Article pubs.acs.org/JAFC
Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family during Apple Fruit Ripening and Softening Zongying Zhang,†,‡ Nan Wang,†,‡ Shenghui Jiang,† Haifeng Xu,† Yicheng Wang,† Chuanzeng Wang,§ Min Li,† Jingxuan Liu,† Changzhi Qu,† Wen Liu,∥ Shujing Wu,† Xiaoliu Chen,† and Xuesen Chen*,† †
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State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, China § Shandong Institute of Pomology, Tai’an, Shandong 271000, China ∥ College of Life Science, Linyi University, Linyi, Shandong 276005, China S Supporting Information *
ABSTRACT: Ethylene and xyloglucan endotransglucosylase/hydrolase (XTH) genes were important for fruit ripening and softening in ‘Taishanzaoxia’ apple. In this study, we found it was ACS1-1/-1 homozygotes in ‘Taishanzaoxia’ apple, which determined the higher transcription activity of ACS1. XTH1, XTH3, XTH4, XTH5, and XTH9 were mainly involved in the early fruit softening independent of ethylene, while XTH2, XTH6, XTH7, XTH8, XTH10, and XTH11 were predominantly involved in the late fruit softening dependent on ethylene. Overexpression of XTH2 and XTH10 in tomato resulted in the elevated expression of genes involved in ethylene biosynthesis (ACS2, ACO1), signal transduction (ERF2), and fruit softening (XTHs, PG2A, Cel2, and TBG4). In summary, the burst of ethylene in ‘Taishanzaoxia’ apple was predominantly determined by ACS1-1/1 genotype, and the differential expression of XTH genes dependent on and independent of ethylene played critical roles in the fruit ripening and softening. XTH2 and XTH10 may act as a signal switch in the feedback regulation of ethylene signaling and fruit softening. KEYWORDS: apple, fruit softening, ethylene, XTH, gene expression
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INTRODUCTION
of polyuronides in cell walls by PG is insufficient for fruit ripening and softening.13 Xyloglucan is the predominant hemicellulose of cell walls, and acts as a tether between microfibrils through the formation of hydrogen bonds with cellulose microfibrils.14,15 Xyloglucan endotransglucosylase/hydrolase (XTH), which was previously named xyloglucan endotransglucosylase (XET), is believed to be critical for fruit ripening and softening because of its role in disassembling xyloglucans and loosening the cell wall in preparation for further modifications by other cell wall associated enzymes.16 The XTH enzymes exhibit XET activity, resulting in the integration of newly secreted xyloglucan chains into an existing wall-bound xyloglucan and restructuring of existing cell wall materials. The XTH enzymes also function as a hydrolase to hydrolyze xyloglucan molecules in the absence (or at low concentrations) of xyloglucan oligosaccharides.3 The characterization of apple XTHs has focused on changes in enzyme activity and gene expression levels during fruit ripening and softening.10,16,17 The most abundant transcripts in ripe apples are those of two XTH genes (i.e., MdXTH2 and MdXTH10).16 The increase in XET activity in ethylene-treated apples may be due to the elevated expression of XTH10, which is highly expressed during ripening, and can be induced by ethylene.3 A previous study revealed that the differential
The plant hormone ethylene plays a key role in the ripening and softening of many climacteric fruits, and in its absence the process fails to proceed to completion.1 However, once initiated, fruit ripening and softening is a one-way process and the beneficial aspects of ethylene for generating a highquality product can soon be outweighed by its propensity to stimulate fruit softening and decay.2 Fleshy fruits soften mainly as a consequence of changes in cell wall structures and the disassembly of different cell wall components.3,4 The plant primary cell wall is composed of cellulose, hemicellulose, pectin, and structural proteins. The solubilization of pectin and depolymerization of hemicelluloses are common features of fruit softening, which is an elaborated biochemical process involving coordinated actions of cell wall modifying enzymes and proteins, including pectin methylesterase (PME), polygalacturonase (PG), β-galactosidase (β-Gal), expansin (EXP), and xyloglucan endotransglucosylase (XET).5,6 PG and PME are two major enzymes associated with the solubilization of pectin. The demethylation of pectin by PME makes the cell walls susceptible to further degradation by PG through hydrolytic cleavage of α-(1−4) galacturonan linkages.7,8 Differences in cortical microstructures and cell adhesion are responsible for the textural differences between ‘Scifresh’ and ‘Royal Gala’ apples. These differences are closely related to PME and PG activities.9 Previous studies determined that PG causes the most prominent changes during softening.10−12 Analyses of transgenic tomatoes indicated that the degradation © 2016 American Chemical Society
Received: Revised: Accepted: Published: 429
October 11, 2016 December 21, 2016 December 27, 2016 December 27, 2016 DOI: 10.1021/acs.jafc.6b04536 J. Agric. Food Chem. 2017, 65, 429−434
Article
Journal of Agricultural and Food Chemistry
(containing 1 M NaCl, pH 6.0) and then placed at 4 °C for 24 h. The resuspended sample was centrifuged at 12000 rpm for 10 min (4 °C), and the supernatant was the enzyme mixture used for the determination of XET activity. Determination of XET Activity. XET activity was assayed with a colorimetric method developed by Sulova et al.22 Briefly, the reaction mixture contained 50 μL of tamarind xyloglucan solution (2 mg mL−1; Megazyme, Bray, Ireland), xyloglucan-derived oligosaccharide solution (0.5 mg mL−1; Megazyme), 10 mM sodium phosphate (pH 6.0), and an enzyme mixture, respectively. The blank sample lacked xyloglucan and the control sample lacked the XET enzyme mixture. The mixtures were incubated at 37 °C for 30 min before the reaction was stopped with the addition of 100 μL of 1 M HCl. We added 0.2 mL of iodine− potassium iodide solution and 0.8 mL of 20% (w/v) Na2SO4 to the samples, which were then incubated in darkness for 30 min. The optical density of the samples was measured at 620 nm. The XET activity rate was calculated as the percentage of xyloglucan degradation catalyzed by 1 g fresh weight. Determination of ACS1 Genotype. Genomic DNA was isolated from leaves of three apple cultivars, ‘Fuji’, ‘Golden Delicious’, and ‘Taishanzaoxia’, using plant genomic DNA kit (TIANGEN, Beijing, China). PCR was carried out to identify the ACS1 allelic forms in apple cultivars. The reaction mixture (25 μL) contained 25 ng of template genomic DNA, 1 μL of forward and reverse primer (10 μM), 2.5 μL of 10× buffer, 2 μL of dNTP (2.5 mM), and 0.3 μL of Taq DNA polymerase (5 U μL−1). The amplification program consisted of 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 50 s, with a final 3 min extension step at 72 °C. The PCR products were examined on a 1% agarose gel. Quantitative Real-Time PCR Analysis. Total RNA was isolated from (2 g) leaves, fruit peels, and fruit flesh that had been quickly ground into a fine powder in liquid nitrogen. A 1 μg aliquot of RNA was analyzed by 1% agarose gel electrophoresis as a quality check. Total RNA concentration (ng μL−1) and quality (optical density ratio: 260 nm/280 nm) were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Firststrand cDNA was synthesized from 1 μg of total RNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgene, Beijing, China). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was conducted using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The reaction samples consisted of 1 μL of cDNA (5-fold dilution), 10 μL of TransStart Top Green qPCR SuperMix, 1 μL of forward and reverse primer (10 μM), and 7 μL of double-distilled H2O. The qRT-PCR program was as follows: 94 °C for 30 s and then 40 cycles of 94 °C for 5 s, 58 °C for 15 s, and 72 °C for 10 s. The qRT-PCR assay was completed in triplicate. The Actin gene (for apple) and 18S gene (for tomato) served as an internal control, and the relative quantities of specific transcripts were determined using the 2−ΔΔCt method.23 All primers were designed using Primer Premier 6 software. Transformation of Tomato. The XTH2 and XTH10 genes were individually incorporated into the pBI121 vector at the BamHI and SalI sites. The constructs were inserted into Agrobacterium tumefaciens strain LBA4404 cells using a thermal stimulation method (i.e., incubation in an ice bath for 5 min, liquid nitrogen for 5 min, and at 37 °C for 5 min). The A. tumefaciens cells were then used to transform tomato cotyledons using a dipping method. Transformed materials were cultured three times on Murashige and Skoog medium supplemented with 100 mg L−1 kanamycin to isolate the transgenic material. The plants were grown in the illumination incubator, with a photoperiod of 16 h light (27 °C), 8 h dark (19 °C). Leafs of the transgenic tomato were used for gene expression analysis. Three lines were tested for each gene.
expression of XTH genes may result in fruit textural differences in progenies of a cross between ‘Fuji’ and Malus sieversii f. niedzwetzkyana apples.18 Depending on the fruit species, different modifications may occur and to different extents, so the roles of individual cell wall modifying enzymes during fruit ripening and softening may differ between species. ‘Taishanzaoxia’ is an early ripening apple cultivar with excellent fruit appearance and quality characteristics.19 Unfortunately, a sharp decrease in fruit firmness accompanied by a burst of ethylene production during the fruit late development period lowers its freshness and shelf life.20 Nevertheless, this characteristic makes the cultivar ideal for investigating the ethylene-dependent mechanisms regulating fruit ripening and softening. Previously, we isolated two XTH genes (i.e., XTH2 and XTH10) from suppression subtractive hybridization (SSH) libraries generated for ‘Taishanzaoxia’ apples harvested around the climacteric stage. The upregulation of XTH2 and XTH10 expression levels was significantly correlated with fruit firmness and ethylene production, suggesting that XTH genes may be important for fruit softening in ‘Taishanzaoxia’ apples.21 However, the roles of other XTH genes during fruit ripening and softening in ‘Taishanzaoxia’ apples were unclear. In this study, we investigated the effects of ethylene on the regulation of XTH expression levels, fruit ripening, and softening. Our findings may help characterize the molecular mechanisms responsible for apple ripening and softening, which is critical for optimizing fruit quality during the breeding of new apple cultivars.
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MATERIALS AND METHODS
Plant Materials. ‘Taishanzaoxia’ apples were harvested from the Shandong Agricultural University fruit breeding orchard (36°26′N, 117°29′E) in Tai’an, China. Fruits were collected at six developmental stages [i.e., 40, 50, 60, 65, 70, and 75 days after full bloom (DAFB)] and immediately transferred to our laboratory for the determination of ethylene production and fruit firmness. In postharvest 1-MCP treatment, fruits harvested 70 DAFB were treated with 1 μL L−1 1methylcyclopropene (1-MCP) and stored at 24 °C. Fruit firmness and ethylene levels were measured at 1 day intervals (0, 1, 2, 3, 4, and 5 days after harvest). The apples were then cut into approximately 1 cm2 pieces, frozen in liquid nitrogen, and stored at −80 °C after the determination. Determination of Fruit Firmness and Ethylene Levels. Fruit firmness was measured using a TA.XTplus Texture Analyzer (Stable Microsystems, Godalming, U.K.) with a P/2 columnar probe (2 mm diameter). The pretest, test, and post-test speeds were 2 mm s−1, 1 mm s−1, and 10 mm s−1, respectively. The depth of penetration was 10 mm, with a trigger force of 10 g. The firmness was automatically calculated using Texture Exponent 32. Each fruit was punctured twice near the equator, and six replicates were used to test fruit firmness. Two fruits were sealed in a 1.5 L glass jar and incubated at 24 °C for 6 h. Headspace samples (1 mL) were collected and analyzed with a GC-9A gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. The temperatures of the separation column and detector were 70 and 120 °C, respectively. Nitrogen and hydrogen were used as the carrier gases at 20 mL min−1 and 50 mL min−1, respectively. The rate of ethylene production was calculated based on peak area quantification. The average ethylene concentration from three jars was calculated and used in subsequent analyses. Xyloglucan Endotransglucosylase Activity Assay. Enzyme Extraction. Fruit tissue (1 g) was ground into a fine powder in liquid nitrogen and then added to 800 μL of 10 mM citrate-phosphate buffer (pH 7.0). The extracts were centrifuged at 12000 rpm for 20 min (4 °C). The sediment was washed with 10 mM citrate-phosphate buffer (pH 7.0) twice, and then centrifuged at 12000 rpm for 10 min (4 °C). The sediment was resuspended with 10 mM citrate-phosphate buffer
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RESULTS AND DISCUSSION ACS1 Genotype, Ethylene, and Fruit Softening. In previous study, we found that fruit firmness was on the decline during the fruit development period in ‘Taishanzaoxia’ apple, 430
DOI: 10.1021/acs.jafc.6b04536 J. Agric. Food Chem. 2017, 65, 429−434
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Figure 1. Changes of fruit firmness and ethylene production in ‘Taishanzaoxia’ apple treated with 1-MCP. (A) Ethylene production rate. (B) Fruit firmness. Uppercase letters mean the significance level of 1%, and lowercase letters mean the significance level of 5%.
Figure 2. Band patterns of ACS1 genotypes (A) and the transcription levels of ACS1 during fruit development period in ‘Taishanzaoxia’ apple (B). (A) Lane 1: ‘Taishanzaoxia’, ACS1-1/-1 homozygote (one band of 489 bp length). Lane 2: ‘Golden Delicious’, ACS1-1/-2 heterozygote (two bands of 489 bp, 655 bp length). Lane 3: ‘Fuji’, ACS1-2/-2 homozygote (one band of 655 bp length). M: 1kb plus marker.
Figure 3. Changes of the XET activity and transcription levels of XTH genes during fruit development period in ‘Taishanzaoxia’ apple.
results further confirmed that ethylene is required for normal ripening and softening of many climacteric fruits.2 ACS and ACO are two key enzymes in ethylene biosynthesis.2 In apple, ACS1 is specifically expressed in ripening fruits and closely related to ethylene production and fruit softening.24 Two allelic forms of ACS1 (i.e., ACS1-1, ACS1-2) were isolated from a genomic library of ‘Golden Delicious’.25 ACS1-2 is linked with the low level of ethylene production, as the insertion of a short interspersed nuclear element (SINE) into
accompanied by a burst of ethylene production. The decline of fruit firmness was significantly correlated with ethylene production (r = −0.851*), suggesting that fruit softening in ‘Taishanzaoxia’ apple was dependent on ethylene.21 In this study, we found that 1-MCP delayed the burst of ethylene production and the decline of fruit firmness during 0−3 DAH. Accompanied by the increase of ethylene production, fruit firmness declined quickly during 2−5 DAH (Figure 1). The 431
DOI: 10.1021/acs.jafc.6b04536 J. Agric. Food Chem. 2017, 65, 429−434
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Journal of Agricultural and Food Chemistry
Figure 4. Effect of 1-MCP on the expression of XTH genes in ‘Taishanzaoxia’ apple. ** means the significance level of 1%, and * means the significance level of 5%.
niedzwetzkyana.18 It was similar to the roles of AdXTH7 in kiwifruit and LeEXT1 in tomato,16,28 which may play a role in early softening of fruits allowing access for other cell wall hydrolases during the rapid softening phase. Accompanied by a sharp increase in ethylene levels, XTH2, XTH7, XTH10, and XTH11 were highly expressed during the rapid fruit softening stage and significantly correlated with fruit firmness (negative) and ethylene production (positive), suggesting that they may be involved in fruit ripening and softening via an ethyleneregulated pathway, which was consistent with the results of a study involving ethylene-treated apples.3 This was also supported by the fact that fruit softening was delayed in fruits treated with 1-MCP and XTH2, XTH7, XTH10, and XTH11 expression levels were suppressed 0−2 DAH, but were upregulated 2−5 DAH accompanied by the accumulation of ethylene (Figure 4). Accompanied by the accumulation of ethylene, the expression of XTH3 was upregulated (Figure 3 and 4), and it was significantly correlated with ethylene (negatively), suggesting that it may be negatively regulated by ethylene. XTH4 was mainly expressed during early fruit development (Figure 3), and 1-MCP had little effect on the expression (Figure 4), suggesting that the expression of XTH4 may be independent of ethylene. XTH6 and XTH8 had two expression peaks during early and late fruit development respectively (Figure 3), suggesting that they may play a role in both early and late fruit development. The expression of XTH6 and XTH8 was evidently suppressed by 1-MCP during late fruit development (Figure 4), suggesting that the late fruit softening was dependent on ethylene. Thus, to conclude, the differential expression of XTH genes was critical for the fruit ripening and softening in ‘Taishanzaoxia’ apple. The early fruit softening mainly resulting from the expression of XTH1, XTH3, XTH4, XTH5, and XTH9 was independent of ethylene. The late fruit softening was dependent on ethylene, which was associated
the promoter region results in the sharp decline of the transcription activity of ACS1-2.25−27 ACS1 is inherited in a Mendelian fashion, and the three allelic combinations, ACS11/-1, ACS1-2/-2, and ACS1-1/-2, generally confer high, low, and medium ethylene production, respectively.24,26 Consistent with the results, we found that it was ACS1-1/-1 homozygote in ‘Taishanzaoxia’ apple as there was one band of 489 bp length (Figure 2A). The transcription levels of ACS1 in ‘Taishanzaoxia’ apple were elevated during the fruit development period (Figure 2B) and significantly correlated with ethylene production (rACS1 = 0.949**). In previous study, we found that the upregulation of ACO1 was also correlated with ethylene production.21 Therefore, the burst of ethylene production in ‘Taishanzaoxia’ apple was closely associated with higher transcription activity of ACS1 determined by ACS11/-1 genotype, and ACO1 also played important roles in ethylene production. Roles of XTH Genes in Fruit Ripening and Softening. The XET activity level increased throughout the fruit development period in ‘Taishanzaoxia’ apple, with a distinct surge during the rapid fruit softening stage (Figure 3). The XET activity was significantly correlated with ethylene production (r = 0.852*) and fruit firmness (r = −0.888*), suggesting that the elevated XET activity dependent on ethylene played important roles in fruit ripening and softening. We previously isolated XTH2 and XTH10 from an SSH library for the ‘Taishanzaoxia’ apple. Their elevated expression levels suggested that they played important roles during fruit ripening and softening.21 In this study, we observed differences in the XTH expression patterns (Figures 3 and 4). The decreased XTH1, XTH5, and XTH9 expression levels during the fruit development period were significantly correlated with fruit firmness (positive), suggesting that they may play a role in the early period of fruit softening, which was consistent with results in the soft/crisp strains of Malus sieversii f. 432
DOI: 10.1021/acs.jafc.6b04536 J. Agric. Food Chem. 2017, 65, 429−434
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Figure 5. Characterization of the expression of genes involved in fruit ripening and softening in tomatoes overexpressing XTH2 and XTH10. ** means the significance level of 1%, and * means the significance level of 5%.
specific MADS2.1 alleles are significantly associated with qualitative assessments of fruit texture.32 Ripening characteristics, such as starch degradation and ethylene-modulated ripening traits, are inhibited in MADS8/9-suppressed apples.33 We previously identified a MADS gene in an SSH library, which was closely associated with fruit ripening and softening and may act as a negative regulator.21 Similar to RIN, the MADS genes are important for regulating flesh formation and softening of fleshy fruits, potentially through the activation of ethylene biosynthesis genes. Therefore, confirming the role of MADS genes during the regulation of ‘Taishanzaoxia’ apple ripening and softening, and determining whether their functions depend on ethylene may be of value.
with upregulation of XTH2, XTH6, XTH7, XTH8, XTH10, and XTH11. The role of XTHs was further verified in transgenic tomatoes overexpressing XTH2 and XTH10. Interestingly, not only was the expression of XTH2 and XTH10 significantly upregulated in transgenic tomatoes, but also the expression of fruit softening related genes (PG2A, XTHs, Cel2 and TBG4) was upregulated (Figure 5). Fruit softening is a genetically programmed development process, involving changes in the structure and composition of the fruit cell wall, which are resulted from the coordinated and interdependent action of a range of hydrolytic enzymes including PG, β-Gal, and XTH.4,29 XTH enzymes are thought to play a key role in fruit softening by loosening the cell wall in preparation for further modification by other cell wall associated enzymes and through disassembly of xyloglucan.16 Therefore, XTH2 and XTH10 may act as a switch in fruit softening through activating the expression of softening-related genes. Additionally, the expression of ethylene biosynthesis and signaling pathway genes (ACS2, ACO1, and ERF2) was also upregulated in transgenic tomato, suggesting that there may be a feedback regulation mechanism between ethylene biosynthesis and fruit softening. To conclude, XTH2 and XTH10 may act as a signal switch triggered off the increase of ethylene production and initiating the process of fruit softening. Therefore, it will be our focus to further identify the function mechanism of XTH2 and XTH10 in the process, which is critical for a more comprehensive characterization of the mechanisms regulating fruit quality and breeding programs aimed at optimizing fruit quality. Other Regulatory Factors in Fruit Ripening and Softening. Fruit softening is a one-way process, and once initiated, the process cannot be completely inhibited by inhibitors of ethylene action (e.g., 1-MCP).2 This implies that there may be regulatory factors upstream of ethylene or other regulatory factors that are part of ethylene-independent pathways. In tomato, RIN, which is a member of the MADSbox family of transcription regulators, is essential for fruit ripening. Its encoded protein acts as an upstream regulator of ethylene, and interacts with the promoters of genes involved in fruit softening, including ACS, PG2A, and EXP1.30,31 In apple,
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04536. Correlation analysis, primers, and differential expression of XTH genes in ‘Taishanzaoxia’ apple (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-538-8249338. E-mail:
[email protected]. ORCID
Zongying Zhang: 0000-0001-9440-3289 Funding
This research was funded by the Special Fund for Agroscientific Research in the Public Interest of China (to Xuesen Chen, Grant No. 201303093, http://www.most.gov.cn/), Natural Science Foundation of China (to Xuesen Chen, Grant No. 31171932, http://www.nsfc.gov.cn/), and National Key Basic Research Program of China (to Xuesen Chen, Grant No. 2011CB100606, http://www.most.gov.cn/) of the Ministry of Science and Technology and Agriculture of the People’s Republic of China. Notes
The authors declare no competing financial interest. ‡ Z.Z. and N.W. are co-first authors. 433
DOI: 10.1021/acs.jafc.6b04536 J. Agric. Food Chem. 2017, 65, 429−434
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DOI: 10.1021/acs.jafc.6b04536 J. Agric. Food Chem. 2017, 65, 429−434
