Plant material
All Arabidopsis thaliana plants are in the Columbia (Col-0) background. Col-0 and AtAHL20 T-DNA insertion mutants, ahl20-1 (Salk_144620) and ahl20-2 (Salk_148971) seeds used in this study were obtained from the Arabidopsis Biological Resource Center (ABRC). Camelina plants Camelina sativa (L.) Crantz var Calena) were grown in a greenhouse (16 h under the light and 8 h in the dark) at 25°C. Camelina seeds were provided by Dr Scot Hulbert of Washington State University, who obtained them from Dr. Stephen Guy at Washington State University (Guy et al., 2014)
Cloning and generation of transgenic Arabidopsis and Camelina plants
Arabidopsis thaliana
AtAHL20 overexpression
Gateway compatible entry plasmids containing Arabidopsis AHL gene coding sequences as well as other genes used in this study were obtained from ABRC. To overexpress AtAHL20, Gateway entry vector, pENTR223, was used in Gateway LR reactions (Invitrogen, Carlsbad, CA) together with a destination vector pEarlyGate100 binary vector (35S constitutive promoter) (Earley et al., 2006). The binary vectors carrying AtAHL20 cDNA were used to transform Col-0 wild-type plants via the floral dip method (Clough and Bent, 1998). To generate point mutations in AtAHL20’s AT-hook domain we used a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) using Gateway compatible primers (Table 2). pENTR223 vector carrying AtAHL20 cDNA was used as a template during the site-directed mutagenesis reaction. The resulting construct was sequenced to confirm the successful mutation of the arginine residues in the respective coding sequences.
GUS constructs
AtAHL20’s 1335 bp long promoter region was PCR amplified using Gateway-compatible primers (Table 2) and cloned into the Gateway compatible entry vector pDONR221 via a BP reaction (Invitrogen, Carlsbad, CA). Following the BP reaction, the resultant entry vector was sequenced to confirm the absence of mutations. pDONR221 entry vectors carrying AtAHL20 promoter were cloned into the Gateway-compatible destination vector pMD163 (ABRC) via the Gateway LR reaction to generate a promoter:GUS expression binary vector.
Transgenic Arabidopsis plants expressing the above mentioned constructs were generated in the wild type Col-0 background via the floral-dip method (Clough and Bent, 1998). Transgenic seeds were screened on 0.5× Linsmaier and Skoog modified basal medium supplemented with appropriate antibiotics containing 1.0% (w/v) phytagel (Sigma-Aldrich), 1.5% (w/v) sucrose and under continuous white light at 25ºC in a Percival E-30B growth chamber.
Camelina sativa
Overexpression of CsAHL20
CsAHL20 coding sequence was extracted from the NCBI database after a BLAST search using AtAHL20 sequence as a query. Primers (Table 2) were designed from the extracted sequence and were used to amplify CsAHL20’s coding sequence. The amplified PCR product was cloned into pDONR221 entry vector via Gateway BP clonase II (Invitrogen, Carlsbad, CA) reaction to generate the pDONR221-CsAHL20 entry vector. A Gateway LR clonase II (Invitrogen, Carlsbad, CA) reaction between pDONR221-CsAHL20 and the destination vector pUSH21 was performed to generate pUSH42-2. In this construct expression of CsAHL20, coding sequence and the selection marker DsRed were separately driven by CaMV 35S promoters. The binary vector was transformed into Agrobacterium tumefaciens strain GV3101 and used for plant transformation via the floral-dip protocol (Lu and Kang, 2008). T1 seeds harvested from transformed plants were illuminated with a green LED light and fluorescent seeds were visually detected under a red filter (Lu and Kang, 2008). Single insertion T-DNA T2 mutants were identified by screening for plants that produced 3:1 fluorescent: nonfluorescent seeds. Homozygous T3 pUSH42-2-CsAHL20 plants from single locus insertion lines were used for qRT-PCR analysis.
Yeast-Two-hybrid plasmids
A GAL4-based Y2H system was used in protein-protein interaction assays (Weber et al., 2005). Yeast strain L40ccU3, bait vector (pBTM116-GW-D9) with TRP1 reporter marker and prey vector (pACT2-GW) with LEU reporter marker were obtained from Dr. Hanjo Hellmann’s lab (Washington State University, Pullman, WA). Gateway entry vectors carrying AtAHL6, AtAHL19, AtAHL20, AtAHL22 and AtAHL29’s coding sequences genes were used in LR reactions to clone the respective open reading frames into the bait and prey vectors (pBTM116-GW-D9) and (pACT2-GW), respectively. Competent yeast cells were transformed with bait and prey plasmid constructs using a standard lithium acetate protocol. Transformed yeast competent cells were incubated for three days at 28°C on SD minimal medium supplemented with Leu and His (SDII). Four randomly selected colonies were diluted 1:2,000 in autoclaved distilled water before 20 μL were simultaneously dropped on both SDII and SDIV lacking tryptophan, leucine, histidine and uracil and containing predetermined levels of 3-amino-1, 2, 4-triazol (3-AT). Yeast was incubated at 28°C for 3-6 days.
RNA extraction, cDNA synthesis, qRT-PCR, semi-quantitative PCR and data analysis
Total RNA was extracted from 10-day old camelina seedlings grown on ½ × MS medium using Plant RNA mini kit (Qiagen, Valencia, CA) according to manufacturer’s recommendations. For Arabidopsis, total RNA was extracted from rosette leaves collected from 21-day old adult plants (analyzed to quantify FT, TSF, AGL8 and SPL3 via qRT-PCR), as well as from 7-day old seedling roots, whole 7-day old seedling, adult plant rosette leaf, 7-day old seedling hypocotyls, flowers and siliques (used for semi-quantitative PCR for AtAHL20 tissue specific expression). On-column DNAse treatment was performed to digest any potential contaminating genomic DNA. Complementary DNA (cDNA) was synthesized from total RNA (500 ng) using the iScript Reverse Transcription Super mix (Bio-Rad, Hercules, CA). qRT-PCR was carried out using Bio-Rad’s SSO Advanced Universal SYBR Green Super Mix (Bio-Rad, Hercules, CA) and 10-fold diluted cDNA templates (synthesized above) on a Bio-Rad’s CFX96 Touch Real-Time PCR Detection System. Melting curves of SYBR green wells were cross checked to eliminate nonspecific amplification. Data are normalized to MDAR4 messenger ribonucleic acid (mRNA) expression (internal control), and fold changes are displayed relative to control plant lines. Error bars represent standard deviations of technical replicates (n = 3). Three biological replicates were used from each plant line.
RNA-Seq library preparation
Total RNA was extracted from rosette leaves harvested from 20-day old growth-chamber-grown plants using MagJET Plant RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA). The Dynabeads mRNA DIRECT Kit (Thermo Fisher Scientific, Waltham, MA) was used for purification of intact polyadenylated (polyA) mRNA. RNA-Seq libraries used were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies, Carlsbad, CA) following the manufacturer’s protocol.
RNA-Seq datasets were analyzed using CLC Genomics Workbench software (Qiagen, Valencia, CA). RNA-Seq libraries were constructed from RNA extracted from rosette leaf tissue pooled from three independent plants. Following Kal’s Z-test (Kal et al., 1999), genes were classified as differentially expressed with a False Discovery Rate (FDR) adjusted p-value < 0.05 and a fold-change absolute value > 3.
Read mapping and differential expression
Reads which already had adaptor sequences removed by the Torrent Suite ver 4.2.1 sequencing software (Thermo Fisher Scientific, Waltham, MA), were quality trimmed using the default setting in CLC Genomics Workbench 7.5 (Qiagen, Valencia, CA). After preprocessing the RNA-Seq data, the reads were mapped to the TAIR10 version of the Arabidopsis genome using CLC Genomics Workbench 7.5 (Qiagen, Valencia, CA). Read counts for each gene were quantified using the RNA-Seq Analysis tool using the default settings. Differential expression of original values was determined with the proportions statistical analysis tool, using Kal’s Z-test with FDR correction.
Gene ontology enrichment
Gene ontology analysis was performed using the PLAZA 3.0 Dicots Workbench Analysis tool (Proost et al., 2015)
Histochemical GUS analysis
GUS analysis was performed as described by (Zhang et al., 2009) on six-day old seedlings, rosette leaves from 20-day old and floral structures from flowering plants grown in the greenhouse.
Flowering time analysis
It has been observed that transplanting seedlings to soil can cause stresses that can alter flowering time. Consequently, all seeds were directly sown in pots containing a pre-watered soil mix Sunshine 50 Mix4 (Aggregate) LA4, Green Island Distributers Inc.; Riverhead, N.Y). These pots were subsequently incubated in darkness for seven days at 4ºC to induce near-uniform germination. After that, pots were transferred to growth chambers under the following conditions: white light (200 μmol m-2 sec-1), 21ºC and 60-70 % humidity. Once the seedlings were several days old, they were thinned to one per pot by clipping using small scissors. Experience in the lab suggests that removal of whole seedlings causes root damage to neighboring seedlings, which in turn can cause damage/stress that can potentially lead to altered flowering time. This approach gives the most uniform and repeatable flowering time results for each genotype. To measure flowering time, we counted the number of days from germination time until the floral stem was 0.5 cm above the basal rosette.
Seedling growth conditions and hypocotyl measurement
Seedling growth conditions and hypocotyl measurements were performed as described in (Zhao et al., 2013).
Sequence alignment
AtAHL20, AtFT, CsAHL20 and CsFT nucleotide and protein sequences were downloaded from NCBI database (Data S4). Both nucleotide and protein sequence alignment were aligned using
BOXSHADE public server https://embnet.vital-it.ch/software/BOX_form.html.
Statistical analysis
Results are presented as mean values for the combined data. Error bars represent the standard error of the mean (SEM) or standard deviation (SD). For group means multiple comparison to the wild type, we used a One-way ANOVA Dunnett’s multiple comparison test or T-test using GraphPad Prism statistical software. **** = p-value < 0.0001, *** = p-value 0.0001 to 0.001, ** = p-value 0.001 to 0.01, * = p-value 0.01 to 0.05, ns = p-value ≥ 0.05.