Physiological , Histological , and Molecular Analyses of Avocado Mesocarp Fatty Acids During Fruit Development

Fatty acids are important components of the avocado mesocarp, so a better understanding of how their change during fruit development will contribute to improving the quality of avocado fruits and their nutritional value. The changes in fatty acids, lipid droplets, and expression of some key genes and regulators participating in late glycolysis and fatty acid biosynthesis were analyzed at different stages of the development of avocado mesocarp. The total fatty acid contents of the avocado mesocarp increased during fruit development, with an increase by a factor of seven (from 1,628.04 to 11,116.30 mg/100 g dry weight) in the late stage of fruit maturation, this was confirmed by the changes observed in the lipid droplets. The composition of the main fatty acids varied at four developmental stages of fruit development. Palmitic, palmitoleic, oleic, and linoleic acid contents generally increased during fruit development, reaching maxima at Harvest, with percentages of total fatty acids of 50%, 9%, 31%, and 8%, respectively. Meanwhile, the amount of PaWRI1, PaACP4-2, and PapPK-β1 expressed consistently increased by up to 4-fold during fruit development. This comprehensive analysis has indicated that the changes in the expressions of PaWRI1, PaACP4-2, and PapPK-β1 were consistent with those in the total fatty acid contents, so they might have key roles in the accumulation of oil in the avocado mesocarp.


Introduction
The fatty acids in avocado fruit are crucial constituents of their taste and possess the critical influence in keeping their quality and ensuring their nutritional value (Dreher & Davenport, 2013).Any changes in the essence and concentrations of these compositions in avocado fruit are important because of their effect on sensory properties.Hence, plant physiologists and molecular biologists express an interest in examining these changes during the development of the edible portions of the fruit, owing to their impact on the market quality of the food product (Donetti & Terry, 2014;Kilaru et al., 2015).
Avocado is among the most economically important subtropical/tropical fruit crop in the world, and increases in production are apparent throughout the world, with Mexico, the USA, Indonesia, Chile, Spain, Israel, Colombia, South Africa and Australia's growth in production being considerable (Schaffer et al., 2012).In terms of oil content, avocado fruit is surpassed only by oil palm and olive fruits (Knothe, 2013).The lipid content of avocado can range from 1% to 30% of the fruit, depending on the season, cultivar, and planting conditions (Rodríguez-Carpena et al., 2011;Vinha et al., 2013;Galvão et al., 2014).Avocado fruit lipids contain from 50% to 60% monounsaturated fatty acids and 10% to 15% polyunsaturated, which are good for human health (Donetti & Terry, 2014;Galvão et al., 2014;Pedreschi et al., 2016).
Today, increasing numbers of consumers are not only seeking a smooth-tasting avocado variety, but also health-motivating substances, such as fatty acids.Even so, although several studies have detected and analyzed the fatty acid contents of various avocado varieties (Vinha et al., 2013;Galvão et al., 2014;Ge et al., 2017aGe et al., , 2017bGe et al., , 2017c)), there is little information on the changes of fatty acids at different stages of avocado mesocarp development.Hence, the objective of the present research was to evaluate the variations in the content and type of fatty acids, visualize the lipid droplets, and analyze the expression of some key genes and regulators participating in fatty acid and late glycolysis biosynthesis at four stages of avocado mesocarp development.Our results will offer available data to help understanding the changes of avocado mesocarp fatty acids during fruit development from physiological, histological, and molecular analyses.

Plant Material, Reagents, and Sample Preparation
The avocado variety 'Guikenda No. 2' (Persea americana var.guatemalensis) were collected from six 10-year-old trees from the Chinese Academy of Tropical Agricultural Sciences (Danzhou city, Hainan province, China: location 19.52° N, 109.57°E, altitude = 200 m).In these trees, fruits corresponding to the main flowering season, which occurred during Feb. 2017, were marked.The marked fruit samplings were collected at different stages of fruit development, based on their days after pollination (DAP), which were from the maximum of one week after the end of second physiological fruit-dropping to physiological fruit maturity (defined as the ability to ripen after harvest).The four stages of fruit development were 65, 85, 105, and 125 DAP at harvest point, when the mesocarp dry matter was ≥ 21.5% based on the criterion according to Medina-Carrillo et al. (2017).The biological replicates included three units, each with two plants, and three biological replicates were measured in each developmental stage.The 18 marked fruit samples in each biological replicate for each developmental stage were randomly collected and immediately transported to the laboratory in standard foam boxes used for export packaging.The fruit samples were maintained at 5 °C.The mesocarp was separated from the fruit, homogenized using a domestic blender, and then stored at 4 °C for a maximum of one week before analysis.

Morphological Characteristics and Mass Assay
The morphological measurements was length and diameter of fruits.Dry weights of avocado mesocarp were measured from fresh mesocarp samples separated and placed in an air dry oven (GZX-9146 MBE, Shanghai, China) at 105 ºC for 6 h..The measurements were performed on 18 fruits in each biological replicate with three biological replicates for each developmental stage.

Extraction and Determination of Fatty Acids
The oil was extracted and fatty acid profiles were detected according to Ge et al. (2017a).The oils extracted from avocado pulp and seed (40 L) were saponified at 80 °C for 30 min after addition of 5 mL NaOH-MeOH (0.2 mol/L).After cooling, the solutions were mixed with 2.5 mL BF 3 -MeOH (14%) and incubated at 80°C for 30 min to produce methyl esters of the fatty acids.Following this, 2 mL of saturated NaCl and 4 mL n-hexane were added, and the resulting solutions were refluxed for 15 min.The upper layers were then removed, filtered through 0.22 μm membranes, and used for fatty acid GC-MS analyses.The analyses were performed using an Agilent 7890B-7000B GC-MS (Santa Clara, CA, USA) equipped with a DB-5MS column (60 m × 0.25 mm i.d., 0.25-m film thickness) using helium (1.3 mL/min) as the carrier gas.The fatty acid methyl esters (FAMEs) were identified by comparing the retention times of the peaks with those of commercial standards and comparing the respective ion chromatograms with those reported in the NIST 2011 library.Methyl nonadecanoate was added as an internal standard and the FAMEs were quantified using the calibration curves of the standards (R 2 ≥ 0.995).The contents of the FAMEs, expressed as mg/100 g DW, were presented as the mean ± standard deviation of three biological replicates with two technical replicates of each.

Histological Analyses
The tissue samples were sectioned to a thickness of 10 to 15 m using a vibratome (CV5030, Leica Biosystems, Wetzlar, Germany), washed three times with 0.2 M phosphate-buffered saline then stained with 0.5 g/mL Nile red for the pulp.To prepare the Nile red solutions for staining, Nile red powder (10 mg) was dissolved in 10 mL acetone then diluted to concentrations of 0.5 and 5 g/mL with distilled water.The tissue samples were observed using confocal microscopy (Zeiss LM510, Carl Zeiss AG, Oberkochen, Germany) with laser excitation at 543 nm and a 40× objective lens and the lipid droplets in the mesocarp were identified 2.5 RNA Extraction and Quantitative Real-Time PCR Total RNA was extracted from the mesocarp at the four stages using RNAiso Plus Reagent (TaKaRa Bio Inc., Kusatsu, Japan) based on the manufacturer's protocol, then treated with RNase-free DNase I (New England Biolabs, Ipswich, MA, USA) to eliminate all contaminating DNA.The resulting RNA was applied for first strand synthesis by the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa Bio Inc.).Five genes were selected for gene expression analysis: PaWRI1 (wrinkled1), PaWRI2 (wrinkled2), PapPK-β1 (plastidic pyruvate kinase beta subunit 1), PaACP4-1 (acyl carrier protein 4-1), and PaACP4-2 (acyl carrier protein 4-2).PaActin7 was used as an endogenous control for standardization.The sequences of these genes were chosen from Additional file 2: Data S1 (Kilaru et al., 2015).Gene-specific primers were developed through the Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) and were synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA).The primers were developed to amplify a product of no more than 170 bp with melting temperatures (T m ) of 55 °C (Table 1).The concentration of cDNA was determined and diluted to 12.5 ng/µL.PCR was performed using QuantStudio 7 Flex Real Time PCR System (Applied Biosystems, Foster City, CA, USA).The reactions were arranged in a total volume of 20 µl including 2 µl cDNA, 10 µl of SYBR Premix Ex Tap II (Tli RNaseH Plus) (TaKaRa Bio Inc.), 1.0 µl of each 10 µM primer, and 6 µl distilled water.The quantitative real-time PCR conditions were as follows: one cycle of 95 °C for 5 min, 33 cycles of 95 °C for 25 s, T m of each primer for 50 s, and 72 °C for 3 min.The final elongation was executed at 72 °C for 10 min.The relative expression value of each gene was counted according to the 2 -ΔΔCt method for relative quantification (Livak & Schmittgen, 2001).Data are presented as means ± SD of three biological reactions possessed in different 96-well plates, each having two replicates in each plate.The primers used for quantitative real-time PCR were presented in Table 1.
Table 1.Primers used for quantitative real-time PCR Note.Gene IDs came from the Additional file 2: Data S1 (Kilaru et al., 2015).

Statistical Analyses
Data analyses were conducted through SPSS Statistics, version 20.0 (IBM Corp, Armonk, NY, USA).Significant differences were verified by one-way analysis of variance, and Duncan's multiple comparison test was used at 95% confidence level.The PCA were obtained using NTSYS pc 2.1 software.

Changes in Morphological Characteristics During Fruit Development
Dry weight in the mesocarp increased significantly all the time from 9.45 to 54.75 g with fruit growth and development (Table 2).Length and diameter increased significantly with fruit growth and development (

Changes in Total Fatty Acid and Lipid Droplets During Fruit Development
Total fatty acid in the mesocarp increased with fruit development, with significant differences in content between the four stages of fruit development (Table 3).Total fatty acid content slowly increased in the mesocarp from 65 to 105 DAP, and then increased sharply, reaching the maximum of 11,116.30mg/100 g DW at 125 DAP.From 105 to 125 DAP, total fatty acid content in the mesocarp rose significantly from 1,628.05 to 11,116.30mg/100 g DW, a 7-fold increase, this was also found in the late developmental period of oil palm fruit (Bourgis et al., 2011;Tranbarger et al., 2011).Oil palm fruit possessed lipid-rich mesocarp tissue, which had 6-to 7-fold increases in the total fatty acids in the mid-to late stage of fruit development (Bourgis et al., 2011;Tranbarger et al., 2011;Dussert et al., 2013).Our results suggested that the period between 105 DAP and 125 DAP was a key time for forming fatty acids in the avocado mesocarp.The histological analyses were carried out to visualize the lipid droplets in the mesocarp during fruit development.The total fatty acid content increased from 1.00 to 1.63% of dry mass from 65 to 105 DAP, respectively (Table 3).Small lipid droplets (approximately 2 m in diameter; Figures 1A, 1B, and 1C) were present throughout the periphery of the cells.At 125 DAP, a great many large lipid droplets (10-25 m in diameter; Figure 1D) had formed, occupying the volume of the most cell, when the total fatty acid content had risen to 11.12% of dry mass (Table 3).Our histological analyses verified the trend in total fatty acid content detected using GC-MS in the avocado mesocarp during fruit development in the present study.Our results agreed with those of Tranbarger et al. (2011) and Dussert et al. (2013) in that lipid droplets (approximately 2 m  During fruit growth and development, the concentration of palmitic and palmitoleic acids gradually increased from 65 to 105 DAP followed by a rapid increase until 125 DAP, reaching maxima of 5,597.97 and 1,019.14mg/100 g DW, respectively.The oleic acid content started to increase after 85 DAP, and then increased dramatically by 70 times from 51.27 mg/100 g DW at 85 DAP to 3,413.14 mg/100 g DW at 125 DAP.The linoleic acid content fluctuated during fruit development, ultimately achieving a maximum of 932.22 mg/100 g DW at 125 DAP.From 65 to 85 DAP, the linoleic acid content was higher than those of the other fatty acids, but the linolenic acid content declined constantly during fruit development and ultimately could reach a minimum of 15.58 mg/100 g DW at 125 DAP. The present study suggested that the main fatty acid components of the avocado mesocarp were also reported that these the same acids were often the dominant fatty acids in mature avocado fruit (Ferreyra et al., 2016;Pedreschi et al., 2016;Rohman et al., 2016;Ge et al., 2017aGe et al., , 2017bGe et al., , 2017c;;Zhu et al., 2017).Previous studies have demonstrated that the palmitic acid content usually declined, while oleic and linoleic acids usually increased during the development of almonds (Zhu et al., 2017), walnuts (Chen et al., 2016a(Chen et al., , 2016b)), and borage (Guo et al., 2016), which results did not agree with those of this study.Our results demonstrated that the content of palmitic, oleic, and linoleic acids all increased during the four stages of fruit development, reaching maxima at the late stage.
The PCA results of eight fatty acids in avocado mesocarp at four stages of fruit development were obtained using NTSYS pc 2.1 software and were shown in Figure 2. PCA generalized eight fatty acids to two principal components which accounted for 96.83% of the total variation.The first component F1 explained 83.12% of the total variation and was mainly associated with palmitoleic acid, palmitic acid, stearic acid, oleic acid, arachic acid, linoleic acid, and linolenic acid.The second component F2 accounted for 13.71% of the total variation and was fundamentally defined by myristic acid.Our results indicated that the pattern of changes in the expression of PaWRI1 was consistent with those of PaACP4-2 and PapPK-β1, which also agreed with Kilaru et al. (2015) where transcriptome analysis of avocado mesocarp indicated that the expressions of PaWRI1, PaACP4, and PapPK-β1 had the same pattern of changes.Therefore, it could be inferred that PaWRI1 might up-regulate PaACP4-2, and PapPK-β1 in the avocado mesocarp in this study.

Conclusion
The content of total fatty acid of the avocado mesocarp increased during fruit development, and a remarkable increase in the total fatty acids was found in the late stage.For the main fatty acid compositions, palmitic, palmitoleic, oleic, and linoleic acids contents generally increased during fruit development, reaching maxima at the late stage.The expression amount of PaWRI1, PaACP4-2, and PapPK-β1 consistently increased by up to 4-fold during fruit development.The comprehensive analysis has indicated that the changes in the expression of PaWRI1, PaACP4-2, and PapPK-β1 were consistent with those in the total fatty acid content, and PaWRI1 gene could possess a key action in regulating the biosynthesis of fatty acids in the avocado mesocarp.

Table 2 .
Schaffer et al. (2012)meter increased rapidly during 65-105 DAP, and showed a relatively slow growth trend during 105-125 DAP.Length/diameter decreased in the earlier-middle stage and increased in the late stage (Table2).Length/diameter reached the minimum value (1.33) at 105 DAP.Therefore, the growth of avocado fruit was mainly horizontal thickening during 65-105 DAP, and mainly longitudinal elongation during 105-125 DAP.Schaffer et al. (2012)suggested that the fruit shapes of the avocado cultivars was exceedingly rich around the world, such as Bacon and Hass for ovate, Ettinger for pyriform, and Fuerte for pyriform with a distinct neck, etc.Based on the fruit length/diameter in the present study, the mature fruit shape (1.40) of the avocado commercial variety "Guikenda No. 2" was almost ovate.Dry masses and fruit size and fruit length/diameter at four stages of fruit development Note.Error bars indicate standard deviation from 18 fruits in each biological replicate with three biological replicates.Means with the different letters indicate significant differences (Waller-Duncan, P ≤ 0.05) among four developmental stages of each morphological trait.

Table 3 .
Total fatty acid contents (mg/100 g DW) in avocado mesocarp at four stages of fruit development

Table 4 .
Fatty acid composition (mean value ± standard deviation, mg/100 g DW) of avocado mesocarp at four stages of fruit development Error bars indicate standard deviation from three biological replicates with two technical replicates of each.Means with different letters in the same horizontal row indicate significant differences (Duncan, P ≤ 0.05).nd = not detected.