PpNAC187 Enhances Lignin Synthesis in ‘Whangkeumbae’ Pear (Pyrus pyrifolia) ‘Hard-End’ Fruit

A disorder in pears that is known as ‘hard-end’ fruit affects the appearance, edible quality, and market value of pear fruit. RNA-Seq was carried out on the calyx end of ‘Whangkeumbae’ pear fruit with and without the hard-end symptom to explore the mechanism underlying the formation of hard-end. The results indicated that the genes in the phenylpropanoid pathway affecting lignification were up-regulated in hard-end fruit. An analysis of differentially expressed genes (DEGs) identified three NAC transcription factors, and RT-qPCR analysis of PpNAC138, PpNAC186, and PpNAC187 confirmed that PpNAC187 gene expression was correlated with the hard-end disorder in pear fruit. A transient increase in PpNAC187 was observed in the calyx end of ‘Whangkeumbae’ fruit when they began to exhibit hard-end symptom. Concomitantly, the higher level of PpCCR and PpCOMT transcripts was observed, which are the key genes in lignin biosynthesis. Notably, lignin content in the stem and leaf tissues of transgenic tobacco overexpressing PpNAC187 was significantly higher than in the control plants that were transformed with an empty vector. Furthermore, transgenic tobacco overexpressing PpNAC187 had a larger number of xylem vessel elements. The results of this study confirmed that PpNAC187 functions in inducing lignification in pear fruit during the development of the hard-end disorder.


Background
The hard-end disorder in pear fruit occurs in many pear-growing regions [1,2]. The disease is frequently found in the USA in pear varieties such as 'Anjou' (Pyrus communis L), 'Winter Nelis' (P. dimorphophylla) and 'Comice' (P. communis) [3,4]. In recent years, the hard-end disorder also appeared in some Asian pear varieties, including 'Whangkeumbae' (P. pyrifolia) and 'Xueqing'(P. nivalis), and other varieties may also be affected [5,6]. Hard-end of pear is a physiological disorder. Pear varieties grafted on Japanese pear (P. serotina Rehd) rootstocks often exhibit this disorder due to scion-rootstock compatibility problems that cause water imbalance problems and low Ca content and a low Ca/K ratio [6,7,2]. Fruits with hard-end are culled out during grading and packing, and severely misshaped fruits are deemed as unmarketable. The rst symptom of hard-end disorder is observed as an abnormally green or yellow color at the blossom end when fruits have grown to one third or half of their full size. A readily apparent protrusion of the calyx forms due to the delayed development of the surrounding tissues. The epidermis over the calyxend of the fruit appears tight and shiny, and the esh near the calyx turns dry and hard due to an accumulation of lignin [5].
We previously reported that lignin content and the number of stone cells increases signi cantly during hard-end development in 'Whangkeumbae' fruit [5]. These hard-end pear fruit also contain high levels of enzymes that are involved in lignin synthesis; including phenylalanine ammonia lyase (PAL), 4coumarate: coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD). As a result, the development of hard-end in pear fruit is correlated with lignin accumulation. Lignin is a phenylpropanoid-derived polymer that is deposited in secondary cell walls to increase the mechanical strength of xylem tissues in vascular plants and to provide defense against attacks from pathogens [8].
Several transcription factors involved in lignin biosynthesis, such as MYB (myeloblastosis), NAC (NAM, ATAF, and CUC), bHLH (basic helix-loop-helix), and others, have been identi ed and characterized in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and loquat (Eriobotrya japonica) fruit [11][12][13][14]. The NAC transcription factor family is one of the largest families of plant-speci c transcription factors and they participate in several physiological processes [15]. Most proteins containing a NAC domain are located at the upstream end of a regulatory network. In Arabidopsis thaliana, AtNST1 and AtNST2 promote secondary wall thickening in the endothecium of anthers, and knock-out mutants of NST1 and NST3 lose secondary-wall deposition in stems [16]. Several studies have reported that NAC genes function as master switches in the biosynthetic pathways for cellulose, xylan, and lignin by initiating a transcriptional signaling network that either affects MYB transcription factors or regulates the expression of structural genes [17,18]. A study on loquat chilling-induced ligni cation demonstrated that EjNAC3regulated expression of the EjCAD-like gene; which is a key gene in ligni cation [19].
'Whangkeumbae' pear fruit was previously reported to accumulate lignin during the development of hardend fruit. In the present study, DEGs regulated by NAC transcription factors were identi ed in hard-end and normal 'Whangkeumbae' fruit using RNA-seq. The association between NAC and lignin accumulation in hard-end tissues was evaluated using transient expression of the identi ed genes in 'Whangkeumbae' fruit and the analysis of transgenic tobacco overexpressing the respective genes.

RNA-Seq analysis
Total of 545 DEGs were identi ed in the comparison of hard-end and normal fruit in samples collected on the day of harvest (120 d after anthesis). A KEGG pathway enrichment analysis placed these DEGs into six pathways which include protein processing in endoplasmic reticulum; ribosome; glycine, serine and threonine metabolism; phenylalanine metabolism; phenylpropanoid biosynthesis; and starch and sucrose metabolism (Fig. 1). The phenylpropanoid biosynthesis and ribosome were enriched in 7 genes; which was the highest among all of the identi ed pathways. The DEGs in the phenylpropanoid biosynthesis pathway were annotated as PpCCR, PpC3H, PpF5H, Pp4CL, PpCOMT, PpPOD (GDR accession No., PCP040222, PCP022543, PCP016311, PCP044725, PCP024172, PCP007841, PCP013947, and PCP030808). Lignin-related genes, PpCCR, Pp4CL, PpCOMT, PpCAD1, and PpCAD2 also exhibited signi cantly higher levels of expression in hard-end fruit, relative to normal pear fruit (Fig. 2).
The phylogenetic analysis of PpNACs andtheir expression pattern Transcription factor family genes were among the genes represented in the transcriptome data. NAC transcription factor genes were selected from the identi ed transcription factors for further analysis.
Three PpNAC genes were found to be DEGs, and PCP044783 was the same sequence with PpNAC138. The other two genes of PCP044783 and PCP012487 were named as PpNAC186 and PpNAC187 according to the reference (Fig. 3) [38]. The three NAC genes that were identi ed as DEGs were either upor down-regulated (Log 2 FC>2 or Log 2 FC<-2) in hard-end fruit, relative to normal pear fruit.
The expression patterns of PpNAC138, PpNAC186 and PpNAC187 in hard-end and normal fruit during fruit development and postharvest storage were analyzed by RT-qPCR. Results indicated that the relative transcript abundance of PpNAC187 exhibited a signi cant increase of expression in hard-end fruit at 90 and 120 d after anthesis, while PpNAC138 and PpNAC186 were all down-regulated in hard-end fruits during fruit development (Fig. 4). The relative transcript abundance of PpNAC187 increased gradually in hard-end fruit during postharvest storage, while no signi cant changes in expression were detected in normal fruit. The transcript abundance of PpNAC187 was consistently higher in hard-end fruit than in normal fruit. The relative abundance of PpNAC138 exhibited some greater level of expression in hard-end fruit then in normal fruit at 60 d, while PpNAC186 only exhibited a higher level of expression at 120 d after harvest in hard-end pear fruits (Fig. 4). Since PpNAC187 exhibited the greatest difference in expression in hard-end vs. normal fruit, it was selected for further analysis.

Subcellular localization of PpNAC187
A pCambia1300-PpNAC187 vector, carrying a Green Fluorescent Protein (GFP) reporter protein, was constructed and subsequently inoculated into onion scales to determine the subcellular localization of PpNAC187. An empty vector was used as a control. When viewed under a uorescent microscope, the cytomembrane and nucleus in living onion epidermal cells infected with the pCambia1300 empty vector exhibited green uorescence. In contrast, only the nucleus exhibited green uorescence in onion epidermal cells infected with the pCambia1300-PpNAC187. These results indicate that the PpNAC187 transcription factor is expressed and localized in the nucleus of onion epidermal cells (Fig. 5). Transient expression of PpNAC187 in 'Whangkeumbae' pear esh Transient expression analysis of PpNAC187 was conducted in 'Whangkeumbae' pear esh by injecting pCambia1300-PpNAC187 into fruit esh, while the injection of an empty vector served as a control ( Fig.   6a) with each 50 fruit. No obvious changes in the pear fruit surface were observed over a three-day period following injection with either vector of all the fruit. Subsequently, however, the color around the inoculation site of fruit injected with Agrobacterium harboring the pCambia1300-PpNAC187 vector changed to dark green by day 5 of 16 fruit among 20 fruit, and the green color progressively deepened by the 10 th d after inoculation. In contrast, the control fruit inoculated with the empty vector exhibited no signi cant change in color over the ten-day post-injection period. Results of the lignin staining indicated no obvious differences between fruit inoculated with empty vector vs. pCambia1300-PpNAC187 after 3 d, but the level of staining was noticeably higher in pCambia1300-PpNAC187 inoculated fruit than control fruit at 5 d and 10 d post-injection (Fig. 6b). The results on the transient expression of PpNAC187 are in agreement with increased lignin accumulation observed in 'Whangkeumbae' hard-end fruit.
The expression of PpNAC187 and lignin-synthesis-related genes was also analyzed in fruit tissues surrounding the injection site. In comparison to fruit injected with the empty vector control, the relative expression level of PpNAC187 was higher in fruit injected with the pCambia1300-PpNAC187 vector. There was a signi cant increase in both PpCAD1 and PpCAD2 expression of fruit injected with the pCambia1300-PpNAC187 vector compared to empty vector after 3 d post-injection. The expression pattern of PpCCR was analogous to PpNAC187, which exhibited an increase in expression at 3 d postinjection. The expression of Pp4CL and PpCOMT increased after 10 d and 5 d post-injection respectively (Fig. 7). Thus, it was concluded that the expression of PpNAC187 (TF) and PpCCR, Pp4CL and PpCOMT (lignin biosynthesis genes)was correlated withthe ligni cation of esh tissues in pears and re ected what occurred during the normal development of hard-end pear fruit.

Functional veri cation of PpNAC187 in transgenic tobacco
Transgenic tobacco plants overexpressing PpNAC187 were generated using an Agrobacterium-mediated transformation method. Insertion of PpNAC187 into the tobacco genome was con rmed byPCR analysis. Results revealed that a band in the size-range of PpNAC187 was ampli ed in PpNAC187 transgenic tobacco but not in plants transformed with the empty vector (Additional File 2: Fig. S1). Two independent PpNAC187 transgenic lines (#1 and #3) were selected. PpNAC187 was highly-expressed in #1 and #3 PpNAC187-overexpressingtransgenic plants and not expressed at the empty vector line (Fig. 8 a-b). The level of stem's lignin staining was higher in PpNAC187-overexpressing lines than that in empty vector plants (Fig. 8 c). Lignin content in stem tissues of PpNAC187-overexpressing lines was notably higher than in empty vector line (Fig. 8 d). When auto uorescence was examined under a uorescence microscope, more cell layers of xylem elements were observed in the stem in PpNAC187-overexpressing plants compared to the control plants (Fig. 8 e). The auto uorescence within the stem sections in PpNAC187-overexpressing plantswas also more pronounced compared to the control tobacco plants transformed with an empty vector (Fig. 8 e). Meanwhile, lignin content in leaf tissues of PpNAC187overexpressing lines also higher than in empty vector line. And the leaf vein of PpNAC187-overexpressing plants have more cell layers of xylem elements were observed than the control plants, The auto uorescence within the leaf veins in PpNAC187-overexpressing plantswas more pronounced than the empty vector line (Fig. 9 a). The expression patterns of the tobacco endogenous lignin-related genes were also conducted, the genes of NtCCR2, Nt4CL and NtCAD1 were upregulated in #1 and #3 lines separately when compared with empty vector line (Fig. 10). PpNAC187-overexpressing plants also grew more brous roots relative to the S1 line (Additional File 3: Fig. S2 a). There was no apparent difference in lignin content in root tissues between PpNAC187-overexpressing and empty vector plants (Additional File 4: Fig.  S2 b).

Discussion
The hard-end disorder of 'Whangkeumbae' fruit is a major problem in the pear industry. Hard-end fruits contain signi cantly more and larger sclerotic cells in the calyx-end of the fruit compared to normal pearfruit; as well as a higher level of synthesis and deposition of lignin [5]. Several genes, including PAL, 4CL, CCR, COMT, CAD, are components of the phenylpropanoid pathway are associated with lignin synthesis [27,28,29,30,25]. Among these genes, PpCAD1 and PpCAD2 are continuously expressed at high levels during fruit development in fruit exhibiting hard-end symptoms [5]. CCR and CAD family genes are also responsible for the regulation of lignin synthesis and stone cell development in pear fruit [31]. In the present study, KEGG analysis of transcriptome data from hard-end and normal 'Whangkeumbae' pears revealed several DEGs that are part of the phenylpropanoid biosynthesis pathway. Based on RPKM values, the expression level of these genes was notably higher in hard-end fruit than in normal fruit.
These results indicate that the lignin synthesis pathway is more highly-activated in hard-end fruit than in normal 'Whangkeumbae' pear fruit and are in accordance with our previous research showing lignin accumulation during the development of hard-end symptoms [5].
Many physiological activities of plants are regulated by the activity of transcription factors [32]. Previous studies have reported that NAC transcription factors are involved in lignin synthesis in fruits. In fruits, EjNAC1 expression was induced in response to low temperature but inhibited by a heat treatment (HT); the latter of which also inhibited ligni cation [33]. In the present study, the conducted RNA-seq analysis revealed that three pear NAC genes are expressed at signi cantly different levels in hard-end 'Whangkeumbae' pear fruit, relative to normal pear fruit. In particular, the expression level of PpNAC187 was signi cantly higher during the development of hard-end fruit than in normal fruit, as well as during postharvest storage. PpNAC187 was localized in nuclei, con rming that PpNAC187 is a functional transcription factor. When a vector containing PpNAC187 was injected into pear esh tissues, the relative expression level of PpNAC187 was signi cantly enhanced, concurrent with the lignin biosynthesis-related genes(PpCCR, Pp4CL, and PpCOMT). And in PpNAC187-overexpressing transgenic tobacco, the expression level of lignin biosynthesis-related genes (NtCCR2, Nt4CL and NtCAD1) was also dramatically increased (Fig. 7). We suggest that lignin synthesis is potentially in uenced by the NAC transcription factor. Several NAC genes, including AtVND and AtNST, have been previously reported to be involved in the regulation of phenylpropanoid biosynthesis and these NAC TFs also play a role in secondary xylem development and/or secondary wall formation in A. thaliana [34][35][36][37]. A greater number of xylem vessel elements were observed in our present study in transverse sections of stems and leaf veins in PpNAC187overexpressing transgenic tobacco lines, relative to tobacco plants transformed with an empty vector (positive control). Extra cell layers of vessel elements were also observed in the PpNAC187overexpressing transgenic tobacco and individual elements were larger in diameter, relative to xylem elements in tobacco plants transformed with empty vector. Our previous studies demonstrated that PpCAD2-overexpressing tobacco plants also produced a greater number of xylem vessel elements in stem and leaf veins than in WT tobacco plants [25]. In the current study, lignin content was signi cantly higher in stem and leaf tissues of PpNAC187-overexpressing transgenic tobacco. Collectively, the data indicate that PpNAC187 plays a role in enhancing lignin accumulation by inducing the expression of PpCCR, Pp4CL and PpCOMT in 'Whangkeumbae' pear fruit during the development of hard-end symptoms. The ectopic expression of NACs in PpNAC187-overexpressing transgenic tobacco activated biochemical and metabolic processes resulting in a greater number of vessel elements, sclerotic cells, and a higher level of lignin accumulation.

Conclusion
In the present study, we demonstrated that the expression of lignin biosynthesis-related genes including 4CL, CCR, COMT, CAD exhibited signi cantly increased in 'Whangkeumbae' pear 'hard-end' fruit. Furthermore, transient overexpression of PpNAC187 in 'Whangkeumbae' pear esh induced the expression of lignin synthesis related genes PpCCR, Pp4CL, and PpCOMT and the degree of ligni cation. In PpNAC187-overexpressing transgenic tobacco, lignin biosynthesis-related genes (NtCCR2, Nt4CL and NtCAD1) also exhibited increased. And thelignin content in both stem and leaf of PpNAC187overexpressing transgenic tobacco was increased. These results suggest that PpNAC187 enhances lignin synthesis by regulating the expression of lignin synthesis related genes in 'Whangkeumbae' pear 'hardend' fruit.

Materials And Methods
Plant material 'Whangkeumbae' pear fruit were picked in orchards located in Wulong and Laiyang, Shandong province, People's Republic of China. Hard-end fruit were picked from ten-year-old 'Whangkeumbae' pear trees in one orchard, and normal pears were harvested from healthy trees in another orchard. Normal and hardend fruit were sampled at 60, 90 and 120 days after anthesis, and sampled again at 0, 60 and 120 days after harvest when storaged under 0 °C. Three biological replicates comprised of ten fruits each were used for each condition (normal vs. hard-end) and at each sampling timepoint. The fruit tissues near the bottom third of the calyx end were taken. After removal of the peel and/or seed, eshy tissues were sliced into small pieces (approximately 1 cm 3 ) and immediately frozen in liquid nitrogen. Sample were stored at −70 °C until further analysis.

RNA-seq analysis
The calyx pulp of normal and hard-end fruit at 120 days after anthesis were RNA-seq analysis. Total RNA was extracted using an RNA extraction kit (Omega, Georgia, USA) according to the manufacturer's instructions. The integrity and quality of the total RNA was evaluated using a 2100 Bioanalyzer RNA Nano chip device (Agilent, Santa Clara, CA, USA). The poly A -mRNA fraction was enriched by treatment of the extracted RNA with oligo (dT) beads and was then reverse-transcribed into rst strand cDNA for use in preparation of the sequencing libraries.
The cDNA libraries were sequenced using an Illumina HiSeq 2500 system at the Biomarker Technologies Corporation (Beijing, China). Raw reads were rst ltered to remove adaptors and low quality sequences, and then mapped to the pear reference genome (https://www.rosaceae.org/species/pyrus/pyrus_communis/genome_v1.0) using TopHat software. A false discovery rate (FDR) < 0.01 and a fold change of ⩾2 were used to identify differentially expressed genes (DEGs). The predicted product of each unigene sequence was aligned to a set of proteins retrieved from the NCBI Nr, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Cluster of Orthologous Groups of proteins (COG) databases. The Reads Per Kb per Million Fragments (RPKM) was used to determine the expression level of genes. The total number of reads for each unigene and gene length were normalized by RPKM. The formula used to calculate was as follows: RPKM = total exon reads / (mapped reads (millions)×exon length (KB)). KEGG pathway enrichment analysis was performed using KOBAS software and utilized an adjusted P-value of <0.05. Transcription factors were identi ed and classi ed into different families by reference to the NCBI Nr, Swiss-Prot, and COG databases. The raw sequences generated for 'Whangkeumbae' in this study were deposited in NCBI (NCBI BioProject Accession: SRP063324, http://www.ncbi.nlm.nih.gov/bioproject/PRJNA294723).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from pear esh tissue using RNAplant Reagent (TianGen, Shanghai, China) according to the manufacturer's instructions. Tobacco leaf RNA was extracted using an EASYspin Plant RNA Kit (Yuanpinghao, China) and genomic DNA was removed by treatments with DNase (Fermentas, Vilnius, Lithuania). The cDNA was synthesized by reverse transcription using the Prime Script™ RT reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions and was subsequently used as template in the RT-qPCR analyses. RT-qPCR was performed on a Light Cycler® 480 instrument (Roche, Switzerland). The protocol included annealing at 94 °C for 5 min, followed by 40 cycles of 94 °C for 15 s, and 60 °C for 1 min. Actin genes from pear and tobacco were used for the normalization of transcript levels. Gene-speci c primers used in the RT-qPCR analyses were designed with Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) software and are listed in Additional File 1: Table S1. Mean expression level was calculated using the 2 -ΔΔCt method [39]. The expression level in normal fruit at 60 days after anthesis was set as 1 in the RT-qPCR analyses conducted on samples collected during fruit development, and the day of harvest was set as 1 in the post-harvest analyses. Three biological and three technical replicates were used in the RT-qPCR analysis of each gene at each timepoint.

Cloning of PpNAC187
Total RNA isolation and cDNA synthesis followed the same protocol used in the RT-qPCR analyses. The PCR primers used to clone PpNAC187 are shown in Additional File 1: Table S2. The PCR program was: 94 ºC for 5 min, 35 cycles of 94 ºC for 30 s, 60 ºC for 1 min, and 72 ºC for 1 min, followed by an extension cycle at 72°C for 10 min and a nal cycle at 4 ºC. PCR products were cloned into PMD19-T vectors (Takara, Dalian, China). The open reading frame (ORF) of PpNAC187 was ampli ed and cloned using Phusion® High-Fidelity DNA Polymerase (Thermo scienti c, Lithuania, EU).

Sequence alignment and phylogenetic analysis
The amino acid sequence alignment analysis of NACs was conducted using DNAMAN software. A phylogenetic tree was reconstructed with Figtree (http://tree.bio.ed.ac.uk/software/ gtree/) online software. The amino acid sequence alignment analysis of pear NACs were refered to Ahmad [20].

Construction of the expression vector
The ORF of PpNAC187 was ligated into the expression vector, pCambia1300, under the control of a 35S promoter. The ORF fragment isolated by digestion with KpnI and Hind was inserted into the expression vector, pSuper1300, under the control of a 35S promoter. The vectors, pCambia1300-PpNAC187 and pSuper1300-PpNAC187, were transferred into Agrobacterium tumefaciens EHA105 using the freeze-thaw method [21]. The sequences of primers used to construct the expression vector are listed in Additional File 1: Table S2.

Subcellular localization of the PpNAC187 transcription factor
The subcellular localization of gene expression was determined using the method described by Sun with some modi cations [22]. After incubation for 24 h at 28 °C in the dark, fresh onion scales (1.5×1 cm) were placed on a 9 cm plate with their inner surface submerged in a 10 mL Agrobacterium solution (OD 600 = 0.6-0.8) supplemented with 20 mg acetosyringone/L for 15-20 min. The onion scales were then transferred to a 1/2 MS solid medium amended with 20 mg acetosyringone/L and cultured for 16-24 days at 28 °C. The onion scales were subsequently rinsed with water and epidermal cell layers were peeled and directly transferred to glass slides. Agrobacteria harboring the pCambia1300-PpNAC187 or the empty pCambia1300 vector were used in the analysis of subcellular localization. The GFP of onion scales inoculated with these vectors were observed under a confocal laser scanning microscope (TCSSP5 , Leica, Germany).

Transient expression of PpNAC187 in 'Whangkeumbae' pear
The method of transient expression of PpNAC187 in pear 'Whangkeumbae' followed the method described by Spolaore with some modi cations [23]. Holes were punched on the calyx end of hard-end fruits on the harvest day using a sterile syringe needle. One ml of Agrobacterium solution (OD 600 = 0.6-0.8) was then injected into the fruit via the holes using a syringe without a needle and the injected fruit was stored in the dark. Fruits inoculated with pSuper1300-PpNAC187 (treated) or the empty pSuper1300 vector (control) were photographed at the sampled timepoints. Samples were taken at 1, 3, 5 and 10 days after the injection, and were immediately frozen in liquid nitrogen and stored at −70 °C until further processing.

Agrobacterium-mediated transformation of tobacco with PpNAC187
The empty pSuper1300 vector and pSuper1300-PpNAC187 were independently transformed into tobacco plants using the Agrobacterium-mediated transformation method as described by Zheng with some modi cations [24]. Portions of tobacco leaves without veins were cut into discs (1×1 cm) and pre-cultured on MS solid medium for 2 days at 28 °C in the dark. The leaf discs were then submerged in 15 mL of Agrobacterium solution (OD 600 = 0.6-0.8) supplemented with 20 mg acetosyringone/L for 15-20 min.
Transgenic tobacco plants were generated on selection media after a 24 h light treatment following the method of Wang [25]. DNA was extracted from tobacco leaf tissue using DNAplant Reagent (TianGen, Shanghai, China) according to the manufacturer's instructions. PpNAC187 primers listed in Additional File 1: Table S2 were used to verify the presence of PpNAC187. The PCR program utilized was: 94 ºC for 5 min, 35 cycles of 94 ºC for 30 s, 60 ºC for 1 min, and 72 ºC for 1 min, followed by a 10 min extension at 72°C and a nal cycle at 4 ºC.

Wiesner staining and microscopy
Wiesner reagent (phloroglucinol/HCl) staining of plant tissue for 5 min was used to visualize ligni cation [5]. Two grams of phloroglucinol were dissolved in 100 ml of 95% alcohol and then ltered into 40 ml of concentrated hydrochloric acid. A razor blade was used to dissect leaf tissue prior to observation.
Ligni ed structures appeared pink or fuchsia in color. Auto-uorescence within stem sections was observed with the aid of an EVOS smart uorescence microscope (Thermo Fisher, America).

Lignin assay
Lignin content was assayed using the method described by Dyckmans with some modi cations [26]. Samples were washed three times in a 10 ml solution (100 mM K2HPO4/KH2PO4, 0.5%Triton X-100, 0.5% PVP, PH 7.8), followed by an additional three washes in 100% methanol. The samples of fruit tissues were then dried overnight and tissue samples were then transferred into 1ml of solution composed of 2 M HCl and 0.1ml thioglycolic acid. Lignin was extracted in this solution by placing samples in a boiling water bath for 4 h. Pellets obtained by centrifugation were resuspended 2 ml 1M NaOH followed by agitation for 18 h. After the addition of 0.2 ml HCl, the mixture was incubated for 4 h at 4 °C. The end product was dissolved in 1 ml 1M NaOH and absorbance at 280 nm was recorded to estimate lignin content. All measurements were performed in triplicate.

Statistical analyses
Two-tailed t-test were performed to determine the statistical signi cance of differences between samples.  Table S1 Gene-speci c primer sequences used in the RT-qPCR analysis of gene expression. Table S2 Primer sequences used in the cloning of PpNAC187 and transgenic plant validation.
Additional File 3: Fig. S2 The morphology and lignin content of roots in PpNAC187-overexpressing transgenic tobacco plants. Figure 1 KEGG enrichment analysis and the annotation of DEGs in control vs. hard-end fruit. The yellow columns indicate cellular processes, the red columns indicate genetic information processing, the blue columns indicate organismal systems, the green columns indicate metabolism, and the purple columns indicate environmental information processing. The gure displays the number of annotated genes in each category.

Figure 2
The relative expression of ligni cation-related genes during the period of fruit development. Asterisks indicate signi cant differences between control and hard end fruit (**, P<0.01; two-tailed t-test).
Page 17/23  The relative expression of NAC transcription factors during fruit development and postharvest storage.
Asterisks indicate signi cant differences between control and hard end fruit (*, P<0.05; **, P<0.01; twotailed t-test). Subcellular localization of a PpNAC187-GFP fusion protein in onion epidermal cells. The laser-scanning confocal microscopy was used to obtain light, uorescent, and merged images. Dark images represent uorescent images, bright represent light images, and merged represent merged dark and light images.  The relative expression of ligni cation-related genes in pear fruit surrounding the site of in ltration.
35S:PpNAC187 represent 'Whangkeumbae' pear fruit in which PpNAC187 was transiently expressed. Empty vector represent fruit that were in ltrated with the pCAMBIA1300 empty vector. The x-axis represents time after in ltration, and the y-axis represents relative expression. Asterisks indicate signi cant differences between empty vector and 35S:PpNAC187 fruit (*, P<0.05; **, P<0.01; two-tailed ttest).