Chiang Mai J. Sci. 2017; 44(2) 427 Chiang Mai J. Sci. 2017; 44(2) : 427-437 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Isolation and Expression Analysis of the Flavanone 3-Hydroxylase Genes in Lotus (Nelumbo nucifera gaertn.), Waterlily (Nymphaea sp.) and Transient Silencing in Waterlily Chaivarakun Chaipanya [a, b], Kanjana Saetiew [b], Sumay Arunyanart [b] and Nonglak Parinthawong* [b] [a] Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand. [b] Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. *Author for correspondence; e-mail: [email protected] Received: 2 September 2014 Accepted: 9 November 2015 ABSTRACT Flavanone 3-hydroxylase (F3H) converts flavanones from dihydroflavonols, which leads to production of flavonoid compounds via the anthocyanin biosynthesis pathway in plants. In this study, the F3H gene was isolated from lotus (Nelumbo nucifera Gaertn.) cv. Buntharik (white petal lotus), cv. Satabankacha (pink petal lotus), and waterlily (Nymphaea sp.) var. St. Louis Gold by reverse transcription PCR (RT-PCR). The open reading frames (ORF) of three cultivars’ genes were 1,134 bp in length, encoding a predicted protein of 377 amino acids. Their nucleotide sequences were identical, and the amino acid sequence shared high homology to F3H from different plant species. Expression of F3H was specifically regulated in petals and stamens, while less expression was found in leaf tissue of waterlily variety St. Louis Gold. The correlation of F3H expression according to specific colouration was performed in waterlily. The F3H gene was more highly expressed in decreasing order of red, purplish blue, and yellow petals when compared using semi quantitative PCR (sqPCR). Gene regulation according to flowering stage and pigmentation was determined in lotus. The F3H expression was slightly diminished in petals of cultivar Satabankacha at the fully-opening stage, whereas it was detected in the cultivar Buntharik only when white petals were tinted with pink. An RNAi gene-silencing vector, pJA8F3H, encoding a hairpin F3H RNA, was introduced to waterlily petals using the Agrobacterium infiltration method, and the F3H expression was analysed at 1 and 3 days post infiltration (dpi) by sqPCR. The results showed that the F3H expression was down-regulated at 3 dpi in flowers tested of the red petal variety and purplish blue petal variety compared to controls. The results confirmed that pJA8F3H is efficient and could be used as a transformation vector to transiently suppress F3H expression in waterlily or lotus. Keywords: anthocyanin, flavanone 3-hydroxylase (F3H), Nelumbo, Nymphaea, RNAi Chiang Mai J. Sci. 2017; 44(2) 428 1. INTRODUCTION Flavonoids accumulate in all higher plants. and Torenia hybrid [8] by disruption of CHS, They are important to plants, as they are CHI, F3H, F3′5′H, and DFR expressions, involved in many biological processes. It is respectively. A cDNA for F3H was first known that flavonoids play roles in signalling isolated from Antirrhinum majus (snapdragon) between plants and microbes, pollen [9]. The gene functions at an early step of development, and protection from UV light, the flavonoid pathway and codes for the and they act as defence molecules against enzyme that catalyses the formation of pathogens. Some flavonoid pigments provide dihydroflavonols from flavanones and an array of colours, which, therefore, attracts provides precursors for many classes of pollinators and seed dispersers, facilitating flavonoid compounds. Therefore, this enzyme reproduction [1]. These secondary metabolites is essential in the flavonoid pathway. have been divided into different categories of Disruption or down-regulation of F3H structure including flavones, flavonols, expression resulted in the decrease of isoflavones, flavanones, chalcones, aurones, flavonoid content and change of petal proanthocyanidins, and anthocyanins, among colour [10-13]. others [2]. The flavonoid biosynthetic Lotus (Nelumbo nucifera Gaertn.) is grown pathway is part of the phenylpropanoid widely in Thailand, as it is a symbolic in pathway with the flavonoid precursors Buddhism. However, there are only two phenylalanine, from the shikimate pathway, natural colours of lotus flowers available- and malonyl-CoA, which is derived from white and pink. Therefore, generation of a citrate produced by the tricarboxylic acid new colour line using a conventional (TCA) cycle [3]. Studies on different mutants breeding program is rather impossible. affecting flavonoid synthesis have been To modify flower colour, an understanding established, and genes encoding flavonoid of the function of genes in the flavonoid biosynthetic enzymes have been cloned in a pathway is essential. Waterlily (Nymphaea sp.) variety of plant species such as petunia, has diverse petal colours including white, maize, snapdragon (Antirrhinum majus), and yellow, pink, and red to dark purple; by Arabidopsis [4]. exploiting this, waterlily was used as a tool to The flavonoid pathway starts with the monitor changes in flavonoid biosynthetic formation of the C15 backbone or chalcones pathway modifications. Therefore, the goal by chalcone synthase (CHS), which are further of the research was to down-regulate the converted to a range of other flavonoids expression of F3H gene in lotus and waterlily. by chalcone isomerase (CHI), flavanone 3- Here, we report the characterisation of the hydroxylase (F3H), flavonoid 32-hydroxylase F3H genes in lotus and waterlily. Expression (F3′H), dihydroflavonol 4-reductase (DFR), analysis using sqPCR suggested that F3H and anthocyanidin synthase (ANS) [3]. The was transcribed in various flower stages and most prominent class of products is the petal colours. In addition, F3H was transiently anthocyanins, providing colour to petals. silenced using RNAi, and its level of expression Mutation of genes involved in the flavonoid is described. biosynthetic pathway resulted in changes of petal colour in many ornamental flowering 2. MATERIALS AND METHODS plants, such as Gentiana triflora [5], Nicotiana 2.1 Plant Materials tabacum [6], Dianthus caryophyllus cv. Eilat [7], The waterlily lines used in this study, Chiang Mai J. Sci. 2017; 44(2) 429 Nymphaea sp. var. St. Louis Gold, var. Royal 5′ RACE GSP1. The PCR products were Red, var. Teri Dunn, and var. Lueang Garnjana, diluted and subjected to the second PCR were obtained from germplasm of Dr. reaction using 5′ nested and 5′ RACE NGSP1 Sermlap Wasuwat and Primlap Chukiatman primers in order to obtain the 5′-gene region. Wasuwat. Two cultivars of lotus (Nelumbo The 3′-region was created using an oligo-dT nucifera Gaertn.) including Satabankacha and primer and a gene-specific F3H8 primer. Buntharik, whose petals are pink and white, The sequences of all primers used are listed respectively, were obtained from ponds at in Table 1, and the PCR reactions were King Mongkut’s Institute of Technology conducted according to the manufacturers’ Ladkrabang. Plant material was harvested, protocols. The 5′- and 3′- fragments obtained immediately frozen in liquid nitrogen, and were cloned into the pTZ57R/T cloning ground to a powder under liquid nitrogen vector (Thermo Scientific, USA) and with a mortar and pestle. For RNA isolation, sequenced. Gene-specific oligonucleotide the petals, stamens, and leaves were harvested primers were designed (F3H stop) and used separately. together with the UPM primer located at the 5′-adaptor region in PCR to synthesise a 2.2 Isolation of DNA, RNA and cDNA full-length F3H gene. In order to analyse the Synthesis F3H gene structure, the genomic DNA was Petals were collected for genomic DNA used as template in the PCR reaction where preparation using DNeasy Plant Mini Kit primer F3H SenseF and F3H stop were (Qiagen, USA). Petals, stamens, and leaves present. The obtained nucleotide sequence of of lotus were extracted for total RNA using F3H gene were analysed by Freeware program a modified Gomez-Gomez method [14], BioEdit [15], BLASTN (www.ncbi.nlm.nih. while InviTrap® (Invitek, Germany) was used gov/Blast.cgi), and the phylogenetic tree was to isolate total RNA from the small amount generated using MEGA5 program [16]. of transient transformed petals of waterlily. First strand cDNA was synthesised from 2.4 Constructions of pJA8F3H 1 μg of DNase-treated total RNA and A 308-bp gene fragment was synthesised 0.5 μL RevertAid reverse transcriptase using F3H SenseF and F3H SenseR (Fermentas, USA). Full-length cDNA was as oligonucleotide primers for PCR. synthesised from 1 μg total RNA according The fragment was cloned into pCR8 to the manufacturer protocol of the (Invitrogen, USA) and then introduced SMARTer™ RACE cDNA amplification kit into pJAWOHL8 using the Gateway system (Clontech, Canada). (Invitrogen, USA). The pJAWOHL8, an RNAi binary silencing vector, carries 2.3 Cloning and Sequencing of the F3H ampicillin and Basta resistance genes as Gene bacterial and plant selective markers, The cDNA derived from SMARTer™ respectively. After construction, the obtained RACE was used as a template for nested-PCR pJA8F3H plasmid was then introduced in combination with the following primers: into Agrobacterium tumefaciens strain GV3101 the UPM primer, which located at the and used for transient silencing assays in 5′-adaptor region, and gene-specific primer, waterlily petals. Chiang Mai J. Sci. 2017; 44(2) 430 Table 1. Primers used in this study. Primer name Primer sequence (5′ → 3′) UPM primer CTA ATA CGA CTC ACT ATA GGG C 5′RACE GSP1 ATC CAC GTC TTG CCG CCG TCC CTG GTG G 5′RACE NGSP1 ACC AGG TCC TGC AGC AGC AGG GTG ATG G F3H8 CCT GGA CAT GGA CCA GAA G F3H stop TCA GGC CAG AAT TTC ATT GA F3H SenseF GAG AAG CTC CGG TTC GAC AT F3H SenseR TAG TTC ACC ACC ATC TTC TGG F3H14F GGA GAT CGT GAC GTT CTT CT F3H10R GGA TTC TGG AAC GTG GCT A CHSF AAG AGC TCC CGT CAA GAG ACT CA CHSR AAG GAT CCC AGA AAA TTG AGT TC 18-SF GGC TCG AAG ACG ATC AGA TAC CG 18-SR GTA CAA AGG GCA GGG ACG TAG TCA A 2.5 Agrobacterium-mediated technique with a combination of F3H14F Transformation and F3H10R primers for F3H or CHSF and As recommended in Zottini et al.