Metabolite profiling reveals a role for intercellular dihydrocamalexic acid in the response of mature Arabidopsis thaliana to Pseudomonas syringae

Kempthorne, Christine J.; Nielsen, Alexander J.; Wilson, Daniel; McNulty, James; Liscombe, David K.

doi:10.1016/j.phytochem.2021.112747

Cited by 8 publications

(5 citation statements)

References 78 publications

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“…In our earlier studies also, silencing of WsSQS of phytosterol pathway and silencing and overexpression of WsWRKY1 involved in regulation of phytosterol and withanolides formation, showed positive correlation with expression of PR genes 14 , 15 . Furthermore, AtCYP76C2 and AtCYP71A12 from Arabidopsis were found to be associated with defense mechanism against P. syringae infection 40 , 41 . Hence, it is clear that the three identified genes are involved in bacterial defense, however, how the change in expression of WsCYP450s affected PR gene expression is quite intriguing and interesting and needs further investigation.…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng

Shilpashree

Sudharshan

Shasany

et al. 2022

Sci Rep

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The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (~ 450%) and reduced withanolide A (~ 50%), its overexpression drastically led to enhanced withanolide A (> 250%) and withanolide B (> 200%) levels and reduced 12-deoxywithastramonolide (~ 60%). Whereas WsCYP76-vigs led to reduced withanolide A (~ 60%) and its overexpression increased withanolide A (~ 150%) and reduced 12-deoxywithastramonolide (~ 60%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (~ 50%) and withanolide A (~ 60%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress.

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Section: Resultsmentioning

confidence: 99%

Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng

Shilpashree

Sudharshan

Shasany

et al. 2022

Sci Rep

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“…It is interesting to note that these results are similar to our previous characterization of three CYPs from W. sominifera indicating that cytochrome P450 enzymes play a crucial role in defense in this plant (Shilpashree et al 2022). Moreover, AtCYP76C2 and AtCYP71A12 from Arabidopsis which were found to be associated with defense mechanism against Pseudomonas syringae infection (Godiard et al 1998; Kempthorne et al 2021). Hence, it is clear that WsCYP71B35 is involved in bacterial defense, however the exact mechanism of signaling needs further investigation.…”

Section: Discussionmentioning

confidence: 99%

The cytochrome P450 enzyme WsCYP71B35 fromWithania somniferahas a role in withanolides biosynthesis and defense against bacteria

Shilpashree,

Narayanan,

Kumar

et al. 2023

Preprint

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The medicinal properties of Ashwagandha (Withania somniferaL. Dunal) are attributed to withanolides, which belong to the triterpenoid steroidal lactones class of compounds. Though it is proposed that intermediates of the universal phytosterol pathway are utilized by cytochrome P450 (CYP450) enzymes to form withanolides, studies on functional characterization of these enzymes has been sparse. This study reports the functional characterization of a CYP450 candidate fromW. somnifera(WsCYP71B35) that exhibited induced expression in response to methyl jasmonate treatment and showed higher expression in tissues that accumulate withanolides. Biochemical assay with yeast microsomal fraction expressing recombinant WsCYP71B35 indicated no activity when phytosterols and their intermediate 24-methylene cholesterol were used as substrates. However, WsCYP71B35 catalyzed product formation with withaferin A, withanolide A, withanolide B, and withanoside IV among the tested substrates. Moreover, virus-induced gene silencing (VIGS) and transient overexpression ofWsCYP71B35inW. somniferaleaves modulated the levels of withaferin A, withanolide A, and withanolide B, indicating the role ofWsCYP71B35in withanolides pathway. Furthermore, VIGS ofWsCYP71B35inW. somniferareduced its tolerance toPseudomonas syringae(DC3000) infection, whereas overexpression enhanced the tolerance to the bacterium inW. somniferaand transgenic tobacco. Overall, these results provide insights into the role ofW. somniferaWsCYP71B35 in withanolides biosynthesis and bacterial defense.

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“…It is interesting to note that these results are similar to our previous characterization of three CYPs from W. sominifera , indicating that cytochrome P450 enzymes play a crucial role in defence in this plant (Shilpashree et al, 2022). Moreover, AtCYP76C2 and AtCYP71A12 from Arabidopsis were found to be associated with a defence mechanism against Pseudomonas syringae infection (Godiard et al, 1998; Kempthorne et al, 2021). Hence, it is clear that WsCYP71B35 is involved in bacterial defence, however, the exact mechanism of signalling needs further investigation.…”

Section: Discussionmentioning

confidence: 99%

“…with a defence mechanism against Pseudomonas syringae infection (Godiard et al, 1998;Kempthorne et al, 2021). Hence, it is clear that F I G U R E 8 Proposed role of characterized WsCYP450s in withanolides biosynthesis.…”

mentioning

confidence: 99%

The cytochrome P450 enzyme WsCYP71B35 from Withania somnifera has a role in withanolides biosynthesis and defence against bacteria

Shilpashree,

Narayanan,

Kumar

et al. 2024

Physiologia Plantarum

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The medicinal properties of Ashwagandha (Withania somnifera L. Dunal) are attributed to withanolides, which belong to the triterpenoid steroidal lactones class of compounds. Though it is proposed that intermediates of the universal phytosterol pathway are utilized by cytochrome P450 (CYP450) enzymes to form withanolides, studies on the functional characterization of these enzymes have been sparse. This study reports the functional characterization of a CYP450 candidate from W. somnifera, WsCYP71B35 that exhibited induced expression in response to methyl jasmonate treatment and showed higher expression in tissues that accumulate withanolides. Biochemical assay with yeast microsomal fraction expressing recombinant WsCYP71B35 indicated no activity when phytosterols and their intermediate 24‐methylene cholesterol were used as substrates. However, WsCYP71B35 catalyzed product formation with withaferin A, withanolide A, withanolide B, and withanoside IV among the tested substrates. Moreover, virus‐induced gene silencing (VIGS) and transient overexpression of WsCYP71B35 in W. somnifera leaves modulated the levels of withaferin A, withanolide A, and withanolide B, indicating the role of WsCYP71B35 in withanolides pathway. Furthermore, VIGS of WsCYP71B35 in W. somnifera reduced its tolerance to Pseudomonas syringae (DC3000) infection, whereas overexpression enhanced the tolerance to the bacterium in W. somnifera and transgenic tobacco. Overall, these results provide insights into the role of W. somnifera WsCYP71B35 in withanolides biosynthesis and bacterial defence.

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Metabolite profiling reveals a role for intercellular dihydrocamalexic acid in the response of mature Arabidopsis thaliana to Pseudomonas syringae

Cited by 8 publications

References 78 publications

Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng

Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng

The cytochrome P450 enzyme WsCYP71B35 fromWithania somniferahas a role in withanolides biosynthesis and defense against bacteria

The cytochrome P450 enzyme WsCYP71B35 from Withania somnifera has a role in withanolides biosynthesis and defence against bacteria

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