BrNAC055, a Novel Transcriptional Activator, Regulates Leaf

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Agricultural and Environmental Chemistry

BrNAC055, a Novel Transcriptional Activator, Regulates Leaf Senescence in Chinese Flowering Cabbage by Modulating Reactive Oxygen Species Production and Chlorophyll Degradation Zhong-qi Fan, Xiao-li Tan, Jian-wen Chen, Zong-li Liu, Jianfei Kuang, Wang-jin Lu, Wei Shan, and Jian-ye Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02309 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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Journal of Agricultural and Food Chemistry

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BrNAC055, a Novel Transcriptional Activator, Regulates Leaf Senescence in

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Chinese Flowering Cabbage by Modulating Reactive Oxygen Species

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Production and Chlorophyll Degradation

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Zhong-qi Fan,† Xiao-Li Tan,† Jian-wen Chen,‡ Zong-li Liu,† Jian-fei Kuang,† Wang-jin Lu,† Wei

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Shan,†* and Jian-ye Chen†*

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†

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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

State

Key

Laboratory

for

Conservation

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China Agricultural University, Guangzhou 510642, China

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‡

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University, Guangzhou 510642, China

and

Utilization

of

Subtropical

Department of Crop Science and Technology, College of Agriculture, South China Agricultural

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ACS Paragon Plus Environment


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ABSTRACT: Both NAC transcription factors (TFs) and reactive oxygen species (ROS) are

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known to be involved in leaf senescence. However, how NAC TFs modulate ROS metabolism

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associated with leaf senescence remains largely uncharacterized, especially during leaf

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senescence of postharvest economically leafy vegetables such as Chinese flowering cabbage.

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Here, we found that expression levels of two genes BrRbohB and BrRbohC-like encoding

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ROS-producing enzymes respiratory burst oxidase homologues (RBOHs) were increased

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consistently with the progression of postharvest leaf senescence, exhibiting a good correlation

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with ROS accumulation and chlorophyll degradation, as well as expressions of two chlorophyll

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catabolic genes (CCGs), BrNYC1 and BrNYE1. Significantly, a novel, nuclear-localized

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transcriptional activator BrNAC055 was identified, and observed to show a similar expression

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pattern with BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1. Further gel mobility shift and dual

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luciferase reporter assays confirmed that BrNAC055 bound directly to the NAC binding sequence

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(NACBS) in BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters, and activated their

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activities. Moreover, transient over-expression of BrNAC055 in tobacco leaves made an increased

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ROS level and accelerated chlorophyll degradation via the up-regulation of NbRbohA and

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NbSGR1, resulting in the promoted leaf senescence. Based on these findings, we conclude that

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BrNAC055 acts as a transcriptional activator of ROS production and chlorophyll degradation by

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activating the transcriptions of RBOHs and CCGs, and thereby accelerates leaf senescence in

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Chinese flowering cabbage.

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KEYWORDS: Chinese flowering cabbage, ROS, leaf senescence, NAC, chlorophyll degradation

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INTRODUCTION

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Chinese flowering cabbage (Brassica rapa ssp. parachinensis), a highly nutritious vegetable

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traditionally cultivated in China, is becoming increasingly popular in Asia and Western countries.

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Rapid senescence with yellowing leaves causes major post-harvest loss of this vegetable during

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transportation and storage.1 Therefore, understanding the physiological and molecular

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mechanisms regulating postharvest leaf senescence is extremely important for maintaining the

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shelf-life and economic value of this important leafy vegetable.

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Leaf senescence is a complicated biological process, which is regulated by many factors such

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as age, abiotic and biotic stresses, plant hormones and transcription factors (TFs).2,3 There are

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numerous reports on the involvement of phytohormones including ethylene, jasmonic acid (JA),

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gibberellins (GAs) and abscisic acid (ABA), and TFs such as WRKY and NAC, in leaf

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senescence.2−6 Additionally, reactive oxygen species (ROS) has been implicated to participate in

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leaf senescence. Obvious increase in ROS generation in senescent leaves has been observed in

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Arabidopsis.7,8 The over-production of ROS causes an imbalance in cell redox that may

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accelerate senescence leading to cell death.7,8 ROS are produced by multiple enzymes, among

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which respiratory burst oxidase homologues (RBOHs), belonging to plant NADPH oxidases, are

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the most thoroughly studied enzymes generating ROS.9,10 Ten RBOH homologues from

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Arabidopsis, AtRbohA-J, form a small multigene family, which are grouped into three classes

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based on their distribution in different plant parts.11 There are many studies to explore the

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mechanism of RBOH gene action in association with ROS generation in many plant biological

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processes including cell growth, plant development and stresses responses.9,10 For example, it has

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been shown that AtRbohD and AtRbohF are the prominent members as they are necessary for 3



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ROS production during ABA-mediated stomatal closure.12 In tobacco (Nicotiana benthamiana),

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NbRbohA and NbRbohB are associated with H2O2 accumulation in response to phytophthora

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infestans infection.13 Recently, it has been reported that Rboh genes are regulated by specific TFs.

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For instance, NTL4, a drought-responsive NAC TF, stimulates ROS production and induces leaf

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senescence in Arabidopsis.14 It is proposed that a MAPK-WRKY pathway is required for

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R3a/AVR3a-triggered immunity ROS burst by the transactivation of NbRbohB.15 These reports

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reveal that transcriptional regulation is an important mechanism in governing ROS production.

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NAC TFs are one of the largest TF families with more than 100 members in Arabidopsis, rice

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and other plant species.16,17 Many NAC proteins such as Arabidopsis AtNAP,18 ORE1,19

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ANAC016,20 ANAC072,21 ATAF1,22 rice OsNAP,23 and OsNAC224 are positive regulators of

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leaf senescence, since over-expression of these genes resulted in accelerated senescence, whereas

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blocking or reducing their expression delayed senescence. In contrast, Arabidopsis JUB1,25

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VNI2,26 and rice ONAC106,27 have negative effects on leaf senescence. Moreover, downstream

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genes of these NAC TFs have been identified, clearly proving that NAC TFs function in leaf

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senescence by regulating senescence-associated genes (SAGs) including chlorophyll catabolic

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genes (CCGs) PAOs, NYCs/NYEs and SGRs, as well as genes related to hormone biosynthesis

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and signaling like ABA biosynthetic genes ZEPs, NCEDs and AAOs, and ABA catabolic gene

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ABA8ox.28 Interestingly, Arabidopsis ANAC032,29 oilseed rape (Brassica napus L.)

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BnaNAC55,30 BnaNAC5631 and BnaNAC8732 are recently demonstrated to act as regulators of

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ROS-mediated cell death and leaf senescence.

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Although NAC TFs and ROS are well-documented to be engaged in leaf senescence,

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TFs-mediated RBOHs associated with ROS production and leaf senescence, especially in

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economically important leafy vegetables, remains to be elucidated. In this study, we found that

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ROS accumulation was increased substantially during leaf senescence of Chinese flowering

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cabbage, paralleling with the induced expressions of BrRbohB and BrRbohC-like. Intriguingly,

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we identified a novel NAC TF BrNAC055, which showed a similar expression pattern with

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BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1, suggesting that it may be a potential regulator of

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ROS accumulation and chlorophyll degradation during leaf senescence. Accordingly, further

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assays verified that BrNAC055 could bind to and activate the promoters of BrRbohB,

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BrRbohC-like, BrNYC1 and BrNYE1. Finally, transient over-expression of BrNAC055 in tobacco

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caused accumulation of ROS and early leaf yellowing. We thus propose that BrNAC055 likely

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acts as a positive regulator of Chinese flowering cabbage leaf senescence by directly activating

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the expressions of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1, and consequently promotes

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ROS production and chlorophyll degradation.

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MATRIALS AND METHODS

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Plant Materials and Samples. Chinese flowering cabbage (Brassica rapa var. parachinensis)

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used in our experiments were grown in a local commercial plantation near Guangzhou, southern

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China. Harvested cabbages were cooled and transported to the lab immediately. Uniform

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non-flowering cabbages with no mechanical damage were selected randomly and placed into

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plastic baskets (10 per box) packed with polyethylene perforated plastic bags to prevent water

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loss. Then, the packed cabbages were kept in dark at 25°C. On the 0, 1, 3, 5 and 7th days of

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storage, the third leaves from the bottom of ten cabbage plants were sampled for analyzing total

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chlorophyll content, chlorophyll fluorescence, and ROS level, as well as temporal expression

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patterns of genes. On the 5th day, the third leaves from the bottom of one cabbage plant was 5



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detached, dissected into three sections and sampled respectively for investigating localized

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expression of genes in a senescing leaf. All sampled leaves were frozen in liquid nitrogen

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immediately and stored at -80°C for further analysis.

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Assessment of Total Chlorophyll Content and Fv/Fm Ratio. The total chlorophyll from leaf

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tissue was extracted in 80% acetone by overnight incubation in dark at 4°C and quantified

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spectrophotometrically at 663 and 645 nm as described earlier.33 The ratio of variable to maximal

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fluorescence (Fv/Fm) was determined non-invasively through a chlorophyll fluorometer

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(Imaging-PAM-M series, Heinz Walz) following the manufacturer’s instruction, which also can

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capture highly resolved digital images of the emitted fluorescence by a charge-coupled device

120

(CCD) camera.

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DAB and NBT Staining. The leaves of Chinese flowering cabbage or tobacco were stained detecting

ROS

accumulation

as

described

previously.34

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for

Leaves

in

DAB

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(3,3’-diaminobenzidine) [1 mg/ml DAB (pH 3.8)] and NBT (nitro blue tetrazolium) staining

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solution [0.5 mg/ml NBT (pH 7.8)] were incubated in darkness for 6 h. Leaves were boiled in

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95% ethanol to eliminate chlorophyll, and then they were stored in fresh 95% ethanol and imaged

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with a camera.

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RNA Extraction, Gene Sequence Analysis and qRT-PCR. Total RNA was isolated from

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cabbage or tobacco leaves using RNeasy Mini kit (Qiagen) according to the user’s manual.

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Reverse transcriptase M-MLV (TaKaRa) was used to synthesis first-strand cDNA. According to

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the sequence deposit in genome database of Chinese cabbage chiifu (http://brassicadb.org/brad/),

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the full-length of BrNAC055 was cloned, sequenced and blasted in the NCBI database. The

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theoretical isoelectric point (pI) and mass value of BrNAC055 protein were predicted at the

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website (http://web.expasy.org/compute_pi/). Multiple-alignment of NACs were performed by

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CLUSTALW (version 1.83) and GeneDoc software. A phylogenetic tree was constructed by

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MEGA program (version 5.0) with the Neighbor-Joining algorithm.

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Quantitative real-time polymerase chain reaction (qRT-PCR) using GoTaq qPCR master mix

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kit (Promega) was carried out with gene-specific primers on the Bio-Rad CFX96 Real-Time PCR

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System. The BrActin1 was used as an internal control.35

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Subcellular Localization of BrNAC055 in Epidermal Cells of Tobacco Leaves. The coding

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sequence of BrNAC055 was inserted into pEAQ vector to fuse with the green fluorescent protein

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(GFP).36 The pEAQ-BrNAC055-GFP or the control pEAQ-GFP plasmids were introduced into

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the Agrobacterium tumefaciens strain EHA105, and then transformed into of tobacco (Nicotiana

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benthamiana) leaves as previously described.33,36 After 2 days of infiltration, epidermal cells were

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observed on a Zeiss fluorescence microscope under a bright field and GFP channel.

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Transcriptional Activation Assay in Yeast Cells. BrNAC055 was cloned into the pGBKT7

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vector (Clontech) to fuse with GAL4 DNA-binding domain. Constructs including

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pGBKT7-BrNAC055, positive control (pGBKT7-53 + pGADT7-T) and negative control

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(pGBKT7 vector) were transferred into yeast strain AH109, following streaking on SD medium

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without tryptophan (SD/-Trp), or tryptophan, histidine, and adenine (SD/-Trp-His-Ade) plates.

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The transcriptional activation of BrNAC055 was assessed based on the growth status and

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α-galactosidase activity of yeast cells.

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Recombinant Protein Preparation and Gel Mobility Shift Assay. To prepare the

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recombinant BrNAC055 protein, the N-terminal fragment of BrNAC055 was amplified and

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cloned into pGEX-4T-1. The GST-BrNAC055-N was introduced into Escherichia coli strain

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Transetta (DE3), and E.coli cells were grown to an OD600 of approximately 0.6. Recombinant

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GST-BrNAC055-N

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isopropyl-b-D-thiogalactopyranoside (IPTG) (0.3 mM ) for 3 hours at 37ºC with gentle shaking.

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Cells were collected and lysed by ultrasonic cell crusher (Sonics) and cleared by concentration.

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Recombinant GST-BrNAC055-N fusion protein was purified using Glutathione-Superflow Resin

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(Clontech) according to the manufacturer’s protocols. The resulting protein was confirmed

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according to the size and purity by SDS-PAGE and Coomassie Brilliant Blue staining.

protein

expression

was

then

induced

with

162

Gel mobility shift assay was performed as described previously.33 The probes containing NAC

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binding sequence (NACBS) (T[G/A]CGT or CACG)37 derived from BrRbohB, BrRbohC-like,

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BrNYC1 and BrNYE1 promoters were labeled with biotin at the 5’ end, and were incubated with

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GST-BrNAC055-N protein (~1 µg) at room temperature for 25 min in a binding buffer. Cold

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probes with unlabeled wild type or mutant DNA fragments were used as competitors. Free and

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protein-DNA complexes were separated using 6% native polyacrylamide gel electrophoresis, and

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detected by a ChemiDoc™ MP Imaging System (Bio-Rad).

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Dual-Luciferase Reporter Assay. For trans-activation assay of BrNAC055, its coding

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sequence was cloned into the modified pBD vector as effectors.38 The reporter construct was

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modified based on pGreenII 0800-LUC reporter vector, which contains 35S::GAL4-firefly

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luciferase (LUC) and 35S::renilla luciferase (REN) as internal control.39 For the assay of

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BrNAC055 on the activation of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters, the

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BrNAC055 coding region was cloned into pEAQ vector to generate effector construct, while

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promoters of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 were introduced into pGreenII

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0800-LUC vector as reporter constructs. Different pairs of effector and reporter constructs were

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co-transferred into tobacco leaves mentioned above.

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After 48 h of transfection, LUC and REN activities were quantified using the dual luciferase

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assay kit (Promega) on a 96-well Luminoskan Ascent Microplate Luminometer (Thermo Fisher

180

Scientific). The ratio of LUC/REN was recorded to indicate the transcriptional ability of

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BrNAC055.

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Transient Over-expression Assay in Tobacco Leaves. Transient over-expression experiments

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were conducted in tobacco leaves using the pEAQ construct carrying BrNAC055 as described

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above. Empty vector used as the control was also infiltrated in a different set of leaves. At 1 and

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3th days of infiltration, the infiltrated tobacco leaves were collected for the detection of

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chlorophyll fluorescence, ROS production and expressions of NbrbohA and NbSGR1.

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Data Analysis. All experimental data were recorded as averages of three or six independent

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biological replicates. Statistical significance between samples was examined by student’s t-test

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(*P < 0.05, **P< 0.01).

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Primers. All primers used in this work were designed using Primer 3 online

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(Http://bioinfo.ut.ee/primer3/), and are shown in Table S1.

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RESULTS

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Evaluation of Chinese Flowering Cabbage Postharvest Leaf Senescence Process. Leaf

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yellowing is the most apparent appearance of senescence, which is caused by chlorophyll

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degradation mediated by CCGs. As shown in Figure 1a, harvested Chinese flowering cabbage

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senesced rapidly at 25°C. Cabbage leaves started yellowing on the 3th day of storage, and it

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became more apparent with the duration of storage time. Consistently, two senescence-associated

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physiological parameters including total chlorophyll content and Fv/Fm ratio, declined 9



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significantly following leaf yellowing progression. The values of total chlorophyll content and

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Fv/Fm ratio on 5th day of storage were ~36.7% and 46.1% of day 0, respectively (Figure 1b). On

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the contrary, the transcription levels of two CCGs BrNYC1 and BrNYE1 related to chlorophyll

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degradation, increased gradually during leaf senescence (Figure 1c).

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ROS Accumulation and Expression of BrRbohs during Leaf Senescence. DAB and NBT

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staining can clearly display H2O2 and O2•− accumulation, respectively. As shown in Figure 2a,

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along with the progression of postharvest leaf senescence of cabbages, both DAB and NBT

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staining intensity obviously increased, suggesting that more ROS were produced in senescent

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leaves. Two genes encoding ROS-generating enzymes RBOHs were notified to be induced

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during leaf senescence from our previous transcriptome database. qRT-PCR was performed to

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quantify their transcription levels, which showed that the expressions of BrRbohB and

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BrRbohC-like were significantly up-regulated during senescence, reaching about 3.2- and

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24.3-fold of the initial level at the 7th day of storage, respectively (Figure 2b).

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Similar with Arabidopsis leaf senescence, cabbage leaf senesces serially from the tip to the

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base. The representative third leaf from the bottom of the cabbage at the 5th day of storage was

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divided into three sections across the leaf axis (Figure 2c). Compared with the leaf basal section,

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the transcript levels of BrRbohB and BrRbohC-like were higher in the senescing middle and tip

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section (Figure 2d). Thus, these data indicate that BrRbohB- and BrRbohC-like-mediated ROS

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production might be related to Chinese flowering cabbage leaf senescence.

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Identification of BrNAC055. Previously, Arabidopsis ANAC055 and oilseed rape BnNAC55

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are reported to be involved in leaf senescence.30,40 A homologue of ANAC055 and BnNAC55,

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was also found in Chinese cabbage genome and NCBI database (XM_009117250.2), and we

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therefore designated it as BrNAC055. Homology research revealed that BrNAC055 exhibited the

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highest similarity to ANAC055 (86%). The Opening Reading Frame (ORF) length of BrNAC055

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is 954 bp, and encodes a protein of 318 amino acids, with calculated molecular weight of 78.11

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kDa, and a pI value of 5.08. The published senescence-associated NACs in Arabidopsis28 were

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used for constructing a phylogenetic tree. As illustrated in Figure 3a, the senescence-associated

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NACs fall in group I-IV. BrNAC055 is clustered with ANAC055, ANAC019, ANAC072,

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ANAC047 and NAP, which are belonged to NAC IV. Moreover, alignment of BrNAC055 with a

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few senescence-associated NACs showed that these proteins include a NAC conserved domain in

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their N-terminus regions with five sub-domains (A-E) (Figure 3b).

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Molecular Characterization of BrNAC055. To explore the possible association of

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BrNAC055 with leaf senescence, its expression pattern was investigated by qRT-PCR. As shown

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in Figure 4a, accompanying with leaf senescence progression, the expression of BrNAC055 was

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obviously increased, reaching about 4.7- and 43.3-fold of the initial level at the 5th and 7th day of

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storage, respectively. In addition, localized expression of BrNAC055 in a senescing leaf showed

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that it was highly expressed in the tip and middle sections, but marginally in the basal section

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(Figure 4b). In addition, BrNAC055 was significantly up-regulated by methyl jasmonate (MeJA)

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and abscisic acid (ABA) (Figure S1), which could accelerated leaf senescence of Chinese

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flowering cabbage,41 while was repressed by gibberellin (GA3) (Figure S1) that delayed leaf

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senescence33. A nuclear localization signal (NLS) is included in BrNAC055 sequence (Figure 3b),

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indicating that BrNAC055 maybe a nucleus protein. To confirm this speculation, BrNAC055 was

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fused in-frame with GFP driven by CaMV35S promoter, and transiently expressed in tobacco leaf

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epidermal cells. Compared with GFP positive control that was uniformly dispersed throughout

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the whole cell, BrNAC055-GFP was predominantly observed in the nucleus (Figure 4c). We also

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investigated the transcriptional activity of BrNAC055 using the GAL4-responsive reporter

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system in yeast cells and dual-luciferase reporter system in tobacco leaves. As shown in Figure

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4d, the transformed yeast cells expressing either BrNAC055 or the positive control (pGBKT7-53

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+ pGADT7-T) grew well on SD/-Trp-His-Ade plates, and showed α-galactosidase activity,

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whereas negative control cells (pGBKT7 vector) showed no α-galactosidase activity.

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Transcriptional activation of BrNAC055 was further validated in tobacco leaves, showing that

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co-expression of BrNAC055 or the activator control VP16 with the reporter could significantly

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elevated the ratio of LUC/REN (Figure 4e). Together, these results imply that BrNAC055 is a

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nuclear-localized transcriptional activator that positively associated with leaf senescence.

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BrNAC055 Activates the Transcriptions of BrRbohB, BrRbohC-like, BrNYC1 and

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BrNYE1 by Binding to NACBS in Their Promoters. Considerable similarity of gene

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expression between BrNAC055 and BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 suggests that

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BrNAC055 may be involved in regulating the expression of these genes during senescence.

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Importantly, the presence of NACBS (T[G/A]CGT or CACG) in the promoter regions BrRbohB,

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BrRbohC-like, BrNYC1 and BrNYE1 (Text S1) further indicates that these genes might be the

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direct targets of BrNAC055.

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To determine whether BrNAC055 could bind to the promoters of BrRbohB, BrRbohC-like,

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BrNYC1 and BrNYE1 in vitro, gel mobility shift assay was performed. For this, GST-BrNAC055

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fusion protein was produced and purified (Figure 5a). DNA fragments harboring the NACBS

263

were biotin-labeled and used as probes. Unlabeled cold or mutated probes served as competitors.

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As presented in Figure 5b, BrNAC055 protein was able to bind the BrRbohB, BrRbohC-like,

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BrNYC1 and BrNYE1 promoter fragments and caused a mobility shift. Moreover, an excess

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amount of unlabeled probe (cold probe) reduced BrNAC055 binding capacity, while unlabeled

267

mutant probe could not affect the binding. In addition, the shift band was not observed when

268

these promoter fragments were incubated with empty GST protein (Figure 5b), indicating that the

269

binding of BrNAC055 to the BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters is

270

specific.

271

Transient dual-luciferase reporter assay in tobacco leaves was further carried out to test

272

whether BrNAC055 directly activating or suppressing the expressions of BrRbohB, BrRbohC-like,

273

BrNYC1 and BrNYE1. The results showed that LUC/REN ratio was significantly increased when

274

the BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 pro-LUC reporter construct was co-expressed

275

with BrNAC055, compared with the control that was co-transfected with the empty construct

276

(Figure 5c), indicating that BrNAC055 activated the transcriptions of BrRbohB, BrRbohC-like,

277

BrNYC1 and BrNYE1 in vivo. These findings provide evidences to support that BrNAC055 acts

278

as an activator of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 transcription through direct

279

binding to their promoters.

280

Transient Over-expression of BrNAC055 Induces ROS Accumulation and Promotes

281

Chlorophyll Degradation in Tobacco Leaves. To gain more evidence of the involvement of

282

BrNAC055 in leaf senescence by controlling ROS production and chlorophyll degradation, it was

283

transiently over-expressed in tobacco leaves since it is difficult to perform stable transformation

284

of Chinese flowering cabbage. The results showed that compared with the left blade of the leaves

285

that were over-expressed with the empty vector, the BrNAC055-transformed leaves (the right

286

blade of the leaves) showed enhanced ROS accumulation evidenced by the increased intensity of

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287

NBT and DAB staining at 1th and 3th day of infiltration respectively (Figure 6a). At the same

288

time, more yellow lesions were observed in the BrNAC055-transformed leaves at 3 dpi (Figure

289

6a). Accordingly, over-expression of BrNAC055 resulted in a significant decrease in Fv/Fm ratio

290

and total chlorophyll content (Figure 6b). Furthermore, expressions of endogenous tobacco genes

291

related to ROS production and chlorophyll degradation were examined. We found that the

292

transcript levels of NbrbohA and NbSGR1 were substantially increased in BrNAC055-expressing

293

leaves at 3 dpi (Figure 6c). These results further support the inference that BrNAC055

294

transcriptionally regulates leaf senescence in Chinese flowering cabbage through up-regulating

295

RBOHs and CCGs, leading to accumulation of ROS and degradation of chlorophyll.

296

DISCUSSION

297

NACs comprise one of the largest plant-specific TF classes, and have been demonstrated to

298

play critical roles in senescence of model plants such as Arabidopsis and rice.3,4,28 Approximately

299

35 Arabidopsis NAC genes including ORS1,42 ANAC016,20 JUB125 and VNI226 have been shown

300

to be up-regulated during natural leaf senescence. Further studies reveal that NAC TFs can act as

301

positive or negative regulators of leaf senescence. For instance, over-expression of ANAC016

302

caused precocious leaf yellowing, while mutant anac016 remained green.20 ANAC032,

303

ANAC046, ANAC072 and AtNAP are also reported to be positively involving in regulating

304

developmental and dark‐induced senescence,18,21,28,29,43 as well as rice OsNAP,23 OsNAC224 and

305

ONAC011.44 In addition, several NAC TFs including Arabidopsis JUNGBRUNNEN1

306

(JUB1)/ANAC04225 and rice ONAC10627 are found to negatively regulate leaf senescence.

307

Moreover, many senescence-associated genes (SAGs) related to chlorophyll degradation, and

308

ABA biosynthesis and signaling are identified as targets of these NAC TFs.28 For example, 14


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Arabidopsis ANAC016 directly binds to the SGR1 promoter and activates its expression.20

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OsNAP and OsNAC2 directly activates transcription of OsSGR, OsNYCs and OsRCCRs,23,24

311

whereas ONAC106 down-regulates transcription of OsSGR and OsNYC1.27 ROS like hydrogen

312

peroxide (H2O2) have been considered as important signaling components for the regulation of

313

senescence. Notably, at least 15 senescence-associated NAC TFs including AtNAP,

314

ANAC092/ORE1, ORS1, ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR1

315

(ATAF1), ANAC032 and JUB1 are ROS-responsive.25,42 Intriguingly, over-expression of JUB1

316

obviously inhibits senescence and suppresses intracellular H2O2 production.25 However, how

317

JUB1 and other NAC TFs control ROS production during leaf senescence is not clear, especially

318

in economically important leafy vegetables. In the present work, we found that two RBOH genes

319

BrRbohB and BrRbohC-like showed enhanced expression during leaf senescence in Chinese

320

flowering cabbage, paralleling with ROS accumulation (Figure 2). More meaningfully, a

321

senescence-induced transcriptional activator BrNAC055 (Figure 4), could directly bind to

322

BrRbohB and BrRbohC-like promoters and up-regulate their transcription (Figure 5).

323

Additionally, like its homologues Arabidopsis ANAC055, BrNAC055 targeted BrNYC1 and

324

BrNYE1 promoters and activated their expression (Figure 5). Unlike JUB1, transient

325

over-expression of BrNAC055 in tobacco leaves induced ROS production and promoted

326

chlorophyll degradation, resulting in accelerated-leaf senescence (Figure 6). Similarly, a NAC TF

327

NTL4 mediates drought-induced leaf senescence by inducing AtRbohC and AtRbohE and

328

promoting ROS level.14 Our present study, together with previous works, suggest that NAC TFs

329

act as positive or negative regulators of leaf senescence by activating or repressing

330

RBOH-mediated ROS production.

15



331

Nowadays, increasing evidences reveal that leaf senescence is complicated biological process

332

tightly controlled by many TFs.3,4 Besides NACs, EIN3/EIL, WRKYs, bHLHs and MYCs are

333

also demonstrated to be important transcriptional regulators of leaf senescence.3,4 Indeed, these

334

TFs consists of a hierarchical and coordinated regulatory cascade/network. For example, the

335

MYC2-ANAC019 interaction in Arabidopsis synergistically enhances NYE1 transcription, which

336

is contributed to JA-induced chlorophyll degradation during leaf senescence.45 Both Arabidopsis

337

EIN3 and ORE1 can target NYE1 and NYC1, and they co-activate their expression to advance

338

ethylene-mediated chlorophyll degradation.46 Surprisingly, although over-expression of oilseed

339

rape BnaNAC55,30 BnaNAC56,31 BnaNAC8732 and BnaNAC10347 induces ROS accumulation,

340

none of these four NACs can target any RBOHs. Indeed, an oilseed rape WRKY TF BnaWGR1 is

341

found to activate RbohD and RbohF expression, leading to ROS accumulation and promoting leaf

342

senescence.48 Previously, we identified two WRKY TFs BrWRKY6 and BrWRKY65, and

343

showed that they were associated with leaf senescence of Chinese flowering cabbage via the

344

up-regulation of BrNYCs and BrSGRs.33,49 Therefore, elucidating whether BrNAC055,

345

BrWRKY6 and BrWRKY65 can form a complex or a coherent regulatory loop to control the

346

expression of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1, as well as screening other

347

interaction proteins and target genes, will help us to explore the intricate transcriptional

348

regulatory cascade/network during leaf senescence in Chinese flowering cabbage.

349

In conclusion, the present work showed that expression of BrRbohB and BrRbohC-like

350

exhibited a good correlation with ROS accumulation and chlorophyll degradation during

351

postharvest leaf senescence in Chinese flowering cabbage. Equally significantly, a

352

senescence-inducible transcriptional activator BrNAC055 was identified and characterized,

16


Page 16 of 36

Page 17 of 36


353

which directly bound to BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters, and enhanced

354

their transcriptions. Transient over-expression of BrNAC055 in tobacco leaves induced ROS

355

production and promoted chlorophyll degradation, resulting in accelerated-leaf senescence. Based

356

on these data, we propose that BrNAC055 acts as a transcriptional activator of ROS production

357

and chlorophyll degradation by activating the expressions of RBOHs and CCGs, and thereby

358

accelerates leaf senescence in Chinese flowering cabbage. Broadly, our findings are providing

359

more new insights into the intricate transcriptional regulatory network of leaf senescence in

360

Chinese flowering cabbage concerning direct connections of NAC TFs, RBOHs-mediated ROS

361

production and CCGs-mediated chlorophyll degradation.

362

ASSOCIATED CONTENT

363

Supporting Information Available

364

Figure S1. qRT-PCR analysis of BrNAC055 in response to MeJA, ABA and GA3 treatments.

365

Table S1. List of primers used in this study.

366

Text S1. Promoter nucleotide sequences of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1.

367

AUTHOR INFORMATION

368

Corresponding Author

369

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

370

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

371

ORCID

372

Wei Shan: 0000-0002-6789-0082

373

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

374

Funding 17



375

This research is supported by the National Natural Science Foundation of China (31671897).

376

Notes

377

All authors declare that they have no conflict of interest.

378

ACKNOWLEDGEMENT

379

We express our sincere gratitude to Dr. George P. Lomonossoff (Department of Biological

380

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

381

Prakash Lakshmanan (Sugar Research Australia) for critically editing the manuscript. This

382

research is supported by the National Natural Science Foundation of China (31671897).

383

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oxygen species accumulation and cell death in plants. Biochem. Biophys. Res. Commun. 2014,

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454, 30−35.

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(48) Yang, L.; Ye, C. F.; Zhao, Y. T.; Cheng, X. L.; Wang, Y. Q.; Jiang, Y. Q.; Yang, B. An

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oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in

523

Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF.

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Planta 2018, doi.org/10.1007/s00425-018-2868-z.

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(49) Fan, Z. Q.; Tan, X. L.; Shan, W.; Kuang, J. F.; Lu, W. J.; Chen, J. Y. BrWRKY65, a WRKY

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527

flowering cabbage. Int. J. Mol. Sci. 2017, 18, pii: E1228.

528 529 530 531 532 533 534 535 536 537 538 539 540

25



541

Figure captions

542

Figure 1. Evaluation of Chinese flowering cabbage postharvest leaf senescence process. (a)

543

Appearance and chlorophyll fluorescence imaging (Fv/Fm) of cabbage leaves during senescence.

544

(b) Changes in Fv/Fm and total chlorophyll content during senescence. (c) Relative expressions

545

of BrNYC1 and BrNYE1 during senescence. Data presented in (b) and (c) are the mean ± S.E. of

546

three biological replicates.

547 548

Figure 2. ROS production and expression patterns of BrRbohs during leaf senescence in Chinese

549

flowering cabbage. (a) Appearance, chlorophyll fluorescence imaging (Fv/Fm) and ROS

550

production of cabbage leaves during senescence. The third leaves from the bottom of cabbage

551

plants were subjected to DAB and NBT staining, which indicates the accumulation of hydrogen

552

peroxide and superoxide radicals respectively. (b) Temporal expression patterns of BrRbohB and

553

BrRbohC-like during senescence. (c-d) Localized expressions of BrRbohB and BrRbohC-like in a

554

senescing leaf. The third leaves from the bottom of cabbage plants on the 5th days was detached

555

and separated into three parts as illustrated. B basal part, M middle part, T top part. Data

556

presented in (b) and (d) are the mean ± S.E. of three biological replicates.

557 558

Figure 3. Sequence and phylogenetic analysis of BrNAC055 protein. (a) Phylogenetic

559

relationships of BrNAC055 with other senescence-associated NACs. BrNAC055 was highlighted

560

with black circle. The phylogenetic tree was created with neighbor-joining test using MEGA

561

program (version 5.0). (b) Multiple alignment of BrNAC055 protein with other NAC members.

562

The following proteins were used for analysis: ANAC055 (NP_188169.1), ANAC019

26


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Page 27 of 36


563

(NP_175697.1), ANAC072 (NP_567773.1) and OsNAC5 (XP_015617286.1). The five highly

564

conserved domains A to E were indicated by black lines. The nuclear localization signal (NLS)

565

was up lined with red.

566 567

Figure 4. Molecular characterization of BrNAC055. (a) Temporal expression pattern of

568

BrNAC055 during senescence. (b) Localized expression of BrNAC055 in a senescing leaf. The

569

third leaves from the bottom of cabbage plants on the 5th days was detached and separated into

570

three parts as illustrated in Fig. 2C. Data presented in (a) and (b) are the mean ± S.E. of three

571

biological replicates. (c) Subcellular localization assay in epidermal cells of tobacco leaves. A

572

plasmid harboring GFP or BrNAC055-GFP was transformed into Nicotiana benthamiana leaves

573

by A. tumefaciens strain EHA105. GFP signals was observed with a fluorescence microscope

574

after 48 h of infiltration. Bars, 25 µm. (d) Transcriptional activation of BrNAC055 in yeast cells.

575

The coding region of BrNAC055 was inserted into the pGBKT7 (GAL4DBD) to create the

576

pGBKT7-BrNAC055

577

pGBKT7-BrNAC055 plasmids were grown on SD plates without tryptophan (Trp-) or without

578

tryptophan, histidine, and adenine (Trp-His-Ade-) for 3 days at 28°C, then followed by the

579

α-galactosidase assay (X-Gal staining). pGBKT7 and pGBKT7-53 + pGADT7-T were used as

580

negative and positive control respectively. (e) Trans-activation of BrNAC055 in Nicotiana

581

benthamiana leaves. The trans-activation ability of BrNAC055 was demonstrated by the ratio of

582

LUC to REN. Data are means ± S.E. of six independent biological replicates. Asterisks represents

583

significant differences at 0.01 level by student’s t-test, compared to pBD.

construct.

The

yeast

cells

of

27


strain

AH109

harboring

the


584

Figure 5. BrNAC055 targets BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters, and

585

activates their transcriptions. (a) SDS-PAGE gel stained with Coomassie brilliant blue, presenting

586

affinity purification of the recombinant GST-BrNAC055 protein. (b) Gel mobility shift assay

587

showing GST-BrNAC055 binding to NACBS in the BrRbohB, BrRbohC-like, BrNYC1 and

588

BrNYE1 promoters. GST protein alone was considered as a negative control. “-” and “+”

589

represent absence or presence, respectively. The probe sequence is shown on the top of image,

590

with WT and mutant BrNAC055 binding site marked with red and underlined. Triangles indicate

591

increasing amounts of unlabelled probe for competition. Arrows indicate the shifted bands

592

position. (c) Dual-luciferase transient expression assay in Nicotiana benthamiana leaves showing

593

that BrNAC055 activates the transcriptions of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1.

594

The reporter and effector vectors are illustrated in the top panel. Data are means ± S.E. of six

595

independent biological replicates. Asterisks indicate significant differences by student’s t-test (P

596

< 0.01)

597 598

Figure 6. Transient over-expression of BrNAC055 induces ROS accumulation and promotes

599

chlorophyll degradation in Nicotiana benthamiana leaves. (a) Appearance, Fv/Fm imaging and

600

histochemical staining (DAB and NBT) of tobacco leaves infiltrated with empty vector (left side

601

of leaf) and BrNAC055 (right) carried by Agrobacterium at days 1 and 3 post-infiltration (dpi). (b)

602

Fv/Fm ratio and total chlorophyll content in Nicotiana benthamiana leaves expressing vector and

603

BrNAC055 at 1 and 3 dpi. (c) Transcript levels of NbrbohA and NbSGR1 in empty and

604

BRNAC055 over-expressed Nicotiana benthamiana leaves at 1 and 3 dpi. Data in b and c

28


Page 28 of 36

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605

represent the means ± S.E. of three biological replicates. Asterisks indicate significant differences

606

by student’s t-test (*P < 0.05 **P < 0.01).

29



Figure 1. Evaluation of Chinese flowering cabbage postharvest leaf senescence process. (a) Appearance and chlorophyll fluorescence imaging (Fv/Fm) of cabbage leaves during senescence. (b) Changes in Fv/Fm and total chlorophyll content during senescence. (c) Relative expressions of BrNYC1 and BrNYE1 during senescence. Data presented in (b) and (c) are the mean ± S.E. of three biological replicates. 165x251mm (300 x 300 DPI)


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Figure 2. ROS production and expression patterns of BrRbohs during leaf senescence in Chinese flowering cabbage. (a) Appearance, chlorophyll fluorescence imaging (Fv/Fm) and ROS production of cabbage leaves during senescence. The third leaves from the bottom of cabbages were subjected to DAB and NBT staining, which indicates the accumulation of hydrogen peroxide and superoxide radicals respectively. (b) Temporal expression pattern of BrRbohB and BrRbohC-like during senescence. (c-d) Localized expression of BrRbohB and BrRbohC-like in a senescing leaf. The third leaves from the bottom of cabbages on the 5th days was detached and separated into three part as illustrated. B basal part, M middle part, T top part. Data presented in (b) and (d) are the mean ± S.E. of three biological replicates. 165x166mm (300 x 300 DPI)



Figure 3. Sequence and phylogenetic analysis of BrNAC055 protein. (a) Phylogenetic relationships of BrNAC055 with other senescence-associated NACs. BrNAC055 was highlighted with black circle. The phylogenetic tree was created with neighbor-joining test using MEGA program (version 5.0). (b) Multiple alignment of BrNAC055 protein with other NAC members. The following proteins were used for analysis: ANAC055 (NP_188169.1), ANAC019 (NP_175697.1), ANAC072 (NP_567773.1) and OsNAC5 (XP_015617286.1). The five highly conserved domains A to E were indicated by black lines. The nuclear localization signal (NLS) were up lined with red. 165x52mm (300 x 300 DPI)


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Figure 4. Molecular characterization of BrNAC055. (a) Temporal expression pattern of BrNAC055 during senescence. (b) Localized expression of BrNAC055 in a senescing leaf. The third leaves from the bottom of cabbage plants on the 5th days was detached and separated into three parts as illustrated in Fig. 2C. Data presented in (a) and (b) are the mean ± S.E. of three biological replicates. (c) Subcellular localization assay in epidermal cells of tobacco leaves. A plasmid harboring GFP or BrNAC055-GFP was transformed into Nicotiana benthamiana leaves by A. tumefaciens strain EHA105. GFP signals was observed with a fluorescence microscope after 48 h of infiltration. Bars, 25 µm. (d) Transcriptional activation of BrNAC055 in yeast cells. The coding region of BrNAC055 was inserted into the pGBKT7 (GAL4DBD) to create the pGBKT7BrNAC055 construct. The yeast cells of strain AH109 harboring the pGBKT7-BrNAC055 plasmids were grown on SD plates without tryptophan (Trp-) or without tryptophan, histidine, and adenine (Trp-His-Ade-) for 3 days at 28°C, then followed by the α-galactosidase assay (X-Gal staining). pGBKT7 and pGBKT7-53 + pGADT7-T were used as negative and positive control respectively. (e) Trans-activation of BrNAC055 in Nicotiana benthamiana leaves. The trans-activation ability of BrNAC055 was demonstrated by the ratio of LUC to REN. Data are means ± S.E. of six independent biological replicates. Asterisks represents significant differences at 0.01 level by student’s t-test, compared to pBD. 165x109mm (300 x 300 DPI)



Figure 5. BrNAC055 targets BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters, and activates their transcriptions. (a) SDS-PAGE gel stained with Coomassie brilliant blue, presenting affinity purification of the recombinant GST-BrNAC055 protein. (b) Gel mobility shift assay showing GST-BrNAC055 binding to NACBS in the BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1 promoters. GST protein alone was considered as a negative control. “-” and “+” represent absence or presence, respectively. The probe sequence is shown on the top of image, with WT and mutant BrNAC055 binding site marked with red and underlined. Triangles indicate increasing amounts of unlabelled probe for competition. Arrows indicate the shifted bands position. (c) Dual-luciferase transient expression assay in Nicotiana benthamiana leaves showing that BrNAC055 activates the transcriptions of BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1. The reporter and effector vectors are illustrated in the top panel. Data are means ± S.E. of six independent biological replicates. Asterisks indicate significant differences by student’s t-test (P < 0.01) 165x168mm (300 x 300 DPI)


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Figure 6. Transient over-expression of BrNAC055 induces ROS accumulation and promotes chlorophyll degradation in Nicotiana benthamiana leaves. (a) Appearance, Fv/Fm imaging and histochemical staining (DAB and NBT) of tobacco leaves infiltrated with empty vector (left side of leaf) and BrNAC055 (right) carried by Agrobacterium at 1 and 3 days post-infiltration (dpi). (b) Fv/Fm ratio and total chlorophyll content in Nicotiana benthamiana leaves expressing vector and BrNAC055 at 1 and 3 dpi. (c) Transcript levels of NbrbohA and NbSGR1 in empty and BRNAC055 over-expressed Nicotiana benthamiana leaves at 1 and 3 dpi. Data in b and c represent the means ± S.E. of three biological replicates. Asterisks indicate significant differences by student’s t-test (*P < 0.05 **P < 0.01). 165x287mm (300 x 300 DPI)



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BrNAC055, a Novel Transcriptional Activator, Regulates Leaf

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