*Article* **Symbiotic and Asymbiotic Germination of** *Dendrobium o*ffi*cinale* **(Orchidaceae) Respond Di**ff**erently to Exogenous Gibberellins**

## **Juan Chen \*, Bo Yan, Yanjing Tang, Yongmei Xing, Yang Li, Dongyu Zhou and Shunxing Guo \***

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China; yanbo1823@gmail.com (B.Y.); yanjingtang1@gmail.com (Y.T.); ymxing@implad.ac.cn (Y.X.); 15114605567ly@gmail.com (Y.L.); zhoudy651@gmail.com (D.Z.) **\*** Correspondence: chenjuan@implad.ac.cn (J.C.); sxguo@implad.ac.cn (S.G.); Tel./Fax: +86-10-57833231 (S.G.)

Received: 7 August 2020; Accepted: 19 August 2020; Published: 25 August 2020

**Abstract:** Seeds of almost all orchids depend on mycorrhizal fungi to induce their germination in the wild. The regulation of this symbiotic germination of orchid seeds involves complex crosstalk interactions between mycorrhizal establishment and the germination process. The aim of this study was to investigate the effect of gibberellins (GAs) on the symbiotic germination of *Dendrobium o*ffi*cinale* seeds and its functioning in the mutualistic interaction between orchid species and their mycobionts. To do this, we used liquid chromatograph-mass spectrometer to quantify endogenous hormones across different development stages between symbiotic and asymbiotic germination of *D. o*ffi*cinale*, as well as real-time quantitative PCR to investigate gene expression levels during seed germination under the different treatment concentrations of exogenous gibberellic acids (GA3). Our results showed that the level of endogenous GA<sup>3</sup> was not significantly different between the asymbiotic and symbiotic germination groups, but the ratio of GA<sup>3</sup> and abscisic acids (ABA) was significantly higher during symbiotic germination than asymbiotic germination. Exogenous GA<sup>3</sup> treatment showed that a high concentration of GA<sup>3</sup> could inhibit fungal colonization in the embryo cell and decrease the seed germination rate, but did not significantly affect asymbiotic germination or the growth of the free-living fungal mycelium. The expression of genes involved in the common symbiotic pathway (e.g., calcium-binding protein and calcium-dependent protein kinase) responded to the changed concentrations of exogenous GA3. Taken together, our results demonstrate that GA<sup>3</sup> is probably a key signal molecule for crosstalk between the seed germination pathway and mycorrhiza symbiosis during the orchid seed symbiotic germination.

**Keywords:** orchid mycorrhiza; plant hormone; symbiosis germination; gene expression

## **1. Introduction**

Orchidaceae is among the largest families of flowering plants, one that is fascinating and rich in species diversity, with diverse pollination mechanisms and a unique mycorrhizal symbiotic relationship [1]. Orchid mycorrhizae differ from other major types of mycorrhizae in that, besides mineral nutrients (e.g., P and N), this type of fungus supplies carbohydrates to the plant, especially in the early stages of seed germination and seedling development [2]. Some orchid species are obligate symbiotic partners with fungi during their whole life cycle (e.g., mycotrophic orchid *Gastrodia* spp.), while some epiphytic and terrestrial orchids can alternately depend on fungi in their adult stages [3].

Orchid seeds are numerous, ranging from 1300 to 4,000,000 seeds per capsule, but they are extremely small and dust-like, with an undifferentiated embryo, limited storage reserves and lacking an endosperm [4]. Accordingly, seed germination and the subsequent development of the protocorm of almost all orchids is dependent either on mutualistic symbiosis with a compatible fungus, such as the member of Tulasnelloid, Sebacinaoid, and Ceratobasidiaceae under natural field conditions (symbiotic germination, SG) [5] or the replacement of the fungus by an exogenous nutrient substance in medium under controlled conditions (asymbiotic germination, AG) [6]. Thus, symbiotic germination is acknowledged as being a unique and important topic of orchid seed biology.

Morphological and cytological studies have shown that fungi enter the embryo of orchid seeds through the suspensor end, then form hyphae coil (pelotons) in the cortical cells of the embryo and finally the pelotons are digested by the embryo cell while the embryo undergoes dramatic development: from being swollen, turning light green (stage 1) to a ruptured seed coat (stage 2), then forming green protocorms (stage 3) and, finally, having expanded leaves on a developed young seedling (stage 4 and stage 5) [7,8]. Arrays of microtubules and actin microfilaments are reportedly involved in the infection droplet release and symbiosome development during legume–rhizobia interactions and establishment of arbuscular mycorrhiza, even probably in the peloton's lysis of orchid mycorrhizae [9]. In addition, the reserve substance of the embryo also undergoes extreme changes; for example, the lipid body and protein in the embryo cell is gradually degraded, and starch grains appear at the beginning of fungal inoculation but these are gradually depleted with the symbiotic germination progress of *Dendrobium o*ffi*cinale* seeds [8]. All this recent research has sketched a relatively clear outline from a cell ultrastructure perspective of the symbiotic germination process of orchid seeds. Yet the molecular mechanism establishing the mycorrhizal relationship between fungi and orchid seeds at their early germination stage remains unclear.

As is well-known, plant hormones, especially gibberellins (GAs) and abscisic acids (ABA), play crucial roles not only in seed germination but also in mycorrhizal establishment. The key steps in the signal transduction pathway for GAs' biosynthesis, metabolism, and seed germination regulation have been demonstrated clearly, and the involved genes encoding key regulatory enzymes related to GAs' biosynthesis have been identified, such as gibberellin 20 oxidase (GA20ox) and GA3-oxidase (*GA3ox*) related to GA biosynthesis, and gibberellin 2-oxidase (GA2ox) that catalyzes the degradation of GAs [10]. DELLA proteins are reportedly central players in hormone-mediated crosstalk and they can interact through the N-terminal domain with the GA receptor encoded by GID1, through which GAs promote DELLA degradation. In addition, DELLA proteins are recognized as common components of the mycorrhizal signaling pathway and mutations to them can cause rice to fail to form mycorrhizal relationships [11,12]. Thus, we hypothesized that biosynthesis and signal transduction pathway of GAs contribute to crosstalk with the common symbiotic pathway (CSP)—a putative signal transduction pathway shared by arbuscular mycorrhizas and the rhizobium-legume symbiosis—transducing glomeromycotan or rhizobial signal perception from the plasma membrane into the nucleus during the symbiotic germination of *D. o*ffi*cinale* seeds [13].

To address our hypothesis, we took the *D. o*ffi*cinale* (an epiphytic orchid) inoculated with *Tulasnella* sp. as a model system, because *D. o*ffi*cinale* is among the Chinese traditional medicinal plants whose genomes have been sequenced [14], and the genome of the fungus *Tulasnella calospora* (a orchid mycorrhizal fungus) is sequenced [15]. The aims of this study were twofold. (1) To identify the differentially expressed genes (DEGs) involved in the biosynthesis and signal transduction of plant hormones and profile their expression patterns based on transcriptomic data. (2) To quantify and analyze the endogenous hormones' level in the orchid at different germination stages between AG and SG and analyze the effect of exogenous GA<sup>3</sup> upon AG and SG of *D. o*ffi*cinale* seeds. This study provides a new insight for better understanding orchids' seed biology and their symbiotic mechanism and provides important data for cultivation of *D. o*ffi*cinale* and other medicinal orchid plants via mycorrhizal techniques.

#### **2. Results**

## *2.1. Determination of Endogenous Hormones' Level at Di*ff*erent Germination Stages between SG and AG of D. o*ffi*cinale*

In the previous study, we experimentally demonstrated that the seed germination of *D. o*ffi*cinale* on the oatmeal agar (OMA) medium with fungi is faster than seed germination on 1/2 MS medium without fungi [7]. *D. o*ffi*cinale* seeds usually take 10 days to develop up to stage 2 in SG, compared to 16 days in AG (Figure 1). After 2 weeks of sowing seeds, more than 50% of the seeds formed the protocorm structures (stage 3) in SG while the protocorm formation took at least 3 weeks in AG. After about 20 days, seeds in SG can develop seedling stage (stages 4), compared to 30 days in AG. It took approximately 5 weeks to finish the germination process in SG and at least two months in AG.

To understand the dynamic changes of endogenous hormones' content during seed germination of *D. o*ffi*cinale*, using liquid chromatograph-mass spectrometer (LC-MS/MS), we quantified the five kinds of endogenous hormones—GA3, ABA, indole-3-acetic acid (IAA), *trans*-zeatin (ZT), and jasmonic acid (JA)—on the free-living fungus and the differently developed seeds in SG and AG, respectively (stage 0, no germination; stage 2, early germination; stage 3, protocorm; stage 4, seedlings) (Figure 1). These results showed that ungerminated seeds have the highest ABA content (12.78 ng/g·FW), but ABA content decreased as seed germination progressed (Table 1). The GA<sup>3</sup> content rose at the early germination stage (stage 2) but declined as seeds developed. The ABA and GA<sup>3</sup> contents were similar between AG and SG at the same stage, but the ratio of GA3/ABA was significantly higher in SG than AG (*p* < 0.05) (Figure 2). Interestingly, IAA was dramatically increased in the protocorm stage of SG (25.91 ng/g·FW) when compared to AG (0.48 ng/g·FW). This result likely explains the faster differentiation rate in the protocorm stage in SG (2 weeks) than AG (3 weeks) during *D. o*ffi*cinale* seed germination. Additionally, minute amounts of ZT (0.0075~0.014 ng/g·FW) were detected in both the free-living mycelium of fungus and ungerminated seeds. For ungerminated seeds, JA could not be detected and the free-living mycelium of *Tulasnella* sp. (S6) featured a low JA content (1.63 ng/g·FW), but JA peaked most in the early germination stage (stage 2) in AG (Table 1). Further, all five kinds of hormones were detected in free-living mycelium of mycorrhizal fungus *Tulasnella* sp., albeit their context ranged almost 10-fold (0.44~4.29 ng/g·FW) (Table 1).

#### *2.2. E*ff*ect of Exogenous GA<sup>3</sup> Treatment on Phenotypic Changes in D. o*ffi*cinale Seeds under SG and AG Conditions*

Different concentrations of GA<sup>3</sup> (0, 0.05, 0.1, 0.5, 1 µM) were added exogenously in medium to observe its effect on the SG and AG groups, respectively (Figure 3). These results showed that GA<sup>3</sup> affected the establishment of the mycorrhizal relationship between fungus and seeds in a dose-dependent manner. Namely, seed germination did not significantly change in the low GA<sup>3</sup> treatment concentration (0.05 µM), though germination was inhibited slightly by the middle GA<sup>3</sup> concentration (0.1 µM), yet germination was completely inhibited when high concentrations of exogenous GA<sup>3</sup> (0.5 µM, 1 µM) in SG compared to the control (SG without any GA<sup>3</sup> treatment) (Figure 3A–T). During the 4 weeks after sowing seeds, their effective germination rate gradually decreased from 40% to 0%, while more exogenous GA<sup>3</sup> was applied in SG. The resulting morphological characters examined under a light microscope showed the clear presence of pelotons in the embryo cell of SG seeds (Figure 3U–W). Seed germination was achieved to seedling differentiation stage (stage 4) under low GA<sup>3</sup> treatment concentration (0.05 µM) in SG (Figure 3B,G,L). No fungal mycelium colonized the seed embryo in the 0.1 µM GA<sup>3</sup> treatment in SG, indicating that exogenous GA<sup>3</sup> at a high concentration probably inhibited the signal recognition that normally occurs between the fungus and the seed, leading to failed fungal colonization (Figure 3S–Y). Neither seed germination in 1/2 MS medium (without fungus) nor the mycelium growth of the fungus on PDA were inhibited or displayed conspicuous morphological changes at any exogenous GA<sup>3</sup> concentration (Figure 3F–J,U1–Y1,A1–F1).

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 23

**Figure 1.** Morphological characters and seed developmental stages of *Dendrobium officinale. (***Left column**): symbiotic germination (**A**,**C**,**E**,**G**,**I**); (r**ight column**): asymbiotic germination (**B**,**D**,**F**,**H**,**J**). (**A**,**B**), stage 1: embryo swollen, turned light green, no germination; (**C**,**D**), stage 2: continued embryo enlargement, rupture of testa (germination); (**E**,**F**), stage 3: appearance of protomeristem (protocorm); (**G**–**H**), stage 4: emergence of first leaf (seedling); (**I**,**J**), stage 5: elongation of the first leaf. Scale bar = 0.5 mm. **Figure 1.** Morphological characters and seed developmental stages of *Dendrobium o*ffi*cinale. (***Left column**): symbiotic germination (**A**,**C**,**E**,**G**,**I**); (**right column**): asymbiotic germination (**B**,**D**,**F**,**H**,**J**). (**A**,**B**), stage 1: embryo swollen, turned light green, no germination; (**C**,**D**), stage 2: continued embryo enlargement, rupture of testa (germination); (**E**,**F**), stage 3: appearance of protomeristem (protocorm); (**G**–**H**), stage 4: emergence of first leaf (seedling); (**I**,**J**), stage 5: elongation of the first leaf. Scale bar = 0.5 mm.

*Conditions*

**Table 1.** The content of five kinds of endogenous plant hormones during the seed germination of *Dendrobium o*ffi*cinale* (*n* = 3). Abbreviation: FW: fresh weight; U0: ungerminated seeds; SG: symbiotic germination; AG: asymbiotic germination; 2, 3, 4 means stage 2, stage 3 and stage 4 during the germination of *D. o*ffi*cinale* seed, respectively; S6 means the free-living mycelium on PDA of our mycorrhizal fungus; GA<sup>3</sup> : gibberellic acids; ABA: abscisic acids. IAA: indole-3-acetic acid; ZT: *trans*-zeatin; JA: jasmonic acid. mycorrhizal fungus; GA3: gibberellic acids; ABA: abscisic acids. IAA: indole-3-acetic acid; ZT: transzeatin; JA: jasmonic acid. **Sample ABA (ng/g·FW) GA3 (ng/g·FW) IAA (ng/g·FW) ZT (ng/g·FW) JA (ng/g·FW)**  U0 12.78 ± 2.87 b 0.99 ± 0.46 abc 9.00 ± 1.2 ab 0.014 ± 0.00 ab 0 a

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 23

**Table 1.** The content of five kinds of endogenous plant hormones during the seed germination of

germination; AG: asymbiotic germination; 2, 3, 4 means stage 2, stage 3 and stage 4 during the germination of *D. officinale* seed, respectively; S6 means the free-living mycelium on PDA of our


Note: Different letters a, b, c, d represent significant difference (*p* < 0.05) of content of phytohormone in different development stage of *D. o*ffi*cinale* seeds. The analysis was performed using the Duncan method in SPSS 17.0 software. method in SPSS 17.0 software.

**Figure 2.** Endogenous hormone content change in different development stages during symbiotic and asymbiotic germination of *Dendrobium officinale* seeds. (**A**) GA3/ABA ratio in different stage between symbiotic and asymbiotic germination; (**B**) IAA/ABA ratio; (**C**) ZT/ABA ratio; (**D**) JA/ABA ratio. Mean and SE values were calculated from at least three replicates. Asterisks indicate significant differences in same development stage between asymbiotic and symbiotic germination according to the *t*-test (\* *p* < 0.05 and \*\* *p* < 0.001). AG, asymbiotic germination; SG, symbiotic germination. **Figure 2.** Endogenous hormone content change in different development stages during symbiotic and asymbiotic germination of *Dendrobium o*ffi*cinale* seeds. (**A**) GA<sup>3</sup> /ABA ratio in different stage between symbiotic and asymbiotic germination; (**B**) IAA/ABA ratio; (**C**) ZT/ABA ratio; (**D**) JA/ABA ratio. Mean and SE values were calculated from at least three replicates. Asterisks indicate significant differences in same development stage between asymbiotic and symbiotic germination according to the *t*-test (\* *p* < 0.05 and \*\* *p* < 0.001). AG, asymbiotic germination; SG, symbiotic germination.

*2.2. Effect of Exogenous GA3 Treatment on Phenotypic Changes in D. officinale Seeds under SG and AG* 

conspicuous morphological changes at any exogenous GA3 concentration (Figure 3F–J,U1–Y1,A1–F1).

Different concentrations of GA3 (0, 0.05, 0.1, 0.5, 1 μM) were added exogenously in medium to observe its effect on the SG and AG groups, respectively (Figure 3). These results showed that GA3 affected the establishment of the mycorrhizal relationship between fungus and seeds in a dosedependent manner. Namely, seed germination did not significantly change in the low GA3 treatment concentration (0.05 μM), though germination was inhibited slightly by the middle GA3 concentration (0.1 μM), yet germination was completely inhibited when high concentrations of exogenous GA3 (0.5 μM, 1 μM) in SG compared to the control (SG without any GA3 treatment) (Figure 3A–T). During the 4 weeks after sowing seeds, their effective germination rate gradually decreased from 40% to 0%, while more exogenous GA3 was applied in SG. The resulting morphological characters examined under a light microscope showed the clear presence of pelotons in the embryo cell of SG seeds (Figure 3U–W). Seed germination was achieved to seedling differentiation stage (stage 4) under low GA3 treatment concentration (0.05 μM) in SG (Figure 3B,G,L). No fungal mycelium colonized the seed embryo in the 0.1 μM GA3 treatment in SG, indicating that exogenous GA3 at a high concentration probably inhibited the signal recognition that normally occurs between the fungus and the seed, leading to failed fungal colonization (Figure 3S–Y). Neither seed germination in 1/2 MS medium

**Figure 3.** Effect of exogenous GA3 concentration on mycorrhizal fungi colonization in symbiotic and asymbiotic germination of *Dendrobium officinale* at 4 weeks after sowing seeds. (**A**–**J**). seed germination **Figure 3.** Effect of exogenous GA<sup>3</sup> concentration on mycorrhizal fungi colonization in symbiotic and asymbiotic germination of *Dendrobium o*ffi*cinale* at 4 weeks after sowing seeds. (**A**–**J**). seed germination when inoculated with *Tulasnella* sp., or without the fungus, at different GA<sup>3</sup> treatment concentrations. (**K**–**T**). morphological characters of symbiotic or asymbiotic germination at different GA<sup>3</sup> treatment concentrations under a stereomicroscope; (**U**–**Y1**). morphological characters of symbiotic or asymbiotic germination at different GA<sup>3</sup> treatment concentrations under a light microscope; (**A1**–**F1**). Colony of free-living mycelium of mycorrhizal fungus on PDA medium at different GA<sup>3</sup> treatment concentrations. Scale bars: (**A**–**J**) = 1 cm; (**K**–**T**) = 1 mm; (**J**–**W**), (**U1**–**Y1**) = 10 µm; (**S**–**Y**) = 5 µm; (**A1**–**F1**) = 1 cm; SG, symbiotic germination; AG, asymbiotic germination.

#### *2.3. Identification of Hormone-Related Genes and Common Symbiosis Pathway-Related Genes and Their Expression Profiles in SG and AG of D. o*ffi*cinale Seeds*

Based on previous RNA-Seq transcriptomic data of *D. o*ffi*cinale* seeds inoculated with *Tulasnella* sp., we screened upregulated genes involved in GA biosynthesis and signal transduction in SG, including those encoding ent–kaurene synthase (KS), ent–kaurene oxidase (KO), GA 20 oxidase (GA20ox), GA 3-beta dioxygenase (*GA3ox*), GA 2-oxidase (GA2ox), and DELLA protein (Table 2). In addition, the expression of predicted key enzyme genes involved in ABA biosynthesis (9-*cis*-epoxycarotenoid dioxygenase, NCED), metabolism (abscisic acid 8'-hydroxylase), and signal transduction (ABA responsive element binding factor) were also induced in SG of *D. o*ffi*cinale* seeds. Compared with AG, the genes related to IAA biosynthesis (YUCCA family monooxygenase and SAUR family protein) had upregulated expression in the SG stage (fold-change > 2.0 and false discovery rate (FDR) < 0.001) (Table 2).

The key genes involved in CSP were upregulated in SG in our transcriptomic database, such as calmodulin-like protein (Dendrobium\_GLEAN\_10048053) and the calcium-dependent protein kinase (Dendrobium\_GLEAN\_10016982) related to Ca2<sup>+</sup> signal transduction. Putative mycorrhizal-induced genes, including those encoding bidirectional sugar transporter protein, chitinase, fatty acid desaturase, and aspartic proteinase were all significantly upregulated in SG compared to AG (Table 2).

Expression levels of putative genes involved in GAs biosynthesis such as *GA2ox (DoG2ox), GA3ox (DoGA3ox), GA3ox* (*DoGA3ox*) and encoding UDP-glucosyl transferase (*DoSGT*), G-box-binding factor (*DoGBF*), probable inactive receptor kinase (*DoIRK*), 9-*cis*-epoxycarotenoid dioxygenase (*DoNCED*), YUCCA family monooxygenase (*DOIPM*), SAUR family protein (*DoSAUR71*), calmodulin-like protein (*DoCML19*), calcium-dependent protein kinase (*DoCDPK26*), and nodulation signaling pathway protein (*DoNSP2-1, DoNSP2-2*) were validated by real-time quantitative PCR (qPCR); as were the mycorrhiza-induced genes encoding lysosomal pro-X carboxypeptidase (*DoPRCP*), hevamine A-like (*DoHAL*), glucan endo-1,3-beta-glucosidase (*DoGGLU*), bidirectional sugar transporter (*DoSWEET14*), beta-1,3-glucanase (*DoGLU*), and aspartic proteinase CDR1-like (*DoCDR1*) (Figures 4–8). After seed germination (stage 2, stage 3, stage 4), all of these genes were usually highly expressed and upregulated in protocorm (stage 3) and seedling development stages (stage 4 and stage 5) in SG compared to AG of *D. o*ffi*cinale*.
