Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica)

Abstract

Background: Neem tree (Azadirachta indica) is one of the richest sources of skeletally diverse triterpenoids and they are well-known for their broad-spectrum pharmacological and insecticidal properties. However, the abundance of Neem triterpenoids varies among the tissues. Here, we delineate quantitative profiling of fifteen major triterpenoids across various tissues including developmental stages of kernel and pericarp, flower, leaf, stem and bark using UPLC-ESI(+)-HRMS based profiling. Transcriptome analysis was used to identify the initial genes involved in isoprenoid biosynthesis. Based on transcriptome analysis, two short-chain prenyltransferases and squalene synthase (AiSQS) were cloned and functionally characterized.

Results: Quantitative profiling revealed differential abundance of both total and individual triterpenoid content across various tissues. RNA from tissues with high triterpenoid content (fruit, flower and leaf) were pooled to generate 79.08 million paired-end reads using Illumina GA ΙΙ platform. 41,140 transcripts were generated by d e novo assembly. Transcriptome annotation led to the identification of the putative genes involved in isoprenoid biosynthesis. Two short-chain prenyltransferases, geranyl diphosphate synthase (AiGDS) and farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) were cloned and functionally characterized using transcriptome data. RT-PCR studies indicated five-fold and ten-fold higher relative expression level of AiSQS in fruits as compared to leaves and flowers, respectively.

Conclusions: Triterpenoid profiling indicated that there is tissue specific variation in their abundance. The mature seed kernel and initial stages of pericarp were found to contain the highest amount of limonoids. Furthermore, a wide diversity of triterpenoids, especially C-seco triterpenoids were observed in kernel as compared to the other tissues. Pericarp, flower and leaf contained mainly ring-intact triterpenoids. The initial genes such as AiGDS, AiFDS and AiSQS involved in the isoprenoids biosynthesis have been functionally characterized. The expression levels of AiFDS and AiSQS were found to be in correlation with the total triterpenoid content in individual tissues.

Fig. 1

Skeletal diversity of Neem triterpenoids. Basic triterpenoids have azadirone, azadiradione, and gedunin type of skeletons. C- Seco triterpenoids have nimbin, salannin and azadirachtin type of skeletons

Fig. 2

Predicted triterpenoid biosynthetic pathway, various Neem tissues and their total triterpenoids content in different tissues; (a) Initial genes involved in triterpenoid biosynthesis. b Different tissues of Neem and physical characteristics of Neem fruits from various stages. c Amount of triterpenoid extracts obtained from various tissues of Neem

Fig. 3

Quantitative abundance of major triterpenoids in different tissues of Neem. Basic and C-seco triterpenoids are highly abundant in Pericarp and Kernel respectively as compared to other tissues

Fig. 4

Functional annotation of transcriptome; (a) Based on Blastx analysis 80 % (32,856) transcripts had homologous proteins in NCBI nr database. b Based on KAAS analysis only 15.2 % (6281) transcripts were assigned 2749 KO numbers. c Based on virtual ribosome analysis 66.5 % (27,368) transcripts had ORF region length more than 100 amino acids and 0.001 % (67) Transcripts did not show ORF region. d Based on Pfam analysis 69.1 % (18,907) transcripts were assigned Pfam IDs

Fig. 5

Total ion chromatograms (TICs) of AiGDS, AiFDS and AiSQS assays and relative expression level of AiSQS; (a) TICs of AiGDS assays; (1) Standard Nerol, (2) Standard geraniol, (3) Co-injection of standard nerol and geraniol, (4) Substrate control, (5) Enzyme control, (6) AiGDS enzyme assay with IPP and DMAPP as substrates, (7) Co-injection of standard geraniol with AiGDS enzyme assay extract. b TICs of AiFDS assays; (1) Standard (E,E)-farnesol, (2) IPP and DMAPP substrate control, (3) Enzyme control, (4) AiFDS enzyme assay with IPP and DMAPP as substrates, (5) Co-injection of standard (E,E)-farnesol and extract of AiFDS enzyme assay with IPP and DMAPP as substrates, (6) Extract of AiFDS enzyme assay with GPP and IPP as substrates. c TICs of AiSQS assays; (1) Standard squalene, (2) Substrate control, (3) Enzyme control, (4) Extract of full length AiSQS enzyme assay with FPP as substrate and NADPH as co-factor, (5) Co-injection of standard squalene and AiSQS enzyme assay extract, (6) Extract of truncated AiSQS enzyme assay with FPP as substrate and NADPH as co-factor and (7) Co-injection of standard squalene and truncated AiSQS enzyme assay extract

Fig. 6

Real-time PCR analysis. a Neem_transcript_10001 showed very high expression in flower. b AiGDS was highly expressed in leaf. c AiFDS has higher expression level in seeds. d Relative expression levels of AiSQS was very high in seeds as compared to other tissues. Error bars represents standard error