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Chemistry and Biology of Aroma and Taste

Identification of Two Transcriptional Activators MabZIP4/5 in Controlling Aroma Biosynthetic Genes during Banana Ripening Yu-fan Guo, Yun-liang Zhang, Wei Shan, Yong-jian Cai, Shumin Liang, Jian-ye Chen, Wang-jin Lu, and Jian-fei Kuang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Journal of Agricultural and Food Chemistry

Identification of Two Transcriptional Activators MabZIP4/5 in Controlling Aroma Biosynthetic Genes during Banana Ripening Yu-fan Guo†, Yun-liang Zhang†, Wei Shan, Yong-jian Cai, Shu-min Liang, Jian-ye Chen, Wang-jin Lu, Jian-fei Kuang*

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Key Laboratory for Postharvest Science, College of Horticultural Science, South China Agricultural University, Guangzhou 510642, PR China

*Corresponding author: Phone: +86-020-85285523 Fax: +86-020-85285527 Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: The transcriptional regulation of aroma formation genes remains poorly understood in

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banana. In this work, we found that the expressions of a subset of aroma biosynthetic genes including

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MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT1 and BanAAT, as well as two bZIP

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genes MabZIP4 and MabZIP5, were down-regulated when pre-stored at 7 °C compared to those

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pre-stored at 22 °C during the ripening process of banana. Furthermore, MabZIP4 and MabZIP5

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were shown to be able to activate the transcription of these aroma biosynthetic genes. Importantly,

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MabZIP4 directly binds to BanAAT promoter, while MabZIP5 binds to the promoters of MaMT1,

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MaACY1, MaAGT1 and BanAAT via the G-box motif, implicating the diverse functional

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significances of MabZIPs in controlling aroma biosynthesis in banana. Overall, this work sheds new

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insights on the understanding of transcriptional regulatory mechanisms associated with aroma

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formation during banana ripening.

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KEYWORDS: aroma biosynthetic genes, banana fruit, bZIP, gene expression, transcriptional

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regulation, ripening

14 15 16 17 18 19 20 21 22 2


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INTRODUCTION

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Aroma compounds in plants are vital secondary metabolites which provide important cues to

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pollinators and seed dispersers, thus enabling plant reproductive and evolutionary success.1 In fruits,

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aroma is a key trait that strongly associates with fruit quality by affecting the sensory perception and

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consumer preference. Moreover, aroma is also used as an important indicator for assessing fruit

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ripening, as specific volatiles appear at different ripening stages. The aroma compounds of fruits

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belong to several chemical groups, including esters, alcohols, aldehydes, acids, ketones and terpenes,

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depending on the variety, developmental stage, postharvest handling and storage conditions.2 For

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example, in ripe banana more than 250 aroma compounds have been found, with the majority of

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which are esters such as 3-methylbutyl acetate and 2-methylpropyl acetate, contributing to the major

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characteristic fruity odors of banana.3

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The biosynthesis of esters has been extensively studied in fruits. For example, in apples, the most

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abundant volatile compounds are straight chain esters derived from free fatty acids including linoleic

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(18:2) and linolenic (18:3) acids and branched chain esters originated from amino acids such as

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valine, leucine and isoleucine.4 Fatty acid metabolism comprises two processes, β-oxidation and

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lipoxygenase (LOX) pathway. In general, long-chain saturated fatty acids form shorter chains via the

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β-oxidation, whereas polyunsaturated fatty acids yield volatile ester precursors through the LOX

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pathway. Both fatty acid and amino acid pathways can give rise to the production of aldehydes,

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followed by reduction to alcohols by alcohol dehydrogenase (ADH).5 The last step of ester

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generation is required the action of alcohol acyl transferase (AAT), a multi-gene family that shows

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substrate specificity.6 In banana, the branched-chain esters and related alcohols are derived from

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amino acids such as leucine and valine, while the straight-chain esters are produced via fatty acid 3



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metabolic cascade and enzymatic oxidation of linoleic and linolenic acids.7 A better understanding of

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genes responsible for aroma formation is essential for manipulating fruit flavor. Genes encoding

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enzymes for the important steps of aroma production in banana have been identified, which include

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BanLOX, BanPDC, BanHPL, BanBCAT, BanADH and BanAAT.6,8 A recent study has revealed a

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series of genes involved in aroma and flavor compound production, including those associated with

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ester formation from amino acids, saturated and unsaturated fatty acids such as lipoxygenases,

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transferases and acetyltransferases.9 It is suggested that the production of fruit esters is controlled at

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least partially by the expression of aroma biosynthetic genes. More recently, protein-protein

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interaction between MaDof23, a transcriptional repressor, and MaERF9, a transcriptional activator,

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antagonistically affects the transcription of 10 fruit ripening-related genes including the aroma

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biosynthesis genes MaCAT and MaPDC.10 Additionally, two ERF-type transcription factors namely

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CitAP2.10 and CitERF71 acted as transcriptional activators of CitTPS genes that were involved in

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monoterpene E-geraniol synthesis in sweet orange fruit.11,12 These findings suggest that

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transcriptional regulation of aroma formation genes could be a potentially effective strategy to

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improve fruit aroma.

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Transcriptional regulation mediated by transcription factors (TFs) is a pivotal control hierarchy for

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various plant biological processes.13 Based on their DNA-binding domains, plant TFs can be

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categorized

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2/ethylene-responsive element binding factor (AP2/ERF), basic helix-loop-helix (bHLH), WRKY,

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basic leucine zipper (bZIP), and so on.14 The bZIP TFs are a kind of proteins structurally defined by

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a motif consisting of a basic DNA-binding domain and heptad repeats of leucine or other

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hydrophobic amino acids for dimerization.15 Numerous studies have indicated that bZIP TFs played a

into

different

families,

such

as

NAM/ATAF1/CUC2

4


(NAC),

APETALA

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vital role in plant development and adaptation to biotic and abiotic stresses.16 Moreover, some bZIP

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TFs were shown to participate in hormone signaling mediated by ABA,17 GA18 and BR.19 More

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specifically, several members of bZIP TFs are involved in fruit ripening. For example, activity of

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VvABF2, a bZIP TF from grape, was found to be increased during berry ripening and was

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up-regulated by ABA treatment. Over-expression of VvABF2 in tomato resulted in reduced fruit

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firmness and accelerated ripening.20 In addition, tomato fruits transformed with SlbZIP1 and SlbZIP2

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driven by a fruit-specific E8 promoter exhibited higher content of sugar and amino acids such as

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asparagine and glutamine.21 However, whether bZIP proteins are involved in aroma formation during

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fruit ripening is yet to be elucidated.

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In the present study we found that banana pre-stored at 7 °C showed abnormal ripening symptoms

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manifesting as incomplete softening and off-flavor. Moreover, we examined the expression of several

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genes encoding aroma biosynthetic enzymes during the ripening of bananas pre-stored at 7 °C and

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22 °C, respectively. More importantly, we identified two bZIP TFs, namely MabZIP4 and MabZIP5,

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which operate as transcriptional activators of the aroma biosynthetic genes in banana. Molecular

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characterization of MabZIP4/5 suggests a controlling mechanism underlying the regulation of

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banana aroma biosynthesis, which might be a potential strategy for flavor improvement in banana

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fruits.

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MATERIALS AND METHODS

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Chemicals. Sporgon that was used for fungicide was purchased from Bayer Crop Science

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Company (Hangzhou, China). Analytical grade of ethylene gas was purchased from Junduo Gas

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Company (Guangzhou, China). The anlaytical grade of Tris base, boric acid, EDTA were purchased 5



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from Sigma-Aldrich (Shanghai, China).

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Plant materials and treatment. Mature green banana fruit (Musa spp. AAA Group, cultivar

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‘Brazil’) were harvested from a local orchard in Guangzhou (China). Fruits were cut into individual

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fingers, and then dipped in 0.05% Sporgon for 3 min to control fruit rot. The fruits were selected for

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uniformity of size and weight, and then were divided into two groups, in which one group (chilling

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treatment group) were stored at 7 °C for 3 days to induce the development of chilling injury

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symptoms, and the other group (non-chilling treatment group) were held at 22 °C for 3 days as

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control. Subsequently, these two groups of bananas were treated by 100 µL L–1 ethylene at 22 °C for

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16 h to initiate fruit ripening, and then stored at 22 °C until fully ripe. Samples of both groups were

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taken at 0, 1, 3, 5 and 7 days of storage on the basis of their ripening progression, and then frozen

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with liquid nitrogen and kept at -80 °C for subsequent use. Each treatment had three replicas.

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Assessment of ripening parameters. Fruit ripening was assessed by three parameters in terms of

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peel color, pulp firmness and ethylene production. Peel color was determined by measuring the

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lightness, chromaticity and hue angle with a Minolta chroma meter (model CR-300), as reported

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previously.22 Fruit firmness was analyzed by a penetrometer (model no.5542; Instron) fitted with a

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cylindrical flat-surfaced plunger (6 mm diameter), according to the previous works.22 For ethylene

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evaluation, a 1-mL sample was taken from the headspace of the jars and injected into a Shimadzu gas

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chromatograph (model no.GC-17A) equipped with a FID detector and an activated alumina column

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(200 cm × 0.3 cm), according to Han et al. (2016).22

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RNA preparation and gene cloning. Total RNA was prepared according to the hot borate

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procedure.23 Genomic DNA contaminants were cleared away from total RNA by means of

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RNAse-free DNase (Promega, USA). And then the DNA-free total RNA was reverse-transcribed for 6


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producing the first-strand cDNA. According to the previous findings,6,9 MaOMT1 (Ma04_t20350),

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MaMT1

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(Ma03_t12120), MaAGT1 (Ma04_t20850) and BanAAT (AX025506) genes which were found to be

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increased in the ripening stage were selected in this study. In addition, two bZIP genes

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(Ma08_t11830 and Ma05_t26820), which were also increased in the ripening phase on the basis of

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our transcriptomic dataset,24 were also chosen in the current work, and they were named as MabZIP4

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and MabZIP5 after the name of MabZIP1-3 in our previous findings.25 Comparison of amino acid

118

sequences and phylogenetic tree was conducted using Clustal W (version 1.83), GeneDoc and

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MEGA6.0 software. The protein sequences for constructing phylogenetic tree are listed in Text S1.

(Ma04_t32060),

MaGT1

(Ma11_t21870),

MaBCAT1

(Ma08_t04930),

MaACY1

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Quantitative real-time PCR. The cDNA was generated as described above, and quantitative

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real-time PCR (RT-qPCR) reactions were carried out as depicted earlier.26 The gene-specific

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oligonucleotide primers for RT-qPCR assays are illustrated in Table S1. Banana MaRPS2 (ribosomal

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protein 2) was used as an endogenous control.26 The relative expression levels of target genes were

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calculated according to the formula 2–∆∆Ct. Three independent biological repeats were used in the

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analysis.

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Subcellular localization. The open reading frames of MabZIP4/5 were subcloned into the

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pEAQ-HT-GFP vector to generate MabZIP4/5-GFP, respectively. Sequences of primers used are

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provided in Table S1. The fusion plasmids and control vector (pEAQ-GFP) were separately

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introduced into Nicotiana benthamiana leaf by Agrobacterium-mediated infiltration according to

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described procedures.27 Fluorescence was visualized after 2 days of infiltration with a fluorescence

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microscope (Zeiss Axioskop 2 Plus).

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Dual-luciferase transient expression assay. The dual reporter vector which involves a native

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GAL4-LUC (firefly luciferase), and an internal control REN (renilla luciferase) driven by the 35S 7



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promoter, was used according to our previous reports.24 To test the transcriptional activities of

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MabZIP4/5, the full-length regions of those genes were cloned and gave rise to the re-constructed

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pEAQ-BD-MabZIP4/5 as effectors. Expression of pEAQ-BD-VP16 served as the positive control.

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To assess the interaction of MabZIP4/5 with the promoters of aroma formation-related genes

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MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT1 and BanAAT, their promoters were

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each inserted into pGreenII 0800-LUC vector, while MabZIP4/5 was cloned into the pEAQ vector as

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effectors, as described.28 The related primers are displayed in Table S1. The constructed reporter and

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effector plasmids were delivered into Agrobacterium tumefaciens strain EHA105, and co-transfected

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into tobacco leaves. After 2 days of infiltration, LUC and REN activities were evaluated using the

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dual-luciferase assay reagents (Promega) according to the manufacturer’s instructions. Six

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independent biological replicates were measured in each test.

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Protein purification and gel shift assay. The C-terminus and full-length coding sequences of

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MabZIP4/5 which contain the DNA-binding domains were cloned into pGEX-4T-1 to produce

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GST-MabZIP4-C and GST-MabZIP5, respectively. The recombinant proteins were then expressed in

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Escherichia coli strain BM Rosetta (DE3) after 6 h induction at 30 °C with 1 mM isopropyl

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β-D-1-thiogalactopyranoside, and affinity purified by glutathione Sepharose 4B resin (Clontech)

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following the manufacturer’s guidelines. DNA fragments (59 bp) containing G-box (CACGTG) in

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the promoters of aroma production genes were labeled with biotin using PierceTM Biotin 3' End DNA

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Labeling Kit (Thermo Scientific). Double-strand biotin-labeled, competitive or mutative probes were

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yielded by annealing the labeled, unlabeled or mutated sequences, respectively. DNA-protein

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interactions were performed in 20-µL reactions that included 2.0 µL 10 × binding buffer (100 mM

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Tris HCl, 250 mM KCl, and 10 mM DTT), 1 µg poly dI-dC, 2.5% glycerol, 0.05% Triton X-100, 5

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mM MgCl2, 10 mM EDTA, 10 ng DNA, and 0.5 µg protein and were then incubated at room

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temperature for 25 min. For the competition assays, unlabeled wild-type or mutant oligonucleotides

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(50- and 100-fold of labeled probes) were added to the reactions, respectively. The reaction products 8


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were run on 6% native polyacrylamide gel in 0.5 × Tris-borate/EDTA at 100 V. Gels were transferred

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to a positively charged nylon membrane (PALL) using a wet transfer cell (Bio-Rad), and the DNA

161

was cross-linked to the membrane using a UV linker. The DNA signal was determined using the

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Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific) in accordance with the

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manufacturer’s protocol. The DNA primer sequences for this assay are listed in Table S1.

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RESULTS

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Effects of chilling treatment on banana fruit ripening. Banana generally develops visual

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chilling injury symptoms such as browning and pitting when preserved at 7 °C for 3 days. In this

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study, banana fruit pre-stored at 7 °C (chilling) or 22 °C (non-chilling) were treated with 100 µL L–1

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ethylene to promote ripening. To compare the physiological parameters related to fruit ripening

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between chilling and non-chilling groups, the peel color, fruit texture and ethylene emission were

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monitored. Figure 1 shows that banana pre-stored at 7 °C for 3 days displayed a delay in ripening

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compared to those pre-stored at 22 °C. Ripening occurred from the 3rd day in storage for the

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non-chilling treated banana, while the chilled fruit started to ripen after 5 days storage, as indicated

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by the changes of peel color (Figure 1a). The lightness and chromaticity in non-chilled banana were

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increased with the progression of ripening and peaked at the 5th day with maximal levels of 73.88

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and 49.78, respectively. Although in the chilling treated fruit the change of lightness and

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chromaticity exhibited similar patterns, their values were significantly lower than in non-chilling

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treated group (Figure 1b). Moreover, the hue angle value which is associated with the change of peel

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color from green to yellow, decreased from 114.45 to 81.80 with 7 days in storage in non-chilling

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fruit, but the value changed at a lower rate in fruit with chilling treatment, indicating an inhibition of

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chlorophyll breakdown in chilled fruits during ripening (Figure 1b). Similar to the change in hue 9



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angle, the fruit firmness in non-chilling treated fruit had a gradual decline with storage and it

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decreased to its lowest value on day 7, while the reduction of fruit firmness in chilling-treated fruit

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occurred at a much lower pace (Figure 1b), suggesting that chilling induced the inhibition of banana

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fruit softening. In addition, the ethylene production peaked on days 3 and 5 with non-chilling and

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chilling treatments, with maximal levels of 4.65 and 2.35 µLg-1h-1, respectively (Figure 1b). It should

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be pointed out that the total ethylene production in chilling treated banana was lower than that in

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non-chilling treated ones, particularly on day 3. The ethylene production in non-chilled fruit was

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1.98-fold higher than that of fruit treated with chilling on day 5, signifying the variation in ripening

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caused by chilling treatment.

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Expression of aroma formation related genes in banana fruits affected by chilling treatment.

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We tested the dynamic changes of the transcript levels of aroma biosynthesis genes including

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MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT19 and BanAAT6 in chilling and

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non-chilling treated bananas during fruit ripening. During the ripening of banana without chilling

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treatment, the expression of MaOMT1, MaMT1, MaACY1 and MaAGT1 were continuously increased

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throughout the storage period, while MaBCAT1 and MaGT1 began to increase at day 1 after ethylene

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treatment and maintained high levels until day 5 followed by a slight decline. Moreover, BanAAT

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was found to be increased at day 1 and peaked at day 3 but decreased dramatically afterwards

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(Figure 2). Notably, pre-treatment of banana at chilling temperature for 3 days significantly

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suppressed gene expression during fruit ripening, though their overall expression pattern showed an

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increased tendency during the experiment.

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Characterization of MabZIP4/5. The expression pattern of MaOMT1, MaMT1, MaGT1,

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MaBCAT1, MaACY1, MaAGT1 and BanAAT suggestS an association of these genes with the aroma 10


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emission during fruit ripening. To ascertain the possible upstream regulators of these genes, we

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carefully checked the promoter sequences of these genes, and found that most of them contained one

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or two G-box motifs which are typical bZIP TFs binding cis-elements (Text S2). Based on these

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findings, we screened our transcriptomic database related to banana fruit ripening24 and found two

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bZIP TFs named as MabZIP4 and MabZIP5, which were markedly up-regulated during banana

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ripening. Phylogenetic analysis of MabZIP4/5 with the bZIP gene family from Arabidopsis reveals

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10 subfamilies, namely A, B, C, D, E, F, G, H, I and S,15 in which MabZIP4 belongs to subfamily I

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while MabZIP5 resides to subfamily A (Figure S1). Amino acid alignments showed that both

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MabZIP4 and MabZIP5 contain a conserved bZIP domain comprising a 25-amino acid basic region

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and a leucine zipper consisting of three leucine repeats (Figure S2). Using RT-qPCR, we examined

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the expression profile of MabZIP4 and MabZIP5 in bananas pre-treated with or without chilling

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during the ripening. In accordance with our transcrptome data,24 the RT-qPCR results showed that

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MabZIP4 and MabZIP5 were gradually increased during ripening of non-chilled bananas (Figure 2).

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Pre-treatment of bananas with chilling temperature resulted in similar trends but with a relative lower

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accumulation of MabZIP4 and MabZIP5 transcripts throughout the whole storage period studied

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(Figure 2), indicating that chilling temperature treatment may greatly impact the expression of

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MabZIP4 and MabZIP5.

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To test whether MabZIP4/5 function as transcription factors, we examined the subcellular

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localization of MabZIP4/5 by fusing their coding regions in front of the green fluorescent protein

223

(GFP) following transient expression in Nicotiana benthamiana leaves. A construction with GFP

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alone served as a negative control. The results showed that the two 35S::MabZIP4/5-GFP proteins

225

were both exclusively localized in the nucleus in contrast to the free GFP showing distribution 11



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uniformly in the whole cells as expected (Figure 3a), suggesting that MabZIP4/5 are nuclear proteins.

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To study the transcriptional activity of MabZIP4/5, we used the luciferase-based transient expression

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systems in Nicotiana benthamiana leaves. The reporter construct contains five repeats of the GAL4

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DNA-binding element and minimal 35S promoter fused to the Firefly luciferase (LUC) reporter as

230

well as a 35S promoter-driven Renilla luciferase (REN) reporter gene as an internal control, while

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effector constructs include 35S promoter-driven transcriptional activator GAL4 DNA binding

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domain fused with MabZIP4 and MabZIP5, respectively (Figure 3b). VP16 fusion construct which

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functions as a transcriptional activator was used as a positive control. Co-expression of either

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MabZIP4 or MabZIP5 with the reporter significantly induced the expression of reporter gene (Figure

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3b), with a 3.8- and 7.9-fold of relative LUC/REN value higher than that of pBD alone, which

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implies that MabZIP4/5 may function as activators in controlling gene expression.

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MabZIP4/5 activate the promoters of aroma biosynthetic genes. To investigate the capacity of

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both MabZIP4 and MabZIP5 to regulate the transcription of the aroma biosynthetic genes, a dual

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luciferase reporter system-mediated transient expression assays were performed. Promoter fragments

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corresponding to ~1 kb upstream of the start codon of MaOMT1, MaMT1, MaGT1, MaBCAT1,

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MaACY1, MaAGT1 and BanAAT were first isolated and sequenced based on the Musa genome public

242

database.29 We then generated six Promoter::LUC reporter constructs in which the promoter

243

fragments of these six genes drove LUC reporter gene expression (Figure 4a). The coding regions of

244

MabZIP4/5 which were expressed under the control of 35S promoter were used as effectors (Figure

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4a). The transient expression assays in tobacco leaves showed that expression of either MabZIP4 or

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MabZIP5 gave rise to apparently increased expression of LUC reporter gene which was driven by the

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promoters of MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT1 and BanAAT, respectively 12


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(Figure 4b). These results suggest a regulation of these genes by MabZIP4 and MabZIP5.

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MabZIP4/5 directly binds to the promoters of several aroma biosynthetic genes. To validate

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the interactions between MabZIP4/5 and promoters of these aroma formation genes, gel shift assays

251

were used. Recombinant MabZIP4/5 proteins fused to glutathione S-transferase (GST) were

252

expressed and purified from E. coli, respectively (Figure S3). As shown in Figure S4, GST-MabZIP4

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recombinant protein was able to form a protein complex with the G-box containing probe derived

254

from BanAAT promoter, while GST-MabZIP5 protein could recognize the G-box sequences in the

255

promoters of MaMT1, MaACY1, MaAGT1 and BanAAT. Moreover, the protein complexes could be

256

competed with the excess of cold probes, but not with the mutated probes indicating the specificity

257

of the DNA-protein interaction (Figure 5). Overall, these results suggest that MabZIP4/5 directly

258

interact with the G-box motifs in the promoters of several aroma formation genes.

259 260

DISCUSSION

261

Refrigerated storage is one of the most effective strategies for preserving fresh fruits and

262

vegetables, but it might affect their quality such as aroma compounds when holding them in very low

263

temperature for a certain period.31 For example, low temperature storage inhibits the aroma emission

264

of different fruits such as tomato,31 peach,32 durian,33 mango,34 papaya,35 and banana.30 In the current

265

work, banana pre-stored at 7 °C for 3 days showed uneven ripening symptoms, such as dull-grey

266

appearance in peel and incompletely softening in pulp (Figure 1), which might be attributed to low

267

ethylene production by chilling injury. Previous study found that the major esters responsible for the

268

unique aroma of banana such as 3-methylbutyl acetate and 3-methylbutyl butanoate were sharply

269

decreased in response to cold treatment.30 It is generally accepted that aroma production is regulated 13



270

at least in part by the expression of aroma biosynthetic genes. In this study, several genes involved in

271

banana aroma biosynthesis such as MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT19

272

and BanAAT6 were increased following fruit ripening, while low temperature pre-storage that might

273

reduce aroma production inhibited the expression of these genes (Figure 2). It seems likely that

274

inhibition of aroma production in chilling-injuried bananas during ripening was associated with

275

down-regulation of MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1 and MaAGT1. Similarly,

276

tomato fruit lost flavor-associated volatiles during cold storage, presumably due to the reduced

277

activity of genes encoding enzymes involved in volatiles production by low temperature, which is

278

attributed to the enhanced status of DNA methylation of these genes.31 In banana, DNA methylation

279

and whether the aroma biosynthetic genes are regulated by other epigenetic modifications such as

280

histone acetylation or deacetylation need to be investigated in future.

281

The transcriptional regulation of genes involved in aroma formation is emerging as a current

282

theme in plant metabolic biology for product quality trait improvement.36 This involves the

283

interaction of transcription factors with the promoters of specific structural genes related to aroma

284

production and the strategies for manipulating those genes for the desired product quality. For

285

example, in Petunia hybrida flower, ODO1 (ODORANT1), a member of the R2R3-type MYB TF

286

family, activated the promoter of the 5-enol-pyruvylshikimate-3-phosphate synthase gene, thus

287

controlling the synthesis of volatile benzenoids.37 In kiwifruit, several members of NAC and EIL TFs

288

were able to promote the transcription of AaTPS1, an important gene involved in monoterpene

289

production.38 More recently, CitERF71 induced the transcription of CitTPS16, a gene contributing to

290

catalyzing E-geraniol synthesis of sweet orange, via directly binding to its promoters through

291

ACCCGCC and GGCGGG motifs.12 In the current work, we identified two bZIP TFs MabZIP4/5 14


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from banana which are nucleus-localized transcriptional activators (Figure 3). Expression of

293

MabZIP4/5 was increased during fruit ripening, but was inhibited in chilled banana during ripening

294

(Fig. 5). In banana genome, there are at least 121 members of bZIP TFs, among which 109 MabZIPs

295

were differentially expressed by abiotic stresses such as cold, salt and osmotic stress.39 In one of our

296

earlier studies, a stress-responsive bZIP from banana fruit, MabZIP3 has been identified, which was

297

shown to physically interact with MaSAP, a protein associated with plant defense against fungal

298

pathogens.25 These findings suggest that most bZIP proteins are involved in stress response, and their

299

roles in secondary metabolism such as aroma formation need to be studied more extensively. In this

300

study MabZIP4/5 were found to activate the promoters of several aroma formation genes including

301

MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT1 and BanAAT (Figure 4). It is

302

noteworthy that in spite of the activation of these aroma biosynthetic genes by MabZIP4/5, the

303

regulatory mechanisms underpinning those gene activation are different, suggesting the diverse roles

304

of MabZIP4/5 in aroma formation of banana fruit. Based on the EMSA results MabZIP4 directly

305

binds to BanAAT promoter, and MabZIP5 binds to the promoters of MaMT1, MaACY1, MaAGT1 and

306

BanAAT, via the G-box motif (Figure 5). However, the activation of MaOMT1, MaMT1, MaGT1,

307

MaBCAT1, MaACY1 and MaAGT1 by MabZIP4, which is not caused by direct binding of MabZIP4,

308

is probably due to the interactions between MabZIP4 and other bZIP TFs. It was reported that bZIP

309

proteins interact with other bZIPs or itself to form heterodimer or homodimer.16 Further investigation

310

of characterizing bZIP proteins that interact with MabZIP4/5 will provide more insights into the

311

transcriptional regulatory network mediated by MabZIPs in aroma production in bananas.

312

In conclusion, bananas pre-stored at chilling temperature decreased the expression of a subset of

313

aroma formation genes in the course of ripening. Moreover, two bZIP TFs MabZIP4 and MabZIP5, 15



314

which are induced by fruit ripening promoted the transcription of MaOMT1, MaMT1, MaGT1,

315

MaBCAT1, MaACY1, MaAGT1 and BanAAT, the genes encoding aroma formation enzymes in

316

banana. Importantly, MabZIP4 was capable of binding directly to BanAAT promoter, while MabZIP5

317

could bind to the promoters of MaMT1, MaACY1, MaAGT1 and BanAAT, suggesting the functional

318

diversity of MabZIPs in controlling banana aroma production. To the best of our knowledge, this is

319

the first report of bZIP TFs involved in controlling aroma formation during fruit ripening. Our study

320

provides novel insights into the transcriptional control of aroma formation in banana during fruit

321

ripening.

322 323

ASSOCIATED CONTENT

324

Supporting Information Available

325

Figure S1. Phylogenetic tree of MabZIP4/5.

326

Figure S2 Amino acid alignments of MabZIP4/5.

327

Figure S3 Affinity purification of the GST-MabZIP4-C and GST-MabZIP5 recombinant proteins

328 329 330

used for gel shift assays. Figure S4 Gel shift assays of MabZIP4/5 binding to the promoters of aroma biosynthetic genes via G-box.

331

Table S1 Primers used in this study.

332

Text S1: Amino acid sequences of MabZIP4/5 and Arabidopsis bZIPs used for phylogenetic tree.

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Text S2: Probes in promoters of aroma biosynthetic genes containing G-box from banana genome.

334

AUTHOR INFORMATION

335

Corresponding Author 16


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*Telephone: +86-020-85285523. Fax: +86-020-85285527. Email: [email protected].

337

ORCID

338

Jian-fei Kuang: 0000-0001-9192-2155

339

Jian-ye Chen: 0000-0002-8975-6941

340

Author Contributions

341

†

342

Funding

343

This work was funded by the National Key R&D Program of China (grant no. 2016YFD0400103),

344

Zhujiang New Star of Science and Technology of Guangzhou City (201506010080), the National

345

Natural Science Foundation of China (grant no. 31772021) and China Agriculture Research System

346

(grant no. CARS-31-11).

347

Notes

348

The authors declare no competing financial interest.

349

ACKNOWLEDGEMENTS

350

We thank Professor George P. Lomonossoff (Department of Biological Chemistry, John Innes Centre,

351

Norwich Research Park) for the generous gifts of the transient expression vectors, and pEAQ vectors,

352

respectively. We also thank Dr. Prakash Lakshmanan (Sugar Research Australia) and Dr. Mingchun

353

Liu (Sichuan University) for their helps with the English language editing.

Yu-fan Guo and Yun-liang Zhang contributed equally to this work.

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Figure legends

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Figure 1. The ripening progression of bananas pre-stored at 22 °C (Non-chilling treatment) or at

492

7 °C for 3 days (Chilling treatment) (a), changes in peel color, pulp firmness and ethylene production

493

(b) during ripening. Each value represents the mean ±SE of three replicates. The ** and * represent

494

significant differences at 0.01 and 0.05 levels (Student’s t-test), respectively.

495 496

Figure 2. Expression of aroma biosynthetic genes and MabZIP4/5 in bananas with chilling and

497

non-chilling treatments during fruit ripening. The expression levels of each gene are expressed as a

498

ratio relative to 0 d of chilling treatment, which was set at 1. Each value represents the mean ±SE of

499

three replicates. The ** and * represent significant differences at 0.01 and 0.05 levels (Student’s

500

t-test), respectively.

501 502

Figure 3. Subcellular localization and transcriptional activation ability of MabZIP4/5. (a)

503

Subcellular localization of MabZIP4/5 in tobacco (N. benthamiana) leaf epidermal cells. GFP

504

fluorescence was detected with a fluorescence microscope. Bar=25 µm. (b) Transcriptional activation

505

ability of MabZIP4/5 in tobacco leaves. The transcription activation ability of MabZIP4/5 is

506

presented as the ratio of LUC/REN. Each value is the mean ± SE of six biological repeats. The **

507

and * represent significant differences at 0.01 and 0.05 levels (Student’s t-test), respectively.

508 509

Figure 4. Transcriptional activation of aroma biosynthetic genes by MabZIP4/5 in transient

510

expression system. (a) Schematic program of the reporter and effector constructs used in the

511

dual-luciferase reporter assay. (b) MabZIP4/5 activate transcription of aroma biosynthetic genes. The 24


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512

activation of aroma biosynthetic genes by MabZIP4/5 was shown by LUC/REN. The value of

513

LUC/REN of the empty vector plus promoter reporter was set as 1. Value presented as mean ± SE of

514

six biological repeats. The ** and * represent significant differences at 0.01 and 0.05 levels

515

according to Student’s t-test, respectively.

516 517

Figure 5. The binding of MabZIP4/5 to the promoters of several aroma biosynthetic genes in gel

518

mobility shift assays. The GST-MabZIP4 or GST-MabZIP5 protein was incubated with probes

519

containing the G-box binding site motifs derived from the promoters of BanAAT (a, e) MaMT1 (b),

520

MaACY1 (c) and MaAGT1 (d); − and + represent absence or presence, respectively, and +++ shows

521

increasing amounts of cold or mutant probes for competition.

522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542

25



Figures Figure 1

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Figure 3

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Figure 5

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Figure 1 169x270mm (300 x 300 DPI)


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Figure 2 167x135mm (300 x 300 DPI)



Figure 3 177x296mm (300 x 300 DPI)


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Figure 4 118x47mm (300 x 300 DPI)



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Identification of Two Transcriptional Activators ... - ACS Publications

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