Metabolite and Transcriptome analyses indicate the involvement of

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Metabolite and Transcriptome analyses indicate the involvement of lignin in programmed changes in peach fruit texture Ying Wang, XinFu Zhang, Shaolan Yang, and Yongbing Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04284 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 15, 2018

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Journal of Agricultural and Food Chemistry

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Metabolite and Transcriptome analyses indicate the involvement of lignin in programmed changes in peach fruit texture

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Ying Wang • Xinfu Zhang • Shaolan Yang∗• Yongbing Yuan∗

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Qingdao Key Laboratory of Genetic Improvement and Breeding in Horticultural Plants,

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College of Horticulture, Qingdao Agricultural University, No. 700 Changcheng Road,

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Chengyang, Qingdao City 266109, Shandong Province, China

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∗Authors

to whom correspondence should be addressed; E-mails: [email protected]

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(Shaolan Yang); [email protected] (Yongbing Yuan)

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Tel: 0086-0532-86080018

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Fax: 0086-0532-86080018

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ACS Paragon Plus Environment


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ABSTRACT

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Texture is an important component of peach fruit quality. In the present study, an analysis of

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metabolite and transcriptome profiles of a non-melting flesh cultivar, ‘Baili’, and a melting flesh

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cultivar, ‘Hongli’, during storage was conducted to explore the molecular mechanisms underlying

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different fruit textures in peach. Results indicated that higher levels of anthocyanins were present

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in ‘Hongli’ peach, while lignin monomers and ethylene precursors were higher in ‘Baili’. A

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transcriptome analysis indicated that genes associated with lignin synthesis were more highly

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expressed in ‘Baili’ than in ‘Hongli’, especially Pp4CL2, Pp4CL3, and PpCOMT2. Texture

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differences between the two varieties may be the result of differential expression of two branches

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of the phenylpropanoid metabolic pathway. One branch regulates flavonoid metabolism and was

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highly active in ‘Hongli’ fruit, while the other branch regulates lignin synthesis and was more

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highly active in ‘Baili’ fruit.

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Keywords: Peach, Transcriptome, Metabolomics, Lignin

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INTRODUCTION

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Texture, an important component used to determine peach fruit quality1, not only affects fruit

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taste and consumer choice but also affects the fruit storage and transportation; and thus receives a

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great deal of attention by the peach fruit industry2,3,4. Changes in the texture that occur in peach

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fruit after harvest can be divided into processes involved in softening and lignification5.

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Softening is the most noticeable texture change that occurs during ripening and is a component

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of the maturation of all fruits6. Previous research has indicated that changes in the cell wall and

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the regulation of plant hormones are the most important aspects controlling fruit softening.

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Enzymes involved in cell wall degradation are closely associated with softening7.

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Polygalacturonase (PG) activity becomes greatly elevated during the postharvest ripening of

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peach fruit and is considered a dominant aspect of peach fruit softening. Qian et al. 8 reported that

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the difference in the softening characteristics of ‘Qin Wang’ vs ‘Qian Jian Bai’ peach fruit was

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related to the level of activity of various PG enzymes, the higher the level of PG a fruit contained,

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the more rapidly it would soften. As with PG, pectinesterase (PE) activity has been detected in a

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very wide range of fruit. Abu-Sarra et al.

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activity during the softening of mango, and found that PE activity increased continuously as

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mango fruit softened, indicating that PE plays a major role in determining the rate of mango fruit

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softening. Hayama et al.11 reported on the expression pattern of three expansin genes in

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‘Akatsuki’ (melting) and ‘Manami’ (non-melting) peach during softening and found that PpExp3,

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rather than PpExp1 or PpExp2, played a role in fruit softening of ‘Akatsuki’ peach. The

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regulation of plant hormones is also considered to be an important factor of the ripening process12.

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In particular, ethylene is considered to be the most important hormone in the regulation of fruit

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softening. In general, fruit softens faster with the application of increasing concentrations of

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ethylene13, and the expression of Pp-ACS1 and Pp-ACO1 genes was demonstrated to play a key

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role in the regulation of ethylene biosynthesis during the promotion of ripening in ‘Hakuho’

9,10

studied the changes in cell wall pectinesterase (PE)



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peach14. And ABA also could accelerate the fruit ripening and softening, as Luo et al15 reported

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that endogenous ABA initiated cherry fruit ripening by regulating maturation-related metabolic

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pathways such as anthocyanin synthesis. Moreover, Soto et al.16 indicated that ABA regulated

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ripening and softening progress of peach fruit by regulating ethylene biosynthesis and signaling

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genes (ACS1, ACO1, ETR2, ERF2)15.

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To date, most research studies have focused on peach fruit softening and less on the

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lignification of peach fruit, even though lignification is also an important aspect affecting the

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texture of fruit. A report on loquat fruit indicated that lignification was induced by chilling injury

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and during senescence17. Cai et al analyzed loquat fruits stored at room temperature and found

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that loquat fruit firmness increased during storage and was positively correlated with the

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accumulation of lignin in the fruit flesh; in a process that was termed senescence lignification18.

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In a subsequent study, the accumulation of lignin and an increase in firmness in the flesh of

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loquat fruit occurred whether fruit was stored at room temperature (senescence lignification) or

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subjected to chilling injury by storage at 0◦C19. Phenylalanine ammonialyase (PAL), caffeic acid

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5-hydroxyferulic acid O-methyltransferase(COMT),4-Coumarate: coenzyme A ligase (4CL),

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cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) are all enzymes that are involved

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in lignin synthesis, and increased during storage of loquat fruit, and were positively correlated

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with the accumulation of lignin20. This phenomenon has also been reported to occur in pear21 and

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mangosteen22. PpCAD1 and PpCAD2 all exhibited higher levels of expression in a hard pear fruit

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cultivar, ‘Whangkeumbae’ relative to normal pear fruit. Additionally, transgenic tobacco

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overexpressing PpCAD2 increased the accumulation of lignin23. Therefore, lignin accumulation

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in fruit changes fruit texture and increases its firmness. Although lignification of peaches occurs

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in response to chilling injury24, less is known about senescence lignification in peach fruit.

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The combination of transcriptomic and metabolomics was analyses is an effective way to identify

metabolic-related

functional

genes25.

Yu

et


al.26

used

a

combined

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transcriptomic/metabolomic approach to examine the response of Lemna aequinoctialis during a

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time course (0, 3 and 7 days) study of nitrogen starvation and exogenously administered sucrose.

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They reported finding that genes involved in nitrogen metabolism were the earliest transcriptomic

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response to nitrogen starvation. The results of the transcriptomic and metabolomic analyses were

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in agreement with each other, thus supporting the reliability and accuracy of the obtained data25.

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Wang et al27 performed a combined transcriptomic/metabolomic analysis of ‘Hujingmilu’ peach

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stored at either a constant 0◦C or LTC (pre-storage at 8◦C for 5 days before storage at 0◦C)

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conditions, and found that LTC can alleviate chilling injury in peach fruit. Metabolomic data can

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provide information on a large number of different metabolites, thus providing a good reference

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for the analysis of transcriptome data28.

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The peach varieties, ‘Baili’ and ‘Hongli’, used in present manuscript are called by a joint name

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as ‘Feitao’ produced in Feicheng county, Shandong Province, China. ‘Feitao’ has been famous

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for more than 1000 years in China in term of big, sweet and juicy. The eating quality of ‘Hongli’

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is usually considered better than that of ‘Baili’ because ‘Hongli’ flesh is more juicy and soluble

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while ‘Baili’ flesh is firmer. Now days it is this traditional good quality character of ‘Hongli’ that

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is not good for handling during postharvest. In this manuscript, the mechanism of different

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texture changes during storage of two peach varieties was worked out by metabolite and

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transcriptome analysis. Our objective was to identify important factors that impact the texture of

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these two varieties. The metabolomic and transcriptomic analyses were conducted on fruit stored

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over a 10-day period at 20 ◦C. Differentially expressed genes (DEGs) and metabolites (DEMs)

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were identified and categorized by functional classifications and metabolic pathways. These

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classifications were further validated by examining the expression of specific enzyme genes.

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MATERIALS AND METHODS

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Plant Material and sampling



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Two varieties of peach ‘Baili’ and ‘Hongli’ were harvested on Aug 26, 2016 at about 120 days

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after anthesis (Total soluble solid reaches 12 %) from a forty-year-old orchard in Feicheng,

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Shandong, China. Standard and identical irrigation and fertilization management programs were

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used throughout the orchard during the growing season. Fruit that was uniform in appearance,

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without visual defects, were harvested and stored at 20◦C. Fruit firmness, rate of ethylene

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production, respiratory rate, cellulose content, and lignin content were measured after harvest,

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Three biological replicates, consisting of ten pooled fruit for each biorep, were sampled for each

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variety at each time point. The mesocarp was sliced into approximately 1 cm3 cubes, immediately

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frozen in liquid nitrogen, and then stored at -80◦C until further analysis. According to the

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difference in the firmness decline of the two varieties, we selected the 0, 2 and 6 DAH of the

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‘Hongli’ fruit (firmness rapid decline period 0-2 d and decline in stationary period 6 d after

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harvest), and the 0, 4 and 6 DAH of the ‘Baili’ fruit (change of firmness in the ‘Baili’ is steady,

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we have chosen representative days of 0, 4 and 6 d after harvest), which were 18 analyzed

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samples (two varieties x three storage time points x three biological replicates)for metabolomic

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and transcriptomic analysis. The samples of ‘Baili’ collected at 0, 4, 6 DAH designated as B0, B4

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and B6, and ‘Hongli’ at 0, 2, 6 DAH designated asH0, H2 and H6, respectively.

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Measurement of ethylene, respiration, and firmness

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The protocol used for measuring ethylene levels was performed as described by Yang et al29.

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Nine fruits were placed in three 6 L sealed containers (three fruits one container) for 1h and the

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extracted gas was measured for ethylene content, with measurements repeated three times for

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each sampling time point. The rate of ethylene production (μL · kg−1· h−1) was calculated as the

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released amount of ethylene per unit time per unit fresh weight (FW) of fruits. Ethylene was

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measured with a GC-2010 gas chromatograph (Shimadzu) and chromatographic conditions

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included the use of a FID detector, GDX-502 capillary column (10 m × 0.53 mm × 1 μm), a split

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ratio of 10, hydrogen carrier gas, 120 ◦C column temperature, 200 ◦C detector temperature, and a


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1 mL injection volume. .

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To assess the rate of respiration, nine fruit were placed in three 6 L sealed containers (three

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fruits one container) for 1 h and the rate of respiration was measured with a COMBO 580

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portable analyzer (Italy). The oxygen concentration should be adjusted to 20.8% when used the

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machine, recording the carbon dioxide concentration at this time (C1), and then inserted the

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needle connected to the instrument into the sealed container for measurement. After a few

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minutes of stabilization, recording the concentration of carbon dioxide (C2) and oxygen at this

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time, using the formula: Respiration rate (mg / (kg·h)) = carbon dioxide release (C2-C1) (mg) /

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(fruit weight (kg) · time (h)). Firmness of the flesh on both sides of the fruit equator was

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measured using a CT3-4500 Texture Analyzer (Brookfield, USA) (probe diameter 2.0 mm). Six

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fruits were measured for each variety at each of the sampled time points. The firmness, ethylene

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release rate and respiration rate were measured every two days after harvest.

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Lignin content

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Lignin content was assessed using an ELISA kit according to a previously published method30,

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which was measured by Shanghai BIO-TECH CO, LTD (Shanghai, China). The supernatant was

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centrifuged at 1000 x g for 20 minutes to obtain a supernatant for detection. First, all Standards

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and Samples were added in duplicate to the Microelisa Strip plate and set standard wells, testing

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sample wells. Added 50μl standard to standard well, 10μl testing sample and 40μl Sample Diluent

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to testing sample well, Blank well didn’t add anything. Added 100μl of HRP-conjugate reagent to

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each well, covered with an adhesive strip and incubated for 60 minutes at 37 ◦C. Aspirated each

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well and wash, repeating the process four times for a total of five washes. Washed by filling each

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well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. After

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the last wash, removed any remaining Wash Solution by aspirating or decanting. Inverted the

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plate and blotted it against clean paper towels. Added chromogen solution A 50μl and chromogen



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solution B 50μl to each well. Gently mixed and incubated for 15 minutes at 37 ◦C (Protect from

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light). Added 50μl Stop Solution to each well then the color in the wells should change from blue

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to yellow. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15

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minutes. Lignin content was expressed as l/mL and three biological replicates were used for

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each sample (variety) at each time point. Three biological replicates were used for each sample at

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each time point.

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Lignin staining

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Lignin was stained with Weisner reagent (phloroglucinol/concentrated hydrochloric acid)

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according to the method described by Blanco-Portaleset al31. The flesh was cut to a thickness of

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approximately 2 mm, longitudinally from the fruit pedicel, and stained with the Weisner reagent

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for 10 min. Three biological replicates were used for each sample at each time point.

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RNA Isolation and cDNA library preparation

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The ‘Baili’ fruit flesh at 0, 4 and 6 DAH and ‘Hongli’ fruit flesh at 0, 2 and 6 DAH were

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selected for total RNA extract, which using the Trizol reagent kit (Invitrogen, China) according

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to the manufacturer’s instructions. And three biological replicates were used for each sample at

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each time point. The extracted total RNA was then treated with RNase-free DNase I (Takara,

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Dalian, China) for 15 min at 25◦C to remove residual DNA. RNA quality was verified by RNase-

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free agarose gel electrophoresis and total RNA concentration of each sample was measured on an

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Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) at 260 and 280 nm. Total

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RNA was then enriched for mRNA utilizing Oligo (dT) beads. The enriched mRNA was

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subsequently fragmented into short fragments using a fragmentation buffer and reverse-

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transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA

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polymerase I, RNase H, dNTP and buffer. The cDNA libraries were prepared for sequencing

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using a NEBNext Ultra RNA Library Prep Kit. Prepared libraries were sequenced using an


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Illumina HiSeq TM 2500 platform at Gene Denovo Biotechnology Co. (Guangzhou, China).

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RNA-seq data processing and analysis

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Reads containing adapters, sequences with more than 10% unknown nucleotides (N), and a

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quality rating less than 50% (Q-value ≤20) were first removed from the data set. Bowtie232 was

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then used to map the reads to a ribosome RNA (rRNA) database. All sequences with rRNA

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mapped reads were then removed from the data set and the remaining reads were used in the

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transcriptome analysis. The reads remaining after filtering were mapped to the peach reference

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genome (“Lovell”, https://www.ncbi.nlm.nih.gov/genome/?term=prunus+persica)by TopHat233

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(version 2.0.3.12). Novel transcripts were identified with Cufflinks34 and TopHat2 software.

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Identification of differentially expressed genes (DEGs)

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Transcript abundance was quantified using RSEM software35 and gene expression levels were

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normalized using the FPKM (Fragments Per Kilobase of transcript per Million mapped reads)

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method. The EdgeR package (http://www.r-project.org/ ) was used to identify DEGs across

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samples or groups36. Genes with a fold change ≥2 and a false discovery rate (FDR) 0.7 or < -0.7, P-value < 0.05) are

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presented in Figure 9 and Supplementary Table S4.In total, 20 lignin synthesis-related genes, 25

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ethylene metabolism genes, and 22 cell wall remodeling-related genes were analyzed. The

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relationship between the majority of lignin synthesis-related and cell-wall remodeling genes was

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negatively correlated. In contrast, the correlation between ethylene metabolism-related and cell

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wall remodeling-related genes was variable.

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We inferred that the high lignin content in ‘Baili’ fruit may be induced by the high level of

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ethylene production. Ethylene has been extensively reported to play a dominant role in promoting

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fruit softening and senescence. Ma, however, also provided evidence that suggested the

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APETALA2/ethylene response factor (AP2/ERF) family may be involved in the biosynthesis of

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lignin in Isatis indigotica Fortune65,66. Additionally, exogenous treatment with ethylene has been

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reported to promote ethylene release in bamboo shoots, increases PAL, CAD, and POD activity,

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and significantly increases lignin content67. Wang et al.68 isolated three ethylene receptor genes

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EjETR1, EjCTR1, and EjEIL1 from ‘Luoyangqing’ loquat fruit that exhibited increased

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expression in response to low temperature. The expression of EjETR1, and EjEIL1 were

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especially linked to chilling injury in ‘Luoyangqing’ fruit, which confirmed a role for ethylene in

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the regulation of lignin synthesis in loquat fruit, as increased lignification is a direct consequence


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of chilling injury. In the current study of peach, the rate of ethylene production was higher in

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‘Baili’ fruit than in ‘Hongli’ fruit (Figure 1c); the former of which also exhibited higher levels of

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lignification. The network analysis also indicated a positive correlation between the expression

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level of ethylene metabolism-related and lignin synthesis-related genes (Figure 9b). These data

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indicate that ethylene may affect lignin synthesis which contributes to the higher level of firmness

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in ‘Baili’ fruit. A systematic analysis of all these components will need to be conducted, however,

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to further validate this premise.

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Transcriptome and metabolite profiles were used in a combinatorial analysis to provide

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information on the underlying basis regulating the different texture of ‘Baili’ vs ‘Hongli’ peach.

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A comparison of the differences in the level of metabolites and gene expression in the two

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cultivars identified 644 metabolites and 4664 pathway annotations. The analysis identified the

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metabolites and genes that may regulate ripening and softening in peach. The results suggested

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that lignification may be a major factor impacting the contrasting texture of ‘Baili’ vs ‘Hongli’

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peach fruit. Results also indicated that ethylene may have the double effect on fruit texture in

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peach. This study provides a foundation for studying the regulation of peach ripening and

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softening. In addition, the provided transcriptomic and metabolomic data also greatly enhance the

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molecular and metabolite information that are now available for ‘Baili’ and ‘Hongli’ peach fruit.

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Our initial investigation has provided a solid framework of data and helped to clarify the specific

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studies that will be needed to fully understand the underlying mechanism of the differences in

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texture between ‘Baili’ and ‘Hongli’ peach fruit.

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ABBREVIATIONS USED

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DEMs, differentially expressed metabolites; DEGs,differentially expressed genes; 4CL, 4-

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Coumarate: Coenzyme A Ligase; COMT, caffeic acid 5-hydroxyferulic acid O-methyltransferas;

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CAD, cinnamyl alcohol dehydrogenase; POD, peroxidase; Nr, NCBI protein database;GO, Gene



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Ontology; KEGG, Kyoto Gene and Genome Encyclopedia; UPLC-QQQ-MS/MS, ultra-high

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performance liquid chromatography coupled to a triple quadrupole mass spectrometer; SA,

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salicylic acid; MeJA, methyl jasmonate; CHS, chalcone synthase; F3H, flavanone 3-hydroxylase;

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AP2/ERF,

APETALA2/ethylene


response

factor;

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CONTRIBUTION

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Shaolan Yang and Yongbing Yuan conceived and designed the experiments, Ying Wang and

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Xinfu Zhang performed the experiments and analyzed the data, Ying Wang and Shaolan Yang

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wrote the manuscript. All authors read and approved the manuscript.

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ACKNOWLEDGMENTS

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This work was supported by the National Key Research and Development Program of China

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(2016YFD0400100), the Project of Shandong Natural Science Foundation (ZR2017MC006), and

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Shandong Modern Fruit Technology Industry System project (SDAIT-06-06).

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CONFLICT OF INTEREST

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The authors declare no conflict of interest.

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Supporting Information.

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Fig. S1 Principal component analysis (PCA) of peach metabolite profiles obtained from 18

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independent samples collected from ‘Baili’ and ‘Hongli’ at 0, 4(0), and 6 d.

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Figs. S2 and S3 Heat maps of different metabolite clusters and the clustering of all metabolites.

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Heat maps of DEMs based on normalized FPKM values in three time points. Green indicates

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lower expression, and red indicates higher expression.

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Fig. S4 The postharvest decay rate in ‘Baili’ and ‘Hongli’ fruit.

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Fig. S5 GO enrichment of DEGs between ‘Baili’ and ‘Hongli’ fruit.

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Fig. S6 and Fig. S7 KEGG pathway analysis of DEGs.



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Table S1. List of gene-specific primers used in the qRT-PCR analysis.

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TableS2. RNA-seq tabular data (number of reads, filtered reads, clean reads, etc.).

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Table S3. Mapping results of all 18 RNA-seq libraries to the peach reference genome.

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Table S4. Pearson correlation coefficients between lignin synthesis-related, cell wall remodeling-

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related, and ethylene metabolism-related gene expression.

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804 805

FIGURE CAPTION

806

FIGURE 1. Physiological characteristics of ‘Baili’ and ‘Hongli’ peach fruit. A. Appearance of

807

‘Baili’ and ‘Hongli’ peach fruit at harvest (day 0). ‘Baili’ (above) and ‘Hongli’ (below). B.

808

Firmness measurements of ‘Baili’ and ‘Hongli’ peach fruit over the course of the storage period.

809

C. Respiration rate of ‘Baili’ and ‘Hongli’ peach fruit over the course of the storage period. D.

810

Ethylene generation in‘Baili’ and ‘Hongli’ peach fruit over the course of the storage period.

811

FIGURE 2. Lignin and cellulose content in ‘Baili’ and ‘Hongli’ peach over the course of the

812

storage period. A. Phloroglucinol-HCl staining of ‘Baili’ and ‘Hongli’ mesocarp fruit tissues. B.

813

Lignin (a) and cellulose (b) content in ‘Baili’ and ‘Hongli’ fruit over the course of the storage

814

period.

815

FIGURE 3. Differentially expressed metabolites (DEMs) and KEGG pathway annotations of

816

metabolites. A. DEMs in ‘Hongli’ and ‘Baili’ fruit. B0 represents ‘Baili’ fruit at 0 days after

817

harvest (DAH), H0 represents ‘Hongli’ fruit at 0 days DAH. B4 represents ‘Baili’ fruit at 4 DAH,

818

H2 represents‘Hongli’ fruit at 2 DAH. B6 represents ‘Baili’ at 6 DAH. H6 represents ‘Hongli’

819

fruit at 6 DAH. B. KEGG pathway annotation of ‘Baili’ and ‘Hongli’ peach at 0, 4(2) and 6 DAH.

820

FIGURE 4. Identification of the major DEMs (adjusted P< 0.05, and absolute log2 Fold

821

Change> 1) present in ‘Baili’ and ‘Hongli’ fruit. A. Metabolites involved in the flavonoid and

822

anthocyanin biosynthesis pathway. B. Metabolites involved in the synthesis of monolignols. C.

823

Metabolites involved in the biosynthesis of plant hormones. D. Metabolites that are precursors of

824

ethylene. The rows in the Heatmap represent metabolites, and the columns indicate samples. The

825

colours of Heatmap cells indicate scaled expression level of genes across different samples. The

826

colour gradient, ranging from green, through black, to red represents low, middle and high values

827

of the FPKM values over the course of the storage period.



828

FIGURE 5. Summary of differentially expressed genes (DEGs) in ‘Baili’ and ‘Hongli’ over the

829

course of the storage period. A. DEGs in ‘Hongli’ vs ‘Baili’ fruit. B. Venn diagrams of DEGs. C.

830

Principal component analysis (PCA) of DEGs in ‘Hongli’ and ‘Baili’ fruit over the course of the

831

storage period. B1 represents‘Baili’ fruit at 0 days after harvest (DAH), H1 represents ‘Hongli’

832

fruit at 0 DAH, B2 represents ‘Baili’ fruit at 4 DAH, H2 represents ‘Hongli’ fruit at 2 DAH. B3

833

represents ‘Baili’ fruit at 6 DAH, H3 represents ‘Hongli’ fruit at 6 DAH.

834

FIGURE 6. KEGG pathway analysis of DEGs and overview of differentially expressed

835

metabolites and genes between pairwise comparisons of ‘Baili’ and ‘Hongli’ fruit at 0, 2(4), 6

836

days after harvest (DAH). A. KEGG pathway analysis of DEGs. Red box indicates

837

phenylpropanoid metabolism pathway. B. Overview of differences in the metabolites and genes

838

expressed in ‘Baili’and ‘Hongli’ fruit. Metabolites pathways and genes that are up- or down-

839

regulated in ‘Baili’ and ‘Hongli’ are labeled in magenta, aqua blue, jade green,or dark gray,

840

respectively. The colors represent up-regulated pathway (log>2) in ‘Baili’, up-regulated pathway

841

(2>log>1) in ‘Hongli’, down-regulated pathway (log

Metabolite and Transcriptome analyses indicate the involvement of

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