Differences in Physiological Characteristics and Gene Expression Levels in Fruits between Japanese Persimmon (Diospyros kaki Thunb.) ‘Hiratanenashi’ and Its Small Fruit Mutant ‘Totsutanenashi’

Abstract

Japanese persimmon ‘Totsutanenashi’ (TTN) is a spontaneous small fruit mutant derived from ‘Hiratanenashi’ (HTN). To characterize the small fruit phenotype of TTN, we carried out a histological analysis, plant growth regulator treatments, and a transcriptome analysis using Illumina sequencing. The parenchymal cell number in TTN fruit was significantly less than in HTN fruit, and the parenchymal cell size in TTN fruit was also significantly smaller than that in HTN fruit at the later growing stage. However, the fruit size of TTN recovered by cytokinin treatments [50 or 200 ppm N-(2-chloro-4-pyridyl)-N′-phenylurea]. Thus, diminished cytokinin activity in TTN fruits may lead to less cell division in the early growing stage and less cell enlargement in the later growing stage. A large-scale transcriptome analysis was conducted using Illumina sequencing to determine the differences in gene expressions between TTN and HTN fruits. Illumina sequences were processed, resulting in 21,662,190 read pairs from HTN and 23,195,203 read pairs from TTN. After assembly of all sequences from HTN and TTN, 118,985 contigs (referred to as unigenes hereafter) ranging from 201 to 11,954 bases, with an average length of 915 bases, were obtained. Digital expression analyses revealed that the expression levels of 164 unigenes were significantly higher in HTN than in TTN, while the expression levels of 265 unigenes were significantly higher in TTN. A parametric analysis of gene set enrichment using the expression levels of unigenes showed that the biological process Gene Ontology categories of “cell cycle” and “regulation of cell cycle” were significantly down-regulated in TTN. The cell cycle-related differentially expressed genes included D3-type cyclin and Mitogen-Activated Protein Kinase Kinase Kinase. Based on the obtained results, the possible involvement of cell cycle-related genes in regulating the small fruit phenotype in TTN is discussed.

Introduction

Fruit size is a commercially important trait for many fruit species. Generally, a larger fruit is preferred by both growers and consumers, therefore, producing larger fruit is a major breeding goal. However, fruit size is assumed to be regulated by polygenes and environmental factors, and the molecular mechanism regulating fruit size is not fully understood. Artificially transformed plants are often used to investigate the molecular mechanisms of various events in model plants such as T-DNA insertion lines in Arabidopsis. However, generating artificial mutants in fruit tree species is challenging because of transformation difficulties and a low regeneration efficiency. Instead, bud mutations, which are natural mutations that occur often on a single branch or spur in an orchard, are effectively used in molecular analyses in many fruit tree species. Because a bud mutant and its original parent are genetically nearly identical, except for the mutated genes, a bud mutant is very useful for studying molecular mechanisms such as fruit size control. Recently, a small fruit mutant of Japanese persimmon (Diospyros kaki Thunb.), ‘Totsutanenashi’ (TTN), which originated from ‘Hiratanenashi’ (HTN), was discovered in Mr. Masaharu Kondo’s orchard in Sado, Niigata Prefecture, Japan. The scions were grafted, and the plantlets were grown. Then, TTN was characterized by a comparison with HTN, and not only fruit size, but also vegetative growth, and fruit maturation appeared to be affected by the mutation in TTN (Yamane et al., 2008, 2009a, b). In addition, TTN fruit matures earlier than HTN, and contains more sugars than HTN fruit, raising the possibility that TTN could meet a market demand for small fruit (Tao et al., 2013). TTN fruits are now commercially available and tentatively sold as baby-persimmon, a trademark for peeled TTN fruits in a plastic package (http://www.baby-persimmon.com/). To date, how mutations occur in TTN is still unclear; however, because the genetic identity between TTN and HTN was suggested by a simple sequence repeat analysis (Yamane et al., 2008), TTN is suitable for studying the genetic and molecular bases of fruit size control in Japanese persimmon. Our final goal is to isolate key genetic factors that control fruit size.

Final fruit size is generally determined by a coordinated series of cell divisions and expansions during fruit growth and development. Therefore, the small fruit of TTN results from lower numbers of parenchymal cells and/or smaller cell sizes in the fruit mesocarp. However, cell numbers and sizes in TTN fruit compared with those in HTN have not been investigated. The duration of cell division in HTN was about 4 weeks after bloom (WAB) (Hamada et al., 2008; Hirata and Hayashi, 1978), and the fruit diameter of TTN was smaller than that of HTN from 1 WAB onward (Yamane et al., 2008), raising the possibility that cell division activity decreases as the cell division period progresses in TTN. In addition, as the growth rate of TTN fruit after the cell division period seemed to be lower than that in HTN fruit (Yamane et al., 2008, 2009a, b), the cell enlargement in TTN fruit may be less than in HTN.

In general, cell division and/or cell enlargement during fruit growth and development are controlled by plant hormones, such as auxin, gibberellin (GA), and cytokinin (CK) (Gillaspy et al., 1993; Srivastava and Handa, 2005). In Japanese persimmon, Hirata et al. (1978) reported that the levels of auxins (indole-3-acetic acid- and indole-3-butyric acid-like substances), GA [a gibberellic acid (GA3)-like substance], and CK-like substances were high in the active cell division stage of HTN, suggesting that these three plant hormones are highly associated with cell division. Kojima et al. (1999) found that the concentration of GA-like substances in fruitlets of HTN increased between 41 and 122 days after flowering, and suggested that GA is necessary for cell expansion. In addition, applications of a synthetic CK, N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) significantly increase the fruit size of Japanese persimmons (Hamada et al., 2008; Itai et al., 1995). Recently, a genetic factor that strongly affects fruit size was discovered in tomato (Solanum lycopersicum L.), which is a model plant for fruit growth and development. The major quantitative trait loci associated with fruit weight, FRUIT WEIGHT 2.2 (FW2.2), have been identified and characterized (Grandillo et al., 1999). FW2.2 encodes a member of the Cell Number Regulator (CNR) family, and negatively regulates cell division during early fruit development (Frary et al., 2000). The FW2.2/CNR family of genes controls organ size by modulating cell numbers in other plant species such as avocado (Persea americana Mill.; Dehan et al., 2010) and sweet cherry (De Franceschi et al., 2013). In avocado, the expression level of Pafw2.2-like was higher in the smaller fruits of ‘Hass’ trees than in normal fruits (Cowan et al., 1997; Dehan et al., 2010; Zilkah and Klein, 1987). In sweet cherry, De Franceschi et al. (2013) reported that PavCNR12, a CNR gene, is located within the confidence interval of the major fruit size G2 quantitative trait loci, which is postulated to affect fruit size by controlling mesocarp cell numbers (Zhang et al., 2010), thereby, controlling fruit size (Olmstead et al., 2007). If the fewer cell numbers in TTN fruits lead to the smaller fruit size, then the expression intensities of the FW2.2/CNR family genes between TTN and HTN during the cell division period should be interesting. However, histological comparisons and gene expression analyses have not been conducted in TTN fruit.

The objective of this study was to elucidate the molecular mechanism of the small fruit phenotype in TTN. First, we determined parenchymal cell numbers and sizes during fruit growth and development, and then, the effects of plant growth regulators (PGRs) on fruit growth were investigated. To compare gene expression levels between TTN and HTN, Illumina sequencing for the transcriptome analysis was performed. Finally, based on the data obtained from the fruit transcriptome analysis, histological analysis, and PGR treatments, the molecular mechanism of small fruit phenotype in TTN is discussed.

Materials and Methods

Plant materials

Pot-grown two Japanese persimmon cultivars, TTN and HTN, and field-grown trees from the experimental farm of Kyoto University were used in this study. Cultural practices, such as pruning, fertilization, pest management, and irrigation, were carried out according to standard procedures for HTN during the experimental period. In the 2005, 2008, and 2009 growing seasons, three uniform fruitlets on pot-grown trees in each cultivar were selected at 1 WAB, and fruit diameters were determined weekly until 22 WAB.

Determinations of numbers and sizes of parenchymal cells during fruit growth and development

In the 2005 growing season, three fruits from pot-grown trees of each cultivar were sampled weekly from 1 to 4 WAB and then biweekly until 22 WAB. In the 2008 growing season, fruits were sampled at 1, 6, and 18 WAB. Equatorial diameters were determined using a caliper. Small blocks of tissue (one block per fruit) were quarried from the equatorial region of the mesocarp, and then fixed in formaldehyde:acetic acid:70% ethanol (1:1:18, v/v). After dehydration, using a butanol series, the blocks were embedded in paraffin wax and sliced to ~10 μm thickness using a rotary microtome. The sliced tissues were stained with 0.5% toluidine blue, and observed under a light microscope. The photographic image was recorded with a digital camera (DP50; Olympus, Tokyo, Japan), and then analyzed by ImagePro plus (Media Cybernetics, Rockville, MD, USA) to determine the size and number of parenchymal cells. In the 2005 growing season, average cell area was determined, whereas both average cell area and average cell number per unit area were determined in the 2008 growing season. The total numbers of cells in transverse sections of fruits were estimated, using a transverse sectional area in the equatorial region by calculating from the equatorial diameter, and average cell area.

PGR treatments

In the 2008 growing season, three types of PGRs were applied, including a synthetic CK, CPPU, two GA isomers (GA₃ and GA₄₊₇), and a synthetic auxin, 1-naphthaleneacetic acid (NAA). CPPU (Fulmet; Kyowa Hakko Bio, Tokyo, Japan) was applied at 50 and 200 ppm, GA₃ (Kyowa Hakko Bio) and GA₄₊₇ (Phyto Technology Laboratories, Shawnee Mission, KS, USA) was applied at 10 and 100 ppm, and NAA was applied at 50 ppm as each treatment. Tween 20, as a surfactant, was added to all of the PGR solutions at a final concentration of 0.05%. A solution containing only 0.05% Tween 20 was used as a control. CPPU was sprayed weekly on the flowers or fruitlets of pot-grown trees of both cultivars using a hand-held sprayer from 2 weeks before bloom to 2 WAB. Similarly, GAs (GA₃ and GA₄₊₇) were sprayed weekly on fruitlets of pot-grown trees of both cultivars from 13 to 17 WAB. NAA was sprayed weekly on fruitlets of field-grown trees of both cultivars from 2 to 4 WAB. In the 2009 growing season, 10 and 100 ppm of CPPU was sprayed on fruitlets of TTN pot-grown trees twice, once at 1 WAB and once at 2 WAB. The diameters and lengths of three fruit from each treatment of each cultivar were determined periodically.

RNA extraction

During the 2011 growing season, fruits of both cultivars were sampled for RNA extraction at 1, 3, 6, 12, and 20 WAB. All fruits were cut, and immediately frozen in liquid N₂, and stored at −80°C until use. Total RNA was extracted using the hot borate method (Wan and Wilkins, 1994). RNA concentrations and qualities were determined by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and agarose gel electrophoresis.

Sequencing, sequence preprocessing, and assembly

Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) paired-end (2 X 100 bp) sequencing was performed for fruit, as per the custom service provided by TaKaRa BIO Inc.’s Dragon Genomics Center (Yokkaichi, Japan). In short, we mixed the same quantity of RNAs from 1, 3, 6, 12, and 20 WAB, and sequencing libraries of each cultivar were constructed using the mRNA-Seq Sample Prep (Illumina) and Small RNA Sample Prep (Illumina) kits following the Directional mRNA-Seq Library Prep Pre-Release Protocol Rev. A (Illumina). Then, the quality of library construction was assessed with Agilent 2100 Bioanalyzer (Agilent). Using the constructed libraries as templates, clonal clusters from molecule fragments were constructed on a illumina flow cell using cBot (Illumina) and TruSeq PE Cluster Kit v3 (Illumina). Sequencing was performed using the TruSeq SBS Kit v3–HS (Illumina). After sequencing, the obtained reads were processed with a Perl script as follows: i) low-quality regions were masked and ii) read pairs in which either read contained more than 10% masked regions were removed. Then, the preprocessed read pairs were assembled by Trinity (Grabherr et al., 2011) with the default settings. In addition, to reduce redundancy and chimeric contigs, the contigs obtained by Trinity were further assembled by CAP3 (Huang and Madan, 1999), and then, the contigs with opposite directional high scoring segment pairs were removed as probable chimeric contigs using the BLASTX results described below.

Functional annotation

To annotate the unigenes, they were used as a query to search the refseq protein database from the National Center for Biotechnology Information (NCBI) and the Arabidopsis protein database (TAIR10) from the Arabidopsis Information Resource (TAIR) using the BLASTX program (Altschul et al., 1990) with an E-value cutoff of 1e-6. Gene Ontology (GO) terms were assigned to the unigenes by Blast2GO (Conesa et al., 2005) using the nr annotations. After GO annotation, WEGO (Ye et al., 2006) was used to classify GO functions for all of the annotated unigenes.

Digital expression analysis

A digital expression analysis was conducted using the perl scripts included in the Trinity package. The HTN and TTN read pair sets were individually mapped to the unigenes using Bowtie (Langmead et al., 2009) and the expected counts, and the fragment per kilobase of transcript per million values were estimated using RSEM (Li and Dewey, 2011). The obtained expected counts were used as inputs in edgeR (Robinson et al., 2010) for statistical analyses. Unigenes with less than a 5% false discovery rate were taken as differentially expressed between HTN and TTN. A Parametric Analysis of Gene set Enrichment (PAGE) (Kim and Volsky, 2005) analysis using the fold change values and the TAIR10 top hit annotations of the unigenes was conducted using agriGO (Du et al., 2010).

Searching for homologs of the CK metabolism genes

We searched for homologs of the CK metabolism genes using the BLASTX algorithm (cutoff e-value < 1e-30) with the Arabidopsis isopentenyltransferase (IPT) 1 (AT1G68460.1), IPT2 (AT2G27760.1), IPT3 (AT3G63110.1), IPT4 (AT4G24650.1), IPT5 (AT5G19040.1), IPT6 (AT1G25410.1), IPT7 (AT3G23630.1), IPT8 (AT3G19160.1), IPT9 (AT5G20040.3), cytochrome P450 735 A1 (CYP735A1; AT5G38450.1), CYP735A2 (AT1G67110.1), LONELY GUY (LOG) 1 (AT2G28305.1), LOG2 (AT2G35990.1), LOG3 (AT2G37210.2), LOG4 (AT3G53450.1), LOG5 (AT4G35190.1), LOG6 (AT5G03270.1), LOG7 (AT5G06300.1), LOG8 (AT5G11950.1), LOG9 (AT5G26140.1), cytokinin oxidase (CKX) 1 (AT2G41510.1), CKX2 (AT2G19500.1), CKX3 (AT5G56970.1), CKX4 (AT4G29740.2), CKX5 (AT1G75450.1), CKX6 (AT3G63440.1), and CKX7 (AT5G21482.1) protein sequences as queries. Then, the unigenes encoding IPT, CYP735A, LOG, and CKX were subjected to a digital expression analysis.

Results

Fruit size, and parenchymal cell numbers and sizes during fruit development

In the 2005 growing season, the average parenchymal cell area increased during fruit development in both cultivars (Fig. 1a). There was no significant difference in the average parenchymal cell areas between HTN and TTN until 10 WAB, except at 3 WAB. However, the average parenchymal cell area of HTN was significantly greater than that of TTN from 12 to 22 WAB, except at 18 WAB (Fig. 1a). The total numbers of parenchymal cells in transverse sections of TTN were significantly less than those of HTN fruit throughout fruit growth and development, except at 3 WAB (Fig. 1b). Similarly, total cell numbers in transverse sections of TTN were significantly less than those of HTN throughout the 2008 growing season. Among the three time points analyzed, the average cell areas and numbers per unit area were similar between the two cultivars at 1 and 6 WAB, whereas they were significantly lower in TTN than in HTN at 18 WAB (Table 1).

Table 1

Average cell areas and cell numbers in ‘Totsutanenashi’ (TTN) and ‘Hiratanenashi’ (HTN) fruits in the 2008 growing season.

Fig. 1

The seasonal changes in the average parenchymal cell area (a) and the total numbers of cells (b) in transverse sections of ‘Hiratanenashi’ (HTN) and ‘Totsutanenashi’ (TTN) fruits. Vertical bars represent SE (n = 3).

PGR treatments

In the 2008 growing season, CPPU treatments increased both the fruit diameters and lengths of TTN significantly (Fig. 2a, b). Although the lengths and diameters of TTN fruits were lower than those of HTN fruit at 1 WAB, changes in fruit lengths and diameters of CPPU-treated TTN fruit after 1 WAB showed similar patterns to those of control HTN fruit, and the final fruit size of CPPU-treated TTN fruits recovered to a size similar to HTN fruits (Fig. 3). There were no differences in the sizes of CPPU-treated TTN fruit at different CPPU concentrations, except for those at 17, 18, 23, and 24 WAB. The fruit diameters of CPPU-treated HTN also increased in the later growing stage, and the HTN fruit diameter in the 200 ppm CPPU treatment was higher than that in the 50 ppm CPPU treatment. CPPU treatments in the next growing season also increased fruit diameters and lengths in TTN fruits, with a greater contribution at 100 ppm compared with 10 ppm (data not shown). However, GA treatments did not affect fruit sizes in either cultivar (data not shown). For NAA treatments, fruit size was unable to be determined because all of the NAA-treated fruits were abscised by 9 WAB.

Fig. 2

The seasonal changes in the fruit diameter (a) and the fruit length (b) of N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU)-treated ‘Hiratanenashi’ (HTN) and ‘Totsutanenashi’ (TTN) fruits. Vertical bars represent SE (n = 3).

Fig. 3

N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU)-treated ‘Hiratanenashi’ (HTN; upper) and ‘Totsutanenashi’ (TTN; bottom) fruits at the maturation stage.

Sequencing, assembly, and annotation

Using Illumina HiSeq 2000 sequencing, 23,935,707 raw read pairs from a HTN fruit library and 25,601,520 raw read pairs from a TTN fruit library were generated. All of the obtained sequences from HiSeq 2000 reads are available from the DDBJ Sequence Read Archive (DRA003812; http://trace.ddbj.nig.ac.jp/dra/index.html). After the preprocessing steps, we obtained 21,662,190 read pairs from HTN and 23,195,203 read pairs from TTN. These read pairs were mixed, and assembled by Trinity. Then, the contigs were assembled by CAP3, chimeric contigs were removed using BLASTX results, and 118,985 unigenes ranging from 201 to 11,954 bases, with an average length of 710 bases, were obtained (Table 2). When the unigene sequences were queried against the NCBI refseq protein and Arabidopsis (TAIR10) protein databases using BLASTX (1e-6), 49,110 (42.3% of total unigenes) and 41,687 (35.0% of total unigenes) unigene sequences received at least one hit against the NCBI refseq and TAIR10 protein databases, respectively. The GO distribution indicated that genes having a wide range of functions were expressed in Japanese persimmon fruit (data not shown).

Table 2

Results of the Japanese persimmon library assembly and annotation.

Digital expression analysis

For the digital expression analysis, the expected counts by mapping using Bowtie in TTN and HTN were estimated by RSEM. Differentially expressed genes (DEGs) were selected based on the false discovery rate (cut-off < 0.05). Comparisons of the expected counts showed that the expression levels of 369 unigenes were significantly different between HTN and TTN fruit. Among these, the expression levels of 164 unigenes were significantly higher in HTN than in TTN, while the expression levels of 265 unigenes were significantly higher in TTN. A PAGE analysis using the expression levels of unigenes showed that the biological process GO categories cell cycle and regulation of cell cycle were significantly down-regulated, while photosynthesis was significantly up-regulated, in TTN (Table 3). The cell cycle-related DEGs included D3-type cyclin (CYCD3) and Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) (Table 4). However, the FW2.2/CNR family of genes were not included in the DEGs.

Table 3

Gene ontology terms selected by parametric analysis of gene set enrichment (PAGE).

Table 4

Expression levels of cell cycle-related differentially expressed genes [false discovery rate < 0.05, ‘Hiratanenashi’ (H) > ‘Totsutanenashi’ (T)].

Expression levels of CK metabolism-related genes

From the search results, using the BLASTX algorithm with Arabidopsis IPT1-9, CYP735A1, CYP735A2, LOG1-9, and CKX1-7 protein sequences as queries, and the TAIR10 annotations, 11 IPT-like unigenes (divided into five types), 1 CYP735A1-like unigene, 5 LOG1-like unigenes, and 12 CKX-like unigenes (divided into five types) were identified, but their expression levels were not significantly different (data not shown).

Discussion

Seasonal changes in parenchymal cell sizes and numbers in TTN and HTN fruit

Japanese persimmon TTN is a spontaneous mutant of HTN with small fruit, but the mechanism behind this phenotype is still unknown. Because the final fruit size depends on cell division and expansion during fruit growth and development, we first measured parenchymal cell sizes and numbers during fruit development to determine whether the small fruit of TTN is due to cell size or cell number. Although the fruit diameter of TTN was significantly smaller than that of HTN from 1 WAB onward throughout fruit growth and development, the average parenchymal cell areas of the cultivars were not significantly different during the early stages of fruit growth, while the total numbers of parenchymal cells in transverse sections of TTN were significantly less than those of HTN fruit from 1 WAB onward. This indicates that a decrease in fruit size in TTN during the early growing stage is mainly due to a reduction in the parenchymal cell number. Therefore, it is likely that the level of cell division in the early growing stage may decrease TTN fruit size. However, the average parenchymal cell area of HTN was greater than that of TTN in the later growing stage. Therefore, it is possible that a lower rate of cell enlargement in the later growing stage also contributes to the small fruit phenotype in TTN.

Effects of PGR treatments on HTN and TTN fruit size

To determine whether lower cell division activity and/or lower cell enlargement is due to an alteration in the plant hormone contents and signaling pathway in TTN fruit, the effects of a synthetic CK (CPPU), GA (GA₃ and GA₄₊₇), and auxin (NAA) on TTN fruit size were investigated. Among the three types of PGRs, only CPPU treatments recovered the fruit size of TTN. GA treatments did not have any significant effect on fruit size, and all of the fruit was abscised by the auxin treatments. CPPU treatments also increased the fruit size of HTN. An increase in the HTN fruit size using CPPU treatments was reported previously (Hamada et al., 2008; Hasegawa et al., 1991; Itai et al., 1995; Sugiyama and Yamaki, 1995). Hamada et al. (2008) suggested that the increases in HTN fruit diameter caused by CPPU were closely related to the increases in lengths of parenchymal cells, but not cell division. Indeed, increases in the HTN fruit diameter by CPPU treatments were observed only in the later growing stage (after 12 WAB) in this study, in accordance with Hamada et al. (2008). However, TTN fruit diameters with CPPU treatments were slightly increased at 2 WAB and then exhibited a similar pattern to that of the control HTN fruit. Considering that the duration of cell division in HTN was about 4 WAB (Hamada et al., 2008; Hirata and Hayashi, 1978), CPPU treatments might affect not only parenchymal cell enlargement in the later growing stage, but also cell division in the early growing stage. The finding that the effect of CPPU treatments on cell division was observed in TTN and not in HTN is intriguing. A possible explanation is that the endogenous levels of CKs in HTN are sufficient for maximum cell division activity, while those in TTN are insufficient. Indeed, the histological analysis also suggested that cell division activity was significantly decreased in TTN compared with HTN. In addition, cell enlargement in the later growing stage was also affected by CPPU treatments. Because the diameter of CPPU-treated fruit also exhibited a similar pattern to non-treated control HTN fruit during the cell enlargement period (after 10 WAB), endogenous levels of CKs in TTN may be also insufficient during the cell enlargement period. Our results indicated that CPPU applications complement the mutation of fruit size in TTN, suggesting that an alteration of CK biosynthesis and/or metabolism throughout the growing season may be involved in the lower cell division activity in the early growing stage and possibly also in the lower cell enlargement activity in the later growing stage.

Transcriptome analysis of HTN and TTN fruits

To test the hypothesis that the mutations in CK synthesis and/or catabolism are involved in the small fruit phenotype of TTN, we conducted a large-scale transcriptome analysis of HTN and TTN fruits using Illumina sequencing. We obtained 118,985 unigenes ranging from 201 to 11,954 bases with an average length of 710 bases. Among these unigenes, 49,110 (42.3%) had homologous genes in GenBank. Recently, Luo et al. (2014) reported a large-scale transcriptome analysis of Chinese pollination-constant, non-astringency persimmon fruit using 454-pyrosequencing. They obtained 83,898 unigenes with an average length of 579 bases, and annotated 54,719 unigenes (65.2%) using the BLASTX algorithm against the nr database. The differences in unigene number and length between our results and Luo et al. (2014) are probably due to the different sequencing strategies, that is, the sample preparation, sequencing platforms and assembly programs. It was reported that the expression level of Pafw2.2-like was higher in smaller fruits than in normal fruits of ‘Hass’ in avocado (Cowan et al., 1997; Dehan et al., 2010; Zilkah and Klein, 1987). FW2.2/CNR family genes are known to be associated with the regulation of fruit cell number in tomato (Frary et al., 2000) and sweet cherry (De Franceschi et al., 2013). Although our studies also suggested that the difference in fruit size between HTN and TTN attributed mainly to different cell numbers, the expression levels of FW2.2/CNR family genes did not significantly differ. These results suggested that, in contrast to avocado, the fewer cell numbers in TTN fruits is regulated by other pathways rather than those where FW2,2/CNR was significantly involved.

Our PAGE analysis revealed that the expression levels of unigenes related to the cell cycle were significantly lower in TTN. The unigenes encoding CYCD3 and MAPKKK were included in the DEGs with lower expression levels in TTN (Table 4). CYCDs are G1 proteins, regulating the progression through the G1 phase and the G1/S transition. In Arabidopsis, the CYCD family includes 10 genes divided into seven subgroups (Menges et al., 2007; Vandepoele et al., 2002). Among them, the CYCD3s, which are induced by CKs, contribute to the control of cell numbers in leaves by regulating the duration of the mitotic phase and the timing of the transition to endocycles (Dewitte et al., 2007; Riou-Khamlichi et al., 1999). In fruits, Kvarnheden et al. (2000) reported that the CYCD3s in tomato are probably involved in transducing the signals leading to fruit development by cell divisions. In addition, the expression levels of CsCYCD3;1 and CsCYCD3;2 were abundant in cucumber pollinated ovaries, and were induced by CPPU treatment (Fu et al., 2010). Therefore, CKs likely control the fruit cell number by regulating the expression levels of the CYCD3s. Thus, CYCD3s may play important roles in early fruit development by promoting cell division in Japanese persimmon fruit. Therefore, it is possible that the smaller number of cells in TTN fruit results from the lower expressions of CYCD3. Furthermore, we also found that the expression level of the unigene encoding ANP1, which is a member of the MAPKKK gene family, was not detected in TTN. A study using mutants carrying knockout alleles of ANP1 and its close relatives, and functionally redundant homologs ANP2 and ANP3, supports a model in which the ANP family of MAPKKKs positively regulates cell division and growth (Krysan et al., 2002). However, no exogenous hormone treatment, including CK, rescued the mutant phenotype of the anp2/anp3 double mutants (Krysan et al., 2002). Because the fruit size of TTN was recovered by CPPU treatments, the lower expression level of ANP1 may not be a major reason for the TTN phenotype.

Expression of CK synthesis-related genes in TTN fruit

The small fruit phenotype of TTN may be caused by an alteration in CK levels in TTN fruit. To test this hypothesis, we examined the expression levels of CK biosynthesis-related genes. The initial step of CK synthesis is catalyzed by isopentenyltransferase (IPT), producing isopentenyladenine (iP) nucleotides from ATP/ADP/AMP and dimethylallyl diphosphate as CK precursors (Sakakibara, 2006). The iP-nucleotides are not only used for the synthesis of iP, but are also converted into trans-zeatin nucleotides by the cytochrome P450 monooxygenases (CYP735A1/CYP735A2) (Takei et al., 2004). To activate these precursors, they are converted into the free-base form by CK riboside 5'-monophospate phosphoribohydrolase, namely LOG, which was recently identified in Arabidopsis and rice (Kurakawa et al., 2007; Kuroha et al., 2009). The active forms of the CKs are degraded by CKX, which removes CK unsaturated isopentenyl chains (Nishiyama et al., 2011). Searching using the BLASTX algorithm revealed 11 IPT-like unigenes (divided into five types), 1 CYP735A1-like unigene, 5 LOG1-like unigenes, and 12 CKX-like unigenes (divided into five types); however, our in silico analysis suggested that their expression levels were not significantly different between HTN and TTN fruits. However, our transcriptome analysis did not exclude the possibility that these CK synthesis-related genes are involved in the small fruit phenotype of TTN, and further studies are required. Presently, we are analyzing the CK contents in TTN fruits and the allele-specific expressions of CK metabolism-related genes, and we are also conducting genomic studies to find mutated regions.

In conclusion, we demonstrated that smaller cell numbers during the early stage of fruit development mainly contribute to the small fruit phenotype of TTN, and the smaller cell size in the later stage may also result in the small fruit phenotype. To support this finding, we found decreases in cell-cycle regulation in TTN fruit. Furthermore, we found that CPPU complemented the small fruit mutation in TTN, and led to the hypothesis that alterations in CK metabolism may be involved in the small fruit phenotype. Although our in silico analysis could not find significant differences in expression levels of CK metabolism-related genes, we are continuing studies to understand the small fruit phenotype of TTN and the genetic basis for regulating fruit size in the Japanese persimmon.

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