Identification and Characterization of Phospholipase D Genes Putatively Involved in Internal Browning of Pineapple during Postharvest Storage

Phospholipase D (PLD) in plants plays vital roles in growth, development, and stress responses. However, the precise role of PLDs in pineapple remains poorly understood. In this study, 10 putative PLD genes, designated as AcPLD1–AcPLD10, were identified based on the pineapple genome database. The 10 AcPLDs could be clustered into five of the six known PLD families according to sequence characterization. Their deduced amino acid sequences displayed similarities to PLDs from other plant species. Expression analyses of PLD mRNAs from pineapple pulp were performed. The 10 PLDs exhibited differential expression patterns during storage periods of fruits treated with hexaldehyde (a specific PLD inhibitor) which could alleviate internal browning (IB) of pineapple after harvest. Functional subcellular localization signaling assays of two PLD proteins (AcPLD2 and AcPLD9) were performed by fluorescence microscopy. To further detect the potential action mechanism underlying PLD involved in the IB defense response, PLD, hydrogen peroxide (H2O2) and H2O2 associated with antioxidative enzymes such as superoxide dismutase, catalase, NADPH, and ascorbate peroxidase were quantified by enzyme-linked immunosorbent assay. This report is the first to provide a genome-wide description of the pineapple PLD gene family, and the results should expand knowledge of this family.

An increasing number of studies have indicated that PLD, which is considered an important signaling enzyme in various organisms and is widespread in plants (Jang et al., 2012), participates in diverse physiological processes, such as development (Lu et al., 2016); hormone signaling mediated by abscisic acid (Peters et al., 2010), salicylic acid (Janda et al., 2015), jasmonic acid (Zhao et al., 2013), and auxin ; reactive oxygen generation (Wu et al., 2010); and in the response to abiotic and biotic stresses such as drought (Lu et al., 2012), high salinity (Yu et al., 2010), low temperature (Kargiotidou et al., 2010), and fungal infection (Liu et al., 2013). The diverse cellular functions of PLD suggest that the enzyme is subjected to complex regulation in the cell.
Although the functions of PLD in many plants have been studied extensively, the specific role of different plant PLDs remains unknown. Moreover, the involvement of PLDs in relation to quality of fruits after harvest has received minimal attention. Pineapple (Ananas comosus L.), which is the second most important harvest crop after banana and tasty tropical fruit, is cultivated in over 80 countries worldwide and produced annually with a gross production value approaching nine billion dollars (FAO, 2014). China produced approximately 1.89 million tons of pineapple in 2014, accounting for 7.4% of worldwide production (FAO, 2014). Under natural conditions, pineapples undergo internal browning (IB) during postharvest storage (Zhang et al., 2015). Therefore, the quality and storage potential of this fruit must be improved. Effective methods to delay the development of IB include 1-methylcyclopropene treatment (Selvarajah et al., 2001), controlled atmosphere (Nimitkeatkai et al., 2006), waxing (Hu et al., 2012), and exogenous abscisic acid (Zhang et al., 2015), but the mechanism of the IB remains unclear. The recent completion of the pineapple genome has allowed for the genome-wide identification of a number of important gene families (Ming et al., 2015). Three different PLD inhibitors, namely, 1-butanol (Pinosa et al., 2013), lysophosphatidyl ethanolamine (Li et al., 2015), and hexaldehyde (Paliyath et al., 2003), can all delay the development of IB in pineapple after harvest. Thus, PLDs may be the involved in IB. However, the functions of PLDs in pineapple need to be elucidated. This study was conducted to address the detailed structural analyses of PLDs in pineapple, as well as determine their phylogenetic relationship with other species. The potential regulatory mechanisms of PLD involvement during IB in pineapple are also discussed.

Plant Materials and Treatments
Pineapple (A. comosus cv. 'Comte de Paris') at stage 3 of maturation as proposed by Selvarajah et al. (2001) was obtained from a commercial orchard in Xuwen County, Guangdong Province in winter. Fruits of uniform size, color, and maturity, as well as being free of physical injury or disease symptoms, were selected, with the crown intact. Subsequently, fruits were immersed for 2 min in 0.05% (w/v) benomyl aqueous solution to control decay, air-dried at 25 • C for 2 h and treated as follows. The selected pineapple fruits were immersed into 0.1% (v/v) hexaldehyde containing 0.01% Tween 80 for 5 min. The control fruits were immersed with distilled water, which also contained 0.01% Tween 80. Each treatment was applied to three replications, and each replication contained 100 fruits. Following treatment, all fruits were placed into unsealed plastic bags (0.04 mm thick), and stored at 25 • C in the dark with relative humidity at 95% for 12 days. Samples of pulp tissues were collected at days 0, 2, 4, 6, 8, 10, and 12 frozen in liquid nitrogen; and stored at −80 • C for future use.

Evaluation of IB Intensity
For IB assessment, at least six fruits for each treatment were halved longitudinally to observe the incidence and severity of IB injury. IB intensity was visually scored from 0 (no IB) to 5 (maximum IB) according to Teisson (1979).

Identification of PLD and Sequence Analysis
A putative pineapple PLD cDNA was identified by searching the pineapple genome database. The homology of predicted PLD protein sequences was checked using Clustal W, with the default parameters set by the DNA Data Bank of Japan 1 . Domain and motif searches were conducted on the protein sequences in SMART 2 . Blast searches were also performed against nucleic acid sequence data repositories at the National Center for Biotechnology Information (NCBI 3 ). A phylogenetic tree was generated using Clustal Omega online tools 4 . The logos of the conserved domains were constructed using MEME program 5 with the default parameters. The theoretical isoelectric point (pI) and molecular weight of the pineapple PLD proteins were calculated using PeptideMass program 6 . Transmembrane helix number was predicted by HMMTOP online tools 7 .

Quantitative Real-Time PCR (qRT-PCR) Analysis
Total RNA was extracted from pineapple pulp using the hot borate method of Wan and Wilkin (1994). RNA purification and first-strand cDNA synthesis were performed as described in our previous paper . qRT-PCR analyses were conducted to detect gene expression patterns of the PLD family during the development of IB in fruits treated with PLD inhibitor (hexaldehyde). The oligonucleotide primers for qRT-PCR analysis were designed using Primer 5.0 software and Acactin (accession number HQ148720) was used as a reference gene. Reactions were carried out using SYBR R premix Ex Taq TM II (Takara, Shiga, Japan) on a LightCycler R 480 II (Roche, Switzerland). The relative expression levels of AcPLDs were calculated using the method of Livak and Schmittgen (2001). Values represented the average of three biological replicate. The sequences of all primers are listed in Supplemental File 1.

Subcellular Localization Analysis
The coding sequences of AcPLD2 and AcPLD9 without the stop codon were amplified by PCR (primers are listed in Supplemental File 2) and subcloned into the pEAQ-GFP-HT vector to form fusion constructs AcPLD2::GFP and AcPLD9::GFP, under the control of the CaMV 35S promoter. The fusion constructs and a control vector were maintained in Agrobacterium tumefaciens strain EHA105 by electroporation. Transient transformation of tobacco leaves was carried out according to the method described by Sainsbury et al. (2009). Localizations of AcPLD2::GFP and AcPLD9::GFP were investigated with a fluorescence microscope (Zeiss Axioskop 2 Plus). All transient expression assays were repeated three times.

Measurements of Superoxide Dismutase (SOD), Catalase (CAT), NADPH, Ascorbate Peroxidase (APX), PLD, and Hydrogen Peroxide (H 2 O 2 ) Contents
The pineapple pulp samples (1.0 g) were homogenized in 9.0 ml of phosphate buffered saline (PBS) (pH 7.4) and then centrifuged at 4000 × g for 15 min. The supernatant solution was collected for analyses of SOD, CAT, NADPH, APX, PLD, and H 2 O 2 contents, which were quantified by enzyme-linked immunosorbent assay (ELISA). ELISA was carried out as follows: (1) The 96-well microplates were coated with 100 µL of the captured specific antibody at 1:2000 dilution in coating buffer and incubated overnight at 4 • C.
(2) Plates were washed three times with PBS to remove unbound antibody, and each well was filled with 300 µL of blocking buffer and incubated for 30 min at 37 • C. (3) Followed by washing, 100 µL sample solution mentioned above was added to each well and incubated for 30 min at 37 • C. (4) After washing three times, 100 µL horseradish peroxidase-conjugated the specific antibody was added to each well and incubated at 37 • C for 30 min. (5) The plate was washed again, and 100 µL of solution 3,3/5,5/-tetramethylbenzidine was added to each well. (6) After developing the color for 30 min at 37 • C, the reaction was terminated after the addition of 50 µL of 2 M H 2 SO 4 per well. (7) The absorbance values at 450 nm were determined, and the values of ELISA were given in relative units (ng.mg −1 protein). Protein content was measured by the method of Bradford (1976) using bovine serum albumin as the standard protein.

Statistical Analysis
The experiment was performed in triplicate (n = 3). Data were presented as the mean ± standard error. Least significant difference at the 5% level was analyzed by SPSS software (version 13.0, SPSS Inc., Chicago, IL, United States).

IB Intensity
Control fruits showed signs of IB after 6 days of storage, whereas no sign of IB was detected in fruits treated with hexaldehyde. When the treated fruits began to exhibit the IB at day 8 of storage, the IB intensity reached 1.7 in control samples. IB progressively increased as storage duration increased. The IB intensity was higher for control samples than for treated fruits, and a noticeable difference was observed between them after 6 days of storage (Figure 1).

Identification of PLD Gene Family Members in Pineapple
Ten putative PLD genes were identified in pineapples by searching against the pineapple genome database. These genes were located on chromosomes 1, 2, 3, 5, 7, 8, 11, 18, 19, and 25, and their temporary names were designated as FIGURE 1 | Changes in IB intensity of fruits treated with hexaldehyde. Each value represents the mean ± standard error of three replicates. AcPLD1-AcPLD10 based on their order on the pineapple chromosome from 1 to 25 (Table 1). AcPLD1-AcPLD10 were predicted to encode proteins of 852,813,905,814,977,1032,850,1126,516, and 874 amino acids, respectively, with calculated molecular weights of 96.6, 91.2, 101.7, 92.4, 111.0, 114.5, 96.3, 127.8, 57.3, and 98.7 kDa, respectively, and pI values of 8. 00, 5.88, 6.57, 5.80, 6.73, 6.91, 6.78, 6.50, 7.15, and 6.79, respectively. Alignment of the full-length deduced proteins of the AcPLDs clearly showed that the proteins contained two conserved HKD domains (HKD1 and HKD2) in their sequences. HKD1 regions displayed more diversity than HKD2 regions (Figures 2, 3). The two HKD domains were separated by 320 amino acids, except for PLD3, PLD5, PLD8, and PLD9, which demonstrated longer or shorter spacing sequences. The "IYIENQYF" motif, which was found among the 10 pineapple PLDs, with the exception of PLD9, was the most conserved domain with the least diversity. The C2 domain was present in pineapple PLDs, except PLD8 and PLD9. The PH domain was present in pineapple PLD8 but not in other pineapple PLDs (Figures 2, 3 and Tables 1, 2). Alignment analysis indicated that the similarities of the deduced amino acids of AcPLDs varied from 10% (AcPLD4 and AcPLD9; AcPLD8, and AcPLD9) to 73% (AcPLD5 and AcPLD10; Table 3).
To examine the phylogenetic relationship between the AcPLD proteins and other plant species, a phylogenetic tree was constructed on the basis of their translated amino acid sequences. The whole plant PLD proteins could be grouped into six types: α, β, γ, δ, ε, and ζ (Qin and Wang, 2002). Based on the tree, the 10 PLD genes in pineapple were clustered into five types. AcPLD2, AcPLD4, and AcPLD7 were clustered to α type, and AcPLD6 was clustered to β or γ type. AcPLD1, AcPLD3, AcPLD5, and AcPLD10 were δ type, whereas AcPLD8 and AcPLD9 were ζ type (Figure 4).
To investigate the potential function of the AcPLD proteins, the transmembrane helix number was analyzed via topology   prediction using HMMTOP online tools. The resulting data showed that zero, one and two transmembranes existed in the associated AcPLDs (Table 1). These data suggested that AcPLD1-AcPLD10 exhibited great structural diversity and possibly exerted different physiological functions.

PLD Gene Expression in Pineapple during IB
To understand the possible role of AcPLD1-AcPLD10 in pineapple during IB, the expression patterns of AcPLD1-AcPLD10 in pulp of fruits treated with hexaldehyde were investigated by qRT-PCR. As shown in Table 4, among the 10 AcPLD genes, AcPLD2 (1.6-to 5.7-fold) was strongly induced after hexaldehyde treatment. By contrast, the transcript level of AcPLD9 (1.9-to 4.0-fold) decreased following hexaldehyde treatment. AcPLD7 and AcPLD8 showed similar expression patterns, and they decreased by 1.3-to 2.5-fold and 1.1-to 2.3-fold in treated fruits, respectively, and did not substantially change between the treated and non-treated fruits. In addition, AcPLD1 and AcPLD3 transcripts remained nearly unchanged, and AcPLD4-6 fluctuated for both control and treated fruits during storage.

Subcellular Localization of AcPLD2 and AcPLD9 Proteins
AcPLD2 and AcPLD9 were selected for subcellular localization analysis due to their gene expression patterns with obvious  characteristics during IB. To detect the potential role of the two proteins (AcPLD2 and AcPLD9), we assayed their localizations in the cell. The results indicated that the fluorescent signals from the AcPLD2-GFP and AcPLD9-GFP constructs were observed in the plasma membrane of tobacco leaf epidermal cells, whereas the fluorescence from the GFP control was distributed within the cells (Figure 5).

Contents of H 2 O 2 and Enzymes Related to H 2 O 2
To further investigate the potential regulatory mechanism of hexaldehyde treatment alleviating IB, we analyzed the contents of H 2 O 2 and enzymes related to H 2 O 2 . As shown in Table 5, the SOD content was decreased before the eighth day of storage and then increased in the treated fruits compared with the control samples. The contents of CAT, NADPH, and APX abundances showed similar patterns; they increased and peaked after 6 days of storage and then decreased in treated and non-treated fruits. However, the CAT, NADPH, and APX levels were suppressed at day 2, and their contents were induced in treated fruits than in non-treated samples. The H 2 O 2 content clearly fluctuated after hexaldehyde treatment during storage, increased at days 4 and 6, decreased at day 8, and increased again at day 12 in treated fruits. A significant difference was observed between the treated and non-treated fruits.

PLD Content
Hexaldehyde-treated fruits showed slightly higher PLD contents than control samples on day 2. However, on day 4, the PLD content was significantly higher in the former than in the latter. Hexaldehyde-treated fruits had lower PLD content than control samples, and treated fruits had relatively higher PLD content than non-treated fruits. Significant differences were noted between the groups ( Table 5).

DISCUSSION
Phospholipase D is an important signaling enzyme that has been identified in various species. A total of 12, 17, 11, and 18 PLD members have been characterized in Arabidopsis (Qin and Wang, 2002), in rice (Oryza sativa) , grape (Vitis vinifera), and poplar (Populus trichocarpa) (Liu et al., 2010) genomes, respectively. Genome-wide identification of PLD genes in pineapples has not been reported. The recent completion of pineapple genome sequencing allows us to identify all PLD protein coding genes in pineapples via homology analyses.
In the present study, 10 putative proteins in the pineapple genome database exhibited significant sequence identity with PLD homolog upon comparison with the NCBI data bank. We performed comprehensive analyses of the 10 AcPLD proteins with 23 other species such as rice and Arabidopsis of PLD protein for reference. Phylogenetic analysis indicated that the 10 identified AcPLD proteins could be classified into five major orthologous types (α, β, γ, δ, and ζ). The results of our study were inconsistent with previous reports on rice (α, β, κ, δ, ϕ, and ζ)  and Arabidopsis (α, β, γ, δ, ε, and ζ) (Pinosa et al., 2013) based on sequence similarity, thereby indicating the evolution of PLDs in different species. Phylogenetic analysis also revealed the evolutionary relationships among the AcPLD proteins, and the δ type contained the largest number (4) of PLD protein family members. The involvement of the PLD gene in various biological processes including seed germination , cold acclimation (Kargiotidou et al., 2010), resistance against pathogens (Zhao et al., 2013), and cold stress responses (Margutti et al., 2017), has been reported. In this study, the AcPLDs exhibited differential expression patterns during IB of pineapple. Among the 10 AcPLD genes, AcPLD6, AcPLD7, AcPLD8, AcPLD9, and AcPLD10 were induced prior to IB (6 days). Furthermore, AcPLD9 was downregulated by hexaldehyde treatment, which implied that AcPLD9 protein functioned as a positive regulator during IB. Our results were similar to the findings a previous report on PLD in Arabidopsis that showed a positive effect on pathogen defense (Pinosa et al., 2013). Notably, FIGURE 5 | Subcellular localization of AcPLD2 andAcPLD9 in tobacco (N. benthamiana) leaf epidermal cells. Tobacco epidermal cells were transiently transformed with constructs containing either fusion plasmids (AcPLD2-GFP and AcPLD9-GFP) or control (GFP alone) via Agrobacterium inoculation, GFP fluorescence was detected 24 h after inoculation. Bar = 50 µrn. AcPLD2 was upregulated in the presence of hexaldehyde, thereby indicating that AcPLD2 protein could function as a repressor in response to hexaldehyde treatment in pineapple. Similarly, Yamaguchi et al. (2009) demonstrated that OsPLDβ1 negatively regulates resistance against pathogens. PLD proteins are suggested to act as a membrane protein (Pinosa et al., 2013) and most isolated PLD proteins are localized in the plasma membrane (Wang and Wang, 2001;Pinosa et al., 2013). Further subcellular localization analyses demonstrated that AcPLD2 and AcPLD9 proteins were localized in the membrane. Thus, the two proteins may function within the plasma membrane. A growing number of studies have suggested that PLD plays vital roles in response to environmental stresses in plants (Li et al., 2004;Wang, 2005;Liu et al., 2013). Activated PLD can hydrolyze phosphatidylcholine to produce PA, which stimulates the NADPH oxidase complex to generate H 2 O 2 (Stahelin et al., 2003). Additionally, positive feedback exists between PLD and H 2 O 2 (Xiao et al., 2005). However, little is known about the relationship between PLD and H 2 O 2 during IB of pineapple. In this study, we showed that the production patterns of PLD and H 2 O 2 were similar. Further hexaldehyde treatment suppressed PLD and H 2 O 2 production in pineapple during late storage. These similarities suggested that PLD may inhibit H 2 O 2 production. Similarly, PLD activity could specifically suppress H 2 O 2 -induced apoptosis in rats (Lee et al., 2000) and cell death in Arabidopsis (Zhang et al., 2003). Stahelin et al. (2003) reported a similar pattern of NADPH and H 2 O 2 changes, thereby suggesting that NADPH may be a key enzyme in the induction of production H 2 O 2 production. In view of the present observations, we propose that hexaldehyde treatment resulted in decreased PLD gene expression and diminished PLD enzyme abundance, decreasing H 2 O 2 generation. However, our findings require verification.
In summary, a total of 10 putative PLD gene family members were identified in the pineapple genome. The 10 PLD genes were characterized based on their sequence identity, gene structure, conserved domains, phylogenetic analysis and gene expression profiles during IB by qRT-PCR. Data from this current study will assist in identifying AcPLD genes and provide a foundation for further study on the biological functions of PLD proteins in pineapples.

AUTHOR CONTRIBUTIONS
KH carried out the experiment. LZ participated in the research design, analyzing the data and drafting the manuscript, and initiated the project, designed the research framework, coordinated the research and reviewed the manuscript. RZ, BH, KS, and ZJ participated in data analysis and supported technically. All authors read and approved the final manuscript.