Pineapple (Ananascomosus cv Tainong 17) fruits were cut longitudinally. Pulps of three translucent fruits were mixed as a sample. Normal fruits were treated as the same method. The samples were frozen in liquid nitrogen and stored in −80˚C refrigerator.

Protein extraction, trypsin digestion, tandem mass tags (TMT) labelling, HPLC fractionation and LC-MS/MS were performed according to the method published [9]. MS/MS data were processed using Maxquant search engine (v1.6.6.0). Tandem mass spectra were searched against Ananas_comosus_4615_PR_20200330 (23,408 sequences) concatenated with reverse decoy database. Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Functional descriptions of protein domains were annotated by InterProScan (http://www.ebi.ac.uk/interpro/). Functional enrichment and clustering were performed according to the paper published [9]. Cluster membership were visualized using “heatmap.2” function (https://www.rdocumentation.org/packages/gplots/versions/3.0.1).

Two grams of frozen pulp were ground in liquid nitrogen. Eighteen microliters of 90% ethanol were added and homogenated. The mixture was stored in 4˚C for 24 hours. The supernatant was concentrated until it was ropy. Pure water was added to the total volume was 10 ml. One milliter of solution was filtered using filter membrane (0.45 µm). The concentrations of sugar, glucose, fructose in the solution were analyzed using High Performance Liquid Chromatography (Pump: Hitachi pump L-2130, Detector: Hitachi RI Detector L-2490, Column: Water Sugar-Pak I). The concentrations of carbohydrates apoplastic were analyzed using the method published [10].

Fresh pulp was cut into cubes of which side length was 1 cm. The cubes were washed with pure water to remove outworn tissues and cells. Ten cubes were put into a 50-ml tube. Pure water was added into the tube until the total volume was 20 ml. The liquid was transferred into another tube and the liquid volume was measured. The volume measured was marked as A. Sample volume was the differenve value between 20 ml and A. It was marked as B. Sample was centrifugated for 30 minutes at 300 g, 4˚C. Supernatant volume was measured and marked as C. The value of C/B was the volume of apoplastic liquid.

Electrolyte leakage and CWI activity of pineapple fruit flesh were determined according to the paper published [1]. Ethylene content in pineapple flesh was measured according to the paper published [11]. Ethanol content in fruit pulps were measured according to the paper published [12]. NMR was performed using Bruker Avance DPX 300 instrument (operating at 300 MHz for 1H).

Pulps were put into liquid nitrogen and transferred into envelope. The envelopes uncorked were put into freeze drier. After water was removed from pulps completely, pulps were ground into powder using liquid nitrogen. One gram of powder was put into pot and put in oven in which temperature was 200˚C for 1 hour. And then, they were transferred into 550˚C for 2 hours. The samples were put in temperature for 1 hour. Five milliter 2 M hydrochloric acid was mixed with the sample and shaken for 5 minutes. Twenty milliter deionized water was added and filtered with Whatman 42# paper. Deionized water was added into the filtrate until the total volume was 25 milliter. One milliter 5% lanthanum chloride and 3.9 ml deionized water was mixed with 0.1 ml filtrate. Calcium in the solution was measured using Atomic-Absorption Spectroscopy. Calcium content = Conc measured × dilution factor × 25 ml/dry matter weight.

Normal pineapple flesh appeared gold and compact (Figure 1). There were much liquid in intercellular space of translucent flesh, which showed water-soaked (Figure 1). The contents of sucrose, glucose, fructose and total sugar in translucent flesh were similar with those corresponding values in normal flesh (Figure 2). However, the apoplastic contents of these sugars in translucent flesh were significantly higher than those in normal flesh (Figure 3), demonstrating that more photosynthesis products accumulated in intercellular space of translucent flesh than those in normal flesh. The accumulation of sugars in apoplast of fruit can lead to pineapple translucency.

The activity of CWI in translucent flesh was 15.03 ± 2.47 µmole hexose/h/gFW, while the corresponding value in normal flesh was only 9.56 ± 2.61

µmole hexose/h/gFW. CWI can transform sucrose into glucose and fructose [13]. The CWI activity in translucent flesh was higher than that in normal flesh, indicating that more sucrose was transformed into glucose and fructose in translucent fruit than that was transformed in normal fruit.

Pulps of translucent fruits and normal fruits were collected for proteome analysis respectively. Five thousands and eighty proteins were identified and 3956 proteins were quantified. The expressions of the proteins identified were determined (Figure 4). There were 205 proteins of which the expressions in translucent flesh were higher than those in normal flesh. The top 10 proteins of which the expressions in translucent flesh versus those in normal fruit (W/C) were listed in Table 1. EF-hand domain-containing protein can bind with calcium ions [14]. 1-aminocyclopropane-1-carboxylate oxidase (ACO) was an essential enzyme for synthesizing ethylene. The W/C values of EF-hand domain-containing protein and ACO was the highest and the second highest respectively (Table 1), suggesting that pineapple translucency had close relationship with calcium and ethylene in fruit.

Universal Stress Protein (USP) regulates H₂O₂ level in intercellular space under hypoxic condition and transduces the oxygen-deficient signal into the downstream defense signaling pathway [15]. Phosphofructokinase is an important regulatory enzyme in glycolytic pathway [16]. Pyruvate decarboxylase (AcPVDC) and alcohol dehydrogenase (AcADH) were important enzymes in anaerobic respiration for synthesizing ethanol [17]. These proteins accumulated in translucent fruit, indicating that anaerobic respiration is the main form of respiration in translucent fruit. Translucent fruit might contain less ATP than

normal fruit.

For studying the functions of the proteins expressing significantly differentially between translucent flesh and normal flesh, proteins were enriched according to domain classification. Results showed that most of the proteins enriched had Cystathionine b-synthase (CBS) domain, serine carboxypeptidase, universal stress protein family, PBI domain, phosphofructokinase, oxidoreductase NAD-binding domain, and rhodanese-like domain (Figure 5). CBS proteins can regulate H₂O₂ content in cell [18]. H₂O₂ can shear polysaccharides and liposome [19]. Cell wall and membranes can be degraded because of these [20]. Proteins containing CBS domains accumulated in translucent flesh, demonstrating that many cells in translucent flesh have been cracked.

ACO was an important enzyme for synthesizing ethylene in plant [21]. Translucent flesh contained much more ACO than normal flesh, suggesting that pineapple translucence had some relationship with ethylene. Ethylene in pineapple flesh was measured in this study. Results showed that ethylene released from translucent flesh was 0.47 ± 0.02 µl/kg/h, while that corresponding value from normal flesh was only 0.18 ± 0.02 µl/kg/h. Translucent fruit contained more ethylene than normal fruit. The expressions of respiratory burst oxidase can be induced by ethylene [22]. Oxygen moleculars can be trsnformed into reactive oxygen species (ROS) through respiratory burst oxidase [23]. These ROS can attack membranes or cell wall, leading to more pores and larger pores in membranes and cell wall [19]. Electrolyte leakage of translucent flesh will be enhanced. More liquid will flow off from sink cells and accumulate in intercellular space of flesh. The synthesis of ethylene can be induced by cold [14]. This can

explain that fruits on which temperature was lower than 15˚C 3 months before harvest had higher incidence of translucence.

The expressions of AcPVDC and AcADH in translucent flesh were more than those in normal fruit, suggesting that translucent fruit synthesized more ethanol. Results showed that ethanol content in translucent fruit with just two eyes yellow was 13.25 ± 0.12 g/L. No ethanol was found in normal fruit with only two eyes yellow. Ethanol content in normal fruits with all eyes yellow was 11.73 ± 0.07 g/L, while the value in translucent fruit with all eyes yellow was 15.37 ± 0.16 g/L. Translucent fruit synthesized more ethanol than normal fruit. Some translucent fruit with heavy symptom have alcohol smell, which might be due to that the expressions of AcPVDA and AcADH were enhanced and more alcohol was synthesized.

The W/C value of EF-hand domain-containing protein was the highest among the proteins expressing differentially between translucent and normal fruit, suggesting that pineapple translucence had relationship with calcium content. In this study, it was found that calcium content in translucent flesh was 0.0735% ± 0.0152%. The corresponding value in normal flesh was 0.1533% ± 0.0326%. Calcium in translucent flesh was lower than that in normal flesh. EF-hand domain-containing protein can bind with calcium ions [24]. If calcium was deficient, pectin molecules cannot be linked [25]. Cell wall tissues cannot be formed [26]. There will be more pores in cell wall of fruit. The diameters of pores in cell wall of flesh will be larger. More liquid will flow out from sink cells of flesh. Pulp cell wall will be degraded [25].

If cell wall and membrane were attacked by H₂O₂, or pectin molecules had not been linked, more pores will form in cell wall and cell membrane. More liquid will percolate from sink cells through these pores. For identifying this speculation, electrolyte leakage and liquid content in intercellular space were measured. Results showed that electrolyte leakage of normal flesh was 65.8% ± 3.1%, while that value of translucent flesh was 75.4% ± 2.7%. The apoplastic liquid content in normal fruit was 170.1 ± 11.2 µl/cm³. The corresponding value of translucent fruit was 195.1 ± 10.5 µl/cm³ (Table 2). Translucent fruit had higher electrolyte leakage and accumulated more liquid in intercellular space than normal fruit. Cells in translucent flesh did have more pores and the pores were larger than those in normal flesh. Translucent flesh was saggy, while normal flesh was compact. That also demonstrates that cell wall and membranes in translucent fruit had been attacked and degraded.

Integrating all of the information above, a possible mechanism leading to pineapple translucence was proposed. When much sugar was transferred into fruit pulp and accumulated in intercellular space, water will be absorbed from cells around and translucence formed. If leaves were removed before harvest, less sugar will be synthesized and transferred into fruit. Translucence incidence will be decreased.

When pineapple fruits developed and chilling wave passed, the synthesis of ethylene was induced, resulting in that the expressions of respiratory burst oxidase were enhanced. Oxygen moleculars were transformed into reactive oxygen ROS, which attacks membranes or cell wall, leading to more and larger pores in membranes and cell wall. If less calcium ions were transported into sink cells of fruit, pectin molecules will not be formed. More pores and larger pores will also form in cell wall.