Transcriptional Controls for Early Bolting and Flowering in Angelica sinensis

The root of the perennial herb Angelica sinensis is a widely used source for traditional Chinese medicines. While the plant thrives in cool-moist regions of western China, early bolting and flowering (EBF) for young plants significantly reduces root quality and yield. Approaches to inhibit EBF by changes in physiology during the vernalization process have been investigated; however, the mechanism for activating EBF is still limited. Here, transcript profiles for bolted and unbolted plants (BP and UBP, respectively) were compared by transcriptomic analysis, expression levels of candidate genes were validated by qRT-PCR, and the accumulations of gibberellins (GA1, GA4, GA8, GA9 and GA20) were also monitored by HPLC-MS/MS. A total of over 72,000 unigenes were detected with ca. 2600 differentially expressed genes (DEGs) observed in the BP compared with UBP. While various signaling pathways participate in flower induction, it is genes associated with floral development and the sucrose pathway that are observed to be coordinated in EBF plants, coherently up- and down-regulating flowering genes that activate and inhibit flowering, respectively. The signature transcripts pattern for the developmental pathways that drive flowering provides insight into the molecular signals that activate plant EBF.

Due to an increasing demand for traditional Chinese medicines, A. sinensis is farmed to meet commercial demand [3]. For industrialized planting, seeds are sown in early summer, plants are collected in Fall and overwinter indoors; the following spring, seedlings are planted for vegetative growth and are either harvested in Fall of the second year to obtain non-lignified roots or kept in the field till mid-summer of the third year for seed collection ( Figure S1) [10]. Early bolting and flowering (EBF) occurs in the second year in up to 40% of the plants, substantially reducing root yield and quality due to lignifications of roots and degradations of bioactive compounds [1,11,12]. For the EBF to occur, the plant must experience vernalization and long-day (LD) conditions; thus, avoiding vernalization or LD Plants 2021, 10,1931 3 of 14 the shade for approximately 15 days and then stored in a natural-rain-proof environment for the winter.
On April 3, 2019, the stored seedlings (root tip diameter 4.5-5.0 mm) were transplanted into pots (diameter 17 cm, depth 20 cm; one seedling per pot) with nutrition matrix and seedlings were greenhouse grown with controlling matrix volumetric moisture content of 60-70%, light condition of 10-12 h per day and air temperature 15-22 • C. No additional fertilizer was applied after the transplant. With plant growth and development, some plants began to initiate flower bud differentiation and then BF, other plants kept on vegetative growth with NBP. On 3 July 2019, samples including the second-tip leaves and lateral roots (1:1, g/g fresh weight) from BP and UBP ( Figure S2) were collected (n = 20 plants with homogeneous growth potential) and then flash frozen in liquid nitrogen for transcriptomic analysis and GA metabolite analysis.

Total RNA Isolation and Illumina Sequencing
Total RNA samples were isolated according to our previous literature [29]. RNA sequencing was conducted using an Illumina HiSeq TM 4000 platform by Gene De novo Biotechnology Co., Ltd. (Guangzhou, China).

Sequence Filtration, Assembly and Unigene Expression Analysis
Sequence filtration, assembly, and unigene expression analysis were conducted according to our previous literature [29]. Briefly, raw reads were filtered by removing reads containing adapters, unknown nucleotides and low-quality bases. De novo assembly of clean reads used a Trinity software [30]. The expression level of each transcript was normalized to reads per kb per million (RPKM) value [31]. In this study, the level of differential expression for each transcript with a criterion of |log 2 (fold-change)| ≥ 1 and p value ≤ 0.05 to identify DEGs between BP and UBP.

Basic Annotation of DEGs and Gene Cluster Analysis
Unigenes were annotated against the databases including: NCBI non-redundant protein (NR), Swiss-Prot protein, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic orthologous groups of proteins (KOG), and gene ontology (GO) by using a BLASTx procedure with an e-value ≤ 10 −5 [32]. Molecular Evolutionary Genetics Analysis (MEGA) 7.0 was used for the gene cluster analysis ( Figure S3).

qRT-PCR Validation
The relative expression levels (RELs) were validated according to our previous literature [29]. Briefly, total RNA was extracted using a plant RNA kit. Primer sequences of the 40 DEGs (Table S1) were designed in primer-blast of NCBI. First-strand cDNA was synthesized using a FastKing RT kit. PCR amplification was carried out using a SuperReal PreMix. Actin was used as an internal reference and the REL was calculated using a 2 −∆∆Ct method [33].

GA Quantification and Identification
Samples were ground into powder in liquid nitrogen, 1.0 g sample was soaked in acetonitrile (10 mL) and agitated at 4 • C for 8 h, then centrifuged at 13,000 r/min and 4 • C for 5 min. Following exhaustive extraction (×3), the upper portion was pooled and dried with nitrogen gas in the dark. Dried residue was re-dissolved in methanol (400 µL) and filtered with 0.22 µm durapore membrane. The reference standard of the 5 GAs (GA 1 , GA 4 , GA 8 , GA 9, and GA 20 ) was dissolved in methanol to make concentrations 0.1, 0.2, 0.5, 2, 5, 20, 50, and 200 ng/mL. Samples (2 µL) were quantified and identified using a HPLC (Agilent1290, USA)-MS/MS (QTRAP 6500, AB SCIEX, USA) by Shanghai Biotree biotech Co., Ltd. (Shanghai, China). Methanol (A) and H 2 O (B) were the gradient elution: 0-1 min 20% A, 1-9 min A increasing up to 80%, 9-10 min 90% A, 10-10.1 min A decreasing down to 20%, and 10.1-15 min 20% A. Representative chromatograms of reference standard of the  5 GAs are shown in Figure S4, and representative chromatograms of the BP and UBP are shown in Figure S5. The content of the 5 GAs was calculated based on calibration curves (Table S2).

Soluble Sugar Measurement
Soluble sugar was measured using a sulfuric acid-phenol protocol [34]. A dried powder (1.0 g) was soaked in 95% EtOH (25 mL) for 72 h at 22 • C and then centrifuged (4 • C, 8000 r/min, 10 min). Extracts (30 µL) were added into 9% phenol reagent (1 mL), sulfuric acid (3 mL) was added after oscillation and then reacted at 22 • C for 30 min. Absorbance was measured at 485 nm, soluble sugar content was evaluated based on mg of Suc.

Statistical Analysis
All the measurements were performed using three replicates. A t-test for independent samples was performed and SPSS 22.0 was used, with p < 0.05 as the basis for significant differences.

Global Gene Analysis
A robust data set was collected ( Figure S6) and after data filtering, 60.7 and 52.4 million high-quality reads were obtained for the BP and UBP, respectively; 44.7 and 37.4 million unique reads as well as 7.8 and 6.4 million multiple reads were mapped. From the 72,502 compiled genes and annotated against the databases including NR, SwissProt, KEGG, KOG, and GO (Tables 1 and 2, Figures S7-S11), 2645 DEGs were obtained ( Figure S12). Of these 2645 DEGs, 369 genes were unidentified by SwissProt, KOG, GO, and KEGG databases. Of the 2276 identified DEGs, 1584 genes with known function were partitioned into being 738 UR and 846 DR. Based on biological function and physiological characteristics, genes were divided into 11 categories: photosynthesis/energy (79), primary metabolism (285), secondary metabolism (80), hormone biosynthesis (34), bio-signaling (201), cell morphogenesis (197), polynucleotide biosynthesis (87), transcription factor (167), translation (119), transport (233), and stress response (102) (Figure 1). Based on flower driving genes characterized in higher plants [20], 40 DEGs (29 UR and 11 DR) were identified as potential regulatory genes for EBF ( Figure 1).  Figure S4, and representative chromatograms of the BP and UBP are shown in Figure S5. The content of the 5 GAs was calculated based on calibration curves (Table S2).

Soluble Sugar Measurement
Soluble sugar was measured using a sulfuric acid-phenol protocol [34]. A dried powder (1.0 g) was soaked in 95% EtOH (25 mL) for 72 h at 22 °C and then centrifuged (4 °C, 8000 r/min, 10 min). Extracts (30 µL) were added into 9% phenol reagent (1 mL), sulfuric acid (3 mL) was added after oscillation and then reacted at 22 °C for 30 min. Absorbance was measured at 485 nm, soluble sugar content was evaluated based on mg of Suc.

Statistical Analysis
All the measurements were performed using three replicates. A t-test for independent samples was performed and SPSS 22.0 was used, with p < 0.05 as the basis for significant differences.
Eleven DEGs associated with sucrose pathway including: Suc metabolism (SUS1, SUS3, SUS7, INVA, INVB, INVE, and INV Inh) and starch metabolism (AMY1.1, BAM1, BAM3, and BAM9) ( Table 3) were transcriptionally regulated so as to favor flowering in BPs. The RELs were consistent with RPKM values, with down-regulated 0.3-fold for the INV Inh gene, and up-regulated 1.3-to 6.1-fold for the other 10 genes in the BP compared to the UBP ( Figure 2B). INV Inh gene, and up-regulated 1.3-to 6.1-fold for the other 10 genes in the BP compared to the UBP ( Figure 2B).

Flower-Regulating DEGs Inarticulately Expressed with EBF
Since GA accumulation can promote flowering, transcripts that encode for GA biosynthesis were screened for up-regulation in EBF plants. The 7 DEGs that are associated with GA signals include: GA biosynthesis (KO, GA2OX1, GA2OX6, GA2OX8, and GA20OX1) and GA mediated signaling pathway (GAI and GAIP) ( Table 3). The RELs of the 7 genes were consistent with RPKM values, with up-regulated 1.1-, 1.02-, 2.3-, 5.2-, and 1.3-fold for the genes KO, GA20OX1, GA2OX6, GA2OX8, and GAIP, respectively, in the BP compared to the UBP, and with down-regulated 0.9-and 0.7-fold for the genes GA2OX1 and GAI in the BP ( Figure 3A).

Sucrose and GA Accumulation
Flowering can be initiated by the accumulation of active GAs including GA1, GA3, GA4, and GA7. Interestingly, GA4 and GA1 as well as the up-stream precursors GA9 and GA20 had a 3.0-, 1.3-, 5.4-, and 4.2-fold increase in BP while the down-stream inactive forms of GA4 and GA1, GA8 had a 1.5-fold increase in UBP ( Figure 4A). Since GA1 and GA4 exhibit higher floral induction activity than other GAs that are produced in plants [20], an elevated level of GA1 and GA4 may promote EBF. In contrast, an almost 2-fold decrease in soluble sugars in the BP was unexpected as elevated sugar is usually a driver of flowering [28] (Figure 4B).

Sucrose and GA Accumulation
Flowering can be initiated by the accumulation of active GAs including GA 1 , GA 3 , GA 4, and GA 7 . Interestingly, GA 4 and GA 1 as well as the up-stream precursors GA 9 and GA 20 had a 3.0-, 1.3-, 5.4-, and 4.2-fold increase in BP while the down-stream inactive forms of GA 4 and GA 1 , GA 8 had a 1.5-fold increase in UBP ( Figure 4A). Since GA 1 and GA 4 exhibit higher floral induction activity than other GAs that are produced in plants [20], an elevated level of GA 1 and GA 4 may promote EBF. In contrast, an almost 2-fold decrease in soluble sugars in the BP was unexpected as elevated sugar is usually a driver of flowering [28] ( Figure 4B).

Sucrose and GA Accumulation
Flowering can be initiated by the accumulation of active GAs including GA1, GA3, GA4, and GA7. Interestingly, GA4 and GA1 as well as the up-stream precursors GA9 and GA20 had a 3.0-, 1.3-, 5.4-, and 4.2-fold increase in BP while the down-stream inactive forms of GA4 and GA1, GA8 had a 1.5-fold increase in UBP ( Figure 4A). Since GA1 and GA4 exhibit higher floral induction activity than other GAs that are produced in plants [20], an elevated level of GA1 and GA4 may promote EBF. In contrast, an almost 2-fold decrease in soluble sugars in the BP was unexpected as elevated sugar is usually a driver of flowering [28] (Figure 4B).

Discussion
The SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) can integrate signals from the photoperiodism, vernalization, sucrose and GA pathways and regulate the expression of LFY, which links floral induction and floral development, when associated with other MADS box genes [35]. MADS box proteins regulate different developmental processes including flowering time, floral meristem identity, and floral organ development [36]. MADS8, which is structurally related to the AGL2 family, is involved in

Discussion
The SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) can integrate signals from the photoperiodism, vernalization, sucrose and GA pathways and regulate the expression of LFY, which links floral induction and floral development, when associated with other MADS box genes [35]. MADS box proteins regulate different developmental processes including flowering time, floral meristem identity, and floral organ development [36]. MADS8, which is structurally related to the AGL2 family, is involved in controlling flowering time [37]. AGL8 promotes early floral meristem identity in synergy with AP1 and CAULIFLOWER [38]. AGL12 acts as promoter of the flowering transition through up-regulation of SOC, FT, and LFY [39]. DEFICIENS (DEFA) is involved in the genetic control of floral development [40]. APETALA1 (AP1) and AP2 are required for the transition of an inflorescence meristem into a floral meristem and promote early floral meristem identity, with AP1 regulating positively AG in cooperation with LFY, while AP2 represses AG by recruiting the transcriptional corepressor TPL and HDA19 [41,42]. AINTEGUMENTA (ANT), a member of the AP2-like family, is involved in flower organs initiation and development and mediates AG down-regulation [43,44]. Previous studies on A. sinensis found that the SOC1 was down-regulated and the AG was up-regulated in 2-year-old plants during transition from vegetative to flower bud differentiation [28]; the AGL62, PMADS1, and DEFA were up-regulated in 3-year-old plants at different growth and development stages [29]. In this study, positive regulators of flowering in the floral development pathway were observed to be up-regulated in EBF plants, while genes that disfavor flowering (AP2 and ANT) were down-regulated, suggesting that transcription regulation of these genes may well be a driver for A. sinensis EBF.
Suc and its cleavage products glucose (Glc) and fructose (Fru) are central molecules for cellular biosynthesis and signal transduction throughout a plant's life cycle [45]. In this Plants 2021, 10, 1931 9 of 14 study, Suc synthases (SUSs) that are encoded by three SUS1, SUS3, and SUS7 genes catalyze a reversible conversion of Suc and UDP to UDP-Glc and Fru [46,47]; Alkaline/neutral invertases (INVs) that are encoded by three INVA, INVB, and INVE genes catalyze an irreversible hydrolysis of Suc to Glc and Fru [48][49][50]; and the invertase inhibitor (INV Inh) inhibits the INV activity by forming a complex with INV [51]. Two kinds of amylase enzymes including α-amylase (AMY) and β-amylase (BAM) could respectively produce α-maltose and β-maltose through the hydrolysis of amylopectin and amylose [52]. In this study, four DEGs encoding amylase enzymes include: AMY1.1, which can increase enzyme activity via accessory binding sites on the protein surface, BAM1 and BAM3, which play important roles in starch degradation and maltose metabolism, and BAM9, which is inactive due to lack the conserved Glu active site [52][53][54]. The SUS6 and AMY2 were found to be up-regulated in 3-year-old plants of A. sinensis at different development stages [29]. Here, since the genes (SUS1, SUS3, SUS7, INVA, INVB, INVE, AMY1.1, BAM1, BAM3, and BAM9) that favor flowering were up-regulated and the INV Inh gene that disfavors flowering was down-regulated, transcriptional regulation of sucrose pathway is consistent with EBF.
While genes associated with GA biosynthesis and GA mediated signaling were differentially regulated in BP versus UBP, the genes did not exhibit coherent transcriptional regulation with EBF, suggesting that transcriptional regulation of GA mediated genes is not a driver of early bolting. Previous studies on A. sinensis found that the GA20OX had no difference change during transition from vegetative to flower bud differentiation [28]; while the GA2OX1 and GA2OX8 were down-regulated at different growth and development stages [29]. For example, with GA mediated signaling, DELLA proteins GA-INSENSITIVE (GAI) and GAIP function as inhibitors by interacting in large multiprotein complexes that repress transcription of GA-inducible genes [55][56][57]. Inconsistent with promoting flowering, the GAIP is transcriptionally up-regulated in BP versus UBP. Inconsistency is also observed in genes that encode GA biosynthesis with a subset of genes up-regulated such as KO, which catalyzes the conversion of ent-kaurene to kaurenoic acid early in the biosynthetic pathway [58] as well as GA20OX1, which converts GA 12 /GA 53 to GA 9 /GA 20 [59] later in the pathway ( Figure S13), while GA2OX catalyzes 2-beta-hydroxylation of GA precursors, rendering them unable to be converted to active GAs is up-regulated under the same condition that promotes flowering (BP). This incoherent transcriptional regulation of GA biosynthesis and signaling for EBF suggests that early bolting may be regulated by events downstream of flowering signaling such as GA and/or sugar accumulation.
While CONSTANS-LIKE (COL) genes are regulators in the photoperiod pathway and flowering, transcripts in this pathway were also inconsistently induced providing an inarticulate signal for plant flowering, which was in accordance with previous findings with the CO, COL2, and COL16 up-regulated while the COL4 and COL10 down-regulated in A. sinensis [28,29]. For example, while both CO3 and COL3 function as floral activators, the two genes were transcriptionally up-and down-regulated, respectively, when comparing BP with UBP. Specifically, CO3 up-regulates the expression of Heading date 3a (HD3A) and FLOWERING LOCUS T-LIKE (FTL) under LD conditions [60,61]. FT-interacting protein 1 (FTIP1) is an essential regulator required for the export of FT protein from the phloem companion cells to sieve elements through the plasmodesmata under LD conditions [62] and was observed to be up-regulated in BP. The FT protein acts as a long-distance signal to induce flowering [63] and FLOWERING LOCUS D (FD) interacts with FT protein to activate the downstream floral meristem identity genes AP1 to initiate floral development [64,65]. While this is consistent with flower induction that is observed with BP, there are several transcriptional responses that are not down-regulated as expected. For example, AS1, a positive regulator of flowering that binds to the promoter of FT [66], was found to be down-regulated in BP. CDF2, a transcriptional repressor that delays flowering by repressing CO transcription under LD conditions [25], was found to be up-regulated almost 4-fold in BP compared with UBP. MIP1A and MIP1B, which repress flowering by forming heterodimeric complexes that sequester CO and COL proteins into non-functional complexes [26], were also found to be up-regulated in BP. Another inconsistent transcriptional response for flowering is up-regulation of HEADING DATE REPRESSOR 1 (HDR1), a flowering suppressor that up-regulates HD1 in LD conditions [67]. Previous studies on A. sinensis also found that the FTIP1, CDF2, MIP1A, and MIP1B were up-regulated at different growth and development stages [29]. Again, inconsistent regulation of photoperiod pathway transcripts associated with flowering in BP suggests down-stream signaling involvement in early bolting. Among the 40 DEGs associated with flowering, 29 genes showed coherent transcriptional regulation with EBF, while 11 genes were incoherent including: GA2OX6, GA2OX8, GAIP, HDR1, COL3, COL16, AS1, CDF2, MIP1A, MIP1B, and EFM. Extensive experiments have demonstrated that gene expression depends on the plant organ and even on the tissues in each organ [68][69][70]. In this study, the total RNA samples were extracted from the equivalent weight of the leaves and roots from BP and UBP, in theory, the level of gene expression obtained in the experiments is an average value of the expression in the leaves and roots, which could explain the incoherent transcriptional regulation of GA pathway and photoperiodic induction for EBF. For the 11 incoherent genes, their regulatory mechanisms need further validation by detecting gene expression in single organ.
Flowering is a process in which plants transition from vegetative to reproductive growth via a complex pathway of signaling networks. The DEGs observed comparing BP and UBP suggests transcription-based regulation of EBF. Specifically, genes associated with floral development and sucrose signaling are transcriptionally correlated with bolting ( Figure 5). For the floral development, SOC1 can integrate signals from the photoperiodic, GA and sucrose pathways to initiate early floral meristem identity by regulating the over-expression of LFY; meanwhile, AP1 in synergy with MADS, AGL8, and AGL12 that are repressed by AP2 and ANT, promote early floral meristem identity. Lastly, the early floral meristem identity induces early bolting and flowering of A. sinensis plants. For sugar signaling, over-expression of genes AMY1.1, BAM1, and BAM3 enhances starch degradation while differential expression SUSs, INVs, and INV Inh cleavage Suc to Glc and Fru can also promote SOC1 expression.

Conclusions
The DEGs observed comparing BP and UBP suggests transcription-based regulation of EBF. This transcriptomic and analysis focuses on four pathways that can mediate a

Conclusions
The DEGs observed comparing BP and UBP suggests transcription-based regulation of EBF. This transcriptomic and analysis focuses on four pathways that can mediate a transition from vegetative to reproductive growth: photoperiodic, GA signaling, autonomous, and floral development. While genes associated with EBF have been identified and mapped here, a causative role of these genes in activating and/or regulating EBF will require the knocking out of specific genes via a CRISPR/Cas 9 system.