Functional analysis of ARF1 from Cymbidium goeringii in IAA response during leaf development

Plant materials and treatments

Plant tissues were collected from 2-year-old C. goeringii ‘Songmei’ plants grown under the conditions of 70% relative humidity, a temperature of 22 °C and natural light. The C. goeringii samples used in this study came from the Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou, China. After collection, the samples were frozen in liquid nitrogen and stored at −80 °C until use. For the exogenous auxin treatment, leaves of C. goeringii were sprayed until dripping with 10 μM IAA and were sampled at 0 h, 2 h, 4 h, 6 h, 1 2 h, 24 h and 48 h.

Arabidopsis with a Columbia ecotype (Col-0) background was used for gene overexpression. This plant material was grown at (22 ± 1) °C under 75% relative humidity and a 16 h day/8 h night photoperiod in an artificial climate chamber. N. benthamiana used for transient transformation was grown in a greenhouse maintained under a 16 h day/8 h night cycle with temperatures of 26 °C and 22 °C, respectively. For the exogenous auxin treatment, leaves of Arabidopsis were sprayed until dripping with 10 μM IAA and were sampled at 0 h, 12 h, 24 h and 48 h.

Cloning of the CgARF1 gene

Total RNA was isolated from plant tissue using a MiniBEST Universal RNA Extraction Kit (Takara, Dalian, China) following the manufacturer’s instructions. The RNA was then reverse transcribed into cDNA using FastKing gDNA Dispelling RT SuperMix (Tiangen, Beijing, China). The CgARF1 gene was amplified with the primer pairs listed in Table 1 and then cloned into the pBI121 vector between two restriction sites (Xba I and Sma I) to construct a recombinant vector. Gene amplification was performed by PCR as follows: 94 °C for 3 min; 35 cycles of 98 °C for 10 s, 60 °C for 15 s, and 72 °C for 30 s; and a final step at 72 °C for 5 min. The vector was then transformed into Trelief™ 5α chemically competent cells (Tsingke, Beijing, China), and the positive clones were tested.

Sequence analysis

The conserved domain of CgARF1 was predicted online at the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Lu et al., 2020). ExPASy (https://web.expasy.org/) was used to predict the physicochemical properties and hydrophobic properties of the CgARF1 protein (Gasteiger et al., 2005). Signaling peptides were predicted by using the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP-4.0/) (Petersen et al., 2011). Transmembrane domains were predicted using TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (Krogh et al., 2001). Secondary structure prediction was performed using the SOPMA server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html). Multiple sequence alignments were analyzed by ClustalX 2.1 software (Larkin et al., 2007). The phylogenetic relationships between CgARF1 and 19 sequences from other species identified previously were analyzed using Mega 6.0 software with the neighbor-joining (NJ) method and a bootstrap value of 1,000 (Kumar, Stecher & Tamura, 2016). The amino acid sequences of the CgARF1 homolog proteins were downloaded from the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Quantitative real-time PCR analysis

RNA was extracted and reverse transcribed as described above. Quantitative real-time PCR (qRT-PCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a StepOnePlus real-time PCR system (Thermo Fisher, Waltham, MA, USA). The procedure was as follows: 95 °C for 5 min and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The 18S RNA of C. goeringii was used as the internal reference gene when analyzing the expression levels of CgARF1. The specific primers used for this experiment are shown in Table 1. qRT-PCR data were analyzed using the 2^−ΔΔCt method. Three technical replicates of each sample were performed for qRT-PCR.

Subcellular localization assay

A subcellular localization prediction tool, WoLF PSORT, was used to predict subcellular localization (Horton et al., 2007). The ORF of the CgARF1 gene (without a stop codon) was inserted into a modified pCambia1300:GFP vector at double restriction sites (XbaI and SmaI) (Table 1). This vector contains the green fluorescent protein (GFP) reporter gene driven by the CaMV 35S promoter, and allows the expression of recombinant proteins fused to GFP at its N-terminus. Then, the recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method (Weigel & Glazabrook, 2005), and the bacteria were then grown in Luria-Bertani (LB) media with both kanamycin and rifampicin at 28 °C. Bacterial cells were harvested by centrifugation and resuspended in an infiltration solution (0.15 mM acetosyringone, 10 mM MgCl₂, 10 mM MES, pH of 5.6) at a final OD₆₀₀ of 0.8. The Agrobacterium suspension mixtures were infiltrated into 6-week-old leaves of N. benthamiana (tobacco) using a needleless syringe (Zhao et al., 2019). Three leaves from each tested plant were injected, and three plants were infected by each gene. The fluorescence signals were observed 48–72 h after injection under a confocal laser scanning microscope (LSM710; Carl Zeiss, Jena, Germany).

Plant transformation in Arabidopsis

pBI121-35S::CgARF1 was transformed into Arabidopsis via the Agrobacterium tumefaciens-mediated floral dipping method (Clough & Bent, 1998). Inflorescences of Arabidopsis were infected for 45–60 s and then incubated in the dark for 20 h. Transgenic seeds were screened on MS media containing 50 mg L⁻¹ kanamycin, and the T3 plants were used for further experiments.

Determination of chlorophyll contents and endogenous phytohormones

Acetone-ethanol solvent extraction was applied for the determination of chlorophyll contents. Arabidopsis leaves collected from the same part of the main stem were soaked in a 1:1 mixture of acetone and ethanol in darkness, and the samples were shaken periodically until the green color completely faded from the leaves. Then, the absorbance values at 663, 646, and 470 nm were determined, and the chlorophyll content was calculated according to Porra’s formula (Porra, 2002). Three biological replicates and three technical replicates of each line were performed for this experiment.

The contents of endogenous hormones in Arabidopsis leaves were determined using enzyme-linked immunosorbent assay (ELISA). Samples were ground in 10 mL of 80% (v/v) methanol extraction medium containing 1 mM butylated hydroxy toluene (BHT) as an antioxidant. The extract was incubated at 4 °C for 4 h and centrifuged at 4,000 rpm for 15 min. The supernatant was passed through C-18 columns and washed with 80% (v/v) methanol, 100% (w/v) methanol, 100% (w/v) ether and 100% (w/v) methanol successively. Then the hormone fractions were dried under N₂ and dissolved in phosphate buffer saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin for analysis.

The monoclonal antigens and antibodies against ABA, GAs (GA₁ + GA₃) and BR used in ELISA were produced at the Phytohormones Research Institute (China Agricultural University; see He, 1993). ELISA was performed on a 96-well microtitration plate. Each well was coated with 100 μL of coating buffer (1.5 g/L Na₂CO₃, 2.93 g/L NaHCO₃, and 0.02 g/L NaN₃) containing 0.25 μg/mL antigens against the hormones, and then incubated for 30 min at 37 °C. After washing four times with PBS containing 0.1% (v/v) Tween 20, each well was filled with 50 μL of sample extracts and 50 μL of 20 μg/mL antibodies, and then incubated and washed as above. Next, 100 μL of color-appearing solution containing 1.5 mg/mL 0-phenylenediamine and 0.008% (v/v) H₂O₂ was added to each well. The reaction was stopped by 12 mol/L H₂SO₄ per well. Color development was detected at 490 nm using an ELISA Reader (Model EL310; Bio-TEK, Winooski, VT, USA). Hormone contents were calculated following Weiler, Jordan & Conrad (1981). Three biological replicates of each hormone were performed for this experiment.

IAA treatment of detached leaves of transgenic plants

True leaves from 10-day-old seedlings were cut with scissors on an ultraclean table and then placed on MS medium, MS medium with 0.2 mg·L⁻¹ IAA or MS medium with 0.5 mg·L⁻¹ IAA. On day 12, the rooting rate, the average number of roots and the average length of roots were calculated under a stereomicroscope. The rooting rate was measured ten leaves per time for each line, the average number of roots was measured five leaves per time for each line, and the average length of roots was measure three roots per time for each line. Every measurement was performed for five times independently.

Cloning and sequence analysis of CgARF1

The CgARF1 gene sequence was obtained from C. goeringii transcriptome data. The open reading frame (ORF) of CgARF1 was cloned from the ‘Songmei’ cultivar of C. goeringii; it was 2,049 bp in length and encoded 682 amino acids (Fig. S1A; Table S1). The CgARF1 protein was acidic and exhibited a molecular weight of 76,416.43 and a theoretical isoelectric point of 6.17. This protein exhibits an instability index greater than 40, indicating that it is unstable (Guruprasad, Reddy & Pandit, 1990). Hydrophobicity analysis showed that the CgARF1 protein might be a hydrophobic protein due to its grand average hydropathicity (GRAVY) value of −0.456 (Fig. S1B). No signaling peptides or transmembrane domains were found in this protein (Figs. S1C and S1D). The secondary structure consisted of 19.50% α-helices, 15.25% extended strands, 4.84% β-turns and 60.41% random coil structures (Fig. S1E).

The amino acid sequence of CgARF1 was aligned with those of 19 homologous ARFs from NCBI, and the results showed that they all contained three typical domains: the B3 DNA binding domain, Aux_resp domain and AUX/IXX family domain (Fig. S2). To study the evolutionary relationships between CgARF1 and the 19 ARF transcription factors (TFs) from other species, a phylogenetic tree was constructed (Fig. 1). The results showed that the 20 ARF TFs were divided into three subfamilies, among which CgARF1 showed the closest evolutionary relationships with three ARF7 sequences from the orchids C. sinense, Phalaenopsis equestris and Dendrobium catenatum. This suggests that these ARF genes might have similar biological functions.

Expression pattern of CgARF1 in different tissues and under IAA treatment

Gene expression patterns are typically closely correlated with gene functions. To better understand the function of CgARF1, its expression levels in different tissues (roots, pseudobulbs, leaves, and flowers) of C. goeringii were first examined. The results showed that this ARF gene was expressed in all examined tissues and showed different expression levels in these four organs (Fig. 2A). The highest expression was found in the leaves, while the lowest level was observed in the pseudobulbs (P < 0.05). This suggests that CgARF1 might be involved in leaf development.

As an auxin response factor, CgARF1 responded strongly to exogenously supplied auxin (IAA), as shown in Fig. 2B. The expression level of CgARF1 sharply increased after spraying with IAA and peaked at 2 h after treatment, while its expression at 12 h was similar to that at 2 h. A significant decrease was observed after 12 h. This up-down-up-down expression pattern indicates the complexity of the regulatory relationship between CgARF1 and exogenous auxin.

Subcellular localization of the CgARF1 protein

To investigate the function of a gene at the protein level, it is important to determine its possible site of residency in the cell. The online analysis tool WoLF PSORT predicted that the CgARF1 protein showed the greatest probability of being located in the nucleus. To determine the subcellular localization of CgARF1, we constructed a CgARF1::GFP fusion protein. Through Agrobacterium-mediated infiltration, this fusion protein was transiently expressed in N. benthamiana. Under a laser confocal microscope, the nuclear DNA was stained with fluorescent blue DAPI. CgARF1::GFP fluorescence largely overlapped with the blue fluorescence, indicating that the CgARF1 protein was mainly located in the nucleus (Fig. 3).

Identification and phenotypic observation of transgenic plants overexpressing CgARF1

To confirm the overexpression of the CgARF1 gene in transgenic plants, total genomic DNA (gDNA) was extracted from the mature leaves of Arabidopsis. PCR detection was conducted with the 35S forward primer and the CgARF1 reverse primer using wild-type (WT) DNA as the negative control. As shown in Fig. S3, an approximately 2,000 bp product was amplified from transgenic Arabidopsis, while no target PCR product was amplified from WT. Quantitative real-time PCR results also showed that the expression level of the CgARF1 gene in the transgenic lines was much higher than that in the wild type (Fig. 4).

Three independent transgenic lines from the T3 generation were randomly selected for phenotypic observation. By observing the entire growth cycle of Arabidopsis, it was found that the CgARF1-overexpressing transgenic plants mainly displayed earlier flowering (Fig. 5A) and earlier senescence of rosette leaves (Figs. 5B and 5C) than WT plants. On day 40 after transplantation into soil, the leaf shape of the transgenic plants showed no change, but leaf etiolation was obvious. In response to this phenomenon, the chlorophyll contents were measured. The results showed that the overexpression of CgARF1 had significant effects on the contents of chlorophyll a, chlorophyll b and total chlorophyll (Figs. 5D–5F). However, there was no obvious effect on chlorophyll a/b between the transgenic and wild-type plants (Fig. 5G).

Growth of detached leaves under IAA treatment

Individual leaves from WT and overexpressing CgARF1 Arabidopsis plants were excised and inoculated onto MS medium supplemented with different concentrations of IAA. On the hormone-free MS medium, wild-type leaves developed symptoms of yellowing and wilting, whereas most of the detached leaves from transgenic plants remained green (Fig. 6A). With an increase in the concentration of hormones, transgenic leaves gradually showed an upward curling phenotype (Fig. 6B). Additionally, their rooting rate, rooting number and rooting length were all obviously increased compared with those of leaves without hormone treatment (Figs. 6C–6E). However, the WT leaves still grew poorly and slowly.

Changes in endogenous phytohormone contents under IAA treatment

The contents of endogenous hormones, including IAA, ABA, GAs, and BR, were also detected in the mature leaves of Arabidopsis (Fig. 7). The results showed that the hormone levels in transgenic Arabidopsis and WT showed the same trend under IAA treatment, but the levels in the former were almost lower than those in the latter, especially for GAs. In addition, at the later stage of IAA treatment, the content of GAs in transgenic plants increased significantly after 24 h, while the ABA contents also showed similar changes after 12 h.