Involvement of WRKY Transcription Factors in Abscisic-Acid-Induced

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Involvement of WRKY Transcription Factors in ABA-Induced Cold Tolerance of Banana Fruit Dong-lan Luo, Liang-jie Ba, Wei Shan, Jian-fei Kuang, Wang-jin Lu, and Jian-ye Chen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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Journal of Agricultural and Food Chemistry

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Involvement of WRKY Transcription Factors in ABA-Induced Cold Tolerance of

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Banana Fruit

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Dong-lan Luo†, §, #, Liang-jie Ba†, §, #, Wei Shan†, Jian-fei Kuang†, Wang-jin Lu†, and Jian-ye Chen†, *

4 5

†

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Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and

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Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, China

8

§

9

Processing, Guiyang College, Guiyang 550003, China

State

Key

Laboratory

for

Conservation

and

Utilization

of

Subtropical

School of Food and Pharmaceutical Engineering/Guizhou Engineering Research Center for Fruit

10 11 12 13 14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


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ABSTRACT: Phytohormone abscisic acid (ABA) and plant-specific WRKY transcription factors

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(TFs) have been implicated to play important roles in various stress responses. The involvement of

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WRKY TFs in ABA-mediated cold tolerance of economical fruits, such as banana fruit, however

26

remains largely unknown. Here, we reported that ABA application could induce expressions of ABA

27

biosynthesis-related genes MaNCED1 and MaNCED2, increase endogenous ABA contents and

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thereby enhance cold tolerance in banana fruit. Four banana fruit WRKY TFs, designated as

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MaWRKY31, MaWRKY33, MaWRKY60, and MaWRKY71, were identified and characterized. All

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these four MaWRKYs were nuclear localized and displayed trans-activation activities. Their

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expressions were induced by ABA treatment during cold storage. More importantly, gel mobility

32

shift assay and transient expression analysis revealed that MaWRKY31, MaWRKY33, MaWRKY60,

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and MaWRKY71 directly bound to the W-box elements in MaNCED1 and MaNCED2 promoters,

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and activated their expressions. Taken together, our findings demonstrate that banana fruit WRKY

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TFs are involved in ABA-induced cold tolerance by, at least in part, increasing ABA levels via

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directly activating NECD expressions.

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KEYWORDS: ABA biosynthesis, banana fruit, cold tolerance, WRKY, trans-activation

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INTRODUCTION

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Banana (Musa acuminate), one of the most popular fruits worldwide, is a typical climacteric fruit

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with a very short shelf-life due to rapid softening.1-3 Low temperature storage is commonly used to

48

extend the shelf-life and to maintain post-harvest qualities of banana fruit. However, the fruit is

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susceptible to chilling injury (CI), causing browning or blackening of skin and failure of ripening,

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resulting in considerable economic loss.4-7 Genetic variation for cold tolerance in banana is very

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limited and improving it through conventional breeding is very difficult. Considering the critical

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requirement of low temperature storage and the cold sensitivity of banana fruit, we are studying the

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molecular mechanism(s) of the cold response in banana fruit with the ultimate objective of genetic

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improving cold tolerance, fruit quality and storage potential. Our previous studies have clearly

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demonstrated that exogenous application of methyl jasmonate (MeJA) or propylene, a functional

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ethylene analogue, greatly alleviates CI of banana fruit.8-10 The involvement of other plant hormones,

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especially stress hormones, such as abscisic acid (ABA) in cold response of banana fruit is likely but

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remains unclear.

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ABA acts as an important regulatory signal to influence multiple plant processes including cold

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stress response.11-13 ABA accumulation under cold condition correlates with increased ABA

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biosynthesis in many plants.14,15 Exogenous application of ABA induces cold tolerance, and ABA

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mutants showed altered cold resistance.11 In addition, exogenous ABA treatment has been implicated

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in the alleviation of CI of horticultural fruits such as grapefruit,16 zucchini squash,17 pineapple fruit18

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and litchi.19 Generally, the endogenous ABA level in plants is finely controlled by the dynamic

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balance of the activity of biosynthesis-related genes NCED (9-cis-epoxycarotenoid dioxygenase),

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catabolism genes CYP707A (ABA 8’-hydroxylase), and reactivation-related genes BG/GT 3



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(β-glucosidases/glucosyltransferases) at the transcriptional level.11,13,20-24 Many transcription factors

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(TFs) such as bZIP (BASIC LEUCINE ZIPPER), WRKY (WRKY DNA-BINDING PROTEIN) and

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MYB (MYELOBLASTOSIS) are reported to be involved in mediating ABA action13,23,25-28

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WRKY constitutes one of the largest families of TFs in plants, characterizing by the highly

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conserved WRKY domain composed of the amino acid sequence WRKYGQK at the N-terminus,

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and the C2H2 or C2HC zinc-finger motif at the C-terminus.29-31 It has been well documented that

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WRKY TFs not only function as an important regulator of plant biotic stress responses, but also of

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abiotic stress tolerance like cold.27,31-33 More noticeably, emerging evidence reveal that WRKY TFs

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are the key hubs in ABA-responsive signalling networks, as they target many well-known

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ABA-responsive genes such as ABFs/AREBs (ABA-responsive element binding factors) and ABIs

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(ABA Insensitive).25,27 Recently, WRKY57 has been shown to directly activate NCED3 expression in

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Arabidopsis, resulting in the elevation of ABA levels and improved drought tolerance.34 In spite of

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such progress, how WRKY TFs regulate cold response through alteration of ABA-dependent manner

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is largely unknown, especially in major commercially important fruits such as bananas.

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In this study, we showed that ABA treatment alleviated banana fruit CI. Four ABA-responsive

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WRKY genes termed MaWRKY31/MaWRKY33/MaWRKY60/MaWRKY71 were identified from

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banana fruit. More importantly, the four WRKY TFs can directly bind to the promoters two NCED

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MaNCED1/MaNCED2, and activated their expressions. Our findings clearly suggest that banana

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fruit WRKY TFs act as positive regulators of ABA-mediated cold tolerance, which is associated with

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their involvement in modulating ABA synthesis by activating NCED expressions. The results

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presented here expands our understanding of the regulatory network of ABA-mediated cold tolerance

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in economically important fruits. 4


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MATRIALS AND METHODS

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Fruit and ABA treatment. Banana fruit (Musa acuminata, AAA group, cv. Cavendish) at green

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mature stage (~12 weeks after anthesis) were harvested and separated into individual fingers. Fruit

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with uniform weight and shape, and free of visual defects, were collected and randomly divided into

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two groups, one for control and the other for ABA treatment, respectively. Each group contained 100

94

individual fingers. For ABA treatment, fruit were soaked into 10 L of distilled water containing 0.1

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mM ABA for 30 min at about 0.1 MPa pressure as described previously.4,8 Fruit immersed in

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distilled water under the same conditions as ABA treatment were used as control. The two groups of

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fruit were subsequently stored at 7 °C for 7 d, and sampled at 0, 1, 3, 5 and 7 d of storage

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respectively. For each sample, banana peel from 5 individual fingers were mixed thoroughly, frozen

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immediately in liquid nitrogen, and stored at -80 °C for further assays. At least three biological

100

replicates were used for all treatments in all experiments.

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Chilling injury (CI) assessment. Chilling injury index and relative electrolyte leakage were used

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to evaluate the CI of banana fruit. These two parameters were determined as described in our

103

previous studies. 4,8

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Quantification of endogenous ABA content. ABA accumulation in banana fruit peel was

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determined using ELISA method as previously described.35 Briefly, 1.0 g of peel was ground and

106

homogenized in the ABA extraction solution (80% v/v methanol). After centrifugation and elution

107

using a Sep-Pak C18 cartridge (Waters), the supernatant was analyzed using the ELISA kit for ABA

108

(China Agricultural University) according to the manufacturer's instruction.

109

Gene isolation and expression analysis. Total RNA was isolated from the banana fruit peel using

110

the hot borate method. The cDNA used for PCR amplification was synthesized from the total RNA 5



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via HiScript II Q RT SuperMix kit (Vazyme) following manufacturer’s instruction. Five NCED and

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four WRKY genes, were selected based on the banana genome (http://banana-genome.cirad.fr/) and

113

our RNA-seq database (unpublished data), and blasted in NCBI. ClustalW and MEGA5 were

114

employed for sequence alignment and construction of a phylogenetic tree, respectively. Gene

115

expression was detected through the real-time quantitative polymerase chain reaction (RT-qPCR) on

116

a Bio-Rad CFX96 Real-Time PCR System as previously described.7 The PCR program included an

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initial denaturation step at 94 °C for 5 min, followed by 40 cycles of 94 °C for 10, 60 °C for 30 s,

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and 72 °C for 30 s. No-template controls and melting curve analyses were included in every reaction.

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Specific primers are designed using Primer Premier 5 software and are listed in Supporting

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Information, Table S1.

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Promoter isolation. The promoter sequences of banana NCED genes was obtained by PCR

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(primers are listed in Supporting Information, Table S1) using genomic DNA as template, which was

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extracted from banana fruit peel using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA).

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Putative

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(http://www.dna.affrc.go.jp/PLACE/signalscan.html) database.

cis-acting

elements

in

the

promoter

were

identified

using

PLACE

126

Subcellular localization analysis. To determine the subcellular localization of MaWRKYs, the

127

coding sequence of MaWRKYs was amplified and subcloned into the pEAQ-GFP vector (primers are

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listed in Supporting Information, Table S1) to produce fusion construct pEAQ-MaWRKYs-GFP

129

using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturer’s instructions, and was

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verified by further sequencing. The pEAQ-MaWRKYs-GFP construct and the control GFP vector

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(pEAQ-GFP) were then electroporated into the Agrobacterium tumefaciens strain GV3101, and

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co-infiltrated into the abaxial side of 4- to 6-week-old tobacco (Nicotiana benthamiana) leaves as 6


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described in our previous study.7 GFP fluorescence was captured with a fluorescence microscope

134

after 48 h of infiltration. At least triple replicates were performed.

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Gel mobility shift assay. The C-terminal of MaWRKYs cDNA sequences were cloned in frame

136

with GST in the pGEX-4T-1 vector (Primers are shown in Supporting Information, Table S1). The

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GST-MaWRKYs fusion proteins were expressed in the E. coli strain BM Rosetta (DE3), purified

138

with Glutathione-Superflow Resin (Clontech) and used for gel mobility shift assay. Purified

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GST-MaWRKYs fusion proteins were incubated with biotin-labeled MaNCED promoter fragments,

140

and the protein-DNA fragments were separated by SDS-PAGE following detection via a Lightshift

141

Chemiluminescent EMSA kit (Thermo) according to our previous work.36

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Dual luciferase transient transfection analysis. To determine the transcription activities of

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MaWRKYs, the coding sequence of MaWRKYs was cloned into 35S promoter driving-pBD vector

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as effector, and the double-reporter vector was constructed with a GAL4-firefly luciferase (LUC)

145

by TATA box and an internal control renilla reniformis luciferase (REN) driven by the 35S promoter.

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For the analysis of trans-activation of MaWRKYs to the MaNCED promoters, the MaNCED

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promoters were cloned into pGreenII 0800-LUC double-reporter vector,37 and MaWRKYs was

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inserted into the pEAQ vector as effector.38 These respective reporter and effector plasmids were

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co-transformed into tobacco leaves by Agrobacterium tumefaciens strain GV3101, and LUC and

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REN luciferase activities were measured using the dual luciferase assay kit (Promega) on a

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Luminoskan

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trans-activation ability of MaWRKYs was indicated by the LUC to REN ratio. At least six

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independent repeats were provided for each combination. The primers used in this analysis are listed

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in Supporting Information, Table S1.

Ascent Microplate

Luminometer

(Thermo)

7


as

previously

described.7

The


155

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Statistical analyses. All data presented here are means of at least three independent biological

156

replicates.

The

data

were

evaluated

by

Student’s

157

to compare the statistical difference at 1% or 5% level.

158

RESULTS AND DISCUSSION

t-test

using

SPSS

18.0

software

159

ABA treatment induces cold tolerance of banana fruit. As shown in Figure 1a, when control

160

banana fruit stored at 7 °C for 3 d, CI symptoms, such as pitting and brown patches, began to appear

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in peel, and it became sever with storage. Thus the CI index values gradually increased with

162

increasing storage time (Figure 1b). Application of ABA at 0.1 mM alleviated the CI symptoms, and

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delayed the progression of CI index. CI index in ABA-treated fruit was ~53.6% and 69.2% of the

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control on the 5th and the 7th day of storage, respectively (Figure 1b). Relative electrolyte leakage is

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an indicator of the membrane damage under stress conditions.5 Relative electrolyte leakage in both

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control and ABA-treated fruit gradually increased with storage, but its progression was considerably

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delayed by ABA treatment (Figure 1c). Relative electrolyte leakage in control fruit was ~13.3% and

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~14.2% higher than ABA-treated fruit on day 5 and 7, respectively (Figure 1c). Collectively, these

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data demonstrate that ABA treatment induces cold tolerance of banana fruit, providing further

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evidence of ABA involvement in plant's responses to abiotic stresses.11,13

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ABA treatment increased endogenous ABA contents and induced expressions of ABA

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biosynthesis-related genes MaNCEDs during cold storage. A range of abiotic stresses such as

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drought, salinity and high/low temperature trigger ABA accumulation, which plays a crucial role in

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the plant stress responses.13,14,23,34 To determine whether ABA levels were affected during cold

175

storage, the ABA contents in control and ABA-treated banana fruit peel were quantified by ELISA. 8


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As shown in Figure 2a, in agreement with previous studies, under the cold storage condition, the

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endogenous ABA content started to increase on day 1 in control and ABA-treated banana fruit,

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reaching a peak on day 5, and decreased thereafter. Moreover, ABA-treated fruit accumulated

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significantly higher levels of ABA than in the control, recording ~0.6- and ~0.3-fold increase in

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ABA-treated fruit on 3 and 5 d of storage, respectively (Figure 2a).

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ABA is biosynthesized from β-carotene involving in zeaxanthin epoxidase (ZEP, known as

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ABA1 in Arabidopsis), ABA aldehyde oxidase (AAO3), MoCo sulfurase (ABA3) and

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9-cis-epoxycarotenoid dioxygenase (NCED),11,13,20-24 among which NCED is the key rate-limiting

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enzyme23,35,39-41 Five NCED genes, termed MaNCED1-MaNCED5, were found in banana genome,

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and their expressions during cold storage were investigated by qRT-PCR. Except for MaNCED3,

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transcripts of all other four MaNCEDs, especially MaNCED1 and MaNCED2 increased in control

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banana fruit with storage (Figure 2b). Compared to the control fruit, accumulations of MaNCED1

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and MaNCED2 transcripts were higher in ABA-treated fruit (Figure 2b). Therefore, the accumulation

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of endogenous ABA in banana fruit during cold storage was correlated with the expressions of ABA

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biosynthesis-related genes.

191

Molecular

characterization

of

four

banana

fruit

WRKY

TFs

192

MaWRKY31/MaWRKY33/MaWRKY60/MaWRKY71. Even though WRKY TFs are well known

193

to be the key nodal points for ABA-responsive signalling networks,27,31-33 their involvement in

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ABA-induced cold tolerance of economical fruits such as bananas, needs to be explored. 147 WRKY

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TFs were identified in banana genome.42 Based on our RNA-seq database of banana fruit stored

196

under cold stress (unpublished data), four WRKY TFs that are most up-regulated were selected and

197

cloned.

Since

the

sequence

of

these

four

WRKY TFs

9


(GSMUA_Achr2G15200_001,


198

GSMUA_Achr6G32720_001, GSMUA_Achr7G05200_001 and GSMUA_Achr1G04770_001 in the

199

banana genome) shared highest similarity with AtWRKY31 (GenBank accession number,

200

NP_567644.1) (46%), AtWRKY33 (NP_181381.2) (43%), AtWRKY60 (NP_180072.1) (46%) and

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AtWRKY71 (NP_174279.1) (34%), so they were designated as MaWRKY31, MaWRKY33,

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MaWRKY60 and MaWRKY71, respectively. Multiple sequence alignment showed that

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MaWRKY33 contains two highly conserved amino acid sequences WRKYGQK, called WRKY

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domain, characteristic of WRKY TFs,43 while MaWRKY31, MaWRKY60 and MaWRKY71 each

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has one WRKY domain (Supporting Information, Figure S1). A phylogenetic tree demonstrated that

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MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71 belongs to Group IIb, Group I, Group

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IIa and Group IIc of WRKY family, respectively (Supporting Information, Figure S2).

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WRKYs are usually nuclear proteins and possess transcriptional activity.32,33,44,45 Nuclear

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localization sequence (NLS) (Supporting Information, Figure S1) was found in MaWRKY31,

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MaWRKY33, MaWRKY60 and MaWRKY71, indicating that they may be nuclear proteins. To

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confirm the subcellular location of MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71, we

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fused the GFP with these four WRKY proteins driven by the CaMV 35S promoter, and were

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agroinfiltrated into tobacco leaves for transient expression. As shown in Figure 3a, these four

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WRKY-GFP fusion proteins were specifically detected in the nucleus of tobacco cells, whereas

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fluorescence of the control GFP was present throughout the cytoplasm and the nucleus. We also

216

analyzed the transcriptional activity of MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71

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in plant cells using the dual-luciferase reporter system containing 5× GAL4 DNA-binding elements

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and TATA box fused to LUC reporter and an internal control REN driven by the 35S promoter

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(Figure 3b). Compared with the pBD negative control, all four banana fruit WRKY proteins 10


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MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71, as well as the transcriptional activator

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control VP16, significantly increased the LUC reporter activities (Figure 3c). These data suggest a

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transcriptional activator role for MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71

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proteins.

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To further confirm the possible involvement of MaWRKY31, MaWRKY33, MaWRKY60 and

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MaWRKY71 in ABA-induced banana fruit cold tolerance, their expression patterns in control and

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ABA-treated fruit during cold storage was examined. As shown in Figure 4, MaWRKY31,

227

MaWRKY33, MaWRKY60 and MaWRKY71 were all induced in control and ABA-treated fruit during

228

cold storage. Moreover, they accumulated higher levels in ABA-treated fruit, with ~0.6-, 1.1-, 0.49-

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and 0.58-fold higher than that in control fruit, after 5 d of storage, respectively (Figure 4). These

230

results indicate that MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71 are ABA-responsive

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and might be involved in ABA-induced cold tolerance of banana fruit. Similarly, wheat TaWRKY1

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and TaWRKY33 were responsive to ABA and conferred tolerance to drought stress in transgenic

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plants.46

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MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71 target W-box element

of

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MaNCED1 and MaNCED2 promoters. Previous studies have demonstrated that WRKY TFs

236

regulate their target genes by binding to W-box element (C/T)TGAC(C/T) present in the target

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promoters.29-33 Indeed, several W-box elements were found in the putative promoter regions of two

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ABA biosynthetic genes, MaNCED1 and MaNCED2 (Supporting Information, Text S1). We then

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examine whether these two genes are directly targeted by MaWRKY31, MaWRKY33, MaWRKY60

240

and MaWRKY71 using gel mobility shift assay. Recombinant glutathione S-transferase

241

(GST)-MaWRKY31, MaWRKY33, MaWRKY60 or MaWRKY71 fusion protein was expressed in 11



242

E.coli and successfully purified (Supporting Information, Figure S3). As expected, the

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GST-MaWRKY31, MaWRKY33, MaWRKY60 or MaWRKY71 fusion proteins were all able to

244

bind to labeled MaNCED1 or MaNCED2 promoter fragment and caused mobility shifts. The

245

mobility shift, however, was effectively abolished when unlabeled MaNCED1 or MaNCED2

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promoter fragment used as cold probe was added, in a dose-dependent manner (Figure 5). The

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mobility shift was also not observed when the promoter fragment of MaNCED1 or MaNCED2 was

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incubated with GST alone (Figure 5), indicating that the binding of MaWRKY31, MaWRKY33,

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MaWRKY60 and MaWRKY71 to the MaNCED1 or MaNCED2 promoter is specific. Similar results

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were also reported in Arabidopsis that ABA- and drought-responsive WRKY57 can bind to the

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NCED3 promoter via the W-box element.34 In addition, WRKY33 also binds to NCED3 and NCED5

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promoters.47

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Trans-activation of MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71 on

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MaNCED1 and MaNCED2 promoters. To confirm the results of gel mobility shift assay and

255

determine whether MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71 can activate

256

promoter of MaNCED1 and MaNCED2, as they showed trans-activation abilities (Figure 3c), dual

257

luciferase transient expression analysis was performed (Figure 6a). As shown in Figure 6b, the

258

promoter activities of MaNCED1 and MaNCED2, indicated by LUC/REN ratio, were significantly

259

enhanced when co-transfected with MaWRKY31, MaWRKY33, MaWRKY60 or MaWRKY71,

260

compared with the empty vector, suggesting that MaWRKY31, MaWRKY33, MaWRKY60 and

261

MaWRKY71 can trans-activate promoters of MaNCED1 and MaNCED2. It has been clearly

262

demonstrated that WRKY TFs act as components of ABA signalling at different levels (Rushton et

263

al., 2012; Dong et al., 2013).27, Except NCEDs, many well-known ABA-responsive TFs such as 12


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ABFs, ABIs, and DREBs, as well as some well-characterized stress-inducible genes including

265

RD29A and COR47, are also reported to be the target genes of WRKY TFs and they elicit different

266

effects on their expressions.27,33,48-51 For example, cucumber cold- and ABA-responsive CsWRKY46

267

interacts with the W-box of ABI5 promoter and triggers its activity,51 while Arabidopsis AtWRKY40

268

binds to the W-box of AtABFs promoters and represses their expressions.48 Contrary to our results

269

that MaWRKY31, MaWRKY33, MaWRKY60 and MaWRKY71 activate MaNCED1 or MaNCED2

270

promoter to positively regulate ABA biosynthesis under cold stress, Arabidopsis WRKY33 targets

271

NCED3/NCED5 and decreases ABA levels associated with plant immunity.47 These findings reveal

272

that WRKY TFs can act as transcriptional activators or repressors to be involved in various stress

273

responses. Intriguingly, genome-wide binding and transcriptional profiling analysis demonstrate that

274

Arabidopsis WRKY33 exhibits dual functionality as it acts either as a repressor or as an activator

275

dependent on its target genes.47 Similarly, very recently, our work showed that a banana AP2/ERF TF

276

MaDREB2 also functions as a transcriptional activator or repressor during fruit ripening.3

277

Considering the dual functionality of WRKY TFs, the interaction of MaWRKY31, MaWRKY33,

278

MaWRKY60 and MaWRKY71 in regulating target genes needs to be studied to get a better

279

understanding of the regulatory network of MaWRKYs involved in the ABA-induced cold tolerance

280

of banana fruit.

281

In summary, ABA treatment can induce cold tolerance of banana fruit. Further we identified and

282

characterized four ABA-responsive WRKY TFs, MaWRKY31, MaWRKY33, MaWRKY60 and

283

MaWRKY71 from banana fruit. They are localized in the nucleus and are transcriptional activators.

284

Moreover, they directly bind to the promoters of two ABA biosynthetic genes MaNCED1 and

285

MaNCED2, and trans-activate their expressions. Collectively, our findings clearly suggest that 13



286

banana fruit WRKY TFs are involved in ABA-induced cold tolerance and regulating this response

287

possibly by increasing ABA levels via directly regulating ABA biosynthesis-related genes. These

288

results thus provide insights on the transcriptional regulatory network of ABA-mediated cold

289

tolerance governed by WRKY TFs.

290

AUTHOR INFORMATION

291

#

292

Corresponding Author

293

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

294

Funding

295

This research was supported by National Key Research and Development Program (grant no.

296

2016YFD0400103) and China Agriculture Research System (grant no. CARS-32-09).

297

Notes

298

All authors have no conflicts of interest to declare.

299

ACKNOWLEDGEMENT

300

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

301

Norwich Research Park) for gifting pEAQ vectors. Constructive comments and critical language

302

editing of the manuscript from Dr. Prakash Lakshmanan (Sugar Research Australia) during revision

303

is gratefully appreciated.

304

ASSOCIATED CONTENT

305

Supporting Information Available

These authors contribute equally to this work.

14


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Page 15 of 31


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Figure S1. Sequence alignments of the conserved WRKY domains of MaWRKY31, MaWRKY33,

307

MaWRKY60 and MaWRKY71 with Arabidopsis WRKY proteins.

308

Figure S2. Phylogenetic tree of MaWRKYs with other plant WRKY proteins.

309

Figure S3. Affinity purification of the recombinant GST-MaWRKY proteins used for EMSA.

310

Table S1. Summary of primers used in this study.

311

Text S1. Promoter nucleotide sequences of MaNCED1 and MaNCED2.

312

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Figure Legends

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Figure 1. ABA treatment induced cold tolerance of banana fruit. (a) Photograph of chilling injury

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(CI) symptoms of ABA-treated and control banana fruit during 7 days of storage at 7 °C. CI index (b)

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and relative electrolytic leakage (c) changes in banana fruit treated with ABA and control during cold

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storage. In (b) and (c), each value represents the mean ± S.E. of three replicates. * and ** indicate

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significant differences in values at P

Involvement of WRKY Transcription Factors in Abscisic-Acid-Induced

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