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Article

Characterization of AGAMOUS Ortholog and Promoter from the Ilex verticillata (Aquifoliaceae)

by
Jiayi Li
†,
Yalan Su
†,
Xiangjian Chen
and
Zhixiong Liu
*
College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(10), 1058; https://doi.org/10.3390/horticulturae10101058
Submission received: 15 September 2024 / Revised: 27 September 2024 / Accepted: 30 September 2024 / Published: 3 October 2024
(This article belongs to the Special Issue Propagation and Flowering of Ornamental Plants)
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Abstract

Arabidopsis AGAMOUS (AG) plays a crucial role in specifying stamen and carpel identities, floral meristem identity determination, and repression of the A-function. Ilex verticillata (Aquifoliaceae) is a dioecious shrub, whereby an individual plant has either male or female flowers with vestigial organs of the opposite sex. The molecular mechanism of male and female organ development in I. verticillata remains unknown. In order to identify the possible roles of AG-like genes in regulating floral development in I. verticillata, AG ortholog (IlveAG) and its promoter (pIlveAG) from the male and female flowers of I. verticillata were separately isolated. IlveAG is highly expressed in stamens, pistils, and sepals of male and female flowers. Moreover, obvious GUS staining was observed in the inflorescence and sepals, stamens, and pistils of mature flowers in pIlveAG::GUS Arabidopsis. The 35S::IlveAG Arabidopsis showed obviously early flowering. Moreover, IlveAG could substitute for endogenous AG to rescue the stamen and pistil in the Arabidopsis ag-1 mutant. In addition, expression of IlveAG can inhibit the development of sepals and petals (two outer whorls of floral organs) in wild-type and ag-1 Arabidopsis. Our findings suggest that IlveAG has a conservative C-function and plays key roles in determination of reproductive floral organs (stamen and carpel) identity and meristem determinacy. Our results provide more details to understand the role of AG orthologs in the development of male and female flowers in woody plants.

1. Introduction

Ilex verticillata (L.) A. Gray (Aquifoliaceae) is a deciduous shrub originating from North America and is widely cultivated in many countries in Europe and America. Moreover, Winterberry Holly (I. verticillata) was introduced and widely cultivated in different areas of China for ornamental red winter fruits. I. verticillata is also a very popular ornamental cut flower or potted plant at Chinese Lunar New Year flower markets. In addition, I. verticillata is also an interesting dioecious shrub, whereby an individual plant has either male or female flowers with vestigial organs of the opposite sex. The male flower possesses a vestigial pistil (pistillode), and the female flower possesses vestigial stamens (staminodes) (Figure 1), which make it an excellent model for exploring male and female flower development during origin and evolution of dioecious angiosperm. Moreover, the flowering time is synchronized between male and female trees, resulting in a low fruit setting rate. Improving the pollination and fruit setting rate of I. verticillata requires a better understanding of the molecular mechanisms of the male and female flower development. Previous studies indicated that different floral homeotic MADS transcription factors (TFs) work together to determine the development of different types of floral organs during flower development [1,2].
In Arabidopsis, the C-class MADS-box TF AGAMOUS (AG) plays central roles in determination of reproductive floral organ (stamen and carpel) identity and meristem determinacy [3]. In addition, AG is closely related to the process of sex differentiation, flowering, and fruiting. Previous studies showed that AG orthologs from other rosid species, PrseAG from Prunus lannesiana [4], PPERAG from P. persica [5], KjAG from Kerria japonica [6], and EjAG from Eriobotrya japonica [7], were mainly expressed in stamen and carpel, and also regulated stamen and carpel development, which showed the conservative expression pattern and function of AG orthologs among rosid species.
In this study, we isolated an AG ortholog (IlveAG) and its promoter (pIlveAG) from both the male and female flowers of I. verticillata. Moreover, we analyzed the function of IlveAG and pIlveAG by detecting the expression pattern of the gene and the activity of the promoter, and combining the ectopic expression of the gene and the promoter in Arabidopsis. The flowers of male and female trees of I. verticillata consist of four whorls of floral organs (5–9 sepals in whorl 1, 5–9 petals in whorl 2, 5–9 stamens (staminodes in female flowers) in whorl 3, and 1 pistil (pistillodes in male flowers) in whorl 4 (Figure 1). Our results provide more details for understanding the function of the AG ortholog in male and female flowers of deciduous woody plants.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Male and female floral buds at sequential development stages were separately sampled from male/female trees growing under natural conditions in the campus of Yangtze University (Jingzhou, Hubei, China). Moreover, male and female floral buds were divided in half, and half of them were immediately put into liquid nitrogen and then stored at −80 °C. The remains were fixed in FAA (38% formaldehyde: acetic: 70% ethanol = 1:1:18, by volume). The root, stem, leaf, sepal, petal, stamen, and vestigial pistil of male trees, as well as the root, stem, leaf, sepal, petal, vestigial stamen, pistil, and young fruit of female trees were separately dissected and immediately frozen in liquid nitrogen for RNA isolation. The ag-1 mutant Arabidopsis (CS25) seeds were obtained from the ABRC (Arabidopsis Biological Resource Center) at Ohio State University, Columbus, OH, USA.

2.2. Isolation of IlveAG and IlveAG Promoter (pIlveAG) from I. verticillata

Total RNA was separately extracted from male and female floral buds using the EASYspin Total RNA Kit (Aidlab, Beijing, China) referring to the manufacturer’s protocol. The AG ortholog IlveAG was separately isolated from male and female floral buds by 3′-full RACE Core Set Ver. 2.0 kit (TaKaRa, Shiga, Japan) with forward primer GSPIVAG3 according to manufacturer’s protocol (Table S1). The forward primer was designed based on the transcript (transcript_3468) of DEG (differentially expressed gene) (Ilumina and PacBio sequencing in Biomarker Technologies) (Biomarker Technologies, Beijing, China). Phylogenetic analysis of IlveAG was referenced to Li et al. [8]. All AG homolog proteins from other species were selected for phylogenetic trees obtained from NCBI Genbank (Table S2).
I. verticillata genomic DNA was extracted from leaves using the CTAB Plant Genomic DNA Rapid Extraction Kit (Aidlab, Beijing, China) following the manufacturer’s instructions. The IlveAG promoter was cloned from I. verticillata genomic DNA with the Genome Walking Kit (TaKaRa, Shiga, Japan), referring to the manufacturer’s guidelines. The forward primers GSPilveAG-1R, GSPilveAG-2R, and GSPilveAG-3R (Table S1) were selected for amplifying the first target sequence, and GSPilveAG-4-1R, GSPilveAG-4-2R, and GSPilveAG-3-3R (Table S1) were selected for amplifying the second target sequence. The IlveAG gene and its promoter were sequenced by using Sanger sequencing in Sangon Biotech (Sangon, Shanghai, China). The cis-acting elements lying at the IlveAG promoter (pIlveAG) were searched in the PLACE database [9].

2.3. Characterization of pIlveAG Activity from the Promoter in Transgenic Arabidopsis

The pIlveAG fragment was introduced into the pCAMBIA1300 vector with Xba I (TaKaRa, Shiga, Japan) and Sac I (TaKaRa, Shiga, Japan) by using the ClonExpress® Ultra One Step Cloning Kit (Vazyme, Nanjing, China), referring to the manufacturer’s instructions. The pIlveAG was cloned with the primer pair pIlveAG-F and pIlveAG-R (Table S1). Then, the construct was transformed into Col-0 A. thaliana using the floral-dip method described by Clough and Bent [10]. Transgenic Arabidopsis seedings were obtained and cultivated for histochemical GUS staining according to Liu et al. [11]. The procedure and observation of GUS staining referred to You et al. [12].

2.4. Cytomorphological Examination and Expression Analysis of IlveAG

The male and female floral buds samples of I. verticillata fixed in FAA were separately dehydrated in a graded ethanol series, cleared with a xylene series, infiltrated with molten paraffin, and then embedded into paraffin block, which was serially sectioned with a Leica RM2235 rotary microtome, and then these sections were stained with safranin-fast green using the method suggested by Liu et al. [13]. Each section was microscopically examined using a CAIKON RCK-40C microscope and photomicrographs were taken.
Total RNA of floral buds at sequential developmental stages were separately extracted from male/female trees with EASYspin Total RNA Kit (Aidlab, Beijing, China) according to the manufacturer’s instructions. The first-stand cDNA was synthesized for qRT-PCR with HiScript® II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. IlveAG expression was separately measured in root, stem, leaf, sepal, petal, stamen, and vestigial pistils of male trees, and in root, stem, leaf, sepal, petal, vestigial stamen, pistil, and young fruit of female trees by qRT-PCR, referring to Liu et al. [11], but with primer pair qIlveAGF and qIlveAGR (Table S1). In addition, IlveAG expression was also separately detected in male and female floral buds at sequential development stages. An amplification fragment of the I. verticillata actin gene was selected as the internal control with primer pair qIlveactinF and qIlveactinR. The qRT-PCR was performed with three biological replicates and relative expression levels were detected, referring to Liu et al. [11], but with 30 s annealing at 57 °C.

2.5. Ectopic Expression Analysis of IlveAG in Arabidopsis ag-1 Mutant

Full-length IlveAG cDNA was cloned into the pBI121 vector with Xba I and Sac I using the ClonExpress® Ultra One Step Cloning Kit (Vazyme, Nanjing, China), referring to the manufacturer’s instructions, but with primer pair TIlveAGF2 and TIlveAGR (Table S1). The 35S::IlveAG construct was introduced into the heterozygous AG/ag-1 Arabidopsis with the above method. Transgenic Arabidopsis seeds were screened and seedlings were cultivated in a growth chamber according to Li et al. [14]. Wild-type, heterozygous AG/ag-1, and homozygous ag-1 transgenic Arabidopsis lines were confirmed by the dCAPS finder program described by Neff et al. [15]. The transgenic Arabidopsis phenotypes were analyzed after flowering.

3. Results

3.1. Characterization of IlveAG and IlveAG Promoter (pIlveAG) from I. verticillata

The 1088 bp IlveAG cDNA has a 726 bp ORF (Open Reading Frame), which encodes 241aa (amino acids) (Genbank accession number: PP415536). Phylogenetic tree analysis grouped IlveAG into core eudicots with euAG lineage (Figure 2), and the gene was named as IlveAG (Ilex verticillata AGAMOUS).
A 1669 bp IlveAG promoter (pIlveAG) (−1297/+372) was cloned from I. verticillata (Figure S1). The pIlveAG contains eight POLLEN1LELAT52-box and six GTGANTG10-box [16,17], which are usually located at the promoter of the genes involved in stamen development and suggest that IlveAG may be involved in regulating stamen development in I. verticillata. The pIlveAG also contains two CArG-box for floral homeotic MADS-box TFs [18] and one CCAAT-box for CONSTANS TF binding to promote flowering [19], as well as two AACAAA/TTTGTT motifs for floral homeotic APETALA2 TF binding [20]. All these data suggest that pIlveAG may drive the IlveAG gene to promote flowering and regulate floral organ development. In addition, gibberellin-responsive elements (PYRIMIDINEBOXOSRAMY1A-box) [21], abscisic-acid-responsive elements (MYB1AT) [22], drought-stress-responsive elements (MYBCORE) [23], and low-temperature-stress-responsive elements (MYCCONSENSUSAT) [24] were found in pIlveAG. These data suggest that hormone and stress signaling pathways may also directly induce IlveAG expression.

3.2. Expression Activity Analysis of pIlveAG in Transgenic Arabidopsis

A GUS reporter gene driven by pIlveAG was assayed in transgenic Arabidopsis. GUS staining was examined in the T1 generation of pIlveAG::GUS independent transgenic lines (Figure 3). Intense GUS staining was obviously observed in the inflorescence and sepals, stamens (anther and filament), and pistils (stigma and style) of mature flowers, but was absent in petals. In addition, GUS reporter gene driven by pIlveAG was activated in sepals, filaments, and pistils of floral buds at stage 12, but was absent in petals and anthers. These data suggest that the pIlveAG may drive IlveAG to regulate inflorescence and floral organ development in I. verticillata.

3.3. Expression Analysis of IlveAG in I. verticillata

IlveAG was obviously expressed in all floral organs and young fruits of the I. verticillata, but was absent in roots, stems, and leaves (Figure 4C). In the male/female trees, the expression level of the IlveAG in stamens was significantly higher than that in sepals, petals, pistils, and young fruit (LSD, p < 0.01). The expression level in petals was lowest among the four whorl floral organs (LSD, p < 0.01). In addition, obvious expression of IlveAG was also detected during the male/female floral bud differentiation. Furthermore, the expression level of IlveAG reached the peak at pollen maturation of male floral buds, which was significantly higher than that at other developmental stages (LSD, p < 0.01) (Figure 5A,B). In the female flower buds, the expression of IlveAG decreased in the stage of linear tetrad of megasporocyte (Figure 5A,C).

3.4. Ectopic Expression of IlveAG in ag-1 Arabidopsis Mutant

To evaluate whether IlveAG could complete the loss-of-function AG phenotype in ag-1 mutant Arabidopsis, 35S::IlveAG was transformed into heterozygote AG/ag-1 Arabidopsis to create complementation lines through agrobacterium mediation. Independent transgenic Arabidopsis lines were identified by dCAPS genotyping. Moreover, twelve 35S::IlveAG lines in the wild-type background and eleven independent 35S::IlveAG lines in the homozygous mutant ag-1 background were obtained. In addition, phenotypes of transgenic Arabidopsis lines in the wild-type and homozygous ag-1 mutant backgrounds were separately assayed to evaluate whether IlveAG could mimic endogenous AG in specifying stamen and carpel identity.
Among twelve 35S::IlveAG transgenic Arabidopis lines in the wild-type background, all lines were observed obviously flowering early. Seven (58.33%) lines had floral organ phenotype changes (Figure 6C–I); one (8.33%) showed its sepal converted into a carpeloid organ (Figure 6C); one (8.33%) showed a small sepal, petal, and stamen (Figure 6D); and five (41.67%) showed decreased sepal numbers (Figure 6E–I), which suggested that IlveAG conserved C-function activity and inhibited the development of the outer two whorls of floral organs (sepal and petal).
Among eleven 35S::IlveAG transgenic Arabidopis lines in the homozygous ag-1 mutant backgrounds, one (9.10%) produced flowers only with pistil and carpeloid sepals (Figure 7C), one (9.10%) produced flowers with normal pistils, partial stamens, and one sepal transformed into pistil (Figure 7D). Two (18.18%) lines completely restored stamens and pistils (Figure 7E,F), and one (9.10%) produced partial filaments and carpels (Figure 7G). The infinite development pattern of flowers was terminated. Two (18.18%) lines displayed a small number of stamens within the sepal and petal nested pattern (Figure 7H,I). The remaining four (36.36%) lines were similar to the homozygous ag-1 mutant Arabidopsis flowers (Figure 7B).

4. Discussion

I. verticillata is a dioecious shrub, whereby an individual plant has either male or female flowers with vestigial organs of the opposite sex. The male flower contains pistillodes, while the female flower contains staminodes, which make it an excellent model for exploring male and female flower development during origin and evolution of dioecious angiosperm. In Arabidopsis, AG plays key roles in controlling reproductive floral organ (stamen and carpel) identity and determining floral meristem identity [3]. Moreover, AG and APETALA2 (AP2) represent C- and A-class genes that work in an antagonistic fashion to specify reproductive organs (stamen and pistil) and perianth (sepal and petal), respectively [25]. AP2 binds at the AG second intron and directly restricts AG expression extending to the outer two whorls [20]. The expression of AG begins at stage 3 of flower development, when stamen and pistil primordia arise, and its expression persists throughout the development of the stamen and pistil [26]. In I. verticillata, IlveAG was obviously expressed in all floral organs and was highest in stamens (staminodes). Functional analysis indicated that IlveAG could mimic endogenous AG in determining stamen and carpel identity in ag-1 mutant Arabidopsis. In rosid species, AG orthologs, such as TrAG from T. rupestris [27], MASAKO C1 from Rosa rugosa [28], and EjAG from Eriobotrya japonica [7], were mainly expressed in stamens and pistils. Moreover, ectopic expression of each gene showed similar results with IlveAG and exhibited a conserved function in specifying reproductive (stamen and pistil) organ identity.
In asterids species, PMADS3 from Petunia hybrida was expressed in the stamen, pistil, and ovule. Ectopic expression of PMADS3 could lead to petals being converted into anthers [29]. In Cyclamen persicum, CpAG1 and CpAG2 were expressed in stamen and carpel. CpAG1 is mainly involved in controlling stamen development, while CpAG2 is mainly involved in specifying carpel identity and meristematic activity termination. Both genes work together to complete AG function [30,31]. In Nicotiana benthamiana, NbAG alone is able to specify stamen identity, while the C-function for pistil development needs both NbAG and NbSHP. However, the absence of the less active NbSHP factor only results in mild defects, and only simultaneous expression of NbAG and NbSHP can lead to floral meristem termination [32].
In other core eudicots, such as Fagopyrum esculentum, FaesAG is expressed only in stamens and pistils, and could mimic endogenous AG in determination of stamen and carpel identity in ag-1 mutant Arabidopsis [14]. In basal eudicots, such as Nigella damascene, NdAG1 determines stamen and carpel identities, while NdAG2 participates in carpel development and floral determinacy [33]. In the basal angiosperms, such as Magnolia wufengensis, obvious MawuAG1 expression was observed in the stamen, carpel, ovule, and seed, while MawuAG2 was observed only in the stamen and carpel. Overexpression of MawuAG1 caused sepals to be converted into carpeloid structures, with ovules and petals converted into stamen-like structures. However, overexpression of MawuAG2 was only able to make petals convert into stamens [34]. However, M. stellata AG-like gene, MastAG, plays key role in carpel identity [35]. These data suggest a certain functional conservation and versatility of AG orthologs among different eudicot species.
In I. verticillata, IlveAG is highly expressed in stamens, pistils, and sepals of male and female flowers. Moreover, pIlveAG has CArG-box for the binding of MADS-box TF and AACAAA/TTTGTT motif for recognition and binding by floral homeotic APETALA2 TF [18,20]. In addition, pIlveAG also contains some cis-regulatory elements for recognition and binding by R2R3-MYB TFs [23,24]. In Petunia axillaris, the R2R3-MYB TF EOB2 is a repressor of flower bud senescence by inhibiting ethylene production and is involved in both petal and pistil maturation through regulation of primary and secondary metabolism [36]. The DlR2R3-MYB from Dimocarpus longan tends to play conserved roles in the flowering regulation and stress responses [37]. Moreover, GAMYB plays an important role in pollen development in both poplars and willows [38]. All these suggest that flowering and floral development may be induced by stress through the R2R3-MYB and IlveAG pathways. I. verticillata may regulate flowering or floral organ development through IlveAG. Additionally, pIlveAG can drive GUS to express in sepals, stamens, and carpels of transgenic Arabidopsis. Ectopic expression of IlveAG leads to early flowering in transgenic Arabidopsis. Moreover, IlveAG fully completes floral defects in the ag-1 mutant Arabidopsis, and IlveAG can inhibit the development of sepals and petals. These results suggest that IlveAG can substitute the endogenous AG gene of Arabidopsis ag-1 mutant to participate in the regulation of stamen and carpel development, promote the formation of floral meristem and regulate flowering, and inhibit the activity of A-class genes. IlveAG is a C-class gene for the regulation of flower development in I. verticillata. Our findings reveal the role of the IlveAG gene in the development of male and female flowers of I. verticillata, and provide more details for further analysis of flowering and fruiting of woody plants.

5. Conclusions

I. verticillata is a dioecious shrub, whereby an individual plant has either male or female flowers with vestigial organs of the opposite sex. The male flower contains pistillodes, and the female flower contains staminodes. Furthermore, the development of male and female flower buds was not synchronized. In Arabidopsis, AG plays key roles in specifying reproductive organ (stamen and pistil) identity and floral meristem determinacy, as well as repression of the A-function. However, the mechanism of male and female floral organ development in I. verticillata is still unknown. Here, an AG ortholog (IlveAG) and its promoter (pIlveAG) were isolated and characterized in I. verticillata. IlveAG is highly expressed in stamens, pistils, and sepals of male and female flowers. Moreover, pIlveAG could drive GUS to obviously express in stamens, carpels, and sepals of transgenic Arabidopsis. Ectopic expression of IlveAG leads to early flowering in transgenic Arabidopsis. Moreover, IlveAG could substitute for endogenous AG to rescue stamens and pistils in the Arabidopsis ag-1 mutant. In addition, ectopic expression of IlveAG inhibits the development of the two outer whorls’ floral organs (sepals and petals). Our findings show that IlveAG has a conservative C-function and plays roles in determining the identity of reproductive organs (stamen and pistil) and regulating meristem determinacy. Our results reveal the expression pattern and function of AG orthologs in the male and female flowers of woody plants, and provide a basis for further analysis of the flowering of I. verticillata.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10101058/s1, Figure S1: IlveAG promoter sequence; Table S1: Primers used in this study; Table S2: Information on Sequences selected for alignments and phylogenetic analyses from NCBI GenBank.

Author Contributions

J.L., writing—original draft preparation; J.L., Y.S. and X.C., methodology; Z.L., writing—review and editing, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Female and male flower of Ilex verticillata (L.) A. Gray. (A,B) Female flower with 7 staminodes; (C,D) Male flower with a pistillode. Petal (pet), staminode (stn), pistil (pis), stamen (sta), pistillode (pil). Scale bar = 1 mm.
Figure 1. Female and male flower of Ilex verticillata (L.) A. Gray. (A,B) Female flower with 7 staminodes; (C,D) Male flower with a pistillode. Petal (pet), staminode (stn), pistil (pis), stamen (sta), pistillode (pil). Scale bar = 1 mm.
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Figure 2. Phylogenetic tree of IlveAG and AG-like transcription factors from other species of angiosperms.
Figure 2. Phylogenetic tree of IlveAG and AG-like transcription factors from other species of angiosperms.
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Figure 3. Histochemical GUS staining of pIlveAG promoter in T1 transgenic Arabidopsis thaliana. (A) Inflorescence of wild-type Arabidopsis; (B) flower of wild-type Arabidopsis; (C) floral bud at stage 12 in wild-type Arabidopsis; (D) inflorescence of pIlveAG::GUS transgenic Arabidopsis; (E) flower of pIlveAG::GUS transgenic Arabidopsis; (F) floral bud at stage 12 in pIlveAG::GUS transgenic Arabidopsis. Sepal (sep), petal (pet), anther (ant), filament (fil), stigma (sti), style (sty), ovary (ova); scale bars: (A,B,D,E) 1 mm, (C,F) 500 µm.
Figure 3. Histochemical GUS staining of pIlveAG promoter in T1 transgenic Arabidopsis thaliana. (A) Inflorescence of wild-type Arabidopsis; (B) flower of wild-type Arabidopsis; (C) floral bud at stage 12 in wild-type Arabidopsis; (D) inflorescence of pIlveAG::GUS transgenic Arabidopsis; (E) flower of pIlveAG::GUS transgenic Arabidopsis; (F) floral bud at stage 12 in pIlveAG::GUS transgenic Arabidopsis. Sepal (sep), petal (pet), anther (ant), filament (fil), stigma (sti), style (sty), ovary (ova); scale bars: (A,B,D,E) 1 mm, (C,F) 500 µm.
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Figure 4. The expression of IlveAG in male and female trees of I. verticillata. (A,B) Flower bud structure of male and female trees in I. verticillata; (C) IlveAG expression in the root, stem, leaf, sepal, petal, stamen, pistil, and fruit were measured by qRT-PCR with IlveAG actin as the internal control. Scale bar: (A,B) 500 μm; Different letters indicate a significant difference (p < 0.01, LSD).
Figure 4. The expression of IlveAG in male and female trees of I. verticillata. (A,B) Flower bud structure of male and female trees in I. verticillata; (C) IlveAG expression in the root, stem, leaf, sepal, petal, stamen, pistil, and fruit were measured by qRT-PCR with IlveAG actin as the internal control. Scale bar: (A,B) 500 μm; Different letters indicate a significant difference (p < 0.01, LSD).
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Figure 5. The expression of IlveAG in male and female floral buds of I. verticillata at different developmental stages. (A) Cytomorphological section of floral male buds at different development stages, M1: microspore mother cell formation; M2: microspore mother cell meiosis; M3: microspore tetrad formation; M4: mononuclear microspore in nucleus located aside stage; M5: full-maturity flower buds with pollen maturation. Cytomorphological section of female buds at sequential development stages, F1: microspore mother cell formation and pistil primodium development; F2: ovule appearance; F3: meiosis period of megasporocyte; F4: linear tetrad period of megasporocyte; F5: embryo sac maturation. (B) The expression of IlveAG in different stages of male flower buds; (C) the expression of IlveAG in different developmental stages of female flower buds; scale bar: (A) 100 μm; different lowercase letters show significant differences (p < 0.01 by LSD).
Figure 5. The expression of IlveAG in male and female floral buds of I. verticillata at different developmental stages. (A) Cytomorphological section of floral male buds at different development stages, M1: microspore mother cell formation; M2: microspore mother cell meiosis; M3: microspore tetrad formation; M4: mononuclear microspore in nucleus located aside stage; M5: full-maturity flower buds with pollen maturation. Cytomorphological section of female buds at sequential development stages, F1: microspore mother cell formation and pistil primodium development; F2: ovule appearance; F3: meiosis period of megasporocyte; F4: linear tetrad period of megasporocyte; F5: embryo sac maturation. (B) The expression of IlveAG in different stages of male flower buds; (C) the expression of IlveAG in different developmental stages of female flower buds; scale bar: (A) 100 μm; different lowercase letters show significant differences (p < 0.01 by LSD).
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Figure 6. Phenotypes of 35S::IlveAG transgenic Arabidopsis in the wild-type background. (A) Wild-type Arabidopsis with normal raceme; (B) normal flower of wild-type Arabidopsis thaliana (1st whorl with 4 sepals, 2nd whorl with 4 petals, 3rd whorl with 6 stamens, 4th whorl with 1 pistil); (C) 35S::IlveAG transgenic Arabidopsis flower with one sepal transformed into a carpeloid organ and the number of sepals, petals, and stamens reduced; (D) 35S::IlveAG transgenic Arabidopsis flower with smaller sepals, petals, and stamens; (E) 35S::IlveAG transgenic Arabidopsis flower with smaller sepals and petals; (F) 35S::IlveAG transgenic Arabidopsis flower with fewer sepals, petals, and stamens; (G) 35S::IlveAG transgenic Arabidopsis flower with small sepals; (H) 35S::IlveAG transgenic Arabidopsis flower with short stamens and fewer sepals; (I) 35S::IlveAG transgenic Arabidopsis flower with small stamens. Sepal (sep), petal (pet), anther (ant), pistil (pis), stigma (sti), ovule (ovu). Scale bars: 1 mm.
Figure 6. Phenotypes of 35S::IlveAG transgenic Arabidopsis in the wild-type background. (A) Wild-type Arabidopsis with normal raceme; (B) normal flower of wild-type Arabidopsis thaliana (1st whorl with 4 sepals, 2nd whorl with 4 petals, 3rd whorl with 6 stamens, 4th whorl with 1 pistil); (C) 35S::IlveAG transgenic Arabidopsis flower with one sepal transformed into a carpeloid organ and the number of sepals, petals, and stamens reduced; (D) 35S::IlveAG transgenic Arabidopsis flower with smaller sepals, petals, and stamens; (E) 35S::IlveAG transgenic Arabidopsis flower with smaller sepals and petals; (F) 35S::IlveAG transgenic Arabidopsis flower with fewer sepals, petals, and stamens; (G) 35S::IlveAG transgenic Arabidopsis flower with small sepals; (H) 35S::IlveAG transgenic Arabidopsis flower with short stamens and fewer sepals; (I) 35S::IlveAG transgenic Arabidopsis flower with small stamens. Sepal (sep), petal (pet), anther (ant), pistil (pis), stigma (sti), ovule (ovu). Scale bars: 1 mm.
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Figure 7. Phenotypes of 35S::IlveAG homozygous transgenic Arabidopsis ag-1 mutants. (A) Arabidopsis ag-1 mutant raceme; (B) homozygous Arabidopsis ag-1 mutant flower (sepals in the 1st whorl, petals in the 2nd and 3rd whorls, and reiterations of this pattern in interior whorls); (C) 35S::IlveAG transgenic ag-1 Arabidopsis flower with pistil and carpeloid sepal; (D) 35S::IlveAG transgenic ag-1 Arabidopsis flower with normal pistil, partial stamen, and one sepal transformed into pistil; (E) 35S::IlveAG transgenic ag-1 Arabidopsis flower with restored pistils and stamens; (F) 35S::IlveAG transgenic ag-1 Arabidopsis flower recovered four whorls of floral organs; (G) 35S::IlveAG transgenic ag-1 Arabidopsis flower with complementation of filaments and carpels; (H) 35S::IlveAG transgenic ag-1 Arabidopsis flower with the appearance of filaments; (I) 35S::IlveAG transgenic ag-1 Arabidopsis flower with the appearance of filaments and partial carpels. Sepal (sep), petal (pet), anther (ant), pistil (pis), stigma (sti), ovule (ovu), filament (fil), carpel (car). Scale bars: 1 mm.
Figure 7. Phenotypes of 35S::IlveAG homozygous transgenic Arabidopsis ag-1 mutants. (A) Arabidopsis ag-1 mutant raceme; (B) homozygous Arabidopsis ag-1 mutant flower (sepals in the 1st whorl, petals in the 2nd and 3rd whorls, and reiterations of this pattern in interior whorls); (C) 35S::IlveAG transgenic ag-1 Arabidopsis flower with pistil and carpeloid sepal; (D) 35S::IlveAG transgenic ag-1 Arabidopsis flower with normal pistil, partial stamen, and one sepal transformed into pistil; (E) 35S::IlveAG transgenic ag-1 Arabidopsis flower with restored pistils and stamens; (F) 35S::IlveAG transgenic ag-1 Arabidopsis flower recovered four whorls of floral organs; (G) 35S::IlveAG transgenic ag-1 Arabidopsis flower with complementation of filaments and carpels; (H) 35S::IlveAG transgenic ag-1 Arabidopsis flower with the appearance of filaments; (I) 35S::IlveAG transgenic ag-1 Arabidopsis flower with the appearance of filaments and partial carpels. Sepal (sep), petal (pet), anther (ant), pistil (pis), stigma (sti), ovule (ovu), filament (fil), carpel (car). Scale bars: 1 mm.
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MDPI and ACS Style

Li, J.; Su, Y.; Chen, X.; Liu, Z. Characterization of AGAMOUS Ortholog and Promoter from the Ilex verticillata (Aquifoliaceae). Horticulturae 2024, 10, 1058. https://doi.org/10.3390/horticulturae10101058

AMA Style

Li J, Su Y, Chen X, Liu Z. Characterization of AGAMOUS Ortholog and Promoter from the Ilex verticillata (Aquifoliaceae). Horticulturae. 2024; 10(10):1058. https://doi.org/10.3390/horticulturae10101058

Chicago/Turabian Style

Li, Jiayi, Yalan Su, Xiangjian Chen, and Zhixiong Liu. 2024. "Characterization of AGAMOUS Ortholog and Promoter from the Ilex verticillata (Aquifoliaceae)" Horticulturae 10, no. 10: 1058. https://doi.org/10.3390/horticulturae10101058

APA Style

Li, J., Su, Y., Chen, X., & Liu, Z. (2024). Characterization of AGAMOUS Ortholog and Promoter from the Ilex verticillata (Aquifoliaceae). Horticulturae, 10(10), 1058. https://doi.org/10.3390/horticulturae10101058

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