Genome-Wide Identiﬁcation of Laccase Gene Family from Punica granatum and Functional Analysis towards Potential Involvement in Lignin Biosynthesis

: Laccase (LAC) is the key enzyme responsible for lignin biosynthesis. Here, 57 PgLACs from pomegranate were identiﬁed and distributed on eight chromosomes and one unplaced scaffold. They were divided into six groups containing three typical Cu-oxidase domains. Totally, 51 cis -acting elements in the promoter region of PgLACs are involved in response to ABA, GA, light, stress, etc., indicating diverse functions of PgLACs . The expression proﬁles of 13 PgLACs during the seed development stage showed that most PgLACs expressed at a higher level earlier than at the later seed development stage in two pomegranate cultivars except PgLAC4 . Also, PgLAC1/6/7/16 expressed at a signiﬁcantly higher level in soft-seed ‘Tunisia’; on the contrary, PgLAC37 and PgLAC50 with a signiﬁcantly higher expression in hard-seed ‘Taishanhong’. Combined with their distinguishing cis -acting elements, it was concluded that PgLAC1/6 / 7 may respond to GA via TATC-box and GARE-motif, and PgLAC16 repressed the promotor activity of embryo mid-maturation genes via RY-element so as to contribute to softer seed formation, whereas PgLAC37 / 50 may participate in seed formation and accelerate seed maturity via ABRE and G-box elements. Collectively, the dramatic gene expressions of PgLAC1/6/7/16/37/50 will provide valuable information to explore the formation of soft-and hard-seed in pomegranate.


Introduction
Pomegranate (Punica granatum L.) is a commercial fruit tree with an old cultivation history and belongs to Lytharceae family [1]. Nowadays, pomegranate is grown commercially in many counties, such as Iran, China, Spain, and Turkey. Pomegranate fruit is very popular with consumers worldwide due to its delicious taste and benefits to human health. The increasing research demonstrates that pomegranate fruit, as well as its extracts, contain abundant bioactive components, e.g., flavonoids, anthocyanins, organic acids, ellagitannins, phenolic acids, and possess unique antihelminthic, antimicrobial, and antioxidant effects [2][3][4][5][6]. Generally, the edible part of pomegranate fruit is juicy pulp, named arils, by which seeds are surrounded. According to seed hardness, pomegranate cultivars are divided into four groups: soft seed, semi-soft seed, semihard seed, and hard seed [7]. Seeds in soft-seed pomegranate cultivars are easily swallowed and edible. However, the disadvantage is spitting seeds for hard-seed pomegranate cultivars, which seriously affects the taste and consumer appreciation. As we know, lignin is a principal structural component of cell walls in higher plants, and the pomegranate seed formation is closely related to lignin biosynthesis and metabolism [8].
Lignin significantly influences the physical properties and enhances the strength and hardness of cells in plants [9]. Laccase (LAC) is a copper-containing polyphenol oxidase,

Identification and Physicochemical Properties of PgLAC Family Members
Pomegranate genome data (ASM765513v2) was downloaded from the website (https://www.ncbi.nlm.nih.gov/assembly/GCF_007655135.1, accessed on 12 March 2022). The sequences of 17 AtLAC proteins were obtained from Uniprot website (https://www. uniprot.org/, accessed on 12 March 2022). PgLACs sequences from 'Tunisia' were obtained by Blast Wrapper (E-value < 1×10 −5 ) in TBtools software with AtLACs sequences and were matched with three Cu-oxidase domains (PFAM00394, PFAM07731, and PFAM07732) on the NCBI CDD (Conserved Domain Database; https://www.ncbi.nlm.nih.gov/Structure/ bwrpsb/bwrpsb.cgi, accessed on 18 March 2022). Subsequently, the Genbank Accession Numbers of PgLACs were obtained on NCBI BLAST alignment (https://blast.ncbi.nlm.nih. gov/Blast.cgi, accessed on 18 March 2022). The protein isoelectric point (pI) and molecular weight (MW) were accessed using online Expasy Protparam (https://web.expasy.org/ compute_pi/, accessed on 25 March 2022). The protein isoelectric point (pI) is calculated using pK values of amino acids, and molecular weight (MW) is calculated by the addition of average isotopic masses of amino acids in the protein and the average isotopic mass of one water molecule.

Bioinformation Analysis of PgLAC Family Members
The subcellular localization was predicted on the WoLF PSORT (https://wolfpsort. hgc.jp/, accessed on 16 July 2023). The phylogenetic tree was constructed using Clustal W method of MAGE 7.0 software (Mega Limited, Auckland, New Zealand) and optimized on the online website Interactive Tree of Life (http://iTOL.embl.de, accessed on 19 May 2023). The amino acid sequence alignment was performed with neighbor-joining (NJ), and the parameters were set as maximum composite likelihood, complete deletion, and bootstrap 1000 of MAGE 7.0 software. PgLAC proteins were clustered based on the published LAC proteins from other plant species (details in Table S1). Conserved motifs of PgLAC proteins were analyzed using the MEME online software (https://meme-suite.org/meme/tools/ meme, accessed on 18 May 2023). Exon-intron structures, chromosomal locations, and gene duplication of PgLAC genes were visualized using Gene Structure Shower, Gene Location Visualize, One-Step MCScanX, and Advanced Circos of TBtools software.

Analysis of Cis-Acting Elements and Protein Interaction Networks
The promotor sequences were obtained from 2000-bp upstream sequences from the start codon of PgLAC genes and predicted cis-acting elements on PlantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 18 May 2023), and illustrated with TBtools software. To explore gene co-expression patterns, the protein interaction networks were drawn on the website String (http://cn.string-db.org, accessed on 20 May 2023), and Arabidopsis thaliana was chosen as the species parameter.

RNA Extraction and Quantitative RT-PCR (qRT-PCR) Analysis of PgLAC Family Members
The total RNA of seeds was extracted using a Quick RNA Isolation Kit (0416-50-GK, Huayueyang, Beijing, China), and the cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). qRT-PCR was run on ABI 7500 PCR instrument (Applied Biosystems, Foster, CA, USA) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The PCR reaction was performed at 95 • C for 5 min, 40 cycles of 95 • C for 10 s, and 60 • C for 30 s. The relative expression level was calculated by 2 −∆∆CT method [27]. The PgActin (XM_031530994.1) was used as an internal reference. All the primers were designed on the website Primer-Blast of NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 6 August 2023) and listed in (Table S2). Statistical analysis was carried out with SPSS Statistics v. 20 (IBM, Chicago, IL, USA) with a significant difference of p < 0.05 and p < 0.01. The gene expression level was drawn using GraphPad Prism 8 software (San Diego, CA, USA).

Comparison of Seed Hardness and Lignin Content during Seed Development Stage
Lignin content was an important index to evaluate the seed hardness in pomegranate. Figure 1a presented seed phenotypic characteristics during four seed development stages of the two pomegranate cultivars. From Figure 1b,c, it was found that seed hardness and lignin content both increased steadily in 'Taishanhong' and 'Tunisia' seeds as seed development and were highly significantly lower in 'Tunisia' seeds than in 'Taishanhong' ones (p < 0.01).

Comparison of Seed Hardness and Lignin Content during Seed Development Stage
Lignin content was an important index to evaluate the seed hardness in pomegranate. Figure 1a presented seed phenotypic characteristics during four seed development stages of the two pomegranate cultivars. From Figure 1b,c, it was found that seed hardness and lignin content both increased steadily in 'Taishanhong' and 'Tunisia' seeds as seed development and were highly significantly lower in 'Tunisia' seeds than in 'Taishanhong' ones (p < 0.01). Figure 1. Seed appearance (a), seed hardness (b), and lignin content (c) during four seed development stages from 'Tunisia' and 'Taishanhong'. ** indicated significant differences between groups at p < 0.01, respectively.

Comparison of Seed Hardness and Lignin Content during Seed Development Stage
Lignin content was an important index to evaluate the seed hardness in pomegranate. Figure 1a presented seed phenotypic characteristics during four seed development stages of the two pomegranate cultivars. From Figure 1b,c, it was found that seed hardness and lignin content both increased steadily in 'Taishanhong' and 'Tunisia' seeds as seed development and were highly significantly lower in 'Tunisia' seeds than in 'Taishanhong' ones (p < 0.01). Figure 1. Seed appearance (a), seed hardness (b), and lignin content (c) during four seed development stages from 'Tunisia' and 'Taishanhong'. ** indicated significant differences between groups at p < 0.01, respectively.

Bioinformatic Characteristics of PgLAC Gene Family Members
The amino acids encoded by the PgLAC genes ranged from 397 aa (PgLAC10) to 614 aa (PgLAC36), and only two PgLAC genes (PgLAC10 and PgLAC52) were lower than 500 aa (Table 1). Their MWs ranged from 44.53 to 68.98 kDa, and the theoretical isoelectric points (pI) were from 4.51 to 9.90 (Table 1). Moreover, the subcellular localization displayed that 21 PgLAC proteins were located on chloroplast; secondly, vacuolar membrane and cytosol each with 11 PgLAC proteins, 5 on endoplasmic reticulum, 2 on extracellular, and the least was nucleus and mitochondrion each with one PgLAC protein.
To explore the evolutionary relationships of PgLACs, the phylogenetic tree of the amino acid sequences of 57 PgLACs was constructed with LAC proteins from other plant species (including 17 LACs from Arabidopsis thaliana, 53 from Populus trichocarpa, 27 from Citrus reticulata Blanco, and 48 from Solanum melongena) ( Figure 3). The results demonstrated that 202 LAC proteins were divided into six groups. Group I had 37 LAC proteins, and 33 LACs belonged to Group II. Moreover, Group VI contained the maximum LAC proteins, up to 77 LACs, while only 12 LAC proteins were clustered into Group III. Group IV and V had 25 and 18 LACs, respectively. Also, the maximum LAC proteins from pomegranate were distributed in Group VI, reaching 34, whereas Group III had only one PgLAC51. Considering that AtLAC4, AtLAC11, and AtLAC17 were responsible for lignin polymerization [23], it was concerned that PgLACs (PgLAC4, 5, 6, 7, 15, 38, and 50) were clustered into Group I with AtLAC4 and AtLAC11, furthermore, PgLACs (PgLAC1, 16, 17, 18, 32, and 37) along with AtLAC17 belonged to Group II (Figure 3), which indicated that they had similar function. To explore the evolutionary relationships of PgLACs, the phylogenetic tree of the a acid sequences of 57 PgLACs was constructed with LAC proteins from other plant sp (including 17 LACs from Arabidopsis thaliana, 53 from Populus trichocarpa, 27 from Citr ticulata Blanco, and 48 from Solanum melongena) ( Figure 3). The results demonstrated 202 LAC proteins were divided into six groups. Group I had 37 LAC proteins, and 33 L belonged to Group II. Moreover, Group VI contained the maximum LAC proteins, up LACs, while only 12 LAC proteins were clustered into Group III. Group IV and V had 25 18 LACs, respectively. Also, the maximum LAC proteins from pomegranate were di uted in Group Ⅵ, reaching 34, whereas Group Ⅲ had only one PgLAC51. Considering AtLAC4, AtLAC11, and AtLAC17 were responsible for lignin polymerization [23], it was cerned that PgLACs (PgLAC4, 5, 6, 7, 15, 38, and 50) were clustered into Group I with AtL and AtLAC11, furthermore, PgLACs (PgLAC1, 16, 17, 18, 32, and 37) along with AtLA belonged to Group Ⅱ (Figure 3), which indicated that they had similar function. Subsequently, the amino acid sequences of the above-mentioned 13 PgLACs aligned with the LACs of other species. The results showed that the PgLAC proteins higher similarity with other LAC proteins and contained three Cu oxidase dom namely, Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3 ( Figure 4).  Subsequently, the amino acid sequences of the above-mentioned 13 PgLACs were aligned with the LACs of other species. The results showed that the PgLAC proteins had higher similarity with other LAC proteins and contained three Cu oxidase domains, namely, Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3 (Figure 4). In addition, the LAC family in other species was investigated and listed ( Table 2). The LACs were clustered into five or six subfamilies, except eight subfamilies from S. miltiorrhiza and Solanum melongena. Group I and V from most species contained more LACs. However, P. granatum and S. melongena had the most members in Group VI and VIII, respectively. A total of 93 LACs were identified from Glycine max, ranking first. Secondly, 65 LACs were obtained from S. miltiorrhiza. Among four fruit tree plants, P. granatum had 57 LACs, P. persica for 48, P. bretschneideri for 41, and C. sinensis for 27. At present, the number of LACs from A. thaliana is less, only 17. Based on the phylogenetic relationships, In addition, the LAC family in other species was investigated and listed ( Table 2). The LACs were clustered into five or six subfamilies, except eight subfamilies from S. miltiorrhiza and Solanum melongena. Group I and V from most species contained more LACs. However, P. granatum and S. melongena had the most members in Group VI and VIII, respectively. A total of 93 LACs were identified from Glycine max, ranking first. Secondly, 65 LACs were obtained from S. miltiorrhiza. Among four fruit tree plants, P. granatum had 57 LACs, P. persica for 48, P. bretschneideri for 41, and C. sinensis for 27. At present, the number of LACs from A. thaliana is less, only 17. Based on the phylogenetic relationships, the 57 PgLACs can be divided into six subfamilies (I-VI), and Group I contained 7 members, 6 members in Group II each contained, 1 member in Group III, 4 members in Group IV, 5 members in Group V, and 34 members in Group VI.

Motif Distribution and Exon/Intron Analysis of PgLAC Family Members
The MEME result showed that 10 conserved motifs were presented in PgLACs ( Figure 5). The length of the 10 motifs was 21-50 aa, and the motif sequences were provided in Figure S1, which encoded multicopper oxidase and belonged to typical plant laccases. Among them, motif1, motif5, motif8 encoded multicopper Cu-oxidase_3; motif3 and motif7 encoded multicopper Cu-oxidase; motif2, motif4, motif6, and motif9 encoded multicopper Cu-oxidase_2. As shown in Figure 5, 50 PgLACs all contained the 10 motifs, and most PgLACs ended with the order of motif9, motif6, motif4, and motif2 except PgLAC45, suggesting that PgLAC gene members possessed relatively conserved sequences. Additionally, among 57 members of PgLAC family, PgLAC26, PgLAC33, PgLAC34, and PgLAC35 all contained one more motif 2; motif 5 did not occur in PgLAC10, PgLAC29, PgLAC44, PgLAC46, PgLAC52, and PgLAC56; PgLAC10 and PgLAC46 also missed motif 1 and motif 8. Therefore, the motif distribution displayed the specificity of the gene structure of PgLACs, perhaps resulting in different functions. To better understand the structural characteristics of PgLAC genes, their exon-intron structures were explored. As shown in Figure 5, 57 PgLACs exhibited diverse intron/exon patterns, and the number of exons ranged from 4 to 10. Among PgLACs, 23 and 20 PgLACs contained 7 and 6 exons, respectively.

Analysis of Cis-Acting Elements in PgLACs Promotors
The cis-acting elements were obtained from the 2000-bp upstream sequence of PgLACs, so as to investigate the possible function of PgLACs. As shown in Figure 6, in PgLACs promotors were observed 51 cis-acting elements involving hormone response, stress response, and development response. Among hormone-responsive elements, the abscisic acid responsiveness element (ABRE) was the most common and appeared on the upstream sequences of 48 PgLACs, reaching a maximum of 10 on the upstream sequences of PgLAC12. The second one was MeJA responsiveness elements (CGTCA-motif and TGACG-motif), which existed on the upstream sequences of 42 PgLACs with 1-3, furtherly, 5 PgLACs (PgLAC10, PgLAC11, PgLAC20, PgLAC21, and PgLAC49) contained 4 or 5 ( Figure 6). Also, other important hormone-responsive elements were discovered, such as salicylic acid responsiveness element (TCA-element), auxin responsiveness elements (TGA-element, AuxRR-core, and AuxRE), and gibberellin responsiveness elements (GARE-motif, P-box, and TATC-box). Thereby, abscisic acid and methyl jasmonate may greatly participate in modulating the expression of PgLACs. Among the cis-acting elements involved in stress response, the light-responsive elements were the most abundant in 53 PgLACs promoters and G-box in 51 PgLACs promotors. In particular, the upstream sequences of PgLAC1 and PgLAC53 contained 10 Box 4, respectively, and the upstream sequences of PgLAC50 had 10 G-box. Meanwhile, the low-temperature responsiveness element (LTR), MBSI (involved in flavonoid biosynthesis), drought stress responsiveness element (MBS), defense and stress responsiveness element (TC-rich repeat), etc., were discovered. Regarding plant development, eight cis-acting elements were detected on the upstream sequences of PgLACs. O 2 -site involved in zein metabolism regulation was the most common, existing in 22 PgLACs promotors, while the least for HD-Zip 1, only 1 in PgLAC50 promotor ( Figure 6).

Analysis of Cis-Acting Elements in PgLACs Promotors
The cis-acting elements were obtained from the 2000-bp upstream sequence of PgLACs, so as to investigate the possible function of PgLACs. As shown in Figure 6, in PgLACs promotors were observed 51 cis-acting elements involving hormone response, stress response, and development response. Among hormone-responsive elements, the abscisic acid responsiveness element (ABRE) was the most common and appeared on the upstream sequences of 48 PgLACs, reaching a maximum of 10 on the upstream sequences of PgLAC12. The second one was MeJA responsiveness elements (CGTCA-motif and TGACG-motif), which existed on the upstream sequences of 42 PgLACs with 1-3, furtherly, 5 PgLACs (PgLAC10, PgLAC11, PgLAC20, PgLAC21, and PgLAC49) contained 4 or 5 ( Figure 6). Also, other important hormone-responsive elements were discovered, such as salicylic acid responsiveness element (TCA-element), auxin responsiveness elements (TGA-element, AuxRR-core, and AuxRE), and gibberellin responsiveness elements (GARE-motif, P-box, and TATC-box). Thereby, abscisic acid and methyl jasmonate may greatly participate in modulating the expression of PgLACs. Among the cis-acting elements involved in stress response, the lightresponsive elements were the most abundant in 53 PgLACs promoters and G-box in 51 PgLACs promotors. In particular, the upstream sequences of PgLAC1 and PgLAC53 contained 10 Box 4, respectively, and the upstream sequences of PgLAC50 had 10 G-box. Meanwhile, the low-temperature responsiveness element (LTR), MBSI (involved in flavonoid biosynthesis), drought stress responsiveness element (MBS), defense and stress responsiveness element (TC-rich repeat), etc., were discovered. Regarding plant development, eight cis-acting elements were detected on the upstream sequences of PgLACs. O2-site involved in zein metabolism regulation was the most common, existing in 22 PgLACs promotors, while the least for HD-Zip 1, only 1 in PgLAC50 promotor ( Figure 6).

Analysis of Protein Interaction Networks
The co-expression of 57 PgLAC proteins was predicted using the String protein interaction database; A. thaliana was the model species. The stronger reaction between the two proteins, the thicker the linkage line. As Figure 7a shown, PgLAC proteins were identical to AtLAC1, AtLAC3, AtLAC5, IRX12 (AtLAC4), AtLAC7, AtLAC11, AtLAC14, TT10 (At- Figure 6. Cis-acting elements analysis on the promoter of PgLACs. The numbers represented the number of cis-acting elements in each PgLAC promotor. The blue represents the hormone response, the green the stress response, and the red for the developmental response.

Analysis of Protein Interaction Networks
The co-expression of 57 PgLAC proteins was predicted using the String protein interaction database; A. thaliana was the model species. The stronger reaction between the two proteins, the thicker the linkage line. As Figure 7a shown, PgLAC proteins were identical to AtLAC1, AtLAC3, AtLAC5, IRX12 (AtLAC4), AtLAC7, AtLAC11, AtLAC14, TT10 (AtLAC15), and AtLAC17, respectively. Also, IRX12, AtLAC11, and AtLAC17 had high homology and co-expressed. PgLAC4, PgLAC5, PgLAC6, PgLAC7, PgLAC38, and PgLAC50 were identical to IRX12 and co-expressed with fasciclin-like arabinogalactan-protein FLA11 which was related to secondary cell-wall cellulose synthesis, and galacturonosyltransferase GAUT12 which involved in pectin assembly (Figure 7b). PgLAC1, PgLAC16, PgLAC17, PgLAC18, PgLAC32, and PgLAC37 were identical to AtLAC17, and co-expressed with IRX12, chitinase-like protein CTL2, cellulose synthase A catalytic subunit 4 (CESA4), and cellulose synthase A catalytic subunit 8 (IRX1) (Figure 7c). PgLAC15 was identical to AtLAC11 and co-expressed with DMP2, which is involved in membrane remodeling and xylem cysteine peptidase XCP1 (Figure 7d). Figure 6. Cis-acting elements analysis on the promoter of PgLACs. The numbers represented the number of cis-acting elements in each PgLAC promotor. The blue represents the hormone response, the green the stress response, and the red for the developmental response.

Expression Profiles of PgLACs during the Seed Development
To clarify the potential function of PgLACs, the expression level of 13 PgLACs (PgLAC1, PgLAC4, PgLAC5, PgLAC6, PgLAC7, PgLAC15, PgLAC16, PgLAC17, PgLAC18, PgLAC32, PgLAC37, PgLAC38, and PgLAC50) was assessed during four seed development stages of soft/hard-seed pomegranate using RT-PCR method. The PgLACs exhibited different expression levels during seed development; importantly, a significant difference in expression levels was found between soft-and hard-seed pomegranates (Figure 8). And, the expression of most PgLACs was higher during the earlier stage of seed development (30-70 d after full flowering) than at the later stage of seed development (120 d after full flowering) between the two pomegranate cultivars except PgLAC4. Moreover, 5 PgLACs (PgLAC1/7/32/38/50) had not a significant difference in the gene expression level at 120 d after full flowering, which demonstrated that the formation of seed hardness depended on the earlier stage of seed development. Meanwhile, during the earlier seed development stage, PgLAC1 and PgLAC7 expressed at a significantly higher level in soft-seed 'Tunisia' than in hard-seed 'Taishanhong'; on the contrary, PgLAC50 displayed a significantly higher expression in 'Taishanhong' than in 'Tunisia' (p < 0.01). Similarly, during the whole seed development, PgLAC6 and PgLAC16 expressed at a significantly higher level in 'Tunisia' than in 'Taishanhong', while the expression of PgLAC37 always kept a significantly higher level in 'Taishanhong' than in 'Tunisia' pomegranate (p < 0.01) (Figure 8). Therefore, it was inferred that the soft-seed development might be a close relationship with PgLAC1, PgLAC6, PgLAC7, and PgLAC16; correspondingly, PgLAC37 and PgLAC50 may greatly participate in hard-seed development in pomegranate. In addition, the peak of gene expression at 30 d, 45 d, and 70 d after full flowering each appeared 4 PgLACs gene in 'Tunisia', while in 'Taishanhong', 7 PgLACs at 30 d after full flowering, and 3 PgLACs at 70 d and 2 PgLACs at 120 d after full flowering. Collectively, Combined with Figure 1, the earlier stage of seed development was the key to the formation of seed hardness and lignin accumulation for hard-seed pomegranate. ment. Meanwhile, during the earlier seed development stage, PgLAC1 and PgLAC7 expressed at a significantly higher level in soft-seed 'Tunisia' than in hard-seed 'Taishanhong'; on the contrary, PgLAC50 displayed a significantly higher expression in 'Taishanhong' than in 'Tunisia' (p < 0.01). Similarly, during the whole seed development, PgLAC6 and PgLAC16 expressed at a significantly higher level in 'Tunisia' than in 'Taishanhong', while the expression of PgLAC37 always kept a significantly higher level in 'Taishanhong' than in 'Tunisia' pomegranate (p < 0.01) (Figure 8). Therefore, it was inferred that the soft-seed development might be a close relationship with PgLAC1, PgLAC6, PgLAC7, and PgLAC16; correspondingly, PgLAC37 and PgLAC50 may greatly participate in hard-seed development in pomegranate. In addition, the peak of gene expression at 30 d, 45 d, and 70 d after full flowering each appeared 4 PgLACs gene in 'Tunisia', while in 'Taishanhong', 7 PgLACs at 30 d after full flowering, and 3 PgLACs at 70 d and 2 PgLACs at 120 d after full flowering. Collectively, Combined with Figure 1, the earlier stage of seed development was the key to the formation of seed hardness and lignin accumulation for hard-seed pomegranate.

Discussion
In general, hard-seed pomegranate cultivars are more resistant to cold stress, whereas soft-seed ones are more popular with consumers due to easily swallowed seeds [32,33]. Seed hardness has become the first preference for more customers. In China, 'Taishanhong' (hardseed) and 'Tunisia' (soft-seed) are widely cultivated pomegranate cultivars, which play an

Discussion
In general, hard-seed pomegranate cultivars are more resistant to cold stress, whereas soft-seed ones are more popular with consumers due to easily swallowed seeds [32,33]. Seed hardness has become the first preference for more customers. In China, 'Taishanhong' (hard-seed) and 'Tunisia' (soft-seed) are widely cultivated pomegranate cultivars, which play an important role in promoting the pomegranate industry. In the present study, it was found that seed hardness and lignin content both increased steadily in 'Taishanhong' and 'Tunisia' seeds as seed development, with a significantly lower level in 'Tunisia' seeds than in 'Taishanhong' ones. Furthermore, lignin content at 45 d after full flowering increased by 7.7% than 30 d after full flowering, whereas that at 120 d after full flowering increased by 3.3% than 70 d after full flowering; thereby, more lignin deposition may be conducted at the earlier stage of seed formation. Similarly, Niu et al. also proved that the soft-seeded variety contained lower lignin at the early fruit developmental stage [34]. To explore the formation of seed hardness is essential to breed new soft-seed pomegranate germplasm, furtherly, other fruit trees.
The proteins interaction networks of 57 PgLAC showed PgLAC1, PgLAC16, PgLAC17, PgLAC18, PgLAC32, and PgLAC37 were co-expressed with CTL2, IRX1, and IRX12 involved in cell wall biosynthesis, further supporting the involvement of the PgLACs in lignin biosynthesis. A previous study reported that the three Arabidopsis LACs (AtLAC4, AtLAC17, and AtLAC11) were responsible for lignin polymerization [23]. The phylogenetic tree was constructed and showed that 57 PgLACs were divided into six groups, seven PgLACs (PgLAC4, 5, 6, 7, 15, 38, and 50) in Group I with AtLAC4 and AtLAC11, and six PgLACs (PgLAC1, 16, 17, 18, 32, and 37) in Group II along with AtLAC17. The results implied the 13 PgLACs may possess similar functions to the Arabidopsis LACs. Therefore, the expression profiles of the 13 PgLACs were investigated during the four seed development stages. We provided the evidence that most PgLACs expressed at a higher level during the earlier stage of seed development than at the later stage of seed development in the two pomegranate cultivars except PgLAC4, which was in line with higher lignin content at the earlier seed development stage. However, the distinct difference in the gene expression levels of individual PgLAC existed between cultivars, higher PgLAC1, PgLAC6, PgLAC7, and PgLAC16 in the soft-seed cultivar, whereas higher PgLAC37 and PgLAC50 in the hard-seed pomegranate cultivar.
G-box widely presents on the promoter of many plant genes with a palindromic DNA motif of CACGTG [35]. Previous reports found that G-box participated in the co-expression system for nuclear photosynthetic genes and influenced organ differentiation [36]. In the current study, the gene expression of PgLAC37 and PgLAC50 during the first 70 d after full flowering displayed higher with a significant difference in 'Taishanhong' than in 'Tunisia'; further, the more G-box elements presented in PgLAC37 with 7 G-box and PgLAC50 with 10 ones, suggesting that the two genes were the key candidate gene for the formation of the hard seed in pomegranate. Similarly, more ABRE elements involved in abscisic acid (ABA) responsiveness were also observed in the PgLAC37 (6 ABRE) and PgLAC50 (9 ABRE). ABA is involved in secondary cell-wall formation [37], and the late-wood formation of Pinus radiata and Pinus sylvestris is correlated with an increase in ABA concentration [38,39]. Taken together, the G-box and ABRE elements in the promotors of PgLAC37 and PgLAC50 may be an essential reason for modulating the higher expression of PgLAC37 and PgLAC50 during the earlier seed development stage of 'Taishanhong' pomegranate, thus, the two genes greatly participated in seed formation and accelerated seed maturity.
Gibberellin (GA) is a primarily growth-regulating phytohormone and regulates diverse biological processes. Previous studies revealed that GA induced berry seedless and regulated flower development, berry set, expansion, and ripening in grapes [40][41][42]. TATC-box and GARE-motif are known to both respond to GA. Interestingly, we found TATC-box in only PgLAC1 promotor and GARE-motif in the promotors of PgLAC6 and PgLAC7, as well as PgLAC4 and PgLAC5 among 13 PgLACs. Collectively, higher expression of PgLAC1/4/6/7 in 'Tunisia' may be induced by GA and then produce softer seeds. The five PgLACs played an indispensable role in the formation of softer seeds. RY-element is found predominantly in seed-specific promoters [43] and mediates repression of embryo mid-maturation genes involved in the accumulation of storage compounds [44]. In the current study, RY-element was observed only in the PgLAC16 promotor, which suggests that the dramatically higher expression of PgLAC16 may inhibit the accumulation of storage compound during the seed development stage, thus hindering the formation of seed hardness in soft-seed pomegranate. In conclusion, PgLAC1/4/6/7/16 with higher expression in 'Tunisia' will greatly contribute to exploring the soft seed formation, which is potential candidate gene for breeding soft-seed pomegranate with GA application.

Conclusions
Laccase is the key enzyme on the lignin biosynthesis pathway, closely correlated with seed hardness. The LAC family was first identified from the pomegranate genome. A total of 57 PgLACs were divided into six groups containing typical Cu-oxidase domains. Exon-intron structure and motif analysis predicted that the PgLACs had diverse functions in lignin biosynthesis. Combined with cis-acting elements and the gene expression patterns, PgLAC37 and PgLAC50 were the key candidate genes for the formation of hard seed in pomegranate, attributed to more G-box and ABRE elements in their promotors, which regulated the expression of PgLAC37 and PgLAC50, participated in seed formation, accelerated seed maturity, finally, produced harder seed. In soft-seed pomegranate, higher expression of PgLAC1/4/6/7 may contribute to soft-seed formation responsive to GA via GARE motif and TATC-box. And the PgLAC16 promotor containing RY-element may regulate soft seed development by reducing the accumulation of storage compounds in seeds. Collectively, the results of our study will provide important gene information and a new perspective for breeding hard-and soft-seed pomegranate cultivars.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae9080918/s1, Figure S1: The sequence information for each motif (Motif 1-Motif 10); Figure S2: Segmental duplication of the PgLAC genes in pomegranate; Table S1: LACs information in the phylogenetic tree; Table S2: The primers used in qRT-PCR; Table S3: Segmental duplication pairs of the PgLAC genes within the pomegranate genome; Table S4: The identified tandem duplication pairs of PgLAC genes.