Genome-wide identification and expression analysis revealed cinnamyl alcohol dehydrogenase genes correlated with fruit-firmness in strawberry 1

Abstract

BACKGROUND:

Strawberry fruits are perishable with a short post-harvest life. Cinnamyl alcohol dehydrogenase (CAD) is the key enzyme for lignin biosynthesis strengthening plant cell wall. A systematic characterization of strawberry CAD family is absent and their involvement in fruit firmness is largely elusive.

OBJECTIVE:

Current work aims for a genome-wide identificationof CAD family and its expression correlation with fruit firmness in strawberry varieties.

METHODS:

A genome-scale identification and molecular characterization for CADs were performed in the commercial strawberry (Fragaria×ananassa) and woodland strawberry (F. vesca). qPCR analysis of CAD homoeologs in three cultivars varying with fruit firmness revealed candidate CAD members positively correlated with lignin content and fruit firmness.

RESULTS:

A total of 14 and 24 CAD loci were identified in the genomes of F.vesca var. Hawaii4 and F. ×ananassa cv. Camarosa, respectively.Phylogenetic analysis supported a division of this family into three classes. Class I FvCAD each has four homoeologs in commercial strawberry, while those of Class II and Class III have only one or two homoeologs. Except for FvCAD2 and -6, there exits at least one pair of CADs sharing ∼97% or above amino acid identity between F. vesca and F. ×ananassa.The flesh firmness and lignin content varied greatly among strawberry germplasm. Distinct dynamic changes in fruit lignin content were observed before the large green stage, but fruit firmness displayed a similar decrease profile during fruit development in three varieties. Of the eight genes detected in F.×ananassa, FvCAD3 and -12 did not display a F. vesca-biased expression pattern during fruit development.FvCAD4 of Class I was expressed at levels positively correlated with variation in fruit lignin content at white stage.Transcript abundance of five Class IIgenes including FvCAD3, -8, -10, -11, and -12 was positively correlated with lignin content and fruit firmness, with FvCAD10 and -11 (FaCAD in previous publication) reaching an extremely significant correlation with the genetic variation in fruit firmness across three varieties.

CONCLUSION:

Strawberry Class II CADs were significantly correlated with the genetic variation in fruit firmness, which might expand the potential choices for improving strawberry shelf life.

Keywords

Strawberry cinnamyl alcohol dehydrogenase fruit firmness lignin content gene expression

1 Introduction

Lignin functions in strengthening plant secondary cell walls, as one type of the phenolic polymers heteropolymerized from monolignols. Monolignols include guaiacyl (G)-, syringyl (S)-, and p-hydroxyphenyl (H)- units derived from coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohols, respectively. In loquat (Eriobotrya japonica), mangosteen, and strawberry, lignin contents were directly related with flesh firmness [1 –4]. A visible correlation of lignin content with mechanical stiffness was observed in poplar plants [5].

There are eleven enzyme families implicated in monolignol biosynthesis.Cinnamyl alcohol dehydrogenase (CAD), the key enzyme catalyzing the final step in the synthesis of many monolignols, is active as a dimer and belonging to the zinc-containing alcohol dehydrogenase family in the medium chain dehydrogenase/reductase (MDR) superfamily [6 –8]. The CAD activity was first characterized in Picea abies and Glycine max [9]. The first cDNA-encoding zinc-binding CAD was isolated from tobacco [10]. CAD genes have evolved into a large family in plants with high homology, accompanied with a diversification of biochemical functions [11]. Phylogenetic analysis supported the division of plant CADs into three classes, with Class I being considered as bona fide CADs [8 , 12–17]. CADs are multi-functional, with some members contributing to both developmental lignification and defense responses [6 , 18–20].

There are nine CAD coding genes in Arabidopsis [21]. AtCAD-C and -D (AtCAD4 and -5) of Class I are primarily involved in the synthesis of both coniferyl and sinapyl alcohols (precursors of syringyl and guaiacyl monolignols) [19 , 23]. Several AtCADs of Class II and III are predominantly expressed in vascular (lignin-forming) tissues [24]. Populus genome harbors 15 CAD loci divided into three classes, with Class I members preferentially expressed in lignified tissues [8]. Fifteen LuCADs were identified in flax (Linum usitatissimum L.), and genes of class I and II were proposed to being responsible for lignin synthesis in different organs [25]. In a Rosaceous species pear, 26 CADs were identified and the authentic CAD2 was confirmed to role in lignin synthesis of fruit stone cells in both Pyrus bretschneideri [26] and P.pyrifolia [27, 28].

Commercial strawberry (F. ×ananassa) is cultivated worldwide and appreciated deeply due to its attractive and nutritious fruits with palatable flavor. Most commercial strawberry varieties are heterogenous octoploid with high genetic variations and polymorphism, which has hampered the dissection of molecular mechanisms regulating strawberry agricultural traits for a long time. The diploid woodland strawberry F. vesca has served as a valuable model genomic system for commercial strawberry over one decade [29, 30]. Considering the origin and evolution of strawberry, F. vesca has been identified as the single dominant subgenome provider controlling many agronomic traits in commercial strawberry [31].

Strawberry fruits are highly perishable with a short shelf life due to a quick softening during the late stage of ripening. Cell wall disassembly and an extensive dissolution of the middle lamella between the cortical parenchyma cells are the main causes for strawberry fruit softening [32 –34]. Cell wall disassembly in ripe fruit mainly results from pectin solubilization and depolymerisation, combining with a slight reduction in the molecular weight of hemicellulosepolymers [35 –37]. The coordinated hormonal regulation of the expression of many cell wall degrading enzymes and the massive transcriptional change, forms a complex, genetically programmed network controlling strawberry softening [34 , 38–42].

Strawberry fruit lignification was revealed to start at the apical zone of the achene and gradually progress to the receptacle, and CAD genes FxaCAD1 (U63534) and -2 were supposed to be involved in lignification processes related to both vasculature development and achene maturation in cv. Chandler [43]. A CAD (AAK28509) gene (renamed FaCAD) was highly expressed in ripe fruits of cv. Holiday (a firm cultivar) and cv. Elsanta (an intermediately firm cultivar), while weakly expressed in the soft cv. Gorella [44]. The FaCAD (JX290511 from cv. Elsanta) shared 98.1% amino acid identity with FxaCAD1 from cv. Chandler. A functional study revealed that the enhanced fruit firmness in agroinfiltrated strawberry fruits was positively associated with an increase in lignin production in fruits of cv. Calypso, although the gene silence effect was shielded due to a similar increase of defense lignin biosynthesis triggered by agroinfiltration in both FaCAD-silenced and control fruits [45]. As a multigene family, strawberry CADs in previous studies were fragmentary and limited.

So far, there is short of a genome-wide study of CADs family in both diploid and commercial strawberries. Current work aimed to investigate the potential relationships between CADs family and fruit firmness based on a systematic characterization, subsequently to build a basis for furthering functional dissection of CADs in strawberry. First, a comprehensive identification and phylogenetic study was conducted for CAD-like genes in the genomes of F. vesca and F. ×ananassa. The molecular features of this family were characterized in F. vesca. Then, a survey of phenotypic variation in fruit firmness and lignin content was performed in F. ×ananassa germplasm. Furthermore, an expression profiling was performed for CADs among different genotypes with varying fruit firmness. This work enabled an evaluation of the potential correlation among CADs transcript levels with lignin content and fruit firmness.

2 Materials and methods

2.1 CAD genes identification in the genomes of F. vesca and F. ×ananassa

To identify CAD genes, the HMMER profiles [46] of Alcohol dehydrogenase GroES-like domain (ADH_N, PF08240) and Zinc-binding dehydrogenase domain (ADH_zinc_N, PF00107) were downloaded from http://pfam.xfam.org/ [47]. Next, a local BLAST search against the F. vesca genome v4.0.a2 protein database [48] was conducted for all candidates using the BioEdit program version 7.2.5. PFAM analysis was performed to select FvCAD genes with an E-value lower than 1E-5. Putative CADs in F. ×ananassa were identified through searching genes and transcripts with keywords against the database of F ×ananassa cv. Camarosa Genome Assembly v1.0 & Annotation v1.0.a1 at Genome Database of Rosaceae (GDR) [49]. An inventory of FvCADs and FaCADs was listed in Supplementary Table S1.

2.2 Phylogenetic analysis and in silico characterization

Phylogenetic analysis was performed based on the CAD protein sequences from strawberry and other plant species using a neighbor-joining (NJ) statistical method. The corresponding tree was constructed using 1000 bootstrap replicates in MEGA v7.0 [50] and visualized via the interactive Tree Of Life (iTOL) [51].

Information for the chromosomal locations of FvCADs was collected from Strawberry Genome v4.0.a2 in GDR (www.rosaceae.org/). Based on the relative lengths of all seven chromosomes in F. vesca [52], the chromosomal distribution of FvCAD genes was displayed using TB tools [53] (Supplementary Figure S1). Analysis of the subcellular localization information for deduced FvCAD proteins was performed at http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/. Molecular weight (MW) and theoretical isoelectric point (pI) prediction were acquired at https://web.expasy.org/cgi-bin/compute_pi/pi_tool/.

Information for exon-intron organization with gff3 format was hunt from F. vesca V4.0 genome database in GDR [48] and shown in IBS1.0 (Illustrator for Biological Sequences) [54]. Conserved protein domain module was analyzed at PFAM and illustrated with IBS1.0. Multiple sequence alignment performed at EBI Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustal/), was visualized using Jalview (https://www.jalview.org/) [55]. The percentage of shared amino acid identity among FvCADs was listed in Supplementary Table S2.

2.3 Strawberry materials and fruit firmness analysis

Strawberry materials used in this study were grown in the trial station of Shanghai Academy of Agricultural Sciences (SAAS), located in Zhuanghang Town, Fengxian District, Shanghai. Fruits from a collection of 23 strawberry varieties (seven released cultivars and 16 breeding materials) were included for the survey of fruit firmness variation. For each genotype, at least 12 fruits were measured in one test. Fruit sampling and firmness test were independently repeated three times during Dec. 2020 to Jan. 2021. The FHM-1 type fruit firmness tester with a conical sensor (Japan, Takemura) was used. Two opposite sites on fruit shoulder were targeted for measuring.

For three varieties varying with fruit firmness including ‘Sweet Charlie’, ‘Benihoppe’, and ‘Tokun’, 12 fruits were tested and independently repeated three times in one week, at five different stages corresponding to the small green (G1), large green (G2), white (W), turning (T), and ripe (R) stages. For RNA analysis and lignin analysis, at least eight fruits (receptacles without achenes) of a certain stage from different plants were pooled and immediately stored in liquid nitrogen. Fruits were stored at –74°C until further analysis.

2.4 RNA purification and RT-PCR

Extraction of the total RNA from strawberry fruits and RT-PCR analysis were performed as previously described [56]. Briefly, the OMEGA Plant RNA Kit (OMEGA Bio-tek, Inc. Cat#R6827-2, USA) and the HiScript III RT SuperMix for qPCR kit with gDNA wiper (Vazyme, Lot#R323, Nanjing, China) were used for RNA purification and reverse transcription, respectively. Semi-quantitative RT-PCR was performed on an ETC 821 DNA amplifier (Eastwin, Beijing, China). Real-time quantitative PCR (qPCR) assays were conducted on a Light Cycler 480 (Roche, German) using ChamQ^™ Universal SYBR qPCR Master Mix (Vazyme, Lot#Q711, Nanjing, China).

RT-PCR primers matching both each FvCAD and homoeologous FaCADs were designed based on sequence alignment to avoid single nucleotide polymorphisms. Primer sequence information was shown in Supplementary Table S3. Two reference genes FaGAPDH1 and FaCHP1 were used for the normalization of relative expression [57, 58]. qPCR analysis was performed using the 2^-ΔΔCT method combined with an analysis of primer amplification efficiency and a normalization factor calibrated with geNorm software [59].

2.5 Lignin determination in strawberry fruits

Lignin content was determined by using a kit (Qiyi Bio Tech, Shanghai, Cat. No. QYS-237008) following the producer’s instructions [60]. Briefly, the homogenized flesh (without achenes) powder was dried at 80°C for 7-8 h. About 100 mg dried fruit power were weighed into a 2.0 ml centrifuge tube, and 500μl sulfuric acid and 20μl perchloric acid were added. When preparing for a standard sample, 50 mg lignin was dissolved with 1 ml perchloric acid (stored at 4) and 20μl was used for reaction with sulfuric acid. Then these tubes were thoroughly sealed with plastic film, fully mixed and maintained at 80 in a water bath for 40 min (shaken every 10 min) for acetylation. After these tubes were cooled to room temperature, 500μl NaOH solution was added and fully mixed. Reaction tubes were then centrifuged at 8000 g 10 min, and 20μl supernatant was mixed with 980μl acetic acid. For measuring the lignin content, 200μl sample solution was transferred in to a 96-well plate. The absorbance value of the blank, the measuring and standard tubes was determined at 280 nm by an ELISA instrument (Spark, TECAN, Switzerland). The results were expressed as milligram lignin per gram of dried flesh powder.

2.6 Correlation evaluation

Three biological repeats for fruit samples at certain stage of all three varieties were subjected for the determination of firmness and lignin content, as well as RNA analysis. Relative transcript abundance from qPCR was obtained for CADs in three biological repeats of each variety. A Pearson correlation analysis using IBM SPSS Statistics software (version 20.0) was performed. Among three strawberry varieties at certain developmental stage, a total of six to nine pairs of fruit firmness, lignin content, and relative mRNA values were included for correlation analysis between lignin and firmness, between FvCAD transcript abundance and lignin content, as well as between FvCAD expression level and fruit firmness. The two-tailed test separated all means with a statistical significance at the 0.05 probability level.

3 Results

3.1 Identification and phylogenetic analysis of CAD genes in the genomes of F. vesca and F. ×ananassa

There exists a multiloci family of CADs in both F. vesca and F. ×ananassa. CAD gene identification was first performed in the dominant subgenome provider (F. vesca) of F.×ananassa. A local BLASTp search in Bioed it with the HMMER profiles of Alcohol dehydrogenase GroES-like domain (ADH_N, PF08240) and Zinc-binding dehydrogenase domain (ADH_zinc_N, PF00107) against the FvH4.0.a2 protein database [48] facilitated the identification of 14 CAD genes in the F. vesca genome (for short, FvCAD) (Table 1). To reveal the evolutionary relationships for FvCADs, a phylogenetic tree was generated together with the orthologs from Arabidopsis thaliana and the Rosaceae plant peach (Prunus persica) (Fig. 1A). Following the classification method previously suggested [8, 21], 14 FvCADs were divided into three classes definitely. The nomenclature of FvCADs was largely based on their similarity with the corresponding Arabidopsis orthologs. FvCAD4 and -5 together with three peach CADs were clustered with AtCAD4 and -5 in the bona fide Class I. Class III consisted of AtCAD1, and two orthologs in strawberry (FvCAD1 and -2) and P. persica. The rest 10 FvCADs were grouped into Class II. The shared identity in amino acid sequences ranged from 52.7% to 97.2% for FvCADs of a same class, and from 40.6% to 53% for those among different classes (Supplementary Table S2).

Table 1
The genomic and biochemical information for 14 cinnamyl alcohol dehydrogenase (CAD) genes identified in strawberry (F. vesca) genome v4.0 (annotation v4.0.a2)

Name Gene ID (v4.0.a2) Gene ID (v2.0) Chromosome location DNA-bp mRNA-bp AA^a MW^b PI^c

FvCAD1 FvH4_5g10200 gene01839 Fvb5:5837238–5839686 2449 1059 352 38.0 6.59

FvCAD2 FvH4_6g40323 gene42469 Fvb6:31877868–31880747 2880 1131 376 41.5 7.18

FvCAD3 FvH4_2g38060 gene08569 Fvb2:27578887–27581862 2976 1113 370 40.5 6.16

FvCAD4 FvH4_2g05132 gene26302 Fvb2:4226793–4229438 2646 1158 385 42.3 5.68

FvCAD5 FvH4_1g16790 gene24025 Fvb1:9724572–9726969 2398 1059 352 38.4 5.57

FvCAD6 FvH4_1g19300 gene20702 Fvb1:11515279–11516900 1622 960 319 34.3 6.60

FvCAD7 FvH4_3g46010 gene01573 Fvb3:38203476–38205447 1972 1092 363 39.3 5.66

FvCAD8 FvH4_1g19265 gene20698 Fvb1:11500537–11502494 1958 1110 369 40.3 6.60

FvCAD9 FvH4_1g23790 gene17840 Fvb1:15640330–15643422 3093 1080 359 38.9 6.26

FvCAD10 FvH4_1g19210 gene24967 Fvb1:11431895–11434771 2877 1086 361 38.7 6.46

FvCAD11 FvH4_1g19281 gene20700 Fvb1:11508957–11510826 1870 1086 361 39.2 6.18

FvCAD12 FvH4_7g07240 gene09243 Fvb7:7215483–7218081 2599 1086 361 39.0 6.31

FvCAD13 FvH4_1g19261 gene20696 Fvb1:11484143–11486263 2121 1311 436 47.5 8.0

FvCAD14 FvH4_1g19240 gene24970 Fvb1:11440881–11442866 1986 1095 364 39.3 6.46

Name	Gene ID (v4.0.a2)	Gene ID (v2.0)	Chromosome location	DNA-bp	mRNA-bp	AA^a	MW^b	PI^c
FvCAD1	FvH4_5g10200	gene01839	Fvb5:5837238–5839686	2449	1059	352	38.0	6.59
FvCAD2	FvH4_6g40323	gene42469	Fvb6:31877868–31880747	2880	1131	376	41.5	7.18
FvCAD3	FvH4_2g38060	gene08569	Fvb2:27578887–27581862	2976	1113	370	40.5	6.16
FvCAD4	FvH4_2g05132	gene26302	Fvb2:4226793–4229438	2646	1158	385	42.3	5.68
FvCAD5	FvH4_1g16790	gene24025	Fvb1:9724572–9726969	2398	1059	352	38.4	5.57
FvCAD6	FvH4_1g19300	gene20702	Fvb1:11515279–11516900	1622	960	319	34.3	6.60
FvCAD7	FvH4_3g46010	gene01573	Fvb3:38203476–38205447	1972	1092	363	39.3	5.66
FvCAD8	FvH4_1g19265	gene20698	Fvb1:11500537–11502494	1958	1110	369	40.3	6.60
FvCAD9	FvH4_1g23790	gene17840	Fvb1:15640330–15643422	3093	1080	359	38.9	6.26
FvCAD10	FvH4_1g19210	gene24967	Fvb1:11431895–11434771	2877	1086	361	38.7	6.46
FvCAD11	FvH4_1g19281	gene20700	Fvb1:11508957–11510826	1870	1086	361	39.2	6.18
FvCAD12	FvH4_7g07240	gene09243	Fvb7:7215483–7218081	2599	1086	361	39.0	6.31
FvCAD13	FvH4_1g19261	gene20696	Fvb1:11484143–11486263	2121	1311	436	47.5	8.0
FvCAD14	FvH4_1g19240	gene24970	Fvb1:11440881–11442866	1986	1095	364	39.3	6.46

^aNumber of amino acid residues of deduced CAD proteins; ^bMolecularweight of the amino acid sequence; kDa is kilo Daltons; ^cIsoelectricpoint.

Fig. 1

Phylogenetic relationships of cinnamyl alcohol dehydrogenase (CAD) proteins in strawberry. (A) The phylogeny analysis for CADs from different plant species. A set of 47 CAD proteins including 24 peach PRUPE_CADs from www.rosaceae.org, nine ArabidopsisAtCADsfrom www.arabidopsis.org, as well as 14 FvCADs from GDR were included for constructing the neighbor-joining (NJ) tree using MEGA7 software [50] with 1000 bootstrap re-samplings and visualized using the interactive Tree Of Life (iTOL) [51]. Colors refer to different classes. (B) Orthology between CADs from diploid woodland strawberry and octoploid commercial strawberry. Amino acid sequences of 14 FvCADs and 24 FaCADs were used for analysis.

Analysis of the physical distribution of FvCADs revealed that this gene family is unequally dispersed on all seven chromosomes (Supplementary Figure S1). FvCAD4 and -5 of Class I were located on Fvb2 and Fvb1, respectively. Interestingly, a set of six genes of Class II comprising FvCAD6, -8, -10, -11, -13, and -14 were clustered on a narrow region of Fvb1, suggesting an independent gene duplication event which probably occurred after the disperse of this family in woodland strawberry genome. In silico analysis of FvCAD deduced proteins suggested a uniform subcellular localization in cytoplasm. By contrast, two pear CADs were localized in both cytoplasm and cytoplasmic membrane [26]. The relative molecular masses of FvCADs range from 34.3 kDa (FvCAD6) to 47.5 kDa (FvCAD13). Most FvCADs display an isoelectric point (pI) lower than 7.0 except for FvCAD2and -13. Comparatively, all 24 PRUPE-CADs from peach genome show a deduced pI lower than 7.0 [61].

Considering the genetic complexity and polymorphism in commercial strawberry, the available genome sequence of strawberry cv. Camarosa has been a great foundation and treasure [31]. Combining BLASTp and keywords (cinnamyl alcohol dehydrogenase) searching in GDR, a total of 26 CAD-like loci were identified in cv. Camarosa genome. Pfam analysis at www.hmmer.org confirmed 24 of all 26 loci encoding proteins belonging to CADs (for short, FaCADs) (Supplementary Table S1). Phylogenetic analysis of 24 FaCADs and 14 FvCADs assigned one to four FaCAD homoeologs to each member of whole family except for FvCAD6 (Fig. 1B). Each FvCAD has at least one homoeolog sharing ∼97% or above amino acid identity in cultivated strawberry with the exception of FvCAD2 and -6.Notably, the bona fide FvCAD4 and FvCAD5 share an amino acid identity with four homoeologs in polyploid strawberry ranging 94.21% ∼97.76%, and 93.75% ∼99.42%, respectively. Most members of Class II, namely, FvCAD7, -8, -10, -11, -12, and -14 only have one corresponding homoeolog in commercial strawberry. Intriguingly, most single copy FaCADs are homoeologous to those FvCADs largely clustered on Fvb1 in woodland strawberry (FvCAD8, -10, -11, -14) (Supplementary Figure S1).

3.2 Structural and molecular characterization of FvCADs

In accordance with the observation in populus [8], the organization of exon-intron in FvCADs could largely be grouped into three patterns (Fig. 2A). All Class II FvCADs except FvCAD6 harbor five exons. Most CADs of class II have the third exon being the longest in coding sequences (CDS) aside from FvCAD3 and -6.This type was named as exon-intron Pattern I, widely observed in monocots and eudicots. FvCAD4 and -5 (Class I) were characterized by five exons in CDS region with the fourth exon being the longest. This feature named after exon-intron Pattern II was similarly observed in other eudicots and basal angiosperms [8]. Class III member (FvCAD1 and -2) and FvCAD6 (Class II) each contains six exons with the fourth being the longest in CDS, which was called exon-intron Pattern III, observed in monocots, eudicots, as well as in gymnosperms.

Fig. 2

Molecular features of CADs in F. vesca.(A) Exon-intron organization(B) Deduced protein domain module. The neighbor-joining tree of these genes was constructed based on the amino acid sequences of 14 FvCADs using 1000 bootstrap replicates in MEGA7.0. The gene structure annotation with gff3 format was hunt from F. vesca V4 genome (FvH4.0.a2 version) [48] and shown in IBS1.0 (Illustrator for Biological Sequences) [54]. Conserved protein domain module was identified in PFAM and illustrated with IBS1.0.

Although FvCAD6 contains a truncated Alcohol dehydrogenase GroES-like domain (ADH_N) which was revealed in PFAM (Fig. 2B), sequence alignment indicated the presence of four highly conserved functional motifs in all 14 members of this family (Supplementary Figure S2). Indeed, all predicted FvCAD proteins have these motifs: (1) 44CGXCX[ST]49 (Catalytic important), (2) 63YPXVPGHE70 (Zinc binding motif), (3) 161APLLCAGXTVY171 (NADPH binding motif), and (4) 188GXGGV(L)G193 (NADPH binding motif), harboring critical and conserved residues (44Cys-His69-Cys165) for CAD activity [62]. Taken together, these observations supported that all FvCADs identified in current study belong to plant CAD protein family of the medium-chain dehydrogenase/reductase (MDR) superfamily [63, 64].

3.3 Phenotypic variation and dynamic change of the firmness and lignin content in F. ×ananassa fruit

Flesh firmness is the crucial factor for the shelf-life of strawberry fruits, which is controlled by genetic and epigenetic factors, simultaneously affected by both cultural practices and natural environmental conditions. Still, the phenotypic variation in fruit firmness trait among different strawberry germplasm could be studied under the same production system. In this study, the flesh firmness in ripe fruits of seven commercial cultivars and 16 breeding materials were repeatedly measured in two months (Fig. 3A). The firmness values of ripe fruits from all 23 varieties fell within a range of 0.14–0.50 kg/cm². Fruits of the American cultivar Sweet Charlie were most firm, with a median firmness value around 0.4 kg/cm². Two Japanese cultivars Benihoppe and Tokun were representative of intermediate and soft genotypes, respectively. In current production system, ripe ‘Benihoppe’ fruits had a firmness value around 0.3 kg/cm², while ‘Tokun’ fruits had a firmness value around 0.18 kg/cm².

Fig. 3

Strawberry fruit firmness is highly variable. (A) The plasticity of flesh firmness in strawberry ripe fruits of different genotypes. The box plot was generated in Origin 2017 with the firmness values of 36 fruits from three independent tests accomplished in two months. Arrows indicate three varieties with different fruit firmness selected for further study. (B) Fruit development in F. ×ananassa cv. Sweet Charlie (firm), cv. Benihoppe (intermediate), and cv. Tokun (soft) at the small green (G1), large green (G2), white (W), turning (T), and ripe (R) stages. (C) Comparative dynamic changes in fruit firmness and lignin contents amongthree strawberry varieties. The fruit firmness was determined by a fruit firmness tester (Japan, Takemura, FHM-1 type with a conical sensor) and expressed in kg/cm². The lignin content was measured by using a test kit (Qiyi Bio Tech, Shanghai) on an ELISA instrument and expressed as mg/g dried flesh powder.

The representative firm cv. Sweet Charlie is widely cultivated in South China. The cultivar Benihoppe developing moderately firm fruits has a most widely geographic distribution in China. By contrast, the soft cultivar Tokun is cultivated in a relatively smaller range. As shown in Fig. 3B, after fruit setting, the size of fruits in all three varieties gradually increased throughout the process of development and maturation. Upon ripening, there occurred a white fruit stage followed by a coloring (turning) stage. The soft variety Tokun experienced a typical white stage, and initiated coloring nearly at a final-size stage, clearly later than the other varieties.

The development and maturation of strawberry fruits were accompanied with a continuous decrease in fruit firmness. Dynamic patterns of strawberry flesh firmness were largely similar in three varieties, but a firmer variety obviously experienced a decrease not as sharp as in a softer variety during the early stage (Fig. 3 Cleft panel). In cv. Tokun, the fruit firmness decreased more steeply from G1 to G2 stage than from W to R stage. Accordingly, the inflection point in the dynamic pattern of ‘Tokun’ firmness was observed at G2 stage, significantly earlier than that observed in the firmer varieties (at W stage). A weak difference between firmness change patterns in cv. Sweet Charlie and cv. Benihoppe could be observed during G2 to W stage, with a relatively gentle decrease in the former than in the latter. By contrast, the dynamic pattern of fruit lignin content was not uniform in three varieties (Fig. 3C right panel). Decisively, lignin content in receptacle of different variety peaked at different stage. Although a continuous decrease in lignin concentration was found in cv. Sweet Charlie, a clear increase did occur in the rest two varieties from G1 (small green fruit) to G2 (large green fruit) stage. In cv. Benihoppe and cv. Tokun, fruit lignin content peaked at G2 stage and was followed by a decrease till ripening similarly observed in ‘Sweet Charlie’. The raw data for fruit firmness and lignin content were provided in box charts (supplementary Figure S3). In addition, there existed a significant correlation between fruit firmness and lignin content through out fruit development, although with a minor decrease in the significance degree along fruit ripening in three varieties (supplementary Table S4).

3.4 Comparative expression patterns of CADs in fruits of F. vesca and F. ×ananassa

Expression analysis would provide insights into a potential contribution of CADs to strawberry flesh firmness. At the beginning, we evaluated whether CADs in commercial strawberry were expressed with a F. vesca-biased pattern. RNAseq data for FvCADs family in woodland strawberry fruits were retrieved from previous reports [65, 66]. CADs expression in seed/achene (wall) and flesh related (cortex and pith) samples or whole fruits (Yw-Yellow wonder; Rg-Ruegen) at different stages was summarized in a heatmap (Fig. 4A). Half of whole family members were obviously expressed in fruits, including FvCAD1, -4, -8, -9, -10, -11, -12. FvCAD4 transcript was notably present in fruits, with a higher level in achenes (wall) than in receptacle, peaking approximately at green fruit stage (stage 3–5). FvCAD9 and -10 were expressed with a relatively higher level in achenes than in flesh too. Expression of FvCAD11 and -8 were largely limited in fruits of 22 days post anthesis (DPA), probably corresponding to the white to turning stages. FvCAD12 transcripts were clearly present in achenes at stage 5 (10–13 DPA). By contrast, the rest seven genes were very weakly expressed (FvCAD7, -14 limited in achenes; FvCAD2, -3, -13 in all fruit parts) or even scarcely detected (FvCAD5, -6) in aforementioned fruit samples.

Fig. 4

Comparative expression patterns of CADs in the fruits of F. vesca and F. ×ananassa.(A) RNA-seq data revealed fruit expression of FvCAD genes in woodland strawberry. The heatmap of FvCADgenes expression [66] was hierarchically clustered using the TBtools package [53] with scale and size of circle representing RPKM normalized log²-transformed counts. Blank - non-expressed (RPKM≤0.3). Stage 1, blooming flower; 2, 2∼4 days post anthesis (DPA); 3, 6∼7 DPA; 4, 8∼10 DPA; 5, 10∼13 DPA. 15/22 D, for 15/22 DPA. (B) Semi-quantitative RT-PCR analysis of 14 CADgenes in F. ×ananassa (cv. Tokun). PCR cycles for CADs is 35, and for the internal control FaCHP1 is 28. Fruits at five stages (G1 - small green, G2 - large green, W - white, T - turning; R - ripe) were harvested without achenes.

To reveal CAD genes probably contributing to fruit firmness in all strawberry varieties, the soft fruit cultivar cv. Tokun was first used for CAD expression analysis. For the sake of simplification, before a locus-specific analysis of CADs in strawberry being performed, in current work RT-PCR primers were designed applicable for amplifying one to four homoeologs (if any) in commercial strawberry based on the phylogeny between FvCADs and FaCADs (Supplementary Table S1, S3). The specificity and applicability of RT-PCR primers were tested in a mixed cDNA template from total RNAs of ‘Tokun’ flesh at all five developmental stages. Finally, expression of six genes including FvCAD2, -5, -6, -7, -13, and -14 were not observed in the flesh tissue of fruits, which met with the analysis in F. vesca. Transcripts of the rest eight FvCADs were detected, each with a unique amplicon with an expected size. These genes were studied via semi-quantitative RT-PCR during ‘Tokun’ fruit development and ripening (Fig. 4B). Six genes including FvCAD1, -4, -8, -9, -10, -11 were distinctly expressed in cv. Tokun with patterns largely consistent with those observed in F. vesca. Two additional genes FvCAD3 and -12 did not display a F. vesca-biased pattern. FvCAD3 was scarcely detected in F. vesca fruits, but obviously expressed in cultivated strawberry with an increasing tendency following fruit ripening. FvCAD12, the gene not expressed in F. vesca fruits at white to turning stages (22 DPA), was significantly transcribed in cv. Tokun fruits at these stages.

3.5 Dynamic expression of CAD genes in strawberry cultivars with varying fruit firmness

To reveal the correlation between CAD expression and strawberry fruit firmness, qPCR was further performed in aforementioned three varieties (Fig. 5). In all varieties, FvCAD1 (Class III) was largely stably expressed in fruits with a mild down-regulation in late developmental process. This decrease in FvCAD1 transcript levels occurred most early in the soft cv. Tokun (at white stage), in the less soft cv. Benihoppe at turning stage, and in the firm cv. Sweet Charlie most late at ripe stage. At early green fruit stages, FvCAD4 was the sole gene of Class I differentially expressed in three varieties, with the highest level in cv. Benihoppe. But this gene was similarly expressed in distinct varieties during later developmental stages.

Fig. 5

Quantitative RT-PCR analysis of strawberry CADgenes during fruit development in ‘Sweet Charlie’, ‘Benihoppe’, and ‘Tokun’ representing firm, intermediate, and soft cultivars, respectively. Two reference genes FaCHP1 and FaGAPDH1 were used as internal control. The relative expression levels of FvCADswerereported as the mean of three biological replicates.

Among six Class II genes detected, the expression of FvCAD9 was quite notable due to a continuous down-regulation pattern during fruit development and maturation, similarly in three varieties. Interestingly, the dynamic decrease in FvCAD9 transcript levels was very sharp during G1 to G2 stage in cv. Tokun, and also steep during G2 to T stage in cv. Benihoppe, that was a reminiscence of the dynamic decline of fruit firmness in each variety. By contrast, expression of FvCAD3, -10, -11, and -12 were obviously up-regulated in all three varieties upon ripening and peaked at the ripe or turning stage. FvCAD8 was distinctly expressed among three varieties during fruit development, not significantly changed in ‘Benihoppe’ and ‘Tokun’, but markedly up-regulated in cv. Sweet Charlie. The expression of FvCAD10 exhibited a uniform dynamic pattern during fruit development in all three varieties, first stably expressed but abruptly enhanced upon ripening. Simultaneously, throughout fruit development and maturation, there existed an equal gradient of FvCAD10 expression levels meeting with fruit firmness degree of distinct strawberry varieties.

3.6 Correlation between the transcript abundance of CADs with fruit lignin content and flesh firmness

A correlation analysis will facilitate our understanding in the involvement of FvCADs in lignin synthesis and strawberry fruit firmness. The expression levels of all eight detected FvCADs in developing fruits were studied in association with the genotypic differences in lignin content and fruit firmness across three varieties (Table 2). FvCAD9, which was similarly down-regulated following fruit development and ripening in three cultivars, exhibited a pattern positively correlated with the developmental decrease in fruit firmness, while no matter with the variation in lignin content and firmness across different genotypes.

Table 2
Correlation evaluation for the transcript abundance of strawberry FvCAD genes withlignin content and fruit firmness across ‘Sweet Charlie’, ‘Benihoppe’, and ‘Tokun’

Gene Correlation coefficients

G1^ab G2 W T R

FvCAD1 –0.005/–0.068 0.260/0.160 0.534/0.673* 0.659/0.525 0.178/0.523/

FvCAD3 –0.448/–0.103 –0.628/–0.640 –0.267/–0.624 –0.259/0.270 0.812^**/0.769^*

FvCAD4 0.189/0.536 0.379/0.569 0.004/0.210 0.860^**/0.678 –0.249/0.288

FvCAD8 –0.780^*/–0.462 –0.499/–0.280 –0.918^**/–0.814^* 0.039/–0.430 0.812^/0.773^

FvCAD9 –0.727/–0.623 0.300/0.412 0.067/0.412 0.374/0.131 –0.117/–0.391

FvCAD10 0.764^*/0.905^ 0.901^/0.932^ 0.738/0.966^ 0.631/0.902^** 0.744^*/0.806^**

FvCAD11 –0.500/–0.149 –0.080/0.159 –0.753^/–0.625 –0.149/–0.645 0.785^/0.949^**

FvCAD12 –0.591/–0.306 –0.646/–0.620 –0.504/–0.674 –0.574/–0.834^* 0.755^/0.814^

Gene	Correlation coefficients
FvCAD1	–0.005/–0.068	0.260/0.160	0.534/0.673*	0.659/0.525	0.178/0.523/
FvCAD3	–0.448/–0.103	–0.628/–0.640	–0.267/–0.624	–0.259/0.270	0.812^*/0.769^
FvCAD4	0.189/0.536	0.379/0.569	0.004/0.210	0.860^**/0.678	–0.249/0.288
FvCAD8	–0.780^*/–0.462	–0.499/–0.280	–0.918^*/–0.814^	0.039/–0.430	0.812^/0.773^
FvCAD9	–0.727/–0.623	0.300/0.412	0.067/0.412	0.374/0.131	–0.117/–0.391
FvCAD10	0.764^/0.905^*	0.901^/0.932^	0.738/0.966^**	0.631/0.902^**	0.744^/0.806^*
FvCAD11	–0.500/–0.149	–0.080/0.159	–0.753^*/–0.625	–0.149/–0.645	0.785^/0.949^*
FvCAD12	–0.591/–0.306	–0.646/–0.620	–0.504/–0.674	–0.574/–0.834^*	0.755^/0.814^

^aCorrelation coefficient with lignin content (left) / fruit firmness (right). ^bSignificance (two tailed values) at the 0.05 and 0.01 probability level, was indicated by ^* (P<0.05) and ^**(P<0.01), respectively.

In white fruits, FvCAD1 was expressed at levels significantly correlated with varying firmness levels in three varieties, while not closely correlated with fruit lignin content. The expression of FvCAD4, the Class I bona fide CAD gene, was significantly correlated with the genetic variation in fruit lignin content at turning stage, while not obviously correlated with fruit firmness at any stage. But for the rest genes, overall there occurred a similar correlation among CAD expression with fruit lignin content as well as with fruit firmness. Markedly, FvCAD10 was expressed at levels closely correlated with fruit lignin variation, to a significant level at least in the green and ripe fruits. The expression levels of FvCAD10 in fruits also held an extremely significant positive correlation with fruit firmness differences among three cultivars during whole fruit development process. In ripe fruits, expression of several members including FvCAD3, -8, -11, and -12 in addition to FvCAD10 uniformly showed a significant positive correlation with the phenotypic variation in fruit lignin content and firmness among different varieties. In brief, the genetic variation in ripe fruit lignin content and flesh firmness across three strawberry varieties was positively correlated with a set of five CAD genes of Class II.

4 Discussion and conclusion

Following strawberry fruit set, the flesh part of fruits sequentially goes through cell division, cell expansion, maturation, and finally fruit ripening before getting attractive and pleasant for consumer [67]. In most cultivated strawberries, fruit ripening is also a rapid softening process, which negatively affects shelf-life and makes fruit vulnerable to fungal pathogens such as gray mold. Although many genetic factors related to pectin degradation and metabolism of hemicellulose were known concerning fruit softening [41], it remains a major challenge to improve strawberry fruit firmness. Thus, to screen novel candidate regulators of fruit firmness without a cost of fruit quality and consumer acceptance is critical for a breeding initiative to improve strawberry shelf life.

There is a visible correlation of lignin content with mechanical stiffness in plant cell wall.As the key enzyme for lignin biosynthesis, CAD is encoded by a gene family with nine members divided into three main classes in Arabidops is [21]. In two rosaceous plants, a total of 24 and 26 CADs had been recognized in peach and pear genome, respectively [26, 61]. In current work, a total of 14 and 24 loci for CADs were identified in woodland strawberry F. vesca and commercial strawberry F. ×ananassa, respectively. A relatively larger size of CAD family in rosaceous plants probably hints some neo-functions or enhanced biological importance of CADs for this genus.

In this study, it was revealed that the genetic variation in lignin content was significantly correlated with the difference in strawberry fruit firmness throughout fruit development and ripening. However, it was reasonable that the developmental decrease in fruit firmness could not always be ascribed to the changes in lignin content.Dynamic lignification in strawberry fruit was proposed to start at the apical zone of the achenes and gradually progress to the receptacle [43]. Current work suggested that an effective lignification in receptacle was accomplished at small green fruit stage (G1) in a firm cultivar, decisively earlier than in the soft and intermediate varieties where fruit lignin content peaked at large green fruit stage (G2). Hereafter, all varieties went through a similar decrease in fruit lignin content and firmness till ripening.In receptacle, the peroxidase (the enzyme following CAD in the lignin biosynthesis pathway) activity is mainly localized in the concentric array of the vascular bundles, in the vascular connections with the seeds and in the epidermal cells [68]. A positive correlation between fruit firmness and FaCAD (a specific gene sharing 98% identity with FvCAD11 and maker-Fvb1-1-augustus-gene-164.37 in this study) expression was found in strawberry fruits [44]. However, down-regulation of FaCAD did not reduce fruit firmness in agroinfiltrated fruits as compared with mock fruits of cv. Calypso [45]. This could probably be caused by the compensatory mechanism in a multi-member CAD family, as well as the incorporation of aldehyde precursors (hydroxy cinnamylaldehydes) instead of corresponding monolignols into lignin, since lignin synthesis is highly plastic in plants [23, 69].

Considering the involvement of CAD family with genetic variation in strawberry firmness, the current work supported that at least four aspects together contributed to a firm flesh cultivar: 1) the developmental decrease in some member (FvCAD9) expression was more gentle and late in a firm cultivar than in intermediate and soft variety; 2) some member (FvCAD10) always held a higher expression level in a firmer cultivar throughout fruit development and ripening; 3) more CADs displayed a continuous up-regulation during fruit development and maturation in a firm variety (four members including FvCAD3, -8, -11, and -12 in the firm cv. Sweet Charlie, but only FvCAD12 in cv. Benihoppe and FvCAD3 in cv. Tokun, displayed this pattern); 4) during the final softening process (T to R stage), more CADs showed an up-regulation in a firmer variety (five members including FvCAD3, -8, -10, -11, and -12 in cv. Sweet Charlie, four including FvCAD8, -10, -11, and -12 in cv. Benihoppe, while only FvCAD3 and -10 in the soft cv. Tokun, exhibited this profile). In this study, the transcript abundance of FvCAD10 showed a highly significant correlation with genetic variation in lignin content and fruit firmness. Future functional study on FvCAD10 homoeolog in commercial strawberry (maker-Fvb1-1-augustus-gene-166.21) and its promoter sequences among a collection of strawberry germplasm might provide promising information for regulating fruit firmness. Such knowledge will illuminate the field for extending strawberry shelf life, subsequently avoiding intensive and even injurious horticultural management, decreasing the economic and ecological costs in harvest and post-harvest phases.

Angiosperm CAD proteins are enzymes with a broad diversification of biochemical function not limited to lignin synthesis. The fruit-expressed strawberry CADs associated or not associated with flesh firmness might play diverse roles in flavor and/or defense other than flesh texture. Arabidopsis AtCAD4 and AtCAD5 were confirmed as the primary genes involved in lignin biosynthesis [23], which also functioned in defense responses against a bacterial pathogen [19]. Lignin is a type of insoluble phenols and different phenolic pathways are interconnected. It is comprehensible that CADs possibly determine the contents of lignin, total phenolics and anthocyanins compounds [61]. A parallel increase in anthocyanin and lignin content and CAD expression has been observed in fruits of F. chiloensis. CAD might be involved in the synthesis of cinnamyl alcohol derivatives responsible for fruit flavor [71]. In peach, it was proposed that CADs could macroscopically affect the fruit sensorial qualities in terms of color (anthocyanins) and astringency (polyphenols), conferring relevant health properties due to their antioxidant characteristics [61]. The up-regulation of several members including FvCAD10 during ripening in all three varieties invited a speculation of these CAD genes to be multi functional and potentially contribute to flavor and/or defense. Whether this is true in strawberry needs to be ascertained in the future.

From an evolutionary point of view, the emergence of real lignin is associated with the origin of bona fide CAD, also called authentic or class I CAD [12]. In Arabidopsis, Class I AtCAD4 and -5 are primarily involved in the synthesis of lignin associated with both developmental and defensive processes [19 , 23]. In pear, the authentic CAD2 was confirmed to role in lignin synthesis of fruit stone cells in both Pyrus bretschneideri [26] and P.pyrifolia [27]. Overexpression of pear CAD2 significantly increased both growth vigor and lignin content in tomato [28]. In strawberry, Class I FvCAD each has four homoeologs in commercial strawberry, significantly more than those of Class II and III, hinting the essential role of this class CAD in strawberry. FvCAD4 was the unique Class I member expressed in all fruit samples examined, with distinct patterns dynamically changed following fruit development and ripening in three varieties. Although transcript level of FvCAD4 was not significantly correlated with the firmness variation across different genotypes, this gene was expressed at levels clearly correlated with lignin content at turning stage, supporting that the Class I authentic FvCAD4 might play an essential and conserved role in lignin synthesis in strawberry, as observed in many other angiosperm plants.

Author contributions

K.D., Q-H. G. and D-A.N. conceived this work. P. W. carried out most experiments and data analysis. J. Y., Z-Y. L. and J-J. Z. contributed to sampling and expression profiling. P. W. prepared tables and figures. K.D. wrote the paper. All authors contributed to revising and approved the manuscript.

Footnotes

Acknowledgments

This work was funded by Shanghai Agriculture Applied Technology Development Program, China (Grant No. G2016060104 and No.G2019-02-08-00-08-F01108). Thanks are due to anonymous reviewers for valuable comments which improved this manuscript.

Funding

The authors report no funding.

Conflict of interest

The authors have no conflict of interest to report.

The supplementary material is available in the electronic version of this article: .

References

Cai

, Xu

, Li

, Ferguson

, Chen

. Accumulation of lignin in relation to change in activities of lignification enzymes in loquat fruit flesh after harvest. Postharv. Biol. Technol. 2006;40:163–9.

Dangcham

, Bowen

, Ferguson

, Ketsa

. Effect of temperature and low oxygen on pericarp hardening of mangosteen fruit stored at low temperature. Postharvest Biol. Technol. 2008;50:37–44.

Shan

, Li

, Wang

, Cai

, Zhang

, Sun

, Zhang

, Xu

, Ferguson

, Chen

. Characterization of cDNAs associated with lignification and their expression profiles in loquat fruit with different lignin accumulation. Planta. 2008;227(6):1243–54.

Ring

, Yeh

, Hücherig

, Hoffmann

, Blanco-Portales

, Fouche

, Villatoro

, Denoyes

, Monfort

, Caballero

, Muñoz-Blanco

, Gershenson

, Schwab

. Metabolic interaction between anthocyanin and lignin biosynthesis is associated with peroxidase FaPRX27 in strawberry fruit. Plant Physiol. 2013;163(1):43–60.

Özparpucu

, Rüggeberg

, Gierlinger

, Cesarino

, Vanholme

, Boerjan

, Burgert

. Unravelling the impact of lignin on cell wall mechanics: a comprehensive study on young poplar trees downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD). Plant J. 2017;91(3):480–90.

Boudet

, Hawkins

, Rochange

. The polymorphism of the genes/enzymes involved in the last two reductive steps of monolignol synthesis: what is the functional significance? C R Biol. 2004;327(9-10):837–45.

Youn

, Camacho

, Moinuddin

, Lee

, Davin

, Lewis

, Kang

. Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4. Org Biomol Chem. 2006;4(9):1687–97.

Barakat

, Bagniewska-Zadworna

, Choi

, Plakkat

, DiLoreto

, Yellanki

, Carlson

. The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression. BMC Plant Biol. 2009;9:26.

Lüderitz

, Grisebach

. Enzymic synthesis of lignin precursors. Comparison of cinnamoyl-CoA reductase and cinnamy lalcohol:NADP + dehydrogenase from spruce (Picea abies L.) and soybean (Glycine max L.). Eur J Biochem. 1981;119(1):115–24.

10.

Knight

, Halpin

, Schuch

. Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco. Plant Mol Biol. 1992;19(5):793–801.

11.

Van

Doorsselaere J

, Baucher

, Feuillet

, Boudet

, Van

Montagu M

, Inzé

. Isolation of cinnamyl alcohol dehydrogenase cDNAs from two important economic species: alfalfa and poplar. Demonstration of a high homology of the gene within angiosperms. Plant Physiol Biochem. 1995;33:105–9.

12.

Guo

, Ran

, Wang

. Evolution of the Cinnamyl/Sinapyl Alcohol Dehydrogenase (CAD/SAD) gene family: the emergence of real lignin is associated with the origin of Bona Fide CAD. J Mol Evol. 2010;71(3):202–18.

13.

Chao

, Liu

, Li

, Jiang

, Gai

. Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa. Planta. 2014;240(5):1097–112.

14.

Jin

, Zhang

, Liu

, Qi

, Chen

, Cao

. The cinnamyl alcohol dehydrogenase gene family in melon (Cucumis melo L.): bioinformatic analysis and expression patterns. PLoS One. 2014;9(7):e101730.

15.

Saballos

, Ejeta

, Sanchez

, Kang

, Vermerris

. A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrib6 gene. Genetics. 2009;181(2):783–95.

16.

Tang

, Zhang

, Li

, Xie

. Cloning and in silico analysis of a cinnamyl alcohol dehydrogenase gene in Pennisetum purpureum. J Genet. 2014;93(1):145–58.

17.

Tobias

, Chow

. Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification. Planta. 2005;220(5):678–88.

18.

Kim

, Bae

, Huh

. Transcriptional regulation of the cinnamyl alcohol dehydrogenase gene from sweet potato in response to plant developmental stage and environmental stress. Plant Cell Rep. 2010;29(7):779–91.

19.

Tronchet

, Balagué

, Kroj

, Jouanin

, Roby

. Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Mol Plant Pathol. 2010;11(1):83–92.

20.

Park

, Kim

, Bhoo

, Lee

, Cho

. Biochemical Characterization of the Rice Cinnamyl Alcohol Dehydrogenase Gene Family. Molecules. 2018;23(10):2659.

21.

Kim

, Kim

, Bedgar

, Moinuddin

, Cardenas

, Davin

, Kang

, Lewis

. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc Natl Acad Sci U S A. 2004;101(6):1455–60.

22.

Sibout

, Eudes

, Pollet

, Goujon

, Mila

, Granier

, Séguin

, Lapierre

, Jouanin

. Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants. Plant Physiol. 2003;132(2):848–60.

23.

Sibout

, Eudes

, Mouille

, Pollet

, Lapierre

, Jouanin

, Séguin

. CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell. 2005;17(7):2059–76.

24.

Kim

, Kim

, Cho

, Franceschi

, Davin

, Lewis

. Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations? Phytochemistry. 2007;68(14):1957–74.

25.

Preisner

, Wojtasik

, Kostyn

, Boba

, Czuj

, Szopa

, Kulma

. The cinnamyl alcohol dehydrogenase family in flax: Differentiation during plant growth and under stress conditions. J Plant Physiol. 2018;221:132–43.

26.

Cheng

, Li

, Zhang

, Jin

, Sheng

, Cai

, Lin

. Characterization and analysis of CCR and CAD gene families at the whole-genome level for lignin synthesis of stone cells in pear (Pyrus bretschneideri) fruit. Biol Open. 2017;6(11):1602–13.

27.

Wang

, Zhang

, Yang

, Wang

, Lu

, Wang

, Yang

, Li

. Heterogenous expression of Pyrus pyrifolia PpCAD2 and PpEXP2 in tobacco impacts lignin accumulation in transgenic plants. Gene. 2017;637:181–9.

28.

, Cheng

, Zhang

, Zhou

, Li

, Yang

. Overexpression of Pear (Pyrus pyrifolia) CAD2in Tomato Affects Lignin Content. Molecules. 2019;24(14):2595.

29.

Folta

, Davis

. Strawberry genes and genomics. Crit. Rev. Plant Sci. 2006;25:399–415.

30.

Shulaev

, Sargent

, Crowhurst

, et al. The genome of woodland strawberry (Fragaria vesca). Nat Genet. 2011;43(2):109–16.

31.

Edger

, Poorten

, VanBuren

, et al. Origin and evolution of the octoploid strawberry genome. Nat Genet. 2019;51(3):541–7.

32.

Brummell DA . Cell wall disassembly in ripening fruit. Funct Plant Biol. 2006;33(2):103–19.

33.

Santiago-Doménech

, Jiménez-Bemúdez

, Matas

, Rose

, Muñoz-Blanco

, Mercado

, Quesada

. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening. J Exp Bot. 2008;59(10):2769–79.

34.

Molina-Hidalgo

, Franco

, Villatoro

, et al. The strawberry (Fragaria×ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae. J Exp Bot. 2013;64:1471–83.

35.

Koh

, Melton

. Ripening-related changes in cell wall polysaccharides of strawberry cortical and pith tissues. Postharvest Biol. Technol. 2002;26:23–33.

36.

Rosli

, Civello

, Martínez

. Changes in cell wall composition of three Fragaria x ananassa cultivars with different softening rate during ripening. Plant Physiol Biochem. 2004;42(10):823–31.

37.

Figueroa

, Rosli

, Civello

, Martínez

, Herrera

, Moya-León

. Changes in cell wall polysaccharides and cell wall degrading enzymes during ripening of Fragaria chiloensis and Fragaria×ananassa fruits. Sci Hortic. 2010;124:454–62.

38.

Paniagua

, Blanco-Portales

, Barceló-Muñoz

, García-Gago

, Waldron

, Quesada

, Muñoz-Blanco

, Mercado

. Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening. J Exp Bot. 2016;67(3):619–31.

39.

Paniagua

, Santiago-Doménech

, Kirby

, Gunning

, Morris

, Quesada

, Matas

, Mercado

. Structural changes in cell wall pectins during strawberry fruit development. Plant Physiol Biochem. 2017;118:55–63.

40.

JaraK , Castro

, Ramos

, Parra-Palma

, Valenzuela-Riffo

, Morales-Quintana

. Molecular Insights into FaEG1, a Strawberry Endoglucanase Enzyme Expressed during Strawberry Fruit Ripening. Plants (Basel). 2019;8:140.

41.

Moya-León

, Mattus-Araya

, Herrera

. Molecular Events Occurring During Softening of Strawberry Fruit. Front Plant Sci. 2019;10:615.

42.

Witasari

, Huang

, Hoffmann

, Rozhon

, Fry

, Schwab

. Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening. Plant J. 2019;100(6):1237–53.

43.

Blanco-Portales

, Medina-Escobar

, López-Ráez

, González-Reyes

, Villalba

, Moyano

, Caballero

, Muñoz-Blanco

. Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler). J Exp Bot. 2002;53(375):1723–34.

44.

Salentijn

, Aharoni

, Schaart

, Boone

, Krens

. Differential gene expression analysis of strawberry cultivars that differ in fruit-hardness. Physiol Planta. 2003;118:571–8.

45.

Yeh

, Huang

, Hoffmann

, Mayershofer

, Schwab

. FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.). Front Plant Sci. 2014;5:518.

46.

Finn

, Clements

, Eddy

. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39(Web Server issue):W29–37.

47.

Finn

, Coggill

, Eberhardt

, Eddy

, Mistry

, Mitchell

, Potter

, Punta

, Qureshi

, Sangrador-Vegas

, Salazar

, Tate

, Bateman

. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44(D1):D279–85.

48.

, Pi

, Gao

, Liu

, Kang

. Updated annotation of the wild strawberry Fragaria vesca V4 genome. Hortic Res. 2019;6:61.

49.

Jung

, Lee

, Cheng

, Buble

, Zheng

, Yu

, Humann

, Ficklin

, Gasic

, Scott

, Frank

, Ru

, Hough

, Evans

, Peace

, Olmstead

, DeVetter

, McFerson

, Coe

, Wegrzyn

, Staton

, Abbott

, Main

. 15 years of GDR: New data and functionality in the Genome Database for Rosaceae. Nucleic Acids Res. 2019;47(D1):D1137–45.

50.

Kumar

, Stecher

, Tamura

. MEGA Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–4.

51.

Letunic

, Bork

. Interactive tree of life (iTOL) v an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016;44(W1):W242–5.

52.

Edger

, VanBuren

, Colle

, Poorten

, Wai

, Niederhuth

, Alger

, Ou

, Acharya

, Wang

, Callow

, McKain

, Shi

, Collier

, Xiong

, Mower

, Slovin

, Hytönen

, Jiang

, Childs

, Knapp

. Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity. Gigascience. 2018;7(2):1–7.

53.

Chen

, Chen

, Zhang

, Thomas

, Frank

, He

, Xia

. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant. 2020;13(8):1194–202.

54.

Liu

, Xie

, Ma

, Luo

, Nie

, Zuo

, Lahrmann

, Zhao

, Zheng

, Zhao

, Xue

, Ren

. IBS: an illustrator for the presentation and visualization of biological sequences. Bioinformatics. 2015;31(20):3359–61.

55.

Waterhouse

, Procter

, Martin

, Clamp

, Barton

. Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25(9):1189–91.

56.

Liu

, Ji

, Liu

, Tian

, Gao

, Zou

, Yang

, Dong

, Tan

, Ni

, Duan

. The sugar transporter system of strawberry: genome-wide identification and expression correlation with fruit soluble sugar-related traits in a Fragaria × ananassa germplasm collection. Hortic Res. 2020;7:132.

57.

Clancy

, Rosli

, Chamala

, Barbazuk

, Civello

, Folta

. Validation of reference transcripts in strawberry (Fragaria spp.). Mol Genet Genomics. 2013;288(12):671–81.

58.

Estrada-Johnson

, Csukasi

, Pizarro

, Vallarino

, Kiryakova

, Vioque

, Brumos

, Medina-Escobar

, Botella

, Alonso

, Fernie

, Sánchez-Sevilla

, Osorio

, Valpuesta

. Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle. Front Plant Sci. 2017;8:889.

59.

Vandesompele

, De Preter

, Pattyn

, Poppe

, Van Roy

, De Paepe

, Speleman

. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034.

60.

Cheng

, Raza

, Wang

, Xu

, Lu

, Gao

, Qin

, Zhang

, Ahmad

, Zhou

, Wen

, Yang

, Liu

. Effects of Multiple Planting Densities on Lignin Metabolism and Lodging Resistance of the Strip Intercropped Soybean Stem. Agronomy. 2020;10(8):1177.

61.

Gabotti

, Negrini

, Morgutti

, Nocito

, Cocucci

. Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels. Physiol Plant. 2015;154(3):329–48.

62.

Jun

, Walker

, Kim

, Ralph

, Vermerris

, Sattler

, Kang

. The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4. Plant Physiol. 2017;174(4):2128–45.

63.

McKie

, Jaouhari

, Douglas

, Goffner

, Feuillet

, Grima-Pettenati

, Boudet

, Baltas

, Gorrichon

. A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification. Biochim Biophys Acta. 1993;1202(1):61–9.

64.

Saathoff

, Hargrove

, Haas

, Tobias

, Twigg

, Sattler

, Sarath

. Switchgrass PviCAD understanding residues important for substrate preferences and activity. Appl Biochem Biotechnol. 2012;168(5):1086–100.

65.

Hollender

, Geretz

, Slovin

, Liu

. Flower and early fruit development in a diploid strawberry, Fragaria vesca. Planta. 2012;235(6):1123–39.

66.

Kang

, Darwish

, Geretz

, Shahan

, Alkharouf

, Liu

. Genome-scale transcriptomic insights into early-stage fruit development in woodland strawberry Fragaria vesca. Plant Cell. 2013;25(6):1960–78.

67.

Fenn

, Giovannoni

. Phytohormones in fruit development and maturation. Plant J. 2020. doi: 10.1111/tpj.15112.

68.

López-Serrano

, Ros-Barceló

. Peroxidase in unripe and processing-ripe strawberries. Food Chemistry. 1995;52:157–60.

69.

Humphreys

, Chapple

. Rewriting the lignin roadmap. Curr Opin Plant Biol. 2002;5(3):224–9.

70.

Concha

, Figueroa

, Poblete

, Oñate

, Schwab

, Figueroa

. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol Biochem. 2013;70:433–44.

71.

Aharoni

, Keizer

, Van Den Broeck

, Blanco-Portales

, Muñoz-Blanco

, Bois

, Smit

, De Vos

, O’Connell

. Novel insight into vascular, stress, and auxin-dependent and -independent gene expression programs in strawberry, a non-climacteric fruit. Plant Physiol. 2002;129(3):1019–31.

Gene	Correlation coefficients
	G1^ab	G2	W	T	R
FvCAD1	–0.005/–0.068	0.260/0.160	0.534/0.673*	0.659/0.525	0.178/0.523/
FvCAD3	–0.448/–0.103	–0.628/–0.640	–0.267/–0.624	–0.259/0.270	0.812^*/0.769^
FvCAD4	0.189/0.536	0.379/0.569	0.004/0.210	0.860^**/0.678	–0.249/0.288
FvCAD8	–0.780^*/–0.462	–0.499/–0.280	–0.918^*/–0.814^	0.039/–0.430	0.812^/0.773^
FvCAD9	–0.727/–0.623	0.300/0.412	0.067/0.412	0.374/0.131	–0.117/–0.391
FvCAD10	0.764^/0.905^*	0.901^/0.932^	0.738/0.966^**	0.631/0.902^**	0.744^/0.806^*
FvCAD11	–0.500/–0.149	–0.080/0.159	–0.753^*/–0.625	–0.149/–0.645	0.785^/0.949^*
FvCAD12	–0.591/–0.306	–0.646/–0.620	–0.504/–0.674	–0.574/–0.834^*	0.755^/0.814^