GIBBERELLIN INSENSITIVE DWARF1 Plays an Important Role in the Growth Regulation of Dwarf Apple Rootstocks

in HortScience

Dwarfing rootstocks can improve the plant architecture of apple trees and increase production. Gibberellins (GAs) are crucial for plant growth and dwarfing traits. The receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), plays an important role in the regulation pathway. However, the growth regulatory mechanism of GID1 in dwarf apple rootstock seedlings is not clear. In this study, we selected dwarf apple rootstock ‘SH6’ and its cross parents as materials to clone the GA receptor gene GID1c. There were two different sites in the alpha/beta hydrolase domain. The expression of GID1c in ‘SH6’ was lower than that in Malus domestica cv. Ralls Janet, with the decrease of GA content. We further conducted GA3 treatment and overexpression of GID1c in tissue culture seedlings of ‘SH6’, and the results showed that the expression of GID1c and biosynthesis genes increased and promoted the accumulation of hormone contents, which ultimately regulates the growth of ‘SH6’ dwarf apple rootstock seedlings. Our results suggest that GID1c may affect the plant architecture and dwarf traits of dwarfing rootstock and accelerate its application in orchards.

Abstract

Dwarfing rootstocks can improve the plant architecture of apple trees and increase production. Gibberellins (GAs) are crucial for plant growth and dwarfing traits. The receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), plays an important role in the regulation pathway. However, the growth regulatory mechanism of GID1 in dwarf apple rootstock seedlings is not clear. In this study, we selected dwarf apple rootstock ‘SH6’ and its cross parents as materials to clone the GA receptor gene GID1c. There were two different sites in the alpha/beta hydrolase domain. The expression of GID1c in ‘SH6’ was lower than that in Malus domestica cv. Ralls Janet, with the decrease of GA content. We further conducted GA3 treatment and overexpression of GID1c in tissue culture seedlings of ‘SH6’, and the results showed that the expression of GID1c and biosynthesis genes increased and promoted the accumulation of hormone contents, which ultimately regulates the growth of ‘SH6’ dwarf apple rootstock seedlings. Our results suggest that GID1c may affect the plant architecture and dwarf traits of dwarfing rootstock and accelerate its application in orchards.

Gibberellins (GAs) are increasingly proving to be a crucial regulator of plant growth and a controlling factor of plant architecture. They regulate various physiological and biochemical processes of plant growth and development, promote germination, control plant height and root growth, improve tillering, regulate flowering, and improve seed production (Andres et al., 2014; Qin et al., 2013; Resentini et al., 2015). Key enzymes involved in GA biosynthesis, such as ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), and ent-kaurene synthase (KS), catalyze the early step(s) of the cyclization and oxidation reactions and affect the GA signal pathway, regulating plant growth phenotype (Fambrini et al., 2011; Toyomasu et al., 2016; Yamamura et al., 2018). The GA 20-oxidases can regulate plant stature and disease development by affecting GA biosynthesis in rice (Qin et al., 2013). Mutants defective in GA biosynthesis, such as GA1, GA2, and GA3, present relatively low internal concentrations of GA, resulting in dwarf phenotypes for both monocot and dicot plants; in turn, GA applications promote growth elongation (Lohr et al., 2014). In the GA signal pathway, GIBBERELLIN INSENSITIVE DWARF1 (GID1) acts as the GA receptor, can combine with GA to induce the degradation of DELLA repressor proteins, following the GA-dependent complex formation of GID1-GA-DELLA, and then regulate many physiological and biochemical processes in plants (Griffiths et al., 2006; Nemoto et al., 2017; Schwechheimer, 2012; Yan et al., 2014). Arabidopsis has three gibberellin receptors: GIBBERELLIN INSENSITIVE DWARF1A (GID1A), GID1B, and GID1C. GID1B and GID1C have specific roles in seed development and root elongation. A previous study suggested that GID1A can bind gibberellin in the cytosol to initiate gibberellin signaling and regulate plant growth and fruit set (Ferreira et al., 2018; Gallego-Giraldo et al., 2014; Livne and Weiss, 2014). In wheat, there are three TaGID1 genes, and overexpression of each TaGID1 gene in the Arabidopsis double-mutant gid1a/1c partially rescued the dwarf phenotype (Li et al., 2013). In woody plants, GID1 also plays an important role in growth and in the dwarf phenotype (Cantín et al., 2018); however, the regulation function of GID1 in the determination of the plant growth traits of dwarfing rootstocks is still unclear.

Plant growth is also controlled by the regulation of different hormone signals, including auxin, brassinolide, and abscisic acid (Ren et al., 2018). During the petal elongation growth process, auxin promotes cell elongation and increases the number of cells in the petal basal region, regulating flower growth and development (Wang et al., 2017). Auxin response factor8 interacts with BIGPETALp, which has been shown to limit petal growth by influencing cell expansion, to modulate petal expansion. In the BR signaling pathway, the BR molecule was shown to bind with receptor-like kinase BRI1 and then promote the activation of BES and BZR to regulate multiple aspects of growth and development (Clouse and Sasse, 1998; Kim et al., 2011; Tang et al., 2011; Wang and He, 2004; Yin et al., 2002; Zhu et al., 2013). During plant growth, environmental stress regulates the transcription of ABA-insensitive (ABI) and NCED genes, affecting plant growth and stress responses. In the ABA catabolism pathway, ABI promotes plant growth and fruit development during strawberry ripening; this promotion is regulated by microRNAs involved in posttranscriptional modification (Li et al., 2016). Therefore, crosstalk may occur between different hormones to regulate plant growth and development.

Plant architecture, including plant height, number of tillers, and root system characteristics, which are regulated by both genetic and environmental factors, is the focus of intense research aiming to improve crop performance (Hedden 2003). In modern apple breeding, the application of dwarf rootstocks can reduce vegetative growth and lead to dwarfing phenotype, which improves the fruit yield and quality (Andrea et al., 2006; Cohen et al., 2007; Foster et al., 2017; Greenwood et al., 2010; Jöst et al., 2015; Smolka et al., 2010); however, the biochemical and molecular mechanisms that regulate dwarfing traits in dwarf apple rootstocks have not been elucidated. Specifically, the function of GA signaling in dwarf apple rootstocks as well as the crosstalk between GA and other hormones remain unclear. In this study, we selected the apple dwarfing interstock ‘SH6’ as experimental material. We cloned the GID1c gene and compared both its sequence and expression specificity in ‘SH6’ and its parents. By using transient transformation, we also analyzed the transcript level of related genes and hormone contents in the tissue of ‘SH6’ seedlings, and the results showed that GID1c may promote the biosynthesis and metabolism of GA and other hormones, which further regulate plant growth characteristics. We aim to analyze the regulation mechanism of GID1c on plant architecture and dwarfism characteristics in ‘SH6’ dwarf apple rootstocks and promote its application value in orchards.

Materials and Methods

Plant materials and growth conditions.

‘SH6’, the hybrid offspring of Malus domestica cv. Ralls Janet and M. honanensis cv. S19 (a brachyplast bud mutation variety of Malus honanensis), is widely used as apple dwarfing interstock for its stable dwarfness and resistance. Malus honanensis and ‘S19’ also have the dwarfing and semidwarfing characteristics, but they have poor genetic stability and rootstock-scion affinity. ‘Ralls Janet’ has no dwarfing or semidwarfing characteristics. The leaves of field plants were collected from the Institute of Pomology in Taigu, Shanxi, China. The tissue culture seedlings of ‘SH6’ were cultivated in the Tissue Culture Center of Beijing University of Agriculture on solid medium [Murashige and Skoog (MS) medium] under a 16-h light cycle (1800–2000 Lx) at 23 °C. Rooting cultures were established on medium (1/2 MS medium) under a 16-hlight cycle (1800–2000 Lx) at 23 °C.

Gene cloning.

Leaves (with the same vegetative vigor) in annual shoots of ‘SH6’, M. domestica cv. Ralls Janet, Malus honanensis, and M. honanensis cv. S19 were collected from field plants for gene cloning at 180 d after full bloom (DAFB). The primers used are shown in Supplemental Table 1 and were designed based on the apple genome sequencing in the National Center for Biotechnology Information (NCBI). The total RNA was isolated using an RNA Pure Total RNA Extraction Kit (Aidlab, Beijing, China). After DNase I (Takara, Shiga, Japan) treatment to remove genomic DNA, first-strand complementary DNA (cDNA) was synthesized using a Promega Reverse Transcription System Kit in accordance with the manufacturer’s instructions (Promega, Madison, WI).

Bioinformatic analysis.

Amino acid sequence alignment analysis was conducted with DNAMAN, and the function domain analysis was performed at http://smart.embl-heidelberg.de/. A phylogenetic tree was constructed in accordance with the nearest-neighbor interchange method using the ClustalW program of MEGA5.2 software. Branches corresponding to partitions were produced from 1000 bootstrap replicates. The evolutionary distances were computed using the p-distance method.

Dwarfing traits measurement.

We selected 10 plants of each cultivar for the measurement of dwarfing traits from field plants at 180 DAFB. The tape was used to measure the plant height and crown diameter (m), a vernier calliper was used to measure the tapering (Kr) of annual shoots (diameter of stem apex: the diameter of stem base), and a resistivity detector was used for resistivity detection (Ω·m) of annual shoots (ρ = RS/l; R, resistance; S, cross-sectional area; l, length).

GA3 and paclobutrazol treatments.

To analyze the growth phenotypes, each of 10 tissue culture seedlings of ‘SH6’ (with the same vegetative vigor) were cultivated on solid medium (4.74 g MS + 6 g agar powder + 30 g sucrose) and supplemented with both 50 μM GA3 (Sigma-Aldrich, St. Louis, MO) and 10 μM paclobutrazol (PAC) under a 16-h light cycle (1800–2000 Lx) at 23 °C. The plant materials were collected after 20 d of treatment for the measurement of growth index and hormone contents.

Quantitative real-time polymerase chain reaction.

Gene expression differences were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). The total RNA was extracted from the leaves in annual young shoots of different cultivars and ‘SH6’ tissue culture seedlings. First-strand cDNA was synthesized using a Tiangen Fast Quant RT Kit (Tiangen, Beijing, China) with 1 µg total RNA. qRT-PCR was performed on an ABI7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) using GoTaq qPCR master mix (Promega) in accordance with the manufacturer’s instructions. The conditions were as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s. The relative gene expression was analyzed using 2(−ΔΔCt) values, which were normalized with the 18S gene. The primers used are shown in Supplemental Table 1 and were designed based on the conserved sequence in NCBI. At least three biological replicate samples were used. Differences were considered significant at P < 0.05.

Vector construction and genetic transformation.

The coding sequence of GID1c was inserted into ap35S-1300–green fluorescent protein (GFP) vector containing XbaI and KpnI sites compared with the p35S-1300-GFP empty vector (WT), after which the vectors were transformed into Agrobacterium tumefaciens strain GV3101. To test the function of GID1c in the ‘SH6’ tissue culture seedlings, we performed a transient transformation using the vacuum draw method (Zhang et al., 2016). Then, the seedlings were cultured for 7 d under a 16-h light cycle (1800–2000 Lx) at 23 °C after 3-day shade treatment. The leaves of the seedlings were subsequently collected for PCR and qRT-PCR testing and related index measurement. The data of three biological replicates were used.

Microscopic examination.

The GFP in transgenic ‘SH6’ tissue culture seedlings was examined using an upright fluorescent microscopic (Leica, Wetzlar, Germany) under blue excitation spectra and a Stereo Fluorescence Microscope (Leica) under green excitation spectra. The microscope images were then observed on a computer.

Hormone content measurement.

The leaves in young annual shoots of different cultivars and the leaves of tissue culture seedlings ‘SH6’ with different treatments were collected and ground into powder. Then, 0.2 to 0.5 g powder was added to 4 mL phosphate-buffered saline extracting solution with polyvinylpyrrolidone to measure the hormone contents with an enzyme-linked immunosorbent assay.

Statistical analysis.

Duncan’s statistical analysis for the measurement of growth index, expression level, and hormone content was performed using Origin Pro 8 (Origin Laboratory Corporation, Northampton, MA) and Photoshop (Adobe, San Jose, CA) software were used for image integration. Error bars for each symbol indicate the sd of the reactions of three biological replicates.

Results

Bioinformatic analysis of GID1c in different apple cultivars.

Given the homology with M. domestica sequences (NM_001294351.1), we cloned the CDS of GID1c in ‘SH6’, M. domestica cv. Ralls Janet, M. honanensis, and M. honanensis cv. S19 leaves with the primers Md-GID1-F and Md-GID1-R. The open reading frame of the GID1c gene encodes a deduced amino acid sequence of 343 residues. There was little difference among the four cultivars except two amino acid sites in the alpha/beta hydrolase domain; the sequence in ‘SH6’ was more homologous with that in M. honanensis cv. S19 and M. honanensis, and the sequence in M. domestica cv. Ralls Janet was more homologous with that in M. domestica (Fig. 1). The GID1c sequence difference in different cultivars may have no effect on the GA signal pathway, but it was in accordance with the plant dwarfing traits. The plant height, crown diameter, tapering, and resistivity in dwarfing cultivars were lower than those in ‘Ralls Janet’ (Fig. 2). These findings revealed the closer genetic relationship between ‘SH6’ and M. honanensis cv. S19 and M. honanensis.

Fig. 1.
Fig. 1.

Bioinformatic analysis of GID1c in Malus domestica, M. domestica cv. Ralls Janet, Malus honanensis, M. honanensis cv. S19, and ‘SH6’. (A) Amino acid sequence alignment analysis of GID1c in Malus domestica, M. domestica cv. Ralls Janet, M. honanensis, M. honanensis cv. S19, and ‘SH6’. Two specific sites in the alpha/beta hydrolase domain are indicated with under lining. (B) Phylogenetic analysis of GID1c in ‘SH6’, M. honanensis cv. S19, M. honanensis (MH), M. domestica cv. Ralls Janet (RJ), M. domestica (NM_001294351.1), Pyrus bretschneideri (XM_009337376.2), Prunus persica (XM_007207866.2), and Vitis vinifera (KY765598.1). The genetic distance is closest between ‘SH6’ and M. honanensis cv. S19.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

Fig. 2.
Fig. 2.

The dwarfing traits of Malus domestica cv. Ralls Janet (RJ), Malus honanensis (MJ), M. honanensis cv. S19, and ‘SH6’. (A) The plant height in different cultivars. (B) The crown diameter in different cultivars. (C) The tapering (Kr) in different cultivars. (D) The resistivity in different cultivars. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

The transcript level of GID1c and GAs contents in different apple cultivars.

In different cultivars, the transcript levels of GID1c in ‘SH6’ and M. honanensis cv. S19 were 5- to 7-fold lower than those in M. domestica cv. Ralls Janet (RJ) and M. honanensis (MH) (Fig. 3A). Moreover, the contents of GA3 in ‘SH6’ and M. honanensis cv. S19 were also lower than those in M. domestica cv. Ralls Janet (RJ) and M. honanensis (MH) (Fig. 3B). In addition, the contents of GA4 in ‘SH6’, M. honanensis (MH), and M. honanensis cv. S19 were lower than that in M. domestica cv. Ralls Janet (RJ) (Fig. 3C). These results suggest that GID1c may be an important factor in the regulation of plant growth by promoting GA content. The lower expression of GID1c in ‘SH6’ may result in the dwarfing phenotype of ‘SH6’ compared with ‘Ralls Janet’ (RJ).

Fig. 3.
Fig. 3.

(A) The GID1c transcript levels and gibberellin (GA) content analysis. The transcript level of GID1c in the different cultivars. (B) The GA3 content in the different cultivars. (C) The GA4 content in the different cultivars. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. FW = fresh weight; MJ = Malus honanensis; RJ = ‘Ralls Janet’. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

GID1c transcript levels and GA contents in response to GA3 and PAC treatments.

We treated ‘SH6’ tissue culture seedlings with GA3 or PAC to explore the effects of GA3 and their inhibitor PAC on ‘SH6’ growth phenotypes; normal MS medium served as a control treatment. The results showed that, compared with the control treatment, the GA3 treatment significantly promoted plant height and root growth, whereas PAC clearly inhibited plant height and root growth (Fig. 4A). Moreover, the GA3 treatment also promoted GID1c expression, with an increase of GA (GA3/GA4) contents, whereas PAC inhibited GID1c expression (Fig. 4B–D). These phenomena increased plant height and the number of internodes and promoted the growth of leaves and roots (Table 1). All these results demonstrated that, by increasing the transcript levels of the GA receptor GID1c, GA treatment promoted the growth of ‘SH6’ with the accumulation of GA content.

Table 1.

Growth index of ‘SH6’ on MS solid medium and supplemented with 50 μM GA3 and 10 μM PAC.

Table 1.
Fig. 4.
Fig. 4.

(A) Growth phenotype of ‘SH6’ tissue culture seedlings under different treatments [GA3 and paclobutrazol (PAC)]. (B) Analysis of GID1c transcript levels in response to different treatments. (C) The GA3 content in the different cultivars. (D) The GA4 content in the different cultivars. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

The transcript levels of GA biosynthesis genes in response to different treatments.

We explored the expression patterns of genes related to GA biosynthesis in response to GA3 and PAC treatments in ‘SH6’ tissue culture seedlings. GA3 treatment increased the transcription level of KO 2-fold compared with the effects of MS treatment, and PAC treatment had little effect on the expression of KO (Fig. 5A). The transcription levels of KAO, KS, and GA20ox genes, all of which are also related to GA biosynthesis, were upregulated in the plants treated with GA3 but were downregulated in the plants treated with PAC (Fig. 5B–D). The results showed that GA3 treatment increased the expression of the GA receptor GID1c and upregulated the transcript levels of KO, KAO, KS, and GA20ox, promoting GA biosynthesis and the accumulation of GAs, which are crucial for the growth regulation of dwarf ‘SH6’ apple rootstock seedlings.

Fig. 5.
Fig. 5.

Analysis of the transcript levels of genes related to gibberellin (GA) biosynthesis under different treatment [GA and paclobutrazol (PAC)] conditions. (AD) The transcript levels of KO, KAO, KS, and GA20ox, respectively. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

Transcript levels of relative genes and hormone content analysis in the GID1c overexpression lines.

To further investigate the function of GID1cin plant growth, we constructed a GID1c overexpression (OE) vector and obtained the transient transformation lines of ‘SH6’ tissue culture seedlings (OE-GID1: Line1, Line2, Line3) compared with the empty vector line (WT), which were examined microscopically. The leaves of the OE-GID1 lines presented distinct green fluorescence (Fig. 6A). The transcript levels of GID1c in the OE-GID1c plants were higher than that in the WT plants (Fig. 6B). Furthermore, the expression of KO, KAO, KS, and GA20ox related to the GA biosynthesis pathway increased, which provided an advantage to GA biosynthesis (Fig. 7A–D). At the same time, the transcription levels of the AUX and auxin response factor (ARF) genes, which are related to the auxin signaling pathway, also increased in the OE-GID1 lines (Fig. 7E and F). The expression of BZR, a negative regulator of the BR pathway, decreased in the OE-GID1c lines, whereas the expression of BES, a positive regulator, increased (Fig. 7G and H). Moreover, the expression of the ABI and NCED genes, which are related to ABA signaling, was significantly upregulated in the OE-GID1 plants compared with the WT plants (Fig. 7I and J). As a result, GID1c OE also increased the accumulation of hormones (GA3, IPA, IAA, BR, and ABA) in OE-GID1 ‘SH6’ tissue culture seedlings, but there was no obvious change in GA4 content (Fig. 8). These results indicated that the OE of GID1c may promote the relative gene expression and then increase the hormone contents to regulate plant growth. There may be a crosstalk between GA signals and other hormone (BR, auxin, and ABA) signals, resulting in synergistic effects that ultimately regulate the growth of ‘SH6’ dwarf apple rootstocks, including the control of plant architecture.

Fig. 6.
Fig. 6.

Microscopic examination and GID1c transcript level analysis of transgenic ‘SH6’ tissue culture seedlings. (A) Examination of green fluorescent protein (GFP) in both transgenic lines (OE-GID1: Line1, Line2, Line3) and the p35S-1300-GFP empty vector (WT) line of ‘SH6’ tissue culture seedlings via an upright fluorescence microscopy and stereo fluorescence microscopy. (B) Analysis of the transcript levels of GID1c in transgenic line (OE-GID1: Line1, Line2, Line3) and WT line of ‘SH6’ tissue culture seedlings. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

Fig. 7.
Fig. 7.

Analysis of the transcript levels of genes related to hormone biosynthesis and metabolism in both transgenic line and WT line of ‘SH6’ tissue culture seedlings. (AJ) The transcript levels of KO, KAO, KS, GA20ox, AUX, ARF, BZR, BES, ABI, and NCED, respectively. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

Fig. 8.
Fig. 8.

Analysis of the hormone contents in transgenic line and WT line of ‘SH6’ tissue culture seedlings. (AF) The content of GA3, GA4, IPA, IAA, BR, and ABA, respectively. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

Discussion

Height is a crucial biological characteristic and economic trait of plants. The selection of plant cultivars that present dwarf phenotypes, which allow higher planting densities, has been an important strategy in modern crop production (Jöst et al., 2015). The largest individual increases in harvest index and yield occurred in semidwarf cultivation systems of wheat, maize, and rice; these increases involved GA-insensitive dwarfing alleles (Prassinos et al., 2009; Rebetzke et al., 2012). Apple dwarf plants include both dwarf and semidwarf types; these types can be mainly generated via combinations of dwarf rootstock-tall scion, dwarf interstock-tall scion, and dwarf rootstock-dwarf scion. ‘SH6’ is the hybrid offspring of M. domestica cv. Ralls Janet × M. honanensis cv. S19 (an M. honanensis bud mutation variety that presents short shoots). Owing to its dwarfing interstock properties, ‘SH6’ is widely applied to apple fruit production in China (Fig. 2). During the past few decades, researchers have proposed several hypotheses to explain plant dwarfing mechanisms. In endogenous hormone regulatory pathways, GA signals and the GA receptor GID1 may perform key functions that regulate plant height and architecture (Hollender et al., 2016). In our study, the GID1c expression in the leaves of ‘SH6’ and M. honanensis cv. ‘S19’ was lower than that in the leaves of both M. domestica cv. Ralls Janet and M. honanensis. Moreover, the GA contents in ‘SH6’ and M. honanensis cv. S19 were lower than in the leaves of M. domestica cv. Ralls Janet (Fig. 3). These results demonstrated that the expression of GID1c coordinated with the accumulation of GA, which affected the dwarf traits in ‘SH6’ apple rootstocks.

GA participates in many aspects of plant growth and development, not only in seed germination, stem elongation, and floral development, but also in wood formation, and the differentiation of xylem fibers, in many plant species (Li et al., 2012). A previous study showed that GA biosynthetic and catabolic enzymes adversely influence plant height by regulating cell division and growth (Tyler et al., 2004). In our research, GA treatment also upregulated the expression levels of GID1c, which probably promoted the growth of ‘SH6’ tissue culture seedlings as well as the plant height, number of internodes, and leaf area by increasing the GA contents (Fig. 4 and Table 1). At high GA concentrations, the GID1 receptor binds with DELLA proteins, which leads to the formation of a GID1-GA-DELLA complex; this complex then targets the DELLA proteins for proteasomal degradation, which prevents dwarf phenotypes and impairs germination, flowering, and fertility (Fukazawa et al., 2015). In our study, the OE of GID1c in ‘SH6’ leaves may increase the GID1c protein, which promoted the increase of GID1-GA-DELLA complex, resulting in the upregulation of GA biosynthesis genes and GA accumulation, and ultimately regulated plant growth characteristics (Fig. 5). All these results indicated that GA treatment and OE of GID1c may rescue the apple dwarfing traits by promoting the relative gene expression in GA biosynthesis and metabolism pathways and increasing the accumulation of GAs.

In the plant growth process, the crosstalk between different hormones is an efficient regulatory factor. By directly interacting between DELLAs and BZR1/BES1, BRs and GAs enhance one another’s signals, thereby regulating cell elongation and plant growth (Allen and Ptashnyk, 2017; Jiang et al., 2015; Shahnejat-Bushehri et al., 2016). We performed GID1c transformations to further investigate the regulatory function of GID1c.The results showed that the OE of GID1c resulted in the upregulation of the expression of genes related to GA biosynthesis and the accumulation of GAs (Figs. 7 and 8). At the same time, the OE of GID1c downregulated the expression of BZR1 transcription factors but upregulated the expression of BES1 in the BR signaling pathway, which increased the content of BR. GID1c expression may promote the crosstalk between GA and BR to regulate the plant growth. Auxin is involved in root development, shoot growth, and fruit ripening. Plants can quickly sense and respond to changes in auxin levels; this sensing occurs via several major classes of auxin-responsive genes, including those of the auxin/indole-3-acetic acid (Aux/IAA) family and the ARF family (Luo et al., 2018). The OE of GID1c upregulated the transcript levels of AUX and ARF and also promoted the accumulation of IPA and IAA; therefore, we speculated that GID1c expression may affect the crosstalk between the GA and auxin signaling pathways to regulate the plant growth. ABA is involved in plant growth processes and abiotic stress responses (Hwang et al., 2018; Upadhyay et al., 2017; Wang et al., 2018). Within the comprehensive core ABA signaling network, GID1c may upregulate the transcript levels of the related genes ABI and NCED to control ABA content and influence plant growth (Figs. 7 and 8). As such, GA treatment and the OE of GID1c in the leaves of the dwarf rootstock seedlings upregulated the expression of GID1c, and then increased the expression of genes related to the GA, auxin, BR, and ABA signal pathways and the hormone contents, which promoted the growth of the dwarf rootstock plants. Based on these data, we hypothesized that the upregulation of GID1c activated the primary genes involved in hormone biosynthesis and metabolism and thereby enhanced the coregulation of different hormones involved in plant growth and inhibited dwarfing traits (Fig. 9). In summary, our results indicated that GID1c is a key regulator in dwarfing plants, and it can be used to improve woody fruit rootstock breeding strategies for dwarf phenotypes and high-planting-density production. However, the rootstock-scion effect and the regulation mechanism in other dwarfing rootstock cultivars require further study.

Fig. 9.
Fig. 9.

Hypothesis model of the GID1c regulation mechanism in fruit dwarf rootstocks. RS = rootstock; DIS = dwarf interstock. The red mark represents gibberellin (GA) signal pathways [ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), ent-kaurene synthase (KS), and GA20-oxidases (GA20ox)], the green mark represents BR signal pathways (BZR and BES), the purple mark represents auxin signal pathways (AUX and ARF), and the blue mark represents ABA signal pathways (ABI and NCED). The arrow indicates a positive effect, and the T-shaped arrow indicates a negative effect.

Citation: HortScience horts 54, 3; 10.21273/HORTSCI13685-18

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  • FosterT.M.McAteeP.A.WaiteC.N.BoldinghH.L.McGhieT.K.2017Apple dwarfing rootstocks exhibit an imbalance in carbohydrate allocation and reduced cell growth and metabolismHort. Res.417009

    • Search Google Scholar
    • Export Citation
  • FukazawaJ.ItoT.KamiyaY.YamaguchiS.TakahashiY.2015Binding of GID1 to DELLAs promotes dissociation of GAF1 from DELLA in GA dependent mannerPlant Signal. Behav.1010E1052923

    • Search Google Scholar
    • Export Citation
  • Gallego-GiraldoC.HuJ.UrbezC.GomezM.D.SuT.P.Perez-Amador MA2014Role of the gibberellin receptors GID1 during fruit-set in ArabidopsisPlant J.79610201032

    • Search Google Scholar
    • Export Citation
  • GreenwoodM.S.DayM.E.SchatzJ.2010Separating the effects of tree size and meristem maturation on shoot development of grafted scions of red spruce (Picea rubens Sarg.)Tree Physiol.30459468

    • Search Google Scholar
    • Export Citation
  • GriffithsJ.MuraseK.RieuI.ZentellaR.ZhangZ.L.PowersS.J.2006Genetic characterization and functional analysis of the Gid1 gibberellin receptors in ArabidopsisPlant Cell1833993414

    • Search Google Scholar
    • Export Citation
  • HeddenP.2003The genes of the green revolutionTrends Genet.1959

  • HollenderC.A.HadiartoT.SrinivasanC.ScorzaR.DardickC.2016A brachytic dwarfism trait (dw) in peach trees is caused by a nonsense mutation within the gibberellic acid receptor PpeGID1cNew Phytol.210227239

    • Search Google Scholar
    • Export Citation
  • HwangS.G.LeeC.Y.TsengC.S.2018Heterologous expression of rice 9-cis-epoxycarotenoid dioxygenase 4 (OsNCED4) in Arabidopsis confers sugar oversensitivity and drought toleranceBot. Stud.5912

    • Search Google Scholar
    • Export Citation
  • JiangJ.ZhangC.WangX.2015A recently evolved isoform of the transcription factor BES1 promotes brassinosteroid signaling and development in Arabidopsis thalianaPlant Cell272361374

    • Search Google Scholar
    • Export Citation
  • JöstM.EsfeldK.BurianA.CannarozziG.ChanyalewS.KuhlemeierC.2015Semi-dwarfism and lodging tolerance in Tef(Eragrostis Tef) is linked to a mutation in the A-tubulin 1 geneJ. Expt. Bot.66933944

    • Search Google Scholar
    • Export Citation
  • KimT.W.GuanS.BurlingameA.L.WangZ.Y.2011The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2Mol. Cell43561571

    • Search Google Scholar
    • Export Citation
  • LiA.YangW.LiS.LiuD.GuoX.SunJ.2013Molecular characterization of three GIBBERELLIN-INSENSITIVE DWARF1 homologous genes in hexaploid wheatJ. Plant Physiol.1704432443

    • Search Google Scholar
    • Export Citation
  • LiD.MouW.LuoZ.LiL.LimwachiranonJ.MaoL.YingT.2016Developmental and stress regulation on expression of a novel miRNA, Fan-miR73, and its target ABI5 in strawberrySci. Rep.628385

    • Search Google Scholar
    • Export Citation
  • LiJ.SimaW.OuyangB.WangT.ZiafK.LuoZ.2012Tomato Sldreb gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesisJ. Expt. Bot.6364076420

    • Search Google Scholar
    • Export Citation
  • LivneS.WeissD.2014Cytosolic activity of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1APlant Cell Physiol.551017271733

  • LohrB.StreitnerC.SteffenA.LangeT.StaigerD.2014A glycine-rich Rna-binding protein affects gibberellin biosynthesis in ArabidopsisMol. Biol. Rep.41439445

    • Search Google Scholar
    • Export Citation
  • LuoJ.ZhouJ.J.ZhangJ.Z.2018Aux/IAA gene family in plants: molecular structure, regulation, and functionIntl. J. Mol. Sci.191E259

  • NemotoK.RamadanA.ArimuraG.I.ImaiK.TomiiK.ShinozakiK.2017Tyrosine phosphorylation of the GARUE3 ubiquitin ligase promotes gibberellin signalling by preventing GID1 degradationNat. Commun.811004

    • Search Google Scholar
    • Export Citation
  • PrassinosC.KoJ.H.LangG.IezzoniA.F.HanK.H.2009Rootstock-induced dwarfing in cherries is caused by differential cessation of terminal meristem growth and is triggered by rootstock-specific gene regulationTree Physiol.29927936

    • Search Google Scholar
    • Export Citation
  • QinX.LiuJ.H.ZhaoW.S.ChenX.J.GuoZ.J.PengY.L.2013Gibberellin 20-oxidase gene OsGA20ox3 regulates plant stature and disease development in riceMol. Plant Microbe Interact.262227239

    • Search Google Scholar
    • Export Citation
  • RebetzkeG.J.BonnettD.G.EllisM.H.2012Combining gibberellic acid-sensitive and insensitive dwarfing genes in breeding of higher-yielding, sesqui-dwarf wheatsField Crops Res.1271725

    • Search Google Scholar
    • Export Citation
  • RenG.LiL.HuangY.WangY.ZhangW.ZhengR.ZhongC.WangX.2018GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxinNew Phytol.2192728742

    • Search Google Scholar
    • Export Citation
  • ResentiniF.Felipo-BenaventA.ColomboL.BlazquezM.A.AlabadiD.MasieroS.2015Tcp14 and Tcp15 mediate the promotion of seed germination by gibberellins in Arabidopsis thalianaMol. Plant8482485

    • Search Google Scholar
    • Export Citation
  • SchwechheimerC.2012Gibberellin signaling in plants: The extended versionFront. Plant Sci.2107

  • Shahnejat-BushehriS.TarkowskaD.SakurabaY.BalazadehS.2016Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signalingNat. Plants216013

    • Search Google Scholar
    • Export Citation
  • SmolkaA.LiX.Y.HeikeltC.WelanderM.ZhuL.H.2010Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in appleTransgenic Res.19933948

    • Search Google Scholar
    • Export Citation
  • TangW.YuanM.WangR.YangY.WangC.Oses-PrietoJ.A.2011PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1Nat. Cell Biol.13124131

    • Search Google Scholar
    • Export Citation
  • ToyomasuT.MiyamotoK.ShentonM.R.SakaiA.SugawaraC.HorieK.2016Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogonBiochem. Biophys. Res. Commun.4803402408

    • Search Google Scholar
    • Export Citation
  • TylerL.ThomasS.G.HuJ.DillA.AlonsoJ.M.EckerJ.R.2004Della proteins and gibberellin-regulated seed germination and floral development in ArabidopsisPlant Physiol.13510081019

    • Search Google Scholar
    • Export Citation
  • UpadhyayR.K.GuptaA.SoniD.GargR.PathreU.V.NathP.2017Ectopic expression of a tomato DREB gene affects several ABA processes and influences plant growth and root architecture in an age-dependent mannerJ. Plant Physiol.21497107

    • Search Google Scholar
    • Export Citation
  • WangJ.J.WangH.B.DingL.SongA.P.ShenF.JiangJ.F.2017Transcriptomic and hormone analyses reveal mechanisms underlying petal elongation in Chrysanthemum morifolium ‘Jinba’Plant Mol. Biol.936593606

    • Search Google Scholar
    • Export Citation
  • WangY.G.FuF.L.YuH.Q.HuT.ZhangY.Y.TaoY.2018Interaction network of core ABA signaling components in maizePlant Mol. Biol.96245263

  • WangZ.Y.HeJ.X.2004Brassinosteroid signal transduction-choices of signals and receptorsTrends Plant Sci.99196

  • YamamuraY.TaguchiY.IchitaniK.UmebaraI.OhshitaA.KurosakiF.2018Characterization of ent-kaurene synthase and kaurene oxidase involved in gibberellin biosynthesis from Scoparia dulcisJ. Nat. Med.72456463

    • Search Google Scholar
    • Export Citation
  • YanA.WuM.YanL.HuR.AliI.GanY.2014AtEXP2 is involved in seed germination and abiotic stress response in ArabidopsisPLoS One91E85208

  • YinY.WangZ.Y.Mora-GarciaS.LiJ.YoshidaS.AsamiT.2002BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongationCell109181191

    • Search Google Scholar
    • Export Citation
  • ZhangJ.TianJ.TaiD.Q.LiK.T.ZhuY.J.YaoY.C.2016An optimized TRV-based virus-induced gene silencing protocol for Malus crabapplePlant Cell Tissue Organ Cult.126499509

    • Search Google Scholar
    • Export Citation
  • ZhuJ.Y.Sae-SeawJ.WangZ.Y.2013Brassinosteroid signallingDevelopment14016151620

Supplemental Table 1.

List of primers used in the study.

Supplemental Table 1.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

We thank the Fruit Tree Key Laboratory of the Beijing University of Agriculture (BUA) and Beijing Nursery Engineering Research Center for Fruit Crops. We are grateful to all the technicians at the BUA Crabapple Germplasm Resource Garden. Financial support was provided by National Natural Science Foundation of China (31772263), Beijing Collaborative Innovation Center for Eco-environmental Improvement with Forestry and Fruit Trees (No. CEFFPXM2016-014207-000038), and 2018 Opening Project of Beijing Key Laboratory of New Technology in Agricultural Application (KF2018023).

These authors contributed equally to this work.

Corresponding authors. E-mail: yaoyc_20@126.com or zzqswu@yahoo.com.

  • View in gallery

    Bioinformatic analysis of GID1c in Malus domestica, M. domestica cv. Ralls Janet, Malus honanensis, M. honanensis cv. S19, and ‘SH6’. (A) Amino acid sequence alignment analysis of GID1c in Malus domestica, M. domestica cv. Ralls Janet, M. honanensis, M. honanensis cv. S19, and ‘SH6’. Two specific sites in the alpha/beta hydrolase domain are indicated with under lining. (B) Phylogenetic analysis of GID1c in ‘SH6’, M. honanensis cv. S19, M. honanensis (MH), M. domestica cv. Ralls Janet (RJ), M. domestica (NM_001294351.1), Pyrus bretschneideri (XM_009337376.2), Prunus persica (XM_007207866.2), and Vitis vinifera (KY765598.1). The genetic distance is closest between ‘SH6’ and M. honanensis cv. S19.

  • View in gallery

    The dwarfing traits of Malus domestica cv. Ralls Janet (RJ), Malus honanensis (MJ), M. honanensis cv. S19, and ‘SH6’. (A) The plant height in different cultivars. (B) The crown diameter in different cultivars. (C) The tapering (Kr) in different cultivars. (D) The resistivity in different cultivars. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    (A) The GID1c transcript levels and gibberellin (GA) content analysis. The transcript level of GID1c in the different cultivars. (B) The GA3 content in the different cultivars. (C) The GA4 content in the different cultivars. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. FW = fresh weight; MJ = Malus honanensis; RJ = ‘Ralls Janet’. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    (A) Growth phenotype of ‘SH6’ tissue culture seedlings under different treatments [GA3 and paclobutrazol (PAC)]. (B) Analysis of GID1c transcript levels in response to different treatments. (C) The GA3 content in the different cultivars. (D) The GA4 content in the different cultivars. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    Analysis of the transcript levels of genes related to gibberellin (GA) biosynthesis under different treatment [GA and paclobutrazol (PAC)] conditions. (AD) The transcript levels of KO, KAO, KS, and GA20ox, respectively. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    Microscopic examination and GID1c transcript level analysis of transgenic ‘SH6’ tissue culture seedlings. (A) Examination of green fluorescent protein (GFP) in both transgenic lines (OE-GID1: Line1, Line2, Line3) and the p35S-1300-GFP empty vector (WT) line of ‘SH6’ tissue culture seedlings via an upright fluorescence microscopy and stereo fluorescence microscopy. (B) Analysis of the transcript levels of GID1c in transgenic line (OE-GID1: Line1, Line2, Line3) and WT line of ‘SH6’ tissue culture seedlings. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    Analysis of the transcript levels of genes related to hormone biosynthesis and metabolism in both transgenic line and WT line of ‘SH6’ tissue culture seedlings. (AJ) The transcript levels of KO, KAO, KS, GA20ox, AUX, ARF, BZR, BES, ABI, and NCED, respectively. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    Analysis of the hormone contents in transgenic line and WT line of ‘SH6’ tissue culture seedlings. (AF) The content of GA3, GA4, IPA, IAA, BR, and ABA, respectively. The data are presented as the means ± sd of three biological replicates with Duncan’s statistical analysis. The asterisks represent the significant difference. *0.01 < P < 0.05, **P < 0.01.

  • View in gallery

    Hypothesis model of the GID1c regulation mechanism in fruit dwarf rootstocks. RS = rootstock; DIS = dwarf interstock. The red mark represents gibberellin (GA) signal pathways [ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), ent-kaurene synthase (KS), and GA20-oxidases (GA20ox)], the green mark represents BR signal pathways (BZR and BES), the purple mark represents auxin signal pathways (AUX and ARF), and the blue mark represents ABA signal pathways (ABI and NCED). The arrow indicates a positive effect, and the T-shaped arrow indicates a negative effect.

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  • AndresF.PorriA.TortiS.MateosJ.Romera-BranchatM.Garcia-MartinezJ.L.2014Short vegetative phase reduces gibberellin biosynthesis at the Arabidopsis shoot apex to regulate the floral transitionProc. Natl. Acad. Sci. USA111E2760E2769

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  • CantínC.M.ArúsP.EduardoI.2018Identification of a new allele of the Dw gene causing brachytic dwarfing in peachBMC Res. Notes111386

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  • FambriniM.MariottiL.ParlantiS.PicciarelliP.SalviniM.CeccarelliN.2011The extreme dwarf phenotype of the GA-sensitive mutant of sunflower, dwarf2, is generated by a deletion in the ent-kaurenoic acid oxidase1 (HaKAO1) gene sequencePlant Mol. Biol.754-5431450

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  • FerreiraL.G.de Alencar DusiD.M.IrsiglerA.S.T.GomesA.C.M.M.MendesM.A.ColomboL.2018GID1 expression is associated with ovule development of sexual and apomictic plantsPlant Cell Rep.372293306

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  • FosterT.M.McAteeP.A.WaiteC.N.BoldinghH.L.McGhieT.K.2017Apple dwarfing rootstocks exhibit an imbalance in carbohydrate allocation and reduced cell growth and metabolismHort. Res.417009

    • Search Google Scholar
    • Export Citation
  • FukazawaJ.ItoT.KamiyaY.YamaguchiS.TakahashiY.2015Binding of GID1 to DELLAs promotes dissociation of GAF1 from DELLA in GA dependent mannerPlant Signal. Behav.1010E1052923

    • Search Google Scholar
    • Export Citation
  • Gallego-GiraldoC.HuJ.UrbezC.GomezM.D.SuT.P.Perez-Amador MA2014Role of the gibberellin receptors GID1 during fruit-set in ArabidopsisPlant J.79610201032

    • Search Google Scholar
    • Export Citation
  • GreenwoodM.S.DayM.E.SchatzJ.2010Separating the effects of tree size and meristem maturation on shoot development of grafted scions of red spruce (Picea rubens Sarg.)Tree Physiol.30459468

    • Search Google Scholar
    • Export Citation
  • GriffithsJ.MuraseK.RieuI.ZentellaR.ZhangZ.L.PowersS.J.2006Genetic characterization and functional analysis of the Gid1 gibberellin receptors in ArabidopsisPlant Cell1833993414

    • Search Google Scholar
    • Export Citation
  • HeddenP.2003The genes of the green revolutionTrends Genet.1959

  • HollenderC.A.HadiartoT.SrinivasanC.ScorzaR.DardickC.2016A brachytic dwarfism trait (dw) in peach trees is caused by a nonsense mutation within the gibberellic acid receptor PpeGID1cNew Phytol.210227239

    • Search Google Scholar
    • Export Citation
  • HwangS.G.LeeC.Y.TsengC.S.2018Heterologous expression of rice 9-cis-epoxycarotenoid dioxygenase 4 (OsNCED4) in Arabidopsis confers sugar oversensitivity and drought toleranceBot. Stud.5912

    • Search Google Scholar
    • Export Citation
  • JiangJ.ZhangC.WangX.2015A recently evolved isoform of the transcription factor BES1 promotes brassinosteroid signaling and development in Arabidopsis thalianaPlant Cell272361374

    • Search Google Scholar
    • Export Citation
  • JöstM.EsfeldK.BurianA.CannarozziG.ChanyalewS.KuhlemeierC.2015Semi-dwarfism and lodging tolerance in Tef(Eragrostis Tef) is linked to a mutation in the A-tubulin 1 geneJ. Expt. Bot.66933944

    • Search Google Scholar
    • Export Citation
  • KimT.W.GuanS.BurlingameA.L.WangZ.Y.2011The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2Mol. Cell43561571

    • Search Google Scholar
    • Export Citation
  • LiA.YangW.LiS.LiuD.GuoX.SunJ.2013Molecular characterization of three GIBBERELLIN-INSENSITIVE DWARF1 homologous genes in hexaploid wheatJ. Plant Physiol.1704432443

    • Search Google Scholar
    • Export Citation
  • LiD.MouW.LuoZ.LiL.LimwachiranonJ.MaoL.YingT.2016Developmental and stress regulation on expression of a novel miRNA, Fan-miR73, and its target ABI5 in strawberrySci. Rep.628385

    • Search Google Scholar
    • Export Citation
  • LiJ.SimaW.OuyangB.WangT.ZiafK.LuoZ.2012Tomato Sldreb gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesisJ. Expt. Bot.6364076420

    • Search Google Scholar
    • Export Citation
  • LivneS.WeissD.2014Cytosolic activity of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1APlant Cell Physiol.551017271733

  • LohrB.StreitnerC.SteffenA.LangeT.StaigerD.2014A glycine-rich Rna-binding protein affects gibberellin biosynthesis in ArabidopsisMol. Biol. Rep.41439445

    • Search Google Scholar
    • Export Citation
  • LuoJ.ZhouJ.J.ZhangJ.Z.2018Aux/IAA gene family in plants: molecular structure, regulation, and functionIntl. J. Mol. Sci.191E259

  • NemotoK.RamadanA.ArimuraG.I.ImaiK.TomiiK.ShinozakiK.2017Tyrosine phosphorylation of the GARUE3 ubiquitin ligase promotes gibberellin signalling by preventing GID1 degradationNat. Commun.811004

    • Search Google Scholar
    • Export Citation
  • PrassinosC.KoJ.H.LangG.IezzoniA.F.HanK.H.2009Rootstock-induced dwarfing in cherries is caused by differential cessation of terminal meristem growth and is triggered by rootstock-specific gene regulationTree Physiol.29927936

    • Search Google Scholar
    • Export Citation
  • QinX.LiuJ.H.ZhaoW.S.ChenX.J.GuoZ.J.PengY.L.2013Gibberellin 20-oxidase gene OsGA20ox3 regulates plant stature and disease development in riceMol. Plant Microbe Interact.262227239

    • Search Google Scholar
    • Export Citation
  • RebetzkeG.J.BonnettD.G.EllisM.H.2012Combining gibberellic acid-sensitive and insensitive dwarfing genes in breeding of higher-yielding, sesqui-dwarf wheatsField Crops Res.1271725

    • Search Google Scholar
    • Export Citation
  • RenG.LiL.HuangY.WangY.ZhangW.ZhengR.ZhongC.WangX.2018GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxinNew Phytol.2192728742

    • Search Google Scholar
    • Export Citation
  • ResentiniF.Felipo-BenaventA.ColomboL.BlazquezM.A.AlabadiD.MasieroS.2015Tcp14 and Tcp15 mediate the promotion of seed germination by gibberellins in Arabidopsis thalianaMol. Plant8482485

    • Search Google Scholar
    • Export Citation
  • SchwechheimerC.2012Gibberellin signaling in plants: The extended versionFront. Plant Sci.2107

  • Shahnejat-BushehriS.TarkowskaD.SakurabaY.BalazadehS.2016Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signalingNat. Plants216013

    • Search Google Scholar
    • Export Citation
  • SmolkaA.LiX.Y.HeikeltC.WelanderM.ZhuL.H.2010Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in appleTransgenic Res.19933948

    • Search Google Scholar
    • Export Citation
  • TangW.YuanM.WangR.YangY.WangC.Oses-PrietoJ.A.2011PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1Nat. Cell Biol.13124131

    • Search Google Scholar
    • Export Citation
  • ToyomasuT.MiyamotoK.ShentonM.R.SakaiA.SugawaraC.HorieK.2016Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogonBiochem. Biophys. Res. Commun.4803402408

    • Search Google Scholar
    • Export Citation
  • TylerL.ThomasS.G.HuJ.DillA.AlonsoJ.M.EckerJ.R.2004Della proteins and gibberellin-regulated seed germination and floral development in ArabidopsisPlant Physiol.13510081019

    • Search Google Scholar
    • Export Citation
  • UpadhyayR.K.GuptaA.SoniD.GargR.PathreU.V.NathP.2017Ectopic expression of a tomato DREB gene affects several ABA processes and influences plant growth and root architecture in an age-dependent mannerJ. Plant Physiol.21497107

    • Search Google Scholar
    • Export Citation
  • WangJ.J.WangH.B.DingL.SongA.P.ShenF.JiangJ.F.2017Transcriptomic and hormone analyses reveal mechanisms underlying petal elongation in Chrysanthemum morifolium ‘Jinba’Plant Mol. Biol.936593606

    • Search Google Scholar
    • Export Citation
  • WangY.G.FuF.L.YuH.Q.HuT.ZhangY.Y.TaoY.2018Interaction network of core ABA signaling components in maizePlant Mol. Biol.96245263

  • WangZ.Y.HeJ.X.2004Brassinosteroid signal transduction-choices of signals and receptorsTrends Plant Sci.99196

  • YamamuraY.TaguchiY.IchitaniK.UmebaraI.OhshitaA.KurosakiF.2018Characterization of ent-kaurene synthase and kaurene oxidase involved in gibberellin biosynthesis from Scoparia dulcisJ. Nat. Med.72456463

    • Search Google Scholar
    • Export Citation
  • YanA.WuM.YanL.HuR.AliI.GanY.2014AtEXP2 is involved in seed germination and abiotic stress response in ArabidopsisPLoS One91E85208

  • YinY.WangZ.Y.Mora-GarciaS.LiJ.YoshidaS.AsamiT.2002BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongationCell109181191

    • Search Google Scholar
    • Export Citation
  • ZhangJ.TianJ.TaiD.Q.LiK.T.ZhuY.J.YaoY.C.2016An optimized TRV-based virus-induced gene silencing protocol for Malus crabapplePlant Cell Tissue Organ Cult.126499509

    • Search Google Scholar
    • Export Citation
  • ZhuJ.Y.Sae-SeawJ.WangZ.Y.2013Brassinosteroid signallingDevelopment14016151620

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