Staining map of stone cell slices 30 d after flowering (DAF) (A), 60 DAF (B), 90 DAF (C), and 120 DAF (D).
Comparison of stone cell mass density of different pollination combinations of fruit at different stages. BY = ‘Bayue’ pear; HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear, YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05.
The contents of stone cells and lignin in fruit of different pollination combinations at different stages: 30 d after anthesis (A), 60 d after anthesis (B), 90 d after anthesis (C), and 120 d after anthesis (D). 1 = ‘Hechengdonghuang’ pear; 2 = ‘Kuerlehuangsuan’ pear; 3 = ‘Qipan’ pear; 4 = ‘Juju’ pear; 5 = ‘Bayue’ pear; 6 = ‘Yelikeamute’ pear; 7 = ‘Kuikeamute1’ pear. Lowercase letters indicate significant differences at P < 0.05.
Comparison of stone cells and lignin content in mature fruit with pollination combinations. BY = ‘Bayue’ pear; HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear; YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05.
Changes of polyphenol oxidase (PPO) and peroxidase (POD) enzyme activities in different pollination combinations at different stages. 1 = ‘Hechengdonghuang’ pear; 2 = ‘Kuerlehuangsuan’ pear; 3 = ‘Qipan’ pear; 4 = ‘Juju’ pear; 5 = ‘Bayue’ pear; 6 = ‘Yelikeamute’ pear; 7 = ‘Kuikeamute1’ pear. A = POD at 30 d; B = POD at 60 d; C = POD 90 at d; D = POD at 120 d; E = PPO at 30 d; F = PPO at 60 d; G = PPO at 90 d; H = PPO at 120 d. Lowercase letters indicate significant differences at P < 0.05; uppercase letters indicate significant differences at P < 0.01.
Changes of phenylalanine ammonia lyase (PAL) activity in different pollination combination. HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear; YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05; uppercase letters indicate significant differences at P < 0.01.
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In this study, the effects of different Xinjiang pear varieties and ‘Korla Fragrant Pear’ pollination on the stone cells and lignin of fruit were investigated. The contents of stone cells and lignin, and the activities of related enzymes [polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonium lyase (PAL)] were analyzed in fruit from different pollination combinations at different growth and developmental stages. Results showed that the stone cell mass density decreased rapidly at 60 to 90 days and 90 to 120 days after flowering. The stone cell and lignin contents, and activities of the three enzymes (PPO, POD, and PAL) decreased rapidly at 60 days after flowering. The stone cell mass density, stone cell and lignin contents, and enzyme activity of fruit from different pollination combinations varied at different timescales. The pear variety ‘Bayue‘ had the lowest stone cell and lignin contents in mature fruit from different pollination combinations. The stone cell content correlated positively with lignin content, stone cell mass density, and enzyme activity.
Stone cells are among the major factors affecting fruit quality and taste. With the development of social economy, the standards set for the appearance and taste of pear fruit are becoming increasingly demanding. Stone cells are the result of secondary thickening of parenchyma cell walls. Coarse peeled and green head fruits are related to an increase in stone cell content during the production of ‘Korla Fragrant Pear’. Currently, research on pear stone cells mainly focuses on the observation of stone cells, synthesis and polymerization of lignin monomers, and analysis of the lignin structure (Ashajiang et al., 2020). After freezing and separation, the stone cells of pears were observed using an optical microscope and were found to be of different shapes. They were deposited by high levels of lignins and cellulose, and were classified as short stone cells (Li and Zhang, 2011). The results showed that many fine characters of the occidental, Baili, and Shali pears have a small stone cell mass and low density. However, the characteristics of some Baili and Shali pears are in contrast to those of most Qiuzi pears (Aalamusa et al., 1994). The final size of the stone cell mass is determined by early and late development of stone cells, and the developmental progress of the stone cells varies according to the variety (Wang, 2014). The secondary wall is mainly formed after lignin deposition, with lignification of the cell wall being closely related to the stone cells. The thick wall formed by the continuous deposition of secondary walls on the primary walls of the parenchymal cells is comprised of stone cells (Yan et al., 2013). The stone cell content correlated positively with the lignin content. The contents of the pulp lignin and stone cell lignin of Xinjiang pear varieties were the lowest among several pear systems (Tian et al., 2017).
In this study, 10 pollination combinations of Xinjiang pear and ‘Korla Fragrant Pear’ were used to analyze changes in stone cell mass density, stone cell and lignin contents, and three enzyme activities in each developmental period of pollination combinations. The relationship between stone cells, lignin, and enzymes was also explored. This study provides a theoretical basis for improving fruit quality and identifying suitable trees for pollination.
The experiment site was located in the fruit tree resource nursery of Luntai County, Bayingoleng Mongolian Autonomous Prefecture in northwestern China’s Xinjiang Province. The climate is warm, temperate, continental, and arid. The annual sunshine duration is long, and the annual sunshine percentage is ≈64%.
Pollinated fruit of seven Xinjiang pear varieties—‘Hechengdonghuang’ (‘HD’), ‘Kuerlehuangsuan’ (‘KH’), ‘Qipan’ (‘QP’), ‘Juju’ (‘JJ’), ‘Bayue’ (‘BY’), ‘Yelikeamute’ (‘YL’), and ‘Kuikeamute1’ (‘KK1’)—and ‘Korla Fragrant Pear’ were studied (Table 1).
The cross section and free-hand sections of the pear fruit were dyed with phloroglucinol–hydrochloric acid using a 1% phloroglucinol configuration method. Four grams of phloroglucinol was dissolved in 100 mL of 95% alcohol. Two big beakers were filled with 1% phloroglucinol solution and 1% hydrochloric acid solution, respectively. The sample pears were then cut transversely into 0.3-cm slices. The samples were placed in the 1% phloroglucinol solution and then in the 1% hydrochloric acid solution for 10 s. After a few seconds, several red dots appeared. A photograph was taken to observe and record the number of dots.
A certain amount of fruit was collected, frozen at –20 °C for 1 d, and subsequently thawed. The edible part of the fruit was separated using the quartering method then put into a tissue blender, and 200 mL of distilled water was added to the homogenate. The homogenate was then slowly transferred to a 1-L beaker and stirred for 1 min using a glass rod. It was stirred again after 5 min, which was repeated twice to remove the suspension, and then rinsed with distilled water. The previously prepared suspensions were then rinsed together. The obtained stone cells were dried and weighed (Wang et al., 2022).
The experimental pears were frozen at –20 °C for over 12 h. The pulp (2 g) was stirred in a tissue blender. The oven temperature was maintained at 60 °C. The stirred fruit pulp was placed in the oven and dried to a constant weight. The dried powder was then crushed. The dry powder was weighed at 0.02 g, washed three times with 95% alcohol, and then washed three times with 1 anhydrous ethanol:2n-hexane and dried. The dried powder was then put into a test tube. In addition, 1.5 mL of 25% bromoacetyl-glacial acetic acid solution and 0.06 mL of 70% perchloric acid solution were added, sealed, and bathed in a water bath at 70 °C for 40 min. Oscillation was performed every 10 min. Subsequently, 2 mol/L NaOH and 4 mol/L ammonium hydroxide were added to 5 and 3 mL, respectively. After comparison, the mix was transferred into a centrifuge tube and centrifuged at 6000 rpm for 10 min. The supernatant was extracted from a 100-mL volumetric flask, and the volume was determined using glacial acetic acid. The solution to be tested was placed in a colorimetric dish, and absorbance was measured at 280 nm to obtain the optical density value.
The effects of calcium and cinnamyl alcohol dehydrogenase on the formation of pear stone cells were examined following the methodology of Song (2018).
Three biological replicates were used for all experiments, and the results were expressed as the mean and se of experiments conducted in triplicate. Data processing and statistical analyses were conducted using Excel 2010 (Microsoft Corp., Redmond, WA) and SPSS 18.0 (version 18.0; IBM Corp., Armonk, NY). GraphPad Prism 8.0.2 (GraphPad Corp., San Diego, CA) was used to draw the graphs.
Sections were stained with phloroglucinol–hydrochloric acid for different periods of pollination. The distribution of the stone cell mass showed a radial shape along the fruit center and extended outward gradually. The relationship between the distribution and location of the stone cell mass was that the stone cell mass near the core was the greatest. There was a greater mass near the pericarp, but less than that near the core, and the stone cell mass in the middle of the fruit was the lowest. The number of stone cell masses decreased with fruit development. Observation of the stone cell mass of pear fruit showed that the stone cell mass near the fruit center had the largest diameter (Fig. 1).
Citation: HortScience 57, 5; 10.21273/HORTSCI16513-22
Figure 2 shows that the stone cell mass density in the fruit decreased with the growth and development of the fruit, decreasing rapidly from 60 to 120 d after flowering (DAF), and decreasing minimally from 30 to 60 DAF. The decrease in the stone cell mass of each pollination variety 30 to 60 DAF was compared. Results showed that the decrease in stone cell masses of the ‘KH’ pollination combination was the least. From 60 to 90 DAF, ‘KH’ decreased the most, followed by ‘BY’, with ‘KK1’ decreasing the least. The variation analysis from 90 d to 120 DAF showed that the ‘BY’ combination had the smallest decrease. The combination of ‘HD’ changed the most from 30 to 60 and from 90 120 DAF. There was no significant difference between the ‘KK1’ and ‘YL’ pollination combinations at 30, 60, 90, and 120 DAF. The difference between ‘HD’ and ‘KH’ was not significant at 30 and 90 DAF, but became significant at 60 and 120 DAF. The two pollination combinations of ‘JJ’ and ‘BY’ showed significant differences at 30 DAF, with no significant differences at 60, 90, and 120 DAF.
Citation: HortScience 57, 5; 10.21273/HORTSCI16513-22
As shown in Fig. 3, the lignin content in the fruit increased slowly from 30 to 60 DAF, and then decreased rapidly from 60 DAF. However, the stone cell content in the fruit showed a downward trend from 30 to 120 DAF, before decreasing rapidly from 60 to 90 DAF. The stone cells and lignin contents of the ‘QP’ pollination combinations were significantly greater than those of other combinations at 30, 60, and 90 DAF. There were significant differences in the stone cell content of each pollination combination at 60 and 120 DAF. There was no significant differences in the stone cell content of ‘KH’ and ‘JJ’ at 90 and 30 DAF. According to the variation range in stone cells at each flowering stage, the variation range of the stone cell content in ‘HD’ for each pollination combination was the greatest from 30 to 60 DAF. The variation range of the stone cell content for ‘QP’ was the greatest from 60 to 90 and 90 to 120 DAF. The combination ‘YL’ had the smallest variation at 30 to 60 d and 60 to 90 DAF, and ‘BY’ had the smallest variation at 90 to 120 DAF. The combination ‘BY’ had the lowest stone cell content at 30, 90, and 120 DAF.
Citation: HortScience 57, 5; 10.21273/HORTSCI16513-22
The lignin content and the stone cell content in the fruit from pollination combination ‘KH’ were significantly greater than others with the same pollination combination. The lignin content and the stone cell content in the fruit of pollination combination ‘BY’ were significantly less than those in the same pollination combination. As shown in Fig. 4, there were no significant differences in lignin content between pollination combination ‘HD’ and pollination combination ‘JJ’. The stone cell content in the fruit of each pollination combination was significantly different.
Citation: HortScience 57, 5; 10.21273/HORTSCI16513-22
PPO, POD, and PAL are key enzymes in lignin synthesis. As shown in Figs. 5 and 6, PPO and POD activity first increased and then decreased rapidly 60 DAF. Meanwhile, PAL activity decreased with fruit growth and development.
Citation: HortScience 57, 5; 10.21273/HORTSCI16513-22
Citation: HortScience 57, 5; 10.21273/HORTSCI16513-22
PPO activity was the greatest in the fruit with the combination ‘QP’ at four stages after flowering. POD activity was the greatest at 30 and 90 DAF, and PAL activity was the greatest at 30 DAF. For the pollination combination ‘JJ’, PPO activity in fruit was the lowest at 120 DAF, and was significantly less than that of other pollination combination. However, the PAL and POD activity of this combination was the greatest at 60 DAF. PAL activity was the greatest at 90 DAF, which was significantly different from that of the combination. In different periods, PPO and PAL activity at 30 DAF, and POD activity at 60 DAF were significantly different among different pollination combinations. There was no significant difference in PPO activity between ‘KH’ and ‘YL’ at 60 and 120 DAF, and POD activity at 30 DAF. There was no significant difference in PPO activity of ‘YL’ and ‘JJ’ combinations at 90 DAF, and POD activity at 30 and 90 DAF. There was no significant difference in PAL activity between ‘HD’ and ‘YL’ at 60 and 90 DAF, and POD activity at 120 DAF. There was no significant difference in PAL activity between the combination ‘QP’ and the combination ‘KK1’ at 120 DAF.
As can be seen from Tables 2 and 3, the stone cell and lignin contents of the different pollination combinations correlated significantly and positively at 30 and 90 DAF. There was a significant positive correlation at 60 and 120 DAF. There was a significant positive correlation between stone cell content and stone cell mass density at 90 DAF. There was a positive correlation at 30, 60, and 120 DAF, but it was not significant. There was a positive correlation between lignin content and stone cell mass density for each period.
There was a positive correlation between stone cell content and PAL activity during the different periods, but it was not significant. There was a significant positive correlation between POD activity and stone cell content at 60, 90, and 120 DAF, but there was no significant positive correlation between them at 30 DAF. There was no significant positive correlation between PPO activity and stone cell content at 30, 60, and 120 DAF, but there was a significant positive correlation at 90 DAF. At 30 DAF, the lignin content in the fruit correlated significantly and positively with the activity of the three enzymes. At 60, 90, and 120 DAF, the lignin content correlated significantly and positively with POD activity. Lignin content correlated significantly and positively with PPO and PAL activity at 60 DAF. At 90 DAF, the lignin content correlated significantly and positively with PPO activity, and correlated positively with PAL activity, but not significantly. At 120 DAF, the lignin content in the fruit did not correlate significantly and positively with PPO activity, but correlated significantly and positively with PAL activity.
The stone cell and lignin contents, and related enzyme activities from different pollination combinations were tested. Results showed that the development of stone cells and lignin differed to varying degrees of differences in different pollination combinations. This was consistent with the experimental results of Wang (2014), indicating that stone cells developed differently among pear varieties. Many studies have found that stone cell mass content first increases and then decreases with fruit growth and development, which is consistent with the changing trend of stone cell content in different pollination combinations during fruit growth and development (Tian et al., 2015). In addition, the results of the phloroglucinol–hydrochloric acid solution staining showed that the distribution of stone cell clusters was radially circular along the center of the fruit before extending outward gradually. The distribution content was near the center of the fruit, near the pericarp, and in the middle of the fruit, which is consistent with the experimental results of Huang et al. (2017). Tian et al. (2017) found that the stone cell content correlated positively with lignin content through an experimental study, and an experiment with Dangshansu pear as the test material also proved that lignin synthesis affected stone cell formation. Liu et al. (2013) also found a significant positive correlation between stone cells and lignin content in fruit. In addition, Yu et al. (2011) proved that POD is involved in the synthesis of lignin, with POD having a strong direct correlation with lignin content and stone cell content. Li et al. (2017) also found that stone cell content was correlated significantly and positively with POD4 gene expression. This experiment also showed that stone cell content, stone cell density, lignin content, and enzyme activity (PPO, POD, and PAL) correlated positively.
In this study, we found that the stone cell content of different pollination combinations increased with varying degrees from 30 to 60 DAF, and the stone cell content decreased rapidly after 60 d. The density of stone cell clusters also decreased with growth and development. The results of phloroglucinol–hydrochloric acid staining showed that the distribution of stone cells was radially circular along the center of the fruit, extending outward gradually. They were mainly distributed near the center of the fruit and near the pericarp in the middle of the fruit. During fruit growth and development, the changing trend of fruit lignin content was consistent with the changing trend of stone cell content, which showed a trend of first increasing and then decreasing, with different degrees of differences in the fruit lignin content in different pollination combinations during different periods. The trends for POD and PPO in fruit growth and development first increased and then decreased, with the trend for PAL decreasing continuously. At each growth stage, stone cell content correlated positively, to varying degrees, with lignin content, POD activity, PPO activity, and PAL activity.
Staining map of stone cell slices 30 d after flowering (DAF) (A), 60 DAF (B), 90 DAF (C), and 120 DAF (D).
Comparison of stone cell mass density of different pollination combinations of fruit at different stages. BY = ‘Bayue’ pear; HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear, YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05.
The contents of stone cells and lignin in fruit of different pollination combinations at different stages: 30 d after anthesis (A), 60 d after anthesis (B), 90 d after anthesis (C), and 120 d after anthesis (D). 1 = ‘Hechengdonghuang’ pear; 2 = ‘Kuerlehuangsuan’ pear; 3 = ‘Qipan’ pear; 4 = ‘Juju’ pear; 5 = ‘Bayue’ pear; 6 = ‘Yelikeamute’ pear; 7 = ‘Kuikeamute1’ pear. Lowercase letters indicate significant differences at P < 0.05.
Comparison of stone cells and lignin content in mature fruit with pollination combinations. BY = ‘Bayue’ pear; HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear; YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05.
Changes of polyphenol oxidase (PPO) and peroxidase (POD) enzyme activities in different pollination combinations at different stages. 1 = ‘Hechengdonghuang’ pear; 2 = ‘Kuerlehuangsuan’ pear; 3 = ‘Qipan’ pear; 4 = ‘Juju’ pear; 5 = ‘Bayue’ pear; 6 = ‘Yelikeamute’ pear; 7 = ‘Kuikeamute1’ pear. A = POD at 30 d; B = POD at 60 d; C = POD 90 at d; D = POD at 120 d; E = PPO at 30 d; F = PPO at 60 d; G = PPO at 90 d; H = PPO at 120 d. Lowercase letters indicate significant differences at P < 0.05; uppercase letters indicate significant differences at P < 0.01.
Changes of phenylalanine ammonia lyase (PAL) activity in different pollination combination. HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear; YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05; uppercase letters indicate significant differences at P < 0.01.
Contributor Notes
This study was supported by the National Natural Science Foundation of China (31860528 and U2003121) and the Bingtuan Science and Technology Program (2021CB055).
J.B. is the corresponding author. E-mail: baobao-xinjiang@126.com.
Staining map of stone cell slices 30 d after flowering (DAF) (A), 60 DAF (B), 90 DAF (C), and 120 DAF (D).
Comparison of stone cell mass density of different pollination combinations of fruit at different stages. BY = ‘Bayue’ pear; HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear, YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05.
The contents of stone cells and lignin in fruit of different pollination combinations at different stages: 30 d after anthesis (A), 60 d after anthesis (B), 90 d after anthesis (C), and 120 d after anthesis (D). 1 = ‘Hechengdonghuang’ pear; 2 = ‘Kuerlehuangsuan’ pear; 3 = ‘Qipan’ pear; 4 = ‘Juju’ pear; 5 = ‘Bayue’ pear; 6 = ‘Yelikeamute’ pear; 7 = ‘Kuikeamute1’ pear. Lowercase letters indicate significant differences at P < 0.05.
Comparison of stone cells and lignin content in mature fruit with pollination combinations. BY = ‘Bayue’ pear; HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear; YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05.
Changes of polyphenol oxidase (PPO) and peroxidase (POD) enzyme activities in different pollination combinations at different stages. 1 = ‘Hechengdonghuang’ pear; 2 = ‘Kuerlehuangsuan’ pear; 3 = ‘Qipan’ pear; 4 = ‘Juju’ pear; 5 = ‘Bayue’ pear; 6 = ‘Yelikeamute’ pear; 7 = ‘Kuikeamute1’ pear. A = POD at 30 d; B = POD at 60 d; C = POD 90 at d; D = POD at 120 d; E = PPO at 30 d; F = PPO at 60 d; G = PPO at 90 d; H = PPO at 120 d. Lowercase letters indicate significant differences at P < 0.05; uppercase letters indicate significant differences at P < 0.01.
Changes of phenylalanine ammonia lyase (PAL) activity in different pollination combination. HD = ‘Hechengdonghuang’ pear; JJ = ‘Juju’ pear; KH = ‘Kuerlehuangsuan’ pear; KK1 = ‘Kuikeamute1’ pear; QP = ‘Qipan’ pear; YL = ‘Yelikeamute’ pear. Lowercase letters indicate significant differences at P < 0.05; uppercase letters indicate significant differences at P < 0.01.
