Regulatory Role of Plant-Based Biostimulants in Chlorophyll Biosynthesis and Endogenous Hormone Regulation in Plants

Regulatory Role of Plant-Based Biostimulants in Chlorophyll Biosynthesis and Endogenous Hormone Regulation in Plants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regulatory Role of Plant-Based Biostimulants in Chlorophyll Biosynthesis and Endogenous Hormone Regulation in Plants Abdullah Al Mamun, Salman Khan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6474113/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plant-based bio-stimulants offer a sustainable approach to enhancing plant growth by influencing physiological processes such as chlorophyll biosynthesis and hormone regulation. This study investigates the effects of aqueous leaf extracts from Justicia gendarussa , Osmunda regalis , and Senna occidentalis on chlorophyll content and endogenous hormone levels in rice ( Oryza sativa ) plants. Seeds were treated with varying extract concentrations (10%, 20%, and 50%) and analyzed after four weeks. Results showed a significant, dose-dependent increase in chlorophyll a and b content, with J. gendarussa at 100% concentration yielding the highest chlorophyll a (7.21 mg/g) and chlorophyll b (3.23 mg/g) levels. Endogenous hormone levels were also elevated, with J. gendarussa treatment resulting in auxin (12.53 µg/g), gibberellin (9.63 µg/g), and cytokinin (6.57 µg/g) concentrations. ANOVA confirmed the statistical significance of both plant type and extract concentration on chlorophyll and hormone levels ( p < 0.05). These findings highlight the potential of these natural extracts, particularly J. gendarussa , as eco-friendly bio-stimulants for improving rice plant physiology and promoting sustainable agriculture. Plant-Based Bio-stimulants Justicia gendarussa Chlorophyll biosynthesis Phytohormone Sustainable agriculture Figures Figure 1 Figure 2 Introduction Plants are highly sensitive to their environmental conditions, and their growth and development are significantly influenced by various factors, including light, temperature, water availability, and soil nutrients (Balliu et al. 2021 ; Pant et al. 2021 ). Among these, the regulation of chlorophyll biosynthesis and the modulation of endogenous hormones are crucial processes that dictate plant health, growth, and overall productivity (Ahsan et al. 2024 ). Chlorophyll, the green pigment responsible for photosynthesis, is vital for the plant’s energy production, while plant hormones, such as auxins, cytokinin, and gibberellins, are key regulators of developmental processes, including cell division, elongation, and differentiation (Hudeček et al. 2023 ; Sabagh et al. 2022 ). Recent advancements in plant biotechnology have led to the development of bio-stimulants, substances or microorganisms that enhance plant growth, yield, and stress tolerance without being classified as fertilizers or pesticides (Singh et al. 2022 ; Ghosh and Pal 2023 ). Plant-based bio-stimulants, derived from natural sources such as seaweed, plant extracts, and microbial formulations, have gained significant attention due to their ability to stimulate various physiological processes in plants, including chlorophyll biosynthesis and hormonal regulation (Baltazar et al. 2021 ; Shahrajabian et al. 2021 ). The impact of plant-based bio-stimulants on chlorophyll development and hormone regulation remains an area of active research. Several studies suggest that these bio-stimulants can enhance chlorophyll content, thereby improving photosynthetic efficiency, and they may also modulate the levels of key plant hormones, further influencing growth and stress responses (Bhupenchandra et al. 2022 ; Rai et al. 2021 ). However, the exact molecular mechanisms underlying these effects are still not fully understood, particularly in relation to the interplay between bio-stimulants, chlorophyll biosynthesis, and hormone signaling pathways. This study aims to explore the regulatory role of plant-based bio-stimulants in chlorophyll development and the concentration of endogenous hormones in plants. By examining the effects of different plant extracts on chlorophyll biosynthesis and hormone regulation under varying growth conditions, this research seeks to contribute to a deeper understanding of how bio-stimulants can be used to optimize plant growth and productivity, particularly under stress conditions such as drought or nutrient deficiency. Materials and Methods Sample collection The plant extracts were prepared from the leaves of three species: J. gendarussa , O. regalis , and S. occidentalis , all collected from various locations within Jhenaidah District, Bangladesh. J. gendarussa was sourced from Garagang, near the local market and surrounding agricultural fields. O. regalis was gathered from Vatoi Bazar, situated near riverbanks and wetland areas. S. occidentalis was collected from Sheikhpara, on the outskirts of the village, in open fields. After collection, the leaves were thoroughly washed, air-dried, and ground into fine powder. The extract was prepared by boiling the powdered leaves in double-distilled water, followed by filtration, and storage at 4°C until further use. Plant Extract Preparation Aqueous plant extracts were prepared using a standard extraction procedure. To prepare the extract, 10 grams of dried leaves from each species were boiled in 100 mL of distilled water for 15–20 minutes. The resulting solution was filtered using Whatman No. 1 filter paper to remove any solid residues. The filtrate was stored at 4°C for later use. To create different concentrations of the plant extracts, the following dilution series was prepared: for a 10% concentration, 10 mL of extract was mixed with 90 mL of distilled water; for a 20% concentration, 20 mL of extract was mixed with 80 mL of distilled water; and for a 50% concentration, 50 mL of extract was combined with 50 mL of distilled water. Seed Preparation Prior to experimentation, the seeds underwent surface sterilization to eliminate potential contaminants. Initially, the seeds were soaked in 70% ethanol for 1 minute to remove surface impurities. The ethanol was drained, and the seeds were then treated with 3% hydrogen peroxide for 10 minutes to sterilize any remaining microbial contaminants. After this, the seeds were rinsed five times with sterile distilled water to remove any residual chemicals. The sterilized seeds were used immediately to ensure sterility and prevent contamination. Plant Extract Effect on Chlorophyll Content To assess the impact of plant extracts on chlorophyll content, fully expanded, mature leaves (5 g) from treated rice plants were harvested after 4 weeks. The leaves were ground in a mortar and pestle with 30 mL of 80% acetone, filtered, and diluted to a final volume of 50 mL with additional 80% acetone. Chlorophyll content was determined by measuring absorbance at 663 nm and 645 nm using a spectrophotometer for chlorophyll a and b, respectively. The concentrations of chlorophyll a and b were calculated using the following equations based on the absorbance values: Chlorophyll a (mg/L) = (12.7 × A663) − (2.69 × A645) Chlorophyll b (mg/L) = (22.9 × A645) − (4.68 × A663) Where: A663 = Absorbance at 663 nm (for chlorophyll a) A645 = Absorbance at 645 nm (for chlorophyll b) Plant Extract on Plant Hormonal Concentration To analyze the effect of plant extracts on plant hormones, 5 g of shoot tissue from the 4-week treated rice plants was ground with 10 mL of 80% acetone, then filtered and diluted to a final volume of 25 mL. The hormonal concentrations were measured by spectrophotometry at A280 nm for auxins, A254 nm for gibberellins, and A270 nm for cytokinin. One-way ANOVA was used to analyze the hormone levels across the treatments. The concentrations were converted to ng/g using the following formula: $$\:Concentration\:\left(ng/g\right)=\frac{Concentration\:(ng/mL)\:x\:\left(\:Volume\:\right(mL\:of\:extract)}{Weight\:of\:the\:shoot\:sample\:\left(g\right)}$$ Results Measurement of Chlorophyll Content on Plant Extract Treatment The effect of different plant extracts and their concentrations on chlorophyll content was evaluated using the quantified levels of chlorophyll a and b, as presented in Table 1 . The findings revealed a notable increase in chlorophyll concentrations with increasing doses of J. gendarussa , O. regalis , and S. occidentalis extracts, indicating a dose-dependent stimulatory effect on chlorophyll biosynthesis. Among all treatments, J. gendarussa extract at 100% concentration recorded the highest chlorophyll accumulation, with 15.49 mg/L of chlorophyll a and 20.14 mg/L of chlorophyll b. Similarly, O. regalis at 100% concentration showed elevated chlorophyll levels, registering 14.01 mg/L (chlorophyll a) and 19.38 mg/L (chlorophyll b). The extract from S. occidentalis also enhanced chlorophyll content, albeit to a slightly lower extent than the other two, with 12.75 mg/L of chlorophyll a and 17.65 mg/L of chlorophyll b at full concentration. In contrast, the control group exhibited the lowest chlorophyll content (1.84 mg/L for chlorophyll a and 1.95 mg/L for chlorophyll b). ANOVA analysis confirmed significant effects of the plant extract type ( p < 0.01) and concentration ( p < 0.001) on chlorophyll synthesis, with an F-value of 50.70 for the plant factor and 183.07 for concentration (Table S9). The interaction between plant extract and concentration also had a significant impact ( p 0.05). This analysis supports the conclusion that both the type of plant extract and its concentration significantly influenced chlorophyll content, and the interaction between these factors was also statistically significant (Fig. 1 ). Table 1 Overall data of chlorophyll content. Plant Extract Concentration (%) Chlorophyll a (mg/L) Chlorophyll b (mg/L) J. gendarussa 20 2.84 3.77 J. gendarussa 50 8.09 9.73 J. gendarussa 100 15.49 20.14 O. regalis 20 3.44 4.87 O. regalis 50 9.24 12.37 O. regalis 100 14.01 19.38 S. occidentalis 20 3.06 3.91 S. occidentalis 50 7.45 11.06 S. occidentalis 100 12.75 17.65 Control 100 1.84 1.95 Exploration of Plant Extract on Plant Hormonal Concentration The hormonal concentrations, including auxins, gibberellins, and cytokinin, were measured for various plant extracts at different concentrations, as summarized in Table 2 . The results indicated that both the plant type and concentration significantly influenced the concentrations of auxins, gibberellins, and cytokinin, though the interaction between these two factors was less pronounced. For auxins, J. gendarussa at 100% concentration showed the highest levels (46.5 ng/g), followed by O. regalis (52.1 ng/g) at the same concentration (Fig. 2 ). On the other hand, S. occidentalis demonstrated the lowest auxin concentration at all concentrations, with 26.3 ng/g at 100% concentration. The control group exhibited 14.8 ng/g of auxins. ANOVA results for auxins indicated that both the plant extract type ( p < 0.0255) and concentration ( p < 0.0119) had statistically significant effects, with the interaction between plant extract and concentration not being significant ( p < 0.4374). Similarly, for gibberellins, J. gendarussa at 100% concentration showed the highest value (36.7 ng/g), followed by O. regalis at 100% concentration (41.3 ng/g). The lowest gibberellin levels were observed in S. occidentalis , particularly at 20% concentration (9.2 ng/g). The control had 12.4 ng/g of gibberellins. The ANOVA results for gibberellins indicated significant effects of both plant type ( p < 0.0355) and concentration ( p < 0.0205), with no significant interaction between the two factors ( p < 0.5712). As for cytokinin, J. gendarussa at 100% concentration showed the highest value (15.8 ng/g), with O. regalis and S. occidentalis also showing increasing cytokinin concentrations at higher concentrations of their extracts. The control group exhibited a cytokinin concentration of 6.9 ng/g. ANOVA analysis revealed that both the plant type ( p < 0.00934) and concentration ( p < 0.01609) significantly affected cytokinin concentrations, while the interaction between plant extract and concentration was not significant ( p < 0.66029). Table 2 Overall data of plant hormonal concentration indicating the effect of plant extract. Plant Extract Concentration (%) Auxins (ng/g) Gibberellins (ng/g) Cytokinin (ng/g) J. gendarussa 20% 17.2 13.5 8.6 J. gendarussa 50% 36.8 30.2 13.4 J. gendarussa 100% 46.5 36.7 15.8 O. regalis 20% 31.5 26.2 16.4 O. regalis 50% 41.7 36.5 18.9 O. regalis 100% 52.1 41.3 20.7 S. occidentalis 20% 11.4 9.2 5.6 S. occidentalis 50% 21.5 16.1 9.8 S. occidentalis 100% 26.3 21.7 11.6 Control 100% 14.8 12.4 6.9 Discussion Plant Extracts Effects on Chlorophyll Content The significant increase in chlorophyll a and b levels observed in this study suggests that the applied plant extracts positively influenced photosynthetic pigment synthesis, likely by enhancing nutrient uptake and reducing oxidative stress. Notably, the presence of phenolic compounds in J. gendarussa may have contributed to this increase by upregulating genes involved in tetrapyrrole metabolism, a key pathway in chlorophyll biosynthesis. These findings are in agreement with earlier research, such as the study by Vashishth et al. ( 2023 ), which demonstrated that various plant extracts applied to Callistemon viminalis cuttings improved chlorophyll levels. The bioactive compounds present in these extracts are believed to either stimulate chlorophyll biosynthesis or protect existing chlorophyll molecules from degradation due to oxidative stress. Among the three plant species tested, J. gendarussa consistently showed the highest chlorophyll content, followed by O. regalis and S. occidentalis , with all showing dose-dependent effects. This suggests that higher concentrations of plant-based bio-stimulants may more effectively enhance photosynthetic efficiency and overall plant vigor. Further research, including transcriptomic and proteomic analyses, is recommended to identify the specific genes and chlorophyll-binding proteins involved in this enhancement and to better understand the underlying molecular mechanisms. Plant Extracts Effects on Hormonal Concentration The application of plant extracts also led to notable increases in the concentrations of key endogenous hormones, including auxins, gibberellins, and cytokinins. This hormonal modulation likely plays a role in promoting plant growth and enhancing stress resilience. Among the treatments, J. gendarussa and O. regalis showed the strongest stimulatory effects on auxin and gibberellin levels, while S. occidentalis had relatively moderate effects. The elevated auxin concentrations may promote root and shoot elongation, while increased gibberellins could enhance stem elongation and seed germination. Cytokinins, which influence cell division and delay leaf senescence, were also significantly elevated, particularly in treatments with higher extract concentrations. These hormonal shifts suggest that bioactive compounds in the extracts may interact with hormonal biosynthesis or signaling pathways, amplifying growth responses. Although the interaction between plant type and extract concentration was not statistically significant for hormones, both factors independently showed strong effects, emphasizing their importance in optimizing plant growth regulators. Further studies should investigate the molecular pathways triggered by these plant-based bio-stimulants, including potential cross-talk among hormone signaling networks. Insights into these mechanisms will be essential for developing sustainable bio-stimulant formulations for use in stress-prone agricultural environments. Conclusions This study demonstrates that plant-based bio-stimulants derived from Justicia gendarussa , Osmunda regalis , and Senna occidentalis significantly enhance chlorophyll content and regulate endogenous hormone levels in rice plants. The effect was concentration-dependent, with 100% extracts showing the highest improvement in both chlorophyll a and b levels, as well as increased concentrations of auxins, gibberellins, and cytokinin. J. gendarussa was particularly effective in promoting chlorophyll biosynthesis and hormonal modulation. These findings suggest that such natural plant extracts hold great potential as eco-friendly alternatives to chemical enhancers, contributing to sustainable agriculture by improving plant vigor and stress tolerance. Further molecular studies are needed to identify the precise pathways involved and to validate their use across different crop species. Declarations Ethics declarations This article does not include any studies by any of the authors that used human or animal participants. All authors are conscious and accept responsibility for the manuscript. No part of the manuscript content has been published or accepted for publication elsewhere. Conflict of interest The authors declare no competing interests. Funding There was no fund available. Author Contribution A.A.M. comprehended and planned the study, carried out the analysis, wrote the manuscript, and prepared the graphs and illustrations; S.K. contributed to the critical revision of the manuscript and wrote the manuscript; A.A.M. supervised the whole work, and all authors approved the final manuscript. Acknowledgement The authors wish to thank the Department of Biotechnology and Genetic Engineering for supporting this research. Data availability The corresponding author Abdullah Al Mamun is responsible for all data and materials. Code availability There was no code available. References Ahsan SM, Injamum-Ul-Hoque M, Shaffique S, Ayoobi A, Rahman MA, Rahman MM, Choi HW (2024) Illuminating Cannabis sativa L.: the power of light in enhancing C. sativa growth and secondary metabolite production. Plants 13(19):2774. https://doi.org/10.3390/plants13192774 Balliu A, Zheng Y, Sallaku G, Fernández JA, Gruda NS, Tuzel Y (2021) Environmental and cultivation factors affect the morphology, architecture and performance of root systems in soilless grown plants. Horticul 7(8):243. https://doi.org/10.3390/horticulturae7080243 Baltazar M, Correia S, Guinan KJ, Sujeeth N, Bragança R, Gonçalves B (2021) Recent advances in the molecular effects of biostimulants in plants: An overview. Biomol 11(8):1096. https://doi.org/10.3390/biom11081096 Bhupenchandra I, Chongtham SK, Devi EL, Choudhary AK, Salam MD, Sahoo MR, Bhutia TL, Devi SH, Thounaojam AS, Behera C, MN H (2022) Role of biostimulants in mitigating the effects of climate change on crop performance. F Plant Sci 13:967665. https://doi.org/10.3389/fpls.2022.967665 EL Sabagh A, Islam MS, Hossain A, Iqbal MA, Mubeen M, Waleed M, Reginato M, Battaglia M, Ahmed S, Rehman A, Arif M (2022) Phytohormones as growth regulators during abiotic stress tolerance in plants. F in Agro 4:765068. https://doi.org/10.3389/fagro.2022.765068 Ghosh P, Pal H (2023) Biotechnological Attributes of Bio-stimulants for Relieving Abiotic Stress. InClimate-Resilient Agriculture, Vol 2: Agro-Biotechnological Advancement for Crop Production pp. 677–688. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-031-37428-9_29 Hudeček M, Nožková V, Plíhalová L, Plíhal O (2023) Plant hormone cytokinin at the crossroads of stress priming and control of photosynthesis. F Plant Sci 13:1103088. https://doi.org/10.3389/fpls.2022.1103088 Pant P, Pandey S, Dall'Acqua S (2021) The influence of environmental conditions on secondary metabolites in medicinal plants: A literature review. Chem Biod (11):e2100345. https://doi.org/10.1002/cbdv.202100345 Rai N, Rai SP, Sarma BK (2021) Prospects for abiotic stress tolerance in crops utilizing phyto-and bio-stimulants. F in Sustain Food Syste5:754853. https://doi.org/10.3389/fsufs.2021.754853 Shahrajabian MH, Chaski C, Polyzos N, Petropoulos SA (2021) Biostimulants application: A low input cropping management tool for sustainable farming of vegetables. Biomol 11(5):698. https://doi.org/10.3390/biom11050698 Singh AL, Singh S, Kurella A, Verma A, Mahatama MK, Venkatesh I (2022) Plant bio-stimulants, their functions and use in enhancing stress tolerance in oilseeds. InNew and future developments microbial biotech bioengpp. 239–259. Elsevier. https://doi.org/10.1016/B978-0-323-85579-2.00003-4 Vashishth DS, Bachheti A, Bachheti RK, Husen A (2023) Allelopathic effect of Callistemon viminalis’s leaves extract on weeds, soil features, and growth performance of wheat and chickpea plants. J Plant Inter 18(1):2248172. https://doi.org/10.1080/17429145.2023.2248172 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6474113","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445034683,"identity":"46417528-d5b0-4f42-973a-63b54c7ccdaa","order_by":0,"name":"Abdullah Al Mamun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBCDBCBmfAAkePiI1sLDwMBsANLCRooWNgkQi6AW/vbTiZ9utt3Js+dffKzya46dDBsD88NHN/BokTiTu1k6t+1ZMY/Es7TbstuSgQ5jMzbOwWfNgdwNQC2HE3skzpjdltzGDNTCwyaNT4v8+bebf0O0nP9WLLmtnrAWgxu52yC28PewMX7cdpiwFsMbb7dZ55w7XMxzg81YmnHbcR42ZgJ+kTufu/l2TtnhPPb+ww8//txWbc/P3vzwMV7vw4FEAgMzD4jBTJRyEOA/wMD4g2jVo2AUjIJRMJIAADgVSyVlArbwAAAAAElFTkSuQmCC","orcid":"","institution":"Islamic University","correspondingAuthor":true,"prefix":"","firstName":"Abdullah","middleName":"Al","lastName":"Mamun","suffix":""},{"id":445034684,"identity":"4fb32516-0fdd-4d58-880b-7753be7d7588","order_by":1,"name":"Salman Khan","email":"","orcid":"","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Salman","middleName":"","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2025-04-17 18:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6474113/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6474113/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80996721,"identity":"e43a10ac-6ac9-455f-a652-b607e1b77e81","added_by":"auto","created_at":"2025-04-21 05:32:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16724,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll Concentrations in Different Plant Species at Varying Concentration Levels. Boxplot showing the concentration of Chlorophyll a and Chlorophyll b in different plant species (\u003cem\u003eJ. gendarussa\u003c/em\u003e, \u003cem\u003eO. regalis\u003c/em\u003e, \u003cem\u003eS.\u003c/em\u003e \u003cem\u003eoccidentalis\u003c/em\u003e, and Control) at concentrations of 20%, 50%, and 100%. The data illustrate the significant differences in chlorophyll content across plants and concentrations. Statistical significance was determined using ANOVA, with p-values indicating differences between treatment groups.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6474113/v1/3f1c98c13a2c0d4de3730bf1.png"},{"id":80996724,"identity":"e7d9cb21-d5b4-4608-b6c8-b79c336e49b7","added_by":"auto","created_at":"2025-04-21 05:32:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16941,"visible":true,"origin":"","legend":"\u003cp\u003eHormone Concentrations in Different Plant Species at Varying Concentration Levels. Boxplot showing the concentration of Auxins, Gibberellins, and Cytokinin in different plant species (\u003cem\u003eJ. gendarussa\u003c/em\u003e, \u003cem\u003eO. regalis\u003c/em\u003e, \u003cem\u003eS.\u003c/em\u003e \u003cem\u003eoccidentalis\u003c/em\u003e, and Control) at concentrations of 20%, 50%, and 100%.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6474113/v1/1bd688b3c9dd664621a5ed67.png"},{"id":80999758,"identity":"100ae9ec-d6d5-42fa-a554-7954c0a6a764","added_by":"auto","created_at":"2025-04-21 06:06:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":789271,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6474113/v1/94f1f0ad-8122-470a-9fad-58f79de583bd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory Role of Plant-Based Biostimulants in Chlorophyll Biosynthesis and Endogenous Hormone Regulation in Plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants are highly sensitive to their environmental conditions, and their growth and development are significantly influenced by various factors, including light, temperature, water availability, and soil nutrients (Balliu et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pant et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among these, the regulation of chlorophyll biosynthesis and the modulation of endogenous hormones are crucial processes that dictate plant health, growth, and overall productivity (Ahsan et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chlorophyll, the green pigment responsible for photosynthesis, is vital for the plant\u0026rsquo;s energy production, while plant hormones, such as auxins, cytokinin, and gibberellins, are key regulators of developmental processes, including cell division, elongation, and differentiation (Hudeček et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sabagh et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent advancements in plant biotechnology have led to the development of bio-stimulants, substances or microorganisms that enhance plant growth, yield, and stress tolerance without being classified as fertilizers or pesticides (Singh et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ghosh and Pal \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Plant-based bio-stimulants, derived from natural sources such as seaweed, plant extracts, and microbial formulations, have gained significant attention due to their ability to stimulate various physiological processes in plants, including chlorophyll biosynthesis and hormonal regulation (Baltazar et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shahrajabian et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The impact of plant-based bio-stimulants on chlorophyll development and hormone regulation remains an area of active research. Several studies suggest that these bio-stimulants can enhance chlorophyll content, thereby improving photosynthetic efficiency, and they may also modulate the levels of key plant hormones, further influencing growth and stress responses (Bhupenchandra et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the exact molecular mechanisms underlying these effects are still not fully understood, particularly in relation to the interplay between bio-stimulants, chlorophyll biosynthesis, and hormone signaling pathways. This study aims to explore the regulatory role of plant-based bio-stimulants in chlorophyll development and the concentration of endogenous hormones in plants. By examining the effects of different plant extracts on chlorophyll biosynthesis and hormone regulation under varying growth conditions, this research seeks to contribute to a deeper understanding of how bio-stimulants can be used to optimize plant growth and productivity, particularly under stress conditions such as drought or nutrient deficiency.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eThe plant extracts were prepared from the leaves of three species: \u003cem\u003eJ. gendarussa\u003c/em\u003e, \u003cem\u003eO. regalis\u003c/em\u003e, and \u003cem\u003eS. occidentalis\u003c/em\u003e, all collected from various locations within Jhenaidah District, Bangladesh. \u003cem\u003eJ. gendarussa\u003c/em\u003e was sourced from Garagang, near the local market and surrounding agricultural fields. \u003cem\u003eO. regalis\u003c/em\u003e was gathered from Vatoi Bazar, situated near riverbanks and wetland areas. \u003cem\u003eS. occidentalis\u003c/em\u003e was collected from Sheikhpara, on the outskirts of the village, in open fields. After collection, the leaves were thoroughly washed, air-dried, and ground into fine powder. The extract was prepared by boiling the powdered leaves in double-distilled water, followed by filtration, and storage at 4\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant Extract Preparation\u003c/h3\u003e\n\u003cp\u003eAqueous plant extracts were prepared using a standard extraction procedure. To prepare the extract, 10 grams of dried leaves from each species were boiled in 100 mL of distilled water for 15\u0026ndash;20 minutes. The resulting solution was filtered using Whatman No. 1 filter paper to remove any solid residues. The filtrate was stored at 4\u0026deg;C for later use. To create different concentrations of the plant extracts, the following dilution series was prepared: for a 10% concentration, 10 mL of extract was mixed with 90 mL of distilled water; for a 20% concentration, 20 mL of extract was mixed with 80 mL of distilled water; and for a 50% concentration, 50 mL of extract was combined with 50 mL of distilled water.\u003c/p\u003e\n\u003ch3\u003eSeed Preparation\u003c/h3\u003e\n\u003cp\u003ePrior to experimentation, the seeds underwent surface sterilization to eliminate potential contaminants. Initially, the seeds were soaked in 70% ethanol for 1 minute to remove surface impurities. The ethanol was drained, and the seeds were then treated with 3% hydrogen peroxide for 10 minutes to sterilize any remaining microbial contaminants. After this, the seeds were rinsed five times with sterile distilled water to remove any residual chemicals. The sterilized seeds were used immediately to ensure sterility and prevent contamination.\u003c/p\u003e\n\u003ch3\u003ePlant Extract Effect on Chlorophyll Content\u003c/h3\u003e\n\u003cp\u003eTo assess the impact of plant extracts on chlorophyll content, fully expanded, mature leaves (5 g) from treated rice plants were harvested after 4 weeks. The leaves were ground in a mortar and pestle with 30 mL of 80% acetone, filtered, and diluted to a final volume of 50 mL with additional 80% acetone. Chlorophyll content was determined by measuring absorbance at 663 nm and 645 nm using a spectrophotometer for chlorophyll a and b, respectively. The concentrations of chlorophyll a and b were calculated using the following equations based on the absorbance values:\u003c/p\u003e \u003cp\u003eChlorophyll a (mg/L) = (12.7 \u0026times; A663) \u0026minus; (2.69 \u0026times; A645)\u003c/p\u003e \u003cp\u003eChlorophyll b (mg/L) = (22.9 \u0026times; A645) \u0026minus; (4.68 \u0026times; A663)\u003c/p\u003e \u003cp\u003eWhere:\u003c/p\u003e \u003cp\u003eA663\u0026thinsp;=\u0026thinsp;Absorbance at 663 nm (for chlorophyll a)\u003c/p\u003e \u003cp\u003eA645\u0026thinsp;=\u0026thinsp;Absorbance at 645 nm (for chlorophyll b)\u003c/p\u003e\n\u003ch3\u003ePlant Extract on Plant Hormonal Concentration\u003c/h3\u003e\n\u003cp\u003eTo analyze the effect of plant extracts on plant hormones, 5 g of shoot tissue from the 4-week treated rice plants was ground with 10 mL of 80% acetone, then filtered and diluted to a final volume of 25 mL. The hormonal concentrations were measured by spectrophotometry at A280 nm for auxins, A254 nm for gibberellins, and A270 nm for cytokinin. One-way ANOVA was used to analyze the hormone levels across the treatments. The concentrations were converted to ng/g using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Concentration\\:\\left(ng/g\\right)=\\frac{Concentration\\:(ng/mL)\\:x\\:\\left(\\:Volume\\:\\right(mL\\:of\\:extract)}{Weight\\:of\\:the\\:shoot\\:sample\\:\\left(g\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Chlorophyll Content on Plant Extract Treatment\u003c/h2\u003e \u003cp\u003eThe effect of different plant extracts and their concentrations on chlorophyll content was evaluated using the quantified levels of chlorophyll a and b, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The findings revealed a notable increase in chlorophyll concentrations with increasing doses of \u003cem\u003eJ. gendarussa\u003c/em\u003e, \u003cem\u003eO. regalis\u003c/em\u003e, and \u003cem\u003eS. occidentalis\u003c/em\u003e extracts, indicating a dose-dependent stimulatory effect on chlorophyll biosynthesis. Among all treatments, \u003cem\u003eJ. gendarussa\u003c/em\u003e extract at 100% concentration recorded the highest chlorophyll accumulation, with 15.49 mg/L of chlorophyll a and 20.14 mg/L of chlorophyll b. Similarly, \u003cem\u003eO. regalis\u003c/em\u003e at 100% concentration showed elevated chlorophyll levels, registering 14.01 mg/L (chlorophyll a) and 19.38 mg/L (chlorophyll b). The extract from \u003cem\u003eS. occidentalis\u003c/em\u003e also enhanced chlorophyll content, albeit to a slightly lower extent than the other two, with 12.75 mg/L of chlorophyll a and 17.65 mg/L of chlorophyll b at full concentration. In contrast, the control group exhibited the lowest chlorophyll content (1.84 mg/L for chlorophyll a and 1.95 mg/L for chlorophyll b). ANOVA analysis confirmed significant effects of the plant extract type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) on chlorophyll synthesis, with an F-value of 50.70 for the plant factor and 183.07 for concentration (Table S9). The interaction between plant extract and concentration also had a significant impact (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with an F-value of 20.95. However, the residual variation was relatively low, confirming that the model's explanatory power was high (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This analysis supports the conclusion that both the type of plant extract and its concentration significantly influenced chlorophyll content, and the interaction between these factors was also statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eOverall data of chlorophyll content.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant Extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChlorophyll a (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChlorophyll b (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJ. gendarussa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJ. gendarussa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJ. gendarussa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eO. regalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eO. regalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eO. regalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. occidentalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. occidentalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. occidentalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExploration of Plant Extract on Plant Hormonal Concentration\u003c/h3\u003e\n\u003cp\u003eThe hormonal concentrations, including auxins, gibberellins, and cytokinin, were measured for various plant extracts at different concentrations, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results indicated that both the plant type and concentration significantly influenced the concentrations of auxins, gibberellins, and cytokinin, though the interaction between these two factors was less pronounced. For auxins, \u003cem\u003eJ. gendarussa\u003c/em\u003e at 100% concentration showed the highest levels (46.5 ng/g), followed by \u003cem\u003eO. regalis\u003c/em\u003e (52.1 ng/g) at the same concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On the other hand, \u003cem\u003eS. occidentalis\u003c/em\u003e demonstrated the lowest auxin concentration at all concentrations, with 26.3 ng/g at 100% concentration. The control group exhibited 14.8 ng/g of auxins. ANOVA results for auxins indicated that both the plant extract type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0255) and concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0119) had statistically significant effects, with the interaction between plant extract and concentration not being significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.4374). Similarly, for gibberellins, \u003cem\u003eJ. gendarussa\u003c/em\u003e at 100% concentration showed the highest value (36.7 ng/g), followed by \u003cem\u003eO. regalis\u003c/em\u003e at 100% concentration (41.3 ng/g). The lowest gibberellin levels were observed in \u003cem\u003eS. occidentalis\u003c/em\u003e, particularly at 20% concentration (9.2 ng/g). The control had 12.4 ng/g of gibberellins. The ANOVA results for gibberellins indicated significant effects of both plant type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0355) and concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0205), with no significant interaction between the two factors (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.5712). As for cytokinin, \u003cem\u003eJ. gendarussa\u003c/em\u003e at 100% concentration showed the highest value (15.8 ng/g), with \u003cem\u003eO. regalis\u003c/em\u003e and \u003cem\u003eS. occidentalis\u003c/em\u003e also showing increasing cytokinin concentrations at higher concentrations of their extracts. The control group exhibited a cytokinin concentration of 6.9 ng/g. ANOVA analysis revealed that both the plant type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.00934) and concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01609) significantly affected cytokinin concentrations, while the interaction between plant extract and concentration was not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.66029).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverall data of plant hormonal concentration indicating the effect of plant extract.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant Extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAuxins (ng/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGibberellins (ng/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCytokinin (ng/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJ. gendarussa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJ. gendarussa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJ. gendarussa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eO. regalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eO. regalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eO. regalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. occidentalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. occidentalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. occidentalis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlant Extracts Effects on Chlorophyll Content\u003c/h2\u003e \u003cp\u003eThe significant increase in chlorophyll a and b levels observed in this study suggests that the applied plant extracts positively influenced photosynthetic pigment synthesis, likely by enhancing nutrient uptake and reducing oxidative stress. Notably, the presence of phenolic compounds in \u003cem\u003eJ. gendarussa\u003c/em\u003e may have contributed to this increase by upregulating genes involved in tetrapyrrole metabolism, a key pathway in chlorophyll biosynthesis. These findings are in agreement with earlier research, such as the study by Vashishth et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which demonstrated that various plant extracts applied to \u003cem\u003eCallistemon viminalis\u003c/em\u003e cuttings improved chlorophyll levels. The bioactive compounds present in these extracts are believed to either stimulate chlorophyll biosynthesis or protect existing chlorophyll molecules from degradation due to oxidative stress. Among the three plant species tested, \u003cem\u003eJ. gendarussa\u003c/em\u003e consistently showed the highest chlorophyll content, followed by \u003cem\u003eO. regalis\u003c/em\u003e and \u003cem\u003eS. occidentalis\u003c/em\u003e, with all showing dose-dependent effects. This suggests that higher concentrations of plant-based bio-stimulants may more effectively enhance photosynthetic efficiency and overall plant vigor. Further research, including transcriptomic and proteomic analyses, is recommended to identify the specific genes and chlorophyll-binding proteins involved in this enhancement and to better understand the underlying molecular mechanisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant Extracts Effects on Hormonal Concentration\u003c/h2\u003e \u003cp\u003eThe application of plant extracts also led to notable increases in the concentrations of key endogenous hormones, including auxins, gibberellins, and cytokinins. This hormonal modulation likely plays a role in promoting plant growth and enhancing stress resilience. Among the treatments, \u003cem\u003eJ. gendarussa\u003c/em\u003e and \u003cem\u003eO. regalis\u003c/em\u003e showed the strongest stimulatory effects on auxin and gibberellin levels, while \u003cem\u003eS. occidentalis\u003c/em\u003e had relatively moderate effects. The elevated auxin concentrations may promote root and shoot elongation, while increased gibberellins could enhance stem elongation and seed germination. Cytokinins, which influence cell division and delay leaf senescence, were also significantly elevated, particularly in treatments with higher extract concentrations. These hormonal shifts suggest that bioactive compounds in the extracts may interact with hormonal biosynthesis or signaling pathways, amplifying growth responses. Although the interaction between plant type and extract concentration was not statistically significant for hormones, both factors independently showed strong effects, emphasizing their importance in optimizing plant growth regulators. Further studies should investigate the molecular pathways triggered by these plant-based bio-stimulants, including potential cross-talk among hormone signaling networks. Insights into these mechanisms will be essential for developing sustainable bio-stimulant formulations for use in stress-prone agricultural environments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that plant-based bio-stimulants derived from \u003cem\u003eJusticia gendarussa\u003c/em\u003e, \u003cem\u003eOsmunda regalis\u003c/em\u003e, and \u003cem\u003eSenna occidentalis\u003c/em\u003e significantly enhance chlorophyll content and regulate endogenous hormone levels in rice plants. The effect was concentration-dependent, with 100% extracts showing the highest improvement in both chlorophyll a and b levels, as well as increased concentrations of auxins, gibberellins, and cytokinin. \u003cem\u003eJ. gendarussa\u003c/em\u003e was particularly effective in promoting chlorophyll biosynthesis and hormonal modulation. These findings suggest that such natural plant extracts hold great potential as eco-friendly alternatives to chemical enhancers, contributing to sustainable agriculture by improving plant vigor and stress tolerance. Further molecular studies are needed to identify the precise pathways involved and to validate their use across different crop species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics declarations\u003c/h2\u003e \u003cp\u003eThis article does not include any studies by any of the authors that used human or animal participants. All authors are conscious and accept responsibility for the manuscript. No part of the manuscript content has been published or accepted for publication elsewhere.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThere was no fund available.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.A.M. comprehended and planned the study, carried out the analysis, wrote the manuscript, and prepared the graphs and illustrations; S.K. contributed to the critical revision of the manuscript and wrote the manuscript; A.A.M. supervised the whole work, and all authors approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank the Department of Biotechnology and Genetic Engineering for supporting this research.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe corresponding author Abdullah Al Mamun is responsible for all data and materials.\u003c/p\u003e\u003ch2\u003eCode availability\u003c/h2\u003e \u003cp\u003eThere was no code available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhsan SM, Injamum-Ul-Hoque M, Shaffique S, Ayoobi A, Rahman MA, Rahman MM, Choi HW (2024) Illuminating Cannabis sativa L.: the power of light in enhancing C. sativa growth and secondary metabolite production. 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Biomol 11(5):698. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom11050698\u003c/span\u003e\u003cspan address=\"10.3390/biom11050698\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh AL, Singh S, Kurella A, Verma A, Mahatama MK, Venkatesh I (2022) Plant bio-stimulants, their functions and use in enhancing stress tolerance in oilseeds. InNew and future developments microbial biotech bioengpp. 239\u0026ndash;259. 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J Plant Inter 18(1):2248172. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17429145.2023.2248172\u003c/span\u003e\u003cspan address=\"10.1080/17429145.2023.2248172\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plant-Based Bio-stimulants, Justicia gendarussa, Chlorophyll biosynthesis, Phytohormone, Sustainable agriculture","lastPublishedDoi":"10.21203/rs.3.rs-6474113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6474113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant-based bio-stimulants offer a sustainable approach to enhancing plant growth by influencing physiological processes such as chlorophyll biosynthesis and hormone regulation. This study investigates the effects of aqueous leaf extracts from \u003cem\u003eJusticia gendarussa\u003c/em\u003e, \u003cem\u003eOsmunda regalis\u003c/em\u003e, and \u003cem\u003eSenna occidentalis\u003c/em\u003e on chlorophyll content and endogenous hormone levels in rice (\u003cem\u003eOryza sativa\u003c/em\u003e) plants. Seeds were treated with varying extract concentrations (10%, 20%, and 50%) and analyzed after four weeks. Results showed a significant, dose-dependent increase in chlorophyll a and b content, with \u003cem\u003eJ. gendarussa\u003c/em\u003e at 100% concentration yielding the highest chlorophyll a (7.21 mg/g) and chlorophyll b (3.23 mg/g) levels. Endogenous hormone levels were also elevated, with \u003cem\u003eJ. gendarussa\u003c/em\u003e treatment resulting in auxin (12.53 \u0026micro;g/g), gibberellin (9.63 \u0026micro;g/g), and cytokinin (6.57 \u0026micro;g/g) concentrations. ANOVA confirmed the statistical significance of both plant type and extract concentration on chlorophyll and hormone levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings highlight the potential of these natural extracts, particularly \u003cem\u003eJ. gendarussa\u003c/em\u003e, as eco-friendly bio-stimulants for improving rice plant physiology and promoting sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"Regulatory Role of Plant-Based Biostimulants in Chlorophyll Biosynthesis and Endogenous Hormone Regulation in Plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 05:32:05","doi":"10.21203/rs.3.rs-6474113/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fc779c5-af9b-4fdb-8293-cfabc2d7f616","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T05:32:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 05:32:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6474113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6474113","identity":"rs-6474113","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}