Introduction

Flower initiation and development are realized through complex networks of exogenous and endogenous signals including light stimuli, hormones, ligand–receptor interactions, signal transduction pathways, and transcription factors cascades. A relevant role in flower formation is played by MADS-box genes, a wide gene family encoding transcription factors able to form homo- and heteromultimeric complexes that can bind DNA. Plant MADS-box genes have evolved mainly through successive gene duplications followed by sub- and/or neo-functionalization, giving rise to five different functional classes (A through E) whose interactions are described by the ABCDE model (Jack 2001; Theissen 2001). In brief, the expression of class A genes drives the development of sepals alone (whorl 1) and, together with the expression of class B genes, of petals (whorl 2); the expression of class C genes determines the development of carpels (whorl 4) alone and, together with the expression of class B genes, of stamens (whorl 3); class D genes are involved in ovule formation; class E genes are necessary for the correct development of all floral organs (Fig. 1a). Even though the ABCDE model, initially developed on Arabidopsis thaliana, works well for most eudicots, slight modifications are required to fit the floral organ formation in some monocots, such as lilies and orchids, in which sepals and petals show similar morphology and are termed tepals (Fig. 1b). The expansion of class B genes expression to whorl 1 is thought to have determined the presence of sepaloid petals (tepals) in these plant groups (Kanno et al. 2007).

Fig. 1
figure 1

Diagram of the ABCDE model (a) and of the expanded ABCDE model of floral development (b)

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Orchidaceae is one of the largest families among flowering plants and includes five subfamilies (Apostasioideae, Vanilloideae, Cypripedioideae, Epidendroideae, Orchidoideae) (Fig. 2) and a large number of tribes and subtribes (Gorniak et al. 2010). This family includes species with greatly diversified and specialized floral morphology, even though a general common structure is detectable. The orchid flower is zygomorphic and includes three outer tepals, two lateral inner tepals, and a median inner tepal (lip or labellum). Male and female orchids reproductive organs are fused into a single structure (column or gynostemium); at the top of the column is the male anther containing packets of pollen (pollinia) and at the base of the column is the ovary.

Fig. 2
figure 2

Time-calibrated simplified phylogeny of the orchid subfamilies, modified from Gustafsson et al (2010). My millions of years

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The recent theory known as “the orchid code” assumes that class B AP3/DEF-like genes had a relevant role in the evolution of the orchid perianth. Two main duplication events generated the current class B AP3/DEF-like genes in orchids. After the duplications, changes in regulatory and coding regions led to sub- and neo-functionalization of AP3/DEF-like genes that are divided into four different clades in orchids, each of which has its own specific expression pattern. High levels of expression of clade-1 and clade-2 AP3/DEF-like genes and low levels of clade-3 and clade-4 specify inner lateral tepals, whereas lip development requires low expression levels of clade-1 and clade-2 AP3/DEF-like genes and high clade-3 and clade-4 expression levels. In contrast to the AP3/DEF-like genes, orchids class B PI/GLO-like genes are documented as single-copy loci in almost all the orchid sub-families (Mondragon-Palomino et al. 2009; Tsai et al. 2005; Xu et al. 2006), while two PI/GLO-like paralogs have been described in Orchidinae (Kim et al. 2007; Cantone et al. 2011). Evolutionary analysis revealed that the two PI/GLO-like paralogs evolved under different selective constraints with a peculiar pattern of purifying selection acting on synonymous sites, suggesting a possible sub-functionalization of these PI/GLO-like genes within Orchidinae (Aceto et al. 2007; Cantone et al. 2009, Cantone et al. 2011). To date, the available data that pertain to the expression pattern of the two PI/GLO-like paralogs are incomplete and obtained by non-quantitative techniques.

In this study, we compared the tissue expression of the PI/GLO-like paralogs in floral tissues of Orchis italica (Orchidaceae, Orchidinae) at different developmental stages using real-time RT-PCR to integrate the evolutionary analysis results with the OrcPI and OrcPI2 gene expression analyses.

Materials and methods

Plant material

Outer and inner tepal, lip, column, and not pollinated ovary tissue of O. italica were examined at two different developmental stages. The earliest stage corresponds to a young inflorescence before anthesis (∼9 mm diameter size) (Fig. 3a), and the latest stage corresponds to a mature inflorescence after anthesis (Fig. 3c).

Fig. 3
figure 3

Floral tissues of O. italica and OrcPI and OrcPI2 gene expression patterns. a, c Immature and mature inflorescence, respectively. b, d Relative expression ratio of the OrcPI and OrcPI2 genes in floral tissues from immature and mature inflorescence, respectively. In inflorescence, Fl floret, Te_out outer tepal, Te_inn lateral inner tepal, Li median inner tepal (lip), Co column. The bars on the graphs represent standard deviation

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Fifteen and five florets from a single immature and mature inflorescence, respectively, were dissected and quickly stored in RNA Later (Ambion) at −20°C until processing. In addition, ten ovaries were manually pollinated, collected 3, 7, and 10 days after pollination (dap) and quickly stored as described before. Leaf tissue was also collected and stored under the same conditions.

RNA extraction and real-time PCR experiments

Total RNA was extracted from the O. italica tissue samples using TRIzol (Ambion), treated with DNase I (Ambion) following the manufacturer’s instructions and quantified by using the BioPhotometer (Eppendorf). Integrity of total RNA was checked by agarose gel electrophoresis.

Based on the cDNA sequence of OrcPI, OrcPI2, and of the actin gene (OitaAct) of O. italica (accession number AB094985, AB537504, and AB630020, respectively), primer pairs that selectively amplify a fragment of each cDNA were designed using the software Primer Express v.3.0 (Applied Biosystems). The primer sequences and the amplicon lengths are listed in Table 1.

Table 1 Primer nucleotide sequences used in real-time PCR experiments
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Total RNA (350 ng) isolated from the different tissues was reverse transcribed using the Advantage RT PCR kit (Clontech) with oligo dT primers. After the reaction, the RT mixture was raised to 35 μl with nuclease-free distilled water. The real-time PCRs were performed using a 7500 real-time PCR System (Applied Biosystems) in the presence of ×1 Power Sybr® Green PCR Master mix (Applied Biosystems) and 0.1 μM of each primer. The thermal protocol was as follows: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. A melting curve of PCR products (95–60°C) was performed to ensure the absence of artifacts. The reactions were conducted using 2 μl of the RT mixture for OrcPI and OrcPI2 gene PCR amplification and 1 μl for OitaAct gene amplification. For all genes examined, two independent biological samples of each tissue were evaluated in technical triplicates, and negative controls were performed without cDNA in the reaction mixture. Two amplicons for each gene were cloned and sequenced using the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems). Sequence reactions were run on a 310 Genetic Analyzer (Applied Biosystems).

Data analysis

PCR efficiency and optimal threshold cycle (C T) for the primer pairs that amplify the target genes, OrcPI and OrcPI2, and the OitaAct endogenous control gene were calculated for each well using the online real-time PCR Miner tool (Zhao and Fernald 2005). In brief, this software defines the exponential phase of each PCR starting from raw fluorescence data and estimates the PCR efficiencies (E) for each well through a nonlinear regression algorithm without the need of a standard curve. The mean PCR efficiency for each gene was used in the subsequent analysis. The relative expression ratios (rER) of the OrcPI and OrcPI2 genes in the different tissues were calculated applying the Gene Expression’s C T Difference (GED) approach (Schefe et al. 2006), using OitaAct as the endogenous control gene and leaf cDNA as the reference sample. For each reaction, the amount of the target cDNA (OrcPI and OrcPI2) relative to the amount of the OitaAct cDNA was measured applying the following formula:

$$ {\text{rER = }}{\left( {1 + {E_{\text{target}}}} \right)^{{ - \Delta {C_{\text{T}}}\,{\text{target}}}}}/{\left( {1 + {E_{\text{actin}}}} \right)^{{ - \Delta {C_{\text{T}}}\,{\text{actin}}}}} $$

where E is the mean PCR efficiency of the gene, ∆C T target is the difference between the C T value of the target gene (OrcPI or OrcPI2) in the tissue of interest and the C T value of the target gene in the reference tissue (leaf), ∆C T actin is the difference between the C T value of the OitaAct gene (endogenous control) in the tissue of interest and the C T value of the OitaAct gene in leaf tissue. Subsequently, mean rER and standard deviation values were calculated for each triplicate and for the two biological samples.

Differences in the relative expression levels of the OrcPI and OrcPI2 genes between and/or among different samples were assessed by the two-tailed t test and ANOVA followed by the Tukey HSD post hoc test, respectively.

Results and discussion

The presence of two transcribed paralogs of the PI/GLO-like locus in orchids is restricted to Orchidinae, whereas the other orchids appear to possess a single PI/GLO-like gene. The duplicated copies present within Orchidinae evolved under selective constraints that were different from those acting on the single copy PI/GLO-like genes of the other orchid lineages. Both paralogs of Orchidinae show relaxation of purifying selection when compared to the single-copy lineages, which is a common pattern after gene duplication events. In addition, a high percentage of sites between the two PI/GLO-like paralogs show differences in selective constraints mainly due to a reduced synonymous substitution rate. Evolutionary analyses suggested possible functional divergence of the two PI/GLO-like genes in Orchidinae. To clarify the expression pattern of the two paralogs in different floral tissues and in different developmental stages and to compare it to the expression profile of the single-copy gene observed in the other orchid lineages, we performed real-time PCR analysis in O. italica.

The melting-curve analysis performed after the real-time PCR amplifications of the OrcPI, OrcPI2, and OitaAct cDNAs showed the effectiveness of the primer pairs used. Sequence analysis of the cloned amplicons confirmed the specificity of the amplified products, corresponding to the selected fragments of the OrcPI, OrcPI2, and OitaAct genes. The mean PCR efficiencies calculated for each gene by real-time PCR Miner tool showed comparable values, ranging from 0.92 ± 0.02 for the OrcPI2 gene to 1.04 ± 0.02 for the OitaAct gene.

Figure 3 shows the relative expression ratio of the OrcPI and OrcPI2 genes in different floral tissues of immature (b) and mature (d) inflorescence. Relative expression analysis revealed the presence of different levels of transcripts of OrcPI and OrcPI2 in the examined floral tissues of O. italica, where the OrcPI2 gene showed a higher expression rate when compared to its paralog, OrcPI. The paired samples t test showed this difference was significant for all tissues, with p ≤ 0.030.

In the immature inflorescence (Fig. 3b), the significantly lowest expression of both genes is detected in the column (p values of the Tukey HSD post hoc test ≤0.003) with an expression ratio OrcPI2/OrcPI of 1.6. The expression level of both genes detected in the outer tepal is not significantly different from that observed in the lip with an expression ratio OrcPI2/OrcPI of 7.05 in the outer tepal and 3.69 in the lip. The highest expression level of the OrcPI and OrcPI2 genes was detected in the lateral inner tepals. In lateral inner tepal, the expression ratio of OrcPI2/OrcPI is 5.65. Even though the difference between this tissue and the outer tepal and lip is not statistically significant for the OrcPI gene, the comparison is statistically significant for the OrcPI2 between the lateral inner tepals and the lip tissue.

In the mature inflorescence (Fig. 3d), the expression pattern of the PI/GLO-like paralogs is different from the profile observed in the immature inflorescence tissues. Both genes continue to show the lowest expression in the column (t test p values ≤0.016) with an expression ratio OrcPI2/OrcPI of 2.6. The amount of OrcPI and OrcPI2 gene transcripts in the outer and lateral inner tepal is higher than what was detected in the column. However, the difference between the OrcPI gene transcript levels in the column and the outer tepal and the difference between the outer and lateral inner tepal are not statistically significant. However, the differences in the OrcPI2 gene expression levels is statistically significant among all the tissues examined and showed higher transcript levels in lateral inner tepals than outer tepals (p = 0.039). The expression ratio of OrcPI2/OrcPI is ∼7 in both tissues. The significantly highest expression level of both PI/GLO-like genes was in the lip tissue with an expression ratio OrcPI2/OrcPI of 8.82.

The pairwise comparison of the OrcPI and OrcPI2 relative tissue expression level between immature and mature inflorescence gave significant results in the lip and column tissues (p ≤ 0.002), which showed an increase in gene expression for both genes in the lip from immature to mature inflorescence and an opposite trend in the column. Specifically, the expression ratio mature/immature inflorescence is 0.16 in the column and 11.77 in the lip for OrcPI and 0.27 in the column and 28.1 in the lip for OrcPI2. The expression level of both genes remains approximately the same in the outer and lateral inner tepals with an expression ratio mature/immature inflorescence of ∼1.

The relative expression profile detected for the OrcPI and OrcPI2 genes appears to follow a common, co-expression pattern in the different floral tissues and in the different developmental stages examined, which is in general agreement with what was observed in some PI/GLO-like single-copy orchid lineages (Mondragon-Palomino and Theissen 2011).

As it was hypothesized the involvement of the PI/GLO-like gene PeMADS6 as negative regulator of the ovary development in the orchid Phalaenopsis equestris (Epidendroideae) (Tsai et al. 2005), relative expression analysis of both OrcPI and OrcPI2 was conducted on ovary tissue of O. italica before and after pollination. In orchids, ovary development is triggered by pollination and in O. italica the complete maturation of the ovary is reached approximately 10 days after pollination (Cozzolino, personal communication). Figure 4 shows the relative expression profile of the OrcPI and OrcPI2 genes in ovary tissue collected from immature and mature inflorescences before pollination and 3, 7, and 10 days after manual pollination. Both genes are weakly expressed in the ovary of the immature inflorescence, whereas significantly high expression is detected in the ovary of the mature inflorescence before pollination. After pollination the expression of both genes rapidly decrease, resembling the expression profile of PeMADS6 in the ovary of P. equestris, which supports the previously hypothesized role of the PI/GLO-like genes as negative regulators of ovary development in orchids. As observed in the other floral tissues examined, expression of the OrcPI2 gene in the ovary is higher than that of the OrcPI gene (p values ranging from 0.003 to 0.019) with an expression ratio OrcPI2/OrcPI of 3.58 in the mature inflorescence ovary.

Fig. 4
figure 4

Relative expression ratio of the OrcPI and OrcPI2 genes in O. italica ovary tissue. Ov ovary; np not pollinated; dap days after pollination. The bars on the graphs represent standard deviation

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The expression analysis of the two PI/GLO-like paralogs in O. italica revealed that the duplicated genes, OrcPI and OrcPI2, are co-expressed. However, in all tissues, the amount of OrcPI2 gene transcripts, ortholog of the single-copy PI/GLO-like gene from other orchid lineages, is higher than the OrcPI gene transcripts. This transcriptional behavior confirms our previous hypothesis of a recent lineage-specific duplication event within Orchidinae, with a functional role of the duplicated gene OrcPI fully or partially redundant relative to OrcPI2. Based on the evolutionary and expression pattern of the two paralogs, the fate of the duplicated locus, OrcPI, might evolve through sub-functionalization which may result in, for example, a partition between the OrcPI and OrcPI2 protein’s capability to bind different products of the four different class-B AP3/DEF-like proteins during the formation of the specific floral quartets, which are necessary for correct floral tissue development.