Hormone analysis and candidate genes identification associated with seed size in Camellia oleifera.

Camellia oleifera is an important woody oil species in China. Its seed oil has been widely used as a cooking oil. Seed size is a crucial factor influencing the yield of seed oil. In this study, the horizontal diameter, vertical diameter and volume of C. oleifera seeds showed a rapid growth tendency from 235 days after pollination (DAP) to 258 DAP but had a slight increase at seed maturity. During seed development, the expression of genes related to cell proliferation and expansion differ greatly. Auxin plays an important role in C. oleifera seeds; YUC4 and IAA17 were significantly downregulated. Weighted gene co-expression network analysis screened 21 hub transcription factors for C. oleifera seed horizontal diameter, vertical diameter and volume. Among them, SPL4 was significantly decreased and associated with all these three traits, while ABI4 and YAB1 were significantly increased and associated with horizontal diameter of C. oleifera seeds. Additionally, KLU significantly decreased (2040-fold). Collectively, our data advances the knowledge of factors related to seed size and provides a theoretical basis for improving the yield of C. oleifera seeds.

Introduction

Camellia oleifera is a small evergreen tree or shrub of the Camellia genus, Theaceae [1]. It is known as one of the world's four largest woody oil plants with oil palm, olive and coconut [2]. The seed of C. oleifera has high oil content with nutrition and healthcare value [3-5]. However, the low yield of C. oleifera seeds has been seriously restricting the healthy development of the C. oleifera industry [6]. Manipulating seed size is an ideal way to solve this problem, because it is reported that seed size is a crucial factor influencing the final seed yield [7]. Currently, some genes have been identified to play a role in determining seed size in Arabidopsis thaliana and rice (Oryza sativa). However, no such studies have been reported on C. oleifera.

The growth and development of seeds begin after pollination and fertilization, and pass through two important periods of cell proliferation and cell expansion [8]. Cell proliferation and cell expansion play key roles in seed growth and development because they determine the number and size of cells in seeds and ultimately affect seed size [9,10]. Among them, the different cell cycles in cell proliferation are regulated by a protein complex composed of cell cycle-dependent kinases (CDKs) and cyclins [11-13]. Current research mainly focused on the regulation of the cell cycle by A- and B-type CDKs (CDKAs and CDKBs) [14,15] and A-, B- and D-type cyclins (CYCAs, CYCBs and CYCDs) [16-18]. However, cell expansion drives the extension of the cell wall and further affects cell size [19]. Therefore, cell size is closely related to the extension of cell wall. Enzymes acting on the cytoskeleton play key roles in cell wall extension [20]. Among them, expansins (EXPs) and xyloglucan endotransglucosylase/hydrolase (XTHs) are important participants in the regulatory framework of the cell wall [21]. Expansins disrupt hydrogen bonding between hemi cellulosic wall components and cellulose microfibrils to facilitate cell wall extension [22]. Unlike expansin, XTHs catalyse the connection of xyloglucan molecules to itself and specifically hydrolyse xyloglucan β-1,4 glycosidic bonds [23,24].

In addition to cell proliferation and cell division, the HAIKU (IKU) pathway, the ubiquitin-proteasome pathway, G-protein signalling pathway and mitogen-activated protein kinase (MAPK) signalling pathways have been discovered to control seed size in Arabidopsis and rice [25]. The ubiquitin-proteasome pathway, G-protein signalling pathway and MAPK signalling pathways have been shown to control seed size by regulating the growth of maternal tissues, while the IKU pathway controls seed size by regulating zygotic tissues [26,27]. Furthermore, phytohormones related to regulating seed size have also been studied, Brassinolide (BR), auxins and salicylic acid (SA) are considered to be important participants [26,28]. In rice, mutants with defects in BR biosynthesis or signal transduction showed shortened seed length, and the overexpression of the BR biosynthesis gene in rice increased seed size [29,30]. This indicates that BR plays an important role in seed size. Auxin has been studied to regulate rice seed size by setting physical constraints via the seed coat [31]. Auxin response factors 2 (ARF2)/MEGAINTEGUMENTA (MNT) is a repressor of cell division and organ growth, and it controls seed size by limiting the cell proliferation of integument [32]. Compared with wild-type seeds, mnt/arf2 mutants increased seed and organ size [26]. SA has also been studied to increase rice yield [33] and regulate cell growth in Arabidopsis [28]. Seed size is also regulated by transcription factors (TFs) [26], such as APETALA 2 (AP2), KLUH (KLU) and ENHANCER3 OF DA1 (EOD3).

Current research on C. oleifera seeds mainly focuses on the genes related to oil content and fatty acid composition [34,35], but there are a few studies on seed size genes. In this study, the content of phytohormone was detected. Gene expressions associated with cell proliferation and cell cycle, cell expansion, known signal pathways and phytohormone at different stages were analysed. Weighted gene co-expression network analysis (WGCNA) was used to analyse the correlation between seed size of C. oleifera and TFs. This mainly provides a theoretical basis for improving the yield of C. oleifera seeds.

Material and methods

Plant materials collection

The plant materials used in this study came from 6-year-old, high-crown-grafted C. oleifera cultivars ‘Huashuo’ that had reached their peak production stage. The sampling site was in the Dongcheng Camellia experimental base in Wangcheng District, Changsha City, Hunan Province (28°05′ N, 113°21′ E), which has a humid mid-subtropical monsoon climate, the annual average temperature is 17°C and the precipitation is 1370 mm. Fifteen trees without significant pests or diseases, that were growing well and had undergone consistent management measures, were selected randomly. The collection of samples was randomly selected from four different directions of 15 trees and repeated three times. The fruits were collected at 210 days after pollination (DAP) (T1), 235 DAP (T2), 258 DAP (T3), 292 DAP (T4) and 333 DAP (T5). The seeds were frozen and stored at −80°C for analysis.

The determination of seed size and volume

Ten Camellia seeds were randomly selected and their vertical diameter and horizontal diameter were measured using a 1/100 vernier caliper. Seed volume was measured according to the method of Li et al. [36]. Briefly, 50 ml water was added to the 100 ml measuring cylinder, and the recording volume was V1. After adding five seeds, the recording volume was V2, so the volume of each seed was V2 - V1/5. When the seeds were small (235 DAP and 258DAP), the seed volume was measured with a 10 ml measuring cylinder.

Analysis of phytohormone content

The determination of phytohormone content refers to the method of Song et al. [37]. Grind 0.5 g of frozen sample was ground with liquid nitrogen and placed in a centrifuge tube. Phosphate buffered saline buffer (pH = 7.4) of nine times the sample volume was added, then centrifuged for 30 min (4°C, 8000 r.p.m.). The supernatant was stored at 4°C for later use. This was repeated three times. The contents of indole-3-acetic acid (IAA), BR and SA were determined using an enzyme-linked immunosorbent assay kit according to manufacturer's instructions.

Co-expression network construction and hub genes selection

All genes were obtained from published Illumina RNA-Seq data of C. oleifera seeds in the National Center for Biotechnology Information database (SRA with accession PRJNA668531; https://dataview.ncbi.nlm.nih.gov/object/PRJNA668531). The genes were screened based on the non-redundant protein database. Weighted gene correlation network analysis was used to construct a co-expression network according to Gong et al. [35] through the default network construction and module detection method. The power of β was set at 8 and the minimum number of module genes was set at 20. The hub gene selection was based on module membership (MM) and intramodular connectivity (K.in) values in the WGCNA results, and the top three genes of MM and K.in value in high correlation modules were selected as hub genes.

Statistical analysis

All graphs were obtained using Origin Pro (version 2018, OriginLab, Northampton, MA), Rstudio (v. 1.3.1056, Boston, Massachusetts, USA) and TBtools (v. 1.0983). The variance analysis was performed using SPSS (v. 19.0, Chicago, USA). All data had three biological replicates. To analyse the difference in multiple comparisons, the values of mean ± s.d. was calculated. Significance was defined at the p < 0.05 level.

Results

Variations in Camellia oleifera seeds size at different developmental stages

The horizontal diameter, vertical diameter and volume changes in C. oleifera seeds at different developmental stages are shown in figure 1. The increasing trends in horizontal diameter, vertical diameter (figure 1a) and volume (figure 1b) of seeds were similar, showing rapid growth from 235 DAP (T2) to 258 DAP (T3) with a slight increase at seed maturity (T4–T5). From 235 to 258 DAP, the horizontal diameter, vertical diameter and seed volume increased by 26.67%, 23.66%, and 5.55%, respectively, which was significantly higher than that in other periods. These results showed that 235 to 258 DAP was the period of rapid growth of C. oleifera seeds (electronic supplementary material, table S6).

Figure 1

Horizontal diameter, vertical diameter and volume of C. oleifera seeds in different developmental periods. The dynamic changes of horizontal diameter, vertical diameter (a) and seed volume (b). The data are the mean ± s.e. of 10 biological replicates and mean values (n = 10) followed by different lowercase letters in each column indicated significant differences at p < 0.05 by Duncan's multiple range test.

Changes in the expression of genes involved in cell proliferation and cell cycle

Genes related to cell proliferation and cell cycle play important roles in the growth and development of plants [10]. As shown in figure 2a and the electronic supplementary material, table S1, CDKs and cyclin-related genes involved in cell proliferation and cell cycle of C. oleifera seeds were analysed. Among CDKs genes, CDKB2-2 was highly expressed from T1 to T3, and its expression level in the T5 period was reduced by 93% compared to the T1 period. The cyclin genes in C. oleifera seeds were mainly A-, B- and D-type cyclins (CYCA, CYCB and CYCD). The expression level of most cyclin genes in the T1–T3 period was significantly higher than that in the T4–T5 period. Among cyclin genes, CYCD3-1, however, showed different trends. The expression of CYCD3-1-1 increased from T1 to T3, which was consistent with the change trend of seed size; however, the expression of CYCD3-1-2 decreased from T1 to T3, which was opposite to the trend change in seed size. Compared to the T1 period, the expression of CYCD3-1-2 was decreased to 6% in the T5 period. It was speculated that CYCD3-1 plays different roles in C. oleifera seed size.

Figure 2

Heatmap comparison of genes related to cell proliferation and cell expansion of C. oleifera seed. The heatmap shows the expression levels of genes related to cell proliferation (a) and cell expansion (b) of C. oleifera seeds in different periods. The red and blue colours represent upregulated and downregulated, respectively. Abbreviations: CDKB, CDKC, CDKD, CDKE: B-, C-, D, E-type cycle-dependent kinases; CYCA, CYCB, CYCD: A-, B- and D-type cyclins; XTHs, xyloglucan endotransglucosylase/hydrolase genes; EXPA, A-type expansins.

Expression level analysis of genes involved in cell expansion

The extension of the cell wall affects seed size by regulating cell size [8,19]. As important genes in the cell wall, EXPs and XTHs gene families were analysed. As shown in figure 2b and the electronic supplementary material, table S1, subfamily EXPA was the main member of EXPs gene family in C. oleifera seeds. The expression levels of EXPA1-2, EXPA10 and EXPA15 were upregulated at first but then reduced at seed maturity (T4–T5). Compared to the T3 period, EXPA1-2, EXPA10 and EXPA15 were reduced by 99.2%, 99.4% and 99.7% in the T5 period, respectively. The expression level of EXPA4-2 was reduced by 99.6% in the T5 period compared to the T1 period. Among XTHs gene family, XTH9 was highly expressed in the T1 period, but the expression in the T4 period decreased to 2%. XTH16 was highly expressed in the T2 period but then decreased. XTH23 showed different trends, XTH23-2 decreased from the T1 to T5 period while the expression of XTH23-3 increased 420-fold in the T4 period compared with the T1 period.

Variations in gene expression in signal pathways associated with the control of seed size

Gene expression levels of the ubiquitin-proteasome pathway, G-protein signalling pathway, MAPK pathway and IKU pathway were analysed in C. oleifera seeds. Among the screened genes related to the ubiquitin-proteasome pathway (figure 3a; electronic supplementary material, table S2), the expression levels of DA1 and PRT2A showed a downward trend. The expression levels of DA1 in the T5 period was reduced by 86% compared to the T1 period. However, the expression levels of DAR1, BBR and PRT2B increased first and then decreased. In the G-protein signalling pathway (figure 3b; electronic supplementary material, table S2), the expression level of GB1-1 in the T5 period was upregulated by fourfold compared to the T1 period, while GB1-2 was highly expressed in the T2 period. Additionally, genes related to the MAPK pathway were analysed (figure 3c; electronic supplementary material, table S2). The expression levels of MKK2, MKK3, MKK9 and MKP1 showed a downward trend. The expression levels of MKP1 were downregulated by 2.7-fold in the T5 period compared to the T1 period. Among the screened genes related to the IKU pathway (figure 3d; electronic supplementary material, table S2), the expression levels of IKU1 and IKU2 in the T5 period was reduced by 50% and 82% compared with the T1 period, respectively.

Figure 3

The expression of genes involved in signalling pathways related to seed size in C. oleifera seed development. The heatmap shows the expression levels of genes related to the ubiquitin-proteasome pathway (a), G-protein signalling pathway (b), MAPK pathway (c), and IKU pathway (d) in C. oleifera seeds at different stages. Red indicates upregulated genes, and blue indicates downregulated genes. Abbreviations: DAR1, DA1-related protein; BBR, BIG BROTHER-related; GPA1, Gαsubunit 1; GB1, Gβsubunit 1; MKK, mitogen-activated protein kinase; MKP, mitogen-activated protein kinase phosphatase; IKU, HAIKU.

Changes and correlation analysis of phytohormones content and related gene expression

The content of IAA, SA and BR in C. oleifera seeds was determined (figure 4a(i), b(i), c(i)). From the T1 to T3 period, the content of IAA, SA and BR showed an increasing trend, but then significantly decreased from the T4 to T5 period. Genes related to phytohormones biosynthesis and signalling pathways were analysed (figure 4a(ii), b(ii), c(ii); electronic supplementary material, table S3). The expression levels of most auxin-related genes were relatively higher in T1–T3 than in T4–T5 (figure 4a(iii)). In the process of auxin biosynthesis, the expression level of YUC4 in the YUC gene family was 1724-fold in the T1 period compared to the T5 period. In the process of the auxin signalling pathway, IAA17 and IAA27-1 were highly expressed in the T1 period, but were reduced by 98% and 96% in the T5 period, respectively. In addition, IAA14 was upregulated during the development of C. oleifera seeds, and in the T4 period, its expression level was 799-fold compared to that in the T1 period. Ten ARFs were expressed during the development of C. oleifera seeds. Among these ARFs, ARF2 was continuously decreased. Most of the genes in the SA biosynthesis and signalling pathways were expressed at a relatively high level in the T1 period (figure 4b(iii)). Compared to the T1 period, PAL-2, PAL1 and PAL6 related to SA biosynthesis were decreased by 99%, 95% and 95% in the T5 period, respectively. As shown in figure 4c(iii), genes related to BR biosynthesis and signalling pathways in C. oleifera seeds were expressed at a relatively high level from T1 to T3, but then decreased, which was consistent with the change trend of BR content.

Figure 4

Changes and correlation analysis of phytohormone contents and related gene expressions in developmental C. oleifera seeds. The content (i) and expression levels of genes (iii) involved in the biosynthetic and signal pathways (ii) of IAA (a), SA (b) and BR (c). Correlation analysis of phytohormone content and related gene expressions with horizontal diameter, vertical diameter and seed volume of C. oleifera seeds (iv). The red and blue colours represent upregulated and downregulated, respectively. Abbreviations: TAR, tryptophan aminotransferase-related; TIR, transport inhibitor response; YUC, YUCCA; AUX/IAA, auxin/IAA-inducible protein; ARF, auxin response factor; GH, Gretchen Hagen; PAL, phenylalanine ammonia lyase; NPR, non-expressor of pathogenesis-related; DWF, dwarf; CPD, constitutive photomorphogenic dwarf; BAK, BRI1-associated receptor kinase; BIN, BR-insensitive; BZR1, brassinazole resistant 1.

Correlation analysis shows that most auxin-related genes were negatively correlated with seed horizontal diameter, vertical diameter and volume of C. oleifera seeds, but positively correlated with IAA content (figure 4a(iv)). Among them, YUC4, IAA17, IAA27-1, ANT and ANT17 were significantly negatively correlated with seed horizontal diameter, vertical diameter and volume of C. oleifera seeds, but IAA14 was significantly positively correlated with them. Among the genes related to SA (figure 4b(iv)), PAL-2 and NPR1-2 were significantly negatively correlated with seed horizontal diameter, vertical diameter and volume of C. oleifera seeds. As shown in figure 4c(iv), all the screened genes related to BR were negatively correlated with seed horizontal diameter, vertical diameter and volume of C. oleifera seeds, but positively correlated with BR content.

Gene expressions of hub genes and selected transcription factors related to seed size

A total of 1880 TFs were identified from the Camellia seed transcriptome according to Gong et al. [35]. In order to screen for important TFs related to horizontal diameter, vertical diameter and seed volume, WGCNA was performed (figure 5). After removing the TFs with reads per kilobase per million mapped reads (RPKM) = 0 at the detected periods of developing seeds, the remaining 1879 TFs were obtained. The TFs were divided into 13 modules and marked with different colours (figure 5a). The number of genes in these modules ranged from 1 to 442 (figure 5b). The correlation analysis showed that yellow, blue, turquoise, red and tan modules had a higher correlation with horizontal diameter (figure 5c; electronic supplementary material, table S4). Yellow, blue, turquoise, red, green and greenyellow had a higher correlation with the vertical diameter. Yellow, blue, red, green and greenyellow had a higher correlation with seed volume (|correlation coefficient| greater than 0.7, p-value less than 0.01). The top three genes with high MM and K.in values in each module were selected as hub genes.

Figure 5

WGCNA identifying TFs related to horizontal diameter, vertical diameter and volume of C. oleifera seeds. (a) Clustering dendrograms of genes. Dissimilarity was based on topological overlap, with assigned module colours. The 13 co-expression modules are shown in different colours. (b) Number of genes in 13 modules. (c) Correlation between 13 modules and seed horizontal diameter, vertical diameter and volume. (d) The expression levels of hub genes in C. oleifera seeds at different developmental stages. (e) Heatmaps show the expression levels of TFs related to seed size. The red and blue colours represent upregulated and downregulated, respectively. Abbreviations: YAB1, YABBY1; BLH2, bel1-like homeodomain2; ABI4, ABA insensitive 4; Gtf3a, transcription factor IIIA; WRKY, WRKY transcription factor; MADS, MADS-box transcription factor; SPL: squamosa promoter-binding-like protein; MYB, MYB family protein; GATA11: GATA transcription factor; NAC014, NAC domain-containing protein 14; NTL9, protein NTM1-like 9; FRS3, fibroblast growth factor receptor substrate 3; GAI1, DELLA protein GAI1; IDD9, protein indeterminate-domain 9; GRF1, growth-regulating factor 1; HSF, heat stress transcription; FAR1, protein FAR1-related sequence; AP2, APETALA 2; GIF, GRF-interacting factor; KLU, KLUH; EOD3, ENHANCER3 OF DA1.

As shown in figure 5d, 21 hub genes were chosen (electronic supplementary material, table S5). Among them, Gtf3a, WRKY44, MADS6, SPL4, MYB44, GATA11, FRS3, SPL12 and GAI1 were hub genes common to the horizontal diameter, vertical diameter and seed volume. Compared with the T1 period, all the common hub genes showed the same trend, and the expressions of SPL4, MADS6, GAI1, MYB44, GATA11, WRKY44, FAR1 and SPL12 were reduced by 99%, 97%, 96%, 86%, 84%, 79%, 76% and 64% in the T5 period, respectively. Additionally, hub genes YAB1 and ABI4 in the tan module related to the horizontal diameter were first significantly upregulated but then decreased. Compared with T1, YAB1 was increased 800-fold in T3 and ABI4 was increased 560-fold in T4.

As shown in figure 5e and the electronic supplementary material, table S5, the expression levels of known seed size-related TFs were also analysed in C. oleifera seeds [26]. The expression levels of AP2, KLU and EOD3 performed significantly. Compared to the T1 period, AP2 and EOD3 were increased 14-fold and 259-fold in the T5 period, respectively. KLU was highly expressed in the T1 period and then decreased. The expression level in T5 was 2040-fold lower than that in the T1 period.

Discussion

Seed size of Camellia oleifera regulate by cell proliferation, cell expansion and signal pathways

The change in seed size is a complicated process regulated by different genes [13]. As an important process of seed development, cell proliferation and cell expansion affect seed size to a large extent. The regulation of the cell cycle on cell proliferation mainly relies on the complex composed of CDKs and cyclins to control G1/S and G2/M phases [38]. During the development of C. oleifera seeds, the expression levels of most CDKs and cyclin genes in the T1–T3 period were significantly higher than those in the T4–T5 period. Among them, CDKB was unique in plants and only expressed in the G2/M process [39]. In Arabidopsis and rice, the degradation of CDKB2 indirectly influenced the mitotic cycle and led to the presence of polyploid cells [40]. Different from the existence of CDKB2-1 and CDKB2-2 in Arabidopsis [41], only CDKB2-2 was highly expressed from the T1 to T3 period of C. oleifera seeds. The D-type cyclin CYCD3-1 restricted the transition of the G1/S phase and overexpressed CYCD3-1 in leaves leading to excessive proliferation of incompletely differentiated polygonal leaf cells in Arabidopsis [42,43]. In C. oleifera seeds, CYCD3-1 showed different trends. CYCD3-1-1 was increased at first but then decreased, while CYCD3-1-2 was significantly decreased. This suggested that CYCD3-1 have different functions in regulating seed size of C. oleifera.

The cell expansion was closely related to the stretch in the cell wall. Among them, XTHs and expansins can loosen cell walls and affect cell expansion [44,45]. During the development of C. oleifera seeds, most of the XTHs and Expansins genes were upregulated from the T1 to T3 period, then decreased. Besides, XTH23 show different treads. The protein sequences of XTH23 in pepper [46], cucumber [47], Arabidopsis [48] and C. oleifera of these species were aligned to construct a phylogenetic tree (electronic supplementary material, figure S1). XTH23-1 in C. oleifera was similar to CaXTH23. In pepper, CaXTH23 was highly expressed in the early stages of fruit development and then decreased gradually with fruit ripening [46], which was consistent with the expression trend of XTH23-1. XTH23-3 was a close relative of CsXTH23, which was related to the rapid expansion of cells in cucumber fruit spines [47]. In C. oleifera seeds, the expression of XTH23-3 increased 420-fold in the T4 period compared with the T1 period in C. oleifera seeds. Combined with the expression levels of XTH23-3 in C. oleifera seeds, it was speculated that it may play an important role in seed development at the nearly mature stage. In Arabidopsis, XTH9 was involved in the expansion of wood cells [49]. XTH9 was highly expressed in the T1 period of C. oleifera seeds and then decreased, which may be related to vigorous cell expansion. Additionally, Expansins genes were involved in cell wall relaxation and extension in Arabidopsis seeds [50]. Suppression of EXPA1 and EXPA10 altered chemical composition of the cell wall [51]. In C. oleifera seeds, most of the Expansins genes were increased first (T1–T3) but then significantly decreased. It was speculated that Expansins genes mainly act on the period of rapid expansion of C. oleifera seeds.

In addition to cell proliferation and cell expansion, the ubiquitin-proteasome pathway, G-protein signalling pathway, MAPK pathway and IKU pathway were identified to be involved in the regulation of seed size [26]. In the ubiquitin-proteasome pathway, DA1 negatively regulates seed size [52]. The larger seeds produced by the da1-1 mutant was the result of enlargement of sporophyte integument in Arabidopsis [53]. In C. oleifera seeds, the expression levels of DA1 were reduced by 86%, which was consistent with the negative regulation of seed size by DA1 in Arabidopsis. Among the genes screened in the MAPK pathway, MKP1 control grain size by restricting cell proliferation in grain hulls and the overexpression of MKP1 leads to small grains in rice [54]. In C. oleifera seeds, the expression levels of MKP1 were downregulated by 2.7-fold in the T5 period. Unlike the ubiquitin-proteasome pathway, the G-protein signalling pathway and MAPK pathway regulated seed size by maternal tissues, the IKU pathway controls seed size by regulating zygotic tissues [26,27]. In Arabidopsis, iku1 and iku2 produced small seeds and led to early cellularization of endosperm, which was similar to the seeds with excessive parental dosage [55]. In the development of C. oleifera seeds, IKU1 and IKU2 were downregulated. Combined with Arabidopsis, IKU1 and IKU2 were important participants in the development of endosperm.

Role of phytohormones in regulating Camellia oleifera seed size

Phytohormone regulates diverse processes in seed growth and development. Auxin, BR and SA have been suggested to play an important role in the regulation of seed size control [28]. In Arabidopsis, YUC4 is a key gene in the process of auxin biosynthesis, and the overexpression of YUC4 influenced auxin homeostasis and seed size [31,56]. In the process of C. oleifera seed development, the expression level of YUC4 was 1724-fold in T1 compared with T5, and its significant downregulation may be related to the accumulation of auxin. AUX/IAA and ARF suppress and regulate auxin signal transduction [57]. Among them, IAA14 was involved in the formation of Arabidopsis lateral roots [58] and IAA17 control tomato fruit size via the regulation of cell size [59]. In C. oleifera seeds, IAA14 was upregulated 799-fold in T4 compared with that in T1. The expression level of IAA17 was reduced by 98% from the TI to T5 period. It suggested that IAA14 and IAA17 regulate C. oleifera seed size by participating in auxin signal transduction. ARF2 inhibits the proliferation of maternal cells [31,60]. In C. oleifera seeds, ARF2 was downregulated and significantly correlated with seed phenotype, which indicates that ARF2 negatively regulates seed size in C. oleifera as in Arabidopsis. Additionally, studies on the change of SA-related genes of C. oleifera seeds showed that PAL-2, PAL1 and PAL6 in the SA biosynthesis pathway were significantly downregulated from the T1 to T5 period, and the gene expression of the signalling pathway did not change significantly in C. oleifera seeds. The downregulation of PAL-2, PAL1 and PAL6 in C. oleifera seeds may be related to the formation of secondary metabolites [61]. In Arabidopsis, BZR1 is an important TF for regulating organ size in the BR signalling pathway. The overexpression of ZmBZR1 in Arabidopsis shows seed enlargement [62], however, BZR1 had not significantly changed in C. oleifera seeds.

The regulation of transcription factors in Camellia oleifera seed size

TFs were also involved in the regulation of seed size [26]. First, the hub genes related to the three traits of C. oleifera seed were analysed by WGCNA. Among the screened hub genes, the SPL gene family plays an important role in plant growth and increases crop yield by regulating the architecture of rice plants [63,64]. In rice, SPL4 could be a key regulator of grain size by acting on cell division control to improve rice yield and the overexpression of SPL4 decreased grain weight and size [65]. Seed-specific overexpression of SPL12 and plant Architecture 1 (IPA1) enhance seed improved seed dormancy grain size in rice [66]. In C. oleifera seeds, SPL4 and SPL12 were decreased to 1% and 36% in T5, respectively, which indicates SPL4 and SPL12 may be important participants in the regulation of seed size. Additionally, WRKY44 controls final seed size by regulating seed coat development in Arabidopsis [67]. FAR1 affected cell proliferation by regulating the cell cycle, and its transcription in the G1 phase was essential for protein accumulation and pheromone-induced cell cycle arrest [68]. In C. oleifera seeds, WRKY44 and FAR1 were decreased to 21% and 24% in the T5 period, respectively. WRKY44 maybe related to seed coat development and FAR1 may be correlated with cell proliferation in C. oleifera seed development. Among the selected hub genes related to horizontal diameter, ABI4 and YAB1 were closely associated with Camellia seed size. The overexpression of ABI4 decreased in plant height and poor seed production in Arabidopsis [69]. The rice YAB1 gene causes dwarfing and affects yield [70]. In C. oleifera seeds, ABI4 and YAB1 were significantly increased 800-fold in T3 and 560-fold in T4, respectively. It indicated that ABI4 and YAB1 may be important participants in the regulation of C. oleifera seed size.

The expression levels of known seed size-related TFs were also screened in C. oleifera seeds [26]. Among them, AP2 affected the development of zygotic embryos and endosperm to inhibit seed size of Arabidopsis. The ap2 mutant produced larger seeds than the wild-type [71]. KLU regulated seed size by promoting the proliferation of maternal cells [72]. EOD3 could act maternally to promote cotyledon cell expansion and thus affects the size of rapeseed [73]. In C. oleifera seeds, AP2 and EOD3 were significantly increased 14-fold and 259-fold in the T5 period, respectively, while KLU significantly decreased (2040-fold), which suggests that these genes may play important roles in the regulation of the C. oleifera seed size.

Conclusion

During seed development of C. oleifera, the seeds horizontal diameter, vertical diameter and volume showed rapid growth from 235 DAP (T2) to 258 DAP (T3) with a slight increase at seed maturity (T4–T5). The content of IAA, SA and BR showed an increasing trend from the T1 to T3 period, but then decreased at seed maturity (T4–T5). By studying genes related to hormone biosynthesis and signalling pathways, we found that most genes related to the auxin signalling pathway were significantly negatively correlated with seed horizontal diameter, vertical diameter and volume. Among them, YUC4 was significantly downregulated (1724-fold). Through WGCNA, 21 hub genes were screened. Among them, SPL4 was significantly decreased and associated with seed horizontal diameter, vertical diameter and volume. ABI4 and YAB1 were significantly increased 800-fold in T3 and 560-fold in T4, respectively. Additionally, KLU significantly decreased (2040-fold). Taken together, these results suggest that these genes are important participants in regulating the size of C. oleifera seeds.

Click here for additional data file.