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Cited by 39Plant biochemistry and biosynthesisPhytochemicals and Medicinal PlantsPlant Gene Expression Analysis

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Comparative temporal metabolomics studies to investigate interspecies variation in three Ocimum species

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Rastogi, Shubhra, Shah, Saumya, Kumar, Ritesh, Kumar, Ajay, Shasany, Ajit Kumar·2020·Scientific Reports
DOI 10.1038/s41598-020-61957-5PMC PMC7089951
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Abstract

Ocimum is one of the most revered medicinally useful plants which have various species. Each of the species is distinct in terms of metabolite composition as well as the medicinal property. Some basil types are used more often as an aromatic and flavoring ingredient. It would be informative to know relatedness among the species which though belong to the same genera while exclusively different in terms of metabolic composition and the operating pathways. In the present investigation the similar effort has been made in order to differentiate three commonly occurring Ocimum species having the high medicinal value, these are Ocimum sanctum, O. gratissimum and O. kilimandscharicum. The parameters for the comparative analysis of these three Ocimum species comprised of temporal changes in number leaf trichomes, essential oil composition, phenylpropanoid pathway genes expression and the activity of important enzymes. O. gratissimum was found to be richest in phenylpropanoid accumulation as well as their gene expression when compared to O. sanctum while O. kilimandscharicum was found to be accumulating terpenoid. In order to get an overview of this qualitative and quantitative regulation of terpenes and phenylpropenes, the expression pattern of some important transcription factors involved in secondary metabolism were also studied.

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Crossref · 10.1038/s41598-020-61957-5

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Figures

Figure 1 Abundant essential oil constituents from six months oil profiling of (A) Ocimum sanctum (OS) (B) Ocimum gratissimum (OG) and, (C) Ocimum kilimandscharicum (OK). Data are given as means ± SD (at least three independent replicates).
Figure 1 Abundant essential oil constituents from six months oil profiling of (A) Ocimum sanctum (OS) (B) Ocimum gratissimum (OG) and, (C) Ocimum kilimandscharicum (OK). Data are given as means ± SD (at least three independent replicates).CC BY · PubMed Central
Figure 2 Trichome densities and their representative microscopic images for six months in three panels for (A) Ocimum sanctum (OS) (B) Ocimum gratissimum (OG) and, (C) Ocimum kilimandscharicum (OK). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01, **P < 0.05 and ***P < 0.1 levels of significance. Arrows in the figure are pointing the glandular trichomes on the abaxial leaf surface; Scale bar = 100 µm. Error bars represent ± SD (at least ten independent plants).
Figure 2 Trichome densities and their representative microscopic images for six months in three panels for (A) Ocimum sanctum (OS) (B) Ocimum gratissimum (OG) and, (C) Ocimum kilimandscharicum (OK). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01, **P < 0.05 and ***P < 0.1 levels of significance. Arrows in the figure are pointing the glandular trichomes on the abaxial leaf surface; Scale bar = 100 µm. Error bars represent ± SD (at least ten independent plants).CC BY · PubMed Central
Figure 3 Pathways leading to the generation of terpenes and phenylpropenes in Ocimum sp. giving an overview of the MVA (mevalonate) and MEP (2-C-methyl-D-erythritol-4-phosphate) pathways localized in the cytosol and the plastids, respectively. DXS, DXP synthase; DXR, DXP reductoisomerase; MCT, 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; CMK, CDP-ME kinase; MCS, CMEPP synthase; HDS, HMBPP synthase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IDI, IPP isomerase; AACT (acetoacetyl-CoA thiolase), HMGS (HMG synthase), HMGR (HMG reductase), MVK, MVA kinase; PMK, phosphome-valonate kinase; PMD, MVA diphosphate decarboxylase; and GGPPS, GGPP synthase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; C3H, p-coumarate 3-hydroxylase; COMT, caffeoyl O-methyl transferase; CC4H, cinnamoyl-CoA 4-hydroxylase; CC3H, p-coumaroyl- CoA 3-hydroxylase; CCOMT, caffeoyl-CoA O-methyl transferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; CAAT, coniferyl alcohol acetyl transferase; EGS, eugenol synthase; EOMT, eugenol O-methyl transferase and CVOMT, chavicol O-methyl transferase. The enzymes for 4- and 3-hydroxylation of CoA esters have not been demonstrated yet and hence [CC4H] and [CC3H] are indicated in brackets to indicate their probable role (Gang et al.56) [adapted from Iijima et al.41 and Rastogi et al.38].
Figure 3 Pathways leading to the generation of terpenes and phenylpropenes in Ocimum sp. giving an overview of the MVA (mevalonate) and MEP (2-C-methyl-D-erythritol-4-phosphate) pathways localized in the cytosol and the plastids, respectively. DXS, DXP synthase; DXR, DXP reductoisomerase; MCT, 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; CMK, CDP-ME kinase; MCS, CMEPP synthase; HDS, HMBPP synthase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IDI, IPP isomerase; AACT (acetoacetyl-CoA thiolase), HMGS (HMG synthase), HMGR (HMG reductase), MVK, MVA kinase; PMK, phosphome-valonate kinase; PMD, MVA diphosphate decarboxylase; and GGPPS, GGPP synthase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; C3H, p-coumarate 3-hydroxylase; COMT, caffeoyl O-methyl transferase; CC4H, cinnamoyl-CoA 4-hydroxylase; CC3H, p-coumaroyl- CoA 3-hydroxylase; CCOMT, caffeoyl-CoA O-methyl transferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; CAAT, coniferyl alcohol acetyl transferase; EGS, eugenol synthase; EOMT, eugenol O-methyl transferase and CVOMT, chavicol O-methyl transferase. The enzymes for 4- and 3-hydroxylation of CoA esters have not been demonstrated yet and hence [CC4H] and [CC3H] are indicated in brackets to indicate their probable role (Gang et al.56) [adapted from Iijima et al.41 and Rastogi et al.38].CC BY · PubMed Central
Figure 4 Relative expression levels of PAL, C4H, 4CL, C3H, COMT, CCR, CAD, EGS and EOMT transcripts in O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK) during six months starting from July’ 2018 till December’ 2018 determined by Real-time RT-PCR. Data are means ± SD (for at least three replicates), and the y-axis represents the relative quotient (RQ). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at P < 0.01* and P < 0.05** levels of significance while ‘ns’ means non-significant.
Figure 4 Relative expression levels of PAL, C4H, 4CL, C3H, COMT, CCR, CAD, EGS and EOMT transcripts in O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK) during six months starting from July’ 2018 till December’ 2018 determined by Real-time RT-PCR. Data are means ± SD (for at least three replicates), and the y-axis represents the relative quotient (RQ). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at P < 0.01* and P < 0.05** levels of significance while ‘ns’ means non-significant.CC BY · PubMed Central
Figure 5 Relative expression levels of bHLH_21387, bHLH_25905, EREB, MADS box_50254, MADS box_43518, MYB2, MYB3, MYB5, MYC, PAP1, TTG1 and WRKY56 transcription factors in O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK) during six months starting from July’ 2018 till December’ 2018 determined by Real-time RT-PCR. Data are means ± SD (for at least three replicates), and the y-axis represents the relative quotient (RQ). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01 and **P < 0.05 levels of significance while ‘ns’ means non-significant.
Figure 5 Relative expression levels of bHLH_21387, bHLH_25905, EREB, MADS box_50254, MADS box_43518, MYB2, MYB3, MYB5, MYC, PAP1, TTG1 and WRKY56 transcription factors in O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK) during six months starting from July’ 2018 till December’ 2018 determined by Real-time RT-PCR. Data are means ± SD (for at least three replicates), and the y-axis represents the relative quotient (RQ). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01 and **P < 0.05 levels of significance while ‘ns’ means non-significant.CC BY · PubMed Central
Figure 6 Enzyme activity of PAL, C4H, CAD quantified in terms of product formed and of 4CL in terms of substrate utilized represented per mg of the total plant protein from O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK) at maturity. L-Phenylalanine, trans- cinnamic acid, p- coumaric acid and coniferyl alcohol were used as the substrates for PAL, C4H, 4CL and CAD, respectively. Activities are given as means of two independent assays ± SD (standard deviation). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01 and **P < 0.05 levels of significance while ‘ns’ means non-significant.
Figure 6 Enzyme activity of PAL, C4H, CAD quantified in terms of product formed and of 4CL in terms of substrate utilized represented per mg of the total plant protein from O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK) at maturity. L-Phenylalanine, trans- cinnamic acid, p- coumaric acid and coniferyl alcohol were used as the substrates for PAL, C4H, 4CL and CAD, respectively. Activities are given as means of two independent assays ± SD (standard deviation). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01 and **P < 0.05 levels of significance while ‘ns’ means non-significant.CC BY · PubMed Central
Figure 7 The content of (A) total phenols, (B) anthocyanin and, (C) chlorophyll determined in mature plant for comparing the three Ocimum species, O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK). Data are means ± SD (for at least three replicates). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01 and **P < 0.05 levels of significance.
Figure 7 The content of (A) total phenols, (B) anthocyanin and, (C) chlorophyll determined in mature plant for comparing the three Ocimum species, O. sanctum (OS), O. gratissimum (OG) and O. kilimandscharicum (OK). Data are means ± SD (for at least three replicates). Statistical analysis was performed using HSD tukey test, standard weighted-means analysis at *P < 0.01 and **P < 0.05 levels of significance.CC BY · PubMed Central

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Research works printed but not linked: 4

  1. Kalita, J. & Khan, M. L. Commercial potentialities of essential oil of Ocimum members growing in North East India. Int. J. Pharm. Life Sci. 4(4), 2559–2567 (2013).
  2. Lal, R. K. et al. High yielding eugenol rich oil producing variety of Ocimum sanctum-‘CIM-Ayu’. JMAPS 25, 746–747 (2003).
  3. Kashyap, C. P., Kaur, R., Arya, V. & Kumar, V. Therapeutic Potency of Ocimum kilimandscharicum Guerke - A Review. Glob. J. Pharm. 5(3), 191–200 (2011).
  4. Sadasivam, S. & Manickam, A. Biochemical methods. 2nd Edition, New age International, New Delhi (1996).

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Metadata

Authors
Rastogi, S., Shah, S., Kumar, R., Kumar, A., & Shasany, A. K.
Affiliations
  • Siksha O Anusandhan University (IN)
  • Central Institute of Medicinal and Aromatic Plants (IN)
Corresponding author
Shasany, Ajit Kumar
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-03-23
Volume
10
Issue
1
Pages
5234-5234
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-61957-5
PMC
PMC7089951
OpenAlex
W3012887410
Cited by
39
References
88
Citations by year
2026: 5 · 2025: 4 · 2024: 7 · 2023: 4 · 2022: 11 · 2021: 7 · 2020: 1
FWCI
1.87
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • DST | Science and Engineering Research Board (YSS/2014/001011)
  • Council of Scientific and Industrial Research (HCP-007, BSC-0203)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Health Sciences › Medicine › Complementary and alternative medicine › Phytochemicals and Medicinal Plants
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant Gene Expression Analysis
Keywords
Ocimum, Ocimum sanctum, Ocimum gratissimum, Ocimum kilimandscharicum, interspecies variation, leaf trichomes, essential oil composition, phenylpropanoid pathway, gene expression, terpenoids, phenylpropenes, secondary metabolism, transcription factors, medicinal plants
MeSH terms
Anthocyanins, Chlorophyll, Enzymes, Oils, Volatile, Plant Proteins, Plants, Medicinal, Transcription Factors, Gene Expression Regulation, Plant, Ocimum, Metabolomics, Trichomes, Secondary Metabolism

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