C annealing temperature, yielded clear bands between 200–400 bp across all species (Fig. 5). Higher DNA concentrations led to smearing, emphasizing the need for precise DNA input to avoid non-specific amplifications. These results align with previous studies showing the critical role of DNA concentration in PCR efficiency.

Role of Glyoxalase I in Stress Tolerance:

Glyoxalase I upregulation is recognized as a response to MG build up during abiotic stress. The presence of Glyoxalase I in Lindernia and Bonnaya species suggests that it contributes to MG detoxification, potentially enhancing drought tolerance. Prior studies in related species have reported similar associations, supporting the hypothesis that Glyoxalase I activity corresponds to increased resilience against oxidative stress.

Comparison with Previous Findings:

While the role of Glyoxalase I in stress tolerance is established in other plant families, this study provides the first insight into its activity within Linderniaceae. The observed amplification efficiency and gene presence suggest that Glyoxalase I could be a critical component of the stress response system in this family, consistent with findings in drought-resistant crops like Arabidopsis and Oryza.

Fig. 4: Isolation and Purification of Genomic DNA

Total genomic DNA was isolated and purified from the leaves of the following plants using the CTAB method: a) Torenia crustacea (L) Cham. & Schltdl, b) Bonnaya ciliata (Colsm.) Spreng., c) Lindernia hyssopioides (L.) Haines, and d) Bonnaya antipoda (L) Druce. The molecular weight marker is indicated (bp = base pairs).

Fig. 5: PCR Amplification of Glyoxalase I Encoding Sequence

This figure shows the PCR amplification of the partial genomic DNA sequence encoding Glyoxalase I from: a) Torenia crustacea (L) Cham. & Schltdl b) Bonnaya ciliata (Colsm.) Spreng., c) Lindernia hyssopioides (L.) Haines, and d) Bonnaya antipoda (L) Druce. The molecular weight marker is included, with Lane 1 & 2 representing a 200 bp PCR amplicon.

CONCLUSION:

The identification of glyoxalase I gene sequences in this study highlights its potential significance in enhancing stress tolerance. The upregulation of glyoxalase I under drought stress conditions in crop plants suggests its future application in developing drought-tolerant varieties. Our findings imply that the drought tolerance observed in plants from the Linderniaceae family could be attributed to the glyoxalase I gene's protective role against methylglyoxal (MG) accumulation, a toxic by-product formed under stress conditions. This gene appears to confer tolerance by boosting the glutathione (GSH)-based detoxification system while minimizing lipid peroxidation. Given the intricate nature of metabolic activities in plant cells, where various biochemical and enzymatic pathways operate in harmony to maintain cellular homeostasis, unravelling and manipulating these pathways could be key in engineering plants with improved stress resilience. Understanding these mechanisms may pave the way for future advancements in crop stress tolerance strategies. The successful amplification of Glyoxalase I in Linderniaceae species highlights its potential as a molecular marker for drought tolerance. This study suggests that Glyoxalase I enables resilience by detoxifying methylglyoxal, a byproduct of stress response. Our findings lay a foundation for future research on genetic interventions aimed at improving crop tolerance to environmental stresses. This study focuses on Glyoxalase I amplification and does not measure gene expression or enzymatic activity under varying stress conditions. Future studies should analyze Glyoxalase I expression patterns and perform functional assays to assess its role in real-time stress adaptation.

ACKNOWLEDGMENTS:

The authors extend their sincere gratitude to Sree Narayana College, Kannur, and Sir Syed Institute of Technical Studies, Kannur, for their support in facilitating this research. Dr. Jeeshna MV acknowledges the University Grants Commission (UGC) for awarding the Junior Research Fellowship (RD A1/177/R.Bot/2020) to Sreelakshmi T., which enabled the successful completion of this study.

AUTHOR CONTRIBUTIONS:

JMV and SMVP conceived of the presented idea, ST and SMVP carried out the experiments and analysed the data, JMV supervised the project, SA , ST analysed the data, and prepared a final manuscript. Authors have read and approved the final manuscript.

FUNDING:

This work was supported by the University Grants Commission [NTA Ref No. 191620007895].

DECLARATIONS:

Ethics approval and consent to participate Fresh plants were collected from a paddy fields, with local regulations and guidelines. The voucher specimen was deposited at the Madras herbarium with accession no.178365, 178366, 178368, 178369.

COMPETING INTERESTS:

The authors decare no competing interests.

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Received on 19.11.2024 Revised on 05.12.2024

Accepted on 21.12.2024 Published on 05.03.2025

Available online from March 11, 2025

Res. J. Pharmacognosy and Phytochem. 2025; 17(1):1-4.

DOI: 10.52711/0975-4385.2025.00001

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