Next Article in Journal
Phenotyping Brown Rot Susceptibility in Stone Fruit: A Literature Review with Emphasis on Peach
Previous Article in Journal
Bruise Damage and Quality Changes in Impact-Bruised, Stored Tomatoes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 7
Issue 5
10.3390/horticulturae7050114
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phenotypic Characterization and Differential Gene Expression Analysis Reveal That Dwarf Mutant dwf Dwarfism Is Associated with Gibberellin in Eggplant

by
Yang Lu
1,2,†,
Shuangxia Luo
1,†,
Qiang Li
1,
Na Li
1,
Wenchao Du
1,
Ping Yu
1,
Xing Wang
1,
Weiwei Zhang
3,
Shuxin Xuan
1,
Xuan Zhou
1,
Jiajia Shen
1,
Jianjun Zhao
1,
Yanhua Wang
1,
Xueping Chen
1,* and
Shuxing Shen
1,*
1
Key Laboratory for Vegetable Germplasm Enhancement and Utilization of Hebei, Collaborative Innovation Center of Vegetable Industry in Hebei, College of Horticulture, Hebei Agricultural University, Baoding 071000, China
2
Hebei University R&D Center for Sericulture and Specialty Enabling Technologies, Institute of Sericulture, Chengde Medical University, Chengde 067000, China
3
College of Life Sciences, Hebei Agricultural University, Baoding 071000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2021, 7(5), 114; https://doi.org/10.3390/horticulturae7050114
Submission received: 26 March 2021 / Revised: 7 May 2021 / Accepted: 13 May 2021 / Published: 16 May 2021
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

:
Dwarfism is a desirable trait in eggplant breeding, as it confers higher lodging resistance and allows simplified management and harvest. However, a few dwarf mutants have been reported, and the molecular mechanism underlying dwarfism in eggplant is completely unknown. Here, we report a dwarf mutant (dwf) isolated from an ethyl methyl sulfonate (EMS)-induced mutant library. The hypocotyl length, plant height, and length of internode cells of dwf were significantly decreased compared to those of the wild-type parent ‘14-345’ (WT). Differential gene expression analysis revealed that GA-related genes, including GA2ox and DELLA, were up-regulated whereas the gibberellin (GA3) content decreased in dwf. Moreover, exogenous GA3 treatment significantly increased the relative growth rate of dwf compared to WT, further indicating the important roles of GA in regulating the dwarf phenotype of dwf. Collectively, our findings shed light on GA-mediated dwarfism in dwf plants and offer a good germplasm that could be used for eggplant dwarfism breeding in the future.

1. Introduction

Eggplant (Solanum melongena L.) is a vegetable crop that is cultivated worldwide and belongs to the genus Solanum in the Solanaceae family. Currently, China is the largest producer of eggplant, with 39.2 million tons and 783,000 ha in 2019 [1]. The vast majority of eggplant varieties have an indeterminate in growth habit and are not suitable for planting at high density. Moreover, the production of indeterminate eggplants is labor intensive.
Dwarfism is an important agronomic trait for crops and is highly associated with agricultural performance, such as higher lodging resistance, yield and harvest index [2,3,4]. Dwarfism or semidwarfism is widely used in plant breeding, such as wheat, rice, and woody species [3,4,5]. The determinate or dwarf varieties make easier management and harvest, with considerable cost savings [6,7]. Therefore, dwarfism is also an important breeding trait in eggplant.
Dwarfism is tightly regulated by plant hormones, among which gibberellin is a crucial signaling molecule involved in dwarfism that has been extensively studied in plants [8,9,10,11]. Genes involved in GA biosynthesis and degradation, including ent-kaurene oxidase (KO) [12], GA 20-oxidase (GA20ox) [13], GA 3-oxidase (GA3ox) [14,15] and GA 2-oxidase (GA2ox) [16], play important roles in determining dwarfism by regulating endogenous bioactive GA contents. One remarkable example is the loss-of-function of GA20ox-2 leading to the rice ‘Green Revolution’ [13]. GIBBERELLIN INSENSITIVE DWARF1 (GID1), which acts as the GA receptor in the signaling pathway, has been demonstrated to control the dwarf phenotype in wheat [17], peach [18] and rice [19]. DELLA is a negative regulator that controls repressors of GA signaling [20,21]. Previous studies showed that if DELLA combines with GAs and GID1 to form a DELLA-GA-GID1 complex, the inhibition of DELLA can be eliminated, and the GA signal can be transmitted downstream [22]. Mutations of DELLA proteins, which impair GA-promoted protein degradation and plant growth, have been documented for many crops including rice [23], barley [24] and tomato [25].
To date, dwarf and semidwarf mutants with short internode lengths have been widely reported for many crops [26,27]. However, few of the dwarf mutants have been documented for eggplant. The creation of new dwarf germplasms and identification of new dwarf genes that can be used for dwarfism breeding in eggplant is a great need. The completion and improvement of eggplant genome sequencing [28], as well as the rapid development of gene cloning, provides convenience for research on the dwarfing mechanism of eggplant.
In this study, we isolated a dwarf mutant dwf obtained from a mutant library in the inbred line ‘14-345’ treated with EMS. We characterized the dwf by performing the plant height and histological comparisons between dwf and WT. We analyzed RNA-seq data and endogenous levels of indole-3-acetic acid (IAA), brassinosteroid (BR), zeatin riboside (ZR) and GA3 from young leaves as well as the effects of exogenous GA3 on plant growth of the dwf and WT. Our results imply a tight relationship between the dwarf phenotype of dwf and GA metabolism in eggplant. The objective of this work is to clarify the dwarfing mechanism of dwf and accelerate dwarf breeding of eggplant.

2. Materials and Methods

2.1. Plant Materials and Growth Condition

The dwarf mutant dwf was isolated from a population generated by EMS mutagenesis from the inbred line ‘14-345’. The dwf mutant was selected at the M2 generation. All seedlings of the dwf mutant and WT were grown in the greenhouse of Hebei Agricultural University in the fall of 2018.

2.2. Plant Height Measurement

The plant height of dwf and WT was measured at the stage when the first true leaf was fully expanded, as well as 30 and 50 days after transplanting (DAT). Measurement was performed using a ruler. Three biological replicates were conducted, each with five plants per genotype.

2.3. Histological Analysis

Longitudinal sections of stems were analyzed by paraffin sectioning. Briefly, the stem samples between the 3rd and 4th leaves were collected when the fourth true leaf was fully expanded and then fixed in FAA (70% ethyl alcohol: glacial acetic acid: formaldehyde = 16:1:1, v/v/v) for more than 24 h. After dehydration and infiltration, all tissues were embedded in paraffin, and 8 μm sections were prepared (Leica RM2145). The sections were affixed to microscope slides, stained with fast green, and observed under a stereomicroscope (DP74, OLYMPUS). Three sections of one sample and approximately 100 cells were observed in our study.

2.4. Hormone Assay

Approximately 0.5 g of leaves at the six-leaf stage was used to determine the contents of GA3, IAA, BR and ZR. Sample treatment and hormone measurement were performed using an enzyme-linked immunosorbent assay (ELISA) as described previously [29].

2.5. RNA-seq Analysis

Young leaves of dwf and WT were collected, frozen quickly in liquid nitrogen and then stored at −80 °C. Total RNA was extracted using an EASTEP Super Total RNA Kit (Promega, Shanghai, China) according to the manufacturer’s instructions, and three biological replicates were designed for each genotype. The quality of RNA was checked by determining the RNA integrity and concentration using RNase-free 1% agarose gel electrophoresis, an RNA Assay Kit in a Qubit2.0 Fluorometer (Life Technologies, South San Francisco, CA, USA) and RNA Nano6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). cDNA library preparation and sequencing were conducted by the Novogene Technology Company in Beijing, China. Sequencing libraries were generated using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, lpswich, MA, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample [30,31]. After generating 125 bp/150 bp paired-end reads sequencing on an Illumina Hiseq platform, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N and low quality reads from raw data aligned to the eggplant reference genome [28] using Hisat2 v2.0.5 [30]. The gene expression level was normalized by the fragments per kilobase of exon per million mapped reads (FPKM) value using the feature Counts v1.5.0-p3 [32]. Differential gene expression analysis was performed using the DESeq2 R package (1.16.1) [33]. Genes with adjusted padj < 0.05 and |log2foldchange| > 1 were determined to be significantly differentially expressed genes (DEGs). Gene Ontology (GO) enrichment was performed using the cluster Profiler R package [34]. GO terms with corrected p-value less than 0.05 were considered significantly enriched.

2.6. Validation of Selected DEGs Using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)

The RNA samples used for RNA-Seq were also used for qRT-PCR analysis. First-strand cDNA was synthesized using RevertAid Premium Reverse Transcriptase (Thermo Scientific™ EP0733) according to the manufacturer’s instructions. GAPDH (GenBank: JX448342.1) was used as the internal control [35], and primer sequences for all genes analyzed are listed in Table S1. The qRT-PCRs reactions were run in a Lightcycler 96 Real-time qPCR detection system (Roche, Indianapolis, IN, USA) with three biological replicates for each type of sample. The program used was: 5 min at 95 °C; 40 cycles of 95 °C (10 s); 58 °C (10 s); 72 °C (30 s). The 2−ΔΔCt method was used to calculate the relative expression levels of the target genes [36].

2.7. Exogenous GA3 Treatments

The shoot tips of dwf and WT at four-true leaf stage were both sprayed with 100 and 200 mg/L GA3. The treatments were continued for seven days with one application per day. The plants treated with water were used as control. The measurement of plant height was performed before treatments and at the 2nd day of the last application of GA3 and water. Increment (plant height after treatment-plant height before treatment) and relative growth rate (Increment/plant height before treatment) were investigated. Three biological replicates were performed to evaluate the plant height, each with five plants per genotype.

2.8. Statistical Analysis

Statistical analyses were performed using Student’s t-test (SPSS21.0) [37]. All data represent mean ± SD. Asterisks denote significant differences (* p < 0.05; ** p < 0.01) as determined by Student’s t-tests.

3. Results

3.1. Phenotypic Comparison of the dwf Mutant and WT

The dwf plant was found in the M2 generation, which showed a dwarfism phenotype (Figure 1A). To further characterize the dwf mutant, we compared the plant height of the dwf mutant and WT at three developmental stages, which were all significantly decreased compared to that of the WT (Figure 1B). Moreover, the plant height difference between dwf and WT increased with the development of plants (Figure 1B). In addition, we investigated the cell length of the internode, which was significantly reduced by 18.82% in dwf (Figure 1C,D).

3.2. Analysis of the Contents of GA3, IAA, BR, and ZR

Given that phytohormones play essential roles in regulating plant height, we further investigated the contents of IAA, BR, GA3 and ZR in dwf and WT. A significant reduction in GA3 content was observed in dwf (Figure 2A), whereas the contents of IAA, BR and ZR did not significantly differ between dwf and WT (Figure 2B–D). These results suggested that GA3 might be associated with dwarfism in dwf plants.

3.3. DEGs and GO Enrichment Analysis

To further characterize the dwf mutant, we performed RNA-seq experiments using total RNA isolated from young leaves of the dwf mutant and WT. The samples were analyzed in three independent biological replicates (six RNA-Seq libraries in total). Summary statistics for each of the RNA-Seq libraries are shown in Table 1. A total of 171.12 and 146.31 million raw reads were obtained from the dwf mutant and WT, respectively. After filtering and removing adapter sequences, low quality reads, and ribosomal reads, 167.54 and 143.52 million clean reads were obtained for the dwf mutant and WT, respectively. More than 90% of the clean reads were mapped to the reference genome (Table 1). The Pearson correlation coefficient analysis showed high reproducibility between the three replicates, ranging from 0.907 to 0.988 (Table S2).
DEG analysis indicated that there were 2682 DEGs between the dwf mutant and WT. Of these, there were 1226 down-regulated and 1456 up-regulated genes in the dwf mutant (Figure 3A; Table S3).
To explore the functions of the DEGs, GO enrichment analysis of biological process (BP) was performed. The top twelve up-regulated GO pathways indicated that DEGs were significantly enriched in pathways related to pollen and reproductive (Figure 3B), whereas the top twenty GO pathways indicated that the down-regulated DEGs were enriched for many cellular-related processes, such as ‘microtubule-based processes’, ‘nuclear division’, ‘cell wall macromolecule catabolic/metabolic process’ and ‘cell cycle process’ (Figure 3C). These results implied that the mutant gene, which is responsible for the dwarfism phenotype of the dwf mutant, had a significant impact on the global gene expression profile in eggplant, especially for genes related to cellular processes.

3.4. DEGs in GA Biosynthesis and Signal Transduction Pathways

Considering that the endogenous GA3 level in dwf was significantly decreased compared to that in WT, we examined the DEGs involved in GA biosynthesis and signal transduction pathways. Four DEGs involved in GA biosynthesis and signal transduction pathways were found. Of these, three genes (Sme2.5_04516.1_g00001.1, Sme2.5_00108.1_g00003.1 and Sme2.5_01907.1_g00015.1) encode GA2oxs and one gene (Sme2.5_00538.1_g00012.1) encodes the DELLA protein. The function of GA2ox is to decrease the levels of active GAs by catalyzing 2β-hydroxylation, and DELLA proteins act as negative regulators in GA signal transduction [38]. Interestingly, the four genes were all significantly up-regulated in dwf, and the expression of these genes showed similar expression patterns in qRT-PCR (Figure 4). The data suggest that the three genes GA2ox and DELLA might play important roles in regulating dwarfism in the dwf mutant.

3.5. The Effects of Exogenous GA3 on dwf and WT Plant Growth

Based on the aforementioned findings, we speculated that the plant height of dwf could be restored by exogenous GA3. Under control conditions, dwf displayed a significantly reduced plant height and increment compared to WT. Interestingly, under both 100 mg/L and 200 mg/L GA3 treatment, dwf significantly increased the relative growth rate compared to WT, whereas the plant height increment of dwf and WT was not different (Figure 5). These findings indicated that exogenous GA3 can partially restore the plant height of dwf.

4. Discussion

Dwarf mutants generally show internode shortening, which is closely correlated with the cell number, cell length and intercellular space of internodes [26,39,40,41]. In the present study, internode cell length of dwf was significantly decreased compared to WT, which could account for, at least partially, the dwarfism of dwf.
Previous studies showed that GA is one of the most important plant hormones in regulating plant height [42,43,44]. GA2ox plays a key role in GA homeostasis by converting active GAs to inactive forms; modification (mutation or overexpression) of GA2ox can decrease GA levels [45] and lead to a dwarf phenotype in many crops, such as Arabidopsis [46], rice [47] and tomato [48]. In the dwf mutant, several genes encoding GA2oxs were up-regulated (the left three of Figure 4) and the content GA3 decreased significantly in dwf compared to WT (Figure 2), indicating that the increased expression of GA2ox promoted GA deactivation in dwf, leading to a low level of GA3 which inhibited internode elongation.
GA-related dwarf mutants are divided into two classes based on their phenotypic response to exogenous GA. One class is GA-deficient mutants, in which the GA content is significantly lower than that of the wild type, and the phenotype of this class of mutants can be rescued by exogenous application of GAs [49]. Several GA-deficient mutants have been found in rice [50], cotton [51] and maize [14]. In our study, GA3 was significantly reduced in the dwf compared to WT. Meanwhile, we found that the dwarf phynotype of dwf could partially restore by exogenous GA3 treatment, and that the plant height increment of dwf after GA treatment increased significantly. These results indicated that the mutant dwf was responsive to GA3.
The other class of GA dwarf mutants is GA-insensitive mutants, the phenotype of which cannot be rescued by exogenous GAs. This class of mutants is mainly caused by mutations in DELLA proteins that act as suppressors of the GA signaling pathway [52]. Gain-of-function or overexpression of DELLA genes gives rise to dwarfism [53]. Studies have shown that mutations or modifications of genes involved in GA synthesis and signaling pathways can lead to dwarfism or semidwarfism [54,55]. However, the up-regulation Sme2.5_00538.1_g00012.1 gene encoding DELLA in dwf seemed to provide evidence that dwf is a GA-insensitive mutant. When the GA level decreased, DELLA will bound the transcription factor (TF) and maintained inhibition, which affected stem growth [56,57], proving the phenomenon that occurred in dwf plants.
As discussed above, dwf is a GA-related dwarf mutant, and the genes Sme2.5_04516.1_g00001.1, Sme2.5_00108.1_g00003.1, Sme2.5_01907.1_g00015.1 encode GA2oxs and Sme2.5_00538.1_g00012.1 encodes DELLA were mainly responsible for dwarfism. The mutant dwf which may offer a good germplasm that could be used for eggplant dwarfism breeding in future.

5. Conclusions

In this study, we isolated a dwarf mutant (dwf), the hypocotyl length, plant height, and length of internode cells of dwf were significantly decreased compared to those of the WT. The GA-related genes, including GA2ox and DELLA were up-regulated expression whereas the GA3 content decreased in the dwf. We treated the seedlings of the WT and dwf with exogenous GA3, and found that the relative growth rate of dwf significantly increased compared to that of WT, indicated that the dwf was a GA-mediated mutant. Our findings offer a good germplasm that could be used for eggplant dwarfism breeding in the future.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/horticulturae7050114/s1.

Author Contributions

Conceptualization, Y.L., S.L. and X.C.; methodology, Y.L., W.D., P.Y. and S.X.; validation, Y.L., S.L. and X.C.; formal analysis, Y.L. and X.W.; investigation, X.Z., J.S. and W.Z.; resources, X.C., J.Z., S.L. and Y.W.; data curation, Y.L. and X.C.; writing—original draft preparation, Y.L. and S.L.; writing—review and editing, Y.L., S.L., Q.L., N.L., J.Z. and X.C.; visualization, Y.L.; supervision, X.C. and S.S.; project administration, X.C. and S.S.; funding acquisition, X.C. and S.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Vegetable Innovation Team Project of the Hebei Modern Agricultural Industrial Technology System (Grant No. HBCT2018030203); the Science and Technology Plan Project of Hebei Province (Grant No. 16226308D); the Key R & D Project of Hebei Province (Grant No. 20326344D); and The Innovation Ability Training Project for Graduate Students of Hebei Province (Grant No. CXZZBS2018114). Technology Innovation Guidance Project-Science and Technology Work Conference of Hebei Provincial Department of Science and Technology.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained in the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Available online: http://www.fao.org/faostat/en/#data (accessed on 10 December 2020).
  2. Hedden, P. The genes of the green revolution. Trends Genet. 2003, 19, 5–9. [Google Scholar] [CrossRef]
  3. Peng, J.R.; Richards, D.E.; Hartley, N.M.; Murphy, G.P.; Devos, K.M.; Flintham, J.E.; Beales, J.; Fish, L.J.; Worland, A.J.; Pelica, F. ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 1999, 400, 256–261. [Google Scholar] [CrossRef]
  4. Sasaki, A.; Ashikari, M.; Ueguchi-Tanaka, M.; Itoh, H.; Nishimura, A.; Swapan, D.; Ishiyama, K.; Saito, T.; Kobayashi, M.; Khush, G.S.; et al. A mutant gibberellin-synthesis gene in rice. Nature 2002, 416, 701–702. [Google Scholar] [CrossRef] [PubMed]
  5. Elias, A.A.; Busov, V.B.; Kosola, K.R.; Ma, C.; Etherington, E.; Shevchenko, O.; Gandhi, H.; Pearce, D.W.; Rood, S.B.; Strauss, S.H. Green Revolution Trees: Semi-dwarfism transgenes modify gibberellins, promote root growth, enhance morphological diversity, and reduce competitiveness in hybrid poplar. Plant Physiol. 2012, 160, 1130–1144. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Jia, Q.J.; Li, C.D.; Shang, Y.; Zhu, J.H.; Hua, W.; Wang, J.M.; Yang, J.M.; Zhang, G.P. Molecular characterization and functional analysis of barley semi-dwarf mutant Riso no. 9265. BMC Genom. 2015, 16, 927–938. [Google Scholar] [CrossRef] [Green Version]
  7. Sun, X.R.; Liu, L.; Zhi, X.N.; Bai, J.R.; Cui, Y.N.; Shu, J.S.; Li, J.M. Genetic analysis of tomato internode length via mixed major gene plus polygene inheritance model. Sci. Hortic. 2019, 246, 759–764. [Google Scholar] [CrossRef]
  8. Santner, A.; Calderon-Villalobos, L.I.A.; Estelle, M. Plant hormones are versatile chemical regulators of plant growth. Nat. Chem. Biol. 2009, 5, 301–307. [Google Scholar] [CrossRef]
  9. Depuydt, S.; Hardtke, C.S. Hormone signaling crosstalk in plant growth regulation. Curr. Biol. 2011, 21, 365–373. [Google Scholar] [CrossRef]
  10. Hu, S.L.; Wang, C.L.; Sanchez, D.L.; Lipka, A.E.; Liu, P.; Yin, Y.H.; Blanco, M.; Lübberstedt, T. Gibberellins promote brassinosteroids action and both increase heterosis for plant height in maize (Zea mays L.). Front. Plant Sci. 2017, 8, 1–17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  11. Xing, M.M.; Su, H.N.; Liu, X.; Yang, L.M.; Zhang, Y.Y.; Wang, Y.; Fang, Z.Y.; Lv, H.H. Morphological, transcriptomics and phytohormone analysis shed light on the development of a novel dwarf mutant of cabbage (Brassica oleracea). Plant Sci. 2020, 290, 110283. [Google Scholar] [CrossRef] [PubMed]
  12. Itoh, H.; Tatsumi, T.; Sakamoto, T.; Otomo, K.; Toyomasu, T.; Kitano, H.; Ashikari, M.; Ichihara, S.; Matsuoka, M. A rice semi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase. Plant Mol. Biol. 2004, 54, 533–547. [Google Scholar] [CrossRef] [PubMed]
  13. Oikawa, T.; Koshioka, M.; Kojima, K.; Yoshida, H.; Kawata, M. A role of OsGA20ox1, encoding an isform of gibberellin 20-oxidase, for regulation of plant stature in rice. Plant Mol. Biol. 2004, 55, 687–700. [Google Scholar] [CrossRef]
  14. Chen, Y.; Hou, M.M.; Liu, L.J.; Wu, S.; Shen, Y.; Ishiyama, K.; Kobayashi, M.; McCarty, D.R.; Tan, B.C. The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol. Plant Physiol. 2014, 166, 2028–2039. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Itoh, H.; Ueguchi-Tanaka, M.; Sentoku, N.; Kitano, H.; Matsuoka, M.M.K. Cloning and functional analysis of two gibberellin 3β-hydroxylase genes that are differently expressed during the growth of rice. Proc. Natl. Acad. Sci. USA 2001, 98, 8909–8914. [Google Scholar] [CrossRef] [Green Version]
  16. Hu, Y.X.; Tao, Y.B.; Xu, Z.F. Overexpression of jatropha gibberellin 2-oxidase 6 GA2ox6) induces dwarfism and smaller leaves, flowers and fruits in Arabidopsis and Jatropha. Front. Plant Sci. 2017, 8, 2103. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Li, A.X.; Yang, W.L.; Li, S.J.; Liu, D.C.; Guo, X.L.; Sun, J.Z.; Zhang, A.M. Molecular characterization of three GIBBERELLIN-INSENSITIVE DWARF1 homologous genes in hexaploid wheat. J. Plant Physiol. 2013, 170, 432–443. [Google Scholar] [CrossRef]
  18. Cheng, J.; Zhang, M.M.; Tan, B.; Jiang, Y.J.; Zheng, X.B.; Ye, X.; Guo, Z.J.; Xiong, T.T.; Wang, W.; Li, J.D.; et al. A signal nucleotide mutation in GID1c disrupts its interaction with DELLA1 and causes a GA-insensitive dwarf phenotype in peach. Plant Biotechnol. J. 2019, 17, 1723–1735. [Google Scholar] [CrossRef] [Green Version]
  19. Piao, R.H.; Chu, S.H.; Jiang, W.Z.; Yu, Y.Y.; Jin, Y.M.; Woo, M.; Lee, J.; Kim, S.; Koh, H. Isolation and characterization of a dominant dwarf gene, d-h, in Rice. PLoS ONE 2014, 9, e86210. [Google Scholar] [CrossRef]
  20. Achard, P.; Gusti, A.; Cheminant, S.; Alioua, M.; Dhondt, S.; Coppens, F.; Beemster, G.T.S.; Genschik, P. Gibberellin signaling control cell proliferation rate in Arabidopsis. Curr. Boil. 2009, 19, 1188–1193. [Google Scholar] [CrossRef]
  21. Arro, J.; Yang, Y.Z.; Song, G.Q.; Zhong, G.Y. RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines. BMC Plant Biol. 2019, 19, 80. [Google Scholar] [CrossRef]
  22. Harberd, N.P.; Belfifield, E.; Yasumura, Y. The angiosperm gibberellin-GID1-DELLA growth regulatory mechanism: How an “inhibitor of an inhibitor” enables flflexible response to flfluctuating environments. Plant Cell 2009, 21, 1328–1339. [Google Scholar] [CrossRef] [Green Version]
  23. Itoh, H.; Sasaki, A.; Ueguchi-Tanaka, M.; Ishiyama, K.; Kobayashi, M.; Hasegawa, Y.; Minami, E.; Ashikari, M.; Matsuoka, M. Dissection of the phosphorylation of rice DELLA protein, SLENDER RICE1. Plant Cell Physiol. 2005, 46, 1392–1399. [Google Scholar] [CrossRef] [PubMed]
  24. Fu, X.; Richards, D.E.; Ait-Ali, T.; Hynes, L.W.; Ougham, H.; Peng, J.; Harberd, N.P. Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor. Plant Cell 2002, 14, 3191–3200. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Carrera, E.; Ruiz-Rivero, O.; Peres, L.E.P.; Atares, A.; Garcia-Martinez, J.L. Characterization of the procera tomato mutant shows novel functions of the SlDELLA protein in the control of flower morphology, cell division and expansion, and the auxin-signaling pathway during fruit-set and development. Plant Physiol. 2012, 160, 1581–1596. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Miao, H.M.; Li, C.; Duan, Y.H.; Wei, L.B.; Ju, M.; Zhang, H.Y. Identification of a Sidwf1 gene controlling short internode length trait in the sesame dwarf mutant dw607. Theor. Appl. Genet. 2020, 133, 73–86. [Google Scholar] [CrossRef] [PubMed]
  27. Sun, Y.Y.; Zhang, H.Q.; Fan, M.; He, Y.J.; Guo, P.A. A mutation in the intron splice acceptor site of a GA3ox gene confers dwarf architecture in watermelon (Citrullus lanatus L.). Sci. Rep. 2020, 10, 14915. [Google Scholar] [CrossRef]
  28. Hirakawa, H.; Shirasawa, K.; Miyatake, K.; Nunome, T.; Negoro, S.; Ohyama, A.; Yamaguchi, H.; Sato, S.; Isobe, S.; Tabata, S.; et al. Draft genome sequence of eggplant (Solanum melongena L.): The representative solanum species indigenous to the old world. DNA Res. 2014, 21, 649–660. [Google Scholar] [CrossRef] [Green Version]
  29. Deng, A.X.; Tan, W.M.; He, S.P.; Liu, W.; Nan, T.G.; Li, Z.H.; Wang, B.M.; Li, Q.X. Monoclonal antibody-based enzyme linked immunosorbent assay for the analysis of jasmonates in plants. J. Integr. Plant Biol. 2008, 50, 1046–1052. [Google Scholar] [CrossRef]
  30. Kim, D.; Langmead, B.; Salzberg, S.L. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 2015, 12, 357–360. [Google Scholar] [CrossRef] [Green Version]
  31. Trapnell, C.; Pachter, L.; Salzberg, S.L. TopHat: Discovering splice junctions with RNA-Seq. Bioinformatics 2009, 25, 1105–1111. [Google Scholar] [CrossRef]
  32. Liao, Y.; Smyth, G.K.; Shi, W. feature Counts: An efficient general purpose program for assigning sequence reads to genomicfeatures. Bioinformatics 2014, 30, 923–930. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [Green Version]
  34. Tarazona, S.; García-Alcalde, F.; Dopazo, J.; Ferrer, A.; Conesa, A. Differential expression in RNA-seq: A matter of depth. Genome Res. 2011, 21, 2213–2223. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Barbierato, V.; Sala, T.; Rinaldi, P.; Bassolino, L.; Barchi, L.; Rotino, G.L.; Toppino, L. A spiking strategy facilitates housekeeping selection for RT-qPCR analysis under different biotic stresses in eggplant. Protoplasma 2017, 254, 2215–2223. [Google Scholar] [CrossRef] [PubMed]
  36. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  37. Mishra, P.; Singh, U.; Pandey, C.M.; Mishra, P.; Pandey, G. Application of student’s t-test, analysis of variance, and covariance. Annu. Card. Anaesth. 2019, 22, 407–411. [Google Scholar] [CrossRef]
  38. Yamaguchi, S. Gibberellin metabolism and its regulation. Annu. Rev. Plant Biol. 2008, 59, 225–251. [Google Scholar] [CrossRef] [PubMed]
  39. Chen, L.M.; Yang, H.L.; Fang, Y.S.; Guo, W.; Chen, H.F.; Zhang, X.J.; Dai, W.J.; Chen, S.L.; Hao, Q.N.; Yuan, S.L.; et al. Overexpression of GmMYB14 improves high-density yield and drought tolerance of soybean through regulating plant architecture mediated by the brassinosteroid pathway. Plant Biotechnol. J. 2020. [Google Scholar] [CrossRef]
  40. Li, C.; Tang, J.; Hu, Z.Y.; Wang, J.W.; Yu, T.; Yi, H.Y.; Cao, M.J. A novel maize dwarf mutant generated by Ty1-copia LTR-retrotransposon insertion in Brachytic2 after spaceflight. Plant Cell Rep. 2020, 39, 393–408. [Google Scholar] [CrossRef]
  41. Sun, L.H.; Yang, W.L.; Li, Y.F.; Shan, Q.Q.; Ye, X.B.; Wang, D.Z.; Yu, K.; Lu, W.W.; Xin, P.Y.; Pei, Z.; et al. A wheat dominant dwarfing line with Rht12, which reduces stem cell length and affects gibberellic acid synthesis, is a 5AL terminal deletion line. Plant J. 2019, 97, 887–900. [Google Scholar] [CrossRef]
  42. Lee, J.; Moon, S.; Jang, S.; Lee, S.; An, S.; Jung, K.-H.; Park, S.K. OsbHLH073 Negatively Regulates Internode Elongation and Plant Height by Modulating GA Homeostasis in Rice. Plants 2020, 9, 547. [Google Scholar] [CrossRef] [Green Version]
  43. Wang, Y.J.; Zhao, J.; Lu, W.J.; Deng, D.X. Gibberellin in plant height control: Old player, new story. Plant Cell Rep. 2017, 36, 391–398. [Google Scholar] [CrossRef] [PubMed]
  44. Chu, Y.L.; Xu, N.; Wu, Q.; Yu, B.; Li, X.X.; Chen, R.R.; Huang, J.L. Rice transcription factor OsMADS57 regulates plant height by modulating gibberellin catabolism. Rice 2019, 12, 38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Schomburg, F.M.; Bizzell, C.M.; Lee, D.J.; Zeevaart, J.A.D.; Amasino, R.M. Overexpression of a novel class of gibberellin 2-Oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell 2002, 15, 151–163. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Magome, H.; Yamaguchi, S.; Hanada, A.; Kamiya, Y.; Oda, K. The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in Arabidopsis. Plant J. 2008, 56, 613–626. [Google Scholar] [CrossRef]
  47. Huang, J.; Tang, D.; Shen, Y.; Qin, B.X.; Hong, L.L.; You, A.Q.; Li, M.; Wang, X.; Yu, H.X.; Gu, M.H.; et al. Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.). JGG 2010, 37, 23–36. [Google Scholar] [CrossRef]
  48. Sun, X.R.; Shu, J.S.; Mohamed, A.M.A.; Deng, X.B.; Zhi, X.N.; Bai, J.R.; Cui, Y.N.; Lu, X.X.; Du, Y.C.; Wang, X.X.; et al. Identification and characterization of EI (Elongated Internode) gene in tomato (Solanum lycopersicum). Int. J. Mol. Sci. 2019, 20, 2204. [Google Scholar] [CrossRef] [Green Version]
  49. Bishop, G.J.; Nomura, T.; Yokota, T.; Harrison, K.; Noguchi, T.; Fujioka, S.; Takatsuto, S.; Jones, J.D.G.; Kamiya, Y. The tomato DWARF enzyme catalyzes C-6 oxidation in brassinosteroid biosynthesis. Proc. Natl. Acad. Sci. USA 1999, 96, 1761–1766. [Google Scholar] [CrossRef] [Green Version]
  50. Ji, S.H.; Gururani, M.A.; Lee, J.W.; Ahn, B.O.; Chun, S.C. Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis. Plant Biol. 2014, 16, 428–443. [Google Scholar] [CrossRef]
  51. Chen, X.S.; Di, J.C.; Xu, N.Y.; Xiao, S.H.; Liu, J.G. The inheritance of an ultra -dwarf plant mutant from upland cotton. Hereditas 2007, 29, 471–498. [Google Scholar] [CrossRef]
  52. Willige, B.C.; Ghosh, S.; Nill, C.; Zourelidou, M.; Dohmann, E.M.N.; Maier, A.; Schwechheimer, C. The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell 2007, 19, 1209–1220. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Lawit, S.J.; Wych, H.M.; Xu, D.P.; Kundu, S.; Tomes, D.T. Maize DELLA proteins dwarf plant8 and dwarf plant9 as modulators of plant development. Plant Cell Physiol. 2010, 51, 1854–1868. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Sakamoto, T.; Miura, K.; Itoh, H.; Tatsumi, T.; Ueguchi-tanaka, M.; Ishiyama, K.; Kobayashi, M.; Agrawal, G.K.; Takeda, S.; Abe, K.; et al. An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol. 2004, 134, 1642–1653. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Wu, Z.G.; Tang, D.; Liu, K.; Miao, C.B.; Zhuo, X.X.; Li, Y.F.; Tan, X.L.; Sun, M.F.; Luo, Q.; Cheng, Z.K. Characterization of a new semi-dominant dwarf allele of SLR1 and its potential application in hybrid rice breeding. J. Exp. Bot. 2018, 69, 4703–4713. [Google Scholar] [CrossRef] [PubMed]
  56. Davière, J.M.; Lucas, M.D.; Prat, S. Transcriptional factor interaction: A central step in DELLA function. Curr. Opin. Genet. Dev. 2008, 18, 295–303. [Google Scholar] [CrossRef]
  57. Davière, J.M.; Achard, P. Gibberellin signaling in plants. Development 2013, 140, 1147–1151. [Google Scholar] [CrossRef] [Green Version]
Horticulturae 07 00114 g001 550
Figure 1. Phenotypic analysis of WT and dwf. (A) Plants of WT and dwf at the fruiting stage. (B) Plant height of WT and dwf at the three developmental stages. I, II and III represent the first true leaf stage, at 30 and 50 DAT. (C) Representative longitudinal sections of stems of WT and dwf. Scale bars = 100 μm. (D) Cell length comparison of WT and dwf. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05), ** indicates Student’s t-test, p < 0.01.
Figure 1. Phenotypic analysis of WT and dwf. (A) Plants of WT and dwf at the fruiting stage. (B) Plant height of WT and dwf at the three developmental stages. I, II and III represent the first true leaf stage, at 30 and 50 DAT. (C) Representative longitudinal sections of stems of WT and dwf. Scale bars = 100 μm. (D) Cell length comparison of WT and dwf. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05), ** indicates Student’s t-test, p < 0.01.
Horticulturae 07 00114 g001
Horticulturae 07 00114 g002 550
Figure 2. The contents of GA3, IAA, BR and ZR in WT and dwf. (A) The GA3 content. (B) The IAA content. (C) The BR content. (D) The ZR content. The four hormones are measured in ng/g·FW. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05).
Figure 2. The contents of GA3, IAA, BR and ZR in WT and dwf. (A) The GA3 content. (B) The IAA content. (C) The BR content. (D) The ZR content. The four hormones are measured in ng/g·FW. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05).
Horticulturae 07 00114 g002
Horticulturae 07 00114 g003 550
Figure 3. Comparisons and biological process GO enrichment of DEGs between WT and dwf. (A) Numbers of up-regulated and down-regulated DEGs. (B) Up-regulated DEGs enriched in thirteen significant BP pathways. (C) Down-regulated DEGs enriched in the top twenty significant BP pathways.
Figure 3. Comparisons and biological process GO enrichment of DEGs between WT and dwf. (A) Numbers of up-regulated and down-regulated DEGs. (B) Up-regulated DEGs enriched in thirteen significant BP pathways. (C) Down-regulated DEGs enriched in the top twenty significant BP pathways.
Horticulturae 07 00114 g003
Horticulturae 07 00114 g004 550
Figure 4. qRT-PCR verified the results of RNA-seq. The gene expression levels were normalized using the relative expression level of the internal control GAPDH, and wild-type expression levels were normalized to 1. The data presented are the means from three biological replicates. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05).
Figure 4. qRT-PCR verified the results of RNA-seq. The gene expression levels were normalized using the relative expression level of the internal control GAPDH, and wild-type expression levels were normalized to 1. The data presented are the means from three biological replicates. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05).
Horticulturae 07 00114 g004
Horticulturae 07 00114 g005 550
Figure 5. Effect of exogenous GA3 treatment on plant growth. (A) Treatment with water. (B) Treatment with 100 mg/L GA3. (C) Treatment with 200 mg/L GA3. In each figure of Plant height, blue indicates plant height before treatment, and yellow indicates the increment. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05).
Figure 5. Effect of exogenous GA3 treatment on plant growth. (A) Treatment with water. (B) Treatment with 100 mg/L GA3. (C) Treatment with 200 mg/L GA3. In each figure of Plant height, blue indicates plant height before treatment, and yellow indicates the increment. * indicates significant differences, compared with the WT (Student’s t-test, p < 0.05).
Horticulturae 07 00114 g005
Table
Table 1. Summary of read numbers in WT and dwf.
Table 1. Summary of read numbers in WT and dwf.
Sample Namedwf-1dwf-2dwf-3WT-1WT-2WT-3
Raw reads58,570,41656,724,56055,828,51448,299,03248,653,90649,359,746
Clean reads57,610,86054,994,77054,936,47447,392,08447,418,85848,711,482
Mapped reads52,295,23149,904,30649,978,93442,883,33742,975,64444,637,625
Ratio of mapped reads (%)90.7790.7490.9890.4990.6391.64
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Lu, Y.; Luo, S.; Li, Q.; Li, N.; Du, W.; Yu, P.; Wang, X.; Zhang, W.; Xuan, S.; Zhou, X.; et al. Phenotypic Characterization and Differential Gene Expression Analysis Reveal That Dwarf Mutant dwf Dwarfism Is Associated with Gibberellin in Eggplant. Horticulturae 2021, 7, 114. https://doi.org/10.3390/horticulturae7050114

AMA Style

Lu Y, Luo S, Li Q, Li N, Du W, Yu P, Wang X, Zhang W, Xuan S, Zhou X, et al. Phenotypic Characterization and Differential Gene Expression Analysis Reveal That Dwarf Mutant dwf Dwarfism Is Associated with Gibberellin in Eggplant. Horticulturae. 2021; 7(5):114. https://doi.org/10.3390/horticulturae7050114

Chicago/Turabian Style

Lu, Yang, Shuangxia Luo, Qiang Li, Na Li, Wenchao Du, Ping Yu, Xing Wang, Weiwei Zhang, Shuxin Xuan, Xuan Zhou, and et al. 2021. "Phenotypic Characterization and Differential Gene Expression Analysis Reveal That Dwarf Mutant dwf Dwarfism Is Associated with Gibberellin in Eggplant" Horticulturae 7, no. 5: 114. https://doi.org/10.3390/horticulturae7050114

APA Style

Lu, Y., Luo, S., Li, Q., Li, N., Du, W., Yu, P., Wang, X., Zhang, W., Xuan, S., Zhou, X., Shen, J., Zhao, J., Wang, Y., Chen, X., & Shen, S. (2021). Phenotypic Characterization and Differential Gene Expression Analysis Reveal That Dwarf Mutant dwf Dwarfism Is Associated with Gibberellin in Eggplant. Horticulturae, 7(5), 114. https://doi.org/10.3390/horticulturae7050114

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop