Virus-Induced Gene Silencing of the Eggplant Chalcone Synthase

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Virus-induced gene silencing of the eggplant chalcone synthase gene during fruit ripening modifies epidermal cells and gravitropism Cuicui Wang, and Daqi Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05617 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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Journal of Agricultural and Food Chemistry

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Virus-induced gene silencing of the eggplant chalcone synthase gene

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during fruit ripening modifies epidermal cells and gravitropism

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Cuicui Wang1, Daqi Fu1,*

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1

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China Agricultural University, Beijing 100083, China.

Fruit Biology Laboratory, College of Food Science and Nutritional Engineering,

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* Corresponding author:

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Email: [email protected];

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Author contributions C.C.W. conceived the project and wrote the article; D.Q.F. supervised the experiments.

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1 ACS Paragon Plus Environment


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Abstract: Eggplant (Solanum melongena L.) fruits accumulate flavonoids in their

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cuticle and epidermal cells during ripening. Although many mutants available in

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model plant species such as Arabidopsis thaliana and Medicago truncatula are

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enabling the intricacies of flavonoid-related physiology to be deduced, the

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mechanisms whereby flavonoids influence eggplant fruit physiology are unknown.

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Virus-induced gene silencing (VIGS) is a reliable tool for the study of flavonoids

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function in fruit, and in this study, we successfully applied this technique to

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down-regulate S. melongena chalcone synthase gene (SmCHS) expression during

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eggplant fruit ripening. In addition to the expected change in fruit color attributable to

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a lack of anthocyanins, several other modifications, including differences in epidermal

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cell size and shape, were observed in the different sectors. We also found that

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silencing of CHS gene expression was associated with a negative gravitropic response

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in eggplant fruits. These observations indicate that epidermal cell expansion during

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ripening is dependent on CHS expression, and that there may be a relationship

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between CHS expression and gravitropism during eggplant fruit ripening.

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Keywords: chalcone synthase, flavonoids, epidermal cells, gravitropism, eggplant

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fruit

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Introduction

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Fruit ripening involves shifts in primary and secondary metabolism that impart

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the characteristic flavors of fruit. During ripening, eggplant fruit undergoes major

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changes in pigment biosynthesis, including chlorophyll degradation and anthocyanin

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and flavonoid accumulation. This increase in flavonoids is tissue specific, since the

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highest concentrations are present in the epidermal peel, with comparatively little

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being distributed in the flesh1. Flavonoids have been widely studied because of their

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potential health benefits and their ubiquitous inclusion in the human diet2, 3.

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To date, flavonoid biosynthesis has been studied extensively at the metabolic and

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molecular levels4, 5. Flavonoid biosynthesis starts from the condensation of

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p-coumaroyl-CoA with malonyl-CoA to generate naringenin chalcone via chalcone

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synthase (CHS). Naringenin chalcone is then rapidly isomerized by chalcone

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isomerase (CHI, Accession: FS057776) to obtain naringenin. Subsequently,

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naringenin is hydroxylated to dihydrokaempferol (DHK) by flavanone 3-hydroxylase

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(F3H,

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hydroxylation of DHK at the 3ʹposition produces dihydroquercetin, whereas flavonoid

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3’5’-hydroxylase (F3’5’H, Accession: X70824)-mediated hydroxylation at the 3ʹ and

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5ʹ positions produces dihydromyricetin. Dihydroflavonol 4-reductase (DFR,

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Accession:

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leucoanthocyanidins, which are further converted into colored anthocyanidins by

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anthocyanidin synthase (ANS, Accession: EU809469)1, 6, 7.

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Accession:

FS038632).

FS075550)

then

Flavonoid

catalyzes

3’-hydroxylase

dihydroflavonols

(F3’H)-mediated

to

colorless

Flavonoids with different physical and biochemical properties can interact with 3 ACS Paragon Plus Environment


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diverse targets in subcellular locations to influence biological activities in plants,

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animals, and microbes8-10. Among other roles, they provide color for flowers, fruits,

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and seeds, regulate reactive oxygen species, protect from ultraviolet radiation, are

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essential for male fertility and participate in plant defense and molecule signaling11.

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Flavonoids can regulate plant hormones or control gene transcription, directly or

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indirectly, thus affecting several important biological processes. In this regard, earlier

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studies proved that flavonoids play important roles in modulating auxin movement

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and degradation12, and thereby could be involved in regulating plant growth13, 14.

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Flavonoids negatively regulate polar auxin transport in plants15,

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, and

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Arabidopsis mutants lacking flavonoids (CHS defective tt4) show elevated levels of

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auxin transport and gravitropism13, 17, 18. Consistent with the influence of flavonoids

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on root gravity response, flavonoid treatment in a gravitropic mutant lacking the gene

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encoding the auxin transport protein, PIN-FORMED2 (PIN2), partially restored the

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formation of lateral auxin gradients in the absence of PIN219.

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Recent evidence has shown that flavonoids have evolved particular roles in the

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expansion of epidermal cells. Micrographs of unstained fruit epicarp sections from

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ripe tomatoes agroinoculated to silence CHS showed that epidermal cells from the

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CHS-silenced sectors were significantly more rounded than those from non-silenced

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sectors in the tomato analyzed20.

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Eggplant (Solanum melongena L) is an agronomically important vegetable that is

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cultivated and consumed in many countries. Consumption of eggplant fruits is

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considered to play an important role in the human diet, as some nutrients in eggplant 4 ACS Paragon Plus Environment

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are deemed to have efficacious anti-disease effects (e.g., anti-inflammatory properties

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or cytostatic effects)10, 21. Moreover, eggplant is ranked among the top ten vegetables

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with regards to oxygen radical scavenging capacity, which is attributable to

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anthocyanins contained in the fruit peels22. However, despite its commercial value,

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comparatively little research efforts have been devoted to the analysis of flavonoids in

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eggplant.

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In the present study, virus-induced gene silencing (VIGS) was applied to induce

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CHS gene silencing in eggplant fruits during ripening, avoiding any indirect effect

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arising from flavonoid depletion in other tissues or during developmental stages.

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Silencing of flavonoid biosynthesis during ripening produced eggplants with

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differentially colored regions—purple (non-silenced) and white (silenced)—which

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allowed their physical isolation and subsequent analysis. We examined the effect of

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flavonoids on epidermal cells and studied the relationship between inhibition of the

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flavonoid pathway and ripe fruit gravitropism. Silenced regions were found to show

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alterations in flavonoid content, epidermal cell shape, and gravitropism. The results of

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our study demonstrate that VIGS using the optimize Agrobacterium-infiltration

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methods can be successfully used to eggplant fruit and represents an effective tool for

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studying CHS gene functions during fruit development.

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Materials and Methods

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Plant materials and treatments

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The eggplant cultivar ‘Zhongnong Changfeng’ was grown in the greenhouse with 5 ACS Paragon Plus Environment


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16/8 h light/dark photoperiod (24–26°C) and added fertilizer regularly when required.

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After 40 days of agro-infiltration, the peel was harvested from the white and purple

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portions of the fruit. All the freshly plant materials were collected and immediately

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frozen with liquid nitrogen, and then stored at −80°C for RNA extraction and other

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analyses.

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Total RNA isolation and qRT-PCR

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Total RNA was extracted from 100 mg eggplant fruit using a Total RNA Isolation

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Kit (Qiagen, Germany) following the manufacturer’s instructions. The integrity and

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concentrations of RNA were determined via 1.5% agarose gel electrophoresis and a

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NanoDrop 2000 spectrophotometer (Thermo, USA), respectively. First-strand cDNA

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was synthesized using a Primescript II 1st strand cDNA Synthesis Kit (Takara, Japan).

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The sequence data have been deposited in the NCBI database with the accession

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number KT119427 (SmCHS). To isolate the open reading frame of the eggplant CHS

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gene, we designed degenerate primers from eggplant (Table S1). The PCR products

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were cloned into a pTRV2 vector and sequenced by the Invitrogen Company (Beijing,

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China).

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Expression pattern analyses were performed using qRT-PCR. Genomic DNA was

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removed from the RNA preparations by digesting with DNase I (Takara, Japan), and

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RNA was reverse-transcribed into cDNA using M-MLV Reverse Transcriptase

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(Promega, USA) according to the manufacturer’s instructions. qRT-PCR was

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performed with an FTC-3000 real-time PCR System (Funglyn Biotech，Canada) using

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the following program: 95°C for 30 s, followed by 36 cycles of 95°C for 20 s, 58°C 6 ACS Paragon Plus Environment

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for 30 s, and 72°C for 30 s. 18S rRNA was used as an internal reference gene since its

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expression was found to be stable in all the analyzed tissues, as was also reported by

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Gantasala23. Results were analyzed using the 2-ΔΔCt method24,

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relative expression levels compared to wild-type eggplant fruit. To detect the presence

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of tobacco rattle virus (TRV) RNA in the infected tissue, RT-PCR was carried out

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using TRV1- and TRV2-specific primers (Table S1).

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Agrobacterium infiltration

25

and reported as

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VIGS was performed using TRV according to methods described in a previous

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study26, 27. A 450 bp fragment of the SmCHS gene was selected using the VIGS tool

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(http://solgenomics.net/tools/vigs) to avoid off-target silencing and then amplified

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from eggplant cDNA using PCR. A pTRV2-SmCHS construct was generated by

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inserting EcoRI-digested and BamHI-digested PCR fragment of CHS into the pTRV2

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vector. Cultures of the GV3101 Agrobacterium strain containing the pTRV1, pTRV2,

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and pTRV2-SmCHS vectors respectively were cultivated at 28°C in Luria-Bertani

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(LB) liquid medium (pH 5.6) with 10 mM 2-(N-morpholino)ethanesulfonic acid

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(MES), 20 μM acetosyringone, and gentamycin, kanamycin, and rifampicin

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antibiotics. After shaking for 12 h, The cells were centrifuged and resuspended in

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infiltration buffer containing 10 mM MgCl2, MES (pH 5.6) and 200 µM

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acetosyringone to a final OD600 = 5.0. Resuspensions of pTRV1 were mixed with

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pTRV2 and pTRV2-SmCHS, respectively, at a ratio of 1:1 and maintained at 25°C for

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3 h. The Agrobacterium strain was infiltrated into eggplant fruit stalks using a 5-mL

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syringe. Eggplant fruits infiltrated with pTRV1 and pTRV2 were used as negative 7 ACS Paragon Plus Environment


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controls. Each inoculation was carried out three times, and 10 fruits were infiltrated

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for each construct. When the VIGS phenotype was visible, eggplant fruits were

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collected and stored.

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Tissue preparation for electron microscopy

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Small pieces of pericarp from three fruits of different plants were collected, and

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immediately fixed in 100 mmol/L phosphate-buffered saline (PBS, pH 7.2) containing

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2.5% (v/v) glutaraldehyde for 3 h at 20°C. After tissue blocks had been washed three

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times with PBS, they were post-fixed with 1% (w/v) OsO4 in PBS for 2h at 4°C,

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followed by a further extensive rinse in PBS, The tissue then was dehydrated in a

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gradient acetone series (30%–100%). The tissue was embedded in SPURR resin (Dow

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Chemical Co., USA) for 24 h at room temperature, and polymerization was conducted

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at 70°C for 8 h. Samples were sliced into ultra-thin (approximately 60–90 nm)

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sections using a microtome (Leica UC6), and then stained with 2% uranyl acetate in

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50% ethanol for 15 min at 25°C and with alkaline lead citrate for 10 min. The stained

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sections were examined using a JEM-1230 electron microscope28.

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Extraction and quantification of flavonoids via liquid chromatography-electr

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-ospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

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The peels from cold-preserved eggplants (600mg) were removed and ground to a

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fine powder using a mortar and pestle. The frozen tissues were then extracted with 70%

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methanol : water (v/v) (the solid : liquid ratio was kept at 1:3 [w/v]). The mixture was

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vortexed for 30 s and thereafter sonicated at 250 W for 15 min in an ice bath,

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followed by centrifugation at 8000 × g for 10 min. The resulting supernatant was 8 ACS Paragon Plus Environment

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collected and a 250 μL aliquot was filtered through a 0.22 μm membrane filter. The

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filtrate was collected and used for subsequent LC–ESI-MS/MS analysis. Extraction

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and quantification of flavonoids were carried out with three biological replicates.

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Quality control samples (QC) were prepared by mixing sample extracts for analysis of

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sample repeatability under the same treatment. During instrumental analysis, a control

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sample was inserted into every 10 test samples to examine the repeatability of the

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analysis29.

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LC–ESI–MS/MS

analyses

included

Ultra

High-Performance

Liquid

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Chromatography (Shim-pack UFLC SHIMADZU CBM20A) and tandem mass

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spectrometry (MS/MS) (Applied Biosystems 4500 QTRAP). A Waters Acquity UPLC

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HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm) was used. The mobile phase was

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ultrapure water (0.1% formic acid added) (A) and acetonitrile (0.1% formic acid

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added) (B) in the organic phase, using a gradient elution of 5%-95% B at 0-20 min

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(linear gradient). The column temperature was set at 40°C and the injection volume

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was 5 µL. After chromatographic separation, the samples were subjected to mass

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spectrometric analysis. Masses were detected in the m/z range of 50 to 1500 D. The

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main parameters of the linear ion trap and triple quadrupole in the API 4500 QTRAP

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LC/MS/MS system were electrospray ionization (ESI) at 550°C, a mass spectrometry

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voltage of 5500 V, and curtain gas of 25 psi, with the collision-activated dissociation

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parameter being set high. In triple quadrupole mode, each ion pair is scanned based on

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optimized declustering potential and collision energy (Table S2). The resulting data

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were processed using MS Analyst 1.6.1 software. 9 ACS Paragon Plus Environment


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Results

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Virus-induced gene silencing of CHS in eggplant fruit

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Three CHS genes are found in eggplant fruit during ripening, SmCHS1

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(Sme2.5_13923.1), SmCHS2 (Sme2.5_02154.1) and SmCHS3 (Sme2.5_01077.1)

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(http://eggplant.kazusa.or.jp/blast.html). The SmCHS1 fragment selected to silence is

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710-1160 bp of the annotated complementary DNA ([cDNA] sequence) sharing 84%

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identity with SmCHS2 and 83% identity with SmCHS3. A search of this fragment

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using

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http://solgenomics.net) did not render high similitude with any other gene in the

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eggplant genome other than the three CHS genes.

the

nucleotide

basic

local

alignment

search

tool

(BLAST)

(at

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To disrupt flavonoid biosynthesis pathways in eggplant fruits, we selected one

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key gene, the chalcone synthase (CHS) gene. We infiltrated 5-cm eggplant fruits with

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GV3101 Agrobacterium cultures carrying pTRV2-SmCHS mixed with agrobacteria

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carrying pTRV1 in a ratio of 1:1 using a needleless syringe. At approximately 20 days

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post-inoculation, typical features of targeted gene silencing, including photobleaching,

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were examined attributable to CHS silencing (Figure 1B), whereas the control plants

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remained purple (Figure 1A). We thereby established that VIGS technology can be

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applied successfully to silence target genes in eggplant fruit. To investigate the effects

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of Agrobacterium infection at the molecular level, we used RNA from fruits collected

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from silenced and control plants respectively to investigate the presence of pTRV1,

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pTRV2-empty and pTRV2- target gene via PCR. We used primers that anneal to the

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outside region of the CHS genes targeted for silencing. All silenced and TRV-infected 10 ACS Paragon Plus Environment

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leaves showed the presence of TRV RNA in the infected tissue (Figure 1C).

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Reductions in CHS mRNA levels ranged from 69% to 90% (Figure 2A). The

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percentage of silencing values shown represent the average of three samples for each

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plant. The relative CHS transcript accumulation obtained thus corroborated the CHS

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silencing phenotype in the inoculated eggplant fruits. Fruit VIGS has more advantages

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than whole-plant VIGS, in that it is a convenient method for studying fruit-specific

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processes, and avoids the potential side effects of gene silencing in vegetative tissue

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or during the process of fruit development.

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Silencing of CHS affects anthocyanin and flavonoids accumulation

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To further study the molecular mechanisms underlying anthocyanin accumulation

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in CHS-silenced eggplants, we investigated the expression profiles of the anthocyanin

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structural genes SmF3’ 5’ H, SmDFR and SmF3H in fruit peels (Figure 2B). Overall,

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the results showed that the expression levels of structural genes were lower in

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CHS-silenced eggplants than in wild-type eggplant. At the metabolic level, we

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measured the concentration of anthocyanins and flavonoids in the silenced and control

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plants via LC–ESI-MS/MS analyses. The main flavonoids are not the same in

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different plant tissues. Naringenin chalcone and the flavonol glycoside rutin are the

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main flavonoid compounds that accumulate in ripe tomatoes30, whereas, delphinidin,

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cyanidin, and dihydroquercetin are the major flavonoids detected in ripe eggplant

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fruits (Figure S1). Anthocyanin levels were lower in the VIGS-CHS white areas than

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in the purple areas. We observed an almost two-fold decrease in the amount of

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naringenin, pelargonidin and delphinidin in the white sectors of treated fruit, but no 11 ACS Paragon Plus Environment


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significant decrease in cyanidin (Figure 3). However, some flavonoids content

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increased in the white sectors. Dihydroquercetin and quercetin levels were

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approximately 11.51% and 61% higher in the silenced plants than in the wild-type

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plants, respectively. No changes in epigallocatechin levels were observed between the

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TRV-CHS-silenced samples and the mock-infected controls (Figure 3). Members of

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the Arabidopsis genus lack chalcone reductase and isoflavone synthase enzymes and

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therefore cannot produce isoflavonoids31. In contrast, isoflavone-genistein has been

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observed in eggplant fruit, increasing to as much as 2-fold higher than that observed

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in mock-infected controls (Figure 4).

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The expression of CHS influences epidermal cell expansion

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Wild-type eggplant fruit peels are purple-red in color. The fruit shape from top to

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bottom is typically uniform and the fruit body is straight, at maturity, fruit length is

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approximately 30 cm. In contrast, we found that CHS-silenced eggplant peels are

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white, the fruit shape is uneven and curved, and the length of the fruit is

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approximately 20 cm, with the shortest recorded length being approximately 15 cm.

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Fruit size is mostly determined by cell expansion, indicating the possibility that

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silencing the CHS gene affects expansion of fruit epicarp cells. Histological

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sectioning of different sectors of fruit epicarp revealed interesting differences. Figure

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5 shows the differences in epidermal cell size and shape observed in the different

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sectors of the two fruit phenotypes studied. A comparison of pTRV2-CHS white

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sectors and pTRV2 purple epicarp revealed that epidermal cells are significantly

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shorter and wider in the white-colored sectors than in the purple sectors. Moreover, 12 ACS Paragon Plus Environment

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the epidermal cells are loosely arranged in the purple areas, whereas, the epidermal

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cells are more compactly arranged in the white areas. These observations indicate that

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epidermal cell expansion during ripening is dependent on CHS expression.

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The expression of CHS plays an important role in the gravitropism of eggplant

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fruit

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To determine whether there were differences in gravitropic responses between

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mock and CHS-silenced fruits, we examined the phenotype of silenced eggplant fruits.

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Representative experiments comparing the gravitropic responses of CHS-silenced and

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mock-treated fruit are shown in Figure 6. To quantify the effect on bending, multiple

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silenced fruits were analyzed. Untreated eggplant showed almost no bending (30–50°),

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whereas the degree of bending in CHS-silenced eggplant was as high as 100–160°.

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Similar differential gravitropic responses have previously been reported in

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Arabidopsis thaliana transparent testa (tt) mutants showing diverse architectural

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phenotypes in root and aerial tissues. The alterations in the skewing angle of root

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elongation were found in the tt1, tt3, tt4, tt8, and tt10 mutants, whereas tt4 and tt10

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mutants also had looping roots, although the looping occurred later in the growth of

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the tt10 seedlings. Further, the tt4 mutants devoid of flavonoids show a delayed

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response to gravity13.

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Discussion

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Accumulation of flavonoids is increased in silenced eggplant

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Flavonoid synthesis is regulated by developmental and environmental signals that

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control the amount and distribution of the various flavonoids found in plants. The 13 ACS Paragon Plus Environment


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synthesis of flavonoids is complex in some species. Like soybeans, eggplant may also

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contain chalcone reductase and isoflavone synthase as it produces isoflavones such as

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genistein. However, Arabidopsis species lack these two enzymes and therefore do not

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produce isoflavones31. Although the concentration of flavonoid compounds in

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eggplant remains unknown, experimentation provides some clues.

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One reason for the accumulation of some flavonoids in the VIGS-CHS portion

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may be that 4-coumaroyl-CoA accumulates when CHS is silenced, leading to

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enhanced biosynthesis of caffeoyl-CoA pathway metabolites (Figure 4). It is also

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possible that selective uptake systems exist for flavonoids, and flavonoids are able to

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move for a long distance in plant tissues

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tt4 mutant, which is defective in flavonoid biosynthesis attributable to a lesion in

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chalcone synthase (CHS), however it preserves the downstream enzymatic machinery

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in the pathway. Perhaps localized enzyme activation and transport mechanisms are

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required for flavonoid accumulation. As flavonoid transport is suppressed by a

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compound that blocks ATP-binding cassette (ABC) transporters, which include the

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newly identified auxin transporters of the multidrug resistance/p-glycoprotein

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(MDR/PGP) class17. With respect to the potential effects of flavonoids on auxin

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transport that allow this flavonoids movement, further experimental evidence is

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required to explain the role of flavonoid movement in controlling plant development.

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These results demonstrate that to understand the localization of flavonoids in eggplant,

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it is necessary to know where the enzymes for flavonoid biosynthesis are made and to

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understand the mechanisms for the movement of some flavonoids from their synthesis

32, 33

. This was best demonstrated using the


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sites. The physiological relevance of selective flavonoid uptake and movement plays

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an important role in plant growth and development.

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CHS-related changes in epidermal cell morphology

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Flavonoids play roles in many facets of plant physiology, including transport12,

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defense34, allelopathy34, structural proteins35, cell physiology36, and modulation of

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reactive oxygen species levels37. There is evidence that flavonoids have evolved

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particular roles in cuticle deposition20, and in recent studies, Heredia analyzed how

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flavonoid accumulation during tomato ripening affected cuticle and epidermal

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properties38. These studies indicate a role for either flavonoids or CHS in altering the

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expression levels of certain genes involved in cuticle biosynthesis; however, it

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remains to be determined which genes are modified.

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During fruit ripening, the fruit cuticular pegs become thickened and cutinization

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of the epidermal and some hypodermal cell walls occurs39, fruit size eventually

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increases due to cell expansion. Flavonoids can become incorporated into the cuticle,

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and there are several flavonoids present in tomato peels40, whereas only naringenin is

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transported and incorporated in the outer cuticle matrix41. In CHS-silenced eggplant

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fruits, the level of naringenin decreased (Fig. 3). The effect of CHS silencing on

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cuticle biomechanics varied among the different plant species. The affinity for a

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certain flavonoid molecule to the cuticle in eggplant needs further research.

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Gravity responses and the auxin connection

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How plants sense and respond to gravity has been a long-standing question in

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biology. Recent genetic and live-imaging studies of shoot gravitropism in the model 15 ACS Paragon Plus Environment


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plant Arabidopsis thaliana have provided new insights into this interesting

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phenomenon. Previous study has shown that basipetal auxin movement provides the

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auxin necessary for gravity responses in Arabidopsis roots42. One possible

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explanation for these phenotypes is the effect that certain flavonoids exert on auxin

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levels in the plant by modulating auxin transport. In this regard, it has been previously

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reported that quercetin is a potent inhibitor of auxin transport43, which is increased in

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CHS-silenced eggplants (Figure 3). The influence of other flavonoid intermediates on

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auxin transport also needs to be assessed44.

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Recently, Kuhn suggested that flavonols affect the transport of auxin by

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modifying the antagonistic kinase/phosphatase equilibrium45. In the past, Maisch

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found that auxin controls its own transport by changing the state of actin filaments46.

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This interaction between auxin and actin has a subsequent effect on gravitropism in

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rice47. Flavonoids negatively affect actin expression in plants, and maybe this could

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explain actin-mediated auxin transport, and the various effects of flavonoids on auxin

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transport given the role of actin in vesicular trafficking and recycling. Li showed that

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rice morphology determinant, an actin-binding protein, mediates the auxin–actin loop

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pathway, thereby affecting cell development48. Another mode of regulation is the

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starch–statolith model, which suggests that gravity is sensed when the statoliths settle

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within the statocyte responding to changes in the orientation of a plant organ49. The

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mechanical stimulus of intracellular amyloplast redistribution is transduced into a

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biochemical signal, triggering a differential gradient of auxin in plant organs50-52.

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Plants perceive gravity and retain the orientation of their organs within the 16 ACS Paragon Plus Environment

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gravitational field through the process of gravitropism. In our study, we found that, in

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addition to roots and stems, eggplant fruit can also sense gravity. When CHS is

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silenced, the flavonoid content is decreased, and the fruit show a depressed

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gravitropic response. Either CHS or certain flavonoids could be related to the fruit

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gravitropic response; however, the mechanisms whereby they regulate fruit

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orientation, which flavonoids interfere with the related signal chains, and the identity

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of their molecular targets remain to be revealed.

348 349

Acknowledgments

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The authors thank Dr. S.P. Dinesh-Kumar (University of California, Davis) for

351

providing the pTRV1 and pTRV2 vectors.

352 353

Abbreviations used

354

CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavonol 3-hydroxylase;

355

F3’H:

356

dihydroflavonol 4-reductase; ANS: anthocyanidin synthase.

flavonol

3’-hydroxylase;

F3’5’H:

flavonol

3’5’-hydroxylase;

DFR:

357 358

Supporting Information

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Liquid chromatography-mass spectrometry chromatogram showing total ion current

360

(Figure S1); List of primers used in virus-induced gene silencing (Table S1);

361

Flavonoids identified in eggplant (Table S2).

362 17 ACS Paragon Plus Environment


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Funding sources

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This work was supported by the National Nature Science Foundation of China (Grant

365

No.: 31571898).

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503

Figures

504 505

Figure 1. Phenotypes of eggplant fruits infiltrated with different VIGS constructs: A:

506

TRV-empty vector; B: TRV-CHS. Scale bar equals 2cm. (C) Confirmation of TRV

507

infection in eggplant fruits by PCR. M means a 2-kb marker, 1: Negative control with

508

TRV alone; 2: The purple portion in CHS-silenced fruits; 3: The white portion in

509

CHS-silenced fruits. Oligonucleotides used are shown in Supplementary S1 Table.

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Figure 2. Gene expression profiles by qRT-PCR.

513

(A) Relative normalized expression of SmCHS1, SmCHS2 and SmCHS3 genes

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detected in the epicarp of agroinoculated VIGS-treated eggplant fruits. 18S

515

rRNA was used as an internal control.

516

(B) Expression of genes involved in the synthesis of flavonoids. According to

517

qRT-PCR data, expression of F3H, DFR , and F3’5’H was down-regulated in

518

CHS-silenced eggplant. Values represent mean ± s.e. (n=3). Student’s t-test was

519

used to assess whether the CHS-silenced eggplant significantly differs from

520

wild-type plants: *P value

Virus-Induced Gene Silencing of the Eggplant Chalcone Synthase

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