*2.4. E*ff*ect of Exogenous GA<sup>3</sup> Treatment on Genes' Expression*

#### 2.4.1. Gene Expression Related to GAs Biosynthesis

After treatment with the exogenous GA<sup>3</sup> concentrations, genes involved in GAs biosynthesis showed diverse expression patterns. We compared the differential expression of the above genes between asymbiotic and symbiotic conditions at a given treatment concentration GA<sup>3</sup> treatment (Figures 4–8). These results showed those genes related to the biosynthesis of GAs (*GA20ox, GA3ox*) were upregulated in seed germination (stage 2), protocorm formation (stage 3), and seedling (stage 4) in the SG, while *DoGA2ox* underwent significantly upregulated expression at the protocorm stage (stage 3) (Figure 4A). After applying exogenous GA3, the expression of *GA3ox* gene in SG was 10.19, 26.42, 74.74, 109.36, and 104.15 times that in AG at 0, 0.05 µM, 0.1 µM, 0.5 µM and 1 µM exogenous GA<sup>3</sup> treatment concentrations, respectively (Figure 4B). In addition, the expression level of GA2ox, the key gene encoding gibberellin oxidase, which catalyzes the degradation of active GAs, was upregulated sharply at a higher GA<sup>3</sup> treatment concentration (0.5 µM) in SG compared to AG (246.17 fold-change). This implied a crosstalk interaction between the biosynthesis and metabolism of GAs and mycorrhizal establishment.


**Table 2.** Putative mycorrhizal induced genes, GA biosynthesis and other hormone gene homologs induced during symbiotic germination of *D. o*ffi*cinale* seeds. Abbreviation: FPKM: fragments per kilobase per million and the value represents the differential expression level; FDR: false discovery rate; A1, A2, A3 represent asymbiotic germination stage 2, stage 3, and stage 4 respectively; S1, S2, S3 represent symbiotic germination stage 2, stage 3, and stage 4, respectively. \* gene expression were validated by qPCR.











*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 12 of 23

**Figure 4.** Expression levels of genes related to GA biosynthesis during symbiotic germination of *Dendrobium officinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination; (**B**). Genes' expression affected by GA concentrations at 4 weeks after sowing seeds. Note the fold-change values are relative to asymbiotic germination. PCR amplifications were performed for three biological replicates and two distinct technical replicates for each sample. Expression levels were calculated by the 2−ΔΔC<sup>т</sup> method normalized against the expression of *EF1-α,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination. **Figure 4.** Expression levels of genes related to GA biosynthesis during symbiotic germination of *Dendrobium o*ffi*cinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination; (**B**). Genes' expression affected by GA concentrations at 4 weeks after sowing seeds. Note the fold-change values are relative to asymbiotic germination. PCR amplifications were performed for three biological replicates and two distinct technical replicates for each sample. Expression levels were calculated by the 2−∆∆C<sup>T</sup> method normalized against the expression of *EF1-*α*,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination.

Treated with exogenous GA3 in the germination experiment, the genes involved in ABA biosynthesis and signaling transduction displayed diverse expression profiles (Figure 5A). For example, the gene DoNCED responsible for ABA biosynthesis was downregulated with a greater GA3 concentration, while the genes involved in the signal transduction of ABA (*DoSGT, DoIRK,* and *DoGBF*) were all upregulated, implying ABA metabolism has a very active response to a changed GA3 concentration (Figure 5B). The expression of genes participating in auxin biosynthesis also displayed a similar profile. Notably, DoIPM, a key gene that belongs to the YUCCA family was upregulated in SG compared to AG under the 0.5-μM GA3 treatment concentration (Figure 6).

2.4.2. Gene Related to ABA Biosynthesis and Signaling Transduction

germination.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 13 of 23

**Figure 5.** Expression levels of genes related to ABA biosynthesis during symbiotic germination of *Dendrobium officinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA3 treatment); (**B**). Genes expression' affected by GA3 concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2−ΔΔC<sup>т</sup> method normalized against the expression of *EF1-α,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic **Figure 5.** Expression levels of genes related to ABA biosynthesis during symbiotic germination of *Dendrobium o*ffi*cinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA<sup>3</sup> treatment); (**B**). Genes expression' affected by GA<sup>3</sup> concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2 <sup>−</sup>∆∆C<sup>T</sup> method normalized against the expression of *EF1-*α*,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 14 of 23

**Figure 6.** Expression levels of genes related to IAA biosynthesis during symbiotic germination of *Dendrobium officinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA3 treatment); (**B**). Genes' expression was affected by GA3 concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2−ΔΔC<sup>т</sup> method normalized against the expression of *EF1-α,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination. 2.4.3. Expression Analysis of Putative Genes Involved in Mycorrhizal Symbiosis and Common **Figure 6.** Expression levels of genes related to IAA biosynthesis during symbiotic germination of *Dendrobium o*ffi*cinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA<sup>3</sup> treatment); (**B**). Genes' expression was affected by GA<sup>3</sup> concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2 <sup>−</sup>∆∆C<sup>T</sup> method normalized against the expression of *EF1-*α*,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 15 of 23

**Figure 7.** Expression levels of genes related to common symbiosis pathway during symbiotic germination of *Dendrobium officinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA3 treatment); (**B**). Genes' expression was affected by GA3 concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2−ΔΔC<sup>т</sup> method normalized against the expression of *EF1-α,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination. **Figure 7.** Expression levels of genes related to common symbiosis pathway during symbiotic germination of *Dendrobium o*ffi*cinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA<sup>3</sup> treatment); (**B**). Genes' expression was affected by GA<sup>3</sup> concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2−∆∆C<sup>T</sup> method normalized against the expression of *EF1-*α*,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 16 of 23

**Figure 8.** Expression levels of putative mycorrhiza-induced genes involved in orchid symbiotic germination of *Dendrobium officinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA3 treatment); (**B**). Genes' expression was affected by GA3 concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2−ΔΔC<sup>т</sup> method normalized against the expression of *EF1-α,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic **Figure 8.** Expression levels of putative mycorrhiza-induced genes involved in orchid symbiotic germination of *Dendrobium o*ffi*cinale* for quantitative qPCR analysis. (**A**). Genes' expression at different development stages between asymbiotic and symbiotic germination (no GA<sup>3</sup> treatment); (**B**). Genes' expression was affected by GA<sup>3</sup> concentrations at 4 weeks after sowing seeds. Expression levels were calculated by the 2−∆∆C<sup>T</sup> method normalized against the expression of *EF1-*α*,* using the expression level of ungerminated seed (stage 0) (**A**) or asymbiotic germination (**B**) as control and the fold change > 2.0 was marked significant differential expression (\*). AG, asymbiotic germination; SG, symbiotic germination.

#### germination. 2.4.2. Gene Related to ABA Biosynthesis and Signaling Transduction

**3. Discussion**  Symbiotic germination of orchid seeds involves the dual process of seed self-development and mutualistic interaction with their mycorrhizal fungi. Thus, the process is quite complex physiologically and ecologically. Orchid seeds are too tiny to perform genetic manipulations and this has inevitably limited the studies on their mechanisms of symbiotic germination, yet recent breakthroughs on arbuscular mycorrhiza have laid the foundation for investigating the SG of orchid seeds [16]. Recent studies show that the mycoheterotrophic symbiosis between orchids and Treated with exogenous GA<sup>3</sup> in the germination experiment, the genes involved in ABA biosynthesis and signaling transduction displayed diverse expression profiles (Figure 5A). For example, the gene DoNCED responsible for ABA biosynthesis was downregulated with a greater GA<sup>3</sup> concentration, while the genes involved in the signal transduction of ABA (*DoSGT, DoIRK,* and *DoGBF*) were all upregulated, implying ABA metabolism has a very active response to a changed GA<sup>3</sup> concentration (Figure 5B). The expression of genes participating in auxin biosynthesis also displayed a similar profile. Notably, DoIPM, a key gene that belongs to the YUCCA family was upregulated in SG compared to AG under the 0.5-µM GA<sup>3</sup> treatment concentration (Figure 6).

mycorrhizal fungi possesses major components shared with mutualistic plant–mycorrhizal symbioses [17]. Many studies have revealed that plant hormones, especially gibberellins, are important factors affecting seed germination [10], and they are also critical for the establishment of mycorrhizal symbiosis [18,19]. In our study, the contents of five plant hormones (GA3, ABA, IAA, ZT, and JA) was determined at four different developmental stages of seed germination of the orchid *D. officinale*. Our results revealed that the mature and ungerminated seed have the highest ABA content

## 2.4.3. Expression Analysis of Putative Genes Involved in Mycorrhizal Symbiosis and Common Symbiosis Pathway

The putative symbiosis-specific expression genes, including *DoHAL, DoPRCP, DoGGLU, DoGLU, DoSWEET, DoCDR1, DoCDPK2,* and *DoNSP2* featured similar expression levels in SG after treatment with different concentrations of GA3. In the SG group with no exogenous GA<sup>3</sup> treatment, the expression level of these genes increased substantially compared to AG, indicating the expression of these genes was induced by mycorrhizal fungi invasion. However, their expression underwent a similar change after imposing the exogenous GA<sup>3</sup> treatment; namely, genes were at first highly expressed in 0.1 µM of exogenous GA<sup>3</sup> but then suppressed as the GA<sup>3</sup> treatment concentration increased (Figures 7 and 8). The expression of *DoCDPK26* was not significantly changed in AG across the GA<sup>3</sup> treatment concentrations but it was significantly and highly expressed in the 0.5-µM GA<sup>3</sup> treatment in the SG group. Similarly, the gene *DoCML19* also was highly expressed in SG yet not significantly changed by exogenous GA3; this implied the expression of these two genes was induced by mycorrhizal fungi but each responded differently to the exogenous GA<sup>3</sup> treatment.

#### **3. Discussion**

Symbiotic germination of orchid seeds involves the dual process of seed self-development and mutualistic interaction with their mycorrhizal fungi. Thus, the process is quite complex physiologically and ecologically. Orchid seeds are too tiny to perform genetic manipulations and this has inevitably limited the studies on their mechanisms of symbiotic germination, yet recent breakthroughs on arbuscular mycorrhiza have laid the foundation for investigating the SG of orchid seeds [16]. Recent studies show that the mycoheterotrophic symbiosis between orchids and mycorrhizal fungi possesses major components shared with mutualistic plant–mycorrhizal symbioses [17]. Many studies have revealed that plant hormones, especially gibberellins, are important factors affecting seed germination [10], and they are also critical for the establishment of mycorrhizal symbiosis [18,19]. In our study, the contents of five plant hormones (GA3, ABA, IAA, ZT, and JA) was determined at four different developmental stages of seed germination of the orchid *D. o*ffi*cinale*. Our results revealed that the mature and ungerminated seed have the highest ABA content (12.78 ng/g·FW) but this declined further along the seed germination process, and is consistent with two other studies [20,21]. A little GA<sup>3</sup> was detected in the early germination stage of SG and AG group but the content is no significant difference between SG and AG group. Exogenous GA<sup>3</sup> negligibly affected asymbiotic germination at all concentrations used in our study, a result supporting early statements by Arditti [6] that, in general, gibberellins appear to have no effect on germinating orchid embryos, in line as well with reported findings on asymbiotic germination testing by Hadley and Harvais [22]. However, exogenous gibberellins did significantly affect symbiotic germination in our study, implying its important role in mycorrhizal establishment. In addition, although the content of GA<sup>3</sup> was similar between the symbiotic and asymbiotic groups, the ratio GA3/ABA changed faster at seedling development stage in SG, indicating fungal infection probably affected the balance of endogenous GAs and ABA. Previous results indicated the gibberellin/abscisic acid balance was capable of governing the seed germination of palm and maize plants [23,24]. In tomato, the level of GAs increases as a consequence of a symbiosis-induced mechanism requiring functional arbuscules that depends on a functional ABA pathway in mycorrhizal symbiosis during the establishment of arbuscular mycorrhiza [25]. Additionally, at least 130 forms of GAs have been identified to date yet only a handful of these (GA1, GA3, GA4, GA5, and GA7) are known to be biologically active [26]. Thus, in our next research project, we plan to quantify other active GAs molecules in *D. o*ffi*cinale* seeds.

The amount of IAA rose dramatically during the seed germination process, but especially during the seedling development stage of the SG group, indicating that IAA production was probably induced by mycorrhizal fungus in SG. Early research has shown that only traces of auxin occur in *Cypripedium* seed but none at all in *Dendrobium* seeds [6,27]; however, in our study, IAA was detected at relatively high content in the ungerminated stage and this content declined in the course of AG. The conflicting

results are likely due the detection methods used. UHPLC is undoubtedly more sensitive for the quantification of trace amounts of plant hormones. Auxin is recognized as a secondary dormancy phytohormone, controlling seed dormancy and germination [28]. In addition, auxin metabolism and signaling also plays a crucial role in the modification of roots growth during their colonization by the ectomycorrhizal fungus *Laccria bicolor* [29]. Our result suggests IAA production was induced greatly during orchid mycorrhizal establishment, which provides a possible explanation for the faster differentiation of embryo when the seed of *D. o*ffi*cinale* was inoculated with the mycorrhizal fungus.

In this study, jasmonic acid (JA) content went undetected in ungerminated seeds and low JA (1.63 ng/g·FW) occurred in the free-living fungus, whereas the most JA was present in the early germination stage (stage 2) in AG (Table 1). JA is widely known to be involved in the response of plants to various stress factors, yet surprisingly little research has been carried out on JA's roles in seed germination [30]. Work by Dave et al. [31] found no massive increase in their contents during seed maturation of *Arabidopsis*, suggesting their accumulation instead occurred during early seed development. A recent study reported crosstalk between JA and ABA contributed to modulating seed germination in bread wheat and *Arabidopsis* [32]. Evidently, more research is required to unravel the molecular mechanisms by which jasmonates regulate the germination of seeds.

Besides inducing plant hormone production, the mycorrhizal fungus itself also produces hormones and this may influence its plant partners in crucial ways. In our study, all five hormones were detected in the mycorrhizal fungus *Tulasnella* sp. As for the dynamic change of hormones in symbiotic germination group of *D. o*ffi*cinale* seeds, whether their production arose from mycorrhizal fungi or from host plant induced by fungus is still unclear and merits further exploration in the future.

Exogenous GA<sup>3</sup> treatment had a dose-dependent effect on the SG of *D. o*ffi*cinale* seeds but did not significantly affect either the AG or free-living mycelium growth in the phenotype. Based on our initial results, we speculate the signal recognition between seed and their mycorrhizal fungi was probably impaired in some way by a higher concentration of GA3. We did not detect fungal invasion (colonization) of the seed embryo when using either 0.5 µM and 1.0 µM exogenous GA3. Under the microscope, we saw the seed embryo enlarged but no germination ensued at these high GA<sup>3</sup> concentrations in the SG group (Figure 3S–Y). Furthermore, high concentrations of GA<sup>3</sup> did not stop free-living mycelium from growing. A previous study has shown that GAs are phytohormones able to inhibit arbuscular mycorrhizal fungal infection by inhibiting arbuscular mycorrhizal hyphal entry into the host root where they suppressed the expression of Reduced Arbuscular Mycorrhization1 (RAM1) and RAM2 homologs that function in hyphal entry and arbuscule formation [19]. A similar scenario probably occurred in SG of *D. o*ffi*cinale* seeds.

Furthermore, after receiving the exogenous GA3, plant hormone-related genes such as biosynthesis and signal transduction of GA, ABA or IAA were characterized by a similar expression profile. Namely, sharply increasing expression in response to 0.5 µM exogenous GA<sup>3</sup> followed by transcriptional downregulation; accordingly, we infer that exogenous GA<sup>3</sup> disturbed the balance of endogenous hormones and crosstalk regulation occurred between GA, IAA, and ABA during the seed germination of *D. o*ffi*cinale* inoculated with the *Tulasnella* sp. fungus. Normally, genes involved in GA and IAA synthesis are highly expressed in SG, especially in the protocorm and seedling stages of orchids. The symbiosis between *Cymbidum goeringii* and a *Rhizoctonia*-like mycorrhizal fungus caused the release of hormones, which were able to promote the growth of *C. goeringii* seedlings [7]. Similarly, it has been demonstrated that auxin promotes *Arabidopsis* root growth by modulating its gibberellin response [33]. We plan to quantify the endogenous hormones to further confirm the relationship between hormone content and gene expression under an exogenous GA<sup>3</sup> treatment during orchid seed germination.

Based on our previous RNA-seq and iTRAQ data, we found four proteins encoding genes involved in the common symbiotic signal pathway, including two genes function-annotated as nodulation signaling pathway protein (*DoNSP2-1* and *DoNSP2-2*) and two Ca2<sup>+</sup> signal-related proteins, a calcium-dependent protein kinase (*DoCDPK26*), and a calmodulin-like protein (*DoCML19*). All these genes were highly expressed in SG but differed markedly. The Ca2<sup>+</sup> signal is a universal second

messenger, and increases in cytosolic Ca2<sup>+</sup> concentration are among the earliest signaling events occurring in plants challenged with mutualistic partners or pathogens [34,35]. CDPK and CML are the two principal protein families of plant Ca2<sup>+</sup> sensors [36]. The gene encoding CDPK was also identified from *D. o*ffi*cinale* roots infected by an orchid mycorrhizal fungus (*Mycena* sp.) by using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) [37]. In our study, the genes encoding CDPK (*DoCDPK26*) and CML (*DoCML19*) exhibited sharply higher expression levels in SG across the applied concentration gradient exogenous GA3, especially under 0.5 µM (for *DoCDPK26*) and 1.0 µM (for *DoCML19*), respectively. However, this expression of *DoCML19* was similar to SG lacking exogenous GA<sup>3</sup> treatment, suggesting gene expression was induced by mycorrhizal fungi and only weakly related to exogenous GA3. Conversely, the gene *DoCDPK26* showed a significant different expression in SG group with versus without exogenous GA<sup>3</sup> treatment, which implied that the CDPK and CML proteins probably participate in this plant–microbe interaction in different ways. Given the difficulty of genetically manipulating orchid seeds and orchid mycorrhizae, in our future research biochemical and physiological methods will be applied to confirm the mechanistic linkage between this plant hormone and Ca2<sup>+</sup> signal during the SG of orchid seed, as well as changed Ca2<sup>+</sup> concentrations across a gradient of exogenous GA<sup>3</sup> during seed germination of *D. o*ffi*cinale*.

We also found that the expression of genes encoding probable mycorrhizal signaling pathway proteins (*DoNSP2-1* and *DoNSP2-2*) (function-annotated as nodulation signaling pathway proteins), both of which encode GARS-family transcriptional regulators, considerably increased under 0.5 µM exogenous GA<sup>3</sup> treatment in SG compared to AG. This result suggests exogenous GA<sup>3</sup> probably affected the mycorrhizal-specific gene expression by controlling the mycorrhizal-signaling pathway. Gibberellin's ability to govern the nodulation signaling pathway in *Lotus japonicus* has been clarified by Maekawa et al. [38], who found that exogenous application of biologically active GA<sup>3</sup> inhibited the formation of infection threads and nodules; hence they suspected GA halted the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. Whether a similar situation, in which GA inhibited the downstream gene expression of the mycorrhizal signaling pathway, occurs in orchid mycorrhiza needs to be confirmed (or not) in a co-culture system of orchid seedlings with its mycorrhizal fungi.

Several typical putative mycorrhizal-fungi-induced expression genes were identified in the SG of *D. o*ffi*cinale* seeds based on our transcriptomic data: *DoCDR1*, *DoGGLU*, *DoGLU*, *DoPRCP*, and *DoSWEET.* For these genes, hardly any expression happened in AG but they were highly expressed in specific ways among different development stages of SG for the *D. o*ffi*cinale* seeds. The gene *DoCDR1* encodes an aspartic protease. Studies have found that the aspartic protease gene in rice, OsCDR1, can induce defense responses in plants and increase plant resistance to bacterial and fungal diseases [39]. *DoCDR1* was also upregulated in different germination stages of SG in the absence of the GA<sup>3</sup> treatment: low concentration of it did not cause this gene's expression to change, but 0.1 µM endogenous GA<sup>3</sup> treatment strongly elevated *DoCDR1*<sup>0</sup> s expression, suggesting that fungi induced it. Exogenous GA<sup>3</sup> probably affected the expression level by interfering with the balance of endogenous hormones.

*DoGGLU* and *DoGLU* are two genes encoding β-1,3-glucanase, belonging to the pathogenesisrelated proteins class that plays an important role in biotic and abiotic stress responses of plants [40]. It has been shown that colonization by mycorrhizal fungi in orchid root does not trigger strong plant defense responses in orchid mycorrhiza of *Serapias vomeracea* with *T. calospora*, given the nonstimulated expression of the plant's defense genes [41]. However, our proteomic analysis showed that fungus invasion activated the plant defense reaction because genes encoding catalase isozyme, L-ascorbate peroxidase, and superoxide dismutase—all of which are enzymes involved in defense mechanisms—were upregulated during the SG of *D. o*ffi*cinale* seeds [7]. High expression levels of β-1,3-glucanase genes suggest the host plant probably produced an antifungal defense reaction, especially in the protocorm stage, via the lysis of pelotons so as to limit the extent of invasion during the SG of *D. o*ffi*cinale*. Finally, since the high GA<sup>3</sup> treatment concentrations triggered the strong expression of *DoGGLU*, *DoGLU*, this indicated the genes respond to exogenous environment stress.

SWEET family sugar exporters in arbuscula mycorrhizal symbiosis in *Medicago truncatula* are known to play a vital role in the transport of glucose across the peri-arbuscular membrane to maintain arbuscular for a healthy mutually beneficial symbiosis [42]. Genes encoding SWEET family proteins are often expressed more in the symbiotic tissues of mycorrhizal protocorms of the orchid *S. vomeracea* with *T. calospora*. In our study, evidence for a similar phenomenon was found. Mycorrhiza-induced genes were specifically expressed in SG and its expression rose sharply under the 0.1-µM exogenous GA<sup>3</sup> treatment; hence, these genes responded to a changed exogenous GA<sup>3</sup> concentration during the SG of *D. o*ffi*cinale* seed. Therefore, we propose that GAs is involved in the crosstalk signal pathway between GAs biosynthesis and common symbiotic signal pathway during *D. o*ffi*cinale* seeds' symbiotic germination and is thereby able to influence the expression of mycorrhizal-induced genes.

## **4. Materials and Methods**

#### *4.1. Plant Materials and Growing Conditions*

Seeds of *D. o*ffi*cinale* were collected from a greenhouse in Jinhua County of Zhejiang Province, China, in November 2015. Mature capsules were surface sterilized, and their axenic seeds were stored at 4 ◦C in wax paper packets inside 1.5-mL sterilized tubes containing sterilized silica gel [7]. A mycorrhizal fungus that was a *Tulasnella* sp. (S6), isolated previously from root of *D. nobile*, was cultured in potato dextrose agar (PDA) medium. Symbiotic germination (SG) testing was carried out in oatmeal agar plates (OMA, 0.25% oat meal and 1% agar) and the asymbiotic germination (AG) testing was performed in 1/2 Murashige & Skoog (1/2 MS) medium without fungi, under a 12-h/12-h light/dark (L/D) cycle at 25 ◦C. In our previous work, we demonstrated this fungus is able to stimulate seed germination of *D. o*ffi*cinale* prior to AG, by reducing time to germination and increasing germination rate [7].

## *4.2. Determination of Endogenous Hormone during Seed Germination of D. o*ffi*cinale*

Endogenous hormones, including gibberellic acid (GA3), abscisic acid (ABA), indole-3-acetic acid (IAA), *trans*-zeatin (ZT) and jasmonic acid (JA), were examined on a total of eight samples at three different developmental stages (stage 2, stage 3, and stage 4) of AG and SG, ungerminated seed, and free-living mycelium of fungus. Each sample consisted of three biological replicates. Standards of ABA, ZT, indole-3-aceticacid, GA3, and JA (Sigma, St. Louis, MO, USA) were used for the quantification of endogenous hormones. Hormone extraction and fractionation followed the description of Kojima et al. [43]. Briefly, 50–200 mg of fresh seeds or fungi were frozen in liquid nitrogen and homogenized with a lysis buffer (methanol:water:formic acid = 7.9:2:0.1) in a 2-mL microcentrifuge tube. The homogenate was kept at 4 ◦C for at least 15 h. After centrifugation at 10,000× *g* for 15 min, the ensuing supernatant was transferred to a new collection tube. The combined eluate was evaporated and then reconstituted with 1 mL of 1 M formic acid, and then the hormone-containing fraction was passed through an MAX column. Quantitative analysis was performed using ultra-high performance liquid chromatography (UHPLC, Agilent 1290 Infinity, Agilent, Santa Clara, CA, USA) coupled with tandem mass spectrometry (MS/MS, Agilent 6490 Triple Quadrupole, Agilent, Santa Clara, CA, USA). Automatic identification and integration of each MRM transition was done under default parameter settings in Masshunter software (Agilent, Santa Clara, CA, USA), but assisted with manual inspections. The mass spectral peak area of the analyte was taken as the ordinate, and a linear regression standard curve drawn with the concentration of the analyte as the abscissa, from which the regression equation was obtained. Then, the mass spectral peak area of the analyte of a given sample was substituted into the linear equation, to calculate the content of each endogenous hormone.
