Article Nitrendipine-Treatment Increases Cork Spot Disorder Incidence in ‘Akituki’ (Pyrus pyrifolia Nakai.) by Altering Calcium Distribution Inside the Fruit

Zhenhua Cui †, Nannan Wang †, Dingli Li, Ran Wang and Chunhui Ma *

College of Horticulture, Qingdao Agricultural University, Qingdao 266109, ; [email protected] (Z.C.); [email protected] (N.W.); [email protected] (D.L.); [email protected] (R.W.) * Correspondence: [email protected] † These authors contributed equally to this work.

Abstract: ‘Akituki’ (Pyrus pyrifolia Nakai.) is a very popular and profitable pear in China. However, its high susceptibility to cork spot disorder has limited its expansion of cultivated area. The mechanisms of cork spot disorder have been discussed extensively, focusing on Ca2+ deficiency, yet no consensus has been made. In this study, we applied nitrendipine (NI) as a Ca2+ uptake inhibitor to explore the role of calcium in cork spot disorder occurrence. Results showed that NI treatment on the fruit remarkably increased the incidence of cork spot disorder; alteration of contents happened at the early developmental stage of the fruit, especially on the outer flesh and the peel of the fruit; and this gap was filled gradually along with the expansion of the fruit. Significant differences in the expression levels of Ca2+ transport-related genes were found in the inner flesh, outer flesh   and peel during the fruit growth period. The observation of free Ca2+ localization indicated the intracellular imbalance of Ca2+ in the NI-treated fruit. In conclusion, NI treatment reduced the Citation: Cui, Z.; Wang, N.; Li, D.; calcium content in the fruit at an early developmental stage, altered the related expression of genes Wang, R.; Ma, C. Nitrendipine- and influenced the cellular Ca2+ balance in the fruit, which prompted the occurrence of cork spot Treatment Increases Cork Spot Disorder Incidence in Pear ‘Akituki’ disorder. Measures for the prevention and control of cork spot disorder should be taken at the early (Pyrus pyrifolia Nakai.) by Altering stage of the fruit development in the field. Calcium Distribution Inside the Fruit. Plants 2021, 10, 994. https://doi.org/ Keywords: Pyrus; cork spot disorder; calcium deficiency; nitrendipine 10.3390/plants10050994

Academic Editor: Vittorio Rossi 1. Introduction Received: 29 March 2021 Cork spot is a physiological disorder of fruit which occurs most in and apples. Accepted: 12 May 2021 The typical characteristics of this disorder in pears are brown desiccated flesh or grayish Published: 17 May 2021 corky lesions beneath the fruit skin [1], and a bumpy fruit surface in some [2]. This disorder in pears initiates in the early developmental stage of the fruit and continues to Publisher’s Note: MDPI stays neutral harvest time, depending on cultivar specificity [3]. ‘Akituki’ (Pyrus pyrifolia), bred in with regard to jurisdictional claims in in 1988, is a very popular cultivar in China for its large fruit size, pretty fruit shape and published maps and institutional affil- taste. However, ‘Akituki’ is very susceptible to cork spot disorder [1], which has caused iations. great economic loss in pear industry and has become a major obstacle to the continuing expansion of the cultivated area [1]. However, the mechanisms of cork spot disorder remain poorly understood; therefore, the management techniques to prevent this disorder are often inadequate. Copyright: © 2021 by the authors. Similar to bitter pits [4] of apples, and hard ends [5] and superficial scalding [6] of Licensee MDPI, Basel, Switzerland. pears, cork spot disorder was generally believed to be closely related to the content of This article is an open access article Ca [7–9]. Foliar spraying of exogenous Ca2+ solution three to five times during the growing distributed under the terms and period could effectively reduce the incidence of cork spot disorder in ‘Anjiu’ pears [10–12]. conditions of the Creative Commons Therefore, cork spot was believed to be a Ca2+-deficiency-related physiological disorder. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ However, a recent study found that spraying a calcium solution, a boron solution or a 4.0/). mixture solution of calcium and boron during the growing period could not reduce the

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Plants 2021, 10, 994 2 of 12 tion or a mixture solution of calcium and boron during the growing period could not reduce the incidence of cork spot-like physiological disorder in apples, and there was no correlation between the incidence of cork spot-like physiological disorder and Ca/B con- tent incidencein fruit or ofleaves cork [13] spot-like. Interestingly, physiological higher disorder levels of in total apples, calcium and there were was found no in correlation cork between the incidence of cork spot-like physiological disorder and Ca/B content in fruit spotted ‘Akituki’ [1] and ‘Chili’ [2] pear fruit than healthy fruit. These findings suggested or leaves [13]. Interestingly, higher levels of total calcium were found in cork spotted that calcium plays an important role in cork spot disorder, but its mechanism is unclear ‘Akituki’ [1] and ‘Chili’ [2] pear fruit than healthy fruit. These findings suggested that and needs further exploration. calcium plays an important role in cork spot disorder, but its mechanism is unclear and When investigating the effect of Ca2+ on cork spot disorder, a supplement of exoge- needs further exploration. nous calcium was applied in most experimental designs [14], but a deliberate calcium When investigating the effect of Ca2+ on cork spot disorder, a supplement of exogenous deficit was rarely induced to analyze the effect of calcium in cork spot development. calcium was applied in most experimental designs [14], but a deliberate calcium deficit was Nitrendipine (NI), as an inhibitor of the Ca2+ transport channel, is mainly used to treat rarely induced to analyze the effect of calcium in cork spot development. Nitrendipine (NI), heart disease [15]. In plants, NI can inhibit the movement of Ca2+ through the L-type Ca2+ as an inhibitor of the Ca2+ transport channel, is mainly used to treat heart disease [15]. In channel in lilies [14] and block the entry of outer Ca2+ into carrot callus cells [16]. It also plants, NI can inhibit the movement of Ca2+ through the L-type Ca2+ channel in lilies [14] has been shown to completely inhibit the entry of Ca2+ into peanut pod cells, and this in- and block the entry of outer Ca2+ into carrot callus cells [16]. It also has been shown to hibitioncompletely was reversible inhibit thewhen entry NI of treatment Ca2+ into was peanut removed pod cells, [14,17] and. Therefor this inhibition NI is wasa suitable reversible 2+ reagentwhen for NI study treatmenting the was impact removed of Ca [14 deficit,17]. Thereforon cork spot NI is development a suitable reagent by inhibiting for studying the the 2+ absorptionimpact of of Ca Ca2+ indeficit pear fruit. on cork spot development by inhibiting the absorption of Ca2+ in 2+ pearThe object fruit. ive of the present study was to investigate the impact of Ca deficit on the 2+ cork spotThe disorder objective occurrence. of the present NI was study applied was in to the investigate study to the inhibit impact the of Ca Ca 2+absorptiondeficit on the in pearcork fruit, spot followed disorder occurrence.by the measurement NI was applied of mineral in the content study to and inhibit the theexpression Ca2+ absorption levels in of Capear2+ transport fruit, followed-related by genes, the measurement and the localization of mineral of contentfree Ca2+ and. The the relationship expression levelsbetween ofCa 2+ Ca2+ transport-relateddeficiency and cork genes, spot and incidence the localization was also ofanalyzed. free Ca2+ Results. The relationship of the study between are ex- Ca2+ pecteddeficiency to provide and valuable cork spot clue incidences for the was exploration also analyzed. of the Results cork spot of the disorder study are’s mecha- expected to nismprovide and to be valuable useful cluesfor the for control the exploration of cork spot of the disorder cork spot in orchard disorder’ss. mechanism and to be useful for the control of cork spot disorder in orchards. 2. Results 2.1. The2. Results Effects of NI Treatment on Cork Spot Incidence and Fruit Quality 2.1. The Effects of NI Treatment on Cork Spot Incidence and Fruit Quality For a two-year consecutive investigation, the cork spot incidence of NI-treated fruit at harvestFor time a two-yearwas much consecutive higher (16%) investigation, than that of the the cork control spot fruit incidence (4%) (Figure of NI-treated 1). The fruit cork atspotted harvest fruit time had was slight much pitting higher on the (16%) surface than compared that of the with control the healthy fruit (4%) fruit (Figure (Fig- 1). ure 2Thea,b,e,f). cork For spotted the anatomical fruit had slightobservation, pitting the on cork the surfacespots in compared disordered with fruit the distributed healthy fruit close(Figure to the2 distala,b,e,f) end. For of thethe anatomicalfruit longitudinal observation,ly and theon the cork outer spots flesh in disordered position of fruit the dis- equatorialtributed cutting close tosurface, the distal respectively end of the ( fruitFigure longitudinally 2g,h). The inner and onflesh the and outer the flesh fruit position core of area therarely equatorial showed cutting cork spot surface, distribution respectively (Figure (Figure 2g,h).2g,h). For Thethe fruit inner treated flesh and with the NI fruit not core showareaing rarelycork spot showed symptoms, cork spot the distribution fruit size had (Figure no change2g,h). For compared the fruit with treated thewith control NI not fruit,showing including cork the spot single symptoms, fruit weight, the fruit vertical size had length no change and horizon comparedtal length with the (Table control 1). fruit, Similarincluding levels of the total single soluble fruit weight,solid, titratable vertical acidity length and horizontalfruit firmness length were (Table also1 found). Similar levels of total soluble solid, titratable acidity and fruit firmness were also found between between the NI-treated fruit and the control fruit (Table 1). the NI-treated fruit and the control fruit (Table1).

Figure 1. The comparison of cork spot incidence between NI-treated fruit and control fruit. Data were collected from 2019 to 2020 and are presented as means ± SE; ** means significant difference at p < 0.01 level.

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Plants 2021, 10, 994 Figure 1. The comparison of cork spot incidence between NI-treated fruit and control3 of fruit. 12 Data were collected from 2019 to 2020 and are presented as means ± SE; ** means significant difference at p < 0.01 level.

Figure 2. ObservationFigure 2. Observation of the cork of the spot cork spotsymptom symptom at atharvest harvest time time (127(127 DAFB). DAFB). (a– d(a)– ared) healthyare healthy fruit; ( efruit;–h) are (e cork–h) spottedare cork spotted fruit; blackfruit; arrows black indicate arrows indicate the cork the corkspotted spotted tissues. tissues.

TableTable 1. Fruit 1. Fruit quality quality analysis analysis ofof control control and and NI-treated NI-treated ‘Akituki’ ‘Aki fruit.tuki’ Both fruit. the control Both andthe NI-treated control and NI-treatedfruit selected fruit selected for analysis for were analysis healthy were fruit healthy without any fruit cork without spot. any cork spot. Total Fresh Vertical Horizontal Titratable Firmness TreatmentsFresh Weight Vertical Horizontal SolubleTotal Soluble Titratable Firmness Treatments Weight (g) Length (cm) Length (cm) Acidity (%) (N) (g) Length (cm) Length (cm)Solids Solids (%) (%) Acidity (%) (N) ControlControl 449.9449.9 ± ±28.728.7 a a 10.510.5 ±± 0.40.4 a a 11.211.2± 0.5 ± a0.5 a 12.8 ±12.80.8 a ± 0.8 0.3 a± 0.020.3 a ± 0.02 9.8 ± a0.9 a 9.8 ± 0.9 a NI-treated 465.0 ± 94.4 a 10.6 ± 0.6 a 11.6 ± 0.8 a 13.1 ± 0.4 a 0.2 ± 0.03 a 10.7 ± 1.3 a NI-treatedData are presented465.0 as± means94.4 a± SE.10.6 The ± same 0.6 lettera in11.6 the same± 0.8 column a means13.1 ± no 0.4 significant a 0.2 difference. ± 0.03 a 10.7 ± 1.3 a Data are presented as means ± SE. The same letter in the same column means no significant dif- ference.2.2. The Effect of NI Treatment on Mineral Elements Content According to the cork spot distribution pattern (Figure2), the fruit inner flesh, outer 2.2. Theflesh Effect and peelof NI were Treatment separately on analyzed Mineral for Elements their Ca, C Kontent and Mg contents during the fruit growth period. The Ca concentrations of both NI-treated and control fruit increased from According67 DAFB to 127 to the DAFB cork in all spot three distribution positions measured pattern in (Fig this studyure. 2), (Figure the fruit3a–c). inner For the flesh, outer flesh innerand peel flesh, were Ca concentration separately showed analyzed a similar for leveltheir for Ca, NI-treated K and Mg and controlcontent fruit,s during from the fruit growth67 toperiod. 127 DAFB, The with Ca concentration an exception, wheres of NI-treatedboth NI-treated fruit had and a higher control concentration fruit increased of from 67 DAFBCa than to 127 control DAFB fruit in at 107all three DAFB positions (Figure3a). measured The values in of Ca/(Kthis study + Mg) (Figur were greatere 3a,b,c). For the in NI-treated fruit than those of control fruit at both 107 and 127 DAFB (Figure3e). For inner flesh, Ca concentration showed a similar level for NI-treated and control fruit, from the outer flesh, NI treatment remarkably reduced the Ca concentration compared with 67 to control127 DAFB fruit, at with 67 DAFB an exception, (Figure3b). where The Ca NI concentration-treated fruit of control had a fruit higher declined concentration to of Ca thana similar control level fruit compared at 107 with DAFB NI-treated (Figure fruit, 3a). along The value with thes of fruit Ca/(K growth + Mg) from were 87 to greater in NI-treated127 DAFB fruit(Figure than3 b)th.ose The of values control of Ca/(K fruit + at Mg) both in control 107 and fruit 127 at both DAFB 67 and (Figure 87 DAFB 3e). For the outerwere flesh, significantly NI treatment higher remarkably than those of NI-treatedreduced the fruit Ca (Figure concentration3e). For the fruit compared peel, the with con- NI-treated and control fruit had similar dynamic patterns of Ca concentration (Figure3c). trol fruitThe controlat 67 DAFB fruit had (Figure higher concentrations 3b). The Ca ofconcentr Ca at bothation 67 and of 107control DAFB fruit compared declined with to a sim- ilar levelNI-treated compared fruit (Figure with3c), NI and-treated a significant fruit difference, along in with Ca/(K the + Mg) fruit was growth only found from at 87 to 127 DAFB67 ( DAFB,Figure with 3b) a. higherThe value values in of control Ca/(K fruit + thanMg) NI-treated in control fruit fruit (Figure at 3bothf). 67 and 87 DAFB were significantly higher than those of NI-treated fruit (Figure 3e). For the fruit peel, the NI-treated and control fruit had similar dynamic patterns of Ca concentration (Figure 3c). The control fruit had higher concentrations of Ca at both 67 and 107 DAFB compared with NI-treated fruit (Figure 3c), and a significant difference in Ca/(K + Mg) was only found at 67 DAFB, with a higher value in control fruit than NI-treated fruit (Figure 3f).

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Figure 3. Analysis of the mineral elements of NI-treated fruit and control fruit during the fruit growth period.Figure (a– c3). areAnalysis Ca content of the ofmineral different elements positions of NI as-treated indicated fruit in and Figure control2;( d fruit–f) are during ratios the of Cafruit to (K +growth Mg) content period. of (a different–c) are Ca positions content asof indicateddifferent positions in Figure 2as. Dataindicated are presented in Figure as2; ( meansd–f) are± ratioSE; s of Ca to (K + Mg) content of different positions as indicated in Figure 2. Data are presented as means ± * means significant difference between control fruit and NI-treated fruit at the corresponding time- SE; * means significant difference between control fruit and NI-treated fruit at the corresponding point at p < 0.05 level. timepoint at p < 0.05 level. 2.3. The Effect of NI-Treatment on the Expression of Genes Related to Ca2+ Transport 2.3. The Effect of NI-Treatment on the Expression of Genes Related to Ca2+ Transport To further investigate the effects of NI treatment on the Ca2+ transport and allocation in To further investigate the effects of NI treatment on the Ca2+ transport and allocation the fruit, Ca2+ transport-related genes such as Ca2+ sensors (PpCMLs), Ca2+/H+ exchangers in the fruit, Ca2+ transport-related genes such as Ca2+ sensors (PpCMLs), Ca2+/H+ ex- (PpCAX) and Ca2-ATPase (PpACA) were selected for the expression analysis. For the inner changers (PpCAX) and Ca2-ATPase (PpACA) were selected for the expression analysis. flesh, PpCML11 had higher expression levels in control fruit than in NI-treated fruit at 67 andFor 87the DAFB; inner however,flesh, PpCML11 its expression had higher increased expression remarkably levels in in NI-treated control fruit fruit than at in 107NI and-treated 127 DAFB fruit (Figure at 67 4 and). PpCML16 87 DAFBhad; however, a higher itsexpression expression level increased in NI-treated remarkably fruit at in 67 DAFB,NI-treated but declinedfruit at 107 quickly and 127 from DAFB 87 to (Figure 127 DAFB. 4). PpCML16PpCML23 hadhad a ahigher higher expression expression level levelin inNI control-treated fruit fruit at at 67 67 DAFB, DAFB and, but decreased declined quickly rapidly from 87 to 127 DAFB.DAFB. NI-treatedPpCML23 had fruita hadhigher higher expression expression level levels in control of PpCML23 fruit atat 10767 DAFB, and 127 and DAFB decreased compared rapidly with from control 87 to fruit.127PpCML25 DAFB. NIexpression-treated fruit stayed had at higher a higher expression level in control levels fruit of PpCML23 from 67to at 127 107 DAFB and 127 comparedDAFB compared with NI-treated with control fruit. PpCML29fruit. PpCML25had higher expression expression stayed levels at a higher in control level fruit in con- fromtrol 67 fruit to 107 from DAFB, 67 to and 127 its DAFB expression compared increased with rapidlyNI-treated in NI-treated fruit. PpCML29 fruit at had 127 higher DAFB. ex- PpCML41pressionhad levels higher in expressioncontrol fruit levels from in 67 NI-treated to 107 DAFB, fruit atand 67 its DAFB expression and in controlincreased fruit rapidly at 87 DAFB,in NI-treated and it showedfruit at 127 relatively DAFB. lowerPpCML41 expression had higher levels expression in both NI-treated levels in NI and-treated control fruit fruitat from67 DAFB 107 toand 127 in DAFB. controlPpCML45 fruit at 87had DAFB, higher and expression it showed levelsrelative inly control lower fruitexpression at both 87 and 107 DAFB compared with NI-treated fruit. PpCML47 had a drastically higher

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levels in both NI-treated and control fruit from 107 to 127 DAFB. PpCML45 had higher Plants 2021, 10, 994 expression levels in control fruit at both 87 and 107 DAFB compared with5 of NI 12 -treated fruit. PpCML47 had a drastically higher expression level at 87 DAFB in control fruit compared with NI-treated fruit. Both PpCML49 and PpACA4 showed higher expression levelsexpression at 87 and level 127 at 87 DAFB DAFB in controlcontrol fruit fruit compared compared with NI-treatedwith NI-treated fruit. Both fruitPpCML49. PpCAX4 had higherand PpACA4 expressionshowed levels higher in expression control fruit levels than at 87 and that 127 in DAFB NI-treated in control fruit fruit at compared 67, 87 and 127 with NI-treated fruit. PpCAX4 had higher expression levels in control fruit than that in DAFB (Figure 4). NI-treated fruit at 67, 87 and 127 DAFB (Figure4).

Figure 4. Expression analysis of Ca2+ transport-related genes of the inner flesh tissues during the fruit growth period. * and Figure **4. mean Expression significantly analysis different of Ca expression2+ transport levels-related within the genes group of at thep < inner 0.05 and fleshp < 0.01,tissues respectively. during the fruit growth period. * and ** mean significantly different expression levels within the group at p < 0.05 and p < 0.01, respectively. For the outer flesh, PpCML11 had a higher expression level at 67 DAFB and lower expression levels at 87 and 127 DAFB in NI-treated fruit compared with control fruit For the outer flesh, PpCML11 had a higher expression level at 67 DAFB and lower (Figure5). PpCML16 had a remarkably higher expression level in control fruit at 87 DAFB expressioncompared levels with NI-treated at 87 and fruit, 127 and DAFB NI-treated in NI fruit-treated showed fruit a higher compared expression with level control of fruit (FigurePpCML16 5). PpCML16at 107 DAFB had compared a remarkably with control higher fruit. expressionPpCML23 had level a higher in control expression fruit level at 87 DAFB comparedand a lower with expression NI-treated level fruit, at 67 and 87NI DAFB,-treated respectively, fruit showed in NI-treated a higher fruit expression compared level of PpCML16with control at 107 fruit. DAFB The onlycompared significant with difference control in fruitPpCML25. PpCML23expression had wasa higher found expression at level107 and DAFB a lower with a higherexpression level inlevel NI-treated at 67 and fruit. 87 Both DAFB, NI-treated respectively, and control in fruitNI-treated had fruit high expression levels of PpCML29 at 87 DAFB, and the control fruit had higher expression comparedlevels of withPpCML29 controlat both fruit 107. The and127 only DAFB significant compared difference with NI-treated in PpCML25 fruit. PpCML41 expression was foundhad at a far107 higher DAFB expression with a higher level at 87level DAFB in inNI control-treated fruit fruit compared. Both NI with-treated NI-treated and control fruitfruit, had and high very expression low levels oflevels its expression of PpCML29 were foundat 87 inDAFB, both controland the and control NI-treated fruit fruit had higher expressionat 67, 107 levels and 127 of DAFB.PpCML2 Even9 at though both higher107 and127 expression DAFB levels compared of PpCML45 withwere NI-treated only fruit. PpCML41observed had at 87 a DAFB far higher in both expression control and NI-treatedlevel at 87 fruit, DAFB a significant in control difference fruit wascompared only with NI-treated fruit, and very low levels of its expression were found in both control and NI-treated fruit at 67, 107 and 127 DAFB. Even though higher expression levels of PpCML45 were only observed at 87 DAFB in both control and NI-treated fruit, a signifi-

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cant difference was only found at 67 DAFB with a higher expression level in control fruit. PpCML47found at showed 67 DAFB a withhigher a higher expression expression level level in NI in-treated control fruit.fruit PpCML47at 87 DAFBshowed. PpCML49 a had a higherhigher expression expression level level in in NI-treated control fruitfruit at compared 87 DAFB. PpCML49 with NI-hadtreated a higher fruit expression at 127 DAFB. No significantlevel in control difference fruit compared of expression with NI-treated level for fruit PpACA4 at 127 DAFB. or PpCAX4 No significant was found difference between the controlof expression fruit and level the for NIPpACA4-treatedor fruitPpCAX4 duringwas the found fruit between growth the period control (Figure fruit and 5). the NI-treated fruit during the fruit growth period (Figure5).

Figure 5. Expression analysis of Ca2+ transport-related genes of the outer flesh tissues during the fruit growth period. * and Figure** 5. mean Expression significantly analysis different of expressionCa2+ transport levels-related within the genes group of at thep < 0.05outer and fleshp < 0.01,tissues respectively. during the fruit growth period. * and ** mean significantly different expression levels within the group at p < 0.05 and p < 0.01, respectively. For the fruit peel, PpCML11 had a lower expression level and higher expression level in NI-treated fruit at 67 and 127 DAFB, respectively, compared with the control fruit (Figure6 ). For the fruit peel, PpCML11 had a lower expression level and higher expression level PpCML16 showed higher expression levels in the control fruit at 67, 87 and 127 DAFB; in and NI-treated a higher fruit expression at 67level and was 127 found DAFB, at127 respectively, DAFB in NI-treated compared fruit. withPpCML23 the controlhad fruit (Figurehigher 6) expression. PpCML16 levels showed in control higher fruit expression at both 67 levels and 87 in DAFB. the control NI-treated fruit fruitat 67, had 87 and 127 DAFBa higher; and expression a higher level expression of PpCML23 levelat 127 was DAFB found than at control 127 DAFBfruit. PpCML25 in NI-treatedhad fruit. PpCML23higher expression had higher levels expressionin control fruit levels at 67, 87 in and control 127 DAFB fruit compared at both with 67 NI-treated and 87 DAFB. NI-fruit.treatedPpCML29 fruit hadhad a higher higher expression expression levels level in controlof PpCML23 fruit at at 67 127 and DAFB 107 DAFB, than and control it fruit. showed a higher expression level in NI-treated fruit at 127 DAFB. PpCML41 kept higher PpCML25 had higher expression levels in control fruit at 67, 87 and 127 DAFB compared expression levels in the control fruit during the whole fruit growth period compared with withthose NI- oftreated the NI-treated fruit. PpCML29 fruit. PpCML45 had highershowed expression a much higher levels expression in control level fruit in control at 67 and 107 DAFB,fruit atand 87 it DAFB showed compared a higher with expression NI-treat fruit. levelPpCML47 in NI-hadtreated higher fruit expression at 127 DAFB levels. in PpCML41 kept higher expression levels in the control fruit during the whole fruit growth period compared with those of the NI-treated fruit. PpCML45 showed a much higher expression level in control fruit at 87 DAFB compared with NI-treat fruit. PpCML47 had higher ex- pression levels in control fruit at 67, 87 and 127 DAFB compared with NI-treated fruit. PpCML49 had higher expression levels in control fruit at 67, 87 and 127 DAFB; and the NI-treated fruit showed a higher expression level of PpCML49 at 107 DAFB. PpACA4 had

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control fruit at 67, 87 and 127 DAFB compared with NI-treated fruit. PpCML49 had higher expression levels in control fruit at 67, 87 and 127 DAFB; and the NI-treated fruit showed a higher expression levels at both 67 and 127 DAFB compared with NI-treated fruit. The higher expression level of PpCML49 at 107 DAFB. PpACA4 had higher expression levels NI-treated fruit showed a higher expression level of PpCAX4 at 67 DAFB compared with at both 67 and 127 DAFB compared with NI-treated fruit. The NI-treated fruit showed a control fruit. The control fruit had higher expression levels of PpCAX4 at both 87 and 127 higher expression level of PpCAX4 at 67 DAFB compared with control fruit. The control fruitDAFB had (Figure higher 6). expression levels of PpCAX4 at both 87 and 127 DAFB (Figure6).

Figure 6. Expression analysis of Ca2+ transport-related genes of the fruit peel during the fruit growth period. * and ** mean 2+ significantlyFigure 6. Expression different expression analysis of levels Ca withintransport the-related group at genesp < 0.05 of the and fruitp < 0.01,peel respectively.during the fruit growth period. * and ** mean significantly different expression levels within the group at p < 0.05 and p < 0.01, respectively. 2.4. Comparison of Free Ca2+ Distribution 2.4. Comparison of Free Ca2+ Distribution Since the cork spot distributed mostly in the outer flesh of the fruit, the free Ca2+ of both intercellularSince the cork and spot intracellular distributed were mostly localized in the to outer analyze flesh the of Cathe2+ fruit,distribution the free inCa the2+ of outerboth fleshintercellular (Figure 7and). At intracellular 87 DAFB, bothwere thelocalized control to fruit analyze and the NI-treated Ca2+ distribution fruit showed in the aouter homogeneous flesh (Figure pattern 7). At of 87 Ca DAFB,2+ distribution, both the andcontrol the fruit Ca2+ andsignal NI- intensitytreated fruit was showed similar a betweenhomogeneous them. pattern At 107 DAFB,of Ca2+ thedistribution, Ca2+ signal and was the not Ca2+ as signal strong intensity as that atwas 87 similar DAFB inbe- controltween fruit,them. and At 107 the CaDAFB,2+ signal the Ca intensity2+ signal of was NI-treated not as strong fruit wasas that even at weaker87 DAFB compared in control withfruit, that and of the control Ca2+ fruit. signal An intensity uneven distributionof NI-treated of Cafruit2+ signalwas eve wasn weaker observed compared in NI-treated with fruitthat at of 107 control DAFB, fruit and. An a much uneven stronger distribution signal wasof Ca shown2+ signal at thewas margin observed of thein NI cells-treated and thefruit cytoplasm at 107 DAFB, had far and weaker a much Ca stronger2+ signals. signal At 127was DAFB, shown both at the the margin control of healthy the cells and and NI-treatedthe cytoplasm healthy had fruit far weaker had very Ca weak2+ signal Ca2+s. Atsignals 127 DAFB, compared both with the thecontrol fruit healthy at 87 and and 107NI DAFB.-treated Ca healthy2+ still fruit distributed had very homogeneously weak Ca2+ signal ins controlcompared fruit, with and the NI-treated fruit at 87 healthy and 107 fruitDAFB showed. Ca2+ still an uneven distributed distribution homogeneously of Ca2+ signal in control with fruit, stronger and intensityNI-treated of healthy Ca2+ at thefruit marginshowed of an the uneven cells. However, distribution for theof Ca NI-treated2+ signal with cork stronger spot disordered intensity tissue, of Ca much2+ at the stronger margin intensityof the cells of Ca. However,2+ was observed for the inNI the-treated whole cork cells spot (Figure disordered7). tissue, much stronger in- tensity of Ca2+ was observed in the whole cells (Figure 7).

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2+ Figure 7. Observation ofFigure free Ca 7.2+ Observationdistribution inof thefree outer Ca fleshdistribution tissues of in control the outer and NI-treatedflesh tissues fruit of bycontrol laser scanningand NI-treated confocal microscope. Greenfruit by fluorescence laser scanning in the confocal merge field microscope indicated. theGreen Ca2+ fluorescencedistribution. in White the merge arrows field indicate indicated the the 2+ 2+ intensified Ca2+ signal;Ca HF meansdistribution healthy. White fruit; DF arrows means indicate cork spot the disordered intensified fruit; Ca scale signal; bars = HF 100 meansµm. healthy fruit; DF means cork spot disordered fruit; scale bars = 100 μm. 3. Discussion 3. DiscussionIn the present study, we explored the relationship between calcium deficiency and corkIn the spot present disorder study, using we NI toexplored inhibitthe the absorption relationship of calcium between in thecalcium fruit ofdeficiency ‘Akituki,’ and 2+ corkwhich spot isdisorder an innovative using method,NI to inhibit as far asthe we absorption know, in the of Ca calciumdeficiency in the related fruit of disorder ‘Akituki,’ study. Separation of the different parts of the fruit for the analysis of mineral elements, Ca2+ which is an innovative method, as far as we know, in the Ca2+ deficiency related disorder transport-related genes expression and free Ca2+ distribution at different growth stages study.improved Separation the reliability of the different of the results parts compared of the fruit with for previous the analysis studies of focusing mineral onelements, the 2+ 2+ Ca whole transport fruit analysis.-related genes expression and free Ca distribution at different growth stages improvedIn our study, the reliability even though of the NI results treatment compared did not influencewith previous the fruit studies attributes, focusing it on the wholeapparently fruit increased analysis. the incidence of cork spot disorder in ‘Akituki’ pears by approximately 12%In our (Figure study,1), indicating even though that NINI altered treatment the Ca did distribution not influence inside the the fruit fruit, attributes, which was it ap- parentlyconfirmed increased by the observation the incidence of free of Ca cork2+ localization spot disorder (Figure in7 ). ‘Akituki’ The cork spot pear distributeds by approxi- matelyclose 12% to the (Figure calyx-end 1), and indicating outer flesh that of fruitNI a (Figureltered 2 the), which Ca distribution was consistent inside with other the fruit, studies of cork spot in pears [1,2] and bitter pits in apples [18]. This also suggests the which was confirmed by the observation of free Ca2+ localization (Figure 7). The cork spot abnormal distribution of calcium inside the fruit. NI-treated fruit had lower Ca content distributed close to the calyx-end and outer flesh of fruit (Figure 2), which was consistent only in the outer flesh and fruit peel, and the inner flesh had similar Ca content to the control withfruit other (Figure studies3). This of maycork bespot because in pear thes inner [1,2] flesh and hasbitter a stronger pits in vascular apples connection[18]. This withalso sug- gestthes the pedicel abnormal compared distribution with the outer of calcium flesh and inside the peel, the and fruit. Ca NI transport-treated was fruit secured had inlower the Ca contentinner only flesh in prior the toouter the otherflesh partsand fruit of the peel, fruit. and As athe result, inner the flesh ratio had of Ca/(K similar + Mg)Ca content had a to the highcontrol value fruit inthe (Figure inner fruit,3). This but itmay was be lower because than in the the inner outer fleshflesh and has the a peelstronger(Figure vascular3) . connectionPotassium with is known the pedicel to be involvedcompared in with cell-expansion-related the outer flesh and process the peel, [19,20 and], and Ca K +transportand 2+ 2+ wasMg securedare knownin the inner to compete flesh prior with Ca to thefor other biding parts sites of at the the fruit. plasma As membrane a result, the [21, 22ratio]. of Ca/(KThe + lowerMg) had Ca concentration a high value at in the the later inner stage fruit, in our but results it was may lower have beenthan duein the to the outer high flesh levels of K+ and Mg2+ during the rapid growth of fruit, which could have led to a reduction and the peel (Figure 3). Potassium is known to be involved in cell-expansion-related in fruit Ca2+ uptake, dilution of the Ca concentration and high susceptibility to cork spot + 2+ 2+ processdisorder [19,20] [23,], and and K also and filled Mg the are gaps known in mineral to compete element contentswith Ca between for biding control sites and at the plasmaNI-treated membrane fruit (Figure [21,22]3).. EvenThe lower though Ca the concentration cork spot occurred at the only later in the stage outer in flesh our and results maystarted have atbe arounden due 100 to DAFBthe high according levels toof our K+ fieldand investigation,Mg2+ during the the disorder rapid mustgrowth initiate of fruit, whichat an could earlier have stage, led where to a reduction the Ca concentration in fruit Ca and2+ uptake, the Ca/(K dilu +tion Mg) valuesof the demonstratedCa concentration andsignificant high susceptibility differences to between cork spot NI-treated disorder and [23] control, and fruit also (Figure filled3b,e). the gaps in mineral el- 2+ ement contentThe observations between of control free Ca anddistribution NI-treated showed fruit intracellular (Figure 3). imbalanceEven though in the the NI- cork spottreated occurred healthy only fruit in the at 107outer and flesh 127 DAFBand started (Figure at7 ),around indicating 100 thatDAFB the according intracellular to our imbalance of Ca2+ happened prior to the formation of cork spots, which was accordant field investigation, the disorder must initiate at an earlier stage, where the Ca concentra- with the analysis of mineral element content above (Figure3). Free Ca 2+ was found to tionbe and enriched the Ca/(K in the + Mg) cell values wall in demonstrated the cork spotted significant pear tissues, differences but the totalbetween Ca content NI-treated andwas control even fruit higher (Figure in the 3b,e). cork spotted tissues [1,2]. In other pear Ca2+ deficiency-related The observation of free Ca2+ distribution showed intracellular imbalance in the NI-treated healthy fruit at 107 and 127 DAFB (Figure 7), indicating that the intracellular imbalance of Ca2+ happened prior to the formation of cork spots, which was accordant with the analysis of mineral element content above (Figure 3). Free Ca2+ was found to be enriched in the cell wall in the cork spotted pear tissues, but the total Ca content was even higher in the cork spotted tissues [1,2]. In other pear Ca2+ deficiency-related disorders,

Plants 2021, 10, 994 9 of 12

disorders, such as superficial scald and hard end [24,25], the disordered fruits also had higher levels of free Ca2+ and Ca2+ mainly concentrated in the cell wall; and no relationship between Ca content and cork spot-like physiological disorder was found in apples [13]. Our result showed cork spotted tissues even had a stronger Ca2+ signal at 127 DAFB (Figure7), which was consistent with the results mentioned above. This could be the result of cell membrane damage and cytoplasmic leakage due to the replacing of Ca2+ by K+ and Mg2+ on the plasma membrane; however, the function of Ca2+ in stabilizing the membrane structure cannot be replaced [21,22]. Early and efficient Ca uptake was considered essential for maintaining normal Ca distribution in the distal part of the fruit and preventing the occurrence of Ca deficiency-related disorder in wax apples [26]. These findings suggested that the Ca deficiency at the early stage of fruit development could be a critical factor contributing to the cork spot development. Early Ca deficiency might influence the Ca delivery inside the fruit and the intracellular distribution in the outer flesh tissues. Therefore, the measures for the prevention and control of cork spot disorder, such as foliar spraying of a Ca2+ solution, should be taken as early as possible in the field. As well as the long-distance transport of Ca2+ via the apoplastic path, the trans- membrane transport of Ca2+ is essential for the intracellular balance of Ca2+, which was mediated by membrane transport , such as CML (probable calcium-binding pro- tein), ACA (auto-inhibited Ca2+-ATPase) and CAX (Ca2+ exchanger) [27]. In our study, NI treatment altered the expression levels of CMLs in different positions of the fruit during the fruit growth period (Figures4–6). In Arabidopsis thaliana, CML are mainly stress- responsive genes, which are up-regulated under different abiotic stresses (McCormack et al., 2015, Zhu et al., 2015). PpCML25 and PpCML47 were up-regulated in the outer flesh of NI-treated fruit at 107 and 87 DAFB, respectively, which were the critical timepoints for the onset of cork spot disorder. Higher expression levels of CMLs were also shown to associate with the occurrence of other Ca2+ deficiency-related disorders in pear fruit, such as superficial scald [25] and hard-end [24]. In apples, the expression levels of ACA and CAX increased as the severity of bitter pits increased [18,28]. MdCAX11 and MdCAX5 were also identified to bind to the tonoplast and transport Ca2+ from the cytoplasm to the vacuole, which increased the incidence of bitter pit [18]. Interestingly, our study found that NI-treated fruit have altered expression levels of both PpACA4 and PpCAX4 in the inner flesh and the peel. However, no significant alterations of PpACA4 and PpCAX4 expression levels were found in the outer flesh (Figure5), which is different from other studies. The different treatment in our study may have caused such results, and the differential gene expression in the inner flesh could influence the normal expression of the genes in the outer flesh. Taken together, NI treatment altered the expression levels of Ca2+ transport-related genes and further promoted the incidence of cork spot in ‘Akituki’ pear.

4. Materials and Methods 4.1. Plants Material and Nitrendipine Treatment Ten-year-old ‘Akituki’ trees grafted on the rootstock of , located at 36◦530 N and 118◦420 E in Weifang, Shandong Province, China, were used for the trial. Trees, spaced at 2 m within rows and 5 m between rows, were trained with a trellis cultivation system and were managed with full nutrition and water. NI (Yuanye Biology, Shanghai, China) was dissolved in lanolin at the concentration of 0.2 mg/mL and homogeneously smeared on the fruit pedicels at 47 days after full bloom (DAFB), and solvent lanolin was used as control to smear on the fruit pedicel. Twenty fruits were selected randomly on each tree for NI treatment and control, respectively, and five trees for each trial were used for the whole trial. After NI treatment on the fruit pedicels at 47 DAFB, fruit samples were taken every 20 days until harvest for different purposes of analysis as described below.

4.2. Investigation of Cork Spot Incidence Cork spot incidence of the fruits selected for the trial were investigated at harvest time (127 DAFB) from 2019 to 2020. The fruit without any cork spots on both equatorial and Plants 2021, 10, 994 10 of 12

longitudinal planes inside the fruit were considered as healthy fruit; otherwise any cork spot observed inside the fruit was considered as cork spot disorder.

4.3. Analysis of Fruit Quality The fruit quality was evaluated at harvest time by measuring the single fruit weight, vertical and horizontal length, total soluble solids, titratable acidity and fruit firmness. All the operations and calculation referred to Cui et al. [2].

4.4. Determination of Ca, K and Mg Contents Fruit of 67, 87, 107 and 127 DAFB were sampled for the Ca, K and Mg content analysis. For each sample, the tissues of fruit peel, outer flesh and inner flesh were separately measured for the Ca, K and Mg contents. Briefly, fruit tissues were dried in an oven at 105 ◦C for 30 min, and then at 75 ◦C until a constant weight was achieved; about 0.5 g of dried tissues were mixed with 2 mL perchloric acids and 10 mL nitric acids. After digestion at room temperature for 10 min, the levels of Ca, K, and Mg were analyzed using an IPC-OES (optima 8000, Perkin Elmer, Cambridge, MA, USA) according to Falchi et al. [28]. The concentrations of measured mineral elements were expressed by the relative content of dry weight. Three replicates were performed for each sample.

4.5. Analysis of Gene Expression Total RNA was extracted with RNA prep Pure Kit (Polysaccharides & Polyphenolics- rich, Tiangen, China) according to the manufacturer’s instructions. RNA quality was checked by both optical density (OD) value (1.9 to 2.1) at 260/280 nm (NanoDrop 2000C, Thermo Scientific, Waltham, MA, USA) and electrophoresis in a 1.5% agarose gel with sharp, clear 28s and 18s rRNA bands stained by ethidium bromide according to [29]. cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s instructions, followed by a five-time dilution to be used as the qPCR template. qPCR was performed in a 20 µL reaction system including 1 µL of cDNA, 0.8 µL of each primer, 10 µL of 2 × chamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) and 7.4 µL of water, which was conducted on a Light Cycler® 480 instrument (Roche, Basel, Switzerland). Gene-specific primers were designed (Table S1) and an actin gene was used as the reference gene for the normalization of the transcript level. Two controls of no-RT and no-template were included. Transcript levels were determined using the 2−∆∆Ct method [30].

4.6. Localization of Free Ca2+ The localization of free Ca2+ was analyzed by fluorescence imaging as previously described by Qiu et al. [31] with some modifications. Briefly, thin slices of flesh were collected from the outer flesh tissues of both NI-treated and control fruit using a razor blade. The flesh tissues were initially washed twice on a glass slide with PBS (pH = 7.4) buffer solution, which were loaded with Fluo-4/AM at 37 ◦C for 1 h and then subsequently washed three times with PBS buffer solution. The Fluo-4 fluorescence maintained on the tissue was visualized under 494 nm excitation wavelength of laser light and 516 nm long- pass emission filter using a laser scanning confocal microscope (TCSSP5II, Leica, Wetzlar, Germany). Ten replicates were performed for each sample.

4.7. Statistical Analysis The statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). Significant difference was determined by Duncan’s multiple range test, and significance was tested at p < 0.05 or p < 0.01 level.

5. Conclusions NI treatment promoted the development of cork spot in ‘Akituki’ pear fruit by altering the expression levels of Ca2+ transport-related genes; the Ca contents in the outer flesh Plants 2021, 10, x 11 of 12

5. Conclusions NI treatment promoted the development of cork spot in ‘Akituki’ pear fruit by al- 2+ Plants 2021, 10, 994 tering the expression levels of Ca transport-related genes; the Ca contents11 in of the 12 outer flesh and peel, especially at early stage (67 DAFB); and finally, the intracellular distribu- tion of Ca2+ in certain tissues, as summarized in Figure 8. As a physiological disorder, cork andspot peel, is affected especially by at genotypes, early stage (67environmental DAFB); and finally, factors the and intracellular management distribution strategies. In areasof with Ca2+ highin certain incidence tissues,s asof summarizedcork spot, cultivars in Figure8 .with As a lower physiological susceptibility disorder, to cork cork spot shouldspot be is utilize affectedd by. D genotypes,rought stress environmental affects the factors soil structure, and management soil pH, strategies. organic Inmatter areas and so on, whichwith high all incidences influence of corkthe Caspot,2+ cultivarsuptake with of the lower root; susceptibility therefore, to appropriate cork spot should irrigation shouldbe utilized.be fulfilled Drought in the stress areas affects with the hot soil summer structure, and soil less pH, rainfall organic. M mattereasures and such so on, as foliar 2+ sprayingwhich of all a influence Ca2+ solution the Ca touptake prevent of the and root; control therefore, the appropriateoccurrence irrigation of cork should spot should be be fulfilled in the areas with hot summer and less rainfall. Measures such as foliar spraying of conducteda Ca2+ solutionas early to as prevent possible and. control Enough the occurrenceCa up-take of corkat an spot early should stage be conductedof the fruit as devel- opmentearly is as essential possible. Enoughto secure Ca a up-take normal at anCa early concentration stage of the fruitand developmentdistribution is and essential reduce the cork tospot secure incidence. a normal CaIn concentrationa future study and on distribution cork spot and disorder, reduce the advanced cork spot incidence.technologies In such as multia future-omics study analysis on cork spotshould disorder, be applied advanced to technologiesexplore the such mechanism as multi-omics of cork analysis spot occur- renceshould at the be molecular applied to explorelevel and the help mechanism propose of cork more spot effective occurrence control at the molecularstrategies. level and help propose more effective control strategies.

Figure 8. A hypothetical summarization of the relationship between Ca2+ deficiency and cork spot occurrence. NI treatment Figure 8.caused A hypothetical the Ca deficiency summarization in the outer fleshof the and relationship fruit peel at anbetween early stage Ca of2+ thedeficiency fruit development and cork (67 spot DAFB). occurrence. Expression NI treat- ment causedlevels the of CaCa2+ dtransport-relatedeficiency in the genesouter then flesh were and altered, fruit peel which at an further early promoted stage of thethe intracellular fruit development imbalance (67 of DAFB) Ca2+ . Ex- pressiondistribution levels of Ca and2+ promptedtransport the-related cork spot genes development. then were DF altered, means corkwhich spot further disordered promoted fruit; HF the means intracellular healthy fruit. imbalance of Ca2+ distribution and prompted the cork spot development. DF means cork spot disordered fruit; HF means healthy fruit. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants10050994/s1. Table S1: Genes selected for expression analysis and primers used for Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Table S1: RT-qPCR. Genes selected for expression analysis and primers used for RT-qPCR. Author Contributions: Z.C. took part in the data analysis and prepared the manuscript; N.W. took Authorpart Contribution in the field investigations: Z.C. took and part data in analysis; the data D.L. analysis provided and important prepared guidance; the manuscript; R.W. reviewed N.W. took part inand the modified field investigation the manuscript; and C.M. data conceived analysis; and D designed.L. provided the experiment, important and guid providedance; fundingR.W. reviewed and modifiedsupport. All the authors manuscript; have read C. andM. conceived agreed to the and published designed version the ofexperi the manuscript.ment, and provided funding support.Funding: All authorsThis study have was read financially and agreed supported to the bypublished the Agriculture version Science of the andmanuscript. Technology of Shandong Province (2019YQ015), China Agricultural Research System (CARS-29-07), Breeding Plan Funding: This study was financially supported by the Agriculture Science and Technology of of Shandong Provincial Qingchuang Research Team (2019) and Foundation of Qingdao Agricultural ShandongUniversity Province PHD. (2019YQ015), China Agricultural Research System (CARS-29-07), Breeding Plan of Shandong Provincial Qingchuang Research Team (2019) and Foundation of Qingdao Ag- riculturalInstitutional University Review PHD Board. Statement: Not applicable. Informed Consent Statement: Not applicable. Institutional Review Board Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the article or Supple- Informedmentary Consent Material. Statement: Not applicable. Data ConflictsAvailability of Interest: Statement:The authors The declare data presented no conflict of in interest. this study The funders are available had no role in inthe the article design or Sup- plementaryof the study; Material in the. collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the man- uscript, or in the decision to publish the results.

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