Transcriptome Dynamics of Floral Organs Approaching Blooming in the Flowering Cherry (Cerasus × yedoensis) Cultivar ‘Somei-Yoshino’

To gain insights into the genetic mechanisms underlying blooming and petal movement in flowering cherry (Cerasus × yedoensis), we performed time-course RNA-seq analysis of the floral buds and open-flowers of the most popular flowering cherry cultivar, ‘Somei-Yoshino’. Independent biological duplicate samples of floral buds and open-flowers were collected from ‘Somei-Yoshino’ trees grown at three different locations in Japan. RNA-seq reads obtained from floral bud and open-flower samples collected in the current study (in 2019) and in a previous study (in 2017) were aligned against the genome sequence of ‘Somei-Yoshino’ to quantify gene transcript levels. Clustering analysis of RNA-seq reads revealed dynamic changes in the transcriptome, with genes in seven modules predominantly expressed at specific time points, ranging from 5 weeks before flowering to 2 weeks after flowering. Based on the identified gene modules and Gene Ontology (GO) terms enriched at different floral stages, we speculate that the genetic mechanisms underlying petal movement and flower opening in cherry involve the processes of development, cell wall organization, reproduction, and metabolism, which are executed by genes encoding transcription factors, phytohormones, transporters, and polysaccharide metabolic enzymes. Furthermore, we propose a method for cherry bloom forecasting, based on gene expression levels at different time points before flowering as RNA markers.

samples collected in the current study (in 2019) and in a previous study (in 2017) were aligned 24 against the genome sequence of 'Somei-Yoshino' to quantify gene transcript levels. Clustering 25 analysis of RNA-seq reads revealed dynamic changes in the transcriptome, with genes in seven 26 modules predominantly expressed at specific time points, ranging from 5 weeks before flowering to 2 27 weeks after flowering. Based on the identified gene modules and Gene Ontology (GO) terms 28 enriched at different floral stages, we speculate that the genetic mechanisms underlying petal 29 movement and flower opening in cherry involve the processes of development, cell wall 30 organization, reproduction, and metabolism, which are executed by genes encoding transcription 31 factors, phytohormones, transporters, and polysaccharide metabolic enzymes. Furthermore, we 32 propose a method for cherry bloom forecasting, based on gene expression levels at different time 33 points before flowering as RNA markers. 34

Introduction 35
Flowering cherry, also known as sakura, typically blooms in the spring and is valued as a popular 36 ornamental flower across the world. 'Somei-Yoshino' (Cerasus × yedoensis), which is presumed to 37 be an interspecific hybrid between C. spachiana and C. speciosa (Takenaka, 1963; Innan et al., 1995; 38 Nakamura et al., 2015), is the most popular cultivar of flowering cherry in Japan. Given its genomic 39 heterozygosity and self-incompatibility,

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Somei-Yoshino' is propagated by grafting (Iketani et al.,  40 2007). Because of its clonal nature, 'Somei-Yoshino' trees planted within a specific location bloom 41 at the same time; however, across the Japanese archipelago, the blooming front progresses from 42 south to north because of differences in environmental conditions. Since the blooming date is 43 important for the tourism industry in the spring season, forecasting methods based on cumulative 44 temperature have been developed to predict the flowering date of cherry blossoms (Aono and  45 Murakami, 2017). 46 Flowering involves two main processes: floral bud initiation and flower opening. have been reported to regulate bud dormancy (Yamane, 2014). However, while the physiological 54 aspect of the flower opening mechanism has been thoroughly investigated, only a few studies have 55 been conducted to explore the genetic basis of this mechanism (van Doorn and Van Meeteren, 2003). 56 The genome sequence of 'Somei-Yoshino' has been determined at the chromosome level, and genes 57 involved in the regulation of dormancy and flowering time have been identified in this cultivar 58 through time-course transcriptome analysis (Shirasawa et al., 2019). Since expression levels of key 59 genes transmit the environmental conditions, such as day-length and temperature, to biological 60 processes such as blooming, the identification of these genes might help predict the blooming date in 61 flowering cherry. Because the transcriptome is affected by environmental conditions (e.g., changes in 62 weather and habitat), biological replications of flowering cherry trees over multiple years and 63 locations would be required for the accurate identification of genes affecting the blooming date. 64 In this study, we aimed to identify genes uniquely expressed before and after flowering, and to obtain 65 insights into the molecular mechanisms underlying blooming in flowering cherry. We  Table S1). 81

RNA-seq Analysis 82
Library preparation and sequencing analysis were performed as described in Shirasawa

GO Enrichment Analysis 134
To identify the GO terms enriched in each module, the ratios of GO terms in each gene module were 135 compared with those of the remaining gene set ( Supplementary Figure S4). 137 At 4-5 WBF (dark-red module), the following GO terms were enriched: biological process (BP) 138 category: 'developmental process' related GO terms including 'aging', 'embryo development', 139 'developmental maturation', 'developmental process', and 'anatomical structure development'; 140 molecular function (MF) category: 'oxidoreductase activity'; and cellular component (CC) category: 141 'plasma membrane' and 'cell periphery'. 142 At 4 WBF (tan module), GO terms enriched in the BP category were not only related to 143 'developmental process' ('anatomical structure formation involved in morphogenesis' and 144 'anatomical structure development') but also 'cellular process' ('sulfur compound metabolic  145 process', 'biosynthetic process', 'developmental process', and 'cell wall organization or biogenesis'

Genetic Mechanisms Underlying Blooming in Flowering Cherry 179
To gain insights into the genetic mechanisms regulating blooming in flowering cherry, we focused on 180 genes categorized in four functional categories (Figure 2, Supplementary Table S3): 1) transcription 181 factor genes; 2) phytohormone-related genes; 3) transporter and aquaporin genes; and 4) cell wall-182 related genes. 183

Transcription factor genes 184
Genes encoding three types of transcription factors that trigger blooming were predominant during 185 the flowering period. The MYB transcription factor genes were overrepresented from 4 to 3 WBF, 186 while the ethylene-responsive transcription factor (ERF) genes and NAC transcription factor genes 187 were expressed at 4 and 3 WBF, respectively. 188

Phytohormone-related genes 189
Genes involved in biosynthesis and signal transduction pathways of gibberellin, ethylene, and 190 cytokinin were enriched at 3-5 WBF, 3-4 WBF, and 3 WBF, respectively. Auxin-related genes were, 191 on the other hand, expressed at 1 WBF and at the flowering days. 192

Transporter and aquaporin genes 193
Genes encoding sugar and inorganic transporters and aquaporins that affect the turgor and osmotic 194 pressure of cells and vacuoles were constitutively expressed from 5 WBF to the day for flowering. 195 Among these, genes encoding 10 types of transporters (ABC transporter G family members, 196 bidirectional sugar transporters, cationic amino acid transporters, lysine histidine transporters, 197 organic cation/carnitine transporters, phosphate transporters, potassium transporters, UDP-galactose 198 transporters, vacuolar amino acid transporters, and zinc transporters) were overrepresented at 3 WBF. 199 Aquaporin genes were overrepresented at 1-2 WAF.  Gene Ontology (GO) terms enriched in the BP category (see Table 1). Boxes indicate the properties 364 of the genes highly expressed in the seven characterized modules (see Supplementary Table S3). 365 process', 'macromolecule modification', 'primary metabolic process', 'regulation of biological process', 'regulation of cellular process', 'response to stimulus', 'cellular response to stimulus', 'biological regulation', 'cell wall organization or biogenesis', and 'organic substance metabolic process'