Pulp lignification in Korla fragrant pear - PubHort · 2019. 11. 11. · 264 E ur o p e a n J o ur n a l o f H o r t i cul t ur a l S c i e n c e Aisajan Mamat et al. | Pulp lignification

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IntroductionIn most species of the Rosaceae family, the fruit flesh is

homogeneous and is predominantly composed of parenchy-ma cells. However, pears are one of the few fruits that have a heterogeneous flesh due to the simultaneous presence of pa-renchyma and stone cells (Brahem et al., 2017). Stone cells, also known as sclereids, are one of the key determinants of pear quality, giving a gritty texture when pears are eaten or processed into juice and jam. The occurrence of stone cells has also been reported in other fruits such as loquat (Cai et al., 2006a; Xu et al., 2015; Zhang et al., 2016), mangosteen (Choehom et al., 2003; Kamdee et al., 2014), and kiwifruit (Yang et al., 2013; Ma et al., 2014; Li et al., 2017; Suo et al., 2018). Stone cells are a type of sclerenchyma cell formed by the secondary thickening of cell walls followed by the depo-sition of lignin on the primary walls of parenchyma cells. The development of stone cells is closely related to the synthesis, transfer, and deposition of lignin (Li et al., 2007; Tao et al., 2009; Cai et al., 2010).

Eur. J. Hortic. Sci. 84(5), 263–273 | ISSN 1611-4426 print, 1611-4434 online | https://doi.org/10.17660/eJHS.2019/84.5.2 | © ISHS 2019

Pulp lignification in Korla fragrant pearAisajan Mamat, Mubarek Ayup, Xiaoli Zhang, Kai Ma, Chuang Mei, Peng Yan, Liqun Han and Jixun WangInstitute of Horticultural Crops, Xinjiang Academy of Agricultural Sciences, Urumqi, China

Original article German Society for Horticultural Science

Significance of this studyWhat is already known on this subject?• Previous reports on pear stone cells describe the

structural characteristics and deposition of lignin. Some present information about lignin monomer composition, enzymes, and genes involved in the monolignol pathway of pear fruit stone cells.

What are the new findings?• In this paper, we determined the pathway for lignin

biosynthesis in Korla pear fruit. We found that lignin biosynthesis mainly occurred during the early stage of fruit development. The acid, aldehyde and alcohol levels indicated that the lignin in fruit pulp is mainly synthesized through the pathway from p-coumaric acid sequentially to coumaroyl-CoA, coniferaldehyde and sinapaldehyde, which then formed coniferyl alcohol and sinapyl alcohol by separate pathways. The coniferaldehyde and sinapaldehyde contents represented more than 99% of the four aldehyde monomers during the entire ripening period. However, the expression patterns of F5H and COMT were completely consistent with the changing trends of lignin production, indicating the existence of a pathway that preferentially reduces coniferaldehyde to sinapaldehyde and sinapyl alcohol.

What is the expected impact on horticulture?• The experimental results may lead to the development

of strategies to reduce or eliminate the probability of stone cell formation.

SummaryKorla fragrant pear (Pyrus & bretschneideri Rehd.)

is a landrace selected from a hybrid pear species of the Xinjiang Autonomous Region in China. One difficulty is the high stone cell content of these pears, which causes the formation of rough skin on the fruit. This leads to decreased fruit quality. Few studies have investigated the path of lignin biosynthesis in pear fruit. The objective of this study was to determine the lignification process in Korla pear fruit. The critical period and the pathway for lignin biosynthesis were determined by analyzing the lignin content, levels of acid, aldehyde and alcohol monomers, enzyme activities, and expression profiles of lignin-associated genes during fruit development. The lignin content in the pear pulp was highest in the early stage (from 10 to 40 days after flowering), decreased sharply, then leveled off 75 days after flowering. In the early stage of fruit development, p-coumaric acid and sinapyl alcohol were the dominant acid and alcohol monomers. However, the coniferyl alcohol and ferulic acid contents increased exponentially in a later stage of fruit development (90 days after flowering). They became the most abundant monomers in the pulp. Coniferaldehyde and sinapaldehyde, on the other hand, represented more than 99% of the four aldehyde monomers during the entire period. In addition, 4-hydroxycinnamoyl CoA ligase (4CL), cinnamoyl CoA reductase (CCR), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) showed higher enzyme activities in the early stage of fruit development, and the expression patterns of the four lignin-related genes (CCR, F5H, COMT, and CAD) showed higher expression levels during the same period. In particular, the expression patterns of F5H and COMT were completely consistent with the changing trends of lignin content. In conclusion, lignin biosynthesis mainly occurred during the early stage of fruit development. The lignin in fruit pulp is primarily synthesized through the pathway from p-coumaric acid sequentially to coumaroyl-CoA, coniferaldehyde and sinapaldehyde, which then formed coniferyl alcohol and sinapyl alcohol by separate pathways.

Keywordsfruit development, lignin biosynthesis, monomers, rough skin fruit, stone cells

264 E u r o p e a n J o u r n a l o f H o r t i c u l t u r a l S c i e n c e

Aisajan Mamat et al. | Pulp lignification in Korla fragrant pear

Lignin is a macromolecular substance characteristic of some vascular plant cell walls. Three phenolic monolignols, namely, p-hydroxycinnamyl, coniferyl alcohol and sinapyl al-cohol, are considered to be the natural precursors of lignin in vascular plants. These precursors are synthesized through the phenylpropanoid pathway, starting from phenylalanine ammonia-lyase (PAL) and leading to the three monolignols through a series of hydroxylations, methylations and reduc-

tions (Boerjan et al., 2003; Wang et al., 2016). The dehydro-genative polymerization of p-hydroxycinnamyl, coniferyl al-cohol and sinapyl alcohol, which is initiated by plant oxidore-ductases (peroxidases and/or laccases), gives rise to p-hy-droxyphenyl (H), guaiacyl (G) and syringyl (S) type lignins, respectively (Bonawitz and Chapple, 2010; Li and Chapple, 2010; Kärkönen and Koutaniemi, 2010). Lignin synthesis involves the coordinated expression of many genes and the

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FIGURE 1. Phenylpropanoid pathway leading to monolignol precursors of lignin. The gray area indicates the current revised pathway, the white area indicates the previous interpretation of the pathway (adapted from W. Boerjan, 2003).

Figure 1. Phenylpropanoid pathway leading to monolignol precursors of lignin. The gray area indicates the current revised pathway, the white area indicates the previous interpretation of the pathway (adapted from W. Boerjan, 2003).



activity of at least ten enzymes (Figure 1). Genes that encode enzymes involved in lignin biosynthesis have been widely re-ported (Xu et al., 2015; Vermerris and Abril, 2015; Wang et al., 2016; Li et al., 2017). Although there is some degree of variation among species in the architecture of the monolig-nol biosynthetic pathway, it is largely consistent across dif-ferent plant groups (Mottiar et al., 2016).

Korla fragrant pear is distinctive for its aroma, rich juicy flesh and crisp texture. Rough skin fruits and hard end fruits, which have a gritty texture, is a physiological disorder of pear fruit and has been frequently observed in the Korla fragrant pear variety in recent years. At present, increasing stone cell content is one of the most important factors that results in decreased quality. In normal fruits, stone cells are almost im-perceptible, but in rough skin fruits, the lignified cell walls of the stone cells do not change during fruit maturation, and they impart a very gritty texture.

Previous reports on pear stone cells describe the struc-tural characteristics and deposition of lignin (Tao et al., 2009; Cai et al., 2010; Yan et al., 2014). Some present information about lignin monomer composition, chemical functional groups and key bond combinations of sclereids in pear fruit (Jin et al., 2013; Yan et al., 2014; Lu et al., 2015; Brahem et al., 2017). There are also some reports about the enzymes and genes that are involved in the monolignol pathway of pear fruit stone cells (Tao et al., 2015; Cao et al., 2016; Wang et al., 2017). Few studies have investigated the path of lignin biosynthesis in pear fruit. To examine the critical period and pathway for lignin biosynthesis in Korla pear fruit, we inves-tigated the changes in lignin content, phenolic intermediates, enzyme activities, as well as the expression profiles of the genes involved in monolignol pathway during fruit develop-ment. The experimental results may lead to the development of strategies to reduce or eliminate the probability of stone cell formation.

Materials and methods

Standards and chemicalsAll chemical agents were analytically or chromatographi-

cally pure. Acetonitrile, methanol and ethanol were obtained from Merck (Darmstadt, Germany). All chemical reference substances (CRS) were obtained from BioBioPha (http://www.biobiopha.com/) and Sigma-Aldrich (http://www.sig-maaldrich.com/united-states.html).

Plant materialFruits of P. bretschneideri cv. Korla fragrant were ob-

tained from 20-year-old pear trees grown on a farm (Korla, Xinjiang, China). The pear fruits were collected at 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 days after flowering. At least thirty fruits of relatively uniform size were collected at the early stage (10–60 days after flowering), and fifteen fruits were collected at the later stage. The fruit skins were peeled with a peeler, and cylindrical samples, 15 mm in diameter, were sampled from the pulp, frozen in liquid nitrogen, and stored at -80°C until further analysis.

Measurement of lignin contentThe lignin content was measured using the Klason meth-

od (Raiskila et al., 2007). Pulp (5.0 g) from 2.0 mm under the peel to 0.5 mm outside the core was collected, oven-dried, ground into a uniform powder and passed through a 200-mesh sieve. The powder was extracted with methanol and oven-dried. A small amount (0.2 g) of this powder was ex-

tracted with 15 mL of 72% H2SO4 at 30°C for 1 h, combined with 115 mL of distilled water and boiled for 1 h. The volume was kept constant during boiling. The liquid mixture was fil-tered and the residue was rinsed with 500 mL of hot water, air-dried and weighed.

Targeted Secondary Metabolite Assay

1. Sample preparation and extraction. The freeze-dried pulp was crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. A total of 100 mg of pow-der was weighed and extracted overnight at 4°C with 1.0 mL of 70% aqueous methanol. Following centrifugation at 10,000 g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China, http://www.an-pel.com.cn/) before LC-MS analysis.2. HPLC conditions. The sample extracts were analyzed us-ing an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC Shi-madzu CBM30A, www.shimadzu.com.cn/; MS, Applied Bio-system 4500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions were as follows: HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm*100 mm); sol-vent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 100:0 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 15.0 min; flow rate 0.40 mL min-1; temperature 40°C; injection volume 5 μL. The effluent was alternatively connect-ed to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS.3. ESI-Q TRAP-MS/MS. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadru-pole-linear ion trap mass spectrometer ((Q TRAP), API 4500 Q TRAP LC/MS/MS system) equipped with an ESI turbo ion-spray interface, operating in a positive ion mode and con-trolled by Analyst 1.6 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 550°C; ion spray voltage (IS), 5,500 V; ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the colli-sion gas (CAD) was high. The instrument tuning and mass calibration were performed with 10 and 100 μmol L-1 poly-propylene glycol solutions in QQQ and LIT modes, respec-tively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were carried out with further DP and CE op-timization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

Enzyme activities4CL, CCR, COMT, F5H, CAD and POD enzyme activities were

tested by ELISA kits from Shanghai mlbio (www.mlbio.cn). The kits were carried out according to the manufacturer’s instructions. The data are expressed on a protein basis and the assays were performed in triplicate.

RNA extraction and qRT-PCR analysisTotal RNA was extracted from the samples using Trizol

reagent (Invitrogen, Shanghai, China) according to the man-ufacturer’s instructions. The total RNA was reverse tran-scribed into cDNA using the Prime Script™ RT reagent Kit (Takara, Japan), and residual genomic DNA was degraded using gDNA Eraser (Perfect Real Time) (Takara, Japan) ac-cording to the manufacturer’s instructions. Each sample was



composed of a total volume of 20 μL, which included 2 μL of cDNA, 0.4 μL of each primer, and 10 μL of 2× SYBR Green PCR Master Mix (Roche). PCR amplification was performed as fol-lows: 50°C for 2 min, 95°C for 30 s, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s.

Results

Lignin and monolignol contents during development The line graph illustrates changes in the contents of lig-

nin, p-coumaryl, coniferyl and sinapyl alcohols in the pulp of Korla pear during fruit development. As shown in Figure 2a, the lignin content increased slowly at the early stage of fruit development, rising from 16.9% on day 10 to 20.9% on day 40 after flowering. It decreased sharply thereafter, dropping to 6.7% on day 60, with the trend starting to level off 75 days after flowering, and reached its lowest level of 3.7% 120 days after flowering, accounting for approximately 18% at the ju-venile fruit stage.

The cinnamyl alcohols are the monomeric precursors of lignin. We therefore analyzed three cinnamyl alcohols (p-coumaryl, coniferyl and sinapyl alcohol). All of these three cinnamyl alcohols were detected in the pulp of Korla pear, indicating the existence of H-G-S units of lignin. The p-cou-maryl and sinapyl alcohol content clearly showed a trend of peaking in the early stage of fruit development, then plunging to its lowest level at day 60 after flowering, with a 7.7- and 10.8-fold decrease, respectively (Figures 2b, d). Being the most abundant cinnamyl alcohol, the sinapyl alcohol content accounted for 77.2% of the total cinnamyl alcohol 10 days af-

ter flowering, while p-coumaryl alcohol and coniferyl alcohol constituted 10.6% and 12.2% of the total cinnamyl alcohol, respectively. However, p-coumaryl and sinapyl alcohol were not detected 60 days after flowering. Conversely, coniferyl alcohol was at its lowest point at day 10 after flowering, and the figures remained nearly stable from 30 to 90 days after flowering, experiencing a slight rise (2.2-fold increase). Then coniferyl alcohol content started to increase exponentially 90 days after flowering and peaked at day 120 after flow-ering with a 26.5-fold increase, and it became the dominant cinnamyl alcohol (Figure 2c).

Phenolic acids in the monolignol pathwayLignin biosynthesis starts with the deamination of phe-

nylalanine to form cinnamic acid, which then undergoes a series of hydroxylation, methylation and reduction reactions. Herein we analyzed six phenolic acids, namely, phenylala-nine, cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid and sinapic acid. The results showed that phenylalanine, cin-namic acid, and p-coumaric acid contents were the highest at day 10 after flowering. The content of these acids subse-quently declined during fruit growth and found with a 2.4-, 9.0- and 2.2-fold decrease at day 120 after flowering, respec-tively. Initially, the phenylalanine content was stable, but a sharp decline was observed 75 days after flowering (Figure 3a). The cinnamic acid content declined steadily in the ear-ly stage (from 10 to 75 days after flowering), followed by a significant increase 75 days after flowering, then decreased rapidly, with a slight rise 105 days after flowering (Figure 3d). Changes in the p-coumaric acid content showed a trend

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FIGURE 2. Changes of lignin and alcohol monomers during fruit development in Korla pear.

Figure 2. Changes of lignin and alcohol monomers during fruit development in Korla pear.



of decreasing initially, then stabilizing and decreasing slowly again (Figure 3f). The caffeic and sinapic acid contents in-creased and peaked at 30 and 50 days after flowering, fol-lowed by a rapid decrease thereafter, and displayed a 2.0- and 1.7-fold decrease at day 120 after flowering, respectively (Figures 3b, e). Ferulic acid, however, was scarcely detect-ed during the early stage of pear development (from 10 to 30 days after flowering) and then slowly increased. Ferulic acid content remained nearly stable during the period from 30 to 90 days after flowering. After this time period, the fig-ure rose sharply and peaked at day120 after flowering, with a 26.3-fold increase (Figure 3c).

Among the four acid monomers (p-coumaric acid, caffeic acid, ferulic acid and sinapic acid), p-coumaric acid was the most abundant monomeric acid, accounting for 87.7% of the total acid monomers at day 10 after flowering, while caffeic acid and sinapic acid represented 10.9% and 1.4% of the to-tal acid monomers, respectively. However, a great disparity was found in this ratio at day 120 after flowering, where fe-rulic acid became the dominant monomeric acid and consti-tuted 86.7% of total acid monomers, while p-coumaric acid, caffeic acid and sinapic acid represented 8.1%, 4.5% and 0.7% of the total acid monomers, respectively.

Aldehyde monomer levels in the monolignol pathwayFour figures are given concerning the cinnamyl aldehyde

contents in the pulp of Korla pear during the fruit develop-ment (Figure 4). The graph reveals that the variation trends of p-coumaraldehyde, caffeylaldehyde, coniferaldehyde and sinapaldehyde were striking similar during the fruit devel-

opment, with highs in the early stage and lows in the later stage. After peaking at day 10, all of the four cinnamyl alde-hydes plunged to their lowest levels at day 120 after flow-ering, with 5.6-, 9.8-, 56.0- and 29.4-fold decreases, respec-tively. Coniferaldehyde and sinapaldehyde were the most abundant aldehyde monomers in Korla pear pulp during the whole process, accounting for 47.6% and 52.1% of the total cinnamyl aldehydes at day 10 flowering, and for 32.4% and 67.6% of total cinnamyl aldehydes at day 120 after flower-ing, respectively (Figures 4a, d). Meanwhile, p-coumaralde-hyde and caffeylaldehyde were scarcely detected at 60 days after flowering (Figures 4b, c).

Enzyme activitiesFigure 5 illustrates the changes in the enzyme activities

of 4CL, CCR, COMT, F5H, CAD and POD in the pulp of Korla pear during the fruit development. Enzyme activities were highest in the early stage of fruit development and lowest in the later stage (Figure 5). 4CL is responsible for the CoA es-terification of the cinnamic acids. Generally, the 4CL activity was stable from 10 to 20 days after flowering. This was fol-lowed by a period of dramatic increase, with 4CL activity in pulp reaching a peak of 0.696 U g-1 at day 40 after flowering. It then decreased to 0.530 U g-1 at day 60 after flowering and remained nearly stable from day 60 to the end of the experi-ment, fluctuating between 0.517 U g-1 and 0.499 U g-1 (Figure 5a).

CCR converts hydroxycinnamoyl CoA esters to their cor-responding aldehydes. CCR activity started to rise 10 days af-ter flowering and peaked at 0.584 U g-1 at 30 days after flow-

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FIGURE 3. Changes of acid monomers in the monolignol pathway during fruit development.

Figure 3. Changes of acid monomers in the monolignol pathway during fruit development.



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FIGURE 4. Changes of aldehyde monomers in the monolignol pathway during fruit development.

Figure 4. Changes of aldehyde monomers in the monolignol pathway during fruit development.

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FIGURE 5. Changes in the activities of enzymes in the monolignol pathway during fruit development.

Figure 5. Changes in the activities of enzymes in the monolignol pathway during fruit development.



ering. Then, the figure dropped rapidly, reaching 0.410 U g-1 at day 40, and remained nearly stable over the subsequent period (Figure 5b).

F5H preferentially converts coniferaldehyde and conifer-yl alcohol to sinapaldehyde and sinapyl alcohol, respectively. Figure 5c indicates that the F5H activity was relatively low at only 0.496 U g-1 10 days after flowering. This figure increased steeply to 0.681 U g-1 at 20 days after flowering and increased again, although more smoothly, to 0.824 U g-1 at 40 days after flowering. The F5H activity then plunged to 0.427 U g-1 at day 60, and the trend was starting to level off 75 days after flow-ering with a slight decrease.

COMT methylates caffeic acid and 5-hydroxyferulic acid, but its preferential substrates are 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol, resulting in sinapaldehyde and sinapyl alcohol, respectively. The highest value of COMT activity appeared in the early stage of fruit development, reaching a peak of 1.560 U g-1 at day 20 before plunging to a low of 0.762 U g-1 at day 40 after flowering. The activity leveled off in the later stage, with only a slight increase from 0.762 U g-1 at day 40 to 0.843 U g-1 at day 120 after flowering (Figure 5d).

CAD functions in the last step of the monolignol pathway, and catalyzes the reduction of cinnamaldehydes to cinnamyl alcohols. The CAD activity in the pulp fluctuated significantly during the pear fruit development (Figure 5e). CAD activity was at its lowest point of 0.495 U g-1 at day 10 after flowering, then rose dramatically over the next thirty days, peaking at 1.148 U g-1 at day 40 after flowering. From day 50, the CAD activity dropped rapidly and reached 0.580 U g-1 at day 75 after flowering. The period between day 75 to day 120 after flowering experienced a slight rise in CAD activity, with the figure reaching 0.835 U g-1 at day 120 after flowering.

POD can promote polymerization of monolignols in the presence of H2O2, resulting in either lignans or lignins. As

shown in Figure 5f, the POD activity was higher in the ear-ly stage of fruit development. There was a slow and steady increase in POD activity from 10 to 40 days after flowering, ranging from 1.453 U g-1 to 1.862 U g-1. This was followed by a sharp decrease to 0.878 U g-1 at day 60 after flowering. After increasing from 0.878 U g-1 at day 60 to 1.076 U g-1 at day 75, the POD activity plunged to a low of 0.615 U g-1 at day 90 af-ter flowering. The latter period brought a sustained decline in this figure.

Expression profiles of CCR, F5H, COMT and CAD genesThe expression patterns of four genes in the monolignol

pathway were assessed during fruit development of Korla pear (Figure 6). The CCR expression in the pulp decreased initially, then increased and decreased again during the fruit development. Expression profile of CCR was relatively higher in the early stage of fruit development (from 10 to 30 days after flowering) and dropped to its lowest point at day 50, with a 5-fold decrease. The next ten days observed a sharp rise, with the amount of CCR expression reaching its peak at day 60 after flowering at which point the figure began to decline and dropped by 0.3-fold at day 120 after flowering (Figure 6a).

As shown in Figures 6 b and c, the COMT and F5H genes exhibited striking similarities in expression patterns during the fruit development. Expression levels of COMT and F5H genes fluctuated considerably. The highest level of COMT expression was observed at 10 days after flowering, whereas that of F5H was observed at 20 days after flowering, transcript levels of both the COMT and F5H genes then plunged to relatively low levels 60 days after flowering. The COMT and F5H genes were scarcely expressed 75 days after flowering. The CAD expression level increased initially and then decreased, but the decreasing trend leveled off 90 days after flowering. CAD showed the lowest amount of expression

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FIGURE 6. Expression profiles of genes in monolignol-specific pathways during fruit development.

Figure 6. Expression profiles of genes in monolignol-specific pathways during fruit development.



10 days after flowering and reached its highest level at day 60 after flowering with a 3-fold increase, followed by a marked decreased. By day 90 after flowering, CAD expression was found with a 2-fold decrease. After that time point, the CAD expression level exhibited no obvious change (Figure 6d).

DiscussionStone cells are one of the key determinants of pear fruit

quality. It has been reported that lignin plays a key role in the formation of stone cells (Tao et al., 2009; Cai et al., 2010; Yan et al., 2014). Our results showed that the lignin content of early-stage fruits were much higher than that of later-stage fruits, indicating that lignin biosynthesis mainly occurs during early stages of fruit development. Similar results have been reported in other pear varieties (Cai et al., 2010; Tao et al., 2015; Wang et al., 2017). The synthesis of lignin involves the coordinated expression of many genes and the activity of at least ten enzymes in order to synthesize pathway intermediates, which serve as substrates for subsequent reactions (Bonawitz and Chapple, 2010).

The conventional model of the phenylpropanoid pathway includes a series of hydroxylation and methylation reactions in which cinnamic acid derived from phenylalanine is converted into a variety of hydroxycinnamic acids (Higuchi, 1981; Humphreys and Chapple, 2002). According to this model, hydroxycinnamic acids are thus intermediates in the synthesis of their corresponding aldehydes and alcohols. However, our experimental results are not in support of this model. We found that in Korla pear, p-coumaric acid was the final hydroxycinnamoyl product of the phenylpropanoid pathway in its strict sense. Although ferulic and sinapic acids were detected in the early stage, the proportions of the corresponding monolignols converted from these two monomeric acids were almost negligible. Our results provided the first indications that not all of the hydroxycinnamic acids were obligatory intermediates in lignin biosynthesis. The last step of the phenylpropanoid pathway is the activation of these acids to their corresponding CoA thioesters. 4CL is responsible for this reaction of 4-coumaric acid (Petersen, 2016). It was reported that 4CL proteins may have distinct functions in the phenylpropanoid metabolic pathway. In Arabidopsis, the 4cl1 mutant shows decreased lignin content. 4CL1 and 4CL2 are expressed in lignifying cells. In contrast, 4CL3 is expressed in a broad range of cell types, and 4CL3 has acquired a distinct role in flavonoid metabolism (Soltani et al., 2006; Saito et al., 2013; Li et al., 2015). In pear, only Pb4CL1 is involved in lignin biosynthesis, whereas Pb4CL2 and Pb4CL4 may participate in flavonoid biosynthesis during the fruit development (Cao et al., 2016). These results are consistent with the results of the 4CL present study. We found that the 4CL activity showed a change tendency similar to that of lignin with overlapping peak times, the rapid decline in p-coumaric acid content was synchronous with the sharp increase in 4CL activity. This indicate that 4CL is key enzyme involved in lignin biosynthesis, and confirms that lignin synthesis in Korla pear pulp starts primarily from p-coumaric acid.

To clarify the specific pathways for lignin biosynthesis in Korla pear fruit, we further investigated the monomers at the aldehyde level. The results showed that peaks of four cinnamyl aldehydes appeared in the early stage of fruit development. Interestingly, the coniferaldehyde and sinapaldehyde content represented more than 99% of the total cinnamyl aldehyde during the entire ripening period. These results indicate that aldehyde monomers for lignin synthesis mostly converted

from feruloyl-CoA. Hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase( HCT) catalyze the production of both caffeoyl shikimate and caffeoyl-CoA (Hoffmann et al., 2003; Wang et al., 2014). CCoAOMT methylates caffeoyl-CoA to produce feruloyl-CoA (Humphreys et al., 2002). CCR converts feruloyl-CoA predominantly to coniferaldehyde (Boerjan et al., 2003; Leple et al., 2007; Koudounas et al., 2015). Together, these data present evidence that lignin in Korla fruit pulp is mainly synthesized through the pathway from p-coumaric acid sequentially to coumaroyl-CoA, coniferaldehyde and sinapaldehyde, which then forms coniferyl and sinapyl alcohols by different pathways. Downregulation of the CCR gene in tobacco, Arabidopsis, poplar, maize and perennial ryegrass significantly reduces the lignin content (Lauvergeat et al., 2001; Leple et al., 2007; Tu et al., 2010). In the present study, we found that the CCR activity was significantly increased 15 days after flowering and reached its peak at day 30, overlapping with the peak time of lignin, and higher expression levels of the CCR gene were observed in the corresponding period, corroborating the conclusion that in pear pulp, lignin biosynthesis may follow the pathway from p-coumaric acid to acyl-CoA ester, followed by the synthesis of G-S-lignin by different pathways.

The cinnamyl alcohols are the monomeric precursors of lignin; therefore, alcohol monomers in fruit pulp shed light on to the structure and metabolic pathways of lignin biosynthesis in pear fruits. We found that sinapyl alcohol was the dominant alcohol monomer in the early stage of fruit development, accounting for 77.2% of total alcohol monomer. F5H and COMT are necessary for syringyl lignin biosynthesis. The downregulation of F5H or COMT eliminates the S subunits, and overexpression of F5H in a tissue-specific manner results in nearly all S subunits (Franke et al., 2002; Goujon et al., 2003). In this study, F5H and COMT showed similar expression patterns during the fruit development, with higher transcript levels in the early stage, consistent with the higher enzymatic activities of F5H and COMT at this period. The transcripts of F5H and COMT were strongly downregulated 50 days after flowering, and correspondingly, the activities of F5H and COMT were also very low and were accompanied by decreases in sinapaldehyde and sinapyl alcohol. It has been reported that the preferential substrate for F5H is not ferulic acid, but coniferaldehyde and coniferyl alcohol (Humphreys et al., 1999). Similarly, the products of F5H-catalyzed hydroxylation, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol, have been proven to be good substrates for COMT, whereas caffeic acid is a poor substrate (Humphreys et al., 1999, 2002; Li et al., 2000; Parvathi et al., 2001). This indicates the existence of a pathway that preferentially reduces coniferaldehyde to sinapaldehyde and sinapyl alcohol.

CAD functions in the last step of monolignol biosynthesis converting cinnamaldehydes into their corresponding alcohols (Hirano et al., 2012). Among the nine CAD-like genes in Arabidopsis, two operate in lignin biosynthesis (Sibout et al., 2003). A double mutant that lacks both of these genes exhibits a 94% reduction in the traditional G and S subunits (Sibout et al., 2003). Previous studies have documented the involvement of CAD in fruit lignification in several fruits, such as loquat (Cai et al., 2006; Saathoff et al., 2011; Xu et al., 2015), pear (Lu et al., 2015; Wang et al., 2017), kiwifruit (Li et al., 2017; Suo et al., 2018) and mangosteen (Kamdee et al., 2014). In aspen, SAD, a CAD homolog, preferentially reduces sinapaldehyde to sinapyl alcohol, but the absence of reads from the WD2 library in this cluster argues against



this hypothesis. Supporting this hypothesis, Kim et al. (2004) did not detect any specific substrate preference among all A. thaliana CADs, and genes with the closest homology to P. tremuloides SAD displayed the poorest ability to use any of the 5 aldehyde substrates tested. Therefore, the higher level of sinapyl alcohol in the early stage of fruit development was due to the substrate preference of F5H and COMT for coniferaldehyde and 5-hydroxyconiferaldehyde.

An interesting observation in this study was the accumulation of ferulic acid and coniferyl alcohol in the later stage of fruit development (90 days after flowering). In this period, the transcript levels of the F5H and COMT genes were scarcely detected and corresponded to the relatively low enzyme activities of F5H and COMT, indicating that the pathway from coniferaldehyde to sinapaldehyde and sinapyl alcohol was weakened or disrupted, and the pathway from coniferaldehyde to coniferyl alcohol was activated, resulting in accumulation of coniferyl alcohol. Correspondingly, the expression level of CAD was relatively high, and the CAD activity tended to increase. Furthermore, the accumulation of ferulic acid might be a degradation product of coniferyl alcohol. Nair et al. (2004) reported that the REF1 gene encodes a functional aldehyde dehydrogenase (ALDH) that is capable of oxidizing both sinapaldehyde and coniferaldehyde to sinapic acid and ferulic acid, respectively. These findings suggest that both ferulic and sinapic acids appear to be largely end products of the pathway, rather than intermediates. This observation is contradicted by the recent model of the pathway in the Arabidopsis. In this model, c4h, 4cl1, ccoaomt1, and ccr1 mutants were observed with accumulated ferulic acid and several ferulic acid derivatives, while in the same mutants, the transcript level of ALDH was reduced or not differential, and the level of coniferaldehyde was reduced (Vanholme et al., 2012). These findings indicates the existence of ALDH-independent pathways toward ferulic acid. The most evident route toward ferulic acid is a one-step reaction from feruloyl-CoA, possibly committed via a thioesterase (Dauwe et al., 2007; Mir Derikvand et al., 2008). Buraimoh et al. (2016) reported that Streptomyces albogriseolus fully utilized coniferyl alcohol as a substrate, leading to the generation of ferulic acid, the generally known immediate metabolic product of coniferyl alcohol degradation, coniferylaldehyde, was not observed in any of the chromatograms, indicating that ferulic acid might be synthesized directly from coniferyl alcohol via an alternative pathway. Metabolic flux analyses are needed to reveal such a pathway, if it exists.

ConclusionsIn this paper, we determined the pathway for lignin

biosynthesis in Korla pear fruit. We found that lignin biosynthesis mainly occurred during the early stage of fruit development. The acid, aldehyde and alcohol levels indicated that the lignin in fruit pulp is mainly synthesized through the pathway from p-coumaric acid sequentially to coumaroyl-CoA, coniferaldehyde and sinapaldehyde, which then formed coniferyl alcohol and sinapyl alcohol by separate pathways. The coniferaldehyde and sinapaldehyde content represented more than 99% of the four aldehyde monomers during the entire ripening period. However, the expression patterns of F5H and COMT were completely consistent with the changing trends of lignin production, indicating the existence of a pathway that preferentially reduces coniferaldehyde to sinapaldehyde and sinapyl alcohol.

AcknowledgmentsThis study was supported by The National Natural

Science Foundation of China (grant 31760565) and the Basic Scientific Research Supporting Program of the Autonomous Region Public Welfare Research Institutes (grant KY2017075).

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Received: Aug. 9, 2018Accepted: May 20, 2019

Address of authors:Aisajan Mamat, Mubarek Ayup, Xiaoli Zhang, Kai Ma, Chuang Mei, Peng Yan, Liqun Han and Jixun WangInstitute of Horticultural Crops, Xinjiang Academy of Agricultural Sciences, 403 Nanchang Road, Urumqi 830091, China

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Pulp lignification in Korla fragrant pear - PubHort · 2019. 11. 11. · 264 E ur o p e a n J o ur n a l o f H o r t i cul t ur a l S c i e n c e Aisajan Mamat et al. | Pulp lignification