Elsevier

Plant Science

Volume 283, June 2019, Pages 116-126
Plant Science

Peach ethylene response factor PpeERF2 represses the expression of ABA biosynthesis and cell wall degradation genes during fruit ripening

https://doi.org/10.1016/j.plantsci.2019.02.009Get rights and content

Highlights

  • PpeERF2 function as a repressor during fruit ripening.

  • PpeNCED2, PpeNCED3 and PpePG1 show ripening-induced expression patterns in peach fruits.

  • PpERF2 negatively affects the expression of PpeNCED2, PpeNCED3 and PpePG1.

Abstract

Ethylene response factors (ERFs) are known to regulate fruit ripening. However, the ERF regulatory networks are not clear. In this study, we have shown that peach (Prunus persica) PpeERF2 regulates fruit ripening through suppressing the expression of two ABA biosynthesis genes (PpeNCED2, PpeNCED3) and a cell wall degradation gene (PpePG1). The transcript levels of PpeERF2 in fruit were opposite to that of PpeNCED2, PpeNCED3 and PpePG1 during ripening and in response to various ripening treatments. PpeERF2 was found to bind to the PpeNCED2, PpeNCED3 and PpePG1 promotors as demonstrated by yeast one-hybrid (Y1H) and EMSA assays; and further found to repress the promoter activities of the three genes in tobacco leaf tissues after Agrobacterium infiltration. Taken together, these results provide new information for a better understanding of the crosstalk network between ethylene signaling, cell wall degradation and ABA biosynthesis during fruit ripening.

Introduction

Ethylene is involved in many plant physiological processes and is essential for the ripening of climacteric fruits [1]. There are two key steps in ethylene biosynthesis. In the first step, ACC (1-aminocyclopropane-1-carboxylic acid) is formed from SAM (S-adenosyl methionine) by the enzyme ACC synthase (ACS). In the second step, ACC is oxidized by ACC oxidase (ACO) to form ethylene [2]. Ethylene is detected and binds to its receptors (ETRs), and its signal is transmitted to several downstream components, including CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), ETHYLENE INSENSITIVE2 (EIN2), and the primary transcription factor EIN3/EIL (EIN3-like). Ethylene response factors (ERFs) act as trans-acting regulators at the last step of the ethylene signaling pathway and can cause an ethylene response by binding to the promoters of many ethylene-inducible genes [3,4].

ERFs are classified to a large family of plant transcription factors because they all contain a highly conserved APETALA2 (AP2) domain of 60–70 amino acids [5]. The AP2 domain binds to conserved DNA element (AGCCGCC, CCGAC, ACCGAC, GCCGAC, ATTTCAAA, and AATTCAAA) present in the promoter of ethylene responsive genes to facilitate the function of ERFs in regulating the expression of these genes [4,[6], [7], [8]]. Many ERFs act as transcription activators and promote the expression levels of ripening-related genes containing AP2 binding site, and thus enhance fruit ripening. For example, apple (Malus domestica) ERF3 (MdERF3) can bind to the promoter of the ethylene biosynthesis gene MdACS1 and enhance its expression [6]. In banana (Musa acuminata), MaERF9 activates the expression of ripening-related genes [9]. Some ERFs may also act as transcription repressors depending on the motif outside of the AP2 domain. For example, kiwifruit (Actinidia deliciosa) AdERF9 represses AdXET2 expression [10]. MdERF2 negatively affects ethylene biosynthesis by suppressing the transcription of MdACS1, which is critical for the biosynthesis of ripening-related ethylene [11].

Fruit softening during ripening is a complex process that occurs as a result of the expression of a number of hydrolase and transglycosylase genes. Among these genes, the role of polygalacturonase (PG) has been studied extensively. An increase in PG activity and mRNA levels has been observed during fruit ripening [12]. Peach cultivars are classified as melting flesh (MF) or non-melting flesh based on their fruit firmness and texture. The differences in softening between MF and non-melting flesh cultivars has been ascribed to partial or complete deletion of the endo-PG gene [13,14]. The stony hard (SH) peaches produce low levels of ethylene and barely soften before or after harvest [15]. The mRNA level of the endo-PG gene is positively regulated by ethylene, and positively correlates with the level of endo-PG enzyme activity and fruit firmness [16]. In addition to PG, pectin methylesterase (PME) is involved in the degradation of pectins. PME is expressed at higher levels in MF than in SH fruit and exhibits softening-associated expression patterns [17]. However, little molecular mechanism is available about ethylene in transcriptional regulation of cell wall metabolism in relation to fruit ripening.

Emerging evidence suggests that fruit ripening processes involve a complex network of multiple plant hormones [18]. In addition to ethylene, the phytohormone abscisic acid (ABA) plays an important role in fruit ripening [19]. ABA biosynthesis requires the cleavage of C40 carotenoids by a series of enzymatic steps to form its direct precursor xanthoxin. The last step is to cleave 9-cis-epoxyxanthophylls by 9-cis-epoxycarotenoid dehydrogenase (NCED) to produce xanthoxin, which is a key rate-limiting step in ABA biosynthesis [19]. Some reports show that ABA could regulate ethylene biosynthesis via the up-regulation of ethylene biosynthesis genes [20]. Perhaps ethylene may also affect ABA biosynthesis by regulating NCED expression [18], but the regulatory mechanism is unclear.

The peach PpeERF2 is expressed in the fruit [21,22]. However, its regulatory role in peach fruit ripening has not been established. In the present study, PpeNCED2, PpeNCED3, PpePG1 and PpePME were found to show ripening-induced expression patterns in peach fruit based on qRT-PCR analyses. Several ethylene response elements were found in the promoters of PpeNCED2, PpeNCED3 and PpePG1. Moreover, the ripening-related PpeERF2 was showed to bind to the promoter of PpeNCED2, PpeNCED3 and PpePG1, and regulate their expression.

Section snippets

Plant materials and treatment

Trees of four peach cultivars used in this study, ‘Zhongyoutao 13’ (CN13), ‘Zhongyoutao 16’ (CN16), ‘Goldhoney 3’ (GH3) and ‘Yumyeong’ (YM), were grown in the experimental orchards of Zhengzhou Fruit Research Institute, Chinese Academy of Agriculture Science, Zhengzhou, China. Fruit sample of ‘CN13’ (melting flesh, MF) were collected at 75, 80, 85, and 90 days after flowering (DAF). ‘CN16’ (stony hard, SH) samples were collected at 70, 75, 80, and 85 DAF. For ‘Goldhoney 3’ (MF) and ‘Yumyeong’

PpeERF2 showed opposite expression pattern to other fruit repining related genes

MF type peach fruit (‘CN13’, ‘GH3’) produces high levels of auxin and ethylene during ripening process and becomes soft and melting in texture at advanced stages of ripening. However, SH type peach fruit (‘CN16’, ‘YM’) shows a low level of auxin and ethylene, as well as very firm and crisp flesh texture during all ripening stages [35].The transcript level of PpeERF2 was not detected in peach fruit at stage S3, and increased to low levels at S4 I and S4 II in both MF and SH type cultivars, CN13

Expression profiles of PpePME, PpePG1, PpeNCED2 and PpeNCED3

In this study, we investigated the expression profiles of PpeERF2, PpeNCED2, PpeNCED3, PpePG1, PpePME, and PpeACS1. PpeERF2 transcript level was dramatically higher in SH cultivars (‘CN16’, ‘YM’) than in the MF cultivars (‘CN13’, ‘GH3’) at stage S4 III. In contrast, the expression level of the other five genes at stage S4 III was dramatically lower in SH cultivars (‘CN16’, ‘YM’) than in the MF cultivars (‘CN13’, ‘GH3’). It is clear that the expression levels of PpeERF2 during fruit ripening are

Acknowledgements

This work was supported by the National Natural Science Foundation of China [No. 31872085], the National Key Research and Development Program [2018YFD1000200], the Agricultural Science and Technology Innovation Program (ASTIP) [CAAS-ASTIP-2019-ZFRI], and Central Public-interest Scientific Institution Basal Research Fund the Preliminary Establishment and Capacity Improvements of the Molecular Breeding Platform for Fruit Trees [Y2019PT19-02].

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