Plant material and growth condition
Sinningia speciosa ‘Espirito Santo’ was obtained from Dr. Cecilia Koo Botanic
Conservation Center, Pingtung, Taiwan. The seeds were cultivated under 16/8 hours (day/night) cycle at 24℃ with 70% relative humidity. Floral bud developmental stage was determined based on dorsal corolla tube length. Floral bud stage 5 which has 8-10 mm length of dorsal tube was used for transcription factor (TF) isolation. Dissected dorsal and ventral petals from floral bud stage 5 were used for expression pattern validation of the dorsal expressed TFs. Finally, the leaves were used for 5’ regulatory region isolation.
All samples were frozen in liquid nitrogen and stored at -80℃.
Prediction of transcription factor
RNA-seq data of S. speciosa ‘Espirito Santo’ floral bud stage 5 was provided and analyzed by Dr. Zhao-Jun, Pan. Based on RNA-seq analysis, 630 genes were found to have dorsi-ventral differential expression (DEGs) (p-value<0.05; log2FC ≥1). In order to find the TFs among these DEGs, TF prediction was performed using iTAK online (v1.6) (http://itak.feilab.net/cgi-bin/itak/online_itak.cgi) for nucleotide sequences. The
(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearc h&LINK_LOC=blasthome) for annotation.
Prediction of TCP binding site
Since SsCYC downstream regulation might be facilitated by the presence of TCP binding sites at the 5’regulatory region of its target genes, screening for the binding sites was done for each of the predicted TF. The 5’ regulatory region of each TF was retrieved from S. speciosa ‘Avenida Niemeyer’ draft genome using RStudio software (Version 1.1.463; RStudio Inc., 2009) and Linux Interface (done by Ya-Chi, Nien). TCP binding consensus was summarized from the paper ‘TCP Transcription Factors: Evolution, Structure, and Biochemical Function’ (González-Grandío and Cubas, 2016) that has compiled most of TCP binding sites found in the in vitro and in vivo experiments in numerous studies. Screening for the presence of each of the summarized TCP binding consensus (Supplementary Table S1) was done for all the predicted regulatory regions using fuzznuc (http://emboss.bioinformatics.nl/cgi-bin/emboss/fuzznuc) for both strands of complementary sequence.
Total RNA extraction and reverse transcription
The total RNA from whole floral bud stage 5 and dissected dorsal and ventral petals of floral bud stage 5 were extracted using Trizol Reagent (Invitrogen, Waltham, MA, USA) according to manufacturer’s protocol. The RNA quality was measure using NanoDrop Spectrophotometer. Synthesis of complementary DNA (cDNA) was done using Superscript IV (Invitrogen, Waltham, MA, USA) according to manufacturer’s protocol.
Isolation of the transcription factor of S. speciosa ‘Espirito Santo’
TFs that have been predicted to contain TCP binding sites at their 5’ regulatory regions were isolated in order to get their full length coding sequences. The sequence of each TF was amplified with PCR using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) (Supplementary Table S2) and the products were purified by gel extraction (Viogene, GP1002), following the manufacturer’s protocol. The purified products were proceed to tailing in order to increase ligation efficiency. A-tailing was done by adding 0.3 μL of TaKaRa Ex Taq DNA Polymerase (Takara Bio, USA), 3 μL of Ex Tag buffer and 0.6 μL of 2mM dATP into 10 μL of purified product and the mixture was then incubated at 72℃ for 1 hour. The A-tailed products were purified by PCR Clean Up system (Viogene, GP1002) and were ligated to T&A™ cloning
Ligation mixture component vector: insert molar ratio 1:3 Vector fragments end conc. 3-30 fmol Insert fragments end conc. 9-90 fmol 10x Ligation Buffer A 2.0 μL 10x Ligation Buffer B 2.0 μL
yT4 DNA ligase 1.0 μL
ddH2O to final volume of 20 μL
The ligation mixture was incubated overnight. The next day, transformation was done using the heat shock method. About 2 μL of vector containing DNA of interest was mixed with 20 μL of competent cell, Escherichia coli HIT-DH5α (Real Biotech Corporation,
Taipei, Taiwan) and was chilled on ice for 20 minutes. Then, the mixture was thawed at 42℃ for 1 minute for heat shock and quickly chilled on ice. After heat shock procedure, 50 μL of LB broth was added to the mixture, followed by incubation at 37℃ for 1 hour.
The bacterial solution was added with 100 μL of 0.1 M IPTG and 20 μL of 80 mg/mL
X-Gal, and spread on LB agar plate contained Ampicillin (100 μg/mL). The plate was incubated for 16-18 hours. Colonies containing the insert were selected by using colony PCR. After confirmation, the colonies containing the correct insertion size of DNA was cultured in 3 mL of LB broth contained Ampicillin (100 μg/mL) by shaking at 37℃ for 16-18 hours. The plasmids were extracted using Mini Plus Plasmid DNA Extraction System (Viogene, GF2002) according to manufacturer protocol and sent to sequencing (Genomics, New Taipei City, Taiwan).
Validation for the expression pattern of the dorsal-expressed TFs of S. speciosa
‘Espirito Santo’
Quantitative real time PCR (qRT-PCR) analysis was done to validate the RNA-seq
data of the dorsal-expressed TFs that have been predicted to contain TCP binding sites at their 5’ regulatory regions. qRT-PCR analysis was performed in Bio-Rad PCR machine (CFX-384) using KAPA SYBR® FAST qPCR Master Mix (2X) Kit (KAPA Biosystem, KR0389) (Supplementary Table S3). The recipe and program were listed below:
qRT-PCR mixture
Step Temperature Time
1 95 ℃ 3 min
After the running of PCR, the obtained data was analyzed using CFX Maestro™ Software for CFX Real-Time PCR Instruments (Version 1.1; Bio-Rad Laboratories Inc, 2017). The expression level of each TF was quantified as relative fold gene expression level (2-ΔΔCT), using 18s as reference gene and ventral petals as the control. The ∆Ct was calculated as Ct (dorsal/ventral) – Ct (reference gene) and the ∆∆Ct was calculated ∆Ct (dorsal petals) – ∆Ct (ventral petals).
Genomic DNA Extraction
Genomic DNA (gDNA) extraction was performed with Hexadecyl trimethyl-ammonium bromide (CTAB) method (Doyle, 1990). The collected leaves were homogenized in liquid nitrogen using mortar and pestle. The homogenized tissue was added with 1 mL of CTAB, 20 mg of PVPP and 5 μL of β-mercaptoethanol, proceed by
incubation at 65℃ for 30 minutes. Next, the mixture was added with 500 μL of PCI (phenol : choloroform : isoamyl alcohol, 25:24:1, pH = 8.0) and inverted for 15 minutes, followed by centrifugation at 13.000 rpm for 10 minutes. The upper layer of the solution was transferred to the new tube, added with 1 μL RNase A and incubated at 37℃ for 20-30 minutes. The solution was added with 500 μL of C:I (choloform: isoamyl alcohol, 24:1)
and inverted for 15 minutes, followed by centrifugation at 13.000 rpm for 10 minutes.
The upper layer was transferred to the new tube and added with one to tenth volume of 3
M NaOAc (pH = 5.5), then precipitated with 0.7 volume of isopropanol. The mixture was
incubated at -20 ℃ for 1 hour, proceed by centrifugation at 13.000 rpm for 10 minutes.
The supernatant was discarded and the pellet was washed by the addition of 1 mL of 70%
ethanol and centrifugation at 13.000 for 5 minutes. The supernatant was discarded and the pellet was air dried. Finally, 30-50 μL of ddH2O was added to dissolve the pellet. The
quantity and quality of extracted gDNA was measured with Nanodrop Spectrophotometer.
The gDNA was stored at -20 ℃.
CTAB buffer (100 mL)
Reagent per reaction
Hexadecyl trimethyl-ammonium bromide (CTAB) 2.0 g
1M Tris (pH = 8.0) 10.0 mL
0.5 Ethylenediaminetetraacetic acid (EDTA, pH = 8.0) 4.0 mL
5 M NaCl 28.0 mL
ddH2O 56.0 mL
The pH was adjusted to 8.0 using NaOH and stored at room temperature
Isolation of the 5’ regulatory region of Sinningia speciosa ‘Espirito Santo’
There were several PCR based approaches used for isolating the 5’ regulatory region of each dorsal-high expressed TF. The regulatory region of Sispe038Scf1202g12026 (SsOFP6) was isolated using pair of primers designed directly
from the predicted regulatory region of S. speciosa ‘Avenida Nieyemer’ (SsAN). The regulatory region of Sispe038Scf1400g01001 (SsCYC) was isolated with forward primer
designed at the known coding sequence (CDS) of S. speciosa ‘Espirito Santo’ (SsES).
Another two regulatory regions, Sispe038Scf1061g02075 (SsERF3) and Sispe038Scf2159g01072 (SsCIB2), were isolated by nested PCR using two sets of primers.
The first set of primers contained the forward primer designed directly from the predicted regulatory region of SsAN and reverse primer designed on the known CDS of SsES. The second set of primers was design to amplify a secondary target within the first run product, thus reducing the non-specific binding in products. For the second round of the nested PCR, the product of the first PCR was diluted to 100 times. All of these three approaches were done with Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) according to manufacturer protocol (Supplementary Table S4). The amplified products were continued to cloning, using the same procedure described for transcription factor isolation and then sent to sequencing (Genomics, New Taipei City,
Taiwan). Last, the regulatory region of Sispe038Scf0228g08027 (SsERF17) was isolated with Thermal Asymmetric Interlaced PCR (TAIL-PCR), whereas for Sispe038Scf0170g01016 (SsRL2), Sispe038Scf2996g00029 (SsNGAL1) and Sispe038Scf5680g00016 (SsERF1), the regulatory regions were isolated with
Self-Formed Adaptor PCR (SEFA-PCR). All the isolated regulatory regions were screened for the presence of TCP binding sites.
Thermal Asymmetric Interlaced PCR (TAIL-PCR)
Thermal Asymmetric Interlaced PCR (TAIL-PCR) is used to amplify the unknown sequence, in this case the regulatory region that is adjacent to the known CDS. It uses two sets of primers which are the gene-specific primers (GS primers) that usually have high melting temperatures and arbitrary degenerate primers (AD primers) (Supplementary Table S5) that usually have low melting temperatures. By using the combination of these
primers, amplification of the expected sequence could be done from the known end and the unknown end, respectively. Specificity is obtained through subsequent rounds of TAIL-PCR, using nested gene-specific primers and alternate of high and low annealing temperatures cycles (Supplementary Fig. S3a). The TAIL-PCR used in this study was referred from Liu et al. (1995) and Liu and Whittier (1995) with modifications. The AD primers were adopted from Singer and Burke (2003). The recipe and program of TAIL-PCR were listed below:
Single reaction for primary TAIL-PCR
Reagent Volume
Phusion DNA polymerase (0.02 units/μL) 0.1 μL 5X Phusion HF or GC Buffer 2.0 μL
Thermal cycle for primary TAIL-PCR
Step Temperature Time
1 94 ℃ 2 min
Single reaction for secondary TAIL-PCR
Reagent Volume
Phusion DNA polymerase (0.02 units/μL) 0.1 μL 5X Phusion HF or GC Buffer 2.0 μL
Thermal cycle for secondary TAIL-PCR Step Temperature Time
1 94 ℃ 10 s
Single reaction for tertiary TAIL-PCR
Reagent Volume
Phusion DNA polymerase (0.02 units/μL) 0.1 μL 5X Phusion HF or GC Buffer 2.0 μL
Thermal cycle for tertiary TAIL-PCR Step Temperature Time
1 94 ℃ 15 s
products which could be seen by the smear appearance on the gel. The expected specific products could usually be observed from the product of 2nd and 3rd round, with the 3rd round product having slight decreased in size. The largest band from the 3rd round product was isolated and continued to cloning, using the same procedure described for transcription factor isolation, then sent to sequencing (Genomics, New Taipei City,
Taiwan) (Supplementary Fig. S3b).
Self-Formed Adaptor PCR (SEFA-PCR)
Self-Formed Adaptor PCR (SEFA-PCR) is developed to overcome the drawbacks of TAIL-PCR, which is the product is usually less than 1.0 kb. It combines the advantages of ligation-mediated PCR in its specificity and of TAIL PCR in its simplicity. It uses four primers that are located sequentially on the known DNA sequences. SP1, SP2, and SP4 are the specific primers designed from the known region and have relatively high annealing temperatures (e.g., 70°C), whereas SP3 (e.g., 5′-TACCCAAAGAAGCAGGAANNNNNNNNGTGAAA-3′) is a partially degenerate primer which plays the key role in the process. First, a single cycle of PCR was carried out at a low annealing temperature (e.g., 35°C) with only primer SP3. At this low annealing temperature, SP3 can prime and elongate at many positions on the DNA template. A position probably exists somewhere downstream of the known DNA sequence
where SP3 primes and extends, thus creating a nascent single strand which has a binding site for SP1. After a single cycle of PCR, the annealing temperature is increased to the point (e.g., 70°C) corresponding to the annealing temperature of SP1. Then, SP1 is added to the reaction mixture. At this high annealing temperature, only SP1 can prime the target site efficiently, thus creating a pool of single-stranded DNA with the SP1 sequence at the 3’ end and the SP3 complementary sequence at the 5’ end. Finally, several cycles of a low annealing temperature (e.g., 55°C) are performed to facilitate the loop-back extension, thus creating an adaptor which contains binding sites for SP1 and SP2. Once the adaptor has been created, the target sequences can be amplified efficiently by SP1. After SEFA PCR, a second round of nested PCR was run with the single primer SP2. A third round of thermally asymmetric PCR was run to improve the specificity with primer SP4 (e.g., annealing at 70°C) and the other short primer, SP5 (e.g., annealing at 60°C), positioned between SP2 and SP3 (Supplementary Table S6; Supplementary Fig. S4a). The SEFA-PCR used in this study was adopted from Wang et al. (2007) with modifications. The recipe and program were listed below:
Single reaction for primary SEFA-PCR
Reagent Volume
Phusion DNA polymerase (0.02 units/μL) 0.2 μL 5X Phusion HF or GC Buffer 4.0 μL
10 mM dNTPs 0.4 μL
5 μM SP3 1.0 μL
gDNA (1000 ng/μL) 1.0 μL
ddH2O add to 20 μL
Thermal cycle for primary SEFA-PCR
Step Temperature Time
1 98 ℃ 30 s
Single reaction for secondary SEFA-PCR
Reagent Volume
Phusion DNA polymerase (0.02 units/μL) 0.1 μL 5X Phusion HF or GC Buffer 2.0 μL
10 mM dNTPs 0.2 μL
5 uM SP2 3.0 μL
1:10 diluted 1st reaction 0.5 μL
ddH2O add to 10 μL
Thermal cycle for secondary SEFA-PCR
Step Temperature Time
1 98 ℃ 30 s
2 98 ℃ 10 s
3 70 ℃ 3 min
4 Go to step 2 for 29 cycles
5 25 ℃ 10 s
Single reaction for tertiary SEFA-PCR
Reagent Volume
Phusion DNA polymerase (0.02 units/μL) 0.1 μL 5X Phusion HF or GC Buffer 2.0 μL
Thermal cycle for tertiary SEFA-PCR
Step Temperature Time
1 98 ℃ 10 s molecular weight, and the desired product is usually expected to be seen in the 3rd product.
Therefore, the largest band from 3rd product was isolated and sent to sequencing
(Genomics, New Taipei City, Taiwan) (Supplementary Fig. S4b). New forward primers were design to amplify the desired regulatory region paired with SP4 primers, using the
same procedure as described in the transcription factor isolation (Supplementary Table S4).
Vector construction for dual-luciferase assay
The PJD301-firefly driven by the 5’ regulatory region of interest was used as the reporter (Supplementary Fig. S5a), whereas PJD301-renilla driven by 35s promoter was used as the internal control to normalized the transfection variability (Supplementary Fig. S5b) (Luehresen et al., 1995). The vector expressing SsCYC tagged with GFP was
served as the effector for the tested group (Supplementary Fig. S6), whereas vector expressing only GFP without SsCYC was used as effector for the control group (Supplementary Fig. S7).The isolated regulatory region sequence of SsRL1, SsERF17, SsOFP6, SsCYC, SsCIB2, and SsNGAL1 were amplified using PCR and cloned into the
BamHI and SalI restriction sites of the PJD301-firefly, whereas SsERF3 and SsERF1 were amplified by PCR to add HincII and NCO1 restriction site and cloned into the AfeI and NcoI restriction site of the vector (Supplementary Table S7). The general recipe for enzyme digestion was described as below:
Recipe for BamHI and Sal1 digestion
Reagent Volume
Recipe for HincII and NcoI digestion
Reagent Volume
Recipe for Afe1 and NcoI digestion
Reagent Volume
The reaction mixtures were incubated overnight and the desired digestion products were purified by gel purification (Viogene, GP1002), following the manufacturer’s protocol.
The purified products were ligated to the PJD301-firefly vector following the recipe described below:
Ligation reaction of PJD-firefly with the desired digestion product PJD301: insert molar ratio 1:3
PJD301 fragments end conc. 3-30 fmol Insert fragments end conc. 9-90 fmol 10x Ligation Buffer A 2.0 μL 10x Ligation Buffer B 2.0 μL
yT4 DNA ligase 1.0 μL
ddH2O to final volume of 20 μL
The ligation mixture was incubated overnight. The next day, transformation was done using the heat shock method into the Escherichia coli HIT-DH5α (Real Biotech
Corporation, Taipei, Taiwan). Amipicillin (100 μg/mL) plate was used as the selection medium. After 16-18 hours of incubation colony PCR was done to select the colony carrying the vector of interest. The colony that has been confirmed to carry the desired vector was cultured into LB contained Ampicillin (100 μg/mL) for maxi plasmid extraction (Viogene, GMV2002).
Protoplast isolation
Protoplast isolation was done according to ‘Arabidopsis mesophyll protoplasts protocol’ (Yoo et al., 2007) with modifications. Nicotiana benthamiana leaves were used
as the source of protoplasts instead of Arabidospsis. The plants were grown under 16/8 hours (day/night) cycle at 27℃ with 70% relative humidity. The leaves from 4-5 weeks-old-plant were chosen and cut into 0.5–1-mm strips from the middle part of a leaf using
a fresh sharp razor blade. The cut leaves were transferred into the prepared enzyme solution and digested in the dark for 3 hours at room temperature. After digestion, the solution was diluted with an equal volume of W5 solution. The enzyme solution containing the protoplasts was filtered through 75-μm nylon mesh into round-bottom tube.
The filtered solution was then centrifuged at 100 g for 2 minutes. The supernatant was removed and the protoplasts were re-suspended with W5 solution at 2 × 105 ml−1 after counting cells under the microscope (× 100) using a hemacytometer. The protoplasts were rested on ice for 30 minutes. After 30 minutes, the W5 solution was removed and the protoplasts were re-suspended in MMG solution 2 × 105 ml−1.
Protoplast DNA-PEG–calcium transfection
The protoplast transfection of vector mixture containing effector, reporter and
internal control was also performed following the method described in ‘Arabidopsis
mesophyll protoplasts protocol’ (Yoo et al., 2007). About 10 μL of vector mixture (the amount of each vector was 10 μg in 10 μL) was added into a 2-ml microfuge tube, followed by 100 μl protoplasts (2 × 104 protoplasts), then the mixture was mixed gently.
About 110 μl of PEG solution was added and the mixture was mixed gently by tapping the tube. The transfection mixture was incubated for 15 minutes at room temperature.
by inverting. The mixture was centrifuge at 100 g for 2 minutes and the supernatant was removed. The protoplasts were re-suspended in 0.5 mL of WI solution in each well of a 12-well tissue culture plate. Incubation was done for 16 hours.
Experimental design for SsCYC and dorsal-expressed TFs interaction analysis
Test Control
Vector Mixture
SsCYC-GFP effector GFP effector
5'regulatory region-PJD301 Firefly 5'regulatory region-PJD301 Firefly
PJD301 Renilla PJD301 Renilla
Dual-luciferase assay.
After 16 hours of incubation, the transfected protoplasts were collected by moving them to 2 mL microfuge tube, followed by centrifugation at 100 g for 2 minutes and the supernatant was removed. The dual-luciferase assay was done in 96-well white flat bottom plate according to the instruction of Dual-Luciferase® Reporter Assay System for product E1960 (Promega Corporation, USA). About 20 μl passive lysis buffer was added into the protoplasts and the mixture was transferred into the well of the plate. After 5 minutes, 100 μl of LAR II reagent was added into the mixture and the firefly luciferase activity was measured by luminometer by 10s measurement using i-control™ Microplate Reader Software (Version 1.8; Tecan, 2011). Then, 100 μL of Stop & Glo® was added and the renilla luciferase activity was measured by 10s
measurement. All reactions were run triplicate. The interaction of SsCYC and its downstream target was determined as normalized fold change (Δfold activity) by calculating the firefly to renilla activity ratio of the tested group divided to the control group. One-Way Analysis of Variance was used to assess the up/down-regulation significance level, using One-Way Analysis of Variance Calculator (https://goodcalculators.com/one-way-anova-calculator/).
Results
34 Transcription factors were predicted among 630 dorsi-ventral DEGs
The RNA-seq data has shown that there were 630 dorsi-ventral DEGs of S.
speciosa ‘Espirito Santo’. In order to screen for the TFs among these DEGs, iTAK was used as the identification and classification tool. Around 34 TFs were identified; 17 of them were the dorsal-expressed TFs and the others 17 were the ventral expressed TFs.
Based on NCBI BLASTX analysis, CYC (SsCYC) which was previously known as the major regulator of floral zygomorphy of A. majus was identified in the dorsal-expressed TF group (Table 1).
Table 1 The list of 34 TFs identified from 630 DEGs and their BLASTX annotations
19 out of 34 TFs were enriched with TCP binding sites at their predicted 5’
regulatory regions
TCP binding site has been known as the important element that mediates gene regulation of TCP TF family. Basically, TCP binding site is classified into two classes
with the consensus of GGNCCCAC for class I and GTGGNCCC for class II. Most of genes that are regulated by TCP-TFs are usually enriched with these binding sites.
with the consensus of GGNCCCAC for class I and GTGGNCCC for class II. Most of genes that are regulated by TCP-TFs are usually enriched with these binding sites.