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Characterization of a Transcriptional Regulator BrWRKY6 that Associates with Gibberellin-Suppressed Leaf Senescence of Chinese Flowering Cabbage Zhong-qi Fan, Xiao-Li Tan, Wei Shan, Jian-fei Kuang, Wang-jin Lu, and Jian-ye Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06085 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Journal of Agricultural and Food Chemistry

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Characterization of a Transcriptional Regulator BrWRKY6 that Associates

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with Gibberellin-Suppressed Leaf Senescence of Chinese Flowering Cabbage

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Zhong-qi Fan, Xiao-Li Tan, Wei Shan, Jian-fei Kuang, Wang-jin Lu, and Jian-ye Chen*

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State

Key

Laboratory

for

Conservation

and

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

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Vegetables/Guangdong Vegetables Engineering Research Center, College of Horticulture, South

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China Agricultural University, Guangzhou 510642, China

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1

ACS Paragon Plus Environment

Utilization

of

Subtropical


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ABSTRACT: Phytohormone gibberellin (GA) and plant-specific WRKY transcription factors

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(TFs) are reported to play important roles in leaf senescence. The association of WRKY TFs with

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GA-mediated leaf senescence of economically important leafy vegetables like Chinese flowering

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cabbage, however remains largely unknown. In this study, we showed that exogenous application

27

of GA3 suppressed Chinese flowering cabbage leaf senescence, with GA3-treated cabbages

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maintaining higher level of maximum quantum yield (Fv/Fm) and total chlorophyll content. GA3

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treatment also led to lower electrolyte leakage and expression level of a series of

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senescence-associated genes (SAGs) including BrSAG12, BrSAG19, and chlorophyll catabolic

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genes (CCGs) BrPPH1, BrNYC1, and BrSGRs. In addition, higher transcript levels of GA

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biosynthetic genes BrKAO2 and BrGA20ox2 were found after GA3 treatment. More importantly,

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a GA-repressible, nuclear-localized WRKY transcription factor (TF) BrWRKY6, a homolog of

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the Arabidopsis AtWRKY6, was identified and characterized. BrWRKY6 was GA-repressible,

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and localized in the nucleus. Further experiments revealed that BrWRKY6 repressed the

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expression of BrKAO2 and BrGA20ox2, while activated BrSAG12, BrNYC1 and BrSGR1,

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through binding to their promoters via the W-box cis-element. Together, the novel GA-WRKY

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link reported in our study provides new insight into the transcriptional regulation of

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GA-suppressed leaf senescence in Chinese flowering cabbage.

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KEYWORDS: Chinese flowering cabbage, GA, leaf senescence, WRKY, transcriptional

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regulation

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INTRODUCTION

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Demand for leafy vegetables such as lettuce (Valeriana olitoria), broccoli (Brassica oleracea),

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Chinese cabbage (Brassica rapa ssp. pekinensis), bok choy (Brassica rapa ssp. chinensis) and

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Chinese flowering cabbage (Brassica rapa ssp. parachinensis) is growing significantly with

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increasing healthy lifestyle, as they are good source of minerals, vitamins and health-promoting

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compounds.1–3 These leafy vegetables, however, possess high metabolic activity after harvest,

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hence are prone to rapid senescence with yellowing leaves, which shortens their postharvest

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shelf-life.3–5 Thus, understanding the regulatory mechanisms of leaf senescence is critical for

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improving the technology to prevent postharvest loss of leafy vegetables.

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Leaf senescence is a complex biological process that is tightly controlled by various factors,

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including developmental stage, phytohormones, nutrients and stresses.6–8 In general, plant

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hormones ethylene, jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA) promote leaf

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senescence, whereas others like cytokinins (CKs), gibberellins (GA), auxins and polyamine (PA)

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have been shown to delay senescence.9–13 Among these phytohormones, transcriptional regulation

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of JA-, ethylene- and ABA-mediated leaf senescence via transcription factors (TFs) has been

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extensively investigated.8,12,14,15 For example, TFs such as ANAC092/ANAC019/055/072,16

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bHLH03/bHLH13/bHLH14/bHLH17,17 and MYC2/MYC3/MYC4,16 are reported to be positively

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or negatively involved in JA-promoted leaf senescence in Arabidopsis. NAC TFs of

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Arabidopsis,18 rice19 and foxtail millet20 are involved in ABA-induce leaf senescence by

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regulating ABA biosynthesis, signaling or chlorophyll degradation. GA has been shown to delay

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leaf senescence of dandelion, banana, nasturtium, rumex, alstromeria and herbaceous perennial

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Paris polyphylla,9,21–24 as well as to delay rose petal senescence.25 Yet, little is known about TFs 3



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associated with GA-mediated leaf senescence.

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WRKYs are plant-specific and constitute the second largest family of senescence-related

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TFs.26 Previous studies have revealed that WRKY TFs like WRKY53,27 WRKY54 and

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WRKY70,28 WRKY57,29 function as important regulatory proteins in leaf senescence. In our

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previous study, we also found that BrWRKY65 from Chinese flowering cabbage may be acting

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as a positive regulator of leaf senescence by activating three senescence-associated genes

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(SAGs).3 Moreover, WRKY TFs are demonstrated to be involved in various physiological

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processes controlled by GA.30 For instance, three WRKY TFs of rice including OsWRKY24, -53,

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and -70, negatively regulate GA signaling in aleurone cells.31 WRKY12 and WRKY13 mediate

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the GA3-controlled flowering time in Arabidopsis.32 Interestingly, Arabidopsis WRKY45 is

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recently reported to be a key regulatory of GA transduction involved in the initiation of leaf

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senescence.33

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Despite the evidence that WRKY TFs are involved in leaf senescence, it remains unclear as to

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whether they are involved in regulation of GA-mediated leaf senescence, especially in rapidly

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senescing leafy vegetables like Chinese flowering cabbage. In the present study, we found that

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exogenous GA3 treatment maintained high expression levels of GA biosynthetic genes BrKAO2

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and BrGA20ox2, while repressed a series of SAGs such as BrSAG12, BrSAG19, and chlorophyll

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catabolic genes (CCGs) BrPPH1, BrNYC1 and BrSGRs encoding pheophytin pheophorbide

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hydrolase (PPH), NON-YELLOW COLOURING (NYC) and STAY-GREEN (SGR) respectively,

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leading to the suppression of leaf senescence in Chinese flowering cabbage. More importantly,

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we further identified a WRKY TF BrWRKY6, a homologue of Arabidopsis WRKY6, which was

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inhibited by GA3, and acted as a transcriptional regulator of BrKAO2, BrGA20ox2, BrSAG12,

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BrNYC1 and BrSGR1 by binding to their promoters. Our results thus provide a novel link

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between BrWRKY6 and GA-mediated leaf senescence in Chinese flowering cabbage.

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MATRIALS AND METHODS

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Plant Materials and GA Treatment. Chinese flowering cabbages (Brassica rapa var.

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parachinensis) were harvested after ~40 days of growth from a commercial vegetable farm near

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Guangzhou, southern China. Harvested cabbages were pre-cooled to 4°C to remove respiratory

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heat, transported to laboratory under low temperature, and treated with GA3 immediately.

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Uniform cabbages without mechanical damage were chosen and randomly divided into two

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groups. For GA treatment, the cabbages were sprayed with 100 µM GA3 (Sigma-Aldrich)

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containing 0.1% tween-20. The cabbages sprayed with distilled water including 0.1% tween-20

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were served as control. Both control and GA-treated cabbages were subsequently placed into

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plastic baskets (20 per box) that packaged with polyethylene perforated plastic bags (0.03 mm

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thickness, with 6 holes of 1 cm in diameter at each side), and stored in the incubators at 15 °C.

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Immediately after GA3 treatment and after 3, 5 and 7 d of storage, the second leaf from the

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bottom of ten cabbages were sampled for physiological and molecular analysis.

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Leaf Senescence Assessment. Senescence-associated physiological parameters, including total

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chlorophyll content, chlorophyll fluorescence such as the maximal PS II quantum yield (Fv/Fm),

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and relative electrolytic leakage were used to assess leaf senescence. Leaf tissues (~2.5 g) was

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ground to a fine powder using liquid nitrogen, and total chlorophyll was extracted with 80% cold

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acetone overnight at 4 °C, and its content was estimated spectrophotometrically at 663 and 645

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nm as previously described.34 Fv/Fm was calculated non-invasively using an Imaging-PAM-M

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series chlorophyll fluorometer (Heinz Walz) integrated with a CCD camera that enables to 5



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capture highly resolved digital images of the emitted fluorescence.35 Relative electrolytic leakage

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was determined as previously described.3

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RNA Extraction, cDNA Synthesis, Gene Cloning and Sequence Analysis. Total RNA of

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sampled tissues was isolated using an RNeasy Plant Mini Kit (Qiagen) according to the

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manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA using a

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PrimeScriptTM RT reagent kit (TaKaRa). Based on the sequence information of Chinese cabbage

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chiifu genome (http://brassicadb.org/brad/), the coding region of BrWRKY6 was cloned using

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gene-specific primers (primers are listed in Supporting Information, Table S1). The sequence of

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PCR product was blasted in the NCBI database for the identification of homologous genes. Basic

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information of BrWRKY6 protein such as theoretical isoelectric point (pI) and mass value were

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calculated using the online database (http://web.expasy.org/compute_pi/). CLUSTALW (version

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1.83), GeneDoc and MEGA5 software were used for the alignment and phylogenetic tree of

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WRKY proteins respectively.

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Detection of Gene Expression with qRT-PCR. Quantitative real-time PCR (qRT-PCR) was

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carried out in a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad) using GoTaq® qPCR Master

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Mix Kit (Promega). The transcript levels of each gene were normalized using EF-1-α

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(GO479260) as the reference gene. Gene-specific primers for qRT-PCR are listed in Supporting

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Information, Table S1.

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Subcellular Localization Analysis. The complete Open Reading Frame (ORF) of BrWRKY6

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was fused with the green fluorescent protein (GFP) gene in the pEAQ-GFP vector. The fusion

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construct pEAQ-BrWRKY6-GFP or the control pEAQ-GFP vector was electro-transformed into

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the Agrobacterium tumefaciens strain GV3101, and transiently expressed in tobacco (Nicotiana

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benthamiana) leaves as described previously.3,36 After 48 h of infiltration, infected leaf tissues

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were analyzed for GFP signal using a fluorescence microscope (Zeiss Axioskop 2 Plus).

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Promoter Isolation and Analysis. DNeasy Plant Mini Kit (Qiagen) was used to isolate

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Genomic DNA of cabbage leaves. The promoters of two GA biosynthetic genes (BrKAO2 and

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BrGA20ox2), and SAGs (BrSAG12, BrPPH1, BrNYC1 and BrSGR1), were amplified with nested

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PCR (primers are listed in Supporting Information, Table S1) using a Genome Walker Kit

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(Clontech). The conserved cis-elements presented in each promoter was predicted in Plant-CARE

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database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

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Electrophoretic

Mobility

Shift

Assay

(EMSA).

The

C-terminal

containing

the

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WRKY-domain of BrWRKY6 (from 290 to 553 aa) was inserted into the pGEX-4T-1 vector, and

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expressed Escherichia coli strain BM Rosetta (DE3), to produce recombinant GST-BrWRKY6

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protein. The induced GST-BrWRKY6 protein was further purified using Glutathione-Superflow

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Resin (Clontech) following the manufacturer’s protocol. Synthesized oligonucleotide probes were

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labeled with biotin at their 5’ ends. EMSA was conducted using the LightShift Chemiluminescent

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EMSA Kit (Thermo Scientific) as previously reported.3 Briefly, GST-BrWRKY6 protein was

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incubated with biotin-labeled probes, then free and protein-DNA complexes were separated by

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6% native polyacrylamide gel electrophoresis, transferred onto nylon membrane and detected by

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a ChemiDoc™ MP Imaging System (Bio-Rad) using the chemiluminescence method. Unlabeled

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and mutated probes, as well as GST protein alone, were used as competitors and negative control

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respectively. Probes used for EMSA are listed in Supporting Information, Table S1.

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Dual-Luciferase Reporter Assays in Tobacco Leaves. For assaying the binding activity of

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BrWRKY6 to the promoters of BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1, their

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promoters were inserted into pGreenII 0800 vector carrying a firefly luciferase (LUC) as

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reporters.37 A 35S promoter-driving renilla luciferase (REN) in the same vector was adopted as

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the internal control. Effector plasmid was constructed by inserting BrWRKY6 into the pEAQ

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vector. The constructed effectors and reporters plasmids with different combinations were

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co-transformed into tobacco leaves mentioned above. LUC and REN luciferase activities were

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measured after 48 h of infiltration on a Luminoskan Ascent Microplate Luminometer (Thermo

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Fisher Scientific) using the dual luciferase assay kit (Promega). The transcriptional ability of

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BrWRKY6 was calculated by the LUC to REN ratio. Six repeats were included for each pair.

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Primers used in this assay are listed in Supporting Information, Table S1.

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Statistical Analysis. Data are presented as mean ± standard errors (S.E.) of three or six

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independent biological replicates. Statistical differences between samples were analyzed by

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Student’s t-test (P < 0.05 or 0.01).

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RESULTS AND DISCUSSION

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Exogenous Application of GA3 Suppresses Leaf Senescence of Chinese Flowering

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Cabbage. As shown in Figure 1a, the leaves of control cabbages began to turn yellow after 5

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days of storage, and by day 7, it became very pronounced. However, GA3-treated cabbage leaves

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remained greener than the control on both day 5 and 7 (Figure 1a). A similar trend was evident

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for noninvasive chlorophyll fluorescence (Figure 1a). The maximum quantum yield (Fv/Fm) and

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total chlorophyll content were significantly higher, while electrolyte leakage was lower in

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GA3-treated leaves compared to control (Figure 1b). We also compared the expression of SAGs

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including BrSAG12, BrSAG19, and chlorophyll degradation genes BrPPH1, BrNYC1, BrSGR1

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and BrSGR2 between GA3-treated and control leaves. The relative expression levels of all SAGs 8


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were increased during leaf senescence, while GA3 application significantly repressed their

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expression compared to control, with a 74.7%, 30.4%, 79.5%, 28.9% and 75.6% decrease for

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BrSAG12, BrSAG19, BrPPH1, BrSGR1 and BrSGR2 respectively, at 7 days of storage.

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Collectively, these results suggest that exogenous application of GA3 suppresses leaf senescence

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of Chinese flowering cabbage, which is similar to the delayed leaf senescence observed in

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GA-treated dandelion, banana, nasturtium, rumex, alstromeria and herbaceous perennial Paris

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polyphylla9,21–24. However, it has been demonstrated recently that exogenous application of GA3

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promotes senescence of Arabidopsis rosette leaves.33,38 These findings indicate that the effect of

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GA on senescence seems contradictory. It has been speculated that the concentration of GA

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applied or the treated leaves situation may lead to opposite regulation of leaf senescence,38 which

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needs to be further investigated, especially in economically important crops.

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Exogenous GA3 Maintains Higher Expression Levels of GA Biosynthetic Genes BrKAO2

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and BrGA20ox2. GA is biosynthesized from geranylgeranyl diphosphate (GGDP) following

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three stages in three subcellular compartments through the activity of a set of enzymes, such as

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terpene synthases (TPSs), ent-kaurene synthase (KS), ent-kaurenoic acid oxidase (KAO) and GA

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20-oxidase (GA20ox), are associated with GA biosynthesis.39–41 We speculated that the

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expression of Chinese flowering cabbage GA biosynthetic genes like BrKS1, BrKAO2 and

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BrGA20ox2, might be affected by GA3 treatment. As shown in Figure 2, qRT-PCR analysis

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showed that transcription levels of BrKAO2 and BrGA20ox2 decreased during leaf senescence,

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while GA3 treatment reversed that decline. Compared with the control leaves, transcription levels

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of BrKAO2 and BrGA20ox2 were higher in GA3-treated leaves (Figure 2). The expression of

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BrKS1 was, however, only slightly affected by GA3 treatment (Figure 2). Thus, our data suggest

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that the suppression of leaf senescence by exogenous application of GA3 may be associated with

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higher transcript levels of GA biosynthesis genes, such as BrKAO2 and BrGA20ox2.

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Identification and Molecular Characterization of BrWRKY6. The plant-specific WRKY

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family is one of the most important senescence-related TFs.26 Previously, Arabidopsis AtWRKY6

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was identified as an important regulator of leaf senescence.42,43 From our RNA-seq transcriptome

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database associated with Chinese flowering cabbage leaf senescence (unpublished data), a

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homologue of AtWRKY6, which showed increased expression during senescence, attracted our

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interest, and we therefore termed this gene as BrWRKY6. The Open Reading Frame (ORF) of

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BrWRKY6 is 1536 bp, encoding a protein of 553 amino acids. Its predicted molecular weight and

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isoelectric point (pI) is 60.49 kDa and 5.71 respectively. Sequence alignment showed that

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BrWRKY6 contains one highly conserved WRKY domain followed by a C2H2-type zinc finger

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motif (Figure 3a), which is the typical characteristic of group II WRKY family.44 To determine

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the phylogeny of BrWRKY6, a phylogenetic tree was constructed, which showed that BrWRKY6

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is a member of WRKY sub-family group IId, and is closely related to AtWRKY31 and

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AtWRKY6 (Figure 3b).

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Consistent with the RNA-seq data, qRT-PCR analysis showed that the BrWRKY6 transcript

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level was increased during leaf senescence. However, compared to the control, GA3 treatment

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reduced the expression of BrWRKY6 by ~59% and 53% on the 5th and 7th days of treatment

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respectively (Figure 4a). To investigate the subcellular localization of BrWRKY6, a BrWRKY6

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and green fluorescent protein (GFP) fusion construct was prepared, and transiently expressed in

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tobacco leaves. Like most reported WRKY TFs that localize in nucleus,3,45 the fluorescence of

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BrWRKY6-GFP was also predominately observed in the nucleus of epidermal cells, and GFP

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signal of positive control was detected both in cytosol and nucleus (Figure 4b). These results

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show that BrWRKY6 is a gibberellin-repressible and nuclear protein.

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BrWRKY6 Regulates the Expression of BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and

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BrSGR1 by Targeting their Promoters through W-box. It is well-documented that WRKY TFs

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specifically bind directly to (C/T)TGAC(C/T) cis-acting element termed as W-box to regulate

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their target genes expression.46,47 EMSA using the purified recombinant BrWRKY6 protein

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(Figure 5a) was performed to investigate whether BrWRKY6 could recognize W-box. As shown

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in Figure 5b, the recombinant BrWRKY6 protein could bind to W-box cis-acting element. As a

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hormone that plays multiple roles in plant development, the biosynthesis of GA must be finely

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regulated. Several TFs such as YABBYs, AT-hook protein of GA feedback1 (AGF1),

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REPRESSION OF SHOOT GROWTH (RSG) and homeodomain-leucine zipper (HD-Zip)

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modulate GA levels by targeting its biosynthetic gene GA20ox2 and GA3ox1,25,48–50 suggesting

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that transcriptional regulation is an important mechanism in controlling GA biosynthesis. In this

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study we have investigated whether BrWRKY6 could regulate the activity of GA biosynthetic

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genes BrKAO2 and BrGA20ox2. Intriguingly, promoter analysis revealed the presence of W-box

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motif in BrKAO2 and BrGA20ox2 promoters (Supporting Information, Text S1), and it was

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shown that the recombinant BrWRKY6 protein could bind to the W-box-containing fragment of

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the BrKAO2 and BrGA20ox2 promoters (Figure 5b). Moreover, increasing amounts of unlabeled

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wild probe obviously reduced BrWRKY6 binding to the biotin-labeled probes, whereas unlabeled

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mutant probes were failed to compete for BrWRKY6 binding (Figure 5b), further indicating that

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BrWRKY6 targets the W-box motif in the BrKAO2 and BrGA20ox2 promoters. WRKY TFs also

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target promoters of SAGs. For instance, Arabidopsis WRKY45 directly binds to the promoter of

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SAG12, SAG13, SAG113 and SEN4.33 Chinese Flowering Cabbage BrWRKY65 can target

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BrNYC1 and BrSGR1.3 We successfully obtained the promoter sequences of BrSAG12, BrPPH1,

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BrNYC1 and BrSGR1, and putative W-box elements were found in the promoters of BrSAG12,

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BrNYC1 and BrSGR1 (Supporting Information, Text S1), suggesting that they may be the targets

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of BrWRKY6. As expected, EMSA results showed that BrWRKY6 bound to the

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W-box-containing fragment of the BrSAG12, BrNYC1 and BrSGR1 promoters (Figure 5b).

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However, it should be pointed out that the direct binding of BrWRKY6 to these promoters is need

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to be further verified by in vivo experiments such as chromatin immunoprecipitation (ChIP)

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assays.

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To further test the effect of BrWRKY6 on BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and

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BrSGR1 expressions after binding to their promoters, we performed a dual-luciferase reporter

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assay in tobacco leaves. For this experiment, the promoter of BrKAO2, BrGA20ox2, BrSAG12,

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BrNYC1 or BrSGR1 was cloned into the pGreenII 0800-LUC vector and co-infiltrated into

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tobacco leaves with BrWRKY6 (Figure 6a). As illustrated in Figure 6b, when BrWRKY6 was

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co-transformed with BrKAO2 or BrGA20ox2 pro-LUC reporter, LUC/REN ratio was remarkably

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reduced, compared with the control that was co-transfected with the empty construct. Further,

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co-transformation of BrWRKY6 with BrSAG12, BrNYC1 or BrSGR1 pro-LUC reporter,

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considerably increased the value of LUC/REN ratio (Figure 6b). These data indicate that

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BrWRKY6 represses BrKAO2 and BrGA20ox2 expression, while activates BrSAG12, BrNYC1

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and BrSGR1, by targeting their promoters, demonstrating that BrWRKY6 may act as a

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transcriptional activator or repressor. Similarly, Arabidopsis WRKY6, WRKY8, WRKY33 and

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WRKY53 have also been reported to be an activator or repressor of gene expression.43,51-53 For

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example, WRKY8 regulates ABI4 positively and ACS6 negatively when plants are subjected to

265

virus infection.51 Importantly, WRKY33 can be a repressor or an activator in a promoter-context

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dependent manner.52 In general, tissue GA levels is precisely controlled through balancing active

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and inactive GAs.54 Active GAs are deactivated by several enzymes such as GA 2-oxidase

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(GA2ox), cytochrome P450 monooxygenase, and GA methyltransferases.54,55 Therefore, whether

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BrWRKY6 can activate GA-deactivation genes needs to be clarified to get a better understanding

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of BrWRKY6’s role in GA homeostasis. In addition, how BrWRKY6 exerts its dual regulatory

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roles in controlling its target genes also requires further research.

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In the past decades, DELLA proteins, a central regulatory molecule of GA signaling and action,

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have been extensively studied.56,57 In the presence of GA, DELLA proteins are degraded via the

274

26S proteasome pathway, leading to the release of TFs involved in the activation of

275

GA-responsive genes.56 More importantly, DELLA proteins interact with TFs to control GA

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homeostasis by direct feedback regulation of the GA biosynthesis genes. For instance,

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Arabidopsis DELLA protein GIBBERELLIN INSENSITIVE (GAI) interacts with GAF1

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(GAI-ASSOCIATED FACTOR1) to form the GAI-GAF1 complex, and co-regulates AtGA20ox2,

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thereby contributes to the feedback regulation of GA biosynthesis.58 Another Arabidopsis DELLA

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protein RGL1 can interact with WRKY45, and repress the activation ability of WRKY45, thus

281

attenuating age-triggered leaf senescence mediated by WRKY45.33 Thus, identifying the

282

interaction partners (like DELLA proteins) of BrWRKY6, as well as their connections in

283

association with GA-mediated leaf senescence of Chinese flowering cabbage, will be our next

284

step in furthering our knowledge of the transcriptional regulation of leaf senescence in Chinese

285

flowering cabbage.

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Taken together, we demonstrated that application of GA3 suppressed leaf senescence of

287

Chinese flowering cabbage. Equally importantly, we characterized a GA3-repressible

288

transcriptional regulator BrWRKY6, which regulated the expression of two GA biosynthetic

289

genes and three SAGs by targeting their promoters. Our findings thus provide novel insights into

290

the transcriptional regulation of GA-mediated suppression of leaf senescence in Chinese

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flowering cabbage involving WRKY TFs.

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AUTHOR INFORMATION

293

Corresponding Author

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

295

Funding

296

This work is funded by grant from the National Natural Science Foundation of China

297

(31671897).

298

Notes

299

All authors have no conflicts of interest to declare.

300

ACKNOWLEDGEMENT

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We would like to thank Dr. George P. Lomonossoff (Department of Biological Chemistry, John

302

Innes Centre, Norwich Research Park) for providing the pEAQ vectors, and Dr. Prakash

303

Lakshmanan (Sugar Research Australia) for critically reading and editing the manuscript.

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ASSOCIATED CONTENT

305

Supporting Information Available

306

Table S1. Summary of primers used in this study. 14


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Text S1. Promoter nucleotide sequences of BrKAO2, BrGA20ox2, BrSAG12, BrPPH1, BrNYC1

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and BrSGR1.

309

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461

Figure legends

462

Figure 1. Application of GA3 delays leaf senescence in Chinese flowering cabbage. (a)

463

Appearance and chlorophyll fluorescence imaging (Fv/Fm) of control and GA3-treated cabbage

464

leaves during senescence. (b) Changes in Fv/Fm, chlorophyll content and relative electrolytic

465

leakage in control and GA3-treated cabbage leaves during senescence. (c) Relative expression of

466

six SAGs including BrSAG12, BrSAG19, BrPPH1, BrNYC1, BrSGR1 and BrSGR2 in control and

467

GA3-treated cabbage leaves during senescence. Data presented in (b) and (c) are the mean ± S.E.

468

of three biological replicates. Asterisks indicate a significant difference in GA3-treated leaves

469

compared with control leaves (student’s t-test: *P < 0.05 and **P < 0.01).

470

Changes in expression of GA biosynthetic genes, including BrKS1, BrKAO2 and

471

Figure 2.

472

BrGA20ox2, in control and GA3-treated cabbage leaves during senescence. Each value represents

473

the mean ± S.E. of three biological replicates. Asterisks indicate a significant difference between

474

control and GA3 treatment by student’s t-test: *P < 0.05 and **P < 0.01.

475 476 477 478 479 480 481 482

22


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483

Figure 3. Sequence and phylogenetic analyses of BrWRKY6. (a) Multiple alignment of the

484

deduced BrWRKY6 protein with other plant WRKY proteins including Arabidopsis AtWRKY6

485

(NP_564792.1), AtWRKY31 (NP_001328882.1) and AtWRKY42 (NP_192354.1), and tomato

486

SlWRKY73 (NM_001247873). Identical and similar amino acids were shaded in black and grey

487

respectively. The conserved WRKY motif is underlined. The C2H2-type zinc finger motif is

488

marked by asterisks. (b) Phylogenetic analysis of BrWRKY6 and WRKYs from different species.

489

BrWRKY6, along with Arabidopsis AtWRKY6 and AtWRKY31, and tomato SlWRKY73 were

490

classified into Group IId of WRKYs. Multiple alignment was carried out using CLUSTALW and

491

the phylogenetic tree was constructed with MEGA5.0 using a bootstrap test of phylogeny with

492

Neighbor-Joining test and default parameters.

493 494

Figure 4. Molecular characterization of BrWRKY6. (a) Relative expression of BrWRKY6 in

495

control and GA3-treated cabbage leaves during senescence. Each value represents the mean ± S.E.

496

of three biological replicates. Asterisks indicate a significant difference in GA3-treated leaves

497

compared with control leaves (student’s t-test: *P < 0.05 and **P < 0.01). (b) Subcellular

498

localization of BrWRKY6 in tobacco leaves. The fusion protein (BrWRKY6-GFP) and GFP

499

positive control were transiently expressed in Nicotiana benthamiana leaves respectively. Images

500

were taken in a dark field for green fluorescence, while the outline of the cell and the merged

501

were photographed in a bright field. Bars, 25 µm.

502 503 504 505

23



506

Figure 5. BrWRKY6 binds to BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1 promoters

507

containing W-box cis-element by electrophoretic mobility shift assay (EMSA). (a) SDS-PAGE

508

gel stained with coomassie brilliant blue demonstrating affinity purification of the recombinant

509

GST-BrWRKY6 protein used for the EMSA. (b) BrWRKY6 binds to W-box cis-element of the

510

BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1 promoters. The sequences of the

511

wild-type probes containing the W-box (red letters) were biotin-labeled. The probes were mixed

512

with the purified GST or recombinant GST-BrWRKY6 protein, and the protein–DNA complexes

513

were separated on native polyacrylamide gels. Competition assays for the labeled probes were

514

performed by adding an excess of unlabeled cold or mutated probes. Arrows indicate the position

515

of shifted bands.

516 517

Figure 6. Dual-luciferase transient expression assay in tobacco leaves showing that BrWRKY6

518

regulates the expression of BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1. (a) Diagrams

519

of the reporter and effector vectors. (b) BrWRKY6 represses BrKAO2 and BrGA20ox2 promoters,

520

and activates BrSAG12, BrNYC1 and BrSGR1 promoters. Each value represents the mean ± S.E.

521

of six biological replicates. Asterisks indicate significantly different values (student’s t-test: **P

522

< 0.01).

24


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Figure 1. Application of GA3 delays leaf senescence in Chinese flowering cabbage. (a) Appearance and chlorophyll fluorescence imaging (Fv/Fm) of control and GA3-treated cabbage leaves during senescence. (b) Changes in Fv/Fm, chlorophyll content and relative electrolytic leakage in control and GA3-treated cabbage leaves during senescence. (c) Relative expression of six SAGs including BrSAG12, BrSAG19, BrPPH1, BrNYC1, BrSGR1 and BrSGR2 in control and GA3-treated cabbage leaves during senescence. Data presented in (b) and (c) are the mean ± S.E. of three biological replicates. Asterisks indicate a significant difference in GA3-treated leaves compared with control leaves (student’s t-test: *P < 0.05 and **P < 0.01). 165x200mm (300 x 300 DPI)



Figure 2. Changes in expression of GA biosynthetic genes, including BrKS1, BrKAO2 and BrGA20ox2, in control and GA3-treated cabbage leaves during senescence. Each value represents the mean ± S.E. of three biological replicates. Asterisks indicate a significant difference between control and GA3 treatment by student’s t-test: *P < 0.05 and **P < 0.01. 164x432mm (300 x 300 DPI)


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Figure 3. Sequence and phylogenetic analyses of BrWRKY6. (a) Multiple alignment of the deduced BrWRKY6 protein with other plant WRKY proteins including Arabidopsis AtWRKY6 (NP_564792.1), AtWRKY31 (NP_001328882.1) and AtWRKY42 (NP_192354.1), and tomato SlWRKY73 (NM_001247873). Identical and similar amino acids were shaded in black and grey respectively. The conserved WRKY motif is underlined. The C2H2-type zinc finger motif is marked by asterisks. (b) Phylogenetic analysis of BrWRKY6 and WRKYs from different species. BrWRKY6, along with Arabidopsis AtWRKY6 and AtWRKY31, and tomato SlWRKY73 were classified into Group IId of WRKYs. Multiple alignment was carried out using CLUSTALW and the phylogenetic tree was constructed with MEGA5.0 using a bootstrap test of phylogeny with Neighbor-Joining test and default parameters. 165x245mm (300 x 300 DPI)



Figure 4. Molecular characterization of BrWRKY6. (a) Relative expression of BrWRKY6 in control and GA3treated cabbage leaves during senescence. Each value represents the mean ± S.E. of three biological replicates. Asterisks indicate a significant difference in GA3-treated leaves compared with control leaves (student’s t-test: *P < 0.05 and **P < 0.01). (b) Subcellular localization of BrWRKY6 in tobacco leaves. The fusion protein (BrWRKY6-GFP) and GFP positive control were transiently expressed in Nicotiana benthamiana leaves respectively. Images were taken in a dark field for green fluorescence, while the outline of the cell and the merged were photographed in a bright field. Bars, 25 µm. 404x130mm (300 x 300 DPI)


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Figure 5. BrWRKY6 binds to BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1 promoters containing Wbox cis-element by electrophoretic mobility shift assay (EMSA). (a) SDS-PAGE gel stained with coomassie brilliant blue demonstrating affinity purification of the recombinant GST-BrWRKY6 protein used for the EMSA. (b) BrWRKY6 binds to W-box cis-element of the BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1 promoters. The sequences of the wild-type probes containing the W-box (red letters) were biotin-labeled. The probes were mixed with the purified GST or recombinant GST-BrWRKY6 protein, and the protein–DNA complexes were separated on native polyacrylamide gels. Competition assays for the labeled probes were performed by adding an excess of unlabeled cold or mutated probes. Arrows indicate the position of shifted bands. 60x23mm (600 x 600 DPI)



Figure 6. Dual-luciferase transient expression assay in tobacco leaves showing that BrWRKY6 regulates the expression of BrKAO2, BrGA20ox2, BrSAG12, BrNYC1 and BrSGR1. (a) Diagrams of the reporter and effector vectors. (b) BrWRKY6 represses BrKAO2 and BrGA20ox2 promoters, and activates BrSAG12, BrNYC1 and BrSGR1 promoters. Each value represents the mean ± S.E. of six biological replicates. Asterisks indicate significantly different values (student’s t-test: **P < 0.01). 165x137mm (300 x 300 DPI)


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TOC 46x41mm (300 x 300 DPI)


Characterization of a Transcriptional Regulator ... - ACS Publications

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