GB has been widely used to alleviate abiotic stress in plants and has shown good results 17,19. GB mitigates abiotic stresses mainly by protecting cell membranes 17, maintaining normal intracellular osmotic pressure 18, reducing photosynthetic system damage, and stabilizing related enzyme activities 21. We found that spraying exogenous GB on eggplant seedlings under normal conditions promoted plant growth, facilitated plant development, and improved photosynthesis in eggplants. In our study, the plant height, shoot fresh weight, and shoot dry weight of eggplant increased significantly after exogenous spraying with different GB concentrations, with 20 and 40 mmol L− 1 being the most effective. It has been shown that when GB is applied exogenously, it is absorbed and quickly moves from the leaves to meristematic tissues, such as buds and stem tips, thus promoting flowering and growth 35,36. Regarding plant appearance and morphology, 40 mmol L− 1 GB significantly increased the flowering time of plants (Figure.1A). However, 60 mmol L− 1 betaine had some inhibitory effect on plant growth (Figure.1A); plant height, stem thickness, root fresh weight, and root dry weight were slightly lower with 0 mmol L− 1 (control). This indicates that exogenous GB can promote growth at low concentrations under normal conditions 23,37.
Conversely, the plant height, stem diameter, shoot fresh weight, and root fresh weight of eggplant plants treated with different concentrations of exogenous cycloleucine decreased significantly with an increase in cyclic leucine concentration compared with 0 mmol L− 1. These results indicated that the exogenous spraying of cycloleucine significantly inhibited the growth and development of eggplant plants. In terms of the plant phenotype, 10 mmol L− 1 cycloleucine delayed the flowering time (Figure.S1), 20 mmol L− 1 and 40 mmol L− 1 cycloleucine directly caused stunted plant growth and development, and even led to the loss of apical dominance and an increase in the number of lateral shoots (Figure.S1, S2). These results are similar to those of Colombarin 30 on potatoes, on potatoes, indicating that plant growth and development were inhibited after cycloleucine application.
Epidermal hairs on plant leaves can effectively reduce leaf water gain 38, protect leaf flesh cells from damage 39, and reduce pathogenic fungal attacks 40. I In this experiment, the density of abaxial hairs on eggplant leaves increased significantly with increasing GB concentrations compared to the control treatment, and the morphology was sharper. However, treatment with cycloleucine spray resulted in a significant morphological change in eggplant leaf abaxial hair, which appeared flattened, and the number of individual hair-like branches decreased. In terms of magnification, 40 × magnification was required to observe leaf abaxial hairs of control plants and plants sprayed with GB, while 110 × magnification was required for cycloleucine-treated plants. This suggests that the morphology of abaxial hairs of leaves may be smaller after cycloleucine treatment.
Previous studies have shown that exogenous GB can significantly improve plant gas exchange properties 41, stabilize photosynthetic pigments and chlorophyll fluorescence, and promote plant growth 23. In this study, Pn and Gs increased and then decreased with increasing betaine concentration. It also validated the improved effect of GB on plant gas exchange parameters and indicated that the main factor of photosynthetic limitation could be the stomatal limitation 22,37. Plants treated with 20 and 40 mmol L− 1 NaCl showed a significant increase in Pn. In addition, according to Gs and Ci, the stomatal opening of leaves was significantly increased after betaine spraying, while Tr was the lowest in plants treated with GB at 20 mmol L− 1 concentration and even significantly lower than CK. Combined with Pn, Gs, and Ci, the main reason for this may be a decrease in the number of stomata on leaves, resulting in a decrease in Tr 42.
Cycloleucine acts as a methylation inhibitor and affects photosynthesis in plants by inhibiting S-adenosylmethionine synthase and, thus, RNA methylation 43. Gas exchange parameters were affected in plants sprayed with cycloleucine. Compared with the control plants, Pn, Gs, and Tr were reduced in cycloleucine-treated plants (Figure.3E, F, and H), whereas Ci levels were elevated. This suggests that the effect of cycloleucine on the gas exchange parameters may be achieved by affecting the uptake and utilization of CO2. This is because, as the concentration of cycloleucine increased, a rising and then decreasing trend was observed in Pn, and Gs, Ci, and Tr showed an increasing trend. This result suggests that cycloleucine may not affect the gas exchange capacity but rather the assimilation capacity of the photosynthetic system for CO2.
Key enzymes of the Calvin cycle (RuBisCO, GAPDH, FBA, FBPase, and TK) play an irreplaceable role in photosynthesis 44,45. RuBisCO and FBPase play important roles in carbon fixation: reduction and RuBP regeneration, respectively. FBA and TK promote CO2 assimilation in leaves and carbon flow in the Calvin cycle, and GAPDH is a key enzyme in glycolysis 45. The exogenous administration of GB has been shown to elevate the activity of key Calvin cycle enzymes, which is similar to our results 46,47. We showed that exogenous spraying of GB increased RuBisCO, GAPDH, FBA, FBPase, and TK activity, whereas plants sprayed with cycloleucine showed decreased RuBisCO, GAPDH, FBA, FBPase, and TK enzyme activity. This suggests that GB can enhance the key activities of the Calvin cycle under normal conditions. Meanwhile, plants sprayed with cycloleucine had decreased activity of key Calvin cycle enzymes, which may be due to the effect of cycloleucine on RNA methylation 30,48, affecting enzyme synthesis and transport, resulting in decreased enzyme activity. Compared to the control treatment, GB increased chlorophyll content (Table 2) 36,49. The leaves of cycloleucine-treated plants turned yellow, and the chlorophyll content decreased. This indicates that cycloleucine may increase the rate of chlorophyll degradation and decrease the synthesis rate 30,49.
Chlorophyll fluorescence provides insights into the cellular response to exogenous substances. It can quantify changes in the donor and acceptor sides of PSII reaction centers, thus providing information about energy uptake, utilization, and transport 50. Compared to the control, GB treatment significantly increased Fv/Fm, qN, and qP (Figure.5A, D, E) and significantly decreased Fv'/Fm' (Figure.5B). Exogenous GB affects Fv/Fm mainly by promoting electron transport from QA to QB in vesicle-like membranes 51,52. Our results showed that exogenous GB elevated plant Fv/Fm (Figure. 5A). However, Fv'/Fm' decreased, indicating that spraying 40 mmol L− 1 GB significantly promoted Fv/Fm compared to CK (p < 0.05), a result consistent with the findings in tomatoes 52.
Similarly, qN increased because of dynamic photoinhibition (Figure.5D). As qN increased, qP decreased (Figure.5D, E), indicating a reduction in the actual photochemical rate of PSII 53. Also, 1-qP decreased in plants treated with 20 and 40 mmol L− 1 GB, indicating that GB could reduce the excitation stress faced by PSII, improve the efficiency of excitation energy capture (Figure.5B), and inhibit the reduction in Y(II) (Figure.5C). In contrast, cycloleucine was responsible for a significant decrease in Fv/Fm, Fv'/Fm,' Y(II), qN, and 1-qP (Figure.5G-J, L), and a significant increase in qP (Figure.5K). This indicates that chronic and dynamic photoinhibition are affected by cycloleucine spraying 44,45. qN decreased while qP was enhanced, indicating that cycloleucine reduced heat dissipation and enhanced photosynthesis. Plants treated with 20 mmol L− 1 cycloleucine showed increased Pn (Figure.3E), which also verifies this result. The 1-qP value decreased significantly (Figure.5L), also indicating that cycloleucine affected the PS II excitation pressure.
Amino acids are the basic units for synthesizing proteins and other metabolites. They are commonly involved in physiological and biochemical processes in plants, such as the regulation of osmotic pressure 54, alteration of enzyme activity, regulation of stomatal opening, and ion transport 55. Glutamate can serve as a carbon pool for δ-aminolevulinic acid during chlorophyll synthesis 56, a and δ-aminolevulinic acid is formed when glutamate reacts with tRNA, indicating the importance of glutamate in chlorophyll biosynthesis 57. In this experiment, both betaine and cycloleucine applications significantly increased the glutamate content of the plants compared to that of the control (Table 3). GB spraying increased the chlorophyll content, verifying that exogenous GB elevates glutamate content 58.
In contrast, treatment with cycloleucine increased glutamate content but decreased chlorophyll content. This suggests that spraying with cycloleucine may have inhibited glutamate utilization by the plants, leading to its formation and accumulation. Serine plays an active role in plant metabolic activities such as phospholipid biosynthesis, photorespiration, and biosynthesis of other amino acids 59. The serine content of the plants sprayed with exogenous GB was significantly higher than that of the controls 60. Meanwhile, the differences between plants treated with 20, 40, and 60 mmol L− 1 GB were insignificant, indicating that the spraying of exogenous GB may have promoted serine synthesis. However, other reasons limited the increase in serine content.
Glycine is also a precursor of GB, which can be oxidized to form serine 59,61. The increase in serine content and the decrease in glycine content after spraying exogenous GB may be due to the fact that exogenous GB inhibited glycine synthesis and promoted the conversion of glycine to serine. The glycine content in cycloleucine treatment increased and then decreased, whereas serine content increased. This indicates that the metabolic synthesis pathways of both serine and glycine were influenced by cycloleucine. Valine, leucine, and isoleucine can act as alternative electron donors in the mitochondrial electron transport chain 62,63. After spraying exogenous GB, valine, leucine, and isoleucine levels increased with increasing GB concentration.
The corresponding photosynthetic and chlorophyll fluorescence parameters improved 42, indicating that GB promoted the synthesis and utilization of valine, leucine, and isoleucine 64. However, photosynthetic and chlorophyll fluorescence parameters decreased with cycloleucine treatment, but leucine and isoleucine content increased, while valine content decreased. This suggests that cycloleucine may have inhibited the utilization of leucine and isoleucine but suppressed valine synthesis. Aspartate is a key amino acid for plant growth, development, and adaptation to environmental challenges, most likely through its interaction with plant hormones, such as growth hormones and ethylene 65. The change in the aspartate content of both exogenous GB and cycloleucine-treated plants was inversely proportional to the IAA content (Figure.6B, E; Table 3), which also verified the above conclusion 66.
Aspartate, glutamate, alanine, and glycine support flowering, whereas threonine and phenylalanine inhibit it 55,67. Combined with the plant phenotypes (Figure.1A), aspartate, glycine, alanine, threonine, and phenylalanine contents were significantly increased after GB spraying compared to the control. However, flowering occurred earlier in plants treated with GB at 40 mmol L− 1. This indicates that, although GB increases amino acid content, it also inhibits the action of threonine and phenylalanine, causing their formation and accumulation. Aspartic acid, glycine, alanine, threonine, and phenylalanine in cycloleucine-treated plants increased with increasing cycloleucine concentrations. However, spraying with cycloleucine significantly affected the reproductive growth of the plants (Figure.1B, S1), indicating that as the plants took up cycloleucine, it directly affected the conversion and utilization of aspartic acid, glycine, alanine, threonine, and phenylalanine, causing their accumulation.
Methionine and threonine are precursors of isoleucine 68,69. This conclusion is also verified by the fact that the trends of methionine, threonine, and isoleucine are the same regardless of whether GB or cycloleucine is sprayed. As for tryptophan, a synthetic precursor of IAA 70, the trends of tryptophan and IAA contents in the plants were consistent after GB application. However, the IAA and tryptophan contents of plants sprayed with cycloleucine decreased. This indicates that cycloleucine affects tryptophan synthesis. Methionine can be used in cellular metabolism as a biomodulatory element for mRNA initiation, translation, protein composition, and S-adenosylmethionine (SAM) 69,71. The high concentration of betaine used in this experiment catalyzed the increase in methionine content 72.
In contrast, methionine content increased with increasing cycloleucine concentration, which worsened plant growth. This suggests that the effect of cycloleucine on methionine content may be consistent with the conversion and utilization of methionine in the metabolic pathway rather than the inhibition of methionine synthesis. Photorespiration is the main source of serine and glycine amino acids in photosynthetic tissues 73,74. The increased serine and glycine content in plants sprayed with cycloleucine indicates enhanced photorespiration and increased energy loss in their leaves 75,76.
Endogenous hormones play important roles in plant growth and development 77,78. They participate in various physiological and biochemical processes in plant cells, tissues, and organs 79, directly affecting plant growth and development 80). GB treatment changes the hormone metabolic pathways in plants and affects the hormone content 81. In this study, the leaf ABA content decreased (Figure.6A), but the IAA content increased (Figure.6B) after spraying with the exogenous GB. The ABA content in leaves affects the opening and closing of stomata, which in turn affects photosynthesis in the leaves 82. After a decrease in ABA content (Figure.6A), there was an increase in both the Pn and Gs (Figure.3A, B), similar to the findings of Chen, et al. 83.
IAA promotes cell growth and differentiation 84 and root development and plant growth 85,86. After spraying exogenous GB, the IAA content increased and directly promoted plant growth and development (Figure.1A), with the best effect observed for GB at 40 mmol L− 1. After spraying cycloleucine, the ABA content decreased (Figure.6D), but the IAA content increased and then decreased (Figure.6E). This indicated that the endogenous hormone level of the plant decreased, and normal plant growth and development were inhibited (Figure.1B) 87,88. Changes in the hormone levels also resulted in delayed floral bud differentiation and flowering 89,90.
In conclusion, the spraying of exogenous GB promoted the growth of eggplant seedlings, increased the fresh and dry weights of the plants, promoted the development of abaxial leaf hairs, promoted the differentiation of flower buds, shortened the flowering time, improved the photosynthetic and fluorescence parameters in eggplant plants, and increased the amino acid content, with the greatest effect at 40 mmol L− 1 concentration of GB. Spraying exogenous cyclic leucine at 20 mmol L− 1 significantly inhibited the growth and development of eggplant, leading to a decrease in the gas exchange parameters of plant leaves, inhibiting the development of abaxial leaf hairs, leading to a decrease in phytohormone content, and affecting the conversion and utilization of amino acids in the plant.