Introduction

Lignins are complex phenolic polymers comprise of guaiacyl (G) units derived from coniferyl alcohol, syringyl (S) units derived from sinapyl alcohol, and p-hydroxyphenyl (H) units derived from p-coumaryl alcohol. Many enzymes are involved in lignin biosynthesis [1]. Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195), which is a key enzyme in monolignol biosynthesis, catalyses the conversion of the corresponding cinnamyl aldehydes to cinnamyl alcohols; this is the last step in the synthesis of monolignols before their polymerization in cell walls. According to these data, CAD displays distinct features between gymnosperms and angiosperms. Gymnosperm CAD is encoded by a single gene, and is believed to be highly specific for the reduction of coniferyl aldehyde [2], and with lower catalytic activity toward sinapyl aldehyde. On the contrary, angiosperm CAD has multiple isoforms that have significant affinity for both coniferyl and sinapyl aldehydes [3].

The relationship between CAD and biotic stress has been well-established, the IbCAD1 gene play a role in the resistance mechanism to biotic and abiotic stresses as well as in tissue-specific developmental lignification [4]. The mRNA level of alfafa MsCADs and ryegrass LpCADs was induced by wounding treatment, and the addition of fungal elicitor to bean cell cultures led to the rapid accumulation of CAD transcripts [3, 5]. Syringyl lignin was found to be accumulated during the hypersensitive resistance response in wheat [6]. Transgenic plants having reduced levels of lignins are more readily attacked by fungi and bacteria [7]. In addition, CAD genes might also be important for the production of biofuels. Some researchers produced gene mutant poplars with reduced CAD activity and observed higher lignin extractability [8]. All of these indicate that the amount and unit composition of lignin is influenced by gene transcription, developmental and environmental conditions. However, little information is yet available on the role of CAD in relation to abiotic stress.

Plants have to develop a broad range of complex defense systems to struggle against pathogenic infections, because they have being confined to the place where they grow. Recently, Ginkgo biloba is one of the most popular functional plants, especially as medicinal plants. Extracts of G.biloba leaves contain active compounds such as flavonoids and terpene lactones(ginkgolides and bilobalide) and can therefore be use to increase peripheral and cerebral blood flow [9, 10]. In addition, Ginkgo has strong diseases and pests resistant, grows in widespread area, which has many good characters, such as disease-resistance, drought, low temperature, barren, and nuclear radiation. Flavonoids metabolism and lignans metabolic both belong to phenylalanine metabolism path branch, lignans synthesis might affect the accumulation of G. biloba flavonoids. Here we report the cloning and characterization of a GbCAD1 encoding a putative CADs protein and also discuss its potential function in resistance mechanism to biotic and abiotic stresses. A combination of biochemical and gene expression analysis indicated that GbCAD1 is the enzyme involved in lignification and defense. Taking into consideration the role of CAD and lignin in imparting disease resistance to the plants, our study was aimed to investigate the possible role of CAD enzyme in imparting disease resistance to the plant, role of lignin deposition in disease resistance against wounding and pathogen infection in Ginkgo and to correlate its transcripts with disease resistance of Ginkgo to pathogen.

Materials and methods

Plant materials and treatments

Fourteen-year old of grafted G. biloba seedlings were growing in a greenhouse in Huanggang (E, 111°54′–112°19′, N, 30°6′–30°39′, Hubei province, central of China) were sampled as plant materials. For gene cloning and tissue expression, diverse tissues including young leaves, mature leaves, ovules, stamens, albumen, gynoecia, stems and roots were collected for DNA and RNA extraction as described by Xu et al. [11]. Tissues were immediately frozen in liquid nitrogen and kept at −80 °C prior to total RNA extraction.

Total RNA was isolated from injured stem tissues of 14 years old grafted G. biloba seedlings after they were attacked by Rhizoctonia solani. For fungal infections blade, the total RNA was isolated from Ginkgo leaves cells punctured with an infected agent isolate of Phyllosticta mortoni Fairman.One year old cuttings from the same genotypic strain of G. biloba were subjected to treatments with UV-B, wounding(WOU), 5-aminolevulinic acid (ALA), abscisic acid (ABA), salicylic acid (SA) and ethephon (ETH). For UV-B treatment, seedlings were exposed to 1,500 μJ/m2 UV-B irradiation in a closed chamber, and the control cuttings were placed in a dark closed chamber. The edges of Ginkgo leaves were cut by about 0.6 cm with scissors for wounding treatment, the intact leaves of Ginkgo was as control. The ALA (100 μM), ABA (10 mM), SA (25 mM), and ETH (20 mM) were dissolved in 0.01 % Tween 20 and sprayed onto young leaves. The control leaves were sprayed with an equivalent volume of 0.01 % (v/v) Tween 20.

Cloning of the full-length cDNA

Given species conservation of the functional domain of the CADs protein and the Ginkgo EST database in GenBank, a pair of degenerate primers, CAF and CAR, was designed according to the conserved peptide sequences, ILGLGGVGHM and NDVRYRFVV, to amplify a conservative fragment of the GbCAD1 gene. The purified PCR product was cloned into the pMD18-T vector (TaKaRa, Dalian China), and the positive clone was confirmed by a BLASTN query of the NCBI Database to verify the homology of this cloned fragment with CAD sequences from other species.

Based on the sequence of cloned GbCAD1 fragments, the specific primer pairs (CA5R and CA3R) and the nested primer pairs (CA5N and CA3N) were designed to amplify the 5′ and 3′ end of GbCAD1 using the SMART™ RACE cDNA Amplification Kit (Clontech, USA). The PCR products were purified and cloned into the pMD18-T vector for sequencing. After comparing and aligning the sequence of 5′RACE, 3′RACE, and the internal fragment, the full-length cDNA sequence of GbCAD1 was obtained.

Southern blot analysis

Genomic DNA (20 μg/sample) was digested overnight at 37 °C with Eco R I, BamH I, Hind III and Kpn I. The digested DNA was fractionated by 0.9 % agarose gel electrophoresis and transferred onto a PVDF membrane (Roche Applied Science, Germany). Specific probes were generated by PCR: GbCAD1 specific probe was obtained using sense CAT1 and antisense CAT2 primers located respectively at positions 161 and 614 on the GbCAD1 cDNA. At total of 50 ng purified amplified fragments were used as a template in a total volume of 20 μl for probe labeling. Probe labeling (DIG), hybridization and signal detection were performed following the manufacturer’s instruction for the DIG High Primer DNA Labeling and Detection Starter Kit II (Roche Applied Science, Germany) [12].

Relative quantification by QRT-PCR

The transcription levels of GbCAD1 were determined in different G. biloba tissues, as well as in young seedling leaf samples collected at different time points after stress and hormone treatments. QRT-PCR was carried out using an ABI PRISM 7,500 Sequence Detection System (Applied Biosystems, American) with SYBR Green PCR Master Mix (Applied Biosystems, American) according to the manufacturer’s protocol. The G. biloba glyceraldehydes-3-phosphate dehydrogenase gene (GbGAPDH, L26924) [13], was used as the reference gene as described by Xu [11].

The gene-specific primers (CAQF, CAQR) and reference primers (GAPF, GAPR) for QRT-PCR were listed in Table 1. The QRT-PCR conditions were: 10 min at 95 °C, and 40 cycles (95 °C for 15 s, 60 °C for 1 min). Before performing QRT-PCR, primer efficiency was evaluated using both GbCAD1 and GbGAPDH at 100, 150, 200, 250 and 300 nM combinations. A 120 nM concentration was chosen as most suitable combination for both genes. For each plant sample, aliquots of 150 ng total RNA was analyzed for each gene and the two genes (GbCAD1 and GbGAPDH) were always analyzed simultaneously. Each sample was amplified three times and all reactions were performed on ABI PRISM 7,500 Sequence Detection System. With a housekeeping gene GbGAPDH, the relative amount of the GbCAD1 transcript is presented as 2(–ddCt) according to the CT method[14]. When comparing the expression of GbCAD1 in different tissues or treatments, the relative expression of GbCAD1 was achieved by calibrating its transcription level to that of the reference gene, GbGAPDH.

Table 1 Primers for GbCAD1
Full size table

Functional expression of the GbCAD1 in Escherichia coli

To clone GbCAD1 into an expression vector, a pair of primers, CAZF and CAZR, were designed and synthesized to amplify the coding region by RT-PCR with the incorporation of a restriction enzyme site and a protective base to simplify later vector construction. After confirmation by sequencing, the resulting recombinant plasmid was introduced into BL21 (DE3) by the heat shock method. A single colony of E.coli BL21 cells harboring the expression plasmid pET28a-GbCAD1 was inoculated at 37 °C in Luria–Bertani medium containing kanamycin (50 mg L−1) and were grown with shaking (150 rpm) at 37 °C until the optical density (OD 600) reached about 0.6. For induction, Isopropyl β-d-thiogalactoside (IPTG) was added at a final concentration of 1 mM and the cells were further cultured at 30 °C for 2 h. The cells were lysed by sonication for 10 s at 4 °C, and centrifuged at 7,000 g for 15 min. Supernatants and pellets were analyzed by SDS-PAGE and Coomassie Brilliant Blue R250 staining. The recombinant GbCAD1 protein from induced cells was purified using Nickel–CL agarose affinity chromatography (Bangalore Genei) and used for in vitro enzyme assays.

Western blotting was carried out to verify expression of a GbCAD1 protein having a His-tag in the N-terminus. After electrophoresis, the proteins were electro-transferred onto a PVDF membrane and detected with antimouse RGS-His antibody (Santa Cruz, American), and a secondary antibody (goat anti-mouse IgG), conjugated to alkaline phosphatase (AP). Western Blue Stabilized Color Substrate for AP (Promega, USA) was used for the color reaction.

Assay of GbCAD1 activity

CAD1 enzyme activity was measured according to the method of Ma [15] with a little modification. The assay mixture contained about 30 μg protein in 500 μl of 0.1 M NaH2PO4/Na2HPO4 buffer at pH 7.5, 0.2 mM NADPH and 0.02 mM coniferyl aldehyde substrate. Reaction were incubated at different temperature or pH for 15 min and then stopped by adding 50 μl 25 % trichloroacetic acid (TCA). For each reaction, 10 min of declination at OD340 was monitored automatically with 1 min intervals. Km and Vmax values were determined by extrapolation from Lineweaver-Burke plots. The effects of pH on enzyme activity were determined after the reaction mixture was incubated in phosphate potassium buffer with the required pH for 15 min. The stability of enzyme was analysed after incubating the reaction mixture at different temperatures.

Lignin content analysis and histo-chemical lignin staining

Lignin content was quantitatively measured by using the Klason method [16]. Briefly, air-dried stem tissues were ground into powder and exhaustively extracted in a Soxhlet apparatus with toluene-ethanol (2:1,v/v), followed by 95 % ethanol and water. Samples were then vacuum dried and 200 mg was hydrolysed in 3 ml of 72 % H2SO4 at 25 °C for 3 h with occasional stirring. The hydrolysate was diluted with the addition of 190 ml H2O and then autoclaved for 1 h. The sample was filtered through a fritted glass crucible, and then washed with hot water. The crucible was dried at 105 °C and weighed. The filtered solution was diluted to 500 ml and A205 was determined spectrophotometrically using a 1 cm long cuvette. The Klason lignin was expressed as a percentage of the cell wall residue. The acid-soluble lignin was calculated by following formula: Acid lignin (g L−1) = A205/110.

For lignin staining observation, hand-cut sections of different sample were treated by 1 % phloroglucinol ethanol and 35 % HCl, and images were captured by Cannon camera [17].

Bioinformatics analysis and molecular evolution analyses

The obtained sequences were analyzed using bioinformatics tools at websites (http://www.ncbi.nlm.nih.gov and http://www.xpasy.org). Vector NTI Suite 10 was used for sequence alignment and analysis. Phylogenetic tree analysis of GbCAD1 and known CADs from other plant species retrieved from GenBank were aligned with Mega 4.0 [18]. The phylogenetic tree was constructed by a neighbor-joining (NJ) method and measured by bootstrap analysis with 1,000 replicates. Model of GbCAD1 were created with SWISS-MODEL [19]. The crystal structure of Arabidopsis AtCAD5 served as the template for 3D modeling(PDB ID 2CF5) and secondary structure prediction [20]. Structural comparison and analysis were performed with Weblab Viewer Lite 3.5 (Accelrys, San Diego, CA, USA). SPSS 17 was used for statistical analysis and graphing.

Results

Characterization of the G. biloba CAD1

A full-length cDNA of the CAD1 gene was obtained from G.biloba leaf tissue by degenerate PCR and RACE method. The cDNA sequence was 1494 bp with a poly A tail, and contained a 1,074 bp open reading frame. A 92 bp 5′ untranslated region was upstream of the start codon, and the coding region was followed by a 328 bp 3′ untranslated region downstream from the stop codon (Fig. 1). The 5′ untranslated region conformed to the predicted translation start site for eukaryotic genes(AXXATGG) [21, 22], and two stop codons(TAA, 1,164 and 1,197 bp) found in the 3′ UTR proved that the cDNA contained the entire ORF. One potential polyadenylation signal AATAAA was found at 267 bp position downstream from the stop codon. The putative GbCAD1 was 357 amino acid with a theoretical molecular weight of 38.7 kDa and a pI value of 5.74. The subcellular location prediction of GbCAD1 did not find any secretory pathway signal peptide.

Fig. 1
figure 1

The partial cDNA sequence and deduced amino acid sequence of GbCAD1 gene. The nucleotides that encode amino acids of GbCAD1 are given in capital letters. Initiation and termination codon are indicated by underlined. Degenerate primers are indicated with “Square box”

Full size image

Multi-alignment of GbCAD1 with other plant CADs and their homologues showed higher similarities to gymnomsperm than other plants (Fig. 2). The amino acid sequence of the GbCAD1 shared 84.9 % identity with that of Picea abies, 84.0 % with Pinus teada, 83.2 % with Pinus pinaster, 82.9 % with Pinus radiata, 70.3 % with Zea mays, 69.5 % with Gossypium hirsutum, Triticum aestivum and Festuca arundinacea, 69.7 % with Liriodendron tulipifera, 69.2 % with Leucaena leucocephala, 67.8 % with Populus nigra, 67.5 % with Citrus sinensis, 67.1 % with Eucalyptus urophylla. Homology analysis with CADs from other plants suggested that GbCAD1 may contain three conserved structural domains. The first domain, GHEVAGVV, was a zinc-binding region (zinc coordinating site: His69) that was highly conserved among the analyzed sequences. The second domain, GENVGVGCIV, was also a zinc-binding region and highly conserved. Particularly, the zinc-binding cysteine residues (Cys100, Cys103, Cys106, Cys114) were almost identical across the analyzed species. The third domain, GLGGVG, was a NADPH-binding region that was highly conserved in the analysis.

Fig. 2
figure 2

Alignment of amino acid sequences of GbCAD1 and other plant CADs. Conserved important regions identified previously are marked as follows: inverted filled triangle denotes catalytic zinc ion coordinating residue, filled circle denotes structural Zn ion coordinating residue, star denotes key residues for substrate specificity. Locations of the Zn1, Zn2 and NADPH binding domains are shown in red boxes. The alignment was performed with the DNAMAN and Vector NTI software programs. The sequence of CADs alignment is homology percentage. Picea abies CAI30877, Pinus taeda CAA86073, Pinus pinaster CBL95258, Pinus radiate AAC31166, Zea mays ACG45271, Liriodendron tulipifera AEN03348, Festuca arundinacea AAK97808, Gossypium hirsutum ABZ01817, Triticum aestivum ADI59734, Leucaena leucocephala ABJ80682, Populus nigra ADN96200, Citrus sinensis ABM67695, Eucalyptus urophylla ACU77870. (Color figure online)

Full size image

The three-dimensional (3D) structure of GbCAD1 (Fig. 1S) was examined by homology modeling using SWISS-MODEL (Swiss Institute of Bioinformatics, Lausanne, Switzerland) [19]. The model predicted that GbCAD1 and AtCAD5 may have similar secondary structures, featuring primarily α-helices and β-sheets intermingled with random coils and β-turns. GbCAD1 was predicted to have six α-helices and 12 β-turns.

The amino acid sequences of CADs from various plants were phylogenetically analyzed by the neighbor-joining method [18]. Figure 3 shows the reconstructed tree; the numbers at the nodes show bootstrap support values (1,000 replicates), and the scale line below the tree represents relative evolutionary distance. The CADs of the analyzed plants were divided into three main groups: dicotyledons, monocotyledons, and gymnosperms.

Fig. 3
figure 3

The phylogenetic tree analysis of CADs from several plants. The tree was constructed by the Neighbor-Joining method, MEGA4.1. The bar represent evolutionary distance, and the numbers at each node represented the bootstrap value, with 1,000 replicates

Full size image

To examine if the GbCAD1 gene belongs to a multigene family, aliquots of 30 μg genomic DNA were digested withEcoR I, BamH I, Kpn I and Hind III, which did not cut within the coding region. These fragments were then hybridized with the coding sequence of GbCAD1 under high stringency conditions. In total, 4–6 specific hybridization bands ranging from 0.5 to 5 kb were recovered in each of the restriction enzyme-digested lanes (Fig. 4), this indicated that GbCAD1 belonged to a multigene family.

Fig. 4
figure 4

Southern blot analysis of GbCAD1 gene. Genomic DNA (25 μg/lane) was digested with EcoR I, BamH I Kpn I and Hind III respectively, followed by hybridization with GbCAD1 coding region sequence as the probe

Full size image

Properties of the recombinant GbCAD1 protein

In order to express GbCAD1 in E.coli, we cloned the coding sequence of GbCAD1 into pET-28a, an expression vector with the T7 promoter and a His-tag, yielding pET28a-GbCAD1. Upon induction by IPTG, GbCAD1 was expressed as a major soluble protein product. The molecular weight of the expressed recombinant protein was estimated as a 35 kDa band with a His-tag: this size was in agreement with that predicted by bioinformatics methods (33.28 kDa). Western blotting of purified recombinant GbCAD1 protein confirmed its specific immune reactivity to anti-His antibodies (Fig. 5).

Fig. 5
figure 5

SDS-PAGE gel and Western blot analysis of GbCAD1 expressed in E.coli BL21 (DE3). After IPTG induction, E.coli BL21 cells containing pET28a-CAD1 were grown at 30 °C for 2 h. M, molecular marker; lane 1, protein of total cells without IPTG induction; lane 2, protein of total cells with IPTG induction for 40 min; lane 3, protein of total cells with IPTG induction for 60 min; lane 4, induction for 90 min; lane 5, induction for 120 min; lane 6, purified recombinant GbCAD1 protein with Nickel-CL agarose affinity chromatography and used for enzyme activity assay; Lane M, protein MW marker; Lane western blot, western blotting of the purified recombinant GbCAD1 protein with an anti-His-tag primary antibody probe

Full size image

GbCAD1 extracted from cell homogenate was purified and cultured in the presence of NADPH or NADH and Coniferyl aldehyde at 25 °C for 1 h. Excessive NADPH was filtered off using microfilters. Absorption spectroscopy revealed that NADPH was bound as a coenzyme to GbCAD1, showing that the enzymatic activity of GbCAD1 is assisted by NADPH instead of NADH.

Because of the potential pH-dependence of plant CAD activities, the enzymatic activity of GbCAD1 was measured (Fig. 6a) at seven points between pH 5.0 and 9.0 (buffered by potassium phosphate) using coniferyl alcohol as the substrate and NADPH as the coenzyme. When the pH was raised from 5.0 to 7.0, the enzymatic activity increased substantially and reached a peak at pH 7.0. When the pH was raised from 7.0 to 8.0, the activity decreased. At pH 8.0, the enzyme retained approximately 60 % of the peak activity recorded at pH 7.0. When the pH was further raised from 8.0 to 9.0, the enzymatic activity decreased dramatically to 7.6 % of the peak activity. These findings indicated that GbCAD1 was a pH-sensitive enzyme. The optimum environment for this enzyme (pH 7.0) was similar to that in the cytoplasm [15].

Fig. 6
figure 6

Effects of pH(a) and temperature(b) on the recombinant GbCAD1 activity. Substrates were coniferyl aldehyde and NADPH, and a 200 mM phosphate potassium buffer was used. In a, the temperature was 25 °C, and in b the pH was 7.0. All reaction mixtures were incubated in water bath for 15 min and then activity was determined. Each value is the mean of three independent samples and vertical bars represent standard errors

Full size image

GbCAD1 was also sensitive to temperature (Fig. 6b). When the experimental temperature was raised from 5 to 25 °C, the enzymatic activity increased only moderately, with a particularly flat slope between 15 and 25 °C. When the temperature was further raised from 25 to 50 °C, the activity decreased sharply to approach zero. These results indicated 15–25 °C to be the optimum temperature for this enzyme.

The order of substrate, GbCAD1, and NADPH addition also affected the catalytic activity (Fig. 7). When coniferyl aldehyde was premixed with NADPH before the mixture was allowed to interact with GbCAD1, the enzymatic reaction showed an S-shaped profile: a slow reaction from 0–6 min, a fast reaction from 6–8 min, and a subsequent slower reaction after 8 min (Fig. 7a). In comparison, when the substrate was premixed with GbCAD1 before interacting with NADPH, the reaction profile was a typical double-reciprocal curve: an initial fast reaction followed by a decreasing reaction rate (Fig. 7b). Moreover, when GbCAD1 was mixed first with NADPH before interacting with the substrate, the reaction rate was almost constant (zero-order reaction) (Fig. 7c). These results substantially differ from those measured from wheat TaCA1 [15].

Fig. 7
figure 7

Analysis of the reaction mechanism catalysed by GbCAD1. Each final reaction mixture contained 100 μM NADPH, 40 mM coniferyl aldehyde, and 1 μg purified recombinant GbCAD1 protein. For each reaction, a two-component mixture was prepared first and a third component was then added. The third (and last) component added was a GbCAD1 protein, b NADPH, and c coniferyl aldehyde. The X axis shows the time lapse after addition of the third component

Full size image

GbCAD1 Expression and CAD activity in different tissue

In order to determine if GbCAD1 gene was related to lignin synthesis or diseases and insect pests defense in Ginkgo, the expression pattern of GbCAD1 was analysed by QRT-PCR. Here we found that GbCAD1 was expressed in the stems, leaves, roots, sclerotesta, infection tissues and wounding tissues of Ginkgo. GbCAD1 is constitutively expressed, but greatly varies in different tissues (Fig. 8). Of all analyzed tissues, GbCAD1 was expressed most abundantly in the root, followed by damaged or fungus-infected stem segments, consistent with the patterns of lignin concentration. In actively-growing one-year-old stem tissues, the lignin concentrations were relatively low, and the GbCAD1 expression levels were high. In contrast to the expression pattern of GbCCR1 (another lignin synthesis-related gene) [23], GbCAD1 was weakly expressed in two- to four-year-old stem tissues and essentially non-expressed in leaf veins. Staining experiments revealed abundant lignin in leaf veins and two- to four-year-old stem tissues. GbCAD1 expression was very low in the episperm and endosperm, consistent with the presence of lignin in these tissues.

Fig. 8
figure 8

The expression of GbCAD1 and enzyme activity in different organs, sections staining with phloroglucinol-HCl. a Annual stem; b Biennial stem; c Triennial stem; d Four years stem; e Petiole. f Root. g Leaf veins of the longitudinal cutting. h Endosperm. i Sclerotesta. j Injured stems; k Diseases stems; l Fungal infections leaves. The arrow indicates histo-chemical lignin staining

Full size image

The data obtained are analyzed by linear regression. The results showed that lignin content has a linear correlation with CAD activity (X1). The correlation coefficient is R 2 = 0.713, the correlation curve equation was as follows (Table 2): Y = (9.632 ± 0.929) + (15.885 ± 0.979)X1. Similarly, the change in lignin content had some linear correlation with the expression level of GbCAD1 mRNA(X2), the correlation coefficient was R 2 = 0.510, the linear equation (Table 3) was Y = (11.917 ± 1.186) + (1.139 ± 0.108)X2. So, the changes of CAD1 expression and the enzyme activity could effect the content of lignin in different tissue of Ginkgo.

Table 2 Relationship between lignin content and CAD activity in different tissue of Ginkgo
Full size table
Table 3 Relationship between GbCAD1 expression level and lignin content in different tissue of Ginkgo
Full size table

UV-B, wounding and hormone regulate the transcription of GbCAD1

To examine the effects of hormones on the expression of GbCAD1, gingko leaves were treated by spraying with ABA, 5-ALA, SA, or ETH. Subsequently, the transcriptional level of this gene was studied by RT-PCR.

ABA spraying induced a sustained elevation of GbCAD1 transcription, but the response was slower compared with other hormone treatments. The elevation was only moderate 4 h after treatment and became noticeable 8 h after treatment. The transcriptional level reached the peak value (360 % higher than the control level) 12 h after treatment and decreased thereafter. At 96 h after treatment, the transcriptional level remained 170 % higher than the control level(Fig. 9a).

Fig. 9
figure 9

Relative quantities of GbCAD1 mRNA at various time points post-treatment with ABA (a), ALA (b), ETH (c), SA (d), UV-B (e), wounding (f). Each sample was individually assayed in triplicate. Values shown represent the mean reading from three treated plants and the error bars indicated the standard errors of the mean

Full size image

Gingko leaves were treated with this concentration of ALA. The GbCAD1 transcription level (Fig. 9b) increased slightly 2 h after treatment, decreased moderately between 8 and 12 h (reaching 70–80 % of the control level), and increased slowly thereafter. Compared with the hormone treatments, ALA spraying failed to significantly affect GbCAD1 expression.

Our previous studies have shown that ethephon treatment could effectively increase flavonoid accumulation in Ginkgo leaves by elevating the expressions of key genes in the flavonoid pathway [11, 12, 24–28]. In the present study, Ginkgo leaves were treated with the same concentration of ethephon. The GbCAD1 transcription level (Fig. 9C) was unchanged 4 h after treatment, started to rapidly increase after 4 h (140 % higher than the control level), and reached a peak value 24 h after treatment (600 % higher than the control level). Subsequently, it slightly decreased and remained at approximately 420 % higher than the control level.

Following, we treated Ginkgo leaves by spraying with a low concentration of SA. The GbCAD1 transcription level (Fig. 9d) increased rapidly to 210 % higher than the control level 4 h after treatment and remained stable until 12 h after treatment. It further increased to 500 % higher than the control level 24 h after treatment and subsequently decreased. At 96 h after treatment, the GbCAD1 transcriptional level was 180 % higher than the control level.

To understand GbCAD1 expression in Gingko leaves under UV-B irradiation, gingko seedlings were treated with 260 nm UV-B at 600 μJ/m2 for different periods of time, and the transcriptional levels were analyzed by RT-PCR (Fig. 9E). During the first 24 h of UV-irradiation, GbCAD1 transcription increased, and then decreased after 24 h. In addition, wound treatment (WOU) also stimulated GbCAD1 expression (Fig. 9f). The transcription of GbCAD1 increased rapidly and reached a peak value 8 h after WOU (220 % higher than the control level). Subsequently, it gradually decreased and reached levels similar to the control 48 h after WOU.

Discussion

Cinnamyl alcohol dehydrogenase is a major rate-limiting enzyme in lignin biosynthesis. It converts cinnam aldehydes into cinnamyl alcohol to impart diversity to formation and structure of lignins. More importantly, the CAD gene family is related to stress responses during plant growth and is also closely associated with fruit and seed maturation and aging.

Bioinformatic analysis

Our analyses revealed that GbCAD1 has high homology to other plant CAD genes, with conserved amino acids throughout the gene sequence. The results suggested that GbCAD1 is a member of the plant CAD protein family. In the present study, GbCAD1 was found to be 84 % homologous to the Pinus taeda CAD gene, suggesting they have similar CAD functions. All functional CADs have a common structurally conserved Zn-binding domain [Zn1 binding labels, CHE(X)2G(X)5G(X)2V] required for enzymatic catalysis. Additionally, these CADs have another Zn-binding region [GD(X)9,10C(X)2C(X)2C(X)7C] and a glycine-rich NADPH-binding region [GXG(X)2G]. These three domains are hallmarks of the alcohol dehydrogenase superfamily [4]. In GbCAD1, the three domains are highly conserved, suggesting that GbCAD1 belongs to the alcohol hydrogenase superfamily. Both SbCAD6 and OsCAD6 have an evolutionarily conserved SKL sequence at the C-terminus, which may serve as a signal peptide sequence to locate these enzymes in plant cell peroxisomes [29]. Our amino acid sequence comparison found no homology between the SKL sequence of GbCAD1 and those of other species, suggesting that GbCAD1 may not be located in peroxisomes.

Phylogenetic analysis found that the examined CADs formed three clusters: gymnosperms, dicotyledonous, and monocotyledonous. GbCAD1 was found in the gymnosperms cluster and diverged earlier than CADs from other plants, consistent with the actual evolutionary history of Ginkgo. The CAD genes in higher plants are encoded by a small multigene family. The Sorghum genome contains more than 14 CAD-encoding genes [29], and the G. hirsutum genome contains more than eight [30]. Similarly, the Ginkgo genome contains multiple homologous CAD genes that belong to a multigene family. Currently, the CAD genes of gymnosperms are believed to exist as single copies. For example, P. taeda has only one CAD isozyme, indicating the presence of a single CAD locus [2]. The southern blot results suggested that, the Ginkgo genome may contain multiple CAD genes. These genes may have different functions or may be expressed in different tissues or at different developmental stages. However, the possibility of pseudogenes also exists.

Expression analysis in different tissue

In higher plants, multiple homologous CAD genes may participate in lignin biosynthesis in different tissues or during different developmental stages in one type of tissue. In graminaceous plants, CAD genes are expressed in vascular bundles and are related to the mechanical properties of the stem tissues [31]. Arabidopsis thaliana has nine functional CAD genes. Of these, only AtCAD4 and AtCAD5 play key roles in lignin biosynthesis. These two genes are expressed in vascular bundles as well as in non-lignified tissues [32, 33]. Tissue-specific expression analysis revealed that AtCAD4 participated in lignin biosynthesis but was only weakly expressed in stems; In contrast, it was strongly expressed in flowers and roots. The AtCAD5 gene was expressed in lignified roots and strongly expressed in pathogen-infected tissues [7], which was probably related to pathogen defenses involving lignin [32]. Studies have shown that, in addition to lignin-synthesizing CAD genes, CAD-like genes also participate in plant defense processes [34]. For example, in Lolium perenne L., LpCAD2 is strongly expressed in the stem and is further stimulated by mechanical damage [5]. In Ginkgo, GbCAD1 is strongly expressed in roots and in stems that are either infected or rapidly growing. Therefore, this gene is involved in both lignin synthesis and pathogen defense. These findings suggest that plant CAD genes may also participate in other unknown functions or have evolved individual functions.

In addition to lignin biosynthesis, stress response, and pathogen defense, plant CAD genes are also implicated in vegetable and fruit tissue development and are closely related to tissue ageing. For example, loquats undergo lignification during storage; during this process CAD transcripts accumulate, stimulated by ethylene [11]. In the present study, GbCAD1 transcription started to increase 4 h after ethephon treatment and remained at high levels for a long period, similar to the elevation of CAD expression in loquats during maturation. Ethephon treatment during Ginkgo leaf maturation may affect flavonoid concentrations and key enzyme activities in the leaves. Ethephon exerts such functions by releasing ethylene to enhance PAL activity [26, 28, 35]. The biosynthesis of lignin and flavonoids in higher plants originates in the phenylalanine metabolic pathway. Therefore, the induction of GbCAD1 expression by ethylene may be related to the increase in PAL activity and subsequent product accumulation.

Induced expression analysis

The CAD gene participates in lignification defense and is induced by a variety of signals, such as MEJA, mechanical wounding, fungal elicitors, stress conditions, and ethylene [32]. Few studies have investigated the relationship between UV exposure and lignin biosynthesis. However, ultraviolet irradiation has been found to induce the expression of genes (e.g., PAL, CHI) related to flavonoid metabolism [12, 27]. Bate et al. [36] have shown that PAL inhibition affected lignin biosynthesis. In our study, UV-B treatment induced an initial steady up-regulation of GbCAD1 transcription followed by a gradual decrease. Based on these observations, the UV-induced GbCAD1 expression may have resulted from cascade reactions following PAL response to UV irradiation. A previous study reported that the sweet potato (Ipomoea batata) IbCAD1 expression was induced by biological and non-biological stresses and responded sensitively to mechanical wounding and chemical signals (SA, JA, ABA) [4]. Another study on the upstream structure of Picea glauca PgCAD promoter found a MYB transcription factor element (TTAGGTT). This element has been frequently observed in the promoters of genes related to plant stress responses. This finding suggested that PgCAD may be responsive to mechanical wounding [37]. Similarly, a MYB-binding cis-element was found in the region upstream of the eucalyptus (Eucalyptus globulus) EgCAD2 promoter. The cis-element has also been frequently observed in genes specifically expressed in the xylem and is inducible by mechanical wounding [38]. In our study, GbCAD1 expression was initially stimulated by mechanical wounding and subsequently returned to the control level, suggesting that this gene participated in the stress response to mechanical wounding and wound repair. These results may be related to the upstream regulatory elements or may be attributed to reaction cascades following the mechanical wounding-induced increase in PAL transcripts.

Salicylic acid is a phenolic hormone that regulates the growth of plants and participates in plant defense against pathogens [39]. Moreover, similar to flavonoid and lignin biosynthesis, SA metabolism is also a part of the phenylalanine metabolic pathway [40]. We previously found that SA treatment could significantly increase the expression of Ginkgo flavonoid-related genes [11, 27]. The expression of flavonoid metabolism-related genes is upregulated in plants in response to pathogen invasion [27, 41, 42]. Salicylic acid is biosynthesized via a branch of the phenylalanine metabolic pathway and begins with the transformation of cinnamic acid [40]. Earlier studies have found the SA-response element in the sweet potato IbCAD1 promoter to be related to SA-induced CAD1 expression [4]. Similarly, defense-related cis-elements have also been identified in the white spruce CAD promoter [37]. Therefore, the SA-induced GbCAD1 expression observed in the current study may be related to the upstream cis-element or indirectly linked to SA-induced defense responses.

Abscisic acid is a hormone in higher plants that is formed through the degradation of carotenoids. Generally, When plants are exposed to osmotic stresses or other stress conditions, ABA can regulate the expression of relevant genes to increase plant adaptability [43]. Earlier studies have found induced expression of IbCAD1 in sweet potato after exposure to biological or non-biological stresses. The IbCAD1 promoter has been confirmed to have response elements to signals such as ABA [4]. The promoter of P. trichocarpa Poptr CAD4 also contains response elements to ABA, mechanical wounding, and light [34]. Studies have suggested that ABA-mediated plant responses to drought stress may be related to the regulation of relevant genes by the MYB transcription factor [44]. Therefore, the ABA-induced GbCAD1 expression observed in our study may be linked to the upstream MYB response elements. This hypothesis, however, must be verified by future cloning and function analyses of the promoter.

Many studies have confirmed that low-concentration ALA treatments enhanced photosynthesis and, consequently, lignin production [45–47]. It also increases the accumulation of products from the phenylalanine metabolic pathway, such as flavonoids, total phenols, and anthocyanins [48]. Previous studies on lignin synthesis-related genes (PoptrCAD4 and PoptrCAD10) in populus have identified cis-elements in the promoter regions related to MYB, Zn-finger binding regions, and photo- and auxin-response factors [34]. In the present study, the ALA treatment produced no CAD induction, suggesting the lack of photo- or auxin-response sequences in the upstream region of GbCAD1.

Ethephon accelerates fruit maturation and coloration. It also regulates the expression of flavonoid metabolism-related genes [11, 12, 27, 49] and is related to flavonoid accumulation in Ginkgo leaves [24, 28]. A previous study found that loquat EjCAD1 expression increased during fruit maturation and ethylene treatment [11]. In the Brassica chinensis, BcCAD1 and BcCAD2 participated in plant ageing, and were also inducible by ethylene [50]. In the present study, the induction of GbCAD1 expression by ethylene may be responsible for the ethylene-induced Ginkgo leaf lignification and secondary metabolite accumulation during leaf maturation. Both flavonoids and lignin are metabolized along parallel branches of the phenylalanine metabolic pathway. These parallel branches interact and cooperate to ensure normal plant development. These results show that plant CAD genes respond to various biological and non-biological stresses, and many of them are inducible by mechanical damage and signals for systemic acquired resistance, suggesting their roles in lignin formation and stress response. However, different CAD genes may respond to different stress signals (i.e., stress specificity). A previous study reported that the sixteen CAD genes could be divided into four groups according to their functions [34, 51]. Group 1 participates in lignin biosynthesis and is strongly expressed in the xylem [51, 52]. Group 2 is strongly expressed in the leaves. Group 3 is expressed in the xylem and preferentially expressed in the leaves [53]. Group 4 is related to stress response, mechanical wounding, fungal elicitation, pathogen defense, and ethylene induction [51]. Based on that study, GbCAD1 is similar to Group 4 with respect to tissue distribution and patterns of induced expression.

In light of these results, we suggest that GbCAD1 may also be involved in lignifications induced by both abiotic and abibotic stresses and in tissue-specific developmental lignification. Further analysis of the GbCAD1 gene and its promoter sequence will provide an insight into the signaling pathway involved in the regulation of biotic and abiotic stresses in lignin biosynthesis as well as into the role of CAD protein during cell division.