1. Introduction
Gibberellins or gibberellic acids (GAs), tetracyclic diterpenoid phytohormones, are widely distributed in higher plants [
1,
2]. They play an important role in seed germination, hypocotyl elongation, flowering time transition, seed and fruit development, and abiotic and biotic stress response [
3,
4,
5,
6]. Since the discoveries of the GA-insensitive dwarf mutants of wheat and rice, one of the most decisive players in ‘Green Revolution’ in the late 1960s which led to a massive increase in grain production, saving millions of lives in developing countries, much effort has been put in place to unravel the molecular mechanism of the GA signal transduction pathway in plants. As a result, the key genes involved in the GA signaling pathway have been isolated, including those encoding for GID1s, DELLAs, and SLYs from various plants [
4,
7,
8].
GID1, a cytosolic gibberellin receptor, was first discovered in rice (
OsGID1) for its ability to sense and bind active GAs, transmit GA signals, and induce downstream reactions [
9]. Subsequently, many other GID1s were also successfully isolated and studied in other plant species [
3,
10], the first of which were the three
OsGID1 homologous genes from Arabidopsis,
AtGID1a,
AtGID1b, and
AtGID1c. All three AtGID1s could strongly bind active GA and physically interact with DELLA proteins in the presence of GA [
11]. They are also functionally redundant because the single mutant has no obvious GA-insensitive phenotype, whilst the triple mutant is dwarfed with a complete loss of response to GA [
11,
12,
13,
14]. However, a recent study showed that, while
AtGID1c positively regulated seed germination,
AtGID1b suppressed germination during seed dormancy in the dark, indicating that these two GID1s function independently during seed germination in Arabidopsis [
15]. Furthermore, a recent study in tomato using CRISPR/Cas9-edited plants showed that, although the three tomato
GID1 genes are functionally redundant, as only the triple-knockout mutant,
gid1TRI, showed the typical GA-insensitive phenotype, this redundancy disappeared under field conditions [
16]; therefore, these three
GID1s play overlapping, yet specific roles in maintaining the optimum growth and development of tomato plant [
16]. However, in peach, the GA-insensitive dwarf mutant
‘shouxing’ is caused by the S191F mutation in PbGID1c, i.e., loss of function of a single GID1 can result in dwarfism [
17]. Therefore, the combined results from these studies clearly demonstrated that GID1s in different plant species participate in different aspects of GA-mediated growth and development either independently or redundantly.
The second GA signaling component, perhaps the most ‘famous’ one is the ‘Green Revolution’ gene,
DELLA, first described in the 1960s but only cloned some 30 years later in the 1990s from wheat [
18]. DELLAs, named after the highly conserved DELLA (Asp–Glu–Leu–Leu–Ala) amino-acid sequence in their N-termini, are negative regulators of GA signaling. They belong to the GRAS (named after the three DELLA proteins
GAI,
RG
A, and
SCR) protein family [
19]. In addition to the DELLA domain, the N-termini contain the TYHYNP (Thr–Tyr–His–Tyr–Asn–Pro) domain, which plays an important role in the transduction of GA signal and maintains the stability of the DELLA protein. Other functional domains are the nuclear localization signal (NLS) and VHIID (Val–His–Ile–Ile–Asp) domain in the middle, and the LZ region (leucine repeat region), PFYRE (Pro–Phe–Tyr–Arg–Glu) domain, repressor domain SAW (Ser–Ala–Trp), and RVER (Arg–Val–Glu–Arg) domain in the C-termini [
20].
DELLAs have been identified in Arabidopsis (
GAI), rice (
Oryza sativa,
SLR1) [
21], wheat (
Triticum aestivum,
Rht-B1b and
Rht-D1b) [
18], corn (
Zea mays,
D8) [
18], barley (
Hordeum vulgare,
SLN1) [
22], and grape (
Vitis vinifera,
VvGAI) [
23]. Mutations/deletions in the DELLA domain can cause GA-insensitive and dominant or semi-dominant dwarf phenotypes in these plants, demonstrating that the integrity of the DELLA motif plays a pivotal role in the function of this family of proteins. For example, the GA-insensitive mutant
gai of Arabidopsis has the N-terminal 17 amino acids which contain the DELLA domain of GAI missing. The mutant has a distinctive phenotype where dwarfism and small, dark leaves are among the most recognizable features, which are also found in the
gid1 triple mutant [
19,
24]. Four
GAI homologous genes,
RGA,
RGL1,
RGL2, and
RGL3, were subsequently identified in Arabidopsis. They participate in seed germination and flower development (
RGA,
RGL1, and
RGL2) and also play roles in stress responses (
RGL1, RGL2, and
RGL3) [
25,
26,
27].
The third important component of GA signaling is the SCF (Skpl/cullin/F-box) of the E3 ubiquitin ligase. SCF plays essential roles in the ubiquitination and degradation of DELLA through the 26S proteasome system. As the subunit of SCF, the F-box protein determines the specificity for substrate DELLA recognition. Therefore, mutation in this protein can block GA signaling and, hence, cause GA insensitivity of the mutant [
28]. For example,
SLY1 (
SLEEPY1) and
SLY2/SNE (
SLEEPY2/SNEEZY) in Arabidopsis and their homologous gene
OsGID2 in rice encode for such F-box proteins [
28,
29]. The Arabidopsis mutant
sly1 is GA-insensitive due to its inability to degrade DELLA [
30]. The fact that the double mutant
sly1sne is more dwarfed and less fertile than the single mutant
sly1 indicates that both
SLY1 and
SNE are functionally redundant in positively regulating the action of GA in growth [
31,
32].
The molecular mechanism of GA signaling in plant has become clearer in recent years owing to the genetic identification of many GA-related mutants in various plant species as mentioned above. It is believed that the active GA molecule first binds to the GA receptor GID1 to form a GA–GID1 complex which can bind to the DELLA domain of the DELLA protein. This results in a conformational change of DELLA. The formation of the GA–GID1–DELLA complex in turn allows the C-terminus of DELLA to interact with the SCF
SLY1/GID2/SNE complex. This leads to the ubiquitination and subsequent degradation of DELLA by the proteasome complex, resulting in the release of GA from the GA–GID1–DELLA complex. GA can then enter the nucleus to activate the expression of downstream genes, thereby promoting GA-related growth and development [
33].
While GA signaling in the regulation of architecture, fertility, and stress is well characterized in Arabidopsis, rice, and other crop plant species, this knowledge in woody plants, such as pear tree, is very limited. Given the important role of DELLAs and other GA signaling components in the regulation of plant height and the lack of dwarf pear rootstocks, we aimed to first identify GID1, DELLA, and SLY homologous genes from the wildtype ‘duli’ pear genome. Subsequently, the evolution and expression profiles of these genes in various tissues of 5 year old ‘duli’ pear trees under normal growth conditions, as well as in tissue-cultured seedlings treated with IAA, GA, PAC, ABA, and NaCl, were assessed in order to dissect their biological functions. The preliminary data obtained in this study could provide valuable information for further research of these genes, which could guide molecular breeding to create the much-desired dwarf ‘duli’ pear rootstocks in future.
2. Results
2.1. The Genome of ‘duli’ Pear Encodes 15 GA Signaling-Related Genes Located on 11 Different Chromosomes
Fifteen homologous genes related to GA signaling were identified from the ‘
duli’ pear genome (
Table 1). Specifically, there were five GID1-encoding genes (
PbGID1s) distributed on four of the 17 chromosomes (Chr3.g17752.m1, Chr4.g40571.m1, and Chr11.g13248.m1 on the third, fourth, and 11th chromosomes; Chr12.g35366.m1 and Chr12.g35281.m1 on the 12th chromosome) (
Figure 1, green). Their coding regions were between 1035 and 1095 bp, encoding proteins of 344–364 amino acids with molecular weights between 38.60 and 41.23 kDa.
Six
PbDELLAs were identified, distributed on chromosomes 2, 9 13, 15, 16, and 17 (
Figure 1, blue). It was noted that the coding regions of these six
PbDELLAs were highly variable in length (1614–2364 bp), number of amino acids (537–787), and molecular weight (58.8–86.3 kDa) of their encoded proteins.
A homologous search also discovered four
SLY genes (
PbSLYs). They were much smaller than both
PbGID1s and
PbDELLAs, whereby the CDS of the largest one was only 720 bp long (Chr8.g54419.m1), located on chromosome 8. It encodes a putative protein of 239 amino acids and 26.3 kDa in molecular weight, while the other three
PbSLYs were located on chromosomes 9, 15, and 17 (
Table 1;
Figure 1, red).
2.2. Phylogenetic Analysis Identified Potential Dwarf-Related Genes from PbGID1s, PbDELLAs, and PbSLYs
Phylogenetically related proteins usually share similar biological functions. Protein sequence alignment of GID1s showed that they fall into two types (
Figure 2). Type A, including Chr12.g35366.m1, Chr12.g35281.m1, and Chr4.g40571.m1, had the closest sequence similarity to peach PpGID1c (XP_007207928), followed by Arabidopsis AtGID1a (AT3G05120) and AtGID1c (AT5G27320), and tomato LeGID1a (Solyc01g098390). Therefore, we named these three PbGID1s PbGID1c-1-1 (Chr12.g35366.m1), PbGID1c-1-2 (Chr12.g35281.m1), and PbGID1c-2 (Chr4.g40571.m1). Type B contained the remaining two members, Chr3.g17752.m1 and Chr11.g13248.m1; they were most closely related to AtGID1b (AT3G63010), LeGID1b1 (Solyc09g074270), LeGID1b2 (Solyc06g008870), and PpGID1b (XP_007200347). Hence, we named them PbGID1b-1 (Chr3.g17752.m1) and PbGID1b-2 (Chr11.g13248.m1).
Similarly, we compared the protein sequences of the six putative PbDELLAs with known DELLAs from other plant species. This led to their classification into three groups. The first group with two ‘
duli’ pear DELLAs, Chr16.g31263.m1 and Chr13.g24360.m1, also contained apple MdRGL1a (DQ007885) and MdRGL1b (DQ007886), Arabidopsis AtGAI (AT1G14920) and AtRGA (AT2G01570), grape VvGAI (AF378125), barley SLN1 (AF460219), wheat Rht-D1a (AJ242531), rice OsSLR1 (AB030956), and maize D8 (AJ242530). Importantly, this group of DELLAs is well known for their regulation of plant height because their knockout mutants exhibit dwarfism, such as wheat
rht-D1a1 and rice
sly1 [
18,
22,
34,
35,
36,
37]. Therefore, we named Chr16.g31263.m1 and Chr13.g24360.m1 as PbGAI1a and PbGAI1b, respectively. The second group included Chr9.g44536.m1 and Chr17.g27556.m1, which were named as PbGAI2a and PbGAI2b because their closest homologous proteins were MdRGL2s (DQ007883, DQ007884) from apple, which were also shown to affect plant height [
38]. The last two members, Chr15.g03238.m1 and Chr2.g41461.m1 in the third group of DELLAs, shared the highest sequence similarity with apple MdRGL3a/b (DQ007887, DQ007888); hence, they were named as PbRGLa and PbRGLb, respectively (
Figure 2).
Lastly, we analyzed the sequences of the four PbSLYs by comparing them with those from Arabidopsis (AtSLY1, AT4G24210 and AtSLY2, AT5G487170) and rice (OsGID2, BAC81428). This resulted in their classification into two groups with each group containing two members. Accordingly, they were named as PbSLY1-1 (Chr8.g54419.m1) and PbSLY1-2 (Chr15.g04246.m1) in the group SLYⅠ, and PbSLY2-1 (Chr9.g46276.m1) and PbSLY2-2 (Chr17.g25753.m1) in the group SLYIⅠ (
Figure 2).
Therefore, the ‘duli’ pear genome has a full repertoire of GA signaling components. Importantly, the potential dwarf-related genes are also present within these three families of genes.
2.3. PbGID1s, PbDELLAs, and PbSLYs Contain the Typical Conserved Domains and Motifs
As shown in
Figure 3a, all PbGID1s contained two conserved domains, the co-esterase (pfam00135) and α/β hydrolase-3 (pfam07859) belonging to the α/β hydrolase family. These PbGID1s also contained 10 conserved motifs (
Supplementary Materials Figure S1a) located in similar positions within the individual PbGID1s (
Figure 3a).
Analysis of the six PbDELLAs showed that they all contained the characteristic conserved DELLA (pfam12041) domain in their N-termini and the GRAS (pfam03514) domain in the C-termini (
Figure 3b). Note that the DELLA (motif 5) showed some variations, where the amino acids EL in PbRGLa and PbRGLb were replaced by GC and GY ((D
GCLA and D
GYLA instead of D
ELLA), respectively. It is also noteworthy that the sequence of PbGAI1b was different from the other five PbDELLAs in that it contained a putative transmembrane domain between amino acids 2 and 24, implying that it may have a different function (
Figure 3b). With the exception of PbGAI2b that did not have motif 9 SAW close to the C-terminus, a typical repressor motif of the GRAS family proteins, all other PbDELLAs had the 10 conserved motifs (
Supplementary Materials Figure S1b).
Conserved domains and motifs of PbSLYs were also analyzed, and the results are shown in
Figure 3c. Only one conserved domain, the F-box (pfam00646) domain, was present. Conserved motifs 1, 2, 3, and 6 were found in the sequences of all four PbSLYs, whilst motif 4 and motif 5 were only found in PbSLY2s and PbSLY1s, respectively, suggesting that these two types of PbSLYs may have different roles in GA signaling.
2.4. Synteny and Duplication Analysis of PbGID1s, PbDELLAs, and PbSLYs of ‘duli’ Pear
Understanding the gene duplication events occurring in the genome and the synteny between different genomes can help understand gene evolution and function. Gene duplication, including tandem duplication, segmental duplication, and whole-genome duplication (WGD), is the major driving force in plant evolution. A multigene family is the result of region-specific duplication of the genome or WGD [
39]. To evaluate the gene duplication events for
PbGID1s, PbDELLAs, and
PbSLYs, synteny and selective pressure analyses were carried out, and the results are presented in
Figure 4a.
PbGID1c-1-1 had a syntenic relationship with
PbGID1c-1-2 and
PbGID1c-2, as did
PbGID1b-1 with
PbGID1b-2. For
PbDELLAs,
PbGAI1a and
PbGAI1b had a syntenic relationship, as did
PbRGLa and
PbRGLb. For
PbSLYs, a syntenic relationship was found between
PbSLY1-1 and
PbSLY1-2, as well as between
PbSLY2-1 and
PbSLY2-2. All these gene pairs were the result of WGD or segmental duplications.
Next, the syntenic relationships between the genes of each family of
GID1s, DELLAs, and
SLYs were compared between ‘
duli’ pear and Arabidopsis. As shown in
Figure 4b,
PbGID1c-1-1,
PbGID1c-1-2, and
PbGID1c-2 had synteny with Arabidopsis
AtGID1c, of which
PbGID1c-1-2 and
PbGID1c-2 also had synteny with
AtGID1a. Both
PbGID1b-1 and
PbGID1b-2 were syntenic with
AtGID1b. For the
PbDELLAs,
PbGAI1a and
PbGAI1b had a syntenic relationship with
AtGAI and
AtRGA, respectively, as did
PbGAI2a with
AtRGL1.
PbSLY2-2 had a syntenic relationship with
AtSLY2/AtSNE.
Therefore, on the basis of the syntenic relationship between these genes in ‘duli’ pear and Arabidopsis, they are divided into two groups. The first group, including PbGAI2b, PbRGLa, and PbRGLb of the PbDELLA family and PbSLY1-1, 1-2, and 2-1 of the PbSLY family, was due to ‘dispersed’ duplication, indicating a separation by other sequences where the genes may have arisen from transposition, such as ‘replicative transposition’, ‘non-replicative transposition’, or ‘conservative transposition’. The second group originated from WGD or segmental duplication, and it included all PbGID1s, PbGAI1a, PbGAI1b, and PbGAI2a of the PbDELLAs, as well as PbSLY2-2.
Ka/Ks represents the ratio between the nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) of protein-coding genes. While synonymous codons produce the same amino acids (synonymous changes), nonsynonymous changes result in different amino acids to be translated. Therefore, the Ka/Ks ratio is used to determine whether there is a selective pressure acting on the protein-coding gene; we found that the values of Ka/Ks for the GA signal transduction-related genes in both ‘
duli’ pear and Arabidopsis were all less than 1 (
Supplementary Materials Table S1). This clearly demonstrates that these genes of ‘
duli’ pear have been subjected to purification selection during the evolution process; hence, the individual gene pairs identified above may have similar functions between Arabidopsis and ‘
duli’ pear.
2.5. Three Types of cis-Acting Elements Were Present in the Promotor Regions of PbGID1s, PbDELLAs, and PbSLYs
Spatial and temporal expression of a gene is very important for its proper biological function. This is determined by the
cis-acting elements in its promoter region. As shown in
Figure 5 and
Supplementary Materials Figure S2, three types of
cis-acing elements were present in the promoter regions of PbGID1s: growth and development regulatory-responsive, hormone-responsive, and stress responsive elements, named here as class I, II, and III, respectively. While the types and numbers of
cis-elements in each type were very similar, the light-responsive element GATA-motif, the 6K protein-binding site Unnamed-1, and the palisade tissue mesophyll cell differentiation element HD-zip1 in class I, as well as the TC-rich repeats in class III, were unique to
PbGID1c-1-1 and
PbGID1c-1-2.
PbGID1c-2 was also very different in that the zeatin metabolism regulatory element O2-site and the auxin response element TGA-element in class II were not present. Interestingly, the promoter of
PbGID1c-2 contained unique
cis-acting elements, the dehydration, low temperature, and salt stress response elements DRE1 and WRE3, and the wound and pathogen response element W box. For the promoter sequences of
PbGID1b-1 and
PbGID1b-2, there were also some unique elements found, such as (1) the regulatory element A-box of class I present in both, (2) the F-box and the rhythm response element circadian present in class I, the GA response element GARE-motif present in class II, and the drought-induced response element MBS in class III unique to
PbGID1b-2, and (3) the light-responsive element AT1-motif, AE-box, and ACE in class I and the salicylic acid-induced
cis-acting element TCA-element in class II unique to
PbGID1b-1.
The common
cis-acting elements found in the promoter regions of all six
PbDELLAs were the G-Box, ABRE, ARE, and W box although the numbers were slightly different (
Figure 5b). However, there were some unique elements in each of the pro
PbDELLAs. For example, the AT1-motif of class I and TGA-element of class II were found in the promoter region of
PbGAI1b, while the p-Box was found in
PbGAI2a. The GCN4-motif and TATC-box were unique to pro
PbGAI2b, the 3-AF1 binding site and the
cis-acting element DRE were unique to pro
PbRGLa, the TCCC-motif and O2-site were unique to
PbRGLb, and chs-CMA2a and WRE3 were unique to pro
PbRGLa and pro
PbRGLb.
The
cis-acting elements found in the promoter regions of all
PbSLYs were Box 4, GT1-motif, and as-1 of class I and ABRE and TGACG-motif of class II, although their numbers were slightly different (
Figure 5c). The TCT-motif and CAT-box in class I, GARE-motif in class II, and MBS in class III were shared by
PbSLY1-1 and
PbSLY1-2, while chs-CMA2a and RY-element in class I were present on both
PbSLY2-1 and
PbSLY2-2. Unique elements, such as Gap-box, I-box, AT1-motif, F-box and transcription factor-binding site AP-1 in class I, as well as LTR and WRE3 in class III, were only found in the promoter region of
PbSLY1-1, while chs-CMA1a and GCN4-motif of class I and TGA-element of class II were only in that of
PbSLY2-1. Furthermore, the TATC-box, p-box, and TCA-element in class II and the WUN-motif in class III were unique to
PbSLY1-2, while the HD-zip1 of class I and TC-rich repeats of class III were unique to
PbSLY2-2.
Taken together, it was found that the common cis-acting elements were present in the promoter regions of the genes in the same family. However, there were also unique ones found in some genes within the same family. This suggests that, while genes in the same family play similar roles, each gene also has its unique function in growth, development, hormone response, and stress response.
2.6. PbGID1s, PbDELLAs, and PbSLYs Were Ubiquitously Expressed with Tissue-Specific High-Level Expression for Some Genes in Each Family
In order to understand the expression profiles of
PbGID1s,
PbDELLAs, and
PbSLYs, real-time RT-PCR was carried out using total RNA isolated from roots, shoots, leaves, flowers, and young fruits of 5 year old trees. The results showed that the transcripts of all candidate genes were present in all the tissues tested; however, their expression levels were different (
Figure 6). For
PbGID1s, with the exception of
PbGID1c-1-2 which was expressed in all tissues at similar levels, other
PbGID1s were expressed at very low levels in the roots and flowers compared to in other tissues. The highest expression levels of
PbGID1c-1-1/1c-2 (Type A) were found in leaves and fruits while those of
PbGID1b-1/1b-2 (Type B) were found in new shoots. However,
PbDELLAs showed opposite trends to
PbGID1s, whereby their expression levels were much higher in roots and flowers, but lower in new shoots.
PbGAI1a, PbGAI1b, and
PbGAI2b were also expressed at high levels in leaves, while
PbGAI2b and
RGLb were expressed at low levels in young fruits. The main expression site for
PbSLYs was in the reproductive tissues. The expression levels of
PbSLYs in flowers were similar to those of
PbDELLAs, but opposite to
PbGID1s.
2.7. Changes in Expression Levels of PbGID1s, PbDELLAs, and PbSLYs in Tissue-Cultured Seedlings Treated with GA3, PAC, IAA, ABA, and NaCl
To see if/how the expression profiles of
PbGID1s,
PbDELLAs, and
PbSLYs changed in response to phytohormones and salt stress, tissue-cultured seedlings were treated with GA
3, PAC, IAA, ABA, and NaCl for different lengths of time. The expression levels of
PbGID1s, PbDELLAs, and
PbSLYs were monitored by qRT-PCR. As shown in
Figure 7, in GA
3-treated seedlings, the highest expression levels of
PbGID1c-1-1,
1c-1-2, and
1c-2 were reached at 12, 120, and 12 h, with 1.8-, 4.2-, and 3.6-fold increases compared to 0 h, respectively. The expression levels of
PbDELLAs and
PbSLYs were increased first and then decreased, with
PbGAI2a/b decreasing to the lowest levels at 3 h after treatment. Similarly, the expression levels of all
PbSLYs were also the lowest at 3 h, with
PbSLY2-1 and
2-2 being the most affected by GA
3.
When the seedlings were treated with PAC, an inhibitor of GA, all PbGID1s, with the exception of PbGID1c-1-2, were downregulated first and then upregulated, with PbGID1c-1-1 and PbGID1c-2 being the most sensitive and reaching the lowest expression levels with 5.3-fold and 6.8-fold decreases at 6h and 3h compared that at 0 h, respectively. On the contrary, PbDELLAs and PbSLYs showed opposite expression patterns to PbGID1s, i.e., their expression levels increased first and then decreased in PAC-treated seedlings. The highest expression levels of PbGAI2a and PbRGLb were reached at 3 h while those of all four PbSLYs were increased at 72 h, with PbSLY2-1 and 2-2 showing the most profound change with increases by 6.3- and 33.6-fold, respectively.
In IAA-treated seedlings the expression levels of all PbGID1s were increased except for PbGID1b-1. PbGID1c-1-1 and PbGID1b-2 were quickly affected, with the highest expression levels with 1.3- and 2.3-fold increases achieved after 3 h, whilst PbGID1c-1-2 was the most affected with a 3.3-fold increase after 120 h. The expression levels of all six PbDELLAs were decreased to start with, before increasing, with PbGAI1a, 1b, and RGLb being the most sensitive to IAA treatment with the lowest expression levels with 10.4-, 1.8-, and 5.5-fold decreases shown after 6 h. All PbSLYs were upregulated when treated with IAA, with PbSLY1-1/1-2 responding the fastest and reaching the highest expression levels at 6–12 h, while PbSLY2-1 and 2-2 were the most affected, with their expression levels increased by 7.4- and 28.1-fold, respectively.
To see if the expression of PbGID1s, PbDELLAs, and PbSLYs was affected by stress, ABA and NaCl were supplemented in the media. The effects of ABA and NaCl on the expression levels of these genes were very similar. In the case of PbGID1s, the initial downregulation of PbGID1b-1 and PbGID1b-2 was the most significant with 4.2- and 6.0-fold (ABA) and 7.1- and 3.8-fold (Nacl) decreases. However, PbDELLAs showed opposite trends, whereby their expression levels first increased and then decreased. This was particularly true for PbGAI2b and PbRGLa/b. All PbSLYs, except for PbSLY1-2 whose transcript level was increased throughout the treatment period, showed an increase then decrease.
Therefore, these combined data indicate that GA signaling plays an important role in the phytohormonal and abiotic stress response during ‘duli’ pear seedling development.
3. Discussion
The ‘duli’ pear is a wild pear species native to China, which is widely distributed in the northern region of the country. While the fruits are small and not edible, its rather well-developed root system, vigorous growth, high tolerance to biotic and abiotic stress, and good compatibility with European and Asian pear trees in grafting make it an ideal rootstock. In particular, a dwarfed rootstock is very desirable in order to achieve high density, uniformed planting, and ease of pesticide spraying and harvest.
Previous studies in wheat, rice, and other crop plants clearly showed that the plant hormone GA plays important roles in regulating plant height and stature [
18,
21,
22,
23]. As such, many GA insensitive dwarf mutants were isolated and used in agriculture to achieve high yield. Therefore, studying the GA signal transduction-related genes, expression patterns, and functions in ‘
duli’ pear could provide valuable information for the molecular breeding of dwarf rootstocks.
3.1. The Genome of ‘duli’ Pear Encodes More Members of Each Family of Genes Related to GA Signal Transduction Pathway Than Other Plant Species
A total of 15 GA signal transduction-related genes were identified from the ‘
duli’ pear genome (
Figure 1,
Table 1). Compared to Arabidopsis, rice, and many other plants, the number of genes in each family was increased. This seems to be correlated to a genome-wide doubling event [
40]. AtGID1s in Arabidopsis have 13 domains, TWVLIS, LDR, FFHGGSF, HS, IYD, YRR, DGW, GDSSGGNI, GNI, MF, LDGKYF, WYW, and GFY, that are responsible for binding to GA or DELLA [
41]. Similarly, our analysis of PbGID1s also identified TWVLIS, FFHGGSF, YRR, GDSSGNI, LDGKYF, and GFY motifs in their sequences (motifs 3, 7, 2, 5, 1, and 4, respectively,
Supplementary Materials Figure S1a), indicating that PbGID1s are most likely typical GID1s, and that these important functional motifs are highly conserved among plant GID1s. It is noteworthy, however, that PbGID1c-1-1 and PbGID1c-1-2 shared the highest similarity of ~94%; the only differences between them were as follows: (1) PbGID1c-1-1 had 20 more amino acids than PbGID1c-1-2 at the N-terminus; (2) only three amino acids were different within the remainder of the sequences. Both genes were also located on the same (12th) chromosome (
Figure 1). Therefore, they are most likely the same gene that has been misannotated. Further cloning and sequencing will confirm this conclusion.
Compared to five in Arabidopsis, only one in rice, and one in tomato, the ‘
duli’ pear genome contained six PbDELLAs. Further analysis showed that they belonged to three groups with two in each group, which is very similar to the DELLAs in the closely related apple genome [
38]. It is worth noting that the predicted protein sequence of PbGAI1a had two DELLA sequences, which were also longer than others (
Figure 3). We were puzzled by this, as this has not been previously reported in the literature. We, therefore, carried out RT-PCR, cloning, and sequencing of
PbGAI1a. The results showed that the deduced protein sequence contained only one DELLA, not two DELLAs (data not shown). Therefore, the two DELLA domains of PbGAI1a in the database were most likely caused by error when the ‘
duli’ pear genome sequence was assembled.
The genome of the ‘
duli’ pear encodes four SLYs compared to two in Arabidopsis and one in rice, respectively [
28,
31]. All four PbSLYs contained the F-box conserved domains and the specific motifs 1, 2, and 3 (F-box, LSL, and GGF, respectively), which were shown to be essential for the function of SLY1/GID2 in Arabidopsis [
30] and rice [
28]. Therefore, the presence of the same motifs in PbSLYs suggests that they are also important for their function in pear. Motif 5 was only found in PbSLY1-1/2 (SLY-I) while motif 4 was found in PbSLY2-1/2 (SLY-II). The study of OsGID2/SLYI in rice showed that it contains a unique VR1 motif in its N-terminus, which is not shared with AtSLY1. However, this motif is not required for its function because both WT-OsGID2 and OsGID2-ΔVR1 can rescue the phenotype of
gid2 [
28]. Therefore, whether the two groups of
PbSLYs function differently in ‘
duli’ pear is worth investigating in the future.
The combined results clearly demonstrated that the ‘duli’ pear genome encodes all three families of GA-related signaling proteins. Despite their small variations in some specific motifs/domains were found compared to their homologous proteins in other plant species, they all contained the conserved domains and motifs important for their proper function in GA signaling.
3.2. Putative Functions of GA Signal-Related Genes in ‘duli’ Pear
In general, GAs are actively synthesized in young developing tissues, such as new shoots and small fruits, while well-developed mature tissues contain low levels of GAs. In line with this, we found that
PbGID1s were expressed at low levels in roots and flowers but high levels in new shoots and young fruits, while
PbDELLAs and
PbSLYs (except for roots) showed an opposite trend (
Figure 6). This indicates that GA can promote the expression of
PbGID1s but inhibit that of
PbDELLAs and
PbSLYs. Similarly, the highest expression level of rice
OsGID2/SLY1 was found in unopened flowers, where the highest level of active GAs was detected [
28]. Different expression levels of
PbGID1s,
PbDELLAs, and
PbSLYs were found when ‘
duli’ pear seedlings were treated with IAA, GA
3, and PAC, as well as ABA and NaCl (
Figure 7). The Type A
PbGID1s were upregulated by IAA and GA3 but downregulated by PAC, indicating that they function in the growth and development of ‘
duli’ pear seedlings. Similar results were found in tomato plants, where the three GID1s were expressed in all tissues but only GID1a responded to GA treatment, confirming the important regulatory role of GID1a in seed germination, stem elongation, and leaf development [
16]. Studies in Arabidopsis and peach showed that the Arabidopsis
gid1a/gid1c double mutant and peach single mutant
ppgid1c were all severely dwarfed, indicating that GID1a and/or GID1c play important roles in regulating stem elongation and, hence, plant height [
13,
17]. These results, together with the results from our homology analysis of these genes (
Figure 2), suggest that the Type A PbGID1s are most likely involved in the regulation of vegetative growth and plant stature in ‘
duli’ pear. On the other hand, the Type B PbGID1s were downregulated in seedlings treated with ABA and NaCl; thus, they may play critical roles in abiotic stress. In supporting this, Illouz-Eliaz et al. found that tomato GID1b, a Type B GID1 maintained its stability when tomato grew in uncontrollable and unfavorable field conditions [
16].
Different DELLA proteins have different roles. For example, in Arabidopsis, AtGAI and AtRGA play a major role in vegetative growth, AtRGA, AtRGL1, and AtRGL2 play a major role in seed germination and flower development, and AtRGL3 plays a major role in stress response [
26,
27]. The three
PbDELLAs,
PbGAI1a,
PbGAI1b, and
PbGAI2b, were highly expressed in roots and leaves but lowly expressed in new shoots, while
PbGAI2a,
PbGAI2b,
PbRGLa, and
PbRGLb were highly expressed in flowers but lowly expressed in young fruits except
PbRGLa (
Figure 6). Treatment with GA
3 resulted in the downregulation of
PbGAI2a and
PbGAI2b (3 h), while PAC caused the upregulation of
PbGAI2a and
PbRGLb (3 h) (
Figure 7). PbDELLAs may also play important roles in stress response. This is because, when seedlings were treated with stress hormone ABA and salt,
PbRGLa and
PbRGLb showed the fastest and most increased expression levels, followed by
PbGAI2a and
PbGAI2b, while
PbGAI1a and
PbGAI1b remained unchanged (
Figure 7). Therefore, these expression data, combined with the results from homology and evolutionary analysis (
Figure 2), indicate that
PbGAI2a, PbGAI2b, PbRGLa, and
PbRGLb function in the regulation of reproduction, as well as in stress, while
PbGAI1a, 1b and
2a play important roles in stem elongation, as well as leaf development, in ‘
duli’ pear. It is worth noting that
PbGAI2a and
PbGAI2b were the only pair of genes without a syntenic relationship (
Figure 2 and
Figure 4a), indicating that there may be some functional differentiation of these two genes.
The expression profiles of
PbSLYs in IAA-, GA3-, PAC-, ABA-, and NaCl-treated tissue-cultured seedlings showed similar trends to those of
PbDELLAs but opposite trends to those of
PbGID1s. Most interestingly, the type II PbSLYs (
PbSLY2-1 and
PbSLY2-2) showed the most significant changes, while type I PbSLYs (
PbSLY1-1 and
PbSLY1-2) showed the fastest changes following different treatments (
Figure 7).
AtSLYs in Arabidopsis also showed different functions because the
sly1sne double mutant a showed more severe dwarf phenotype and significantly reduced fertility compared to the single mutant
sly1 [
32]. Furthermore, overexpression of
SNE (
SLY2) can partially restore the GA-insensitive phenotype of
sly1 mutants [
42,
43]. Therefore,
SNE, a type II SLY, can replace
SLY1, i.e., SLY2 functions redundantly with SLY1. However, overexpression of
SLY1 in the
sly1 mutant can lead to the degradation of AtRGL1 and AtRGL2, while excessive
SNE/SLY2 results in no change in RGL2 and only a reduction in RGL1 levels. This indicates that the functions of SNE/SLY2 and SLY1 are not exactly the same [
31]. Whether the two types of
PbSLYs function in a similar fashion to those in Arabidopsis remains to be explored.
In summary, we identified 15 GA-related signaling genes from the wildtype ‘
duli’ pear genome. Further bioinformatic analysis and expression studies of these genes showed that they are typical and comparable to those characterized from Arabidopsis and other plant species. The type A PbGID1s play a major role in the regulation of growth and development, while type B PbGID1s play a major role in stress. The members of type I and II
PbDELLAs (
PbGAI1a, PbGAI1b, PbGAI2a, and
PbGAI2b) may be involved in stem elongation and leaf (vegetative) development, while type Ⅱ and Ⅲ
PbDELLAs (
PbGAI2a, PbGAI2b, PbRGLa, and
PbRGLb), as well as all four
PbSLYs, may be involved in reproduction and stress response (
Supplementary Materials Figure S3). As such, these preliminary data could guide further research into GA signaling pathways and the identification of desirable genes for ‘
duli’ pear rootstock development.