D. officinale was collected from the Guangxi Zhuang Autonomous Region in China and was identified by professor Jun Duan. All authors comply with the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The D. officinale plants at the reproductive stage were grown in a greenhouse at South China Botanical Garden (Guangzhou, China) in The roots, stems, leaves, flowers and seeds (120 days after pollination) from at least six potted plants were harvested and used for gene expression analysis. Protocorm-like bodies (PLBs, T1 stage), multiple shoots from PLBs (T2 stage) and plantlets (T3, about 2 cm in height; T4, about 8 cm in height) were cultured on half-strength Murashige and Skoog (MS)  medium supplemented with 0.5 mg l-1 1-naphthalene acetic acid (NAA), 1 g l-1 activated carbon, 20 g l-1 sucrose and 6 g l-1 agar at pH 5.4, were grown in a growth chamber under 40 µmol m-2 s-1 light intensity and a 12-h photoperiod. All samples were collected, frozen in liquid nitrogen, then RNA was extracted immediately or placed rapidly at -80 °C.
A. thaliana (Columbia ecotype) obtained from professor Keqiang Wu (South China Botanical Garden, Chinese Academy of Sciences) was used as the wild type (WT) and. Arabidopsis plants were cultivated in a mixture of topsoil and vermiculite (1:3, v/v) under a 16-h photoperiod, 100 µmol m-2 s-1 light intensity, and at 22 °C in a growth chamber. Arabidopsis seeds were sown on half-strength MS medium  containing 15 g l-1 sucrose and 8 g l-1 agar and grown in the same conditions as the Arabidopsis plants.
Identification of LEA gene family in two orchids, and exon-intron prediction
The peptide and General Feature Format (GFF) files of P. equestris and D. officinale were obtained from the National Center of Biotechnology Information (NCBI) provided by Zhang et al. [35-36], respectively. The proteins in the peptide files were annotation using NCBI non-redundant (NR), Gene Ontology (GO), Pfam , Swissprot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The genes were annotated as the LEA genes were considered as candidate LEA genes, then rechecked in NCBI BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
For the prediction of gene structure, the GFF annotation of LEA genes was obtained from GFF files of P. equestris and D. officinale and submitted to the Gene Structure Display Server (GSDS) 2.0 (http://gsds.cbi.pku.edu.cn/)  to generate a diagram of gene structure.
Multiple sequence alignments and phylogenetic analysis
The LEA proteins were aligned using MAFFT version 7 software  to generate a FASTA alignment file. The alignment file was uploaded into MEGA version 7  to construct a phylogenetic tree by the neighbor-joining (NJ) method.
Construction of the 35S::LEA-GFP vector and generation of transgenic lines
The coding sequences of LEA36 and DoLEA43 without a termination codon were amplified by KOD-Plus DNA polymerase kit (Toyobo Co., Ltd., Osaka, Japan) and inserted in the NcoI site of the pCAMBIA 1302 vector using the In-Fusion HD Cloning Kit (Takara Bio Inc., Dalian, China) according to the manufacturer’s instructions. The transformation of Agrobacterium tumefaciens strain EHA105 with the correct construct was performed using the freeze and thaw method . Thereafter, A. tumefaciens harboring the construct was transformed into Arabidopsis by the floral dip method  to generated the overexpression transgenic lines.
The transgenic seeds of 35S::DoLEA36 and 35S::DoLEA43 were surface-sterilized in 1% NaClO for 10 min, washed in sterile distilled water six times, then sown on half-strength MS medium in Petri dishes containing 1.5% sucrose and 0.8% agar (pH 5.7). Plates were incubated at 4 °C in the dark for 2 d for stratification, then cultured in a growth chamber. After stratification, 4 day-old seedlings were used to survey GFP fluorescence and photographed with a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany). Transgenic plants that only contained pCAMBIA 1302 were used as the positive control.
Protein extraction from Arabidopsis
Total protein was extracted according to He et al. . Briefly, one week old seedlings were harvested, cleaned and ground into a fine powder in liquid nitrogen. Thereafter, an extraction buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 20% glycerol, 1% NP-40 and containing a protease inhibitor cocktail (Roche, Mannheim, Germany)] was added then centrifuged at 14,000 g for 20 min. The supernatant containing total proteins was transferred into a proteinase-free 1.5 ml Eppendorf tube and used for Western blot analysis immediately or frozen in liquid nitrogen for 2 min then stored at -80 °C.
One week old seedlings (200 mg) were also used to extract the cytoplasmic proteins. The Cytoplasmic Protein Extraction Kit (BestBio Inc., Shanghai, China) was used to extracted cytoplasmic proteins according to the manufacturer’s protocol. For the extraction of nuclear proteins, 4 g of seedlings were ground in liquid nitrogen into a fine powder, then 20 ml of lysis buffer [20 mM Tris-HCl (pH 7.4), 25% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 250 mM sucrose, 1 mM DTT and 1 mM PMSF] was added, washed six times with a washing buffer [20 mM Tris-HCl (pH 7.4), 25% glycerol, 2.5 mM MgCl2 and 0.2% Triton X-100], and washed once with nuclei resuspension buffer [20 mM Tris-HCl (pH 7.4), 25% glycerol and 2.5 mM MgCl2]. The nuclei storage buffer (200 μL) containing the Roche protease inhibitor cocktail was used to suspend the nuclear proteins. The cytoplasmic and nuclear proteins were used for Western blot analysis immediately or frozen in liquid nitrogen for 2 min then stored at -80 °C within 1 month.
SDS-PAGE and Western blot
The protein samples were degenerated in 1× SDS loading buffer and incubated in boiling water for 10 min, then centrifuged at 12,000 rpm for 2 min. The supernatant, which included the degenerated proteins, was used immediately for SDS-PAGE analysis. SDS-PAGE was carried out in 12% gels, then proteins were transferred from gels to a polyvinylidene fluoride (PVDF) membrane by a wet transfer apparatus (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% skimmed milk for 1.5 h at room temperature, the membrane was incubated with primary antibody for 1.5 h at room temperature, then washed with 1× TBST buffer (Solarbio Science & Technology Co., Ltd., Beijing, China) three times (each wash was 10 min). The membrane was then incubated with secondary antibodies at room temperature for 1.5 h. After washing membranes in 1× TBST buffer three times, the anti-GFP mouse monoclonal antibody (1: 5,000 dilution, Thermo Fisher Scientific, Waltham, MA, USA; cat#GF28R) was used as the probe to detect the level of GFP-tagged proteins. Actin and histone H3 were probed by Anti-Plant Actin Mouse Monoclonal Antibody (A01050; Abbkine, Wuhan, China) and a-H3 (ab1791; Abcam, Cambridge, UK) at 1: 5000, respectively. The goat anti-rabbit IgG-HRP (catalog number sc-2301, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and goat-anti mouse IgG HRP antibody (Thermo Fisher Scientific; #62-6520) were used as the secondary antibodies depending on the primary antibody. Protein bands were visualized in a ChemiDocTM MP Imaging system (Bio-Rad, Hercules, CA, USA). The protein marker used was EasySee® Western Marker (DM201, TransGen Biotech, Beijing, China).
Semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR) analysis
Total RNA was isolated with an RNA extraction kit (RNAout 2.0, Tiandz Inc., Beijing, China) according to the manufacturer’s protocol. One μg of RNA was used to synthesize cDNA by reverse transcription PCR (RT-PCR) of each reaction using a MonScriptTM PTIII all-in-one Mix Reverse Transcriptase Kit (Monad Biotech Co., Ltd., Suzhou, China). cDNA content was diluted to 200 ng μl-1 and 1 μl was used as template for PCR amplification using a 20 μl reaction system, then amplified by 35 cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 1 min. After amplification, 5 μl of each reaction was surveyed by 1% agarose gel electrophoresis and visualized using the Gel Imaging System (GenoSens1880, Shanghai Qinxiang Scientific Instrument Co., Ltd., Shanghai, China). For quantitative real-time PCR (qPCR) analysis, total RNA was extracted, cDNA was generated as indicated above, and qPCR was performed as described by He et al. . All specific primer pairs are listed in Additional file 2 table 2.
Gene expression analysis based on RNA sequencing data
For expression analysis of LEA genes from P. equestris, the transcriptome sequencing data of P. equestris roots (SRR2080194), stems (SRR2080200), leaves (SRR2080202), flowers (SRR2080204) and seeds (SRR3606718) were obtained from the NCBI Sequence Read Archive provided by Niu et al. . The clean reads were mapped to the P. equestris genome by TopHat version 2.0.8 (Kim et al. 2013) and gene expression level was calculated by the fragments per kilobase of exon per million fragments mapped (FPKM) method using HTSeq . For the expression analysis of LEA genes from D. officinale at different developmental stages (see plant materials section), FPKM were obtained from our D. officinale developmental database, which constructed in our laboratory. This data is available upon reasonable request.
In this study, we explored callus formation via two callus induction methods. In the first method, hypocotyls from 6-day-old Arabidopsis seedlings after stratification were cut by disposable knives about 0.5 mm from the cotyledon-hypocotyl junction and at about 0.5 mm from the hypocotyl-root junction. The center of hypocotyls was transferred carefully to Petri dishes containing half-strength MS medium with 1.5% sucrose and 0.8% agar (pH 5.7) with a sterile toothpick, incubated for 2 days in the dark and grown in a growth chamber. In the second method, hypocotyls were cut once at about 2 mm from the hypocotyl-root junction, the hypocotyl-root explants were transferred to Petri dishes containing half-strength MS medium with 1.5% sucrose and 0.8% agar (pH 5.7) and incubated in the dark at 22 °C. Callus was observed at the end of the cut site. Callus on an explant was considered to be induced if the callus was visible on a Leica S8 APO stereomicroscope (Leica Microsystems Ltd., Heerbrugg, Switzerland). Callus induction was quantified as a percentage of explants forming callus. Every experiment was repeated in triplicate. At least 50 seedlings of WT and transgenic lines from each experiment were used.
PLBs growing on half-strength MS medium containing 2.0% sucrose, 0.5 mg/L NAA and 0.6% agar (pH 5.4) were used for the wounding treatment. The PLBs were sliced into 2 mm thick slices and placed on the same medium. Explants at 0, 2, 5, 10, and 25 h after wounding were harvested and total RNA was extracted using the method described above. DoLEA36 and DoLEA43 expression was detected by qPCR analysis. Three biological replicates were performed for each sample.
Prediction of cis-responsive element in DoLEA36 and DoLEA43 promoters
To further explore the gene-responsive factors, 2000 bp upstream of the initiation codon from DoLEA36 and DoLEA43 were obtained from the D. officinale genome and used to predict the cis-responsive elements by PLANTCARE  and PLACE .
All the data of induction rate was analyzed by SigmaPlot12.5 software (Systat Software Inc., San Jose, CA, USA) using one-way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was considered to be statistically significant.