@article { ISI:000360677400002,
	title = {Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica)},
	journal = {BMC Plant Biology},
	volume = {15},
	year = {2015},
	month = {SEP},
	pages = {Article Number: 214},
	publisher = {BIOMED CENTRAL LTD},
	address = {236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLAND},
	abstract = {<p>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.. 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.</p>},
	keywords = {Azadirachta indica, Quantitative profiling, Transcriptome, Triterpenoids},
	issn = {1471-2229},
	doi = {10.1186/s12870-015-0593-3},
	author = {Pandreka, Avinash and Dandekar, Devdutta S. and Haldar, Saikat and Uttara, Vairagkar and Vijayshree, Shinde G. and Mulani, Fayaj A. and Aarthy, Thiagarayaselvam and Thulasiram, Hirekodathakallu V.}
}
@article {47033,
	title = {Limonoid biosynthesis 3: functional characterization of crucial genes involved in neem limonoid biosynthesis},
	journal = {Phytochemistry},
	volume = {184},
	year = {2021},
	month = {APR},
	pages = {112669},
	type = {Article},
	abstract = {<p>Neem (Azadirachta indica L.) is well known for its medicinal, agricultural, and pesticidal applications since ages. The secondary metabolites, limonoids, confer these biological properties, wherein over 150 different limonoids have been reported from neem. To understand limonoid biosynthesis, we analyzed tissue-specific (kernel, pericarp, leaves, and flower) transcriptome that resulted in the identification of one farnesyl diphosphate synthase (AiFDS), one squalene synthase (AiSQS), three squalene epoxidases (AiSQE1, AiSQE2, and AiSQE3), two triterpene synthases (AiTTS1 and AiTTS2), cycloartenol synthase (AiCAS), two cytochrome P450 reductases, and ten cytochrome P450 systems. Comparative tissue-expression analysis indicated that AiFDS, AiSQS, AiSQE3, and AiTTS1 are expressed higher in the kernel than in the other tissues. Heterologously expressed recombinant AiTTS1 produced tirucalla-7,24-dien-3 beta-ol as the sole product. Expression profile data, phylogeny with triterpene synthases from Meliaceae and Rutaceae families, real-time PCR of different tissues, and transient transformation revealed the involvement of tirucalla-7,24-dien-3 beta-ol synthase (AiTTS1) in limonoid biosynthesis. Further, mutagenesis studies of AiTTS1 indicated that Y125 and F260 are probably involved in stabilization of dammarenyl cation. A 2.6-fold increase in production of tirucalla-7,24-dien-3 beta-ol was observed when AiSQE1 was coexpressed with mutant AiTTS1 in a yeast system. Furthermore, we functionally characterized the highly expressed cytochrome P450 reductases and cycloartenol synthase. This study helps in further analysis and identification of genes involved in limonoid biosynthesis in Meliaceae/Rutaceae and their production in a metabolically tractable heterologous system.</p>
},
	keywords = {24-dien-3 beta-ol, Azadirachta indica A. Juss., Cytochrome P450 system, Limonoids, Meliaceae, Tirucalla-7, Transcriptome},
	issn = {0031-9422},
	doi = {10.1016/j.phytochem.2021.112669},
	author = {Pandreka, Avinash and Chaya, Patil S. and Kumar, Ashish and Aarthy, Thiagarayaselvam and Mulani, Fayaj A. and Bhagyashree, Date D. and Shilpashree, H. B. and Jennifer, Cheruvathur and Ponnusamy, Sudha and Nagegowda, Dinesh and Thulasiram, V. Hirekodathakallu}
}
