Physiological and transcriptomic analyses reveal the roles of secondary metabolism in the adaptive responses of Stylosanthes to manganese toxicity

- Physiological and transcriptomic analyses reveal the roles of secondary metabolism in the adaptive responses of Stylosanthes to manganese toxicity.
- However, little is known about the adaptive responses of stylo to Mn toxicity.
- Thus, this study integrated both physiological and transcriptomic analyses of stylo subjected to Mn toxicity..
- In contrast, the activities of enzymes, such as peroxidase (POD), phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO), were significantly increased in stylo leaves upon treatment with increasing Mn levels, particularly Mn levels greater than 400 μ M.
- Transcriptome analysis revealed 2471 up-regulated and 1623 down-regulated genes in stylo leaves subjected to Mn toxicity.
- Numerous genes associated with transcription factors (TFs), such as genes belonging to the C2H2 zinc finger transcription factor, WRKY and MYB families, were also regulated by Mn in stylo leaves.
- Furthermore, the C2H2 and MYB transcription factors were predicted to be involved in the transcriptional regulation of genes that participate in secondary metabolism in stylo during Mn exposure.
- Conclusions: Taken together, this study reveals the roles of secondary metabolism in the adaptive responses of stylo to Mn toxicity, which is probably regulated by specific transcription factors..
- Hence, excess Mn toxicity generally occurs in acid soils due to the accumulation of bioactive divalent Mn (II) [5, 6].
- Consequently, soil amelioration, such as lime applica- tion, is typically conducted to alleviate Mn toxicity by decreasing Mn availability, but this application is costly from both economic and environmental aspects [7].
- For these reasons, breeding crop varieties with superior Mn tolerance represents a sustainable alternative agronomi- cal strategy, but this strategy requires better understand- ing of how plants respond to Mn toxicity.
- Although morphological changes in plants grown under the condi- tion of Mn toxicity vary among plant species, the ap- pearances of Mn toxicity reported in most plants generally include leaf chlorosis, brown spots, crinkled leaves and brown roots, and ultimately plant growth in- hibition [8, 9].
- Adverse impacts caused by Mn toxicity have also been documented in plant cells at physio- logical levels, such as triggering oxidative stress, causing lipid peroxidation, inhibiting enzyme activity, impairing chlorophyll biosynthesis and photosynthesis and disturb- ing the uptake and translocation of other mineral ele- ments [1, 9]..
- Furthermore, secondary metabolic processes and metabolites, such as phenolics, flavonoids and phenylalanine, are regulated by Mn stress in plants suggesting the potential roles of secondary metabolism in the adaptation of plants to Mn toxicity..
- Studying the responses of plants to vary- ing Mn concentrations is useful to determine how plants cope with Mn toxicity..
- Recently, it has been documented that high Mn adaptability in stylo may be achieved by its fine regulation of proteins involved in specific pathways, such as defense response, photosynthesis and metabol- ism [11].
- Furthermore, important roles of organic acids, such as malate, in stylo adaptation to Mn toxicity have been reported.
- Although stylo has considerable potential for Mn tolerance, the effects of excess Mn toxicity on profile.
- alterations in the gene expression of stylo have not been reported, and the molecular responses of stylo to Mn stress remain largely unknown.
- Previous studies have paved the way for the current study dissecting the mo- lecular responses of stylo to Mn toxicity.
- Accordingly, in this study, the effects of various Mn concentrations on the physiological changes in stylo were first investigated..
- Transcriptomic analysis of Mn-responsive genes in stylo leaves was further performed using an RNA-seq ap- proach.
- Leaf chlorosis, a symptom of Mn toxicity, was observed in stylo leaves treated with greater than 200 μM Mn, espe- cially 400 and 800 μM Mn (Fig.
- Consistent with this finding, increases in malondial- dehyde (MDA) levels were observed in stylo leaves sub- jected to high Mn stress.
- Simi- larly, the maximum quantum yield of photosystem II (Fv/Fm) significantly declined in stylo under Mn treat- ments from 200 to 800 μM Mn compared with the controls (Additional file 1: Fig.
- S1), suggesting that photosynthesis was inhibited by Mn toxicity..
- Activities of peroxidase (POD), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were analyzed in stylo leaves ex- posed to Mn treatments.
- In addition, increases in PPO and PAL activities were also found in stylo leaves under Mn stress, especially 400 and 800 μM Mn treatments (Fig.
- PPO and PAL activities in stylo leaves treated with 400 μM Mn were 54.7 and 35.8% increased compared with their con- trols (Fig.
- Transcriptome analysis of stylo leaves responding to Mn toxicity.
- In this study, comparative transcriptomic analysis in stylo leaves subjected to 5 and 400 μM Mn treatments was performed.
- Differentially expressed genes (DEGs) in stylo leaves exposed to Mn treatments were identified based on.
- Among these genes, 2471 genes were up-regulated, while 1623 genes were down-regulated by Mn toxicity (Additional file 3:.
- 2 Plant dry weight and Mn concentrations in stylo under different Mn treatments.
- 4 GO analysis of DEGs in stylo.
- Secondary metabolic pathways in response to Mn toxicity According to GO analysis, a total of 1201 unigenes were predicted to be involved in metabolic processes in the BP categories, including 824 up-regulated unigenes and 377 down-regulated unigenes (Fig.
- Interestingly, a large number of genes in the above pathways were enhanced by Mn toxicity.
- For example, genes encoding PAL, β-glucosidases (GLUs), cinnamoyl-CoA reductase (CCR), cinnamyl-alcohol dehydrogenases (CADs), feruloyl- CoA 6-hydroxylases (F6Hs) and caffeic acid 3-O- methyltransferases (COMTs), were up-regulated in the phe- nylpropanoid biosynthesis pathway (Fig.
- hydroxyisoflavanone dehydratase (HIDH), isoflavone 2- hydroxylase (I2H) and vestitone reductases (VRs) related to the isoflavonoid biosynthesis process was enhanced in stylo under Mn toxicity (Fig.
- Transcription factors involved in stylo responses to Mn toxicity.
- Among them, 11 genes were sig- nificantly increased by greater than 8-fold under Mn toxicity: DREB group protein, protein PPLZ02, dehydra- tion-responsive element-binding protein 1E-like, ethylene- responsive transcription factor 4 and TINY transcription factor belonging to the AP2 family.
- myb-related protein Zm1 belonging to the MYB family.
- transcription factor bHLH18-like belonging to the HLH family.
- hypothetical protein LR48 belonging to the WRKY family.
- To explore the candidate TFs involved in the transcrip- tional regulation of genes associated with secondary me- tabolism in stylo during Mn exposure, the protein-protein interaction networks of DEGs related to secondary meta- bolic pathways and TFs were further constructed.
- These results suggest that the candidate TFs highlighted above may be involved in the regulation of secondary metabolism- related genes in the response of stylo to Mn toxicity..
- 6 DEGs related to secondary metabolism in stylo ’ s response to Mn toxicity.
- 8 DEGs associated with transcription factors in stylo leaves.
- Changes in secondary metabolite levels under Mn stress Subsequently, the concentrations of four secondary me- tabolites, including total phenols, flavonoids, tannins and anthocyanidins, in stylo leaves treated with various Mn concentrations were further detected.
- Compared to their respective controls, the levels of total phenols, tannins and anthocyanidins in stylo leaves increased with 400 and 800 μM Mn treatments, while the concentrations of flavonoids increased with Mn treatments increasing from 200 to 800 μM, peaking at 800 μM (Fig.
- Fur- thermore, the concentrations of total phenols, flavo- noids, tannins and anthocyanidins in stylo leaves subjected to 400 μM Mn treatment were increased and 7.4-fold, respectively, compared with their controls (Fig.
- These results suggest that secondary metabolism may play a key role in stylo’s response to Mn toxicity..
- Mn toxicity not only causes adverse impacts in plant cells but also threatens human health [27, 28].
- However, the transcriptome pro- files of plants in response to Mn toxicity have not been well documented.
- In this study, the molecular responses of stylo to Mn toxicity were investigated through inte- gration of physiological and transcriptomic analyses, providing potential Mn-tolerant strategies in plants for further study..
- Response of stylo to Mn toxicity.
- 1), which was supported by the results obtained from a previous study in stylo [11]..
- RNA-seq analysis was further conducted to identify DEGs in stylo’s response to Mn toxicity.
- 4), suggesting various adjustments in stylo’s response to Mn toxicity.
- This finding is consistent with a previous study demonstrating that homologous genes encoding antioxi- dant enzymes are up-regulated in stylo and citrus (Citrus grandis) in response to Mn toxicity [11, 34].
- Further- more, excess Mn up-regulation of POD and APX tran- scripts may result in increasing POD and APX activities in stylo leaves subjected to excess Mn toxicity (Fig.
- 3 and Additional file 6: Table S5), suggesting that these antioxidant enzymes may participate in ROS scavenging in stylo.
- Homologues of these genes are regulated by Mn or other metal stresses suggesting that overlap between metal stress responses may be con- served in stylo and other plants..
- 6 and 7), suggesting that secondary metabolism is regulated by Mn toxicity.
- In the phenylpropanoid pathway, for example, the expression of PAL was up- regulated by Mn stress in the transcriptome data (Fig..
- 11 Concentrations of secondary metabolites in stylo leaves subjected to different Mn treatments.
- Accordingly, increased PAL transcript levels may enhance its enzyme activity in stylo’s response to Mn toxicity (Figs.
- Furthermore, some genes encoding proteins involved in different steps in the phenylpropanoid biosynthesis path- way, such as CCR, CADs, COMTs, F6Hs and PODs, were all up-regulated in stylo subjected to Mn toxicity (Fig.
- Therefore, these results strongly suggest that the phenylpropanoid biosynthesis process is activated in stylo to counteract Mn stress..
- The flavonoid biosynthesis process is performed by a set of key genes encoding CHSs, CHIs, FLSs and F3,5H, which were found to be regulated by Mn toxicity in this study (Fig.
- Similarly, in this study, significant increases in secondary metabolites, including total phenols, flavo- noids, tannins and anthocyanins, were observed in stylo leaves exposed to 400 and 800 μM Mn (Fig.
- expression might be important for stylo adaptation to Mn toxicity..
- In this study, the identification of 123 transcription fac- tors suggested complex regulation in stylo’s response to Mn toxicity (Fig.
- Among those identified transcription factors, seven DEGs belonging to the C2H2 zinc finger transcription factor family were up-regulated by Mn toxicity in stylo (Additional file 5: Table S4).
- these homologous genes were also up-regulated by Mn toxicity in stylo (Additional file 3: Table S2), sug- gesting that the functions of C2H2 transcription factors may be conserved in Mn detoxification through regula- tion of downstream critical Mn-resistant genes.
- 9), suggesting that these C2H2 transcription factors may also play transcriptional regulatory roles in secondary metabolism in stylo during Mn exposure, which deserves further study..
- Moreover, nine transcription factor genes belonging to the WRKY family were up-regulated by Mn toxicity in stylo (Additional file 5: Table S4).
- Although the function of WRKY transcription factor remains largely unknown in stylo’s response to Mn toxicity, WRKY46 in Arabi- dopsis a negative regulator of ALMT1.
- However, WRKY46 expression in Arabidopsis is suppressed by Al toxicity [52], but WRKY gene transcripts in stylo were enhanced by Mn stress in this study (Additional file 5: Table S4), suggesting that there are other regulatory networks that are mediated by WRKY-mediated gene expression in stylo during Mn toxicity..
- Additionally, 22 transcription factors belonging to the MYB family were identified in this study (Additional file 5: Table S4).
- Thus, future efforts are required to investigate the functions of these transcription factors in stylo sub- jected to Mn toxicity..
- Among them, a set of genes en- coding homologs to ATP-binding cassette (ABC) transporters were regulated by Mn toxicity.
- Therefore, genes encoding ABC trans- porters are probably involved in stylo leaves defense against Mn toxicity..
- Furthermore, genes encoding cation/H + exchangers were found to be up-regulated by Mn toxicity in this.
- For example, the cation/H + exchanger in Arabidopsis, AtCAX2, is involved in conferring tolerance to Mn toxicity when it is heterolo- gously expressed in a Mn-sensitive yeast mutant pmc1vcx1cnb or in tobacco (Nicotiana tabacum) via se- questration of Mn into vacuoles [61].
- In addition, one gene encoding homologs to aquaporin protein, aquaporin TIP1–3-like, was also up-regulated by Mn toxicity in stylo leaves (Additional file 7: Table S6)..
- In addition, a group of genes belonging to transporters for various substrates, including phosphate, potassium and copper, were found to be differentially regulated by Mn in stylo leaves (Additional file 7: Table S6).
- Furthermore, the identified DEGs associated with secondary metabolic pathways suggested that fine-tuned regulation of secondary metabolism might represent an adaptive strategy of stylo to cope with Mn toxicity.
- In addition, activation of various transcription factors may help stylo to tolerate Mn toxicity through transcriptional regulation.
- Taken together, this study not only reveals new insights into the molecular mechanisms underlying stylo’s response to Mn toxicity but also provides candi- date genes that may be useful in the development of.
- O 2 − levels in stylo leaves were detected using the histo- chemical method [66].
- Transcriptome analysis of stylo leaves.
- stylo leaves subjected to control (5 μM) and Mn toxicity (400 μM) treatments was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).
- synthesis kit (Vazyme, China) according to the protocol..
- Determination of secondary metabolite concentrations The concentrations of secondary metabolites, including total phenols, flavonoids, anthocyanins and tannins, in stylo leaves exposed to various Mn treatments were ana- lyzed using commercial assay kits (Nanjing Jiancheng Bioengineering Co., Ltd., Jiangsu, China).
- Microsoft Excel 2003 (Microsoft Company, USA) was used in the data analysis.
- Information of DEGs identified in stylo leaves subjected to control (5 μ M) and Mn toxicity (400 μ M) treatments..
- Transcription factors responding to Mn toxicity..
- Advances in the mechanisms of plant tolerance to manganese toxicity.
- Early induction of Fe-SOD gene expression is involved in tolerance to Mn toxicity in perennial ryegrass

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