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Article

Pb4CL2 Inducing Lignin Accumulation in Superficial Scald ‘Chili’ (Pyrus bretschneideri) Pear Fruit

by
Qian Li
1,†,
Chenxia Cheng
1,†,
Chunjian Zhang
1,
Junxiu Xue
1,
Yong Zhang
1,
Caihong Wang
1,
Ruihong Dang
2 and
Shaolan Yang
1,*
1
College of Horticulture, Qingdao Agricultural University, No. 700 Changcheng Road, Chengyang, Qingdao 266109, China
2
Bioengineering College, Aks Vocational and Technical College, No.41 Xuefu Road, Aks 843000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2022, 12(11), 2650; https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy12112650
Submission received: 25 August 2022 / Revised: 20 October 2022 / Accepted: 25 October 2022 / Published: 27 October 2022
(This article belongs to the Special Issue Plant Stress Responses: Host–Microbe Interactions, Reactive Oxygen Species Signaling, the Role of Phytohormones, and Disease Control)
Browse Figures
Versions Notes

Abstract

:
Superficial scald of pear fruit is a physiological disorder that easily occurs during cold storage and seriously affects pear eating quality and commodity value. It is important to study the mechanism of superficial scald disorder. Our previous study reported that the incidence of superficial scald of calcium chloride (CaCl2)-treated pear fruit during storage was significantly lower than that of untreated fruit. In this study, we found that the accumulation of lignin in CaCl2-treated fruit was significantly lower than that of untreated fruit. The expression of the Pb4CL2 gene in the lignin synthesis pathway was downregulated in the CaCl2-treated fruit. The lignification level of the fruit overexpressing Pb4CL2 was significantly higher than that of the empty vector fruit. Therefore, we speculate that downregulation of Pb4CL2 after CaCl2 treatment plays an important role in CaCl2 inhibiting superficial scald disorder by affecting lignin accumulation in pear fruit.

1. Introduction

Fruit quality is often affected by physiological disorders during cold storage. Superficial scald disorder is one of the most serious physiological disorders, and often occurs in pear (Pyrus communis L. and Pyrus serotine Redh.) and apple (Malus pumila L.) fruit during cold storage [1]. The epidermis of the fruit with superficial scald disorder shows dark brown lesions, accompanied by necrosis of hypodermal cortical cells [1,2,3,4]. The occurrence of superficial scald disorder seriously restricts fruit quality and economic value.
Studies have shown that plant nutrient imbalances are involved in the regulation of plant growth and development; a calcium nutrition imbalance is an important factor caused by fruit biotic and abiotic stress [5,6,7,8]. Many studies have shown that calcium deficiency is one of the causes of various fruit physiological diseases [9,10,11]. Our previous study found that calcium content was reduced in the ‘Chili’ pear fruit with superficial scald disorder and calcium-like (CML) genes were upregulated, suggesting that the occurrence of superficial scald disorder in ‘Chili’ pear was caused by a calcium imbalance [12].
As a metabolite of the phenylpropanoid pathway, lignin plays an important role in cell structural support, water transport, and biotic and abiotic stress involved in plant growth and storage processes, such as the regulation of fruit taste, texture, and physiological disorders [13,14,15,16]. Many reports have shown that the occurrence of many physiological disorders are accompanied by lignin accumulation, such as skin russeting in ‘Golden Delicious’ apple [17], cork spot in ‘Chili’ pear (Pyrus bretschneideri Rehd.) [18], lenticel disorder in ‘Xinli No. 7′ pear (Pyrus bretschneideri Rehd.) [19], blossom-end rot in tomato [20], oleocellosis in citrus [21], and post-harvest granulation in navel orange fruit [22]. High lignin content seriously affects the commercial value of the affected fruit. Therefore, lignin could be used as an important indicator for evaluating the quality of several fruit, such as mangosteen [23], loquat [24], pear [18], and citrus [25]. However, the molecular mechanisms of how lignin and calcium antagonistically regulate superficial scald remains unclear.
Our previous study found that pre-harvest polyethylene (PE) bagging of fruit induced superficial scald during cold storage [12]. The superficial scald of fruit was significantly reduced after CaCl2 treatment [12]. However, the mechanism of superficial scald that was alleviated by CaCl2 is not clear in pear fruit. Based on the RNA-seq data of no-bagging versus PE-bagging fruit, we found that the expression levels of lignin biosynthesis genes were increased in PE-bagging fruit [26]. Moreover, in this study, the transcript abundance of lignin biosynthesis genes was significantly decreased after CaCl2 treatment. Thus, in this study, we focused on exploring the relationship between lignin synthesis and the occurrence of superficial scald. This study will provide a theoretical basis for resolving superficial scald disorder.

2. Materials and Methods

2.1. Plant Materials

Fifteen-year-old ‘Chili’ pear trees in Laiyang Orchard, Shandong Province, China were used in the study. The fruit were bagged with polyethylene (PE) bags on day 65 after anthesis. The fruit were harvested on 28 September 2018, as the PE bags were removed, and a total of 200 disease-free pear fruit of uniform size were selected and pre-cooled for 1 d. These fruit samples were divided into two groups, each with 100 fruit. In the treatment group, the fruit were soaked in 2% CaCl2 for 15 min. In the control group, the fruit were soaked in water for 15 min. After air-drying, fruit were placed at 2 ± 0.5 °C and 50% humidity during cold storage. Superficial scald incidence, soluble solids (TSS), and firmness were measured at 0, 30, and 60 days. The weight loss rate, respiration rate, and ethylene production rate were measured every 15 days. The fruit tissue were frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80 °C [12].
In the second year, 200 PE-bagged fruit were selected for a transient expression test. One hundred fruit were injected with the empty vector pSuper1300, and the other 100 fruit were injected with pSuper1300:Pb4CL2. Then, fruit were placed at 2.0 ± 0.5 °C and 50% humidity during cold storage. TSS, weight loss rate, firmness, respiration rate, and ethylene production rate were measured after injection. The fruit tissue was frozen in liquid nitrogen and stored at −80 °C.

2.2. Determination of Soluble Sugar Content

Five CaCl2-treated fruit were selected at 0, 30, and 60 days, and the fruit that were not treated with CaCl2 were used as controls. The pulp tissue at the equator of the fruit was taken out and squeezed into juice, and TSS was measured with the Handheld Brix Meter (ATAGO PR10, Tokyo, Japan). Data was analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).

2.3. Weight Loss Rate

Nine fruit were selected from different treatments, to determine the weight loss rate during storage time. The weight loss rate was calculated as follows: Weight loss rate (%) = (Weight0d − Weight15/30/60/75/90d)/Weight0d × 100%. Results were analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).

2.4. Respiratory Rate

Nine fruit were selected to determine the oxygen and carbon dioxide content with different treatments, using the carbon dioxide analyzer (F0530, Itsey). The volume of each container was 24.1 L. The temperature was 25 °C and the humidity was 50%. Respiratory rate was calculated as follows: Respiration intensity (mg kg−1 s−1) = Carbon dioxide release (mg)/Weight (kg) × Time (s). Results were analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).

2.5. Ethylene Production Rate

Ethylene production rate was measured with gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan), referring to the method of Yang et al. [27]. Nine fruit was put into three sealed medium containers (three fruit in each container) for 1 h, and the gas was extracted to determine the ethylene content, with three biological replicates in each sampling time point. Data was analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).

2.6. Firmness

Firmness was measured on both sides of the equator (probe diameter was 2.0 mm) using the CT3-4500 texture analyzer (Brookfield, WI, USA) with 0.5 mm s−1 testing speed. Then the correlation analysis of texture characteristics was carried out according to previous research [28]. Five fruit of each treatment was used for measurement and four equidistant points were taken from each fruit. Results were analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).

2.7. Wiesner Staining

Wiesner reagent (phloroglucinol-HCl staining) staining of pear pulp tissue was used to visualize lignification [29]. A razor blade was used to obtain fruit pulp tissue, then pulp sections were kept in the Wiesner reagent for 5 min. The lignin structure was dyed pink or fuchsia in color.

2.8. Lignin Content

The sample was prepared as described by Fan [30]. Lignin content was measured using a kit (G0708W, Suzhou Grace Bio-technology Co. LTD, Suzhou, China), according to the manufacturer’s instructions. Results were analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).

2.9. Quantitative Real-Time PCR (qRT-PCR)

Samples of ‘Chili’ pear pulp tissue was selected for qRT-PCR from each treatment at 0, 30, and 60 d. Total RNA was extracted using an RNA purification kit (TIANGEN, Beijing, China), and then the cDNA reverse transcription from RNA was performed with Prime Script™ RT Reagent Kit (Takara, Tokyo, Japan). Gene-specific primers were designed using NCBI Primer designing tool (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/tools/primer-blast/, accessed on 8 May 2019) for the qRT-PCR analysis (Table S1). Quantitative real-time PCR was carried out with 20 μL reaction solution, composed of 1 μL of each primer, 7 μL H2O, 1 μL cDNA, and 10 μL 2 × chamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). qRT-PCR was measured using Light Cycler® 480 instrument (Roche, Basel, Switzerland). Transcript levels were measured by the 2−ΔΔCt method [31]. The PbActin (GenID: LOC125473976) was used as the housekeeping reference gene because we used this gene as a housekeeping gene in the previous report [28]. The experiments were carried out on three independent biological replicates of each sample. Data are given as mean ± SD (n = 3). Asterisks represent significant differences (** p < 0.01, Student’s t-test) between control and CaCl2 treatment.

2.10. Vector Construction and Transient Expression of Pb4CL2 in Pear Fruit

The method of Pb4CL2 gene cloning and expression vector construction was described by Wang [26]. The fragments of Pb4CL2 were PCR amplified from ‘Chili’ (P. bretschneideri) pear cDNA. Then, Pb4CL2 fragments containing Xba I and Smal I restriction sites were inserted into pSuper1300 vectors to generate the recombinant plasmids used in this study. The transient overexpression of Pb4CL2 in the pear method was described by Li [32]. Agrobacterium cells (GV3101) expressing pSuper1300:Pb4CL2 and empty pSuper1300 were injected into the pear fruit, each with a volume of 1 mL. Photograph were taken at 3, 6, 8, and 10 days after the injection. Fruit tissue was immediately frozen in liquid nitrogen and stored at −80 °C.

2.11. Statistical Analyses

Superficial scald was expressed as superficial scald (%) = superficial scald fruit (number)/(superficial scald fruit (number) + healthy fruit (number)) × 100%. TSS was expressed as soluble sugar × 100% and analyzed by Student’s t-test. Weight loss rate was expressed as weight loss rate (%) = (weight0d − weight15/30/60/75/90d)/weight0d × 100% and analyzed by Student’s t-test. Respiratory rate (%) was expressed as respiration intensity (mg kg−1 s−1) = carbon dioxide release (mg)/weight (kg) × time (s) and analyzed by Student’s t-test. Ethylene production rate was expressed as ethylene production rate (μL kg−1 s−1) = (Sample peak area/Peak area of standard sample) × Concentration of standard sample (16.1 μL/L) × (medium containers volume (L)—fruit volume (L)/weight (kg) × time (s) and analyzed by Student’s t-test. Firmness was expressed as firmness (N) = firmness (g)/102 and analyzed by Student’s t-test. Gene expression was measured by the 2−ΔΔCt method [31] and analyzed by Student’s t-test. Asterisks represent significant differences (** p < 0.01, Student’s t-test).
Figures were composed and significant differences of samples were analyzed using GraphPad Prism software version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results

3.1. The Incidence of Superficial Scald, TSS, Weight Loss Rate, Respiratory Rate, Ethylene Production, and Firmness Rate in Pear Fruit

The incidence of superficial scald was significantly decreased after CaCl2 treatment (Figure 1A). The TSS content of fruit treated with CaCl2 was not significantly decreased compared with untreated fruit during storage (Figure 1B). With the prolongation of storage time, the weight loss rate of fruit gradually increased. At 30 days after storage, the weight loss rate of CaCl2-treated and control fruit was 12.66 and 24.99%, respectively. At 60 days after storage, the weight loss rate of the CaCl2-treated and control fruit were 22.06 and 35.01%, respectively, indicating that the CaCl2 treatment significantly reduced the weight loss rate of the fruit (Figure 1C). As shown in Figure 1D, we found that the control fruit had a respiratory peak at 30 days after storage, whereas the CaCl2-treated fruit had a respiratory peak at 75 days after storage. The trend of ethylene production rate was consistent with the respiration rate (Figure 1E), whereas firmness showed no significant difference between superficial scald-affected and healthy fruit (Figure 1F), indicating that CaCl2 treatment could delay the appearance of respiratory and ethylene peak times.

3.2. Lignin Staining in Pear Fruit

The results of staining the fruit tissue with Wiesner reagent are shown in Figure 2A. The pink part of the fruit treated with CaCl2 was significantly lower than that of the untreated fruit; the occurrence of superficial scald may be accompanied by the accumulation of lignin, and CaCl2 treatment may significantly reduce lignin accumulation. This is consistent with the measurement of lignin content, which showed that CaCl2 treatment reduced lignin content (Figure 2B). We speculate that the occurrence of superficial scald was accompanied by the accumulation of lignin.

3.3. Expression of Lignin-Related Genes

Based on the RNA-seq data of no-bagging versus PE-bagging fruit, seven lignin-related genes with increased transcript abundance in PE-bagging fruit were selected [28]. Quantitative qRT-PCR was used to verify the gene expression levels at 0, 30, and 60 d after CaCl2 treatment. The results showed that the expression patterns of PbCAD and Pp4CL2 were consistent with the finding that CaCl2 treatment significantly reduced lignin accumulation (Figure 3A–G). The expression of PbCAD was significantly lower than in the control fruit at 30 and 60 d after treatment; there was no difference in expression levels at 0 d (Figure 3E). Moreover, Pb4CL2 transcript levels significantly decreased in fruit tissues treated with CaCl2 during storage, compared with control fruit tissues.

3.4. Overexpression of Pb4CL2-Induced Superficial Scald Disorder

To further verify that Pb4CL2 participates in inducing superficial scald disorder via regulating the accumulation of lignin in pear fruit, transient overexpression of Pb4CL2 was conducted in the pear pulp. The color around the injection site of fruit injected with pSuper:Pb4CL2 changed to dark brown at 6 d after inoculation, and the brown color, which is similar to the symptoms of superficial scald disorder, progressively deepened by 10 d after inoculation (Figure 4A). However, the color around the injection site of fruit injected with the empty vector only slightly changed until 10 d after injection (Figure 4A). qRT-PCR analyses showed that the expression of Pb4CL2 was significantly increased in peels of fruit at 6 d after injection (Figure 4B). Wiesner staining of fruit tissues indicated that lignification in fruit inoculated with pSuper:Pb4CL2 gradually increased, and was obviously larger than the fruit injected with the empty vector (Figure 4C).At 10 d after injection, the degree of pulp lignification in fruit inoculated with pSuper:Pb4CL2 was severe, and the middle of the fruit was incomplete during the slicing process. The lignin content in fruit overexpressing pSuper:Pb4CL2 was significantly higher than that of the empty vector fruit (Figure 4D). Thus, we suggest that downregulation of Pb4CL2 after CaCl2 treatment plays an important role in CaCl2 inhibiting superficial scald disorder by affecting lignin accumulation in pear fruit.
The firmness of the peel in fruit overexpressing pSuper:Pb4CL2 was significantly greater than that of the empty vector fruit (Figure 5A). The firmness of the pulp in fruit overexpressing pSuper:Pb4CL2 was also significantly greater than that of the empty vector fruit (Figure 5B). There was no significant differences in TSS, respiration rate, and ethylene production rate (Figure 5C–E), but the weight loss rate in fruit injected with pSuper:Pb4CL2 was significantly higher than that of pSuper1300-transferred fruit after treatment (Figure 5F).

4. Discussion

Superficial scald disorder is one of the most serious physiological disorders, and often occurs on the pear surface. Symptoms of superficial scald disorder on the surface of pear fruit include browning or deep patches, which spreads on the entire surface of fruit [12]. Superficial scald disorder is one of the causes of decline in the commodity value of ‘Chili’ pear fruit. Therefore, it is important to study the mechanisms of superficial scald disorder. Our previous study reported that the incidence of superficial scald in fruit was significantly reduced after CaCl2 treatment [12]. Other studies have shown that the TSS and weight loss were decreased and firmness was increased after CaCl2 treatment in papaya fruit [33]. In addition, 10% gibberellin combined with 3% CaCl2 could effectively reduce weight loss, respiration rate, and ethylene production, as well as maintain high firmness, in mango fruit [34]. However, firmness was reduced by CaCl2 treatment in hard end pear fruit [35]. In this study, we found that the firmness and lignin content of the CaCl2-treated pear fruit was significantly lower than that of the control fruit. Thus, we speculated that lignin accumulation in fruit might be affected by CaCl2.
Lignification is one of the important factors affecting fruit quality, and severely affects the development of the fruit industry [36]. Some studies have shown that the browning of fruit and vegetables may be associated with lignin accumulation [37,38]. The russet of ‘Xiusu’ pear peel has been reported to be regulated by lignin synthesis signals [39]. The browning suberization crisp of early crisp pear has been reported to be associated with lignin accumulation [37]. The browning of cut jicama (Pachyrizus erosus L. Urban) was shown to be related to lignin accumulation [38]. Moreover, the ‘Golden Delicious’ apple skin russet was shown to be the result of lignin accumulation [17]. The symptoms of superficial scald disorder on pear fruit are browning or deeper patches [12]. As expected, our results showed that the occurrence of superficial scald disorder on pear fruit was positively related to lignin accumulation.
Some studies have reported that fruit lignin accumulation is increased by the upregulation of genes related to lignin synthesis, which further promotes russet formation [39,40,41,42]. For example, the Pp4CL1, PpCAD1, and PpCAD2 genes related to lignin synthesis were elevated in hard end pear [32]. The PpPOD1, PpPOD2, and PpPOD3 expression showed no correlation with lignin content during fruit cold storage, but the expression of PpCAD1 and PpCAD2 was positively correlated with lignin accumulation [28]. Based on the RNA-seq data of no-bagging versus PE-bagging fruit, seven lignin-related genes with increased transcript abundance in PE-bagging fruit were selected for qRT-PCR analysis after CaCl2 treatment [26]. The expression of Pb4CL2 and PbCAD was significantly downregulated after CaCl2 treatment. In our previous study, we found similar results in hard end pear fruit; the 4CL activity of pear (Pyrus pyrifolia Whangkeumbae) fruit were significantly different between hard end and CaCl2-treated fruit at 0 d [28]. This indicated that the 4CL activity of pear fruit could be rapidly changed after CaCl2 treatment. In addition, the 4CL gene expression level was positively correlated with 4CL activity. Therefore, it is reasonable that Pb4CL gene expression was significantly lower than in the control at 0 days after CaCl2 treatment. However, the expression pattern of Pb4CL2 was relatively stable, and 4CL has been generally considered to be a key enzyme involved in lignin synthesis [43,44] and determining degree of fruit russet [45]. The Pb4CL2 was chosen for further research because its expression level was lower in the CaCl2-treated group than in the control group, which was consistent with lignin content and superficial scald incidence. Regarding the magnitude of induction between the CaCl2-treated group and control group, it may be that gene expression was inhibited more by CaCl2 on 0 d, and less inhibited during storage time. Another reason may be that the 4CL activity of pear fruit could be rapidly changed after CaCl2 treatment [28]. Combined with the inhibition of superficial scald disorder incidence after CaCl2 treatment, we speculated that Pb4CL2 may function in regulating the process of superficial scald disorder.
Transient overexpression of the Pb4CL2 gene in the pear pulp showed that the surface of fruit injected with pSuper:Pb4CL2 was more sunken, with dark brown spots, than of the fruit injected with the empty vector. The abundance of Pb4CL2 gene expression was the highest at 6 d after injection. The lignin accumulation in fruit injected with the pSuper:Pb4CL2 was significantly higher than in the fruit injected with the empty vector, especially at 8 and 10 d. The main reason for Pb4CL2 gene expression peaking earlier than the lignification peak may be because gene expression usually occurs earlier than the phenotype, and 4CL is mainly upstream of the lignin pathway, ultimately regulating lignin accumulation by affecting the expression of downstream genes. This is consistent with the 4CL potentially participating in lignin synthesis [43,44], and lignin accumulation directly causing pear russet [42]. We infer that CaCl2 treatment may reduce the incidence of superficial scald disorder via inhibition of Pb4CL2 gene expression and lignin accumulation. However, the mechanisms of CaCl2 in modulating 4CL gene expression is not clear, and should be explored in the future.

5. Conclusions

Our previous research results showed that CaCl2 treatment effectively reduced the incidence of superficial scald disorder in pear fruit [12]. In this study, we found that the process of CaCl2 reducing the occurrence of superficial scald disorder was accompanied by a reduction in lignin. Moreover, the expression of Pb4CL2, a gene related to lignin synthesis, was downregulated in fruit treated with CaCl2. Overexpression of Pb4CL2 induced superficial scald disorder via increased lignification. We infer that calcium chloride treatment may reduce the incidence of superficial scald disorder via inhibiting Pb4CL2 gene expression and lignin accumulation.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/agronomy12112650/s1. Table S1. Sequence of primers used in the qRT-PCR amplifications.

Author Contributions

Study design: C.C., R.D. and S.Y.; data collection: Q.L., C.C., C.Z., J.X. and Y.Z.; data analysis: Q.L., C.C., C.W. and S.Y.; data interpretation: C.C.; software: Q.L., C.Z. and Y.Z.; supervision: C.W., R.D. and S.Y.; visualization: Q.L., C.Z. and J.X.; writing—original draft: Q.L. and C.C.; writing—review and editing: Q.L., C.C., R.D. and S.Y.; All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Project of Shandong Natural Science Foundation (ZR2021MC005 and ZR2017MC006), Project of National Natural Science Foundation of China (31201608), Project of Shandong Modern Fruit Technology Industry System (SDAIT-06-06), Project of Improving Agriculture Varieties of Shandong Province in China (2019LZGC008), Breeding Plan of Shandong Provincial Qingchuang Research Team (2019), and Scientific Research Funds for High-Level Personnel of Qingdao Agricultural University (1119046).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effect of CaCl2 treatment on the superficial scald incidence, TSS content, weight loss rate, respiratory rate, ethylene production rate, and firmness of ‘Chili’ pear fruit. Superficial scald incidence (A) and TSS content (B) of fruit at 0, 30, and 60 d after treatment. Weight loss rate (C), respiration rate (D), and ethylene production rate (E) of fruit at 0,15, 30, 45, 60, 75, and 90 d after treatment. (F) The firmness of fruit at 0, 30, and 60 d after treatment. Data are given as mean ± SD (n = 3).
Figure 1. Effect of CaCl2 treatment on the superficial scald incidence, TSS content, weight loss rate, respiratory rate, ethylene production rate, and firmness of ‘Chili’ pear fruit. Superficial scald incidence (A) and TSS content (B) of fruit at 0, 30, and 60 d after treatment. Weight loss rate (C), respiration rate (D), and ethylene production rate (E) of fruit at 0,15, 30, 45, 60, 75, and 90 d after treatment. (F) The firmness of fruit at 0, 30, and 60 d after treatment. Data are given as mean ± SD (n = 3).
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Figure 2. Effect of CaCl2 treatment on lignin accumulation in pear pulp. (A) Transverse sections of the pulp were stained with phloroglucinol-HCl for the detection of lignin at 30 and 60 d after treatment. Scale bar = 2 cm. (B) The lignin content of fruit at 30 and 60 d after treatment. Data are given as mean ± SD (n = 3).
Figure 2. Effect of CaCl2 treatment on lignin accumulation in pear pulp. (A) Transverse sections of the pulp were stained with phloroglucinol-HCl for the detection of lignin at 30 and 60 d after treatment. Scale bar = 2 cm. (B) The lignin content of fruit at 30 and 60 d after treatment. Data are given as mean ± SD (n = 3).
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Figure 3. The expression pattern of lignin-related genes in pear fruit after CaCl2 treatments. (A–G) The expression levels of lignin-related genes were verified by qRT-PCR. Data are given as mean ± SD (n = 3).
Figure 3. The expression pattern of lignin-related genes in pear fruit after CaCl2 treatments. (A–G) The expression levels of lignin-related genes were verified by qRT-PCR. Data are given as mean ± SD (n = 3).
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Figure 4. Transient expression of Pb4CL2 in ‘Chili’ pear fruit. (A) Fruit phenotype of fruit at 3, 6, 8, and 10 d after injection of pSuper:Pb4CL2 or empty vector. Scale bar = 2 cm. (B) The expression of Pb4CL2 in pear fruit surrounding the injection site. (C) Wiesner staining of fruit sections at 3, 6, 8, and10 d after infiltration of pSuper:Pb4CL2 or empty vector. Scale bar = 2 cm. The lignin content (D) in Pb4CL2-infiltrated and empty-vector-infiltrated pear fruit. Data are given as mean ± SD (n = 3).
Figure 4. Transient expression of Pb4CL2 in ‘Chili’ pear fruit. (A) Fruit phenotype of fruit at 3, 6, 8, and 10 d after injection of pSuper:Pb4CL2 or empty vector. Scale bar = 2 cm. (B) The expression of Pb4CL2 in pear fruit surrounding the injection site. (C) Wiesner staining of fruit sections at 3, 6, 8, and10 d after infiltration of pSuper:Pb4CL2 or empty vector. Scale bar = 2 cm. The lignin content (D) in Pb4CL2-infiltrated and empty-vector-infiltrated pear fruit. Data are given as mean ± SD (n = 3).
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Figure 5. Firmness, TSS content, weight loss rate, respiratory rate, and ethylene production rate of ‘Chili’ pear fruit after injection of pSuper:Pb4CL2 or empty vector. Firmness of peel (A) and pulp (B) in Pb4CL2-infiltrated and empty-vector-infiltrated pear fruit. The TSS content (C), respiratory rate (D), ethylene production rate (E), and weight loss rate (F) of fruit at 3, 6, 8, and 10 d after infiltration. Data are given as mean ± SD (n = 3).
Figure 5. Firmness, TSS content, weight loss rate, respiratory rate, and ethylene production rate of ‘Chili’ pear fruit after injection of pSuper:Pb4CL2 or empty vector. Firmness of peel (A) and pulp (B) in Pb4CL2-infiltrated and empty-vector-infiltrated pear fruit. The TSS content (C), respiratory rate (D), ethylene production rate (E), and weight loss rate (F) of fruit at 3, 6, 8, and 10 d after infiltration. Data are given as mean ± SD (n = 3).
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Li, Q.; Cheng, C.; Zhang, C.; Xue, J.; Zhang, Y.; Wang, C.; Dang, R.; Yang, S. Pb4CL2 Inducing Lignin Accumulation in Superficial Scald ‘Chili’ (Pyrus bretschneideri) Pear Fruit. Agronomy 2022, 12, 2650. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy12112650

AMA Style

Li Q, Cheng C, Zhang C, Xue J, Zhang Y, Wang C, Dang R, Yang S. Pb4CL2 Inducing Lignin Accumulation in Superficial Scald ‘Chili’ (Pyrus bretschneideri) Pear Fruit. Agronomy. 2022; 12(11):2650. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy12112650

Chicago/Turabian Style

Li, Qian, Chenxia Cheng, Chunjian Zhang, Junxiu Xue, Yong Zhang, Caihong Wang, Ruihong Dang, and Shaolan Yang. 2022. "Pb4CL2 Inducing Lignin Accumulation in Superficial Scald ‘Chili’ (Pyrus bretschneideri) Pear Fruit" Agronomy 12, no. 11: 2650. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy12112650

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