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A mechanistic insight into hydrogen peroxide-mediated elicitation of bioactive xanthones in Hoppea fastigiata shoot cultures

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Abstract

Main conclusion

Elicitation of xanthones is mediated by ROS where Ca 2+ mediated generation of H 2 O 2 activates the shikimate pathway, a key regulator in early steps of xanthone biosynthesis in H. fastigiata.

Shoot cultures of Hoppea fastigiata upon treatment with yeast extract (YE) accumulate an enhanced amount of 1,3,5-trihydroxy-8-methoxy xanthone. We demonstrated that YE treatment was followed by a rapid burst of reactive oxygen species (ROS, O2 − and H2O2) and subsequent increase in xanthone contents. The antioxidant enzymes (NADPH oxidase, superoxide dismutase (SOD), peroxidase and catalase) followed a similar kinetics as that of ROS, depending on their role in production or degradation. It was observed that shikimate dehydrogenase (SKDH) and shikimate kinase (SK) activities enhanced after 8 h, benzophenone synthase activity continued to rise after elicitation and peaked at 18 h. Activities of phenylalanine ammonia-lyase and 4-hydroxycinnamoyl-CoA ligase remained suppressed and unaffected, respectively, after elicitation. This suggests a possible phenylalanine-independent biosynthesis of xanthones. Successive treatment of shoots cultures with a NADPH-oxidase inhibitor diphenylene iodide and a ROS-scavenger dihydrolipoic acid showed inhibition in ROS (O2 − and H2O2) accumulation. These treatments were also shown to decrease the activities of SKDH and SK, leading to a suppressed amount of xanthones formation. Although O2 − showed continuous increase upon treatment with a SOD inhibitor diethyldithiocarbamic acid, the contents of H2O2 and xanthones were decreased, which correlates well with the reduced activities of SKDH and SK. Treatments with calcium antagonists, such as, lanthanum chloride and EGTA were also shown to block the activities of SKDH, SK, NADPH-oxidase and SOD, and consequently leading to suppressed accumulation of ROS (O2 − and H2O2) and xanthones.

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Abbreviations

BPS:

Benzophenone synthase

DHLA:

Dihydrolipoic acid

DIECA:

Diethyldithiocarbamic acid

DPI:

Diphenylene iodide

PAL:

Phenylalanine ammonia-lyase

SK:

Shikimate kinase

SKDH:

Shikimate dehydrogenase

SOD:

Superoxide dismutase

ROS:

Reactive oxygen species

YE:

Yeast extract

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Acknowledgments

Utkarsh Ravindra Moon thanks University Grant Commission, India for the award of an individual junior/senior research fellowship [F. No. 2-16/98 (SA-I), dated 4-21-2011]. Facilities created from a research grant (4-25/2013/TS-I) of Ministry of Human Resource Development (MHRD) India were utilized in this work. We thank Prof Ludger Beerhues of TU-Braunschweig, Germany for providing 2,4,6-trihydroxybenzophenone as a gift.

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  1. Agricultural and Food Engineering Department, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur, Kharagpur, 721 302, India

    Utkarsh Ravindra Moon & Adinpunya Mitra

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Correspondence to Adinpunya Mitra.

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425_2016_2506_MOESM1_ESM.tif

Fig. S1 Effect of ROS inhibitors, ROS scavenger and calcium antagonists on shoot cultures of H. fastigiata. a NADPH oxidase activity. b O2 − accumulation. c SOD activity. d H2O2 accumulation. All the inhibitors were added 20 min prior to the addition of YE (10 mg ml−1). All the readings were taken after 4 h, 8 h and 16 h of treatment. Each value is the mean ± SD from three independent experiments. Mean values are significantly different at the P ≤ 0.05 level, n = 3 (TIFF 1645 kb)

425_2016_2506_MOESM2_ESM.tif

Fig. S2 Time-course changes of 4CL enzyme activityin shoot cultures of H. fastigiata upon YE treatment (10 mg ml−1). Each value is the mean ± SD from three independent experiments. Mean values are significantly different at the P ≤ 0.05 level, n = 3 (TIFF 1440 kb)

425_2016_2506_MOESM3_ESM.tif

Fig. S3 Effect of calcium antagonists on SKDH activities. a In gel assay of SKDH. b Average area intensity values and their standard deviations for gels destained using appropriate procedure are presented and estimated using ImageJ software. All the antagonists were added 20 min prior to YE treatment (10 mg ml−1). The assay was performed after 16 h of YE treatment. La, lanthanum chloride (TIFF 838 kb)

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Moon, U.R., Mitra, A. A mechanistic insight into hydrogen peroxide-mediated elicitation of bioactive xanthones in Hoppea fastigiata shoot cultures. Planta 244, 259–274 (2016). https://doi.org/10.1007/s00425-016-2506-6

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