Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica).
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.
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. avinash.pandreka@igib.in.|CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, 110007, India. avinash.pandreka@igib.in.
Avinash
A
Pandreka
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. ds.dandekar@ncl.res.in.
Devdutta S
DS
Dandekar
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. s.haldar@ncl.res.in.
Saikat
S
Haldar
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. us.vairagkar@ncl.res.in.
Vairagkar
V
Uttara
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. vg.shinde@ncl.res.in.
Shinde G
SG
Vijayshree
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. fa.mulani@ncl.res.in.
Fayaj A
FA
Mulani
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. aarthyT@ncl.res.in.
Thiagarayaselvam
T
Aarthy
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India. hv.thulasiram@ncl.res.in.|CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, 110007, India. hv.thulasiram@ncl.res.in.
Hirekodathakallu V
HV
Thulasiram
D031601:Azadirachta / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D051228:Geranyltranstransferase / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014315:Triterpenes / Q000378:metabolism*
D010940:Plant Proteins; D014315:Triterpenes; D051228:Geranyltranstransferase; D013186:Farnesyl-Diphosphate Farnesyltransferase