Cancer bush (Lessertia frutescens L. syn. Sutherlandia frutescens L.) is an economically important multipurpose medicinal plant indigenous to southern Africa (Chuang et al. 2014). It is widely distributed in Namibia, Botswana, and South Africa (Chuang et al. 2014; Hamdi et al. 2021). In South Africa, cancer bush is commonly found in the Western Cape, Eastern Cape, Northern Cape, KwaZulu-Natal, and Mpumalanga provinces (Colling et al. 2010; Hamdi et al. 2021). Cancer bush is a small, attractive, perennial woody shrub that grows up to 1 m long (Gibson 2011). The striking scarlet flowers have made it a popular ornamental plant (Gibson 2011; Masenya et al. 2022). It is also reported to be used by British botanists since the early 1990s due to its phytochemical properties (Haffajee 2002; Street and Prinsloo 2013). Although extremely bitter, cancer bush is widely used in traditional medicine in Africa to treat diseases such as cancer, diabetes, infections, and inflammation (Gibson 2011; Mncwangi et al. 2023). In modern medicine, cancer bush is well known as an adaptogenic tonic, and the commercial tablets are popular to counteract the muscle-wasting effects associated with HIV-AIDS in patients and to stimulate appetite (Omolaoye et al. 2021; Mncwangi et al. 2023). The plant’s a.i., such as L-canavanine, D-pinitol, γ-amino-butyric acid, triterpenoid glucoside known as “SU1,” or triterpenoid saponins and flavanol glycosides are reported to possess anti-inflammatory, antioxidant, anticonvulsant, anticancer, antidiabetic, antimutagenic, analgesic, and immunomodulatory properties (Omolaoye et al. 2021; Shaik et al. 2011). Although cancer bush is an important medicinal plant, its growth and productivity are mainly limited by climate change (Hamdi et al. 2021).
Climate change intensifies the abiotic stress that reduces plant production and yield due to frequent drought and flooding, extreme temperatures, high wind speeds, and altered intensity and spectrum of ultraviolet radiation (Liu et al. 2020; Zhang et al. 2023). Heat stress due to high temperature can negatively affect plant growth, development, and more severely the reproductive stages causing a decrease of crop yield (Fahad et al. 2017; Mirón et al. 2023). Seed germination may be inhibited due to high temperatures above 30 to 38 °C (Cetin et al. 2023; Prasad and Djanaguiraman 2014). High temperature stress causes water deficiency in plant tissues, which in turn leads to injury of cell membranes and reduction in rates of transpiration, protein synthesis, and ion uptake and transport (Ahanger et al. 2017; Farooq et al. 2023). In addition, heat stress leads to overproduction of reactive oxygen species (ROS) and inhabitation of photosynthetic enzymes (Mittler et al. 2022; Shaffique et al. 2022; Tamta and Patni 2023), which ultimately results in the loss of cellular organization, cell death, crop failure, and economic losses (Gray and Brady 2016; Srivastava et al. 2023).
SA is a synthetic plant growth regulator (PGR) that serves as a critical signal molecule mediating immunity and plant growth (Kulak et al. 2021; Sakhabutdinova et al. 2003). SA is the major solute involved in flower induction, general growth and development, various enzyme biosynthesis, stomata movements, membrane protections, and cell respiration (Hafez et al. 2019; Sharma et al. 2020). It is also a defense-related plant hormone that plays a key role in resistance to different microbial pathogens such as virus, bacteria, fungi, and oomycetes (Koo et al. 2020). One of the most prominent roles of SA is in stress tolerance of plants, where it acts as a signaling molecule that induces resistance (Sharma et al. 2020). Rady and Mohamed (2015), Zulfiqar et al. (2020), and Guo et al. (2022) reported that 1 mM, 50 mg⋅L−1 and 0.75 mM SA effectively improved growth and yield of common bean (Phaseolus vulgaris L.), gladiolus (Gladiolus grandifloras L.), and maize (Zea mays L.) plants under normal or environmental stress conditions such as salt and heat stress, respectively. Although SA is effective, its excessive application could be associated with water and air pollution (Kaya et al. 2023). The residues of synthetic PGRs in agricultural products could be detrimental to human health due to toxicity (Kaya et al. 2023; Kobayashi et al. 2020), thus the need for green synthesis of SA. Also, little is known about the effect of SA in combination with natural biostimulants on plant performance.
Plant biostimulants such as moringa (Moringa oleifera Lam.) extracts are substances applied as seed soaking and/or foliar spray that positively modify plant growth and productivity with alterations in metabolic processes under normal or environmental stress conditions (Batool et al. 2019; Soliman et al. 2020). Moringa, a multipurpose tree from the Moringaceae family is native to several habitats in South America, Africa, and Asia (Benettayeb et al. 2022; Shakour et al. 2023). Moringa leaves are a rich source of macro and micronutrients, vitamins, antioxidants, phytohormones such as auxins, gibberellins (GAs), cytokinins (CKs) or zeatin, SA, and JA (Azeem et al. 2023; Shakour et al. 2023). These components make moringa a potential natural plant growth stimulant (Azeem et al. 2023; Zulfiqar et al. 2020).
In addition, the preparation of plant-derived biostimulants such as moringa leaf extract (MLE) in ethanol or water is both cost-efficient and eco-friendly and could be used as a natural growth promoter and/or stress-reducing agent for many plant species due to the presence of phytochemicals and phytohormones (Yap et al. 2021; Zulfiqar et al. 2020). Latif and Mohamed (2016) and Yap et al. (2021) reported that the application of MLE (5 to 10 g/L) as a foliar spray effectively improved plant growth and yield of common bean and milk thistle (Silybum marianum L.) under environmental stress such as heat and salinity stresses. However, little is known about the biostimulant potential of moringa seed extract (MSE) to enhance plant productivity under heat stress conditions. To the best of our knowledge, no findings have been reported on the effect of a combination of MSE and SA on the growth and productivity of medicinal plants under environmental stress conditions. The aim of this study was to evaluate the effect of MSE, SA, and their combination on growth, bioactive, and phytohormone attributes of cancer bush plants subjected to heat stress.
Discussion
Cancer bush is an important medicinal plant that is also used as an ornamental plant (Mncwangi et al. 2023). It is commonly harvested in the wild and commercially cultivated by a few farmers (Masenya et al. 2023). With the effect of global climate change, abiotic stresses such as high temperature, drought, saline-alkali, and flooding are the main factors restricting plant growth and development, leading to the loss of yield (Khan and Mehmood 2023). Natural biostimulants, which are more cost effective, safer, and environmentally friendly than common agrochemicals, can be innovative tools for enhancing the quality and yield of medicinal plants (Shahrajabian and Sun 2022). Moringa plant extracts, including leaves and seed extracts are a rich source of nutrients such as macro and micronutrients, vitamins, antioxidants, and phytohormones, including auxins, GAs, CKs, SA, and JA, which are responsible for promoting plant growth and development (Ahmed et al. 2021; Buthelezi et al. 2023). In addition, natural biostimulants enriched with natural or synthetic PGRs such as melatonin, SA, and ABA can promote plant resistance to environmental stresses (Abdel Megeed et al. 2021). Mostly, PGRs are applied at low concentrations and are involved in the hormonal homeostasis and/or signaling network, therefore triggering plant growth, development, metabolic processes, and responses to stress (Shah et al. 2023). Recently, there has been great interest in replacing synthetic agrochemicals with biostimulants.
The results of the current study showed that the application of MSE and MSE + SA significantly enhanced plant growth attributes. This could be attributed to the presence of bioactive compounds in MSE. MSE contains essential minerals, antioxidants, and phytohormones such as IAA, Gas, and CKs (Buthelezi et al. 2023). Phytohormones, such as IAA, GAs, and CKs including zeatin, promote cell division and expansion, plant growth, and yield (Ahmed et al. 2021; Shakour et al. 2023). This diverse composition of MSE indicates that this extract can be used as a potential plant biostimulant. Several studies highlighted the role of moringa plant extracts, particularly MLE, to improve plant growth and development in different crops (Ahmed et al. 2021; Buthelezi et al. 2023; Khalofah et al. 2020; Latif and Mohamed 2016; Mahmood et al. 2022; Rady and Mohamed 2015; Shakour et al. 2023; Yap et al. 2021; Zulfiqar et al. 2020), which is also evident from results of the current study.
Moreover, the increase in the growth attributes of heat-stressed cancer bush plants in response to SA treatment, especially in combination with MSE, could be attributed to the protective role of SA on membranes that might increase the tolerance of plants to heat stress (Kobayashi et al. 2020). SA is a naturalistic plant phenolic compound that contributes as a nonenzymatic antioxidant and an endogenous signaling molecule inducing stress tolerance against abiotic (heat, drought, heavy metal, and salt stress) and biotic (against pest and diseases) stresses in plants (Kaya et al. 2023; Kobayashi et al. 2020). In addition, SA interacts with other hormones in plants that are involved in the regulation of cell division and expansion, such as auxin, GA, and ethylene (ET), to modulate plant growth (Coşkun et al. 2023; Lin et al. 2023).
The increase of total chlorophyll and carotenoid contents in heat-stressed cancer bush plants by the foliar applications of MSE and/or MSE + SA could be attributed to that MSE is rich in phytochemicals such as antioxidants, particularly proline and ascorbic acid and phytohormones including CKs and GAs (Buthelezi et al. 2022, 2023). The MSE components also act to promote the biosynthesis of leaf photosynthetic pigments due to their high content of mineral nutrients and phytohormones, increasing leaf chlorophyll concentrations and consequently the enhanced photosynthesis process produces more assimilates including osmoprotectants (Mahmood et al. 2022; Zulfiqar et al. 2020). The increased total chlorophyll concentration in the leaves of heat-stressed cancer bush plants treated with SA and/or MSE + SA foliar spray could be related to the influence of SA on endogenous CK contents, which may further increase after MSE application (Buthelezi et al. 2023; Kaya et al. 2023). This is further supported by Fig. 1, which shows that total chlorophylls concentrations were higher in plants treated with MSE + SA followed by plants foliar sprayed with MSE compared with control. Also, SA-treated plants synthesize more CKs, which improves chloroplast differentiation and chlorophyll biosynthesis and prevents chlorophyll degradation (Coşkun et al. 2023; Goncharuk and Zagoskina 2023).
The enhanced total chlorophyll content in heat-stressed plants by MSE and/or SA foliar spray might have increased plant growth (Table 1) and productivity because photosynthesis and biomass production are controlled directly by the amount of chlorophyll present in plants (Taffouo et al. 2017). Chlorophyll harvests and converts the energy of absorbed photons to the energy of chemical bonds resulting from photosynthesis, thus enhancing plant growth (Croft et al. 2020). In plants, carotenoids are essential for photosynthesis and photoprotection and play critical roles as light harvesting pigments and structural components of photosystems (Dhami and Cazzonelli 2020). In addition, carotenoids can act as signaling molecules in response to environmental and developmental cues or serve as regulators of plant growth (Rocha Júnior et al. 2023; Sherin et al. 2022). This is further supported by the results of the current study, which shows that plants treated with MSE or MSE + SA had higher total carotenoid contents (Fig. 1), which may have improved plant growth attributes (Table 1). Our results are similar to those of Rady and Mohamed (2015), who stated that the application of 1 mM SA and 3% MLE significantly enhanced the total chlorophyll (1.76 and 1.77 mg⋅g−1 FW) and carotenoid (0.45 and 0.44 mg⋅g−1 FW) concentrations of common bean plants grown on a saline soil (EC = 6.23–6.28 dS⋅m−1) compared with control (1.70 and 0.43 mg⋅g−1 FW), respectively. Also, Batool et al. (2020) stated that foliar application of 3% MLE improved leaf chlorophyll a (51%) and b (61%), and total chlorophyll contents (54%) of moringa seedlings compared with control.
The EL from plant tissues is commonly used as a parameter to evaluate cell integrity and as an indicator of plant stress tolerance (Rady and Mohamed 2015). Because membrane damage often results in an increased leakage of cytosolic constituents to the apoplastic space, higher values of EL imply lower membrane stability (Sujata Goyal et al. 2023). In our study, the treatments with MSE either alone and/or in combination with SA showed the best results under heat stress by reducing the accumulation of EL in cancer bush leaves (Fig. 2). These results suggest that MSE components such as essential mineral nutrients and phytohormones (Buthelezi et al. 2022, 2023) could have easily translocated through leaf stomata to active parts such as biosynthesizing and/or meristematic cells to provide them the ability to overcome the stress conditions (Buthelezi et al. 2023; Shakour et al. 2023). Mineral nutrients enable crop plants to adapt to environmental stress conditions through signaling pathways that affect the adaptive responses of plants to environmental stresses and/or expression and regulation of stress-induced genes that contribute to stress tolerance (Mahmood et al. 2022; Yap et al. 2021). Also, the application of MSE with SA induces phytohormone biosynthesis that further maintains integrity of cellular membranes under environmental stress conditions such as drought and heat stress, which are considered an integral part of the heat stress tolerance mechanism (Batool et al. 2020; Coşkun et al. 2023; Shakour et al. 2023). Our results are similar to those of Rady and Mohamed (2015), who reported that the application of 1 mM SA and 3% MLE effectively reduced EL (8.04 and 8.08%, respectively) compared with control (9.68%).
The higher concentrations of ABA, JA, and IAA were observed in heat-stressed cancer bush plants treated with MSE, which could be due to the presence of bioactive compounds and phytohormones in MSE (Buthelezi et al. 2022, 2023). Phytohormones promote many plant-related physiological processes and signaling networks in plants to modify plant responses to environmental stressors (Iqbal et al. 2023; Singh et al. 2023). ABA is synthesized under environmental stress conditions and triggers an adaptive response by activating a group of genes responsible for stress resistance (Iqbal et al. 2023; Ma et al. 2020). It is involved in the regulation of many aspects of plant performance, including seed germination, embryo maturation, leaf senescence, stomatal aperture, and tolerance to environmental stress (Pal et al. 2023; Singh et al. 2023).
JA is a plant-signaling molecule closely associated with plant resistance to abiotic stress and is usually involved in physiological and molecular responses such as the activation of the antioxidant system, accumulation of amino acids and soluble sugars, and regulation of stomatal opening and closing (Hewedy et al. 2023; Iqbal et al. 2023). IAA regulates growth and developmental processes such as cell division and elongation, tissue differentiation, and apical dominance, and promotes stem and lateral root growth, fruit development, and tolerance to pathogens (Singh et al. 2023; Sosnowski et al. 2023). This is in accordance with our results in which heat-stressed plants treated with MSE had enhanced plant growth attributes (Table 1) and leaf pigments (Fig. 1) and reduced EL (Fig. 2). Moreover, a study by Buthelezi et al. (2023) showed that this extract is rich in phytohormones such as IAA, GA, and CK, which promotes plant growth, productivity, and tolerance to environmental stresses (Khalofah et al. 2020; Shakour et al. 2023).
The water loss caused by drought can induce excessive production of ROS such as O2•−, 1O2, and H2O2, which results in membrane lipid peroxidation, protein denaturing, and cell death (Hamedeh et al. 2022; Kesawat et al. 2023). The application of MSE effectively mitigated the oxidative damage as a result of the decreased ROS production; H2O2 and O2•− (Fig. 3), which could be due to either the presence of antioxidants or direct ROS scavenging capacity of peptides and amino acids in MSE (Buthelezi et al. 2022, 2023). Also, plants treated with MSE + SA showed lower values of H2O2 and O2•− compared with control, which also could be attributed to SA being an important signal molecule that can activate different defense mechanism in plants, thus enhancing plants’ adaptability to abiotic stress conditions (Kaya et al. 2023).
The higher concentration of glutathione observed in heat-stressed cancer bush plants treated with MSE + SA could be because MSE is rich in some antioxidants, proline, and ascorbic acid and phytohormones (Buthelezi et al. 2022, 2023). Also, SA is reported to produce osmolytes such as proline and antioxidants (Coşkun et al. 2023). In addition to crucial roles in defense system and as enzyme cofactors, these components, particularly antioxidants, directly or indirectly scavenge ROS and/or control ROS production and influence plant growth and development by modifying processes from mitosis and cell elongation to senescence and plant death (Altaf et al. 2022; Goncharuk and Zagoskina 2023).
Moreover, glutathione acts as an antioxidant in many ways. It can react chemically with O2•−, OH•, and H2O2, and, therefore, can function directly as a free radical scavenger (Madhu et al. 2023). This is further supported by the results of the current study, which shows that heat-stressed cancer bush plants treated with MSE or MSE + SA had higher glutathione concentrations and lower levels of O2•− and H2O2 compared with control. Abd El Mageed et al. (2023) reported that the enzyme glutathione reductase maintains the glutathione pool in the reduced state that in turn reduces dehydroascorbate to ascorbate, which is a primary antioxidant and direct scavenger of ROS (Hasanuzzaman et al. 2019). It was also reported that increased glutathione reductase expression enhances the tolerance to oxidative stress (Abd El Mageed et al. 2023; Madhu et al. 2023). Our results are similar to those of Khalofah et al. (2020), who reported that glutathione content was significantly higher in cadmium stressed garden cress (Lepidium sativum L.) plants treated with 6% MLE (159.42 U/mg protein) compared with 2% and 4% MLE (151.62 and 156.25 U/mg protein) and control (148.51 U/mg protein). This could be because the antioxidant glutathione plays an important role in the regulation of plant growth, development, and responses to abiotic stresses (Madhu et al. 2023).
Heat-stressed cancer bush plants treated with MSE and MSE + SA showed no symptoms of wilting, which could be attributed to the presence of phytohormones, particularly CKs in MSE (Buthelezi et al. 2023; Shakour et al. 2023). The presence of CKs in MSE (Buthelezi et al. 2022, 2023) prevents premature leaf senescence and maintains higher leaf area for photosynthetic activity and higher chlorophyll concentration in plant leaves (Iqbal et al. 2023; Zulfiqar et al. 2020). SA treatments enhance synthesis of CKs in plants, which promotes chlorophyll biosynthesis or prevents chlorophyll degradation in leaves (Kaya et al. 2023; Kobayashi et al. 2020). This is further supported by the results of this study, which showed that heat-stressed cancer bush plants treated with either MSE or MSE + SA had higher total chlorophyll contents compared with control (Fig. 1). This might have delayed wilting or leaf senescence, which is accompanied by various changes in cell structure, physiological metabolism, and gene expressions (Madhu et al. 2023). In addition, the phytohormones and bioactive compounds present in MSE might have played an important role in genetic modification, which can delay leaf aging, maintain photosynthesis for a long time, and sustain leaf activity, thereby increasing yield (Buthelezi et al. 2023; Zulfiqar et al. 2020).
