EjHAT1 participates in heat-alleviation of loquat fruit lignification by

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Food and Beverage Chemistry/Biochemistry

EjHAT1 participates in heat-alleviation of loquat fruit lignification by suppressing the promoter activity of key lignin monomer synthesis gene EjCAD5 meng xu, meng-xue Zhang, yanna Shi, xiaofen Liu, Xian Li, D. Grierson, and Kunsong Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00641 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

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Journal of Agricultural and Food Chemistry

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EjHAT1 participates in heat-alleviation of loquat fruit lignification

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by suppressing the promoter activity of key lignin monomer synthesis

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gene EjCAD5

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Meng Xu1,2, Meng-xue Zhang1,2, Yan-na Shi1,2, Xiao-fen Liu1,2, Xian Li1,2, Donald

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Grierson1,2,3, Kun-song Chen1,2,*

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Zhejiang University, Zijingang Campus, Hangzhou 310058, PR China

Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology,

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Development and Quality Improvement, Zhejiang University, Zijingang Campus,

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Hangzhou 310058, PR China

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3

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Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom

The State Agriculture Ministry Laboratory of Horticultural Plant Growth,

Plant and Crop Sciences Division, School of Biosciences, University of Nottingham,

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* Corresponding authors:

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E-mail: [email protected](KC)

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Tel: 0086-571-88982461

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ACS Paragon Plus Environment


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Abstract

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Texture attributes such as firmness and lignification are important for fruit quality.

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Lignification has been widely studied in model plants and energy crops but fruit

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lignification has rarely been reported, despite it having an adverse effect on fruit

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quality and consumer preference. Chilling-induced loquat fruit lignification that

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occurs after harvest can be alleviated by heat treatment (HT) applied prior to low

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temperature storage. Enzyme activity assay showed HT treatment could retard the low

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temperature-induced increase in cinnamyl alcohol dehydrogenase (CAD) activity.

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Transcript analysis and substrate activity assays of recombinant CAD proteins

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highlighted the key role of EjCAD5 in chilling-induced lignin biosynthesis. A novel

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homeobox-leucine zipper protein (EjHAT1) was identified as a negative regulator of

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EjCAD5. Therefore, the effect of HT treatment on lignification may be partially due

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to the suppression of the EjCAD5 promoter activity by EjHAT1.

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Keywords: lignification, heat treatment, loquat, cinnamyl alcohol dehydrogenase,

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HD-ZIP

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Introduction

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Texture attributes such as firmness and lignification are important in fruit

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production, not only because they affect taste and consumer preference but also

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because of their influence on fruit storage and transportation.1-2 Fruits such as loquat

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and kiwifruit are susceptible to chilling injury which induces lignin accumulation,

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resulting in the flesh becoming woody and juiceless, and also develops internal

43

browning.3-4 Lignification has been widely studied in model plants and specific

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energy crops but studies on fruit lignification have rarely been reported. Loquat is a

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chilling-sensitive fruit which typically undergoes chilling-induced lignification during

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storage at low temperature. Postharvest treatments have been developed to reduce

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such chilling-induced lignification, such as low temperature conditioning (LTC),5 heat

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treatment (HT)6 and MeJA treatment,7 all of which also provide useful methods for

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exploring the mechanism of lignin biosynthesis in order to improve fruit quality.

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Red-fleshed and white-fleshed loquat fruit respond differently to lignification

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during postharvest storage. The red-fleshed loquat (‘Luoyangqing’) are susceptible to

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chilling induced lignification, which causes increased firmness, while white-fleshed

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loquat (‘Baisha’) do not undergo lignification after harvest. The availability of these

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resources makes loquat a suitable material for exploring the mechanism of lignin

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biosynthesis. According to Shan et al.,8 the content of lignin and the enzyme activities

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of cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) increased in red

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fleshed loquat (‘Luoyangqing’) at postharvest while the lignin content, CAD and POD

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activities in white fleshed loquat (‘Baisha’) did not change, which suggested that

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CAD and POD enzymes may be responsible for lignification in ‘Luoyangqing’ loquat.

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CAD catalyzes the last step in lignin monomer biosynthesis and the mutants CAD4

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and CAD5 in Arabidopsis show a reduction of about 94% in lignin content.9

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Brown-midrib (bm) mutants of maize have altered total lignin content and polymer

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structure, which lead to a reddish-brown color of modified lignin and improved

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digestibility.10 According to Fu et al.,11 the down-regulation of CAD in switchgrass

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resulted in improved sugar release and forage digestibility. These results indicate that

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CAD is a good target for improving energetic value of lignocellulosic biomass from

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crops and also raises the possibility that it may play an important role in fruit

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lignification.

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A number of transcriptional regulators of lignin biosynthesis have been reported,

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including NACs (AtNST1 - AtNST3) and MYBs (such as AtMYB46, AtMYB83) which

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can activate the entire pathway of secondary cell wall biosynthesis,12 and other

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transcription factors such as WRKY, bHLH, HD-ZIP and AP2/ERF.13-16 HD-ZIP

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transcription factors belong to a family unique to plants which have a homeodomain

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with a leucine zipper acting as a dimerization motif. They have been reported to

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participate in a subset of biological processes such as organ formation, stress

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responses and secondary cell wall synthesis.13,17-19

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Materials and Methods

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Plant materials and treatments

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Loquat (Eriobotrya japonica Lindl. cv. Luoyangqing, LYQ) fruit were collected

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in 2011 at Luqiao, Zhejiang province, China. For heat treatment (HT), fruit were

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treated at 40 °C (Hot air, 90-95% RH, in Climacell 404, MMM Medcenter

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Einrichtungen GmbH, Germany) for 4 h and then transferred to 0 °C; other fruit were

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stored at 0 °C as controls. The treatment method was described in Xu et al.20

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Stereomicroscopy in tandem with Wiesner reaction

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A binocular stereomicroscope (Carl Zeiss, Oberkochen, Germany) combined with the

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Weisner reagent test was used for visualizing the distribution of lignin on the

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equatorial plane of loquat flesh at the macroscopic-scale. Loquat fruit stored at 0 °C

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were cut into halves at the equatorial plane. 1% phloroglucinol ethanol solution was

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dropped on the equatorial plane, followed by drops of concentrated HCl to cause the

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Wiesner reaction. The images of half-loquats were merged into one.

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The determination of lignin monomer composition

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Loquat samples were frozen in liquid nitrogen, ground into a powder and

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homogenized in 5 ml washing buffer (100 mM K2HPO4/KH2PO4, 0.5% Triton X-100,

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0.5% PVP, pH 7.8). The mix was shaken at 300 rpm for 30 min, centrifuged and the

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sediment washed once with washing buffer and 100% methanol four times. The pellet

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was dried at 80 °C and 10 mg dried powder was mixed with 500 μl of 2 M NAOH

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containing 25 μl nitrobenzene. The mix was incubated in a hydrothermal reactor and

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extracted twice with dichloromethane and 3-ethoxy-4-hydroxybenzaldehyde was

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added as an internal standard. The aqueous phase was acidified using HCl and

101

extracted

102

N,O-bis-(trimethylsilyl)-trifluoracetamide was added for silylation. The silylated

twice

with

ether.

The

ether

phrases

5


were

dried

and


103

products were analyzed using an Agilent 7890A series GC-MS equipped with HP-5

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column and three biological replications were used for the assay, which was

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performed according to Meyer et al.21

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Crude CAD activity assays

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The fruit flesh samples were ground into a powder in liquid nitrogen and 1 g

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sample powder was extracted using 2 ml Tris: HCl buffer containing 2% PEG, PVPP

109

and 5 mM DTT. The formation of coniferyl aldehyde from coniferyl alcohol was

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monitored using a Thermo Scientific Microplate Reader at 400 nm for 2.5 min and

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three biological replications were used for the assay. One unit of CAD activity was

112

defined as the change in absorbance at 400 nm of 0.01 per min and calculated on a

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protein basis. The enzyme activity method was according to Shan et al.8 Protein

114

content was determined using a BCA protein Assay Kit (Fdbio science).

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Purification and enzyme activity assay of recombinant CAD proteins.

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The full length CAD genes were inserted into pET-28N vector (Clontech) and

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transformed into Escherichia coli strain BL21. Primers are listed in Table S7. The

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transformed cells were cultured to 0.6-0.8 at OD600 and followed by incubation at

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16 °C with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). For protein

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purification, a HisTALONTM Gravity Column (Clontech) was used according to the

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user manual.

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For analysis of the enzyme activities of recombinant CAD proteins in vitro, 200

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μl reaction buffer (50 mM phosphate buffer, pH=6.2) containing 500 μM NADPH

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and 250 μM separate aldehyde substrate with 1 μg purified CAD protein were

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incubated at 30 °C for 5 min and then 50 μl acetonitrile was added to terminate the

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reaction and the mixture was centrifuged at 12,000 g for 5 min, 20 μl of supernatant

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reaction mixture was used for HPLC analysis as described.22 The mobile phases were

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A, water containing 0.1% formic acid and B, acetonitrile containing 0.1% formic acid.

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The reaction mixture was loaded onto a C18 column at 1 mL/min for 2 min using

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10% mobile phase B. over the next 16 min, the gradient was ramped to 40% mobile

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phase B, and held at 40% mobile phase B for 4 min. Finally, the gradient was

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decreased to 10% mobile phase B for 5 min. The reaction product was monitored by

133

HPLC with the standard substance used as control (except p-coumaryl alcohol). Three

134

replicates were used for each enzyme reaction and HPLC analysis. Boiled

135

recombinant EjCAD5 protein was used as the negative control.

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Gene and promoter isolation

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The CAD fragments were obtained from the RNA-seq database and the full

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sequence of all CAD genes obtained using a SMART RACE cDNA Amplification Kit

139

(Clontech), using primers listed in Table S1 and Table S2. The promoters of CAD

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genes were isolated using a Genome Walk Kit (Clontech), using the primers described

141

in Table S3. Coding sequences (CDS) were deposited in Genbank and promoter

142

sequences are shown in Fig S1.

143

Phylogenetic analysis

144

Alignment was performed using the neighbor-joining method with ClastalX

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(v1.8.1), with amino acid sequences of HD-ZIP II and CAD obtained from The

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Arabidopsis Information Resource (TAIR). The phylogenetic tree was constructed

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147

using FigTree (v1.3.1).

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RNA extraction and cDNA synthesis

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Total RNA was extracted from frozen loquat flesh according to the protocol

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described by Xu et al.20 TURBO DNA-free kit (ambion) was used to remove genomic

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DNA and 1 μg of RNA was used to synthesize cDNA according to the user manual

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(BioRad).

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Real-time PCR analysis

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Gene specific primers for measuring expression of CAD genes were designed

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using the online software Primer3 (http://primer3.ut.ee/). The quality and specificity

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of each pair of primers were tested by melting curves and product sequencing. The

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EjACT gene (GeneBank no.JN004223) was chosen as the internal control. Primers for

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real-time PCR are listed in Table S4.

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Dual-luciferase assay

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Dual-luciferase assay was used to analyze the interaction of EjHAT1

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(MK439893) with the EjCAD5 promoter. The full-length of EjHAT1 was cloned into

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EcoR I- and Sal I-digested pGreen II 0029 62-SK vector (SK) and the promoter was

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cloned into pGreen II LUC vector. The primers used are listed in Table S5.

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All recombinant control (SK) and LUC constructs were electroporated into

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Agrobacterium tumefaciens GV3101, which were incubated then diluted to an OD600

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of 0.75 and infiltrated into tobacco (Nicotiana tabacum) leaves with infiltration buffer

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(10 mM MES, 10 mM MgCl2, 150 mM acetosyringone, pH 5.6). Three days after

168

infiltration, the LUC and REN fluorescence intensities were assayed using

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dual-luciferase assay reagents (Promega).

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Yeast one-hybrid screening and assay

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Yeast one-hybrid screening with the EjCAD5 promoter was conducted according

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to the protocol of the MatchmakerTM Gold Yeast One-Hybrid Library Screening

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System (Clontech). The EjCAD5 promoter was constructed into pAbAi vector and the

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CDS of EjHAT1 was inserted into pGADT7 vector. The primers used are listed in

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Table S6. SD medium with aureobasidin but lacking leucine (SD-Leu+AbA) was

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used for yeast one-hybrid assay.

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Statistical analysis

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The statistical significance of differences was calculated using Student’s t-test.

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Least significant difference (LSD) at the 5% level was calculated using DPS7.05

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(Zhejiang University, Hangzhou, China)

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Results

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Lignin distribution and monomer composition in loquat fruit flesh

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To study the overall lignification in loquat fruit after postharvest, the

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macroscopic-scale lignin distribution was visualized using a stereomicroscope after

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phloroglucinol/HCl lignin-staining treatment. As shown in Fig 1A, many pink-red

187

stained

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chilling-induced lignin deposits were widely distributed. Using the nitrobenzene

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oxidation method, the lignin monomer composition in loquat flesh was investigated.

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The guaiacyl (G) and syringyl (S) monomers in loquat flesh were 230 ± 34 nmol g-1

spots

were

observed

throughout

chilled

9


loquat

flesh,

indicating


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FW and 183 ± 34 nmol g-1 FW separately while the proportion of p-hydroxyphenyl

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(H) monomer was about 2% with 14 ± 3 nmol g-1 FW (Fig. 1B). These results

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suggested the lignin composition of loquat fruit is similar to other dicots.

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Analysis of CAD activity in HT and 0 °C treatment

195

The changes in firmness and lignin content of the loquat fruit were described in

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previous study.20 As shown in Fig. 2, the crude CAD activity from loquat fresh at 0 °C

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increased at 1 d and then slightly decreased and remained stable for the next 8 d at

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low temperature while the activity in loquats subjected to HT pretreatment remained

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stable over the first 2 d then declined at 4 d and slightly increased from 4 to 8 d. The

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overall enzyme activity was reduced by HT pretreatment, indicating CAD activity

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was positively correlated with lignin content.

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CAD gene isolation and phylogenetic analysis

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In order to identify the key CAD genes associated with lignification, four new

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loquat CAD genes were obtained by RACE technology and named EjCAD4 to

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EjCAD7 in addition to the three previously reported CAD genes.8,20 Phylogenetic

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analysis indicated EjCAD4 (MK439889) and EjCAD5 (MK439890) were distributed

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in clade I and clustered with AtCAD4 and AtCAD5. EjCAD6 (MK439891) was

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clustered with AtCAD6 and belong to clade II. EjCAD3 and EjCAD7 (MK439892)

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were clustered with AtCAD1, and are members of clade IV, together with EjCAD1

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and EjCAD2 (Fig. 3A). Aligment of loquat and Arabidopsis CAD amino acid

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sequence showed EjCAD3 - EjCAD7 contain two zinc-binding signature domains and

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one NADPH-binding domain and are therefore considerated as bona fide CAD genes,

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although the first zinc-binding signature in EjCAD7 had V82 changed to C82.

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Furthermore, EjCAD1 and EjCAD2 contained only a VTG(X)2G(X)9L(X)5 conserved

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domain similar to AtCAD101 - AtCAD108, which were considerated as CAD-like

216

genes (Fig. 3B).

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Association of CAD expression and lignification in loquat fruit

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Gene expression patterns of the EjCAD genes were analyzed. As shown in Fig. 4,

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EjCAD3 and EjCAD5 transcripts were significantly reduced by HT pretreatment

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compared to 0 °C storage. EjCAD3 was substantially up-regulated over the first two

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days at 0 °C storage while HT pretreatment retarded this increase. EjCAD5 transcripts

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increased during 8 d storage at 0 °C but increased only slightly at 2 d and declined for

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the next 6 d in HT pretreatment samples. The expression pattern of EjCAD3 and

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EjCAD5 were positive correlated with lignin content and firmness.

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The catalytic activity of recombinant CAD proteins in vitro

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In order to explore the function of differentially expressed CAD genes,

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prokaryotic expression experiments were performed to obtain the recombinant CAD

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proteins. The predicted molecular mass of purified recombinant CAD proteins with

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His-tag of EjCAD3 and EjCAD5 were 41.3 kDa

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confirmed by SDS-PAGE

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proteins, 1 μg purified CAD protein was used for the reaction with three lignin

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monomer

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respectively. As shown in Fig 5, EjCAD5 protein had the highest activity with

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coniferaldehyde, sinapaldehyde and p-coumaraldehyde, with a preference for

precursors

and 41.6 kDa, which was

(Fig S2). To analyze the activity of recombinant CAD

sinapaldehyde,

coniferaldehyde

11


and

p-coumaraldehyde,


235

coniferaldehyde and sinapaldehyde. However, EjCAD3 activity with sinapaldehyde

236

was low and had a relative weak activity (about 0.59 fold) with coniferaldehyde, 0.41

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fold for p-Coumaraldehyde compared with EjCAD5 (Table 1). These results it

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suggested EjCAD5 is more important in lignin monomer biosynthesis.

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Interaction between EjHAT1 and the EjCAD5 promoter

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To explore the transcriptional regulatory mechanism of the lignin monomer

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biosynthesis gene EjCAD5, cis-acting element analysis of the EjCAD5 promoter and

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yeast-one hybrid screening was performed. The length of the EjCAD5 promoter used

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for analysis and yeast screening was 1299 bp and contains conserved cis-acting

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elements which can be recognized by NAC, MYB and HD-ZIP factors (Fig. 6). The

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EjCAD5 promoter sequence was inserted into a pAbAi vector to test for

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auto-activation and the result show this could be supressed by AbA 200 ng ml-1 (Fig

247

S3) and was therefore suitable for use in yeast-one hybrid screening with a loquat

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yeast one-hybrid library (Clontech). A novel HD-ZIP protein, named as EjHAT1, was

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obtained in the screening and further interaction experiments indicated EjHAT1 could

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directly interact with the EjCAD5 promoter (Fig. 6).

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Regulatory role of EjHAT1 in controlling lignin monomer biosynthesis gene

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EjCAD5

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The expression of EjHAT1 was analyzed and the results indicated that EjHAT1

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decreased and then remained stable during 0 °C storage apart from a slight increase at

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4 d. Prior HT treatment reduced this decrease (Fig. 7), suggesting EjHAT1 may act as

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a negative regulator of lignin biosynthesis.

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To further study the function of EjHAT1 in regulating EjCAD5, dual luciferase

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assay was performed. As shown in Fig. 8, EjHAT1 could repress the activity of the

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EjCAD5 promoter by approximately 0.6 fold, which was coincident with the

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expression pattern differences between 0 °C and HT treatment.

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Discussion

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Extensive lignin biosynthesis research has focused on Arabidopsis and energy

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crops such as Populus and switchgrass, but detailed information about the regulatory

265

mechanism of lignin biosynthesis in fruit is limited, despite the fact that an improved

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understanding of lignification could improve fruit quality and marketability.23

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Chilling induces lignification (Fig 1) and HT treatment has been reported to be

268

an effective postharvest pretreatment to alleviate loquat fruit lignification during low

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temperature storage.20 The regulatiory mechanism whereby HT alleviates lignification

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in loquat fruit has been investigated previously. Two MYBs (EjMYB1 and EjMYB8)

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and one NAC (EjNAC3) transcription factors have been reported as direct regulators

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of lignin biosynthesis genes20,24-25 and one NAC (EjNAC1) and one HSF (EjHSF3)

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have been reported as indirect regulators.20,26 These genes are all regulators of the

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up-stream phenylalanine pathway gene Ej4CL1, apart from EjNAC3.

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EjCAD5 enzyme contributes to HT alleviated lignification

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Base on the analysis of lignin content and lignin biosynthesis enzyme activity in

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red-flesh and white-flesh loquat after postharvest,8 it suggested that CAD played an

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important role in red-flesh loquat lignification. Our results showed that CAD activity

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was lower after HT pretreatment, which reduced lignification during 0 °C storage.

280

This supports Shan’s results and conclusions’ that CAD in loquat fruit plays an

281

important role in lignification.8 Interestingly, increases in CAD enzyme activity in

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response to temperature have been reported in Medicago truncatula and loss of

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function of MtCAD1 resulted in strikingly different lignin composition and reduced

284

lignin content. Furthermore, CAD mutants are dwarfed when grown at 30 °C while

285

appearing normal under standard conditions.27

286

Enzyme activity assays indicated HT treatment could effectively retard the increase in

287

CAD enzyme activity, which was consistent with HT lowering CAD activity and

288

contributing to alleviation of lignin accumulation (Fig 1). The expression of EjCAD3

289

and EjCAD5 was positive correlated with the change in CAD enzyme activity as well

290

as the accumulation of lignin (Fig 4)，which identified them as the candidate CAD

291

genes expressed in loquat flesh.

292

In Arabidopsis, nine CAD genes have been identified. Among them, AtCAD4 and

293

AtCAD5 had the highest enzyme activities. AtCAD5 could catalyze sinapaldehyde,

294

coniferaldehyde and p-Coumaraldehyde whereas AtCAD4 had a preference for

295

coniferaldehyde and p-Coumaraldehyde.28 Phylogenetic analysis showed EjCAD5

296

clustered with AtCAD4 and AtCAD5. The catalytic activities of loquat CAD enzyme

297

were examined in vitro using recombinant CAD proteins. The results indicated

298

EjCAD5 had higher overall activity towards these substrates and preferred

299

sinapaldehyde and coniferaldehyde, like AtCAD5. The lignin monomer composition

300

of loquat flesh confirmed the preferential production of sinapaldehyde and

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coniferaldehyde (Fig 1B). Moreover, another candidate (EjCAD3) had a similar

302

substrate preference to AtCAD4, but had lower activity (Fig 5).

303

EjHAT1 acts as a repressor of lignin monomer biosynthesis gene EjCAD5

304

HD-ZIP transcription factors are unique to the plant kingdom and have been

305

classified into four subfamilies. The phylogenetic tree indicated that EjHAT1, encodes

306

an HD-Zip protein in subfamily II, and is a homolog of Arabidopsis AtHAT14,

307

AtHAT22 and AtHAT9 (Fig S4), but the functions of these three TFs are still unclear.

308

HD-Zip transcription factors in subfamily II usually respond to changes in

309

illumination condition, and their functions in plant development is associated with

310

light response, shade avoidance and signaling.17 Two HD-ZIP II transcription factors,

311

AtHB4 and AtHAT3 have important functions in controlling leaf development and

312

responding to shade in Arabidopsis.29 HD-ZIP II transcription factor AtHB2 is a

313

negative regulator of paralogous genes in response to illumination conditions that

314

suppresses its own expression by binding its own promoter.30 However, the function

315

of HD-Zip II in the regulation of lignin synthesis has not been reported, although

316

there were several reports indicating that HD-ZIP transcription factors in subfamily

317

III are involved in plant secondary cell wall metabolism. The Populus HD-ZIP III

318

transcription factor POPCORONA regulates cell differentiation during the secondary

319

growth of woody stems.31 AtHB8 is a positive regulator of secondary cell wall

320

synthesis and over-expression of AtHB8 promoted xylem differentiation in

321

Arabidopsis.32 AtHB15 functions as a negative regulator of secondary wall

322

development in Arabidopsis pith.33 These reported HD-Zip TFs belong to subfamily

15



323

III and are involved in the regulation of lignification by acting as indirect regulators of

324

lignin biosynthesis genes.31-33

325

We identified a novel HD-ZIP transcription factor in subfamily II which can

326

directly bind the EjCAD5 promoter in yeast one-hybrid screening assays (Fig 6). The

327

cis-element analysis of EjCAD5 promoter found a conserved HD-ZIP II binding

328

element34 CAATCATTG at -143 bp starting from the ATG, which is consistent with

329

the interaction between EjHAT1 and the EjCAD5 promoter (Fig. 6).

330

The expression of EjHAT1 transcripts was negatively associated with EjCAD5

331

expression, which indicated EjHAT1 may participate in lignin biosynthesis by acting

332

as a repressor (Fig 7) and the demonstration using luciferase assay indicated EjHAT1

333

could suppress the activity of the EjCAD5 promoter (Fig 8). These results indicate

334

EjHAT1 participates in HT alleviation of lignification in loquat by suppressing the

335

activity of the EjCAD5 promoter.

336

Here, we found the increase in CAD activity in loquat flesh at low temperature

337

could be suppressed by HT treatment. Gene expression and enzyme activity analysis

338

of recombinant CAD proteins in vitro indicated the key role of EjCAD5 in the

339

chilling-induced lignin biosynthesis. Further experiments showed a novel

340

transcription factor EjHAT1, which participated in lignin biosynthesis by suppressing

341

the activity of the EjCAD5 promoter, responded to HT treatment. This provides a new

342

insight into ways of improving fruit quality and understanding the mechanism of HT

343

alleviated chilling induced lignification of loquat fruit during postharvest storage.

344

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Acknowledgement

346

Author contributions

347

Kun-song Chen and Yan-na Shi conceived and designed the experiments, Meng Xu

348

and Meng-xue Zhang performed the experiments and analyzed the data, and Meng Xu

349

and Xiao-fen Liu wrote the draft manuscript, Xian Li was be involved the

350

experiments for the MS revision, Donald Grierson improved the manuscript English.

351

Authors would like to thank Prof. Xue-ren Yin for his suggestions to the research.

352

Corresponding author

353

* E-mail: [email protected](KC) Tel: 0086-571-88982461

354

ORCID

355

Kun-song Chen: 0000-0003-2874-2383

356

Funding

357

This research was supported by the National Natural Science Foundation of China

358

(31630067), the 111 Project (B17039).

359

Notes

360

The authors declare no competing financial interest.

361

Supporting Information

362

Fig. S1 Sequences of promoters of lignin biosynthesis-related genes from loquat fruit.

363

Fig S2. SDS PAGE of purified recombinant CAD proteins with 6×his-tag.

364

Fig S3. Yeast one-hybrid analysis of the ability of EjHAT1 to bind the promoter of

365

EjCAD5.

366

Fig S4. Phylogenetic analysis of loquat EjHAT1 and Arabidopsis HD-ZIP II deduced

17



367

amino acid sequences.

368

Table S1-S7. All primer sequences used in this research.

369 370

Reference

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Table 1 Chromatograph information of recombinant CAD activity and product

500

synthesis rate.

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503 504

Fig. 1. Lignin distribution and composition of loquat flesh after chilling. (A) lignin

505

distribution in LYQ loquat at the macroscopic-scale, with enlarged view of the region

506

in the square box. (B) Composition of loquat flesh lignin measured by the

507

nitrobenzene oxidation method. The sum of vanillin (G1) and vanillic acid (G2) for

508

total G monomer. The sum of syringaldehyde (S1) and syringic acid (S2) for total S

509

monomer. The sum of p-hydroxybenzaldehyde (H1) and p-hydroxybenzoic acid (H2)

510

for total H monomer.

511

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512 513 514

Fig. 2. Changes in crude CAD activity in flesh of loquat fruits stored at 0 °C with and

515

without HT pretreatment. The decrease in coniferyl alcohol at 400 nm was monitored

516

and one unit of CAD activity was defined as the change in absorbance of 0.01 per min

517

at 400 nm, calculated on a protein basis.

518

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519 520

Fig. 3. Phylogenetic analysis and aligment of loquat and Arabidopsis CAD sequences.

521

(A) Phylogenetic analysis of loquat and Arabidopsis CAD deduced protein sequences.

522

Amino acid sequences of Arabidopsis CAD genes were downloaded from TAIR

523

(https://www.arabidopsis.org/). The loquat CAD genes are shown in red. (B)

524

Alignment of loquat and Arabidopsis CAD deduced protein sequences. The locations

525

of conserved domains are indicated with horizontal bars. Black shading indicates

526

100% consensus amino acid sequence among the different genes, while the red color

527

represents lower levels of consensus. The conserved zinc-binding domains are

528

GHE(X)2G(X)5G(X)2V and GD(X)10C(X)2C(X)2C(X)7C respectively. The conserved

529

NADPH domain is GXG(X)2G.

530

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531 532

Fig. 4. The expression of loquat CAD transcripts during 0 °C storage with and

533

without HT pretreatment. The error bars were calculated using three biological

534

replicates.

535

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536 537

Fig. 5. HPLC analysis of reaction products of recombinant EjCAD3 and EjCAD5.

538

The chromatograph shows the conversion of substrates into products. Boiled

539

recombinant EjCAD5 protein (denatured) was used as control and results for different

540

recombinant proteins are offset and shown in different colors. Standards were used to

541

identify the reaction product according to retention time.

542 543

29



544

545 546

Fig. 6. (A) The cis-elements analysis of the EjCAD5 promoter. The promoter region

547

used for analysis and yeast one-hybrid assay was 1299 bp. (B) Yeast one-hybrid

548

analysis tested the ability of EjHAT1 to bind the promoter of EjCAD5. The interaction

549

was determined on SD medium lacking Leu in the presence of AbA 200 ng ml-1. The

550

empty pGADT7 vector was used as the negative control.

551

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552 553

Fig. 7. Expression of loquat EjHAT1 at low temperature (0 °C) and with HT

554

treatment. Error bars indicate SE from three biological replicates. LSD indicates

555

least-significant difference at 0.05.

556

31



557 558

Fig. 8. In vivo interaction of EjHAT1 with the promoter of the lignin synthesis

559

EjCAD5 from loquat using dual luciferase assay. The ratio of LUC/REN ﬂuorescence

560

obtained with the empty vector (SK) plus the promoter used as a calibrator (set as 1).

561

Error bars indicate SE from five replicates.

562

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Fig. Table of Contents (TOC) graphic

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EjHAT1 participates in heat-alleviation of loquat fruit lignification by

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