Comparative transcriptome analysis of celery leaf blades identified an

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Omics Technologies Applied to Agriculture and Food

Comparative transcriptome analysis of celery leaf blades identified an R2R3 MYB transcription factor that regulates apigenin metabolism Jun Yan, Li Yu, Shijun Lu, Longying Zhu, Shuang Xu, Yanhui Wan, Hong Wang, Ying Wang, and Weimin Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01052 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Journal of Agricultural and Food Chemistry

Comparative transcriptome analysis of celery leaf blades identified an R2R3 MYB transcription factor that regulates apigenin metabolism Jun Yan

E-mail:[email protected]

Li Yu

E-mail: [email protected]

Shijun Lu


Longying Zhu


Shuang Xu


Yanhui Wan


Hong Wang


Ying Wang


Weimin Zhu*

E-mail: [email protected] ; Telephone number: +86-025-67131604

*Corresponding author Jun Yan and Li Yu contributed equally to this work.

Horticulture Research Institute, Shanghai Academy Agricultural Sciences Key Laboratory of Protected Horticulture Technology Addresses:No.1000 Jin Qi Road, Fengxian District, Shanghai, China

1

ACS Paragon Plus Environment


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Abstract

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Apigenin has been proven to possess many pharmacological properties, but mechanism of regulation

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of apigenin biosynthesis in plants remains unclear. Apigenin is the main flavonoid in celery and is

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mainly accumulated in the middle stage of leaf blade development. In this study, comparative

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transcriptomic analysis revealed a large number of structural genes and transcription factor genes that

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may be involved in the apigenin metabolic pathway. Based on the apigenin content in different

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celery accessions, an R2R3-MYB transcription factor gene, named AgMYB1, was isolated from the

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high apigenin celery accession C014. Bioinformatics analysis indicated that AgMYB1 may be

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involved in flavonoid metabolism. AgMYB1 expression showed a positive relation with the

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expression of the apigenin accumulation marker gene FNSI and with the apigenin content in different

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celery tissues. Moreover, overexpression and antisense expression of AgMYB1 in transgenic celery

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plants significantly increased and reduced the expression of apigenin biosynthetic genes and the

13

apigenin content, respectively. These findings suggest that AgMYB1 is involved in positive

14

regulation of apigenin metabolism in celery.

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Key words: celery; leaf blade; apigenin; comparative transcriptome; R2R3-MYB transcription factor;

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overexpression and antisense expression; positive regulation

17 18 19 20 21 22 23 2


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Introduction

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Celery (Apium graveolens L.) is a widely consumed vegetable that originated from the

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Mediterranean and was introduced into China during the Han dynasty (second century B.C.). The

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Chinese celery variety “Shanghai yellow heart” is distributed in suburban farmlands of Shanghai and

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has light-green slender stalks, yellow interior leaves, a crisp and tender texture, and full-bodied

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flavor. Celery is rich in a variety of bioactive constituents, the principal flavonoid constituents of

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which are apigenin and luteolin1. The apigenin content was much higher than the luteolin content,

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and the apigenin content in the leaf blades was much higher than that in the leaf stalks 2. Marked

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differences in apigenin content have also been observed among different celery varieties 2, 3.

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Apigenin has been demonstrated to have medicinal value in the treatment of epilepsy 4, cancer 5, and

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hyperlipemia6. Compared with other flavonoids (such as quercetin, kaempferol, and luteolin),

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apigenin exhibits the lowest toxicity 7. To date, most studies have focused on the pharmacological

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action of apigenin, while the metabolic mechanisms of apigenin in plants, especially the regulatory

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mechanism, remain unclear.

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Illumina sequencing technology has been employed to elucidate secondary metabolic

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mechanisms in many plant species, such as taxane biosynthesis in Taxus yunnanensis 8, flavonoid

40

biosynthesis in lily (Lilium 'Sorbonne') 9,and saponin biosynthesis in Chlorophytum borivilianum10.

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Celery has 2n=2x=22 chromosomes, with a large (3×109 bp) but poorly characterized genome.

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Recently, Illumina sequencing technology has been used to analyze transcriptomes from the roots,

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stems, petioles, and leaves of celery 11. Transcriptomic analysis of different growth stages

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that lignin accumulation is correlated with the transcript levels of genes involved in lignin

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biosynthesis12. In our previous showed show rapid accumulation of apigenin in the middle stage of

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celery leaf blade development, and the expression levels of CHS, CHI and FNSI were found to be

indicated

3



47 48

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associated with apigenin biosynthesis13. Additionally, it has been confirmed that transcription factors (TFs), such as MYB, bHLH

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proteins, and WD-repeat protein regulate flavonoid accumulation in plants 14. However, the

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regulatory process of apigenin metabolism in plants has not been reported. MYB proteins are a large

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and versatile TF family, and R2R3-MYBs can positively or negatively control the secondary

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metabolism in plants 15, including flavonoid16, phenylpropanoid17 and benzenoid18 metabolism. In

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this study, based on our previous studies, comparative transcriptome analyses were carried out

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among three developmental stages of celery leaf blades to identify candidate genes associated with

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apigenin metabolism. A larger number of unigenes encoding structural genes and TF genes were

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obtained, and the transcript levels of structural genes were consistent with the results of our previous

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research. Therefore, we focused on the transcriptional regulation of apigenin metabolism. Ultimately,

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we discovered an R2R3 MYB that is positively associated with apigenin accumulation. The function

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of this R2R3 MYB gene was then validated.

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Materials and methods

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Plant materials

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Seeds of 28 celery accessions, including 12 high-apigenin accessions and 16 low-apigenin

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accessions2, were sown in 50-well trays containing peat:perlite (3:1, v/v). Seedlings (15 cm in height

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and with four leaves) were transferred into pots (diameter 15 cm, height 25 cm), with one seedling

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planted in each pot. After 65 days, the plants were divided into seven leaf blade stages. The plant

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growth conditions and the seven leaf blade stages were described in our previous report 13.

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Leaf blades at stages 2, 5, and 7 of C014 “Shanghai yellow heart” were selected for

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transcriptome sampling based on an assessment of the apigenin concentrations 13. Seven leaf blade

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stages were sampled from 28 celery accessions for apigenin accumulation and candidate gene 4


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expression. All the collected samples were immediately frozen in liquid nitrogen and stored at -80°C

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until further experimentation. All experiments were performed with three biological replicates.

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Illumina sequencing, functional annotation, and differential expression analysis

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Total RNA from three leaf blade stages of C014 was isolated using TRIzol reagent (Invitrogen,

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Carlsbad, CA, USA) and purified with RNase-free DNaseI (TaKaRa, Japan). Three cDNA libraries

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were constructed using the TruSeqTM RNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA).

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These cDNA libraries were sequenced using Illumina HiSeqTM 2500 platform. After removal of

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adapter sequences and low-quality reads ( stage 5 vs. stage 7, which is

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consistent with the developmental speeds of the three stages. Temporal expression pattern of DEGs

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indicated that three profiles were significant expression clusters which show a down-regulated trend

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with the development of leaf. And most DEGs included in these three profiles participate in various

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kinds of secondary metabolic pathways. These results indicated that secondary metabolism was more

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active in the early stage of leaf development.The DEGs detected by qRT-PCR exhibited high

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similarity with the transcriptomic analysis results (correlation coefficient=0.869), suggesting that the 14


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DEGs identified in this study could be used for further analyses.

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Celery leaf contain an abundance of beneficial bioactive substances, a majority of which have

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been proven to be beneficial for the treatment or alleviation of diseases such as cancer, hypertension,

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hyperlipidemia and arthrolithiasis16. The results of the KEGG pathway and KEGG pathway

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enrichment analysis indicated “biosynthesis of secondary metabolites” to be a very important

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pathway in celery leaf. With the exception of flavonoids, DEGs involved in biosynthetic pathways of

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other bioactive substance were also enriched , such as those involved in “sesquiterpenoid and

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triterpenoid biosynthesis”, “stilbenoid, diarylheptanoid and gingerol biosynthesis”, “linoleic acid

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metabolism”, “carotenoid biosynthesis”, “glucosinolate biosynthesis”, “biosynthesis of unsaturated

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fatty acids”, “ascorbate and aldarate metabolism”, and “limonene and pinene degradation”. These

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findings could be used to explore the metabolism mechanism of these bioactive substances in celery.

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Structural genes and TF genes involved in apigenin metabolism

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PAL, C4H, 4CL, and CHS convert into phenylalanine to chalcones. Then, CHI catalyzes the

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conversion of chalcones to naringenins or flavanones. These upstream steps of the pathway are

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highly conserved and common to all branches 29. Naringenins are converted to apigenins and

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luteolins by FNSI and F3’H, respectively 24. In this study, all DEGs encoding CHS, CHI, and FNSI

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were positively associated with apigenin accumulation, and these DEGs showed significantly higher

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expression in stage 2 than in stage 5 and stage 7. However, the DEGs encoding F3’H were negatively

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associated with apigenin accumulation. These results were consistent with the observed apigenin

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accumulation and with our previous experimental results 13. The accumulation of secondary

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metabolites has been reported to be positively correlated with the expression of structural genes, such

321

as those associated with taxane biosynthesis in T. yunnanensis8, catechin biosynthesis in Camellia

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sinensis 30, and flavonoid biosynthesis in lily 9. 15



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In our previous study, we found that the accumulation of apigenin in celery tissues was

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temporally and spatially controlled by the expression level of structural genes associated with

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apigenin metabolism13. However, the regulatory mechanism of apigenin metabolism is unclear. In

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this study, we identified 181 DEGs encoding TFs, such as MYB, bHLH, WD40, WRKY, NAC and

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AP2/ERF. In plants, MYB, bHLH, and WD40 are able to act individually or in combination with

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other TFs to regulate a series of enzymes involved in flavonoid metabolism in several species

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The other TFs identified among the DEGs are implicated in various biological processes and

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abiotic/biotic stimuli, including organ and tissue differentiation, seed maturation, embryo and

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vascular patterning, cold stress, hormone and sugar signaling, and light responses

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years, WRKY, NAC and AP2/ERF TFs have been shown to participate in the regulation of

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secondary metabolites. For example, WRKY and AP2/ERF can promote artemisinin accumulation in

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Artemisia annua

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expression of structural genes that participate in metabolic pathways. In addition, AP2/ERF was

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found to regulate nicotine biosynthesis in tobacco 39. NAC can modulate carotenoid biosynthesis by

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activating phytoene desaturase genes during fruit ripening 40. In this study, the expression levels of

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52 DEGs encoding TFs were positively or negatively correlated with apigenin accumulation in three

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leaf developmental stages; thus, these TFs may be candidate genes that participate in the regulation

340

of apigenin metabolism.

341

Identification of the AgMYB1 gene

35,36

and vincristine accumulation in Catharanthus roseus

37,38

32-34.

14,31.

In recent

by improving the

342

MYB TFs have been reported to independently regulate the early stage flavonoid biosynthesis

343

genes. For example, in Ginkgo biloba, GbMYBF2 plays a crucial role in restraining transcriptional

344

regulation in flavonoid biosynthesis

345

biosynthesis pathway in soybean

42.

41,

and GmMYB100 negatively regulates the flavonoid

Apigenin is one of the early products of the flavonoid 16


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biosynthesis pathway, which is a simple metabolic pathway. In this study, qRT-PCR analysis of

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accessions with different apigenin content and seven leaf blade developmental stages indicated that

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the expression pattern of unigene 27341, encoding an MYB gene, may regulate the accumulation of

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apigenin in celery. Thus, the sequence of unigene 27341 was used to identify the AgMYB1gene.

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Bioinformatic analysis of AgMYB1

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Based on the sequence of unigene 27341, the 852-bp full-length cDNA of the R2R3-MYB gene

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AgMYB1 was isolated from celery accession C014. At the genomic level, AgMYB1 has three exons;

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the R2 domain encompasses exons 1 and 2, whereas the R3 domain spans exons 2 and 3. The

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genomic structure of AgMYB1 is similar to that of the reported R2R3 MYBs

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sequence of AgMYB1 shares high identity with carrot MYB proteins DcMYB308 and DcMYB3-1

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which were not assigned biological functions to date. The phylogenetic analysis classified AgMYB1

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into Arabidopsis subgroup 5 MYB proteins (AtMYB123), which control the biosynthesis of

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proanthocyanidins in the seed coat

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regulates anthocyanin biosynthesis in vegetative tissues

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might participate in the regulation of flavonoid metabolism in celery.

43.

27.

The amino acid

AgMYB1 was also closely related to subgroup 6, which 44.

These results indicated that AgMYB1

361

Due to the highly variable C-terminal, MYBs have a complex regulatory network. The Malus

362

pumila MYB3 protein can induce anthocyanin accumulation, but is similar to AtMYB3, AtMYB4,

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and AtMYB7, which usually function as repressors 45. In Fagopyrum tataricum, FtMYB15 clustered

364

with Fragaria×ananassa FaMYB1, but shares low amino acid sequence identity at the C-terminal

365

and functions as a positive and negative regulator of proanthocyanidins and anthocyanin biosynthesis,

366

respectively 46. In G. biloba, activation and repression motifs coexist in the C-terminal of GbMYBF2,

367

and GbMYBF2 is a negative regulator of flavonoid biosynthesis41. In this study, the N-terminal of

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AgMYB1 was homologous to both transcriptional repressors, including AtMYB3, AtMYB4, and 17



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AmMYB308, and transcriptional activators, including AtMYB123, ZmC1 and HvMYB1, which

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negatively or positively regulate flavonoid biosynthesis. The sequence alignment indicated that

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AgMYB1 had only 40.56% amino acid sequence identity with AtMYB123. Protein motif analysis

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showed that AgMYB1 does not have a TT2 domain(V[I/V]RT[K/R]A[I/T]RCS), but shares one

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conserved motif with HvMYB1, AtMYB123 and AtMYB5. This phenomenon suggests that

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AgMYB1 has a complex regulatory network, that may has similar regulatory functions as

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AtMYB123, and meanwhile possesses special regulatory mechanisms.

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Expression analysis of AgMYB1 in different celery tissues

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The expression level of AgMYB1 varied among the different celery tissues. According to the

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apigenin biosynthesis pathway, PAL, C4H, 4CL, CHS, CHI, FNSI, F3’H and F3H are the structural

379

genes involved in apigenin biosynthesis24,25. Our previous study indicated that the expression of CHS,

380

CHI and FNSI was positively correlated with apigenin accumulation

381

pattern of AgMYB1 was similar to those of CHS, CHI and FNSI in the different celery tissues. We

382

thus speculated that AgMYB1 may play a positive regulatory role in apigenin biosynthesis in celery.

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Functional characterization of AgMYB1

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To further verify the function of AgMYB1 in apigenin metabolism in celery, AgMYB1 and

385

antisense-AgMYB1 driven by the CaMV 35S promoter were transformed into celery accession C014.

386

Over-expression of AgMYB1 in celery significantly increased apigenin accumulation, but antisense

387

expression of AgMYB1 significantly decreased the apigenin content. These results provide strong

388

evidence of the involvement of AgMYB1 in positive regulation of the apigenin metabolic process.

389

13.In

this study, expression

It was reported that the R2R3-MYB TF gene could regulate secondary metabolite contents by

390

controlling structural genes, such as those involved in flavonoid biosynthesis in G. biloba

391

anthocyanin biosynthesis in Oenanthe javanica

49,

41,

and the phenolic acid biosynthetic pathway in 18


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392

Salvia miltiorrhiza Bge. f. Alba50. The effect of AgMYB1 on the structural genes involved in apigenin

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biosynthesis indicated that compared with WT, the expression of CHS, CHI, and FNSI was

394

upregulated in the AgMYB1-OE lines and downregulated in the AgMYB1-AT lines, while the

395

opposite trend was observed for PAL expression. PAL catalyzes the first step in phenylpropanoids

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biosynthesis, including flavonoid, stilbenoid, and lignin29. However, MYB TFs can regulate the

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accumulation of many kinds of secondary metabolites.For example, over-expression maize

398

R2R3-MYB (ZmMYB31) could promote flavonoid accumulation but repress lignin accumulation 47,

399

and Grape R2R3-MYB (VvMYB5a) could affect the metabolism of anthocyanin, flavone, tannin, and

400

lignin48. Further studies are necessary to elucidate the mechanism of regulation offered by AgMYB1

401

on other phenylpropanoid pathway in celery. Earlier, it has been demonstrated that certain members

402

of MYB family transcription factors are regulators of transcription of structural genes of

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phenylpropanoid biosynthesis42. Therefore, in future, it will be interesting to study whether AgMYB1

404

directly regulates the expression of PAL, CHS, CHI and FNSI genes in celery.

405

Abbreviations

406

TFs: transcription factors; MYB: myeloblastosis; bHLH: basic helix-loop-helix; WD-repeat protein:

407

tryptophan-aspartic acid repeat protein; FPKM: fragments per kb per million fragments mapped;

408

DEGs: differentially expressed unigenes; qRT-PCR: quantitative real time PCR; HPLC:

409

high-performance liquid chromatography; ORF: open reading frame; CaMV 35S: cauliflower mosaic

410

virus 35S; MS: Murashige and Skoog; Km: kanamycin; CTAB: cetyltrimethylammonium bromide;

411

PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumarate CoA ligase;

412

CHS: chalcone synthase; CHI: chalcone isomerase; FNSI: flavone synthase

413

Acknowledgments

414

We thank the editors and reviewers for their critical reading and constructive suggestions. This work 19



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was funded by the National Natural Science Foundation of China (no.31601752), Green Leafy

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Vegetables Industrial Technology System of Shanghai---Breeding Group (Hu nong ke chan zi no.2)

417

and the Shanghai Academy of Agricultural Sciences “Climbing Project” (PG141) and “Excellent

418

team”(ZY1602).

419

Supporting Information

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Table S1 Candidate MYB sequences used for qRT-PCR

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Table S2 Accession numbers of MYB proteins listed in Figure 5 and Fig.S3

422

Table S3 Primers used for qRT-PCR

423

Table S4 KEGG pathways of the assembled transcripts

424

Table S5 KEGG enrichment pathways of all DEGs

425

Table S6 The DEGs involved in flavonoid biosynthesis

426

Table S7 qRT-PCR verification of selected DEG

427

Table S8 The DEGs identified as transcription factors family

428

Fig. S1 qRT-PCR expression assays of putative MYB transcription factors genes in leaf stage 4 of 16

429

low apigenin accessions and 12 high apigenin accessions.

430

Fig. S2 qRT-PCR expression assays of Unigene27341 in seven leaf blades stages of 16 low apigenin

431

accessions and 12 high apigenin accessions Fig. S3 Phyogenetic relationship of AgMYB1 with Arabidopsis MYB transcription factors. Phyogenetic tree was construted online with the IT3F website. The clades of Arabidopsis were classified as previously report (Stracke et al., 2001 and and Dubos et al.,2010 ; highlighted with colored diamonds for each subgroup), and AgMYB1(MH538294) belong to subgroup 5. Accession numbers for all protein sequences are listed in Supplemental Table S2.

20


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Fig. 1 Temporal expression pattern of DEGs. A. Cluster analysis of the expression profiles of the DEGs. Profiles blocks with colored background are significant clusters of the P-value≤0.05. B. A bubble map shows the top 20 significantly enriched KEGG pathways among the DEGs of significantly clustered profiles. The x-axis shows the enrichment factor (the enriched gene number proportion of background gene number) of each enriched pathway. The color of the round dots represents the Q-value of each pathway and the size represents the number of enriched unigenes. Fig. 2 Correlation of gene expression results obtained from qRT-PCR and RNA-Seq Fig. 3 Simplified apigenin metabolism pathway Fig.4 Bioinformatics analysis of AgMYB1 gene. A. Structural organization of AgMYB1 gene. The schematic diagram showing the exons(labeled boxes), introns(labeled solid lines), R2 domain(gray backgrounds), and R3 domain(black backgrounds). B. Multialignment of amino acid sequences of AgMYB1 with others plant R2R3-MYB proteins. Black indicates identical amino acids and gray shows similar amino acids. Lines above the sequence highlight the R2 and R3 domains. C. Schematic diagram of the comparison of AgMYB1 with proteins of the nearest clade in the phylogenetic tree showing the putative motifs. Each box indicates a putative motif. Fig. 5 Phylogenetic relationship of AgMYB1 with other plant MYB transcription factors. Phylogenetic tree was constructed using MEGA7 software with the neighbor-joining method (1000 replicates). The bootstrap values are shown as percentages when greater than 50%. The clades of Arabidopsis were classified as previously report (Stracke et al., 2001and Dubos et al., 2010; highlighted with colored diamonds for each subgroup), and AgMYB1(MH538294) classified with carrot MYB and ZmC1 sequences (marked with blue circles). Accession numbers for all protein sequences are listed in Supplemental Table S2. 27



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Fig. 6 Expression assays of AgMYB1 and apigenin biosynthetic marker gene FNSI in different tissues of celery accession C014 (high apigenin accession) and C024 (low apigenin accession). The data points are mean of three biological replicates±SD. The different letter on each bar show significant differences among different tissues in accession C014 and C024. Fig.7 Transgenic detection. A. PCR detection of the fragments (35S promoter +AgMYB1). M, Marker DL2000; 1, WT; 14, empty vector; 2-8, OE-transgenic lines; 9-13, AT-transgenic lines. This figure only shows a part of the results. B. Major gene transformation processes. a. calluses growing on kanamycin medium, b. survival calluses growing on differentiation medium, c. development of young shoots, d-f. transgenic plant growing on plant medium. Fig. 8 qRT-PCR analysis of AgMYB1 and apigenin biosynthetic structure gene in leaf stage 4 of overexpression and antisense expression AgMYB1 in accession C014. The data points are mean of three biological replicates±SD. The different letter on each bar show significant differences(P

Comparative transcriptome analysis of celery leaf blades identified an

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