Integrating Transcriptomics and Metabolomics to Comprehensively Analyze Phytohormone Regulatory Mechanisms in 
Light is an important environmental signal on which plants depend for survival; however, it is also a source of abiotic stress for plants . UV-B radiation (UV-B) (280–315 nm) light is an intrinsic part of the sunlight that reaches the Earth’s surface. Plants serve as sequestering organisms, and therefore, exposure to UV-B throughout their life cycle is unavoidable. Although the atmospheric ozone layer absorbs most of the UV-B, about 5% of solar UV-B reaches the Earth’s surface . UV-B can be potentially harmful to plants. It not only impairs the photosynthetic system of plants but also reduces chlorophyll content, causes DNA damage and affects DNA replication and transcription, thereby inhibiting plant development and metabolism . Hormones have a major regulatory role in plant growth, development and adaptation to the environment. When plants defend themselves against abiotic stresses, multiple hormones cross-talk and co-regulate. Each hormone has its own unique function in the signal transduction process, and the mechanism of action for regulating plant stress tolerance is also different . Low-temperature stress induces the production of abscisic acid (ABA) to activate ABA-dependent signaling response pathways and increase ABA content in plants, thereby positively regulating plant tolerance to adversity stress . Abiotic stress can induce the expression of genes related to jasmonic acid (JA) synthesis in response to various unfavorable conditions . Salicylic acid is involved in regulating plant response to UV-B stress, and SA serves as a signal to reduce oxidative stress by triggering the up-regulation of antioxidants, thereby promoting growth and photosynthesis . It was found that cytokinin (CK) can cross-talk with ethylene signaling to co-regulate plant response to low-temperature signaling . It has been shown that cold stress causes GA-induced degradation of DELLA deterrent proteins in Arabidopsis thaliana , which controls many key developmental processes and responses to stresses such as cold . Therefore, the study of changes in hormone levels in plants as well as phytohormone signaling networks is essential for understanding plant responses to various types of environmental stresses. Rhododendron chrysanthum Pall. ( R. chrysanthum ) is a rhododendron growing in high-altitude and low-temperature mountainous areas, which is one of the rare germplasm resources in the world . After a long adaptive evolutionary process, R. chrysanthum has evolved resistance to abiotic stresses such as low temperature, drought and strong UV-B, and thus can be used as a good experimental material for exploring plants’ resistance to UV-B . Existing studies on R. chrysanthum have focused on the response of its metabolic pathways to UV-B . Hormonal studies on R. chrysanthum , on the other hand, have focused on the role played by exogenous ABA in regulating the metabolic pathways of R. chrysanthum . Meanwhile, the complete study of changes in various phytohormones, their biosynthesis, and signaling-related genes and metabolites in R. chrysanthum under UV-B stress is less studied. Not only are individual metabolic pathways regulated by environmental factors, but the homeostasis of the entire metabolic network is also affected . Compared to traditional physiological and biochemical studies and genetic phenotyping, histological research techniques allow for a more systematic observation of physiological changes in plants and may offer greater potential for the discovery of new genes . In recent years, the association analysis of various organizational platforms has emerged as a new trend in the field of plant metabolism research . In this experiment, seven important hormones were studied transcriptomically and metabolomically in R. chrysanthum . The present study is a comprehensive study of various types of phytohormones that have been detected in R. chrysanthum , from biosynthesis to signaling, based on previous studies . Changes in metabolite levels and pathway gene expression levels during the defense of R. chrysanthum against UV-B demonstrated the mechanisms by which different hormones regulate plant leaves against abiotic stresses. This is the first complete transcriptomic and metabolomic analysis of phytohormonal regulation during the defense of R. chrysanthum against UV-B radiation. The findings enrich existing insights into the mechanisms of phytohormone action and provide new perspectives in the field of phytohormone response to UV-B.   2.1. Differential Photosynthetic Performance upon UV-B Exposure By comparing the photosynthetic characteristics of UV-B radiation in R. chrysanthum , such as Fv/Fm and Fm, the effect of UV-B radiation on R. chrysanthum was observed. The fluorescence of chlorophyll was measured by IMAGING PAM. The results showed that Fv/Fm and Fm decreased significantly after UV-B radiation . In order to further understand the changes in the electron transport chain of the photosynthetic system without leaves under the same treatment, five JIP assay parameters were selected for reflecting the changes in the activity of the electron transport chain. As shown in , UV-B radiation significantly reduced the performance index of the leaves of R. chrysanthum (PIABS), the quantum yield of the light energy absorbed by the Photosystem II (PSII) reaction centers for electron transport (φEo), the potential activity of PSII (Fv/Fo) and the captured energy per reaction center of PSII (TRo/RC). UV-B radiation likewise led to a significant decrease in the chlorophyll content of R. chrysanthum ( A). Taken together, the changes in chlorophyll fluorescence parameters suggest that UV-B radiation can have severe adverse effects on R. chrysanthum . Therefore, R. chrysanthum will respond to this stress through changes in its own metabolic pathways. This experiment used LC-MS/MS (liquid chromatography–tandem mass spectrometry) to analyze the metabolism of CK and UV-B-irradiated R. chrysanthum leaves ( B). Many different metabolites were detected in the metabolic pathways of amino acids, lipids, and other macromolecules after UV-B radiation of R. chrysanthum . Therefore, we sequenced its leaves. The number of relevant differentially expressed genes in leaves after UV-B radiation was obtained based on sequencing results. The most differential genes (DEGs) were mainly concentrated in the carbohydrate metabolism pathway, with 116 up-regulated genes and 120 down-regulated genes ( C). 2.2. Abscisic Acid (ABA) Production and Signal Transduction in R. chrysanthum in Response to UV-B The role of the above key metabolic pathways in actively responding to UV-B radiation cannot be ignored, and at the same time, these metabolic pathways are also regulated by phytohormone signaling networks. Therefore, the present study focused on comprehensively analyzing the changes in the levels of various hormones, their biosynthesis, and DEGs in signaling pathways in R. chrysanthum under UV-B radiation . This study aimed to present a complete picture of the role of R. chrysanthum phytohormones in the defense of R. chrysanthum against UV-B radiation. The results indicate that during the stage of ABA synthesis, two genes exhibited differential expression. Among them, the expression level of ZEP in R. chrysanthum irradiated by UV-B was higher, while the expression level of CrtZ in control leaves was higher. In the ABA signaling pathway, the expression of PP2C and PYLs was down-regulated, while the expression of SnRK2 was up-regulated in R. chrysanthum after UV-B radiation . 2.3. Gibberellin (GA) Production and Signal Transduction in R. chrysanthum in Response to UV-B In GA biosynthesis, after UV-B radiation, the metabolites GA 53 , GA 19 , GA 20 , GA 24 , GA 1 , GA 3 and GA 4 were down-regulated, GA 15 and GA 9 were up-regulated, CYP701 expression was down-regulated and GA3ox expression was up-regulated. In the GA signal transduction pathway, the expression of GI1D was up-regulated and that of DELLA was down-regulated after UV-B radiation . 2.4. Jasmonic Acid Production and Signal Transduction in R. chrysanthum in Response to UV-B This study examined R. chrysanthum ’s jasmonic acid (JA) biosynthesis and signal transduction pathways to determine the metabolite content and expression patterns of DEGs . Under UV-B radiation, JA-ile, α-linolenic acid and JA contents were higher in the control group (CK group) than in the UV-B group, and OPDA contents were lower than in the UV-B group. Except for PLA , LOX2s , DAD1 , AOS , ACAA1 and MFP25 , biosynthesis genes were up-regulated in leaves after UV-B radiation, which catalyzed a series of reactions in the JA biosynthesis pathway. MYC2 and JAZ expression was down-regulated in response to UV-B radiation, and they are important components of the JA signaling pathway. 2.5. Auxin Production and Signal Transduction in R. chrysanthum in Response to UV-B Radiation The content of Indole-3-acetonitrile was higher and the content of Indole-3-acetate was lower in the leaves of R. chrysanthum under UV-B radiation. A total of seven differentially expressed genes of the auxin pathway were obtained, including TAA1, ALDH , TAA1 , CYP71A13 and amiE family genes. After UV-B radiation, the expressions of DDC and TAA1 were down-regulated, while the expressions of CYP71A13 , ALDH and amiE were up-regulated. Through differential gene screening, two differentially expressed genes were found in the auxin signaling pathway, namely AUX/IAA and ARF family genes. Analysis of gene expression profiles of each family showed that AUX/IAA and ARF gene families were down-regulated in the leaf midlobes of the UV-B group . 2.6. Salicylic Acid (SA) Production and Signal Transduction in R. chrysanthum in Response to UV-B Radiation We studied the content of metabolites, the expression of related genes and the SA signal transduction pathway in the process of R. chrysanthum SA synthesis. The phenylalanine pathway is the earliest discovered pathway for the synthesis of SA. The results show that UV-B radiation increased the contents of phenylalanine and SA in R. chrysanthum leaves, and the expression levels of TAT and HPD genes in R. chrysanthum leaves were higher than those in the CK group. The signal transduction pathway of SA is mainly NPR1-dependent, and the expression level of NPR1 gene was higher in leaves of the UV-B group . 2.7. Cytokinin (CK) Production and Signal Transduction in R. chrysanthum in Response to UV-B Radiation Through the analysis of the level of CK in the leaves of R. chrysanthum before and after UV-B radiation, it was found that the content of other metabolites increased significantly after radiation. In this study, we collected all aspects of genes related to various aspects of CK homeostasis. The results show that the level of CRE1/AHK4 increased in the CK synthesis pathway due to UV-B radiation in plant leaves, while Type-BARR expression decreased in the CK signal transduction pathway . 2.8. Ethylene Production and Signal Transduction in R. chrysanthum in Response to UV-B Radiation It can be seen from that the levels of carboxylic acid and ethylene metabolites in R. chrysanthum leaves decreased under UV-B radiation, and the expression levels of JA mtnK and TAT genes increased in the ethylene biosynthesis pathway, while AMD1 expression decreased. The expression level of CTR1 decreased in the ethylene signal transduction pathway. 2.9. Correlation Analysis of Various Phytohormones of R. chrysanthum with Photosynthetic Indicators In order to explore the relationship between various plant hormones under UV-B radiation and the key genes closely associated with them, their regulatory effects on the R. chrysanthum photosynthetic system were analyzed. The correlation between DEGs in various plant hormones, their pathways and the above photosynthetic physiological indexes was analyzed. The result show that the key factors involved in CK regulation of UV-B radiation in R. chrysanthum may include BARR and CRE genes. The ALDH , CYP71A13 , AUX/IAA and amiE genes in IAA may be related to protection against UV-B radiation. Under UV-B radiation, ethylene and SA have two kinds of DEGs with strong correlation, namely mtnK and CTR1 , and HPD and TAT-1 . GA, ABA and JA all involve a DEG in close association, CYP701-2 , SNRK2 and PAL , respectively. This experiment also found that phytohormones have an effect on plant photosynthesis, and that plants reduce photo-oxidative damage under unfavorable conditions through multiple layers of positive and negative regulation. For example, Ck, SA and JAs are promoters of photomorphogenesis, whereas ethylene, ABA, growth factors and GAs are negative regulators .   By comparing the photosynthetic characteristics of UV-B radiation in R. chrysanthum , such as Fv/Fm and Fm, the effect of UV-B radiation on R. chrysanthum was observed. The fluorescence of chlorophyll was measured by IMAGING PAM. The results showed that Fv/Fm and Fm decreased significantly after UV-B radiation . In order to further understand the changes in the electron transport chain of the photosynthetic system without leaves under the same treatment, five JIP assay parameters were selected for reflecting the changes in the activity of the electron transport chain. As shown in , UV-B radiation significantly reduced the performance index of the leaves of R. chrysanthum (PIABS), the quantum yield of the light energy absorbed by the Photosystem II (PSII) reaction centers for electron transport (φEo), the potential activity of PSII (Fv/Fo) and the captured energy per reaction center of PSII (TRo/RC). UV-B radiation likewise led to a significant decrease in the chlorophyll content of R. chrysanthum ( A). Taken together, the changes in chlorophyll fluorescence parameters suggest that UV-B radiation can have severe adverse effects on R. chrysanthum . Therefore, R. chrysanthum will respond to this stress through changes in its own metabolic pathways. This experiment used LC-MS/MS (liquid chromatography–tandem mass spectrometry) to analyze the metabolism of CK and UV-B-irradiated R. chrysanthum leaves ( B). Many different metabolites were detected in the metabolic pathways of amino acids, lipids, and other macromolecules after UV-B radiation of R. chrysanthum . Therefore, we sequenced its leaves. The number of relevant differentially expressed genes in leaves after UV-B radiation was obtained based on sequencing results. The most differential genes (DEGs) were mainly concentrated in the carbohydrate metabolism pathway, with 116 up-regulated genes and 120 down-regulated genes ( C).   The role of the above key metabolic pathways in actively responding to UV-B radiation cannot be ignored, and at the same time, these metabolic pathways are also regulated by phytohormone signaling networks. Therefore, the present study focused on comprehensively analyzing the changes in the levels of various hormones, their biosynthesis, and DEGs in signaling pathways in R. chrysanthum under UV-B radiation . This study aimed to present a complete picture of the role of R. chrysanthum phytohormones in the defense of R. chrysanthum against UV-B radiation. The results indicate that during the stage of ABA synthesis, two genes exhibited differential expression. Among them, the expression level of ZEP in R. chrysanthum irradiated by UV-B was higher, while the expression level of CrtZ in control leaves was higher. In the ABA signaling pathway, the expression of PP2C and PYLs was down-regulated, while the expression of SnRK2 was up-regulated in R. chrysanthum after UV-B radiation .   In GA biosynthesis, after UV-B radiation, the metabolites GA 53 , GA 19 , GA 20 , GA 24 , GA 1 , GA 3 and GA 4 were down-regulated, GA 15 and GA 9 were up-regulated, CYP701 expression was down-regulated and GA3ox expression was up-regulated. In the GA signal transduction pathway, the expression of GI1D was up-regulated and that of DELLA was down-regulated after UV-B radiation .   This study examined R. chrysanthum ’s jasmonic acid (JA) biosynthesis and signal transduction pathways to determine the metabolite content and expression patterns of DEGs . Under UV-B radiation, JA-ile, α-linolenic acid and JA contents were higher in the control group (CK group) than in the UV-B group, and OPDA contents were lower than in the UV-B group. Except for PLA , LOX2s , DAD1 , AOS , ACAA1 and MFP25 , biosynthesis genes were up-regulated in leaves after UV-B radiation, which catalyzed a series of reactions in the JA biosynthesis pathway. MYC2 and JAZ expression was down-regulated in response to UV-B radiation, and they are important components of the JA signaling pathway.   The content of Indole-3-acetonitrile was higher and the content of Indole-3-acetate was lower in the leaves of R. chrysanthum under UV-B radiation. A total of seven differentially expressed genes of the auxin pathway were obtained, including TAA1, ALDH , TAA1 , CYP71A13 and amiE family genes. After UV-B radiation, the expressions of DDC and TAA1 were down-regulated, while the expressions of CYP71A13 , ALDH and amiE were up-regulated. Through differential gene screening, two differentially expressed genes were found in the auxin signaling pathway, namely AUX/IAA and ARF family genes. Analysis of gene expression profiles of each family showed that AUX/IAA and ARF gene families were down-regulated in the leaf midlobes of the UV-B group .   We studied the content of metabolites, the expression of related genes and the SA signal transduction pathway in the process of R. chrysanthum SA synthesis. The phenylalanine pathway is the earliest discovered pathway for the synthesis of SA. The results show that UV-B radiation increased the contents of phenylalanine and SA in R. chrysanthum leaves, and the expression levels of TAT and HPD genes in R. chrysanthum leaves were higher than those in the CK group. The signal transduction pathway of SA is mainly NPR1-dependent, and the expression level of NPR1 gene was higher in leaves of the UV-B group .   Through the analysis of the level of CK in the leaves of R. chrysanthum before and after UV-B radiation, it was found that the content of other metabolites increased significantly after radiation. In this study, we collected all aspects of genes related to various aspects of CK homeostasis. The results show that the level of CRE1/AHK4 increased in the CK synthesis pathway due to UV-B radiation in plant leaves, while Type-BARR expression decreased in the CK signal transduction pathway .   It can be seen from that the levels of carboxylic acid and ethylene metabolites in R. chrysanthum leaves decreased under UV-B radiation, and the expression levels of JA mtnK and TAT genes increased in the ethylene biosynthesis pathway, while AMD1 expression decreased. The expression level of CTR1 decreased in the ethylene signal transduction pathway.   In order to explore the relationship between various plant hormones under UV-B radiation and the key genes closely associated with them, their regulatory effects on the R. chrysanthum photosynthetic system were analyzed. The correlation between DEGs in various plant hormones, their pathways and the above photosynthetic physiological indexes was analyzed. The result show that the key factors involved in CK regulation of UV-B radiation in R. chrysanthum may include BARR and CRE genes. The ALDH , CYP71A13 , AUX/IAA and amiE genes in IAA may be related to protection against UV-B radiation. Under UV-B radiation, ethylene and SA have two kinds of DEGs with strong correlation, namely mtnK and CTR1 , and HPD and TAT-1 . GA, ABA and JA all involve a DEG in close association, CYP701-2 , SNRK2 and PAL , respectively. This experiment also found that phytohormones have an effect on plant photosynthesis, and that plants reduce photo-oxidative damage under unfavorable conditions through multiple layers of positive and negative regulation. For example, Ck, SA and JAs are promoters of photomorphogenesis, whereas ethylene, ABA, growth factors and GAs are negative regulators .   Light energy induces a series of adversity responses in plants, whereby interactions between phytohormones and ROS can contribute to plant adversity adaptation . In plant leaves, chloroplasts can serve as the first line of defense to protect PSII reaction centers from light damage. For this reason, in terms of chlorophyll fluorescence parameters, we found that the fluorescence intensity of R. chrysanthum decreased successively after UV-B radiation, indicating that UV-B radiation reduced the reduction ability of the fast reducing PQ library and the slow reducing PQ library, and that the receptor side of the PSII reaction center was damaged. UV-B radiation significantly reduced the maximum photochemical efficiency (Fv/Fm) and potential activity (Fv/Fo) of PSII, whereas the decrease in Tro/Rc reflected an increase in the inactivation of the PSII reaction center . φEo represents the quantum yield of light energy absorbed by the reaction center for electron transfer and is able to reflect the conversion efficiency of light energy . UV-B radiation reduced photosynthetic pigments in R. chrysanthum , resulting in a decrease in the captured light energy of the photosynthetic system and the absorbed light energy of the photosynthetic reaction center while damaging the photosynthetic system and reducing the effective photosynthetic efficiency. This may be due to the effect of UV-B radiation on the membrane structure, resulting in fewer antenna complexes distributed in the membrane, and thus lower energy conversion and absorption efficiency . This down-regulation of the PSII reaction center may be a coping mechanism adopted by R. chrysanthum to maintain PSII energy conversion efficiency when they resist UV-B radiation. Ethylene is the only gas in plant hormones, and it plays an important role in plant growth, development and response to stress. Ethylene synthesis consists of two main enzymatic reactions: (1) 1-aminocyclopropane-1-carboxylicacid (ACC) and methionine react together; (2) ACC is converted to ethylene. These two steps are regulated by ACCsynthase (ACS) and ACCoxidase (ACCoxidase), respectively . From the perspective of ethylene signaling to the transcriptional regulation of ethylene downstream response factors, a complex ethylene signaling pathway model has been formed. In the correlation analysis, the ethylene content in R. chrysanthum was negatively correlated with mtnk under UV-B radiation, which may reduce the accumulation of ethylene by increasing gene expression. The CTR1 gene is positively correlated with the ethylene level in R. chrysanthum , and when the ethylene signal is present, ethylene is able to bind to the ethylene receptor (ETR) on the endoplasmic reticulum (ER) membrane and is then transduced by CTR1 and EIN2 . This signal is further amplified in an EIN3-mediated transcriptional activation cascade that activates the expression of ethylene response genes . In the absence of the ethylene signal, the ethylene receptor may deactivate the kinase activity of CTR1 in another way, phosphorylating the C-terminal domain of EIN2 , thereby preventing its participation in ethylene signal transduction . In conclusion, the accumulation and stabilization of the EIN3 protein is conducive to the activation of the ethylene signaling pathway, thus enhancing the role of ethylene in plants. The increase in CTR1 and EBF1/2 can directly or indirectly inhibit the signal transduction of ethylene and block the transmission of the ethylene signal, which plays an important negative regulatory role. These results suggest that CTRs and mtnK may be involved in the effect of ethylene on UV-B resistance in R. chrysanthum leaves. ABA is an important plant hormone which has many physiological functions, such as inhibiting growth, promoting shedding, promoting dormancy, causing stomatal closure, regulating seed embryo development, promoting fruit ripening and increasing stress resistance . Nevertheless, there was no significant change in ABA content after UV-B radiation, which indicates that the leaves of R. chrysanthum had certain resistance to UV-B radiation. A number of signaling intermediates associated with ABA responses have been identified by previous studies, and they are tightly controlled by intracellular signal transduction pathways . Progress is currently being made in exploring ABA signaling contributing to the construction of the PYL-PP2C-SnRK2 signaling model. The co-regulatory network of ABA metabolic pathways in R. chrysanthum showed that SnRK2 was negatively correlated with ABA expression levels . According to previous studies, PYR/PYLs are ABA receptors located at the top of the negative regulatory pathway which inhibit PP2Cs from controlling ABA signaling . Therefore, we speculate that PYL2 may interact with PP2C to inhibit phosphatase activity, allow for SnRK2 activation and target protein phosphorylation, and thus make the leaves have certain resistance to UV-B radiation. Cytokinin (CK) plays critical regulatory roles in phloem differentiation, chloroplast differentiation, microtubule differentiation, leaf senescence, apical dominance regulation and response to adversity . The decrease in CK levels caused by UV-B radiation may be related to the degree of photosynthesis in plant leaves when they are exposed to a larger photosynthetic capacity per unit leaf area. Studies on Arabidopsis thaliana and tobacco showed that the maximum photosynthetic rate, transpiration rate, stomatal conductance and mass per unit leaf area were reduced when the plants were exposed to light treatment, along with CK . The results of this experiment show that the CK content in the leaves of R. chrysanthum was positively correlated with ARR-B, indicating that UV-B radiation can cause some effects on R. chrysanthum . b-type ARRs are a class of transcription factors that activate the transcription of the A-type ARR gene. Previous studies have found that the expression of the a-type ARR gene leads to earlier flowering time, longer root systems, more lateral roots, earlier senescence and reduced CK sensitivity in transgenic plants, and plays a major role among the many transcription factors involved in CK signaling . ARR-B may regulate leaf resistance to radiation through the CK signaling pathway in R. chrysanthum . Auxin is a general term for indole-3-acetic acid (IAA) and its similarly acting analogs that occur naturally in plants . As a signaling compound that promotes and influences plant development and physiological changes, it affects almost the whole process of plant growth and development . UV-B stress changes auxin distribution in plants by affecting the auxin synthesis and transport process and influences downstream genes to make corresponding changes through the auxin signal transduction process. The analysis shows that growth hormone content was negatively regulated by ALDH , CYP71A13 and amiE and positively regulated by AUX/IAA . This indicates that UV-B radiation in the growth hormone biosynthesis pathway reduces growth hormone accumulation by increasing gene expression, and that Aux/IAA proteins are transcriptional repressors that mainly inhibit the activity of ARF transcription factors . In the signaling pathway, when growth hormone is sensed in the nucleus, it binds to the receptor TIR1/AFB , which promotes degradation of the Aux/IAA transcriptional repressor protein, releasing the ARF transcription factor and activating downstream gene expression . Salicylic acid (SA), a naturally occurring small-molecule phenolic in the plant kingdom, plays an important role in plant responses to biotic and abiotic stresses as a signaling molecule for plant disease resistance responses. In most plants, SA is synthesized mainly by the phenylalanine pathway. Phenylalanine is catalyzed by the enzyme phenylalanine dehydrogenase to produce cinnamic acid, which is then converted to SA by benzoic acid . The precursor to many polyphenols, such as flavonoids, is cinnamic acid . Studies have shown that UV-B stress can lead to 7–10 times or more up-regulation of endogenous SA content in plants . The results of this experiment show that SA content increased in the leaves of R. chrysanthum under UV-B radiation. Correlation analysis shows that TAT1 and HPD contents were positively correlated with SA. Therefore, we hypothesized that the higher SA accumulation in the leaves of R. chrysanthum under UV-B radiation might be related to the higher expression of the TAT1 and HPD genes. Certainly, this conjecture needs to be verified by further experiments. Jasmonic acids (JAs) are an important class of lipid-derived phytohormones widely distributed in higher plants and regulate a variety of physiological processes . JAs can act as endogenous signaling molecules to enhance plant resistance to different adversity stresses by regulating gene expression and subsequently accumulating secondary metabolites . JA content in the leaves of R. chrysanthum was reduced under UV-B radiation, and correlation analysis shows that PLA content was positively correlated with SA. PLA is a key enzyme in its metabolic synthesis pathway and is involved in JA biosynthesis. It has been shown that PLA2 with a molecular mass of 48,000 u was purified from the leaf membrane fractions of faba bean, and it was suggested that it has an important role in the production of linolenic acid . Gibberellins (GAs) are diterpenoid phytohormones that play a crucial role in the whole life cycle of plants . In recent years, numerous studies have shown that GAs significantly promote plant seed germination and seedling growth under adverse environmental conditions, as GAs can alleviate the inhibitory effects of stress on plants; currently there are up to a hundred GAs isolated from various types of organisms named in the order of their discovery . According to our results, the total content of GA compounds was detected to be lower in UV-B-irradiated R. chrysanthum leaves versus normal leaves, and correlation analysis shows that the content of the CYP701 gene in the GA synthesis pathway was negatively correlated with GA. The CYP701 gene is a related enzyme in the GA biosynthesis pathway. Therefore, the effect of GA on the resistance of R. chrysanthum leaves to UV-B radiation needs to be further investigated. In order to survive, plants must adapt to a wide range of abiotic and biotic stresses. Unfriendly environmental conditions, for example, increased light, a lack of water or low oxygen levels due to abiotic factors such as waterlogging, temperature extremes, salinity and pollutants, may decrease photosynthesis, leading to excess excitation energy in chloroplasts . Flexible interactions, complementarities and interactions between phytohormones that regulate photosynthesis are required during plant development and growth to optimize plant dynamics and adaptability in a continuously evolving environment. Chloroplast development is dependent on complex interactions between phytohormones, whereby the plant ensures the minimization of any possible photo-oxidative damage in chloroplasts during light-induced de-yellowing. For example, CKs, JAs and DELLA proteins directly or indirectly promote photomorphogenesis . In contrast, ethylene, growth hormone and GAs adversely regulate photomorphogenesis either by pif or by inhibiting B-GATAs . Plant growth and development are regulated by complementary phytohormones, including photosynthesis between different plant tissues and cells. CK positively regulates photosynthesis by expressing photosynthesis-related genes . In contrast, ABA negatively regulates photosynthesis by inhibiting stomatal formation. In addition, interactions between CK and growth hormone regulate plant photosynthesis, whereas cross-talk between growth hormone and ethylene inhibits ethylene biosynthesis and prolongs photosynthesis . Plant tolerance to environmental stresses and the manner in which these photoprotective mechanisms are activated vary from species to species, but typically a range of responses occur in plants, some of which are regulated by phytohormones. In fact, studies to more fully understand the effects of hormones on photosynthesis and photoprotection under abiotic stresses are not only important for a better comprehension of the underlying biological processes but may also contribute to the development of new crops with a more robust photosynthetic system. This study of phytohormonal regulation in R. chrysanthum under UV-B radiation provides valuable insights for enhancing plant stress tolerance. These insights can be applied to develop genetically engineered crops with improved UV-B tolerance and higher yields. Additionally, this study highlights the potential for metabolic engineering to boost the production of secondary metabolites with pharmaceutical value. The identified gene expression patterns can also serve as molecular tools for monitoring UV-B stress in plants. Based on these findings, we hypothesized that engineering modifications to these molecular pathways, such as enhancing CK synthesis or SA signaling, may help plants to better adapt and achieve growth advantages under UV-B radiation. This could not only support plant survival in the natural environment but may also provide a theoretical basis for the development of crop varieties tolerant to adversity stress in agricultural production. Overall, the results lay a foundation for developing climate-resilient plants and sustainable agricultural practices.   4.1. Plant Materials and Treatment R. chrysanthum was preserved in an artificial climate room at 18 °C (14 h light)/16 °C (10 h dark) under white fluorescent light at 50 umol (photon) m −2 s −1 . The tissue culture seedlings of R. chrysanthum , which had the same growth state for 8 months, were selected as the research material. The experimental materials were divided into experimental (UV-B group) and control groups (CK group). The CK and UV-B groups were transplanted into 1/4 MS medium. The test group was irradiated under UV-B treatment for 2 days, 8 h per day. The control group was irradiated under PAR (photosynthetically active radiation) treatment for two days, eight hours per day . To eliminate inter-individual differences, three biological replicates were performed in this study. The PAR irradiation treatment involved the placement of a 400 nm photofilm (Edmund, Filter Long 2IN SQ, Barrington, NJ, USA). The UV-B radiation treatment involved the measurement of filters (Edmund, Filter Long 2IN SQ, Barrington, NJ, USA). The artificial UV-B radiation source was a UV-B TL 20W/01RS fluorescent tube (Philips, UltravioletB TL 20 W/01 RS, Amsterdam, The Netherlands). The effective received irradiance of the samples was UV-B: 2.3 W/m2, PAR: 50 umol/ (m 2 ·s). 4.2. Identification and Quantification of Metabolites We strictly followed the experimental steps of a previous study . Using vacuum freeze-drying technology, we placed the biological samples in a lyophilizer (Scientz-100F, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China), then ground (30 Hz, 1.5 min) the samples to a powder form by using a grinder (MM400, Retsch, Haan, Germany). Next, we weighed 50 mg of sample powder using an electronic balance (MS105DΜ) and added 1200 μL of −20 °C pre-cooled 70% methanolic aqueous internal standard extract (less than 50 mg added at the rate of 1200 μL extractant per 50 mg sample) . Vortexing was performed every 30 min for 30 s for a total of 6 times. After centrifugation (12,000 rpm, 3 min), the supernatant was withdrawn, and the sample was filtered with a microporous membrane with a pore size of 0.22 μm and stored in an injection vial for LC-MS/MS (liquid chromatography–tandem mass spectrometry) analysis. Analysis of sample extracts was performed using an LC-MS/MS system and a tandem mass spectrometer. Metabolite profiling data were obtained from different samples, and the chromatographic peaks of all substances were integrated and corrected. Metabolites with FC (fold change) ≥ 2 and FC ≤ 0.5 were selected as differential metabolites. 4.3. cDNA Library Construction and Transcriptomics Data Analysis Transcriptomics experiments were conducted strictly according to the previous experimental instructions . The total RNA was extracted from the samples using the CTAB method. The total RNA samples were treated by using an rRNA removal method, and the rRNA was hybridized with a DNA probe. The hybrid DNA/RNA chain was digested by RNaseH, the DNA probe was digested by DNaseI and the RNA was purified. The resulting RNA was fragmented by using a disruption buffer, reverse-transcribed with randomized N6 primers, and then synthesized into cDNA duplexes, forming synthetic double-stranded DNA that was sprawlingly phosphorylated at the 5′ end, formed a sticky end with a prominent “a” at the 3′ end, and attached to a bulge-like junction with a prominent “T” at the 3′ end. The ligated product was amplified by PCR with specific primers; the PCR product was heat-denatured to a single strand, and then the single-stranded DNA was cyclized with a bridge primer to obtain a single-stranded circular DNA library, which was finally sequenced on the machine . The transcriptome sequencing in this study was performed using the MGISEQ-2000 platform of BGI Genomics Co., Ltd. (Shenzhen, China) and a total of 58 Gb was sequenced, and 93,034 unigenes were obtained after assembly and de-redundancy. To ensure the reliability of the results, we relied on the filtering software SOAPnuke (v1.4.0)  and used Bowtie2 (v2.2.5)  to compare the clean reads to the reference gene sequences to obtain the comparison results. The gene and transcript expression levels were estimated using RSEM (v 1.2.8) . To identify differentially expressed genes following experimental treatments, the expression levels of each transcript were measured using the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method. Subsequently, the DESeq2 method, based on the negative binomial distribution, was employed to analyze differentially expressed genes in response to the experimental treatments. Genes were identified as differentially expressed when they exhibited an FC (fold change) greater than 1 and q-value (adjusted p -value) < 0.05. Functional annotation and classification of Unigenes were conducted using publicly available database resources, including KEGG, Pfam and SwissProt. The annotation process involved sequence alignment of Unigene datasets against the reference sequences in these databases using BLAST-based comparison algorithms. 4.4. Chlorophyll Fluorescence Measurements The CK and UV-B groups were dark-treated for 20 min before measurement, and finally, chlorophyll fluorescence parameters were obtained using IMAGING PAM m-series (Walz, Effeltrich, Germany) . 4.5. Rapid Fluorescence Detection Our method was in compliance with previous experimental descriptions . In this experiment, the operation steps of the Handy-PEA instrument were as follows: We clamped the leaf clip on the front of the leaf and allowed for dark adaptation for 20 min. In an attempt to minimize the effect of leaf heterogeneity on the detection results, eight detection points were selected for each leaf: four detection points were selected for each leaf along both sides of the main leaf veins, and four detection points were equally spaced from the leaf tip to the leaf base. After dark adaptation, we connected the instrument probe to the blade clamp on the blade and opened the slide switch to expose the measuring hole to the laser light source. According to the pre-set LED light source of 3000 μmol m −2 s −1 , the detection time was 1s for fast fluorescence signal acquisition. Finally, the fluorescence signals of 8 detection points on each leaf were averaged and used as the final fast fluorescence data of the sample. 4.6. Determination of Chlorophyll Content A total of 1.0 g of leaves of R. chrysanthum from the CK and UV-B groups was weighed, ground repeatedly in 80% acetone solution and filtered, and the filtrate was collected. The absorbance of the extracts at 663 nm and 645 nm was determined by using a colorimetric method. This process was repeated three times. The mass concentrations of chlorophyll a and chlorophyll b in the extracts were calculated according to the formula below, and then the chlorophyll content in the leaves was calculated and expressed as the mass of chlorophyll contained in each gram of fresh weight of leaf tissue (mg/g). The formula was calculated as follows, where ρ is the mass concentration of chlorophyll mg/L, calculated by the formula below; V is the total volume of the sample extract in mL; and m is the sample mass. ρ a = 12.72 A 663 − 2.59 A 645 ρ b = 22.88 A 645 − 4.67 A 663 ρ c h l o r o p h y l l c o n t e n t = ρ a + ρ b c h l o r o p h y l l c o n t e n t = ρ ∗ V / m ∗ 1000 m g / g 4.7. Statistical Analysis The trials were executed on three occasions, employing a fully randomized design, and the data were analyzed utilizing IBM SPSS Statistics version 26. A one-way ANOVA was utilized to evaluate the statistical significance of the findings. Upon identifying significant disparities, Duncan’s multiple range test was implemented to pinpoint the specific mean differences at a significance threshold of p < 0.05. Genomic expression data were processed through the interactive data mining system named Dr.Tom . The relationships between plant hormones and differentially expressed genes (DEGs) derived from RNA sequencing were examined using Pearson’s correlation coefficient, with a stringent correlation threshold set at 0.9 and a significance level of p < 0.05. The dataset obtained from the above experiments was previously used to draw conclusions on other issues regarding the response of R. chrysathum to UV-B .   R. chrysanthum was preserved in an artificial climate room at 18 °C (14 h light)/16 °C (10 h dark) under white fluorescent light at 50 umol (photon) m −2 s −1 . The tissue culture seedlings of R. chrysanthum , which had the same growth state for 8 months, were selected as the research material. The experimental materials were divided into experimental (UV-B group) and control groups (CK group). The CK and UV-B groups were transplanted into 1/4 MS medium. The test group was irradiated under UV-B treatment for 2 days, 8 h per day. The control group was irradiated under PAR (photosynthetically active radiation) treatment for two days, eight hours per day . To eliminate inter-individual differences, three biological replicates were performed in this study. The PAR irradiation treatment involved the placement of a 400 nm photofilm (Edmund, Filter Long 2IN SQ, Barrington, NJ, USA). The UV-B radiation treatment involved the measurement of filters (Edmund, Filter Long 2IN SQ, Barrington, NJ, USA). The artificial UV-B radiation source was a UV-B TL 20W/01RS fluorescent tube (Philips, UltravioletB TL 20 W/01 RS, Amsterdam, The Netherlands). The effective received irradiance of the samples was UV-B: 2.3 W/m2, PAR: 50 umol/ (m 2 ·s).   We strictly followed the experimental steps of a previous study . Using vacuum freeze-drying technology, we placed the biological samples in a lyophilizer (Scientz-100F, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China), then ground (30 Hz, 1.5 min) the samples to a powder form by using a grinder (MM400, Retsch, Haan, Germany). Next, we weighed 50 mg of sample powder using an electronic balance (MS105DΜ) and added 1200 μL of −20 °C pre-cooled 70% methanolic aqueous internal standard extract (less than 50 mg added at the rate of 1200 μL extractant per 50 mg sample) . Vortexing was performed every 30 min for 30 s for a total of 6 times. After centrifugation (12,000 rpm, 3 min), the supernatant was withdrawn, and the sample was filtered with a microporous membrane with a pore size of 0.22 μm and stored in an injection vial for LC-MS/MS (liquid chromatography–tandem mass spectrometry) analysis. Analysis of sample extracts was performed using an LC-MS/MS system and a tandem mass spectrometer. Metabolite profiling data were obtained from different samples, and the chromatographic peaks of all substances were integrated and corrected. Metabolites with FC (fold change) ≥ 2 and FC ≤ 0.5 were selected as differential metabolites.   Transcriptomics experiments were conducted strictly according to the previous experimental instructions . The total RNA was extracted from the samples using the CTAB method. The total RNA samples were treated by using an rRNA removal method, and the rRNA was hybridized with a DNA probe. The hybrid DNA/RNA chain was digested by RNaseH, the DNA probe was digested by DNaseI and the RNA was purified. The resulting RNA was fragmented by using a disruption buffer, reverse-transcribed with randomized N6 primers, and then synthesized into cDNA duplexes, forming synthetic double-stranded DNA that was sprawlingly phosphorylated at the 5′ end, formed a sticky end with a prominent “a” at the 3′ end, and attached to a bulge-like junction with a prominent “T” at the 3′ end. The ligated product was amplified by PCR with specific primers; the PCR product was heat-denatured to a single strand, and then the single-stranded DNA was cyclized with a bridge primer to obtain a single-stranded circular DNA library, which was finally sequenced on the machine . The transcriptome sequencing in this study was performed using the MGISEQ-2000 platform of BGI Genomics Co., Ltd. (Shenzhen, China) and a total of 58 Gb was sequenced, and 93,034 unigenes were obtained after assembly and de-redundancy. To ensure the reliability of the results, we relied on the filtering software SOAPnuke (v1.4.0)  and used Bowtie2 (v2.2.5)  to compare the clean reads to the reference gene sequences to obtain the comparison results. The gene and transcript expression levels were estimated using RSEM (v 1.2.8) . To identify differentially expressed genes following experimental treatments, the expression levels of each transcript were measured using the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method. Subsequently, the DESeq2 method, based on the negative binomial distribution, was employed to analyze differentially expressed genes in response to the experimental treatments. Genes were identified as differentially expressed when they exhibited an FC (fold change) greater than 1 and q-value (adjusted p -value) < 0.05. Functional annotation and classification of Unigenes were conducted using publicly available database resources, including KEGG, Pfam and SwissProt. The annotation process involved sequence alignment of Unigene datasets against the reference sequences in these databases using BLAST-based comparison algorithms.   The CK and UV-B groups were dark-treated for 20 min before measurement, and finally, chlorophyll fluorescence parameters were obtained using IMAGING PAM m-series (Walz, Effeltrich, Germany) .   Our method was in compliance with previous experimental descriptions . In this experiment, the operation steps of the Handy-PEA instrument were as follows: We clamped the leaf clip on the front of the leaf and allowed for dark adaptation for 20 min. In an attempt to minimize the effect of leaf heterogeneity on the detection results, eight detection points were selected for each leaf: four detection points were selected for each leaf along both sides of the main leaf veins, and four detection points were equally spaced from the leaf tip to the leaf base. After dark adaptation, we connected the instrument probe to the blade clamp on the blade and opened the slide switch to expose the measuring hole to the laser light source. According to the pre-set LED light source of 3000 μmol m −2 s −1 , the detection time was 1s for fast fluorescence signal acquisition. Finally, the fluorescence signals of 8 detection points on each leaf were averaged and used as the final fast fluorescence data of the sample.   A total of 1.0 g of leaves of R. chrysanthum from the CK and UV-B groups was weighed, ground repeatedly in 80% acetone solution and filtered, and the filtrate was collected. The absorbance of the extracts at 663 nm and 645 nm was determined by using a colorimetric method. This process was repeated three times. The mass concentrations of chlorophyll a and chlorophyll b in the extracts were calculated according to the formula below, and then the chlorophyll content in the leaves was calculated and expressed as the mass of chlorophyll contained in each gram of fresh weight of leaf tissue (mg/g). The formula was calculated as follows, where ρ is the mass concentration of chlorophyll mg/L, calculated by the formula below; V is the total volume of the sample extract in mL; and m is the sample mass. ρ a = 12.72 A 663 − 2.59 A 645 ρ b = 22.88 A 645 − 4.67 A 663 ρ c h l o r o p h y l l c o n t e n t = ρ a + ρ b c h l o r o p h y l l c o n t e n t = ρ ∗ V / m ∗ 1000 m g / g   The trials were executed on three occasions, employing a fully randomized design, and the data were analyzed utilizing IBM SPSS Statistics version 26. A one-way ANOVA was utilized to evaluate the statistical significance of the findings. Upon identifying significant disparities, Duncan’s multiple range test was implemented to pinpoint the specific mean differences at a significance threshold of p < 0.05. Genomic expression data were processed through the interactive data mining system named Dr.Tom . The relationships between plant hormones and differentially expressed genes (DEGs) derived from RNA sequencing were examined using Pearson’s correlation coefficient, with a stringent correlation threshold set at 0.9 and a significance level of p < 0.05. The dataset obtained from the above experiments was previously used to draw conclusions on other issues regarding the response of R. chrysathum to UV-B .   The present study provides a comprehensive analysis of phytohormonal regulation in R. chrysanthum under UV-B radiation, revealing the complex interplay between hormonal signaling and metabolic responses in plant stress tolerance. Our findings indicate that UV-B radiation significantly impacts the photosynthetic capacity of R. chrysanthum , leading to a decline in photosynthesis efficiency and damage to the photosynthetic system. This is accompanied by alterations in the levels of various phytohormones, their biosynthesis, and the expression of related signaling genes, which collectively contribute to the plant’s defense mechanisms against UV-B stress . Additionally, while UV-B radiation primarily imposes stress on the plant, our study also hints at the potential for UV-B exposure to induce adaptive responses that may enhance certain growth-related processes in R. chrysanthum . This aligns with the concept of radiation hormesis, where radiation can stimulate plant growth through the activation of specific hormonal pathways and defense mechanisms. These findings not only expand our understanding of hormonal regulatory mechanisms in plant resistance to UV-B radiation but also lay the groundwork for developing strategies to enhance plant tolerance to adverse environmental stresses.   