Two terpene synthases are responsible for the major sesquiterpenes emitted from the flowers of kiwifruit (Actinidia deliciosa)

Kiwifruit vines rely on bees for pollen transfer between spatially separated male and female individuals and require synchronized flowering to ensure pollination. Volatile terpene compounds, which are important cues for insect pollinator attraction, were studied by dynamic headspace sampling in the major green-fleshed kiwifruit (Actinidia deliciosa) cultivar ‘Hayward’ and its male pollinator ‘Chieftain’. Terpene volatile levels showed a profile dominated by the sesquiterpenes α-farnesene and germacrene D. These two compounds were emitted by all floral tissues and could be observed throughout the day, with lower levels at night. The monoterpene (E)-β-ocimene was also detected in flowers but was emitted predominantly during the day and only from petal tissue. Using a functional genomics approach, two terpene synthase (TPS) genes were isolated from a ‘Hayward’ petal EST library. Bacterial expression and transient in planta data combined with analysis by enantioselective gas chromatography revealed that one TPS produced primarily (E,E)-α-farnesene and small amounts of (E)-β-ocimene, whereas the second TPS produced primarily (+)-germacrene D. Subcellular localization using GFP fusions showed that both enzymes were localized in the cytoplasm, the site for sesquiterpene production. Real-time PCR analysis revealed that both TPS genes were expressed in the same tissues and at the same times as the corresponding floral volatiles. The results indicate that two genes can account for the major floral sesquiterpene volatiles observed in both male and female A. deliciosa flowers

Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance

Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H 2O 2)-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.link_to_subscribed_fulltex

Contribution of CoA ligases to benzenoid biosynthesis in Petunia flowers

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavour, fragrance, cosmetic and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step towards understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases (TPS), pyrophosphatases (PPase), alcohol dehydrogenases (ADH), aldo-keto reductases (AKR), carotenoid cleavage dioxygenases (CCD), alcohol acetyltransferases (AAT) and aldehyde dehydrogenases (ALDH), which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes and acetates. Molecular modeling and docking further supported the role of identified enzymes in aroma formation in Cymbopogon. Also, simple sequence repeats (SSRs) were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition

Fig. 4. A in Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis

Fig. 4. A) 1H and 13C NMR Chemical Shifts of tirucalla-7,24-dien-3β-ol. 13Cδ are shown in blue colour and 1Hδ are shown in black colour. B) RT-PCR analysis of triterpene synthases and squalene epoxidase in different tissues of neem. I) RT-PCR for AiTTS1 shows higher expression in kernel, II) RT-PCR for AiTTS2 shows higher expression in pericarp and kernel, III) RT-PCR for AiCAS shows higher expression in Kernel and flower and IV) RT-PCR for AiSQE1 shows higher expression in kernel and pericarp. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Published as part of <i>Pandreka, Avinash, Chaya, Patil S., Kumar, Ashish, Aarthy, Thiagarayaselvam, Mulani, Fayaj A., Bhagyashree, Date D., B, Shilpashree H., Jennifer, Cheruvathur, Ponnusamy, Sudha, Nagegowda, Dinesh & Thulasiram, Hirekodathakallu V., 2021, Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis, pp. 1-12 in Phytochemistry (112669) 184</i> on page 6, DOI: 10.1016/j.phytochem.2021.112669, <a href="http://zenodo.org/record/10126978">http://zenodo.org/record/10126978</a&gt

Fig. 1 in Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis

Fig. 1. Limonoid biosynthesis in neem. Tirucalla-7,24-dien-3β-ol involves in neem limonoid biosynthesis.Published as part of <i>Pandreka, Avinash, Chaya, Patil S., Kumar, Ashish, Aarthy, Thiagarayaselvam, Mulani, Fayaj A., Bhagyashree, Date D., B, Shilpashree H., Jennifer, Cheruvathur, Ponnusamy, Sudha, Nagegowda, Dinesh & Thulasiram, Hirekodathakallu V., 2021, Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis, pp. 1-12 in Phytochemistry (112669) 184</i> on page 2, DOI: 10.1016/j.phytochem.2021.112669, <a href="http://zenodo.org/record/10126978">http://zenodo.org/record/10126978</a&gt

Fig. 5 in Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis

Fig. 5. Transient expression of AiTTS1 in neem twig leaves. A) Real-time qPCR analysis showing relative expression levels of AiTTS1 in pRI101-ANand pRI101- AN_AiTTS1 infiltrated leaves. Expression levels of these genes were normalized to actin and are represented incomparison with pRI101-AN control. Data represent means ±SE of two independent biological replicates. B) Limonoid profiling of neem transient leaves between control pRI101-AN and pRI101-AN-AiTTS1. Most of the neem limonoids production is increased as compared to vector control which clearly shows that AiTTS1 involved in limonoid biosynthesis.Published as part of <i>Pandreka, Avinash, Chaya, Patil S., Kumar, Ashish, Aarthy, Thiagarayaselvam, Mulani, Fayaj A., Bhagyashree, Date D., B, Shilpashree H., Jennifer, Cheruvathur, Ponnusamy, Sudha, Nagegowda, Dinesh & Thulasiram, Hirekodathakallu V., 2021, Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis, pp. 1-12 in Phytochemistry (112669) 184</i> on page 7, DOI: 10.1016/j.phytochem.2021.112669, <a href="http://zenodo.org/record/10126978">http://zenodo.org/record/10126978</a&gt

Fig. 2 in Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis

Fig. 2. Heat map for the RPKM values of transcripts which involved in isopreniod biosynthesis. Triterpenoid biosynthesis related genes were highly expressed in kernel and pericarp, which is in line with total triterpenoid profiling.Published as part of <i>Pandreka, Avinash, Chaya, Patil S., Kumar, Ashish, Aarthy, Thiagarayaselvam, Mulani, Fayaj A., Bhagyashree, Date D., B, Shilpashree H., Jennifer, Cheruvathur, Ponnusamy, Sudha, Nagegowda, Dinesh & Thulasiram, Hirekodathakallu V., 2021, Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis, pp. 1-12 in Phytochemistry (112669) 184</i> on page 4, DOI: 10.1016/j.phytochem.2021.112669, <a href="http://zenodo.org/record/10126978">http://zenodo.org/record/10126978</a&gt