Next Article in Journal
Enzyme Activities in the Lignin Metabolism of Chinese Olive (Canarium album) with Different Flesh Characteristics
Previous Article in Journal
Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 8
Issue 5
10.3390/horticulturae8050407
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 thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

RhAGL24 Regulating Auxin-Related Gene RhARF18 Affects Stamen Petaloidy in Rose

by
Lin Liu
,
Yanchao Guo
,
Zhicheng Wu
,
Haoran Ren
,
Yunhe Jiang
,
Nan Ma
,
Junping Gao
and
Xiaoming Sun
*
Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, College of Horticulture, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(5), 407; https://doi.org/10.3390/horticulturae8050407
Submission received: 8 April 2022 / Revised: 23 April 2022 / Accepted: 27 April 2022 / Published: 6 May 2022
(This article belongs to the Section Developmental Physiology, Biochemistry, and Molecular Biology)
Browse Figures
Review Reports Versions Notes

Abstract

:
AGAMOUS-LIKE 24 (AGL24) is a key gene regulating floral transition, but its involvement in flower organ identity remains largely unknown. In this study, we found that RhAGL24 is strongly related to petal and stamen development in rose. Its expression increases rapidly at the petal primordium development stage and maintains a high level until the complete differentiation stage. RhAGL24 silencing increases the number of malformed petals and decreases the number of stamens, indicating that this gene affects stamen petaloidy. RhAG (AGAMOUS), a class C gene associated with petal and stamen development, is downregulated in RhAGL24-silenced plants. Moreover, we found that RhAGL24 could directly bind to the promoter region of RhARF18 (AUXIN RESPONSE FACTORS 18), a regulator of RhAG. Our results suggested that RhAGL24-RhARF18 module regulates stamen petaloidy in rose and provide new insights into the function of AGL24 for plants.

1. Introduction

Stamen petaloidy occurs in roses, and the number of petals and stamens is related to ornamental value. Stamen petaloidy increases the diversity and appreciation of rose varieties. Studies on stamen petaloidy have great important biological significance and industrial value. At present, many reports about flower organ development are available, but studies on the mechanism of stamen petaloidy in roses are rare.
Floral organ development has always been a hot topic in plant research. According to the familiar ABCDE model, flowers have four whorls, namely, sepals, petals, stamens, and carpels arranged from the outside to the inside [1]. Four genes, namely, AGAMOUS (AG), APETALA2 (AP2), APETALA3 (AP3), and PISTILLATA (PI), can affect floral organ development but not the location of primordia and are expressed at different locations in cells, resulting in different states of differentiation. The functions of these genes also have overlapping fields in the concentric circles of flower primordia [2]. AP2 plays a role in the two outer whorls, PI and AP3 are involved in the second and third whorls of development, and AG functions in the two inner whorls. The combined gene products determine the four organ types of wild-type flowers. The products of AG and AP2 genes have antagonistic effects. AP2 inhibits AG gene expression in the outer two whorls and AG inhibits AP2 gene expression in the inner whorls [3]. SEUSS (SEU) and LEUNIG (LUG) negatively regulate AG gene expression in the first two whorls of flowers [4]. lug ag double mutations have the same phenotype as ag single mutations, and AG is epistatic to LUG [5]. Stamen-specific patterns are dependent on LUG but not on AP2 [6]. AP3 and PI play a redundant role in floral meristem formation [7]. AP3/PI interacts with other factors in early flower development to directly inhibit AP1 expression [8].
In addition, floral organ development is mainly related to auxin. Auxin plays a key role in flower primordium development [9]. The auxin transport protein PIN-FORMED1 (PIN1) is a transmembrane protein with polar localization and participates in auxin efflux. The inflorescence of the mutant pin1 usually has no flower [10,11]. However, exogenous auxin application on inflorescence of tomato pin1 can induce the formation of floral primordia [12]. This suggests that auxin is required for normal development of floral primordia. The phenotypes of pinoid (pid) and monopteros mutants are similar to the pin1, and neither can produce normal flower buds [13,14]. Auxin response factors (ARFs) are a family of transcription factors that specifically bind to auxin elements to regulate downstream gene expression [15,16]. The auxin response factor family has many members with different functions [17,18]. For example, ETTIN/ARF3 is involved in floral meristem determinism [19], and the mutation of ETTIN/ARF3 can enhance the phenotype of ag mutants [20,21]. ARF4 regulates flowering time [22]. MONOPTEROS/ARF5 promotes bud formation [23] and participates in the development of male and female gametes [24]. ARF18 inhibits auxin transduction [25], regulates angular wall development [26], affects petal and stamen development, and negatively regulates RhAG expression [27]. Significant differences in auxin-related gene expression are also found between the single and double flower transcriptomes of apple species [28].
The flowering pathway is a complex regulatory network that is influenced by various factors. FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), and LEAFY (LFY) are three important regulatory factors in Arabidopsis thaliana [29,30]. Located in inflorescence and floral meristems, AGL24 mediates the communication among these three genes and is activated during floral transition. AGL24 is a transcription factor that dose-dependently promotes flowering, and its overexpression leads to early flowering [31]. AGL24 and SOC1 have similar functions of stimulating flowering and are upregulated through vernalization or by gibberellin to form a positive feedback loop [29]. By contrast, AGL24 mRNA levels are not affected by FLC, so AGL24 is a FLC-independent target in the vernalization pathway [32]. SHORT VEGETATIVE PHASE (SVP), a gene with extremely high homology to AGL24, exhibits completely opposite effects. Although both are important regulatory factors in flower transformation, SVP is the suppressive factor of flowering, and AGL24 is the promoter of flowering [31]. In svp agl24 double mutants, SVP is epistatic to AGL24, and the number and characteristics of flower organs are affected. Yeast hybridization experiments showed that the dimer of AP1-SVP or AP1-AGL24 could bind to LUG-SEU to inhibit AG activity. Moreover, the phenotype of the triple-mutant ap1 agl24 svp is more distinct than that of agl24 svp, suggesting that the three genes have functional redundancy in AG regulation during the early stage of flower development [33].
AGL24 has been studied as a regulator of flowering time in many species, but its relationship with floral organ identity is rarely reported. Here, we found that RhAGL24 is highly expressed during petal and stamen primordia development. Therefore, we aimed to explore the function and regulatory mechanism of RhAGL24 in petal and stamen development. We discovered a RhAGL24-RhARF18 module that regulates stamen petaloidy in rose and provided new insights into the function of AGL24 for plants.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Sterile tissue culture seedlings of Rosa hybrida ‘Samantha’ were grown on Murashige and Skoog (MS) medium containing plant hormones [34]. Upon growing stems and leaves, the tissue culture seedlings were supplemented with the hormone ratio of 1.0 mg L−1 6-benzylaminopurine (6-BA), 0.01 mg L−1 α-naphthalene acetic acid (NAA), and 3 mg L−1 gibberellin acid (GA3). After 1 month of growth, the specimens were transferred to the rooting medium of 1/2 MS medium supplemented with 0.1 mg L−1 NAA. After 1 month of growth, the tissue culture seedlings took root and were transplanted into a pot with a 1:1 mixture of peat and vermiculite. Nicotiana benthamiana was grown under the same conditions as rooting roses. Both plants were reared at 22 ± 1 °C under a 16 h light and 8 h dark photoperiod.

2.2. RNA Extraction and Quantitative RT-PCR Analysis

Total RNA of rose was extracted by thermal borate method [35]. In the reverse transcription experiment, the HiScriptⓇ II Q RT SuperMix for qPCR (+gDNA Wiper) kit (Vazyme, Nanjing, China) was used for 1 μg RNA reverse transcription into cDNA in accordance with the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was performed with StepOnePlus™ Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the KAPA SYBR FAST Universal qRT-PCR kit (Kapa Biosystems, Boston, MA, USA). The specific test steps are carried out in accordance with the instructions. Relative gene expression was calculated by the 2−ΔΔCT method [36]. RhUBI2 gene with stable expression was selected as internal control [37], and the primers used for qRT-PCR were designed in NCBI [38] as shown in Table S1.

2.3. Cloning and Sequence Analysis

NCBI and Primer Premier 5.0 software were employed for primer design (Table S1) using the sequence of RchiOBHm_Chr7g0205071 from ‘Old Blush’ genome as the reference [39]. The open reading frame (ORF) of RhAGL24 was cloned from the cDNA of ‘Samantha’ flower bud and subsequently corrected. The promoter of RhARF18 was cloned and corrected in the same way. The amino acid sequence of RhAGL24 was translated by an online translate website [40]. The protein conserved domains were predicted with NCBI. The cis-acting element was predicted by JASPAR [41], and the cis-acting element on the RhARF18 promoter was detected with FIMO [42].
The protein sequences of target genes were downloaded from NCBI and saved in fasta format. MEGA software [43] was downloaded, Alignment by ClustalW protein sequence was selected for multiple sequence alignment results, and the model with the lowest BIC score was chosen. Phylogeny was selected to construct the evolutionary tree using the neighbor-joining method at 1000 bootstraps.

2.4. Virus-Induced Gene Silencing

Virus-induced gene silencing (VIGS) was performed as previously described [44]. pTRV2-RhAGL24 vector was constructed through the homologous recombination of a specific RhAGL24 gene fragment of 150 bp ORF region and 215 bp 3′-untranslated region linked to pTRV2 using EcoR I and BamH I restriction sites. pTRV1, pTRV2, and pTRV2-RhAGL24 were transformed into Agrobacterium tumefaciens GV3101. Primer sequences are shown in Table S1. pTRV1 and pTRV2 with an absorbance OD600 of 1.0 were mixed as control, and pTRV1 and pTRV2-RhAGL24 were mixed as experimental group and placed in the dark at room temperature for 3 h. More than 100 rooting rose plants were equally incorporated in the two agrobacterium mixtures and placed in a vacuum of −25 kPa for 10 min, and this process was repeated once. Afterward, the plants were washed with deionized water and left in the dark at 8 °C for 3 d before being transplanted to pots with a 1:1 mixture of peat and vermiculite. The plants were then grown at 22 ± 1 °C under 16 h light and 8 h dark photoperiod for 40 d.

2.5. Yeast One-Hybrid Assay and Double Luciferase Assay

pGADTA-RhAGL24 and pAbAi-proRhARF18 vectors were constructed as previously reported [45]. First, the pAbAi-proRhARF18 vector was transformed into Y1H yeast strain and coated into SD/-Ura medium. Different Aureobasidin A (AbA) concentrations were prepared and analyzed to identify the one that could inhibit the activity of pAbAi-proRhARF18. pGADT7-RhAGL24 vector was then transformed into pAbAi-proRhARF18 yeast strain to observe whether it could grow on SD/-Ura/-Leu medium with the above AbA concentration.
In the transactivation experiment of RhAGL24, proRhARF18:LUC reporter plasmid was constructed by inserting the short fragment of RhARF18 promoter into the pGreenII 0800-LUC vector upstream of LUC gene. The RhAGL24 sequence was inserted into pGreenII 62-SK vector to construct pro35S:RhAGL24 plasmid, which was transformed into A. tumefaciens GV3101 containing pSoup plasmid. proRhARF18:LUC (OD600 = 1) and pGreenII 62-SK empty vector were mixed as the negative control, and proRhARF18:LUC and pro35S:AGL24 vector were used as experimental group. For injection, 1-month-old tobacco was selected. After 3 days, fluorescein was applied to the back of the leaves. A CCD imaging device (CHEMIPROHT 1300B/LND, 16-bit; Roper Scientific) was used to capture LUC images at −110 °C. The sequences of relevant primers are shown in Table S1.

2.6. Statistical Analysis

Statistical analysis of all data with at least three biological replicates was performed using student’s t test in GraphPad Prism 8.0.2 software [46]. Significant differences were denoted by p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

3. Results

3.1. Phylogenetic Relationship and Conservation of AGL24 Genes

To investigate the molecular regulatory mechanisms of flower development, we statistically analyzed the differentially expressed genes from transcriptome sequencing of rose organ primordia development at different stages. We found that RchiOBHm_Chr7g0205071 expression was low in the sepal primordia development stage but increased rapidly in the petal and stamen primordia development stage. We identified the gene as RhAGL24 according to the reference genome [39], designed specific primers for this gene using Primer Premier 5.0 software by referring to the ‘Old Blush’ genome and cloned RhAGL24 gene from the cDNA of R. hybrida ‘Samantha’. RhAGL24 has an ORF length of 678 bp and encodes 225 amino acids.
To identify the genetic relationship between RhAGL24 and other species, we downloaded 29 AGL24 protein sequences from NCBI and constructed a phylogenetic tree. The results showed that RhAGL24 has the closest relation to strawberry in Rosaceae (Figure 1A). To study the conserved domain of RhAGL24, we used NCBI as a reference and predicted that this gene has two conserved domains, namely, MADS box and K box region. Multi-sequence comparison with AGL24 of A. thaliana and other species revealed the specific sequence and location of the two conserved domains (Figure 1B). The MADS box family comprises many members and plays an important role in the development of many plant organs, including fruits, roots, stems, leaves, and flowers. As an important member of the MADS family, we further studied RhAGL24 and its role in plant development.

3.2. Expression Analysis of RhAGL24 in Different Developmental Stages and Organs

AGL24 is involved in flower development in plants, especially during transition from vegetative to reproductive stages in Arabidopsis. However, we found significant changes in RhAGL24 expression during the early rose primordia stage. Early rosa development is divided into eight stages: vegetative development; prophase of transition from vegetative growth to reproductive growth; transition from vegetative growth to reproductive growth; sepal primordia differentiation; petal primordia differentiation; stamen primordia differentiation; pistil primordia differentiation; and completion (stages 1 to 8) (Figure 2A). We found that RhAGL24 expression increased along the development stage, from being at a low level in the vegetative to sepal primordium differentiation stages to suddenly increasing at the petal primordium differentiation stage and remaining at a high level throughout the subsequent development stages (Figure 2A). In addition, we detected RhAGL24 expression in various rose organs. The results showed that RhAGL24 gene had the highest expression in leaf organs such as leaves and sepals, had a high expression in flowers (including petals, stamens, and pistils), and had a low expression in roots and stems (Figure 2B). On the basis of these results, RhAGL24 is highly expressed in petals, stamens, and pistils during floral organ development and later flower development. Therefore, RhAGL24 may play an important role in the formation and development of petals, stamens, and pistils.

3.3. Silencing RhAGL24 Affected the Number of Petals and Stamens in Rose

To test the role of RhAGL24 in organ development, we silenced RhAGL24 in rose plants using VIGS technique. The results showed that RhAGL24 expression was significantly reduced in TRV-RhAGL24 lines (Figure 3B) and decreased by 60% in TRV-RhAGL24 plants compared with that in the control. Two changes occurred in the RhAGL24-silenced lines: the number of petals increased, and the number of stamens decreased (Figure 3B).
According to the morphology of rose petals, we can classify them into normal petals (NPs) and malformed petals (MPs) (Figure 3A). NPs are basically symmetrical on both sides, are relatively intact or have slightly asymmetric left and right sides with concave tips and no stamens. MPs are also asymmetrical on both sides but have distinct stamens. We counted the number of NPs, MPs, stamens, and pistils. Compared with that in the control group, the total petal number increased significantly after RhAGL24 silencing (Figure 3A). The number of NPs was almost the same but that of MPs increased significantly, the number of stamens decreased but not significantly (Figure 3C). The number of pistils did not change significantly. These results suggested that RhAGL24 is involved in flower organ development. From a macro point of view, silencing RhAGL24 increases the number of petals and decreases the number of stamens. However, when the petals are categorized, RhAGL24 affects only the number of MPs. Given its high expression in stamen-containing organs (Figure 2B), RhAGL24 may affect the number of petals and stamens by inhibiting the homologous transformation of stamens to petals.

3.4. RhAGL24 Affects the Expression of RhAG and Auxin-Related Genes

Flower development is an extremely complex process and involves many genes, such as AP1, AP2, AP3, PI, and AG, as indicated in the famous ABCDE model [3]. Multiple genes jointly regulate and form a flower development network. The AG of class C gene plays an important role in the development of petals and stamens [47]. In A. thaliana ag mutants, almost no stamens exist because they transform into petals. Most of the genes affecting the number of petals and stamens regulate AG expression and thus cause phenotypic changes. Therefore, we first detected RhAG expression levels in TRV and TRV-RhAGL24 lines. The results showed that RhAG was significantly down-regulated in TRV-RhAGL24 plants (Figure 4). We detected the similarity between the fragment of RhAGL24 used for VIGS and RhAG gene, and ruled out the possibility that this fragment could silence RhAG. Therefore, the results show that RhAGL24 may directly or indirectly affect RhAG expression and thus regulate petal and stamen development.
Plant hormones play an important role in plant development; for example, auxin regulates floral organ development [15,48]. The single and double petal transcriptomes of Malus showed significant difference in auxin-related gene expression [28]. ARF3 is involved in floral meristem determinism, and ARF18 regulates AG transcription [19,27]. Therefore, we selected four auxin-related genes (ARF3, ARF5, ARF18, and PID) related to flower organ development and detected their expression levels in TRV and TRV-RhAGL24 lines. The results showed that in RhAGL24-silenced plants, RhARF3 and RhPID expression decreased, RhARF5 expression did not change significantly, and RhARF18 expression increased (Figure 4). Therefore, we hypothesized that RhAGL24 changes the number of petals and stamens by affecting the expression of many flower development genes and auxin-related genes and participating in the network of flower organ development.

3.5. RhAGL24 Inhibits RhARF18 Transcriptional Activity in Rose

Given that RhAGL24 is a transcription factor, we predicted a recognizable cis-acting element CArG sequence using an online software JASPAR (Figure 5A). We used FIMO to predict the auxin-related gene promoter on the cis-acting element. The results showed that only the promoter of RhARF18 had three CArG sequences; the element in the promoter of the other genes was not predicted (Figure 5B). Hence, we cloned and corrected the promoter sequences of the gene. We applied yeast one-hybrid assay to detect whether RhAGL24 could bind to the RhARF18 promoter in vitro. Considering the three predicted binding sites, we cut the promoter into three segments named P1, P2, and P3 (Figure 5B) with one promoter each. Experimental results showed that when the AbA concentration was 300 ng ml−1, the RhAGL24 protein could bind to RhARF18 promoter P1 and activate the reporter gene expression. However, the other two promoters are self-activated and could not be inhibited by AbA (Figure 5C). Therefore, we need to explore other ways to determine whether these two sites are recognized and bound by RhAGL24. These results suggested that RhAGL24 recognizes and binds to the RhARF18 promoter in yeast and regulates RhARF18 expression at the transcriptional level.
We further verified the above finding by conducting double luciferase assay. The results showed that the fluorescence of RhAGL24 and RhARF18 promoters simultaneously injected was significantly lower than that of the individual RhARF18 promoters injected alone (Figure 5D). Therefore, RhAGL24 inhibits the transcriptional activity of RhARF18 promoter, thus affecting RhARF18 expression and changing the number of petals and stamens.

4. Discussion

Along with SVP, SOC1, LFY, and other genes, AGL24 affects the transformation of plants from the vegetative stage to the reproductive stage, thus influencing the flowering time [30,31]. In addition, this gene is associated with inflorescence development and plays a role in inflorescence determinism [49]. However, AGL24 in floral organ development has rarely been investigated. In this study, we found that RhAGL24 is highly expressed during petal and stamen primordia development and later flower organ development. Its silencing in rose significantly increases the number of petals, decreases the number of stamens, and enhances stamen petaloidy. Rosaceae plants have many double petals, resulting from stamen petaloidy. AGL24 expression also significantly differs in the single and double flower transcriptomes of Malus and is significantly decreased in double flower [28], a result consistent with the present findings. This work provides a new basis for the double petal phenomenon of Rosaceae and offers new directions and theoretical support for the breeding of new rose varieties.
Plant hormones participate in various plant development, and auxin plays an important role in flower organ development [50]. At present, many auxin-related genes have been studied in relation to flower organ development [9]. Different auxin-related genes are enriched in the above-mentioned Malus transcriptome. For example, double-petal plants have increased ARF18-LIKE expression and decreased ARF4-LIKE expression [28]. We previously found that the number of petals decreased after silencing RhARF18 in rose and that RhARF18 could regulate RhAG expression [27]. After the mutation of serine threonine kinase PID, the petals increase in number and appear as a filiform, the sepals and stamens are reduced, and fusion is exhibited by each flower organ [13,48]. In the present study, we found that auxin-related genes were also changed in pTRV2-RhAGL24 strains. For example, RhARF3 and RhPID were downregulated, and RhARF18 was upregulated. This finding suggests that auxin-related genes are involved in the homologous transformation of stamens and petals during flower organ development. In addition, yeast one-hybrid and double luciferase assay showed that RhAGL24 could inhibit regulate RhARF18, which in turn could suppress RhAG transcription. Therefore, the present study complements the regulatory network of petal and stamen development.
The regulatory network of floral organ development is complex and many of its pathways remain undiscovered. Hence, additional experiments are needed to reveal these pathways.

5. Conclusions

RhAGL24 expression increased rapidly at petal primordium development stage and maintained a high level until the complete differentiation stage. This gene also plays an important role in the later flower organ development. Its silencing increased the number of MPs and decreased the number of stamens, indicating that RhAGL24 affects stamen petaloidy. In RhAGL24-silenced plants, RhAG was also downregulated. Moreover, RhAGL24 inhibited the transcriptional activity of the promoter of RhARF18, a regulator of RhAG. Our results suggested that RhAGL24-RhARF18 module regulates stamen petaloidy in rose. This work provides new insights into the function of AGL24 for plants and serves as a reference for the breeding of new rose varieties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8050407/s1, Table S1: Primers used for the experiment.

Author Contributions

Conceptualization, J.G. and X.S.; methodology, Y.G. and H.R.; software, Z.W.; formal analysis, X.S.; investigation, L.L.; data curation, L.L.; writing—original draft preparation, L.L.; writing—review and editing, X.S., Y.J., N.M. and J.G.; funding acquisition, J.G. and X.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant number 31902059, Yunnan academician expert workstation, grant number 202105AF150036, Science and Technology Program of Yunnan Province, grant number 202102AE090001.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We sincerely thank all colleagues who made suggestions on this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Coen, E.S.; Meyerowitz, E.M. The war of the whorls: Genetic interactions controlling flower development. Nature 1991, 353, 31–37. [Google Scholar] [CrossRef] [PubMed]
  2. Bowman, J.L.; Smyth, D.R.; Meyerowitz, E.M. Genes directing flower development in Arabidopsis. Plant Cell 1989, 1, 37–52. [Google Scholar] [PubMed] [Green Version]
  3. Bowman, J.L.; Smyth, D.R.; Meyerowitz, E.M. Genetic interactions among floral homeotic genes of Arabidopsis. Development 1991, 112, 1–20. [Google Scholar] [CrossRef] [PubMed]
  4. Franks, R.G.; Wang, C.; Levin, J.Z.; Liu, Z. SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG. Development 2002, 129, 253–263. [Google Scholar] [CrossRef] [PubMed]
  5. Liu, Z.; Meyerowitz, E.M. LEUNIG regulates AGAMOUS expression in Arabidopsis flowers. Development 1995, 121, 975–991. [Google Scholar] [CrossRef]
  6. Deyholos, M.K.; Sieburth, L.E. Separable whorl-specific expression and negative regulation by enhancer elements within the AGAMOUS second intron. Plant Cell 2000, 12, 1799–1810. [Google Scholar] [CrossRef] [Green Version]
  7. Krizek, B.A.; Meyerowitz, E.M. The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function. Development 1996, 122, 11–22. [Google Scholar] [CrossRef]
  8. Sundström, J.F.; Nakayama, N.; Glimelius, K.; Irish, V.F. Direct regulation of the floral homeotic APETALA1 gene by APETALA3 and PISTILLATA in Arabidopsis. Plant J. 2006, 46, 593–600. [Google Scholar] [CrossRef]
  9. Cheng, Y.; Zhao, Y. A role for auxin in flower development. J. Integr. Plant Biol. 2007, 49, 99–104. [Google Scholar] [CrossRef]
  10. Gälweiler, L.; Guan, C.; Müller, A.; Wisman, E.; Mendgen, K.; Yephremov, A.; Palme, K. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 1998, 282, 2226–2230. [Google Scholar] [CrossRef] [Green Version]
  11. Wisniewska, J.; Xu, J.; Seifertová, D.; Brewer, P.B.; Ruzicka, K.; Blilou, I.; Rouquié, D.; Benková, E.; Scheres, B.; Friml, J. Polar PIN localization directs auxin flow in plants. Science 2006, 312, 883. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Reinhardt, D.; Mandel, T.; Kuhlemeier, C. Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 2000, 12, 507–518. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Bennett, R.; Alvarez, J.; Bossinger, G.; Smyth, D. Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J. 1995, 8, 505–520. [Google Scholar] [CrossRef]
  14. Przemeck, G.K.; Mattsson, J.; Hardtke, C.S.; Sung, Z.R.; Berleth, T. Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 1996, 200, 229–237. [Google Scholar] [CrossRef] [PubMed]
  15. Tiwari, S.B.; Hagen, G.; Guilfoyle, T. The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell 2003, 15, 533–543. [Google Scholar] [CrossRef] [Green Version]
  16. Guilfoyle, T.J.; Hagen, G. Auxin response factors. Curr. Opin. Plant Biol. 2007, 10, 453–460. [Google Scholar] [CrossRef]
  17. Okushima, Y.; Mitina, I.; Quach, H.L.; Theologis, A. AUXIN RESPONSE FACTOR 2 (ARF2): A pleiotropic developmental regulator. Plant J. 2005, 43, 29–46. [Google Scholar] [CrossRef]
  18. Lim, P.O.; Lee, I.C.; Kim, J.; Kim, H.J.; Ryu, J.S.; Woo, H.R.; Nam, H.G. Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity. J. Exp. Bot. 2010, 61, 1419–1430. [Google Scholar] [CrossRef]
  19. Zhang, K.; Wang, R.Z.; Zi, H.L.; Li, Y.P.; Cao, X.W.; Li, D.M.; Guo, L.; Tong, J.H.; Pan, Y.Y.; Jiao, Y.L.; et al. AUXIN RESPONSE FACTOR3 Regulates Floral Meristem Determinacy by Repressing Cytokinin Biosynthesis and Signaling. Plant Cell 2018, 30, 324–346. [Google Scholar] [CrossRef] [Green Version]
  20. Finet, C.; Fourquin, C.; Vinauger, M.; Berne-Dedieu, A.; Chambrier, P.; Paindavoine, S.; Scutt, C.P. Parallel structural evolution of auxin response factors in the angiosperms. Plant J. 2010, 63, 952–959. [Google Scholar] [CrossRef]
  21. Liu, X.G.; Dinh, T.T.; Li, D.M.; Shi, B.H.; Li, Y.P.; Cao, X.W.; Guo, L.; Pan, Y.Y.; Jiao, Y.L.; Chen, X.M. AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy. Plant J. 2014, 80, 629–641. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Dong, X.X.; Li, Y.J.; Guan, Y.H.; Wang, S.X.; Luo, H.; Li, X.M.; Li, H.; Zhang, Z.H. Auxin-induced AUXIN RESPONSE FACTOR4 activates APETALA1 and FRUITFULL to promote flowering in woodland strawberry. Hortic. Res. 2021, 8, 1–12. [Google Scholar] [CrossRef] [PubMed]
  23. Ckurshumova, W.; Smirnova, T.; Marcos, D.; Zayed, Y.; Berleth, T. Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation. N. Phytol. 2014, 204, 556–566. [Google Scholar] [CrossRef] [PubMed]
  24. Liu, Z.N.; Miao, L.M.; Huo, R.X.; Song, X.Y.; Johnson, C.; Kong, L.J.; Sundaresan, V.; Yu, X.L. ARF2-ARF4 and ARF5 are Essential for Female and Male Gametophyte Development in Arabidopsis. Plant Cell Physiol. 2018, 59, 179–189. [Google Scholar] [CrossRef]
  25. Jia, Y.B.; Kong, X.P.; Hu, K.Q.; Cao, M.Q.; Liu, J.J.; Ma, C.L.; Guo, S.Y.; Yuan, X.Z.; Zhao, S.; Robert, H.S.; et al. PIFs coordinate shade avoidance by inhibiting auxin repressor ARF18 and metabolic regulator QQS. N. Phytol. 2020, 228, 609–621. [Google Scholar] [CrossRef]
  26. Liu, J.; Hua, W.; Hu, Z.Y.; Yang, H.L.; Zhang, L.; Li, R.J.; Deng, L.B.; Sun, X.C.; Wang, X.F.; Wang, H.Z. Natural variation in ARF18 gene simultaneously affects seed weight and silique length in polyploid rapeseed. Proc. Natl. Acad. Sci. USA 2015, 112, E5123–E5132. [Google Scholar] [CrossRef] [Green Version]
  27. Chen, J.; Li, Y.; Li, Y.; Li, Y.; Wang, Y.; Jiang, C.; Choisy, P.; Xu, T.; Cai, Y.; Pei, D.; et al. AUXIN RESPONSE FACTOR 18–HISTONE DEACETYLASE 6 module regulates floral organ identity in rose (Rosa hybrida). Plant Physiol. 2021, 186, 1074–1087. [Google Scholar] [CrossRef]
  28. Gul, H.; Tong, Z.G.; Han, X.L.; Nawaz, I.; Wahocho, S.A.; Khan, S.; Zhang, C.X.; Tian, Y.; Cong, P.H.; Zhang, L.Y. Comparative Transcriptome Analysis between Ornamental Apple Species Provides Insights into Mechanism of Double Flowering. Agronomy 2019, 9, 112. [Google Scholar] [CrossRef] [Green Version]
  29. Lee, J.; Oh, M.; Park, H.; Lee, I. SOC1 translocated to the nucleus by interaction with AGL24 directly regulates. LEAF Plant J. 2008, 55, 832–843. [Google Scholar] [CrossRef]
  30. Liu, C.; Chen, H.; Er, H.L.; Soo, H.M.; Kumar, P.P.; Han, J.H.; Liou, Y.C.; Yu, H. Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis. Development 2008, 135, 1481–1491. [Google Scholar] [CrossRef] [Green Version]
  31. Yu, H.; Xu, Y.F.; Tan, E.L.; Kumar, P.P. AGAMOUS-LIKE 24, a dosage-dependent mediator of the flowering signals. Proc. Natl. Acad. Sci. USA 2002, 99, 16336–16341. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Michaels, S.D.; Ditta, G.; Gustafson-Brown, C.; Pelaz, S.; Yanofsky, M.; Amasino, R.M. AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. Plant J. 2003, 33, 867–874. [Google Scholar] [CrossRef] [PubMed]
  33. Gregis, V.; Sessa, A.; Colombo, L.; Kater, M.M. AGL24, SHORT VEGETATIVE PHASE, and APETALA1 Redundantly Control AGAMOUS during Early Stages of Flower Development in Arabidopsis. Plant Cell 2006, 18, 1373–1382. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Chen, C.X.; Hussain, N.; Wang, Y.R.; Li, M.T.; Liu, L.; Qin, M.Z.; Ma, N.; Gao, J.P.; Sun, X.M. An Ethylene-inhibited NF-YC Transcription Factor RhNF-YC9 Regulates Petal Expansion in Rose. Hortic. Plant J. 2020, 6, 419–427. [Google Scholar] [CrossRef]
  35. Zhang, S.; Feng, M.; Chen, W.; Zhou, X.; Lu, J.; Wang, Y.; Li, Y.; Jiang, C.Z.; Gan, S.S.; Ma, N.; et al. In rose, transcription factor PTM balances growth and drought survival via PIP2;1 aquaporin. Nat. Plants 2019, 5, 290–299. [Google Scholar] [CrossRef]
  36. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T) (-Delta Delta C) method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  37. Meng, Y.; Li, N.; Tian, J.; Gao, J.; Zhang, C. Identification and validation of reference genes for gene expression studies in postharvest rose flower (Rosa hybrida). Sci. Hortic. 2013, 158, 16–21. [Google Scholar] [CrossRef] [Green Version]
  38. Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T.L. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 2012, 13, 134. [Google Scholar] [CrossRef] [Green Version]
  39. Raymond, O.; Gouzy, J.; Just, J.; Badouin, H.; Verdenaud, M.; Lemainque, A.; Vergne, P.; Moja, S.; Choisne, N.; Pont, C.; et al. The Rosa genome provides new insights into the domestication of modern roses. Nat. Genet. 2018, 50, 772–777. [Google Scholar] [CrossRef]
  40. Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, L.; Appel, R.D.; Bairoch, A. ExPasy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003, 31, 3784–3788. [Google Scholar] [CrossRef] [Green Version]
  41. Castro-Mondragon, J.A.; Riudavets-Puig, R.; Rauluseviciute, I.; Berhanu, L.R.; Turchi, L.; Blanc-Mathieu, R.; Lucas, J.; Boddie, P.; Khan, A.; Manosalva, P.N.; et al. JASPAR 2022: The 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2022, 50, D165–D173. [Google Scholar] [CrossRef] [PubMed]
  42. Grant, C.E.; Bailey, T.L.; Noble, W.S. FIMO: Scanning for occurrences of a given motif. Bioinformatics 2011, 27, 1017–1018. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  44. Tian, J.; Pei, H.; Zhang, S.; Chen, J.; Chen, W.; Yang, R.; Meng, Y.; You, J.; Gao, J.; Ma, N. TRV-GFP: A modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function. J. Exp. Bot. 2014, 65, 311–322. [Google Scholar] [CrossRef] [Green Version]
  45. Xu, Y.; Zhao, X.; Aiwaili, P.; Mu, X.; Zhao, M.; Zhao, J.; Cheng, L.; Ma, C.; Gao, J.; Hong, B. A zinc finger protein BBX19 interacts with ABF3 to affect drought tolerance negatively in chrysanthemum. Plant J. 2020, 103, 1783–1795. [Google Scholar] [CrossRef]
  46. Swift, M.L. GraphPad Prism, Data Analysis, and Scientific Graphing. J. Chem. Inf. Comput. Sci. 1997, 37, 411–412. [Google Scholar] [CrossRef]
  47. Mizukami, Y.; Ma, H. Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell 1992, 71, 119–131. [Google Scholar] [CrossRef]
  48. Pfluger, J.; Zambryski, P. The role of SEUSS in auxin response and floral organ patterning. Development 2004, 131, 4697–4707. [Google Scholar] [CrossRef] [Green Version]
  49. Smith, H.M.S.; Ung, N.; Lal, S.; Courtier, J. Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development. J. Exp. Bot. 2011, 62, 583–593. [Google Scholar] [CrossRef] [Green Version]
  50. Chandler, J.W. The Hormonal Regulation of Flower Development. J. Plant Growth Regul. 2011, 30, 242–254. [Google Scholar] [CrossRef]
Horticulturae 08 00407 g001 550
Figure 1. Conserved analysis of AGL24 protein sequence. (A) Phylogenetic evolutionary trees of AGL24 from different species; (B) comparison of multiple sequences of AGL24 proteins from different species. Conservative structures are represented above the alignment, and the amino acids that are the same for all species are represented underneath the alignment. At—Arabidopsis thaliana; Br—Brassica rapa; Rh—Rosa hybrida; Md—Malus domestica; Rs—Raphanus sativus; Bn—Brassica napus; Bo—Brassica oleracea; Cs—Camelina sativa; Fv—Fragaria vesca; Pb—Pyrus × bretschneideri; Pd—Prunus dulcis; Pm—Prunus mume; Th—Tarenaya hassleriana.
Figure 1. Conserved analysis of AGL24 protein sequence. (A) Phylogenetic evolutionary trees of AGL24 from different species; (B) comparison of multiple sequences of AGL24 proteins from different species. Conservative structures are represented above the alignment, and the amino acids that are the same for all species are represented underneath the alignment. At—Arabidopsis thaliana; Br—Brassica rapa; Rh—Rosa hybrida; Md—Malus domestica; Rs—Raphanus sativus; Bn—Brassica napus; Bo—Brassica oleracea; Cs—Camelina sativa; Fv—Fragaria vesca; Pb—Pyrus × bretschneideri; Pd—Prunus dulcis; Pm—Prunus mume; Th—Tarenaya hassleriana.
Horticulturae 08 00407 g001
Horticulturae 08 00407 g002 550
Figure 2. Expression analysis of RhAGL24 gene. (A) Relative expression levels of RhAGL24 at different developmental stages of floral organ primordia. One biological sample consisted of 10 floral buds at any stage. All the values are the means ± SD of three biological replicates. Stage 1—vegetative development; stage 2—prophase of transition from vegetative growth to reproductive growth; stage 3—transition from vegetative growth to reproductive growth; stage 4—sepal primordia differentiation; stage 5—petal primordia differentiation; stage 6—stamen primordia differentiation; stage 7—pistil primordia differentiation; stage 8—completion. (B) Relative expression levels of RhAGL24 in different plant organs. All the values are the means ± SD of three biological replicates. RhUBI2 was used as an internal control. Asterisks indicate statistically significant differences by Student’s t-test (* p < 0.05, ** p < 0.01 and *** p < 0.001).
Figure 2. Expression analysis of RhAGL24 gene. (A) Relative expression levels of RhAGL24 at different developmental stages of floral organ primordia. One biological sample consisted of 10 floral buds at any stage. All the values are the means ± SD of three biological replicates. Stage 1—vegetative development; stage 2—prophase of transition from vegetative growth to reproductive growth; stage 3—transition from vegetative growth to reproductive growth; stage 4—sepal primordia differentiation; stage 5—petal primordia differentiation; stage 6—stamen primordia differentiation; stage 7—pistil primordia differentiation; stage 8—completion. (B) Relative expression levels of RhAGL24 in different plant organs. All the values are the means ± SD of three biological replicates. RhUBI2 was used as an internal control. Asterisks indicate statistically significant differences by Student’s t-test (* p < 0.05, ** p < 0.01 and *** p < 0.001).
Horticulturae 08 00407 g002
Horticulturae 08 00407 g003 550
Figure 3. Silencing of RhAGL24 affects the number of petals and stamens. (A) Phenotype of TRV and TRV-RhAGL24 strains. (B) RhAGL24 expression in RhAGL24-silenced (TRV-RhAGL24) and TRV control plants as determined by qRT-PCR. (C) Statistics on the phenotypic data of TRV and TRV-RhAGL24 strains. NP—normal petal; MP—malformed petal; ST—stamen; PI—pistil. All the values are the means ± SD of seven biological replicates. RhUBI2 was used as an internal control. Asterisks indicate statistically significant differences by Student’s t-test (* p < 0.05, *** p < 0.001).
Figure 3. Silencing of RhAGL24 affects the number of petals and stamens. (A) Phenotype of TRV and TRV-RhAGL24 strains. (B) RhAGL24 expression in RhAGL24-silenced (TRV-RhAGL24) and TRV control plants as determined by qRT-PCR. (C) Statistics on the phenotypic data of TRV and TRV-RhAGL24 strains. NP—normal petal; MP—malformed petal; ST—stamen; PI—pistil. All the values are the means ± SD of seven biological replicates. RhUBI2 was used as an internal control. Asterisks indicate statistically significant differences by Student’s t-test (* p < 0.05, *** p < 0.001).
Horticulturae 08 00407 g003
Horticulturae 08 00407 g004 550
Figure 4. Expression of flower organ identity-related genes in RhAGL24-silenced plants. All the values are the means ± SD of seven biological replicates. RhUBI2 was used as an internal control. Asterisks indicate statistically significant differences by Student’s t-test (* p < 0.05, ** p < 0.01).
Figure 4. Expression of flower organ identity-related genes in RhAGL24-silenced plants. All the values are the means ± SD of seven biological replicates. RhUBI2 was used as an internal control. Asterisks indicate statistically significant differences by Student’s t-test (* p < 0.05, ** p < 0.01).
Horticulturae 08 00407 g004
Horticulturae 08 00407 g005 550
Figure 5. Validation of RhAGL24 downstream genes. (A) Prediction of RhAGL24 identifiable sequences. (B) Sequence analysis of RhARF18 promoter. The RhARF18 promoter was divided into three segments of P1, P2, and P3, with one predicted cis-acting element each. MA0555.1 is the cis-acting element of RhAGL24 in (A). (C) Yeast one-hybridization between RhAGL24 and RhARF18 promoter; 10−1, 10−2, and 10−3 are the concentrations of diluted yeast. (D) Dual luciferase assay of RhAGL24 and RhARF18 promoter. The proRhARF18:LUC construct was co-infiltrated with pro35S:RhAGL24 or SK empty vector into N. benthamiana leaves. The picture on the left shows the tobacco in the open field, and the picture on the right shows the tobacco in the fluorescent field.
Figure 5. Validation of RhAGL24 downstream genes. (A) Prediction of RhAGL24 identifiable sequences. (B) Sequence analysis of RhARF18 promoter. The RhARF18 promoter was divided into three segments of P1, P2, and P3, with one predicted cis-acting element each. MA0555.1 is the cis-acting element of RhAGL24 in (A). (C) Yeast one-hybridization between RhAGL24 and RhARF18 promoter; 10−1, 10−2, and 10−3 are the concentrations of diluted yeast. (D) Dual luciferase assay of RhAGL24 and RhARF18 promoter. The proRhARF18:LUC construct was co-infiltrated with pro35S:RhAGL24 or SK empty vector into N. benthamiana leaves. The picture on the left shows the tobacco in the open field, and the picture on the right shows the tobacco in the fluorescent field.
Horticulturae 08 00407 g005
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Liu, L.; Guo, Y.; Wu, Z.; Ren, H.; Jiang, Y.; Ma, N.; Gao, J.; Sun, X. RhAGL24 Regulating Auxin-Related Gene RhARF18 Affects Stamen Petaloidy in Rose. Horticulturae 2022, 8, 407. https://doi.org/10.3390/horticulturae8050407

AMA Style

Liu L, Guo Y, Wu Z, Ren H, Jiang Y, Ma N, Gao J, Sun X. RhAGL24 Regulating Auxin-Related Gene RhARF18 Affects Stamen Petaloidy in Rose. Horticulturae. 2022; 8(5):407. https://doi.org/10.3390/horticulturae8050407

Chicago/Turabian Style

Liu, Lin, Yanchao Guo, Zhicheng Wu, Haoran Ren, Yunhe Jiang, Nan Ma, Junping Gao, and Xiaoming Sun. 2022. "RhAGL24 Regulating Auxin-Related Gene RhARF18 Affects Stamen Petaloidy in Rose" Horticulturae 8, no. 5: 407. https://doi.org/10.3390/horticulturae8050407

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