Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans

doi:10.21203/rs.3.rs-24581/v1

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Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans

https://doi.org/10.21203/rs.3.rs-24581/v1

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published 16 Jul, 2020

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Background: Osmanthus fragrans Lour. is one of the top ten traditional ornamental flower in China. The flower time of once-flowering cultivars in O. fragrans is greatly affected by the relatively low temperature, but there is few reports on its molecular mechanism to date.

Results: In this study, the cell size of adaxial and abaxial petal epidermal cells and ultrastructural morphology of petal cells at different developmental stages were observed. The cell size of adaxial and abaxial petal epidermal cells increased gradually with the process of flower opening. Then the transcriptomic sequencing was employed to analyze the differentially expressed genes (DEGs) under different days’ treatments with relatively low temperatures (19°C) or 23°C. Analysis of DEGs in GO analysis showed that “metabolic process”, “cellular process”, “binding”, “catalytic activity”, “cell”, “cell part”, “membrane”, “membrane part”, “single-organism process”, and “organelle” were highly enriched. In KEGG analysis, “metabolic pathways”, “biosynthesis of secondary metabolites”, “plant-pathogen interaction”, “starch and sucrose metabolism”, and “plant hormone signal transduction” were the top five pathways containing the greatest number of DEGs. DEGs involved in cell wall metabolism, phytohormone signal transduction pathways, and eight kinds of transcription factors were analyzed in depth.

Conclusions: Several unigenes involved in cell wall metabolism, phytohormone signal transduction pathway, and TFs with highly variable expression levels between different temperature treatments may be involved in petal cell expansion during flower opening process in response to the relatively low temperature. These results could improve our understanding of the molecular mechanism of relatively low temperature regulating flower opening of O. fragrans and provide theoretical reference for the prediction and regulation of flowering time and genetic modification in O. fragrans.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Flower opening

Osmanthus fragrans

Petal

Relatively low temperature

Transcriptomic analysis

Osmanthus fragrans Lour. (Oleaceae) is one of the top ten traditional ornamental plant in China with more than 2500 years’ history in cultivation [1]. It belongs to small evergreen trees, grown as ornamental plants for their fragrant edible flowers. Cultivars of O. fragrans could be divided into once-flowering group and recurrent-flowering group according to different flowering habits [2]. The once-flowering cultivars bloom in autumn and the flower time varies greatly in different areas, such as in Hangzhou, Shanghai, Nanjing, and Suzhou, or even in different years in the same area [3]. The researches on the flower time of different cultivars indicated that relatively low temperature before blooming is the most important environmental factor determining the flower opening of O. fragrans [3, 4]. However, the knowledge of molecular mechanism of flower opening in O. fragrans, especially response to relatively low temperature, still remains limited.

In many higher plants, the growth of flower petals is the most remarkable process during flower opening. Flower petals are the most important component of reproductive organs and play vital roles in attracting the correct pollinator(s). Flower color, size, shape and appearance, which are determined by flower petal, are important traits appreciated by the breeders and consumers [5]. One of the traits, the size of flower petal is determined by cell division at early phases of petal growth and cell expansion at later stages of flower opening [6].

Cell expansion is accompanied by a series of process including cell wall loosening, cellulose biosynthesis, polysaccharides conversion into soluble carbohydrate, ion and water uptake, and cytoskeleton rearrangement [7]. Cell wall loosening proteins include expansin (EXP), xyloglucan endotransglycosylase/hydrolase (XTH), endo-1,4-β-D-glucanase, and pectinase [8]. EXP and XTH particularly participate in disrupt the non-covalent bonds between the cellulose microfibrils and the cross-linking glycans of the cell wall to increase the cell-wall creep rate, while pectinase and endo-1,4-β-D-glucanase can degrade the cell wall [9]. In wintersweet (Chimonanthus praecox), the expressions of EXP genes increase during flower opening [10]. The developing petals of carnation show high activities of cellulase and pectin esterase [11]. Soluble carbohydrates depending on the degradation of polysaccharides can act as osmotically active compounds which could lower the osmotic water potential and facilitate water influx in order to allow cell expansion [12]. The concentration of soluble carbohydrates in the petals will increase in the flower opening process such as carnation [13], rose [14], chrysanthemum [15], Tweedia caerulea [16], and lisianthus [17].

Cell expansion is regulated by both external factors, such as temperature, humidity, and the quality and quantity of light, and internal factors, such as the circadian clock and phytohormones [18, 19]. Phytohormones are the most important mediators regulating flower opening and could be affected by circadian factors or environmental factors. As far as the present researches are concerned, most of the phytohormones, such as auxin (AUX), gibberellin (GA), ethylene (ETH), brassinosteroid (BR), jasmonic acid (JA) and abscisic acid (ABA), are proven to affect flower opening [5, 6, 20–23].

In this research, potted plants of O. fragrans ‘Yanhong Gui’ were employed as materials to research the effects of different temperature conditions on flower opening process under controlled relatively low temperatures. Then the transcriptomic sequencing was used to analyze the differentially expressed genes (DEGs) after different days’ treatment under relatively high or low temperatures to figure out the key genes involved in the regulation of flower opening to relatively low temperature. This study would lay foundation on fully revealing the molecular mechanism of relatively low temperature regulating flower opening of O. fragrans and provide theoretical reference for the prediction and regulation of flowering time and genetic modification in O. fragrans.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis

Observation on the cell size of adaxial and abaxial petal epidermal cells with SEM revealed that cell size of adaxial petal epidermal cells increased gradually from S1 to S6 (Fig. 1a, Table S1), while that of abaxial petal epidermal cells enlarged gradually from S1 to S5, reached the peak at S5, and then greatly decreased from S5 to S6 (Fig. 1b, Table S1). What's more, based on TEM observation, it was found that vacuole occupied most area of petal cells in O. fragrans (Fig. 1c, Table S1), and vacuole size increased gradually from S1 to S6. At S2, vacuole size of cells was nearly two times as large as that at S1. With the cell development, vacuole size of cells at S6 was just one time larger than that at S2 (Fig. 1c, Table S1).

Bud appearance with treatments

Before treatment, the buds of all plants were at S1, and they were spindle-shaped, with unfurled outer bud scales and furled inner bud scales. Bud appearance remained the same under 23 °C treatment for 2 d, 4 d, and 6 d (H2, H4, H6), as well as the 19 °C treatment for 2 d and 4 d (L2 and L4). When treated with 19 °C treatment for 6 d (L6), the bud stage reached at S2. Buds of L6 became globular-shaped, the inner bud scales unfurled and the inflorescence was visible.

Illumina sequencing and de novo assembly of sequence reads

Reference transcriptome sequencing generated 164,753,984 raw reads and 110,307,366 clean reads (Table S2). A total of 152,247 transcripts with average length of 751 nt were obtained and 96,920 unigenes with average length of 873 nt were yielded after all clean reads were assembled (Table S3).

Functional annotation and classification

The assembled unigenes were aligned to seven databases (Nr, Nt, Swiss-Prot, KEGG, KOG, GO, and InterPro) in order to obtain the putative annotations (Table S4). Totally, 61,654 (63.61%) unigenes were successfully annotated using at least one database, while 8,150 (8.41%) unigenes were annotated using all seven databases. Total 57,721 (59.56%) of unigenes were annotated using NR database, 59.26% of which have high homology to sequences from Sesamum indicum (Fig. S1). In total, 37,284 unigenes were matched in the Swiss-Prot database, which is about 38.47% of all annotated unigenes (Table S4). There were 40,764 unigenes mapped into 134 KEGG pathways which can be divided into six large pathways including “cellular processes”, “environmental information processing”, “genetic information processing”, “human diseases”, “metabolism”, and “organismal systems” (Fig. S2). The pathways with the highest numbers of unigenes were “metabolic pathways” (Ko01100, 8,561 unigenes, 21%), followed by “biosynthesis of secondary metabolites” (Ko01110, 4,440 unigenes, 10.89%), “plant-pathogen interaction” (Ko04626, 1,876 unigenes, 4.6%), and “plant hormone signal transduction” (Ko04075, 1,444 unigenes, 3.54%) (Table S5). Next, GO analysis was performed and a total of 16,014 unigenes (16.52% of all annotated unigenes) were categorized into 53 GO terms under three main categories: biological process, cellular component, and molecular function (Fig. S3). Proteins related to “metabolic process”, “cellular process”, and “single-organism process” were enriched in biological processes. The “single-organism process” category defines a biological process involving only one organism, and the 4,806 unigenes annotated as such may be related to flowering organisms specifically. In the cellular component category, the “cell”, “cell part”, and “membrane” were the most highly presented GO terms. Under the molecular function category, the “catalytic activity”, and “binding proteins” were the most enriched (Fig. S3). We also performed KOG analysis to evaluate the functions of the assembled unigenes and 43,496 unigenes were assigned 25 KOG classification (Fig. S4). Among the 25 KOG classification, the cluster of “general function prediction only” (11,753 unigenes, 27.02%) represented the largest group, followed by “signal transduction mechanisms” (6,000 unigenes, 13.79%) and “function unknown” (4,761, 10.95%). “Cell motility” has the smallest proportion, only accounted for 0.20% (Fig. S4). Additionally, 46,405 (47.88%) unigenes in the transcriptome library were annotated against InterPro database (Table S4).

RNA-seq Illumina sequencing and mapping to the reference transcriptome database

Eighteen cDNA libraries for RNA-seq analysis were respectively sequenced, generating 23.58–24.14 Mb raw reads (Table S2). Furthermore, we obtained 23.58–24.13 Mb clean reads in 18 cDNA libraries, with the clean data rate of 99.94%-99.97%. Then, these clean reads were respectively mapped to the reference transcriptome database. Total mapped reads percentage and unique match percentage in the 18 libraries were similar. Total mapped reads percentage ranged from 86.39–88.44%, and unique match percentage ranged from 53.7–56.29% (Table S6). The correlation coefficients among biological replications are greater than 0.94 in all samples (Fig. 2, Table S7).

Screening and analysis of DEGs

We performed a detailed comparative analysis of the DEGs (P value ≤ 0.01 and |log₂Ratio| ≥ 1) in three comparisons including L2 vs H2, L4 vs H4, and L6 vs H6. In the L2 vs H2 comparison, there were 7,365 and 15,864 unigenes were down-regulated and up-regulated, respectively. Meanwhile, 8,393 and 22,755 unigenes were down-regulated and up-regulated in the L4 vs H4 combination, respectively. In the L6 vs H6 comparison, 9,551 unigenes were down-regulated and 22,011 unigenes were up-regulated, respectively (Fig. 3a). In total, there were more up-regulated unigenes than down-regulated unigenes during floral opening under relatively low temperature. Additionally, 7,496 up-regulated DEGs were shared by three comparisons, while 4,108, 6,132, and 6,913 up-regulated DEGs were only up-regulated in the L2 vs H2, L4 vs H4, and L6 vs H6 comparison, respectively (Fig. 3b). 2,968 down-regulated DEGs were shared by three comparisons, while 2,954, 2,189, and 3,806 down-regulated unigenes were only down-expressed in the L2 vs H2, L4 vs H4, and L6 vs H6 comparison, respectively (Fig. 3c).

GO enrichment and KEGG pathway analysis of DEGs

GO analysis were used to classify the functions of the annotated DEGs under different temperatures treated for different days. In all comparisons, the most significantly enriched GO terms in biological process were “metabolic process”, “cellular process”, and “single-organism process”. In cellular component, five GO terms including “cell”, “cell part”, “membrane”, “membrane part”, and “organelle” were highly enriched. “Binding” and “catalytic activity” which were in the molecular function were significantly enriched in all comparison’s groups (Table 1). All DEGs were mapped to KEGG pathways in order to investigate the major pathways of them (Table 2). They were enriched in 132, 133, and 134 KEGG metabolic pathways in the L2 vs H2, L4 vs H4, and L6 vs H6 comparisons. In all the comparison group, “metabolic pathways”, “biosynthesis of secondary metabolites”, “plant-pathogen interaction”, “starch and sucrose metabolism”, and “plant hormone signal transduction” were the top five pathways with the largest number of DEGs (Table 2).

Table 1

The top ten most enriched GO terms of DEGs in all comparison groups
GO term hierarchy 1	GO term hierarchy 2	Number of DEGs
GO term hierarchy 1	GO term hierarchy 2	L2 vs H2	L4 vs H4	L6 vs H6
Biological process	metabolic process	1823	2328	2539
	cellular process	1693	2190	2331
	single-organism process	1117	1424	1556
Cellular component	cell	1287	1626	1729
	cell part	1271	1610	1710
	membrane	1162	1395	1613
	membrane part	886	1052	1229
	organelle	866	1117	1187
Molecular function	binding	1628	2135	2266
Molecular function	catalytic activity	1693	2136	2379

Table 2

The top ten most enriched KEGG of DEGs in all comparison groups
Pathway	ID	L2 vs H2		L4 vs H4		L6 vs H6
Pathway	ID	Percent (%)	P-value	Percent (%)	P-value	Percent (%)	P-value
Metabolic pathways	ko01100	22.94	3.69895E-07	23.64	2.02306E-16	23.78	9.35908E-20
Biosynthesis of secondary metabolites	ko01110	12.43	1.83429E-07	13.09	8.08594E-19	13.23	3.08193E-23
Plant-pathogen interaction	ko04626	5.42	2.90029E-05	5.43	4.82036E-07	5.67	1.43759E-11
Starch and sucrose metabolism	ko00500	4.03	9.30016E-08	3.69	3.81391E-05	3.77	8.46098E-07
Plant hormone signal transduction	ko04075	3.96	0.01001351	4.27	6.50429E-07	4.65	2.91758E-15
Protein processing in endoplasmic reticulum	ko04141	3.04	0.03015519	2.82	0.2699489	2.63	0.827178
Carbon metabolism	ko01200	2.84	0.1309717	2.87	0.06273836	3.1	0.000191297
Endocytosis	ko04144	2.72	0.5626473	2.93	0.07581657	2.73	0.5247731
Ribosome	ko03010	2.76	0.006505746	2.94	4.06865E-06	-	-
Phenylpropanoid biosynthesis	ko00940	2.55	2.74168E-13	-	-	-	-
Amino sugar and nucleotide sugar metabolism	ko00520	2.51	0.007218067	2.66	1.27859E-05	2.46	0.003741373
Biosynthesis of amino acids	ko01230	2.45	0.8067985	2.66	0.272135	2.78	0.04693815
Spliceosome	ko03040	-	-	2.82	0.8755484	2.52	0.9998826
RNA transport	ko03013	-	-	-	-	2.56	0.997384

Identification of DEGs involved in the cell wall metabolism

The expansion of petal cells depends on the cell wall loosening and cellulose biosynthesis, soluble carbohydrate allocation, ion and water transport, and cytoskeleton rearrangement [7]. Therefore, we explored the DEGs involved in the cell wall metabolism under different temperatures treated. EXP and XTH are involved in cell expansion, loosening and rearranging the cell wall fibers in growing tissues [8]. In our study, four α-EXP and one β-EXP were significantly different expressed under 23 °C and 19 °C comparisons (Fig. 4, Table S8). Among them, four α-EXP were up regulated under relatively low temperature (19 °C) and one β-EXP was down regulated under the treatment of 19 °C. Eleven unigenes which encoded as XTH protein were significantly expressed, seven of them (CL256.Contig3, CL3252.Contig2, CL10571.Contig1, Unigene1021, Unigene1631, Unigene24045, and Unigene25288) were up regulated under the treatment of 19 °C and four (CL1547.Contig1, CL7741.Contig1, CL8777.Contig1, and Unigene26777) of them were down-regulated under 19 °C treatment (Fig. 4, Table S8). Among them, the expression of CL8777.Contig1 decreased sharply in all three comparisons when the plants were exposed to relatively low temperature.

The expression of unigenes involved in cell wall synthesis, modification or hydrolysis which probably participate in the process of flower opening were also assessed (Fig. 4, Table S8). There were three cellulose synthase (CES) genes, six xylosidase (XYL) genes, two pectin esterase (PE) genes, five polygalacturonase (PG) genes, and two pectate lyase (PL) genes with dramatic change fold in all three comparisons (Fig. 4, Table S8). The expression of all CESs and PLs genes increased when treated with relatively low temperature of 19 °C for different days. Most of the XYLs were up-regulated except Unigene17352 under relatively low temperatures. Three PGs’ expression increased and two of them decreased when exposed to relatively low temperature. One PE gene was greatly up-regulated while one PE gene was down-regulated when the plants were exposed to 19 °C. More genes involved in cell wall synthesis, modification or hydrolysis are up-regulated, suggesting that these genes have vital roles in regulating petal cell expansion.

Aquaporins can facilitate the passage of water and/or small neutral solute fluxes across membranes and can be classified into four subclasses such as plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic membrane proteins (NIPs) and small basic intrinsic proteins (SIPs) based on the sequence homology and cellular localization [24]. Three kinds of aquaporins including two PIPs, two TIPs and three NIPs, were found in our transcriptomes and most of them were significantly up-regulated under relatively low temperature (Fig. 4, Table S8). However, only the expression level of two NIPs (Unigene22675, and Unigene40148) decreased under relatively low temperature. Dehydrins are hydrophilic, thermostable stress proteins and some of them were identified as osmotic stress-responsive protein which could facilitate the inflow and outflow of water [25]. In our research, all the dehydrin genes were down-regulated when exposed to relatively low temperature (Fig. 4, Table S8).

Identification of DEGs involved in the phytohormone signal transduction pathway

There are some reports on phytohormones that can affect the floral opening [19], and therefore, we explored the DEGs involved in eight phytohormone signal transduction pathways. Nineteen unigenes were significantly differently-expressed under different temperatures in AUX signal transduction pathway which included the auxin influx carrier protein (AUX1), auxin/Indole-3-acetic acid (AUX/IAA), auxin response factor (ARF), auxin responsive GH3 family protein (GH3) and small auxin-up RNA family protein (SAUR) (Fig. 5). The majority of them had lower expression in relatively low temperature, especially three ARF members (CL661.Contig9, CL274.Contig1, and CL12908.Contig6), which decreased sharply as the treated days lengthened. Only one AUX1, two AUX/IAA, one ARF, and one GH3 were up regulated when treated with relatively low temperature (Fig. 5, Table S9). In JA signal transduction pathway, most of genes such as COI1, JAZ, and MYC2 were down-regulated, only two JAZ (Unigene43759 and CL8754.Contig3) genes and four MYC2 (CL37.Contig1, CL6240.Contig1, CL3114.Contig2, and CL2658.Contig2) genes were up-regulated under 19 °C treatment (Fig. 5, Table S9). Only six TGA genes in SA signal transduction pathway were expressed differently under different temperatures and two of them (CL2000.Contig1 and CL225.Contig1) were increased after 19 °C treatment (Fig. 5, Table S9).

Nineteen unigenes in the GA signaling pathway were also differentially expressed in different temperatures (Fig. 5). Five of six GID1 were down-regulated under relatively low temperature, with the exception of Unigene22649, which was nearly increased eight thousand times after 2 d treatment by 19 °C. Three DELLA and five PIF3 genes were decreased while two DELLA and three PIF3 genes were increased after relatively low temperature treatment (Fig. 5, Table S9). Only four of twenty unigenes including two PYR/PYL and two ABF (ABA responsive element binding factor) genes in ABA signal transduction pathway were up regulated after 19 °C treatment. However, the expression of one ABF (CL12556.Contig1) was decreased by two hundred times under the relatively low temperature at 19 °C compared with that under 23 °C (Fig. 5, Table S9). Most of the unigenes in CTK signal transduction pathway were down-regulated under relatively low temperature including all AHP, and all B-ARR members, especially the AHP (CL11134.Contig2), which was decreased severely after 6 d treatment by 19 °C (Fig. 5). However, most of the A-ARR members (CL7866.Contig2, Unigene18524, and CL8358.Contig1) were up-regulated when treated by relatively low temperature (Fig. 5, Table S9).

In ETH signal transduction pathway, the expression pattern of some ETR (Unigene14562), CTR1 (CL10648.Contig2), and EIN3 (CL10633.Contig1) genes, were more complicated. For example, the expression level of Unigene14562 and CL10633.Contig1 were decreased when treated by 2 d of relatively low temperature, while they were increased when the treatment time was lengthened. Most of the genes in this pathway were down-regulated after the relatively low temperature’ treatment (Fig. 5, Table S9). Eight of fifteen unigenes in the BR signal transduction pathways including all BRI1, one BSK, one TCH4, and one CYCD3 were increased under 19 °C treatment (Fig. 5, Table S9). The expression level of BRI1, especially Unigene37674 and Unigene35684 were dramatically increased when treated with 19 °C. However, seven of fifteen unigenes in this signal pathway were decreased under 19 °C treatment. The expression level of BZR1/2 were eight-fold under 19 °C compared to 23 °C, and the expression level were continuously increased as the treatment time prolonged. The same was true for TCH4 (CL8777.Contig2).

Identification of DEGs of transcription factors (TFs)

Many TFs changed dramatically under different temperatures, so we improved the screening criteria. The TFs with a |log₂Ratio| ≥ 2 in any comparison were analyzed further. 79 TFs which were divided into eight gene family were selected for further analysis because they may be involved in petal cell expansion according to previous study (Fig. 6, Table S10). In the MYB TF family, only five out of twenty genes (Unigene36971, CL242.Contig2, CL10695.Contig2, Unigene11061, and Unigene30676) were up expressed under relatively low temperature. In the bHLH TF family, three genes (Unigene30441, CL796.Contig2, and CL6865.Contig1) were up expressed after treated by 19 °C. Among them, especially the bHLH79, the expression level of bHLH79 isoform X2 (CL6865.Contig1) increased five hundred times when treated by 6 d of relatively low temperature. However, the expression level of bHLH 117 decreased sharply when exposed to 19 °C. None of the TF genes was up-regulated under relatively low temperature in the AP2 and NAC TF family. In the TCP TF family, two genes (CL9936.Contig2, and Unigene10400) were up-expressed under relatively low temperature treatment. Four out of nine genes were up-regulated in the MADS TF family, specially the AGAMOUS gene, which is the C-function genes, and its expression increased significantly with the 19 °C treatment. In the WRKY TF family, only two genes (CL11608.Contig1, and Unigene860) were up-regulated under relatively low temperature. In Zinc finger TF family, four out of eleven genes’ expression increased when exposed to 19 °C. Generally speaking, only a small proportion of TFs were up-regulated under relatively low temperature.

qRT-PCR analysis

Ten differentially expressed unigenes comprising four genes involving in cell wall metabolism, four genes related to phytohormone signal transduction and two genes of TF were analyzed by qRT-PCR in order to confirm the results of RNA-SEq. All ten genes tested by qRT-PCR confirmed significant differential expression between 19 °C and 23 °C treatments (Fig. 7). Although fold changes of the ten genes between 19 °C and 23 °C treatments were not always similar in RNA-Seq and qRT-PCR results, the overall trend was consistent. Based on qRT-PCR results, the expression levels of CL10934.Contig2 (EXP), CL1473.Contig2 (XYL) and CL6542.Contig1 (PL) at 2d, 4d and 6d, were significantly lower in 23 °C treatment than those in 19 °C treatment, while the expression levels of other seven genes at 2d, 4d and 6d were higher in 23 °C treatment. Similarly, CL10934.Contig2, CL1473.Contig2 and CL6542.Contig1 were down-regulated, while other seven genes were up-regulated in 23 °C treatment, which verified the accuracy of RNA-Seq results.

The flower time of once-flowering cultivars of O. fragrans are greatly affected by the relatively low temperature and they need a certain amount of chilling at autumn before blooming. The sensitivity difference to chilling requirement among different cultivars leads to the variation of flowering time [3]. The early flower cultivar such as ‘Zao Yingui’ would blossom when the daily minimum temperature lower than 21 °C last for after 5–8 days, while the late blossoming cultivar, such as ‘Wan Yingui’, needs 5–8 days of the daily minimum temperature lower than 19 °C [26]. In our unpublished research, the cultivar of ‘Yanhong Gui’ which has finished bud differentiation couldn’t blossom, when the environmental temperature is 23 °C or higher. The relatively low temperature, like 19 °C, helps ‘Yanhong Gui’ blossom in experimental condition. However, the relatively low temperature which can determine the process of flower opening in O. fragrans is quite unclear.

Many phytohormone signal transduction genes participate in the cell division and cell expansion of petal development [18]. In our research, the expression of AINTEGUMENTA (ANT, CL4443.Contig1) and JAGGED (JAG, Unigene22695) decreased sharply (Table S10) when treated with relatively low temperature. Since ANT and JAG genes were reported to positively regulate cell proliferation [27, 28], suggesting that the petal development after S1 are mainly dominated by cell expansion instead of cell division. At the same time, the cell size of adaxial petal epidermal cells increased gradually from S1 to S6 (Fig. 1a), while that of abaxial petal epidermal cells enlarged gradually from S1 to S5, reached the peak at S5, and then greatly decreased from S5 to S6 (Fig. 1b). This results was coincident with Gaillardia grandiflora [29], carnation [30] and T. caerulea [16], the cell division ceased and cell expansion occurred during floral opening process. However, in rose [31] and E. grandiflorum [32] cell division and cell expansion simultaneously appeared during this process. The vacuole occupied most area of petal cells in O. fragrans according to the results of TEM (Fig. 1c). The same condition was occurred in E. grandiflorum [32]. These results indicated that the petal cell expansion was accompanied by the enlargement of vacuole.

The accumulation of osmotically active compounds, water influx, cell wall loosening, cellulose biosynthesis, and cytoskeleton rearrangement are accompanied by cell expansion [7]. The expression level of EXP and XTH homologous genes involved in cell wall loosening, are up-regulated during floral open process in sandersonia [33], Mirabilis jalapa [34], petunia [9], carnation [35], and wintersweet [10]. EXPs consist of four sub families: α-EXP, β-EXP, EXP-like A and EXP-like B and were first discovered to loosen cell walls in pH dependent manner in cucumber [36]. Meanwhile, EXPs are also the molecular indicators of cell elongation in Arabidopsis [37]. The β-EXP is more efficient in disrupting cell wall polymers than α-EXP [34], and however, its function in petal development is not clear. In our research, all the four α-EXP genes were up-regulated when treated with the relatively low temperature, indicating that they may probably participate in the cell wall loosening during flower opening process (Fig. 4, Table S8). However, the only one β-EXP (CL8343.Contig1) gene’s expression decreased with the petal development when plants exposed to 19 °C (Fig. 4, Table S8). The result was coincidence with the results in rose [38] that the expression of β-EXP was down-regulated during the petal development. This result suggested that β-EXP may be involved in petal development and it may own various functions like α-EXP. Plant-specific NAC family genes can regulate plant development, cell division, senescence, and responses to abiotic stress [39]. In recent research, the N-terminal binding domain of RhNAC2 could be bound to the promoter of RhEXPA4 thus regulating of dehydration tolerance during the expansion of rose petals [40]. In our current research, the expression of all the members of NAC family decreased under the relatively low temperature (Fig. 6, Table S10), indicating that the suppression of NAC genes may facilitate the expression of EXPs to promote the elongation of flower petals in O. fragrans. XTHs play vital roles in cell expansion, loosening and rearranging the cell wall fibers during the petal development. In rose, four XTH genes (RbXTH3, RbXTH5, RbXTH6, and RbXTH12) were up-regulated during flower opening and associated with petal movement [41]. OsXTH12 gene can cause rice glume-unclosing after anthesis under high temperature conditions [42]. In our experiment, we cannot found the homologous genes of XTH3, XTH5, XTH6 and XTH12, but other family members were discovered. Most of XTH genes (seven out of eleven) were up regulated under relatively low temperature indicating they may be involved in the flower opening process of O. fragrans and different family members may have different functions.

These cell wall metabolism processes were also influenced by multiple kinds of phytohormones via transcription factors and miRNAs [19]. The expression patterns of phytohormone pathways genes looks different under different temperatures treated by different days in our research. In A. thaliana, both the AUX and JA signal transduction function synergistically during petal development [43]. ARF8 can interacts with BIGPETALp (BPEp) to regulate cell expansion in petals [6] and can trigger the expression of MYB21 and MYB24 to induce the production of JA to promote petal growth [43]. The levels of endogenous AUX were much higher in the basal petals and application of exogenous indole-3-butyric acid (IBA) can enlarge the cell length in chrysanthemum [5]. Many unigenes involved in AUX signal transduction pathway were increasingly expressed with the flower opening of rose, suggesting that AUX signaling participated in petal development [38]. TFs such as JAG can directly repress the expression of PETAL LOSS (PTL) to increase cell proliferation and enlargement [44]. miRNAs have also been found to be involved in petal elongation, such as miRNA319a, which functions by regulating TCP4 which regulates AUX effects [45]. In our research, the expression level of TCP4-like gene (CL9936.Contig2) increased when treated by the relatively low temperature, implying it may regulate the petal development in O. fragrans (Fig. 6, Table S10).

JA and its derivative methyl jasmonate (MeJA) can affect the flower opening of E. grandiflorum [46], rice [47], A. thaliana [21]. BPEp, which can be regulated by JA, may also play a role in JA-mediated petal growth [6, 21]. In our research, only a small proportion of unigenes in JA signal transduction pathway were up-regulated under relatively low temperature, thus these unigenes may function as positive regulators of flower opening in O. fragrans (Fig. 5, Table S9).

GA can regulate seed germination, stem elongation, petal growth and flowering [48], and ABA has an antagonistically role such as in floral transition and fruit development [49]. The same circumstance occurred in the G. hybrida petal growth [20]. Compared to unigenes involved in ABA signal transduction pathway, there were much more unigenes in GA signal transduction pathway which increased sharply under the treatment of 19 °C. This result was consistent with the result in rose [38]. However, whether this kind of antagonistic role exists in petal growth of O. fragrans still needs further demonstration. Overexpression of the MYB62 gene results in a GA-deficient phenotype and can be partially alleviated with the application of exogenous GA in G. hybrida [50]. In our study, we did not find the homologous unigenes to MYB62 in O. fragrans, but some unigenes homologous to other gene family members, such as MYB4 (CL9450.Contig1) and MYB86 (Unigene19015), were expressed differently under different temperatures (Fig. 6, Table S10), indicating they may be involved in the regulation of relatively low temperature on flowering opening in O. fragrans.

The effect of ETH on flower opening may be dose-dependent, species-dependent or cultivar-dependent [19]. ETH can promote flower opening in carnation, Phalaenopsis orchids, and petunia, but it inhibits flower opening in several rose cultivars, although in some rare cultivars it promoted their opening [19]. ETH can induce the expression of DELLA genes via EIN3-3 when it functions as inhibitors during flower opening in rose [22]. TFs, such as NAC100, were induced by ETH in rose petals, which inhibits petal cell expansion to reduce the petal size [51]. In our study, only a small proportion of unigenes in ETH signal transduction pathway were up-regulated under relatively low temperature (Fig. 5, Table S9), suggesting that ETH may function little or have inhibiting effect on flower opening.

Cell elongation is also regulated by BR, which is a plant-specific steroid hormone and longitudinal cell expansion of BR mutants is greatly reduced in A. thaliana [52, 53]. The BR and GA pathways have reciprocal interaction because they have common target transcriptional regulators [37, 52–54]. In G. hybrida, the brassinolide (BL) shows more efficient effect on lengthening the cells in the middle and basal regions of petals than GA [23]. The expression level of BES1/BZR1 was decreased after only 0.5 h BR treatment, indicating its vital role in petal cell expansion [23]. In our study, expression level of the homologous unigene of BZR1 (Unigene45694), decreased dramatically when exposed to relatively low temperature, suggesting the same role in petal development of O. fragrans as that in G. hybrida (Fig. 5, Table S9). Meanwhile, four zinc-finger TFs (CL5412.Contig2, Unigene48369, CL2297.Contig1, and CL7288.Contig2) were up-regulated under relatively low temperature (Fig. 6, Table S10), indicating that transcriptional regulation is also important in O. fragrans petal growth.

The comprehensive transcriptomic dataset relating to flower opening of O. fragrans under relatively low temperature (19 °C) treatment was performed using RNA-seq technology, and a group of DEGs, which may regulate petal growth through pairwise comparison analysis of DEGs between different temperature treatments, had identified, including unigenes involved in cell wall metabolism, phytohormone signal transduction pathway, and TFs. Overall, the resources generated in this study would lay foundation on fully revealing the molecular mechanism of relatively low temperature regulating flower opening of O. fragrans and provide theoretical reference for the prediction and regulation of flowering time and genetic modification in O. fragrans.

Plant materials and treatments

Plants of O. fragrans ‘Yanhong Gui’ were potted and grown in the resource nursery of Zhejiang Agriculture and Forestry University. The development of flower opening process are classified into S1-S6 as described in this paper [3]. When the floral development stage was at S1, potted plants were moved into the growth cabinet with the temperature of 19 °C (relatively low temperature) or 23 °C (relative high temperature), respectively. The relative humidity was about 50–60% and the photoperiod was 12 h light/12 h dark regime with an illumination of 80 µmol·m^− 2·s^− 1. Petals were harvested every two days after treatment until the developmental stage of plants under 19 °C condition reached S2. The samples were named as H2, H4, H6 (treated for 2 d, 4 d, and 6 d under 23 °C treatment, respectively) and L2, L4, L6 (treated by 2 d, 4 d, and 6 d under 19 °C treatment, respectively). All samples were collected at 10:00 am in order to avoid the influence of diurnal rhythm. Three biological replicate samples were collected to generate 18 samples in total. In addition, petals at different developmental stages (S1-S6) were respectively sampled for SEM and TEM analysis.

SEM and TEM analysis

SEM and TEM were carried out according to previous studies [55, 56], and were observed and photographed in Hitachi Model TM-1000 SEM and Model H-7650 respectively in Zhejiang University. Cell size of adaxial and abaxial petal epidermal cells, and vacuole size were manually measured using Image J software (http://rsb.info.nih.gov/ij/, NIH, MD, USA). Data from individual measurements were averaged and analyzed using SPSS Statistics v. 18.0 (Duncan’s test) (SPSS Inc., Chicago, IL, USA).

RNA extraction, cDNA library preparation and sequencing

RNA isolation and RNA purification of 18 samples were carried out as described previously [57]. cDNA library preparation and sequencing of 18 samples of O. fragrans were respectively performed as described previously [58] for RNA-seq analysis. What’s more, a RNA pool mixed from 18 RNA samples was used to construct a cDNA library for reference transcriptome sequencing. The Illumina sequencing was performed at the Beijing Genomics Institute (BGI) (Shenzhen, China) (http://www.genomics.cn/index.php) according to the manufacturer’s instructions (Illumina Inc, San Diego, CA, USA). A total of 18 sets of raw reads were obtained for RNA-seq analysis, corresponding to H2-1, H2-2, H2-3, H4-1, H4-2, H4-3, H6-1, H6-2, H6-3, L2-1, L2-2, L2-3, L4-1, L4-2, L4-3, L6-1, L6-2, and L6-3, while raw reads were obtained for reference transcriptome corresponding to ALL.

Transcriptome data processing and analysis

Raw reads were cleaned by removing the sequence of adaptor, high content of unknown bases and low-quality reads before downstream analysis to decrease data noise. As O. fragrans did not have an appropriate reference genome sequence, the Trinity method [59] was used to de novo assemble all clean reads and the Tgicl method [60] was used to cluster the transcripts to obtain non-redundant unigenes. The assembled unigene sequences were aligned by Blastn [61] to nucleotide database NT, aligned by Blastx [61] or Diamond [62] to protein databases NR, Swiss-Prot, KEGG, and KOG, and aligned by InterProScan5 [63] to protein database InterPro to get the annotations. With the NR annotation, GO annotations of unigenes were obtained using Blast2GO software [64]. FPKM (reads mapped per 1000 bp per million sequenced reads) method was used in calculated expression level [65]. Comparisons of FPKM between different temperatures treated by the same days (L2 vs H2, L4 vs H4, and L6 vs H6 respectively) were performed for each unigene. To identify the DEGs in the two samples, the Audic and Claverie method was employed [66]. Unigene with a P value ≤ 0.01 and a |log₂Ratio| ≥ 1 were considered significant DEGs. All DEGs were mapped to each term of the GO and KEGG databases. For the analysis of transcription factors, |log₂Ratio| ≥ 2 were marked to be significantly different between the two samples.

Quantitative real-time PCR (qRT-PCR) analysis

The extraction of total RNA, first-strand cDNA synthesis, and qRT-PCR were performed as previously described [57]. Ten DEGs were randomly chosen and the primer sequences were listed in Table S11. Gene relative expression levels of those ten genes were normalized relatively to the expression level of an internal control OfACT [67] using the 2^−△Ct method.

ABA: abscisic acid; ARF: auxin response factor; AUX: auxin; BR: brassinosteroid; CES: cellulose synthase; DEGs: differentially expressed genes; ETH: ethylene; EXP: expansin; FPKM: reads mapped per 1000 bp per million sequenced reads; GA: gibberellin; IAA: indole-3-acetic acid; JA: jasmonic acid; NIP: nodulin-26-like intrinsic membrance protein; PE: pectin esterase; PG: polygalacturonase; PIP: plasma membrane intrinsic protein; PL: pectate lyase; qRT-PCR: quantitative real-time PCR; SAUR: small auxin-up RNA family protein; SEM: scanning electron microscopy; SIP: small basic intrinsic protein; TEM: transmission electron microscopy; TIP: tonoplast intrinsic protein; XTH: xyloglucan endotransglycosylase/hydrolase; XYL: xylosidase

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The RNA-Seq datasets used in the current study are available in the Sequence Read Archive (SRA) at NCBI repository.

Competing interest

The authors declare that they have no competing interests.

Funding

This research was supported by Zhejiang Provincial Natural Science Foundation of China (Grant Nos. LY19C160006, LY19C160002, LQ16C160003), Zhejiang Provincial Major Program of New Cultivar Breeding (Grant No. 2016C02056-12), and China Scholarship Council (201808330145 and 201808330146).

Authors’ contributions

HZ designed the research. JF, CZ and HZ drafted the manuscript. JF and CZ performed the transcriptome data processing and analysis. YL performed qRT-PCR detection. TP extracted RNA and constructed cDNA Library. BD, XG and YZ performed SEM and TEM analysis.

Acknowledgements

Not applicable.

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Fig. S1 Species from which the transcript annotation was derived in the NR database

Fig. S2 KEGG pathway annotation of Osmanthus fragrans unigenes

Fig. S3 GO classification of Osmanthus fragrans unigenes

Fig. S4 KOG classification of Osmanthus fragrans unigenes

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Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans

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Journal Publication

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Abstract

Figures

Background

Results

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis

Bud appearance with treatments

Illumina sequencing and de novo assembly of sequence reads

Functional annotation and classification

RNA-seq Illumina sequencing and mapping to the reference transcriptome database

Screening and analysis of DEGs

GO enrichment and KEGG pathway analysis of DEGs

Identification of DEGs involved in the cell wall metabolism

Identification of DEGs involved in the phytohormone signal transduction pathway

Identification of DEGs of transcription factors (TFs)

qRT-PCR analysis

Discussion

Conclusion

Methods

Plant materials and treatments

SEM and TEM analysis

RNA extraction, cDNA library preparation and sequencing

Transcriptome data processing and analysis

Quantitative real-time PCR (qRT-PCR) analysis

Abbreviations

Declarations

References

Supplementary Figure Legends

Supplementary Files

Status:

Journal Publication

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